Electron Transport Proteins of Synechococcus Sp. Pcc 7002

The Pennsylvania State University The Graduate School Department of Biochemistry, Microbiology, and Molecular Biology

ELECTRON TRANSPORT PROTEINS OF SYNECHOCOCCUS SP. PCC 7002

A Thesis in Biochemistry, Microbiology, and Molecular Biology by Christopher T. Nomura

Submitted in Partial Fulfillment of the Requirements for the Degree of

Doctor of Philosophy

May 2001 Date of Signature We approve the thesis of Christopher T. Nomura

Donald A. Bryant Ernest C. Pollard Professor of Biotechnology Professor of Biochemistry and Molecular Biology Chair of Committee

John H. Golbeck Professor of Biochemistry and Biophysics

Ola Sodeinde Assistant Professor of Biochemistry and Molecular Biology

Paul Babitzke Associate Professor of Biochemistry and Molecular Biology

Juliette T. J. Lecomte Associate Professor of Chemistry

Robert A. Schlegel Professor of Biochemistry and Molecular Biology Head of the Department of Biochemistry and Molecular Biology iii

ABSTRACT

Cyanobacteria are photosynthetic, oxygen-evolving prokaryotes that have adapted to a wide range of ecological niches. In particular, cyanobacteria represent interesting organisms to study electron transport in because they have both photosynthetic and respiratory proteins involved in electron transport on the same membrane: the thylakoid membrane. Of particular interest to our lab is the identification and characterization of the minimal conserved number of genes responsible for coding proteins used in electron transport in cyanobacteria. In order to address this issue, a Synechococcus sp. PCC 7002 cosmid library was screened with heterologous probes made from the completely sequenced genome of the freshwater cyanobacterium, Synechocystis sp. PCC 6803.

These heterologous probes were also used to screen Synechococcus sp. PCC 7002 partial genomic libraries in the cases where positive hybridizations could not be identified within the cosmid libraries. In this study, 35 open reading frames in the marine cyanobacterium

Synechococcus sp. PCC 7002 have been identified and sequenced which, either, encode electron transport proteins or encode accessory proteins necessary for the assembly of these electron transport proteins based on BLAST algorithm searches. These open reading frames are represented by the following genes: ndhA, ndhI, ndhG, ndhE, ndhB, ndhC, ndhK, ndhJ, ndhD1, ndhD2, ndhD3, ndhD4, ndhF1, ndhF2, ndhF3, ndhF4, ndhF5, iv ndhH, ndhL, ndbA, ndbB, hypE, hoxE, hoxF, hoxU, hoxY, hypD, hoxH, hoxW, hypA, hypB, hypF, hypC, hypD, petJ1, petJ2, bcpA, ctaCI, ctaDI, ctaEI, ctaCII, ctaDII, and ctaEII. These genes putatively encode subunits for the type I NADH dehydrogenase, two type II NADH dehydrogenases, the subunits and accessory proteins for a bi-

directional hydrogenase, and three putative mobile electron carriers: cytochrome c6, cytochrome c62, and BcpA, and the subunits for two different members of the heme- copper cytochrome oxidase family. Most of these genes bear the highest homology to their respective counterparts in the freshwater cyanobacterial strain Synechocystis sp.

PCC 6803 and a comparison between these minimal conserved sets of genes may represent the smallest number of genes necessary for these organisms to carry out electron transport.

Attempts were made to inactivate several of the electron transfer protein genes identified in this study. These include the ndhB gene, which, encodes a subunit of the

type I NADH dehydrogenase, the petJ1 gene that encodes cytochrome c6, the petJ2 gene that encodes a putative second c-type cytochrome, the bcpA gene which encodes a putative blue-copper protein, the hoxH gene which encodes the large Ni containing subunit of the hydrogenase enzyme, the hoxF gene which encodes the FMN containing subunit of the hydrogenase enzyme, the ctaDI gene, which encodes the large subunit of the type I cyanobacterial cytochrome oxidase, and the ctaDII gene, which encodes the large subunit of a secondary heme-copper oxidase in Synechococcus sp. PCC 7002. This study found that the ndhB and petJ1genes are essential for the viability of Synechococcus sp. PCC 7002 and therefore could not be inactivated. The hoxH, hoxF, petJ2, bcpA, v ctaDI, and ctaDII genes are all non-essential to Synechococcus sp. PCC 702 under normal growth conditions and could be inactivated.

Physiological studies of the hoxH, hoxF, petJ2, and bcpA inactivated strains of

Synechococcus sp. PCC 7002 revealed that there were no significant phenotypes under the conditions tested. However, evidence has been found in this study that the heme- copper oxidase enzymes encoded by the ctaCIDIEI and ctaCIIDIIEII gene clusters have significant roles in respiration, high-light tolerance and oxidative stress responses in

Synechococcus sp. PCC 7002. Table of Contents

List of Figures xiii List of Tables xviii Acknowledgments xx Chapter 1 INTRODUCTION 1 1.1 Electron Transport Proteins 1 1.1.1 Type I NADH dehydrogenase 4 1.1.2 Type II NADH dehydrogenase 7 1.1.3 Hydrogenase 9 1.1.4 Mobile electron carriers 12

1.1.4.1 Cytochrome c6 13 1.1.4.2 Plastocyanin 16 1.1.5 Terminal oxidases 17 1.2 Purpose of the present work 22

Chapter 2 MATERIALS AND METHODS 24 2.1 Bacterial strains and growth conditions 24 2.1.1 Synechococcus sp. PCC 7002 24 2.1.2 Synechocystis sp. PCC 6803 26 2.1.3 Escherichia coli 27 2.2 Standard laboratory methods 28 2.3 Isolation and manipulation of DNA 29 vii

2.3.1 Plasmid and cosmid isolation from Escherichia coli 29 2.3.2 Total DNA isolation from Synechococus sp. PCC 7002 30 and Synechocystis sp. PCC 6803 2.4 Transformation Procedures 31 2.4.1 E. coli transformation procedures 31 2.4.2 Cyanobacterial transformation procedures 32 2.5 Southern blot transfer and hybridization 34 2.6 Cloning and sequencing 35 2.6.1 General cloning strategy 35 2.6.2 Construction of a Synechococcus sp. PCC 7002 genomic library 36 2.6.3 Probes 36 2.6.4 Colony hybridization 38 2.6.5 DNA sequencing and analysis 39 2.7 RNA isolation, Northern-blot hybridization, and RT-PCR analysis 40 2.7.1 RNA isolation 40 2.7.2 Northern-blot hybridization analyses 41

2.7.3 Reverse transcriptase-polymerase chain reaction (RT-PCR) 43 2.8 Overproduction of the rBcpA protein in Escherichia coli 44 2.8.1 Generation of E. coli expression plasmids 44 2.8.2 Protein overproduction in E. coli and isolation of inclusion bodies 45 2.8.3 Purification of rBcpA from E. coli 46

2.9 Cytochrome c6 purification from Synechococcus sp. PCC 7002 47 and amino terminal sequencing 2.10 SDS-polyacrylamide gel electrophoreses, TMBZ staining and 48 immunoblot analysis viii

2.10.1 SDS PAGE 48 2.10.2 3, 3', 5, 5'-tetramethylbenzidine (TMBZ) staining 49 2.10.3 Immunoblot analysis 50 2.11 Determination of chlorophyll a and carotenoid contents 51 2.12 Oxygen evolution and consumption rates 52 2.13 P-700+ kinetic measurements 52 2.14 77K fluorescence emission measurements 54 2.15 Pulse Amplitude Modulated (PAM) fluorescence measurements 54 2.16 Superoxide dismutase (SOD) activity measurements 55 2.17 Detection of hydroperoxides 56 2.18 Catalase activity 57 2.19 Peroxidase activity 57 2.20 Cell viability 58

Chapter 3 RESULTS 60 Cloning of electron transport protein genes 60 3.1. Type I NADH dehydrogenase 66 3.1.1 ndhAIGE 67 3.1.2 ndhB 70 3.1.2.1 Attempted mutagenesis of the Synechococcus sp 73 PCC 7002 ndhB gene by interposon mutagenesis 3.1.3 ndhD1, ndhD2, ndhD3, ndhD4 78 3.1.4 ndhF1, ndhF2, ndhF3, ndhF4, ndhF5 83 3.1.5 Gene organization and phlyogenetic analysis of the ndhD 89 and ndhF genes in cyanobacteria ix

3.1.6 ndhCKJ 95 3.1.7 ndhH 97 3.1.8 ndhL 100 3.2 Type II NADH dehydrogenases 103 3.3 Bi-directional hydrogenase 107 3.3.1 hypE 109 3.3.2 hoxE 109 3.3.3 hoxF 109 3.3.4 hoxU 110 3.3.5 hoxY 111 3.3.6 hyp3 111 3.3.7 hoxH 111 3.3.8 hoxW 112 3.3.9 hypA 112 3.3.10 hypB 113 3.3.11 hypF 113 3.3.12 hypC 116 3.3.13 hypD 116 3.3.14 Interposon mutagenesis of the hoxH and hoxF genes in 117 Synechococcus sp. PCC 7002 3.3.15 Chlorophyll and carotenoid contents of the hoxH- and hoxH-/hoxF- 123 strains 3.3.16 Growth analysis of hoxH- and hoxH-/hoxF- double mutants 124

3.4 Mobile electron carriers 127 x

3.4.1 Cloning and sequencing of the Synechococcus sp. PCC 7002 127

cytochrome c6 gene 3.4.2 Attempted deletion of the petJ1 gene by interposon mutagenesis 138 3.4.3 Attempted functional substitution of the Synechococcus sp. 142 strain PCC 7002 petJ gene with the petE and petJ genes from Synechocystis sp. strain PCC 6803 3.4.4 petJ2 157 3.4.5 Interposon mutagenesis of the petJ2 gene in Synechococcus sp. 159 PCC 7002 3.4.6 Growth analysis of petJ2::aphII 162 3.4.7 cytM 165 3.4.8 bcpA 169 3.4.9 Interposon mutagenesis of the bcpA gene from Synechococcus sp. 172 PCC 7002 3.4.10 Growth rate analysis of bcpA- strains of Synechococcus sp. 176 PCC 7002 3.4.11 Overproduction of the BcpA protein in E. coli 176 3.4.12 Immunoblot analysis of rBcpA 180

3.5 Heme-Copper Oxidases in Synechococcus sp. PCC 7002 186 3.5.1 Screening and Cloning of the ctaI and ctaII gene clusters from 186 Synechococcus sp. PCC 7002 3.5.2 Insertional Mutagenesis of ctaDI and ctaDII from 190 Synechococcus sp. PCC 7002 xi

3.5.3 Expression of the cta gene clusters in Synechococcus sp. PCC 193 7002 3.5.4 Respiratory activity and oxygen evolution activity in 203 Synechococcus sp. PCC 7002 wild type, ctaDI-, and ctaDII- strains 3.5.5 Growth analysis of Synechococcus sp. PCC 7002 wild type, ctaDI-, 206 ctaDII-, and ctaDI- ctaDII- strains 3.5.6 Chlorophyll and carotenoid contents of wild type, ctaDI-, ctaDII- 211 ctaDI- ctaDII- strains 3.5.7 Photoinhibition of whole chain electron transport activity in 213 Synechococcus sp. PCC 7002 wild type and ctaDII-, and ctaDI- ctaDII- strains

3.5.8 Fluorescence emission at 77K of wild-type, ctaDI-, ctaDII- and 215 ctaDI- ctaDII- strains of Synechococcus sp. PCC 7002 3.5.9 P700+ Reduction Kinetics 219 3.5.10 Pulse Amplitude Modulated Fluorescence Measurements 222 3.5.11 Oxidative Stress and Cell Viability of Synechococcus sp. 223 PCC 7002 wild type and ctaD mutant strains 3.5.12 Superoxide Dismutase (SOD) Activity Assays 229 3.5.13 Hydroperoxide levels in Synechococcus sp. PCC 7002 231 3.5.14 Catalase and peroxidase activity in Synechococcus sp. PCC 7002 233

Chapter 4 DISCUSSIONS AND CONCLUSIONS 235 4.1 Genes and gene organization in cyanobacteria 235 4.1.1 Future directions of the gene sequencing project in 237 xii

Synechococcus sp. PCC 7002 4.2 Type I NADH dehydrogenase 238 4.2.1 Future directions for research of the type I NADH dehydrogenease 241 genes in Synechococcus sp. PCC 7002 4.3 Type II NADH dehydrogenases 242 4.3.1 Future directions for research of the type II NADH dehydrogenease 243 genes in Synechococcus sp. PCC 7002 4.4 Bi-directional hydrogenase 244 4.4.1 Future directions for research of bidirectional hydrogenase and 245 hyp genes in Synechococcus sp. PCC 7002 4.5 Mobile electron carriers 247 4.5.1 Future directions for research of the mobile electron carrier genes 252 in Synechococcus sp. PCC 7002 4.6 Terminal Oxidases Present in Synechococcus sp. PCC 7002 253 4.6.1 Effects of Cytochrome Oxidase on Electron Flow Around PSII 257 4.6.2 Cytochrome Oxidase and its Role in High Light Stress 258 4.6.3 Tolerance of Methyl Viologen and High Light Stress in ctaDII- 259 strains of Synechococcus sp. PCC 7002 4.6.4 Future research directions for cta- mutants in Synechococcus 261 sp. PCC 7002 4.7 Concluding Remarks 267 References 270 Appendix A: Restriction maps of electron transport genes 296 Appendix B: ClustalW alignments of electron transport proteins 316 xiii

List of Figures

Figure 1 Depiction of the cyanobacterial thylakoid membrane 2

Figure 2 Schematic illustration of the similarities and difference of 20 four subclasses of the heme-copper oxidase superfamily

Figure 3 Gene organization of ndhAIGE gene cluster in cyanobacteria 68

Figure 4 Gene organization around the ndhB gene in cyanobacteria 72

Figure 5 The ndhB gene of Synechococcus sp. PCC 7002 is 77 required for cell viability

Figure 6 ClustalW alignment of NdhD amino acid sequences from 81 Synechococcus sp. PCC 7002

Figure 7 ClustalW alignment of NdhF amino acid sequences from 86 Synechococcus sp. PCC 7002

Figure 8 Gene organization of ndhF and ndhD genes 91

Figure 9 Phylogenetic analysis of NdhF and NdhD proteins 94

Figure 10 Gene organization of ndhCJK gene clusters 96

Figure 11 Gene organization around the ndhH gene in cyanobacateria 99

Figure 12 Gene organization around the ndhL gene in cyanobacteria 102

Figure 13 Gene organization around the ndbA gene in cyanobacteria 104 xiv

Figure 14 Gene organization around the ndbB gene in cyanobacteria 106

Figure 15 Hydrogenase gene cluster organization 108

Figure 16 HypF and acylphosphatase alignments 115

Figure 17 Interposon mutagenesis of hoxH of Synechococcus sp. PCC 7002 119

Figure 18 Interposon mutagenesis of hoxF of Synechococcus sp. PCC 7002 122

Figure 19 Temperature-shift effects on wild-type and hoxH::aphII 126

Figure 20 SDS PAGE analysis of cytochrome c6 from Synechococcus sp. 130 PCC 7002

Figure 21 Probe design and Southern blot hybridization of petJ from 132 Synechococcus sp. strain PCC 7002

Figure 22 Gene organization around petJ1 in cyanobacteria 134

Figure 23 ClustalW alignment of PetJ1 amino acid sequences 137

Figure 24 Attempted interposon mutagenesis of the petJ1 gene 140

Figure 25 Northern blot analysis of the petJ1 gene 141

Figure 26 Insertion of the Synechocystis sp. PCC 6803 petEgene into 145 the DpetJ::aphII merodiploid strain of Synechococcus sp PCC7002

Figure 27 Attempted mutagenesis of the petJ1 gene in the DpetJ::aphII 147 merodiploid strain Synechococcus sp.PCC 7002/Synechocystis sp. PCC 6803 petE platform vector strain. xv

Figure 28 Insertion of the Synechocystis sp. PCC 6803 petJgene into 149 the petJ::aphII merodiploid strain of Synechococcus sp. PCC 7002.

Figure 29 The petJ gene from Synechocystis sp. PCC 6803 cannot 151 functionally replace the petJ gene from Synechococcus sp. PCC 7002.

Figure 30 RNA blot analysis of the Synechocystis sp. PCC 6803 153 petE gene in the petJ::aphII merodiploid strain of Synechococcus sp. PCC 7002

Figure 31 RNA blot analysis of the Synechocystis sp. PCC 6803 155 petJ gene in the DpetJ::aphII merodiploid strain of Synechococcus sp. PCC 7002

Figure 32 Western blot analysis of the Synechocystis sp. PCC 6803 petE 156 protein produced in the petJ::aphII merodiploid strain of Synechococcus sp. PCC7002

Figure 33 ClustalW alignment of PetJ2 with PetJ amino acid sequences 158

Figure 34 Interposon mutagenesis of the petJ2 gene 161

Figure 35 Growth analysis of the petJ2::aphII strain of Synechococcus sp. 164 PCC 7002

Figure 36 Gene organization around the cytM gene in cyanobacteria 167

Figure 37 ClustalW alignment of CytM amino acid sequences 168

Figure 38 A. BcpA ClustalW alignment 171 B. BcpA/PetE ClustalW alignment xvi

Figure 39 Transcript analysis of petJ1, petJ2, and bcpA 173

Figure 40 Interposon mutagenesis of the Synechococcus sp. 175 PCC 7002 bcpA gene

Figure 41 Growth analysis of the bcpA::aphII strain of Synechococcus sp. 178 PCC 7002

Figure 42 Overproduction of the rBcpA protein 183

Figure 43 Immunoblot analysis of rBcpA 185

Figure 44 Gene organization of the ctaCIDIEI genes from cyanobacteria 188

Figure 45 Gene organization of the ctaCIIDIIEII genes from cyanobacteria 189

Figure 46 Interposon mutagenesis of the ctaDI gene of Synechococcus sp. 192 PCC 7002

Figure 47 Interposon mutagenesis of the ctaDII gene of Synechococcus sp. 195 PCC 7002

Figure 48 Northern blot analysis of the ctaCIDIEI gene cluster of 198 Synechococcus sp. PCC 7002

Figure 49 RT-PCR analysis of the ctaCIIDIIEII gene cluster of 202 Synechococcus sp. PCC 7002

Figure 50 Effect of extreme high light intensity on cell viability wild type 208 and ctaDI- strains of Synechococcus sp. PCC 7002

Figure 51 Growth analysis of analysis of the wild type, ctaDI-, ctaDII-, 210 ctaDI- ctaDII- strains of Synechococcus sp. PCC 7002 grown xvii

under 250 µE m-2 s-1 and 4.5 mE m-2 s-1

Figure 52 Effects of high light intensity on whole chain electron transport 214 in wild type, ctaDI-, ctaDII-, and ctaDI- ctaDII- strains of Synechococcus sp. PCC 7002

Figure 53 Fluorescence emission spectra at 77K of whole cells of Synechococcus 218 sp. PCC 7002 wild-type and cytochrome oxidase mutant strains-

Figure 54 Methyl viologen tolerance and sensitivity in Synechococcus 225 sp. PCC 7002

Figure 55 Growth analysis of analysis of the wild type, ctaDI-, ctaDII-, 227 ctaDI- ctaDII- strains of Synechococcus sp. PCC 7002 in the presence of methyl viologen

2+ Figure 56 Oxidase Mg and CuA binding motif alignments 255

Figure 57 Model for toxicity in ctaD mutant strains grown under 265 4.5 mE m-2 s-1 constant illumination. xviii

List of Tables

Table 1 Nomenclature and properties of type I NADH dehydrogenases 5

Table 2 Primers for Synechocystis sp. PCC 6803 electron transfer protein 37 genes

Table 3 Electron transport proteins from Synechococcus sp. PCC 7002 63

Table 4 Percent identity and similarity of Synechococcus sp. PCC 7002 65 electron transport proteins with homologs from other bacteria and the chloroplast

Table 5 Percent identity and similarity of Synechoccus sp. PCC 7002 NdhD 83 proteins compared to one another

Table 6 Percent identity and similarity of Synechoccus sp. PCC 7002 NdhF 88 proteins compared to one another

Table 7 Protein refolding conditions for rBcpA 183

Table 8 Primers for Synechococcus sp. PCC 7002 ctaCIDIEI and 196 ctaCIIDIIEII genes

Table 9 Oxygen uptake activity of wild type, ctaDI-, ctaDII-, and 204 ctaDI- ctaDII- strains of Synechococcus sp. PCC 7002

Table 10 Doubling times, oxygen evolution rates and Fv’/Fm’ for wild type, 205 ctaDI-, ctaDII-, and ctaDI- ctaDII strains of Synechococcus sp. PCC 7002 grown under various light intensities xix

Table 11 Chlorophyll a and carotenoid contents for wild type, ctaDI-, 212 ctaDII-, and ctaDI- ctaDII strains of Synechococcus sp. PCC 7002 grown under various light intensities.

Table 12 P700+ redox kinetics of wild type, ctaDI-, ctaDII-, and 220 ctaDI- ctaDII- strains of Synechococcus sp. PCC 7002

Table 13 Cell viability of wild type, ctaDI-, ctaDII-, and ctaDI- ctaDII- 228 strains of Synechococcus sp. PCC 7002

Table 14 Superoxide dismutase activities of wild type, ctaDI-, ctaDII-, 230 and ctaDI- ctaDII- strains of Synechococcus sp. PCC 7002

Table 15 Hydroperoxide concentrations of wild type, ctaDI-, ctaDII-, 232 and ctaDI- ctaDII- strains of Synechococcus sp. PCC 7002

Table 16 Catalase and peroxidase activities of wild type, ctaDI-, ctaDII-, 234 and ctaDI- ctaDII- strains of Synechococcus sp. PCC 7002 xx

ACKNOWLEDGMENTS

Thanks to everyone who helped make this reality-

Family: Mom, Dad, Mary, Grandma and Grandpa Ito, Grandma Miki and Grandpa

Henry, Uncle Eric and Auntie Hatsumi, Amy, Adam, Celeste, Joachim, The Ito Family,

The Nomura Family, The Nakadegawa Family.

Old School: (WEST COAST) Douglas Jímenez, Wayne Szeto, Kim Gasuad, Lupe

Garcia, Scottie Henderson, Matt Mills and Miné Berg, Bernice Frankl, Dr. Leo Ortiz,

Carlos Morel, John Lee Hooker, Chris Lopez, Leandra, MBRS, MARC, Kwame

Asamoah, “Chocalate” Terry G, Kiersten and Tony. (EAST COAST) Noah, Jason, Elan,

Ranon, Alex, Jess.

New School: Søren and Suzanne, NUF and Yumiko, Gaozhong Shen, Lena and Ilya,

Juergen, Katja, Toshio, Kaori, Tanja Gruber, Vicki Stirewalt, Wendy Schlucter, Soohee

Chung, Zhao, Frank and Jessica, Joel, Fetchko, Denise, Pete, Nick, Celina, Matt, Laurie,

Crazy Bob, all of the undegrads who do all the work in our lab Melissa, Anne, Ginny,

Matt, Kirstin, John, Mel and everyone else I don’t have room for.

The Committee: Dr. John Golbeck, Dr. Ola Sodeinde, Dr. Paul Babitzke, Dr. Juliette

Lecomte, and of course my advisor, Dr. Don Bryant.

Many thanks for all your support, patience, and encouragement. Sorry if I left you out (thanks to you too!!) Chapter 1

INTRODUCTION

1.1 Electron transport proteins in cyanobacteria

Cyanobacteria are photosynthetic, oxygen-evolving prokaryotes that have adapted to a wide range of ecological niches (Stanier and Cohen-Bazire, 1977). Cyanobacteria represent interesting organisms in which to study electron transport because they have both photosynthetic and respiratory proteins involved in electron transport on a single membrane: the thylakoid membrane (Figure 1). Photosynthetic electron transport in cyanobacteria is similar to that of higher plants (Scherer, 1990). They have a

plastoquinone pool that is reduced by PSII, a cytochrome b6f complex which acts as a plastoquinol-plastocyanin/cytochrome c6 oxidoreductase and is homologous in function to the cytochrome bc complex of mitochondria, and a soluble cytochrome c6 that reduces

PSI (Scherer, 1990). Unlike plants, in which the light harvesting apparatus is made up of integral membrane protein complexes that contain chlorophyll a and chlorophyll b that direct energy to the photosystems, cyanobacteria use light-harvesting complexes called phycobilisomes (Bryant et al., 1990). 2

Figure 1. Depiction of the thylakoid membrane of cyanobacteria adapted from D.A.

Bryant (1994). Photosynthetic and respiratory complexes are identified by the text in the figure. Light energy is represented by hn. The arrows with solid lines represent electron flow and arrows with dashed lines represent proton movement.

Phycobilisome STROMA

h h ADP + Pi ATP + H2O

F F h h E Flvd D + + C NADP + H CF1 or NADPH FNR FNR A B Fd PsaD + + PsaE F + H NADH NAD+ + H+ H F B H CP47 CP47 A b' b Q Cyt b a A QB 6 FX Thylakoid Cyt Cyt PQ r HP b b SIV A Cyt c c CF 559 Pheo 559 NDH 1 c c 0 membrane D1 CP43 CP43 PQH PsaB ox D1 2 o LP Cyt f PsaA A0 P D2 680 D2 FeS P700 Z + + Mn4 Mn4 H+ H 2H +1/2O2 - Cyt h 9 9 e h 553 + 33 33 H2O H H2O + LUMEN 2H +1/2O2 h

Photosystem II Photosystem II NADH Cytochrome b6/f Photosystem I Cytochrome ATP dehydrogenase complex oxidase synthase

Phycobilisomes are composed of water-soluble phycobiliproteins that harvest light and transfer light energy to the photosynthetic reaction centers (Bryant et al., 1990). Light energy is captured by phycobilisomes and chlorophyll and ultimately transferred to a special pair of chlorophyll a molecules (P680) from which the electron is passed through

a series of electron acceptors within PSII to plastoquinone QB. The oxidation of water by the Mn center of the PSII complex provides electrons to reduce the oxidized P680 reaction center. Electrons from reduced plastoquinone are passed through the 3

cytochrome b6f complex, which then transfers electrons to an oxidized mobile electron carrier, either plastocyanin or cytochrome c6, depending on the species of cyanobacteria or environmental conditions (Scherer, 1990; Zhang et al., 1992). The mobile electron carrier then transfers its electron to reduce the oxidized special-pair (P700) chlorophyll a molecules of PSI. Electrons are transferred in the PSI reaction center through a series of cofactors to soluble ferredoxin. Ferredoxin transfers its electrons to ferredoxin:NADP+ oxidoreductase, which in turn forms NADPH by reduction of NADP+. The net result of these reactions is reducing power to fix carbon from carbon dioxide, the production of oxygen from the oxidation of water, and transfer of protons into the lumenal space of the

thylakoid. This proton transport is facilitated by the cytochrome b6f complex, type I

NADH dehydrogenase, and cytochrome oxidase and generates an electrochemical proton gradient and transfer of the protons back into the cytoplasmic space through ATP synthase results in ATP synthesis (Schmetterer, 1994). Although the role of these photosynthetic proteins has been defined by many studies, the roles of many of the other potential electron transfer proteins within cyanobacteria remains open for research.

The recent ability to sequence and identify open reading frames from entire genomes has opened up new avenues to perform comparative analyses between many different organisms (Blattner et al., 1997; Deckert et al., 1998; Kaneko et al., 1996;

Nelson et al., 1999). The genome of the freshwater, unicellular cyanobacterium

Synechocystis sp. PCC 6803 has been completely sequenced and open reading frames corresponding to putative electron transport proteins have been identified (Kaneko et al.,

1996). Our lab is interested in determining the similarities and differences in the number 4 and types of genes that may be involved in electron transport in Synechococcus sp. PCC

7002 and other cyanobacterial species. Of particular interest to our lab is the identification and characterization of the minimal conserved number of genes responsible for coding proteins used in electron transport in cyanobacteria.

1.1.1 Type I NADH Dehydrogenase

The NADH-ubiquinone oxidoreductase (complex I) of mitochondria is an enzyme that consists of more than 40 different subunits (Anderson et al., 1982; Arizmendi et al.,

1992a; Arizmendi et al., 1992b; Walker, 1992a; Walker et al., 1992). The genes for most of the subunits are located in the nucleus and the gene products must be imported into the mitochondria for assembly on the inner membrane (Walker, 1992a; Walker et al., 1992;

Weiss et al., 1991). In vertebrates, 7 of these subunits are encoded by the mitochondria.

These subunits are ND1, ND2, ND3, ND4, ND4L, ND5 and ND6. Complex I is the first major enzyme in the respiratory electron transport chain of mitochondria and is required to generate the proton motive force used for ATP synthesis by the translocation of protons (Weiss et al., 1991).

Homologues of the mitochondrial-encoded complex I genes as well as some of the complex I subunits encoded by the nucleus are also found in plastid DNA suggesting that a NADH dehydrogenase may be located within the chloroplast (Hiratsuka et al.,

1989). The genes for the NADH dehydrogenase of higher plant chloroplasts that are homologous to the mitochondrial genes are named ndhA, ndhB, ndhC, ndhD, ndhE, 5 ndhF, ndhG, ndhH, ndhI, ndhJ, and ndhK (Table 1) (Friedrich et al., 1995; Friedrich and

Weiss, 1997; Schmitz-Linneweber et al., 2000).

Table 1. Nomenclature and properties of homologous of type I NADH dehydrogenase subunits from different organisms adapted from Friedrich and Weiss (1997). The NADH dehydrogenase subunits were identified for E. coli (Weidner et al., 1993), Bos taurus

(Walker, 1992b; Walker et al., 1992), Oryza sativa (Shimada and Sugiura, 1991), and

Synechocystis sp. PCC 6803 (Kaneko et al., 1996).

E. coli B. taurus O. sativa 6803 cofactor(s)/predicted function NuoA ND3 NdhC NdhC NuoB PSST NdhK NdhK 1X[4Fe-4S] NuoC 30(IP) NdhJ NdhJ NuoD 49(IP) NdhH NdhH NuoE 24(FP) not found not found 1X[2Fe-2S] NuoF 51(FP) not found not found NADH-binding; FMN; 1X[4Fe-4S] NuoG 75(IP) not found not found 1X[4Fe-4S]; 1 (2*)X[2Fe-2S] NuoH ND1 NdhA NdhA Ubiquinone-binding NuoI TYKY NdhI NdhI 2X[4Fe-4S] NuoJ ND6 NdhG NdhG NuoK ND4L NdhE NdhE NuoL ND5 NdhF NdhF NuoM ND4 NdhD NdhD NuoN ND2 NdhB NdhB 6803 is Synechocystis sp. PCC 6803, * indicates that there is an additional Fe-S cluster on subunit NuoG of E. coli. 6

Several prokaryotes also possess type I NADH dehydrogenases (Dupuis et al., 1995;

Weidner et al., 1993). The type I NAD(P)H dehydrogenase, or NDH-1, found in prokaryotes is a multi-subunit complex with a minimum of 14 subunits in E. coli

(Weidner et al., 1993) and Rhodobacter sphaeroides (Dupuis et al., 1995). The NDH-1 complex translocates protons across the membrane, has a flavin mononucleotide and iron-sulfur clusters as the prosthetic groups, and is inhibited by rotenone in a manner similar to mitochondrial complex I (Weidner et al., 1993). Thus, biochemically, the functions of the prokaryotic NADH dehydrogenase are similar to those of the mitochondrial enzyme.

The nomenclature used for the chloroplast ndh genes is also used for type I

NADH dehydrogenase gene homologs within cyanobacteria. Cyanobacteria also have another open reading frame, ndhL, associated with the type I NADH dehydrogenase

(Kaneko et al., 1996; Ogawa, 1991; Sugita et al., 1995). The ndhL gene was originally isolated by Ogawa (Ogawa, 1991) and it was designated ictA (for inorganic carbon transport gene A) because a mutation which inactivated ictA was found to be defective in inorganic carbon transport. The NdhL protein is unique to cyanobacteria. In

Synechocystis sp. PCC 6803, the NDH-1 complex was found on both the thylakoid and cytoplasmic membranes (Schmetterer, 1994). Genes for 16 single-copy ndh genes have been identified in the Synechocystis sp. PCC 6803 genome. There are multiple copies of the ndhF and ndhD genes in Synechocystis sp. PCC 6803 (Kaneko et al., 1996).

However, as predicted in chloroplast NDH-1 complexes, there are no genes predicted to encode subunits involved in NAD(P)H/H+ binding (Kaneko et al., 1996) that are encoded 7 by the nuoE, nuoF, and nuoG genes in E. coli (Weidner et al., 1993). This suggests that cyanobacteria and chloroplast type I NADH dehydrogenases may use an electron donor or acceptor other than NAD(P)H/H+ as a donor/acceptor of electrons. Other proteins may have taken over this function in cyanobacteria (see below).

Inactivation of genes encoding individual subunits of the NDH-1 complex in cyanobacteria has shown that the NDH-1 complex is active in both photosynthetic and respiratory processes (Klughammer et al., 1999; Marco et al., 1993; Ogawa, 1991;

Schluchter et al., 1993). Inactivation of the ndhB genes in Synechocystis sp. PCC 6803

(Ogawa, 1991) and in Synechococcus sp. PCC 7942 (Marco et al., 1993). A similar phenotype was also observed when the ndhD3 and ndhF3 genes of Synechococcus sp.

PCC 7002 were inactivated (Klughammer et al., 1999). However, inactivation of the ndhF1 gene in Synechococcus sp. PCC 7002 showed that it is involved in both cyclic electron transport around PSI as well as respiratory electron transport (Schluchter et al.,

1993). These observations suggest that there are multiple types of NDH-1 complexes, possibly consisting of different subunits, in cyanobacteria.

1.1.2 Type II NADH dehydrogenases

A second type of NADH dehydrogenase, the type II NADH dehydrogenase or

NDH-2, is found in prokaryotes. The NDH-2 protein is a single subunit, has no iron- sulfur clusters, and does not appear to have the ability to translocate protons across the membrane. The enzyme also has a flavin adenine dinucleotide (FAD) as a cofactor and 8 unlike NDH-1, is not inhibited by rotenone. This enzyme is found in E. coli (Blattner et al., 1997), Bacillus sp. YN-1 (Xu et al., 1991), Bacillus megaterium (Thiaglingam and

Yang, 1993) and Thermus thermophilus (Yagi et al., 1988). Recently, three open reading frames (slr0851, slr1743, sll1484) were identified in Synechocystis sp. PCC 6803 that exhibited low sequence similarity to the NDH-2 proteins from E. coli, Bacillus sp. YN-1

(Howitt et al., 1999; Kaneko et al., 1996; Xu et al., 1991) and these genes have been denoted ndbA, ndbB, and ndbC. All three putative proteins have characteristic FAD and

NADH binding motifs and, in contrast to the E. coli NDH-2 protein, all three predicted

NDH-2 proteins from Synechocystis sp. PCC 6803 appear to be hydrophilic (Howitt et al., 1999). The predicted protein from the sll1484 reading frame is the only predicted protein product containing a hydrophobic stretch of amino acids that would be long enough to span a membrane. Expression plasmids were made with slr0851 and slr1743 to see if they could complement a strain of E. coli that lacks a functional NDH-2 or

NDH-1 (Howitt et al., 1999). It was shown that slr1743 was able to complement the mutant E. coli strain lacking a functional type II NADH dehydrogenase, thus showing that cyanobacteria contain a functional type II NADH dehydrogenase (Howitt et al.,

1999). Howitt et al. (1999) also made interposon mutations in all three of the NDH-2 reading frames resulting in strains of cyanobacteria that were deplete in one, two, or all three of the NDH-2 proteins. They also deleted these genes in Synechocystis sp. PCC

6803 strains that lacked PSI. They discovered a very unusual phenotype in that PSI- deficient strains that were also Ndb- were able to grow in high light whereas the parental

PSI-deficient strain was unable to grow under this condition (Howitt et al., 1999). The 9 results of this study imply that the type II NADH dehydrogenase may function as an important redox sensor of the membrane plastoquinone pool and the soluble fraction of

NADH within the cell.

1.1.3 Hydrogenase

Hydrogenase enzymes are found in a wide variety of microorganisms, and they

+ - catalyze the reaction: 2H + 2 e Ö H2. The physiological function of most prokaryotic hydrogenases is the oxidation of hydrogen gas coupled to the energy-conserving reduction of electron acceptors (Wu and Mandrand, 1993). Another function of hydrogenase enzymes is the production of hydrogen in a non-energy-conserving manner in order to maintain intracellular pH homeostasis and redox potential balance (Adams et al., 1980).

Hydrogenases can be divided into several classes according to Wu and Mandrand

(1993). Class I consists of the membrane-bound NiFe proteins. These enzymes are heterodimers with a large subunit that has a Ni-Fe active site, and a small subunit that carries multiple Fe-S clusters interacting with the redox, electron transport partners of the hydrogenase. These so-called uptake hydrogenases are found in bacteria such as

Rhodobacter capsulatus (Colbeau et al., 1993), Bradyrhizobium japonicum (Zuber et al.,

1986), and Azotobacter vinelandii (Seefeldt and Arp, 1986), Anabaena sp. PCC 7120

(Carrasco et al., 1995) and Nostoc sp. PCC 73102 (Oxelfelt et al., 1998; Tamagnini et al.,

1997). The structure has been solved for the NiFe-containing hydrogenase from 10

Desulfovibrio gigas (Volbeda et al., 1995) and the structure reveals that the NiFe cluster responsible for hydrogenase activity is covalently coordinated to the protein through four cysteinyl ligands. A mechanism for hydrogen binding and cleavage was proposed to involve an intermediate state in which hydrogen is bridged between both the Fe and Ni at the catalytic site (Volbeda et al., 1995).

Class II consists of the NiFe(Se) hydrogenases that also have two subunits. The large subunit has a Ni-Fe-Se active site and the small subunit, as in Class I, has multiple

Fe-S clusters. The heterolytic cleavage of molecular hydrogen seems to be mediated by the nickel center and the selenocysteine residue. The selenium ligand might also protect the nickel atom from oxidation (Garcin et al., 1999). These enzymes are found in sulfur- metabolizing bacteria such as D. gigas (Malki et al., 1995) and Desulfovibrio fructosovorans (Rousset et al., 1990).

Class III hydrogenases are the anaerobic Fe-only hydrogenases; these are soluble enzymes that are made up of one to four subunits. All Class III hydrogenases have a catalytic subunit composed of 420-580 amino acids (Meyer and Gagnon, 1991). The large, catalytic subunit has a binuclear Fe center at the active site. This type of enzyme is found in anaerobic bacterial species such as Clostridium pasteurianum, where the structure has been determined (Peters et al., 1998). The enzyme has three distinct [4Fe-

4S] clusters, a [2Fe-2S] cluster, and the active site (H-cluster) is an unusual six iron atom cluster consisting of a [4Fe-4S] cubane cluster that is covalently bridged by a cysteinate thiol to a [2Fe] subcluster (Peters et al., 1998). The 76 residues at the N-terminus that form a [2Fe-2S] cluster have a fold similar to plant-type ferredoxins which may shuttle 11 electrons from the redox partners (c-type cytochromes) to the hydrogenase active site and

(Kummerle et al., 1999).

Class IV hydrogenases are the F420-MV NAD reducing NiFe(Se) enzymes found in archaebacteria such as Methanococcus voltae (Muth et al., 1987) and

Methanobacterium fervidus (Steigerwald et al., 1990). These enzymes can reduce either the 8-hydroxy-5-deazaflavin cofactor F420 or methyl viologen. The enzymes comprise 3 subunits; a, b , and g, with molecular masses of 47 kDa, 31 kDa and 26 kDa, respectively.

These subunits form a complex with a subunit stoichiometry of (a1b1g 1)8 (Alex et al.,

1990).

Class V consists of the reversible/bi-directional hydrogenases which are found in

Alcaligenes eutrophus (now Ralstonia eutropha) (Tran-Betcke et al., 1990), Nocardia sp.

(Schmitz et al., 1995), Synechocystis sp. PCC 6803 (Appel and Schulz, 1996; Kaneko et al., 1996), Anacystis nidulans (Synechococcus sp. PCC 6301) (Boison et al., 1996;

Boison et al., 1998), and Anabaena sp. PCC 7120 (Houchins and Burris, 1981; Schiefer and Happe, 1999). In R. eutropha, the enzyme is a heterotetramer that can be divided into two heterodimers that catalyze specific reactions. One heterodimer is responsible for the hydrogenase activity and the other heterodimer is responsible for the diaphorase activity.

Cyanobacteria are known to have two types of hydrogenases: uptake hydrogenases and bi-directional or reversible hydrogenases. The uptake hydrogenase is located on the thylakoid membrane of cyanobacteria and catalyzes the oxidation of hydrogen in the presence of phenazine methosulfate (PMS) or methylene blue (MB) as 12 electron acceptors. However, the uptake hydrogenase of cyanobacteria does not support the methyl viologen-dependent evolution of hydrogen. This type of enzyme is found in

Anabaena sp. (Carrasco et al., 1995), and Nostoc sp. (Oxelfelt et al., 1998; Tamagnini et al., 1997). The second type of hydrogenase found in cyanobacteria catalyzes both the oxidation and evolution of hydrogen and thus is called the bi-directional or reversible hydrogenase. In cyanobacteria, the bi-directional hydrogenases consist of 4-5 subunits.

The enzyme may also be divided into a heterodimeric hydrogenase moiety and a heterodimeric or heterotrimeric diaphorase moiety. The hydrogenase subunits are encoded by conserved gene clusters in cyanobacteria and they share sequence similarity with hydrogenases from other bacteria such as A. eutrophus (Massanz et al., 1998) and

Escherichia coli (Jacobi et al., 1992).

1.1.4 Mobile Electron Carriers

Mobile electron carriers are responsible for the oxidation of the cytochrome b6f complex and the transfer of electrons to either oxidized P700 in PSI or to cytochrome terminal oxidases in cyanobacteria. Two soluble proteins which have been reported to

facilitate this electron transfer in cyanobacteria: cytochrome c6 (PetJ) and the blue copper protein, plastocyanin (PetE) (Ghassemian et al., 1994; Ho and Krogmann, 1984;

Sandmann and Boger, 1981; Zhang et al., 1992). 13

1.1.4.1 Cytochrome c6

Cytochrome c6 (formerly cytochrome c-553) is a small, soluble iron-heme protein from a unique class of cytochromes, the c-type cytochromes. These c-type cytochromes are characterized by a heme cofactor that is covalently bound to the CXXCH-consensus amino acid motif. The fifth and sixth ligands to the Fe of the heme cofactor are a

conserved methionine and histidine in cytochrome c6. Examples of c-type cytochromes include the c-type soluble cytochromes of mitochondria, mobile cytochrome c-555 in anoxygenic green photosynthetic bacteria, and cytochrome c2 in purple photosynthetic bacteria (Dickerson, 1980).

Cytochrome c6 is used in cyanobacteria as a mobile carrier of electrons between the cytochrome b6f complex and P700 of the PSI reaction center (Zhang et al., 1992).

Many eukaryotic algae and cyanobacteria use either cytochrome c6 (PetJ) or the blue copper protein, plastocyanin (PetE) as the mobile carrier of electrons between

cytochrome b6f complex and the P-700 reaction center of PSI (Ho and Krogmann, 1984;

Zhang et al., 1992). Whether the organism (cyanobacterium or eukaryotic alga) uses

plastocyanin or cytochrome c6 as an electron donor to PSI depends on the chemical environment of the cells during growth. If the cells are grown in the presence of copper,

the cells will synthesize plastocyanin for electron transport from cytochrome b6f to PSI; however, in the absence of copper, the cells synthesize cytochrome c6 (Ho and

Krogmann, 1984; Zhang et al., 1992). Cytochrome c6 undergoes a reversible oxidation- reduction reaction similar to that of plastocyanin during photosynthetic electron transport. 14

Cytochrome c6 is similar in size, pI and midpoint potential (335 to 390 mV) to plastocyanin (Kerfeld and Krogmann, 1998).

In addition to its role in photosynthetic electron transport, cytochrome c6 may also play a role in respiratory electron transport in cyanobacteria. Lockau has presented evidence in Anabaena variabilis that both cytochrome c6 and plastocyanin have dual roles as electron carriers for photosynthesis and respiration (Lockau, 1981). Previous

experiments have demonstrated that cytochrome c6-depleted membranes of Nostoc muscorum could be made competent in transferring electrons to either PSI or cytochrome

oxidase by the addition of cytochrome c6 (Stürzl et al., 1982).

The gene encoding cytochrome c6 (petJ) has been insertionally inactivated in

Synechococcus sp. strain PCC 7942 (Laudenbach et al., 1990) or deleted in the case of

Synechocystis sp. strain PCC 6803 (Zhang et al., 1994). These interposon/insertion/deletion mutants were still able to grow at wild-type rates. It was presumed that other physiological electron donors present within these organisms could

replace the function of cytochrome c6. In Synechocystis sp. strain PCC 6803, the primary electron donor is plastocyanin (Briggs et al., 1990; Zhang et al., 1992); cytochrome c6 is expressed only under copper depleted conditions (Zhang et al., 1992). However, it appears that even under copper deplete conditions, Synechocystis sp. strain PCC 6803 is still able to produce a minimal amount of functional plastocyanin, since the Synechocystis sp. strain PCC 6803 can still grow under copper-depleted conditions with the petJ gene inactivated (Zhang et al., 1994). The observation that the petJ- strain was still able to grow was attributed to a possible, third mobile electron carrier. However, if this were the 15

case, a double mutation to inactivate the petJ and petE genes encoding the cytochrome c6 and plastocyanin proteins, respectively, should have been easy to achieve. However,

Manna and Vermaas (1997) found that the petJ and petE genes could not be simultaneously inactivated within the same strain.

Subsequent to the report of Laudenbach, et al. (1990) it has been discovered that

Synechococcus sp. strain PCC 7942 has a petE gene encoding a functional plastocyanin

(Clarke and Campbell, 1996) and as in Synechocystis sp. strain PCC 6803, this mobile electron carrier was probably responsible for the wild-type phenotype seen in the petJ interposon mutant of this organism.

In other cyanobacteria, it has been observed that some species have more than one

isoform of cytochrome c6, suggesting that there may be multiple copies of the gene encoding this protein in cyanobacteria (Ho and Krogmann, 1984). This is similar to the situation in purple bacteria such as R. sphaeroides (Jenney and Daldal, 1993; Jenney et al., 1996) and R. capsulatus (Jenney and Daldal, 1993; Jenney et al., 1996). It has been reported that some cyanobacteria (Nostoc muscorum and Spirulina platensis) have no detectable plastocyanin (Ho and Krogmann, 1984). In these cases, it is assumed that

cytochrome c6 is the sole mobile electron carrier between the cytochrome b6f complex and either PSI or cytochrome oxidase. 16

1.1.4.2 Plastocyanin

Plastocyanin is a small (97-99 amino acids) soluble electron carrier that transfers

electrons from cytochrome b6f complex to Photosystem I (PSI) via the reversible

+ 2+ 2+ + oxidation (Cu ® Cu + 1 e) and reduction (Cu + 1 e-® Cu ) of its active copper center in plants as well as in eukaryotic algae and some cyanobacteria (Ho and Krogmann,

1984). Plastocyanin folds into an eight-stranded, b-sandwich cylinder (Chapman et al.,

1977) and the copper containing active center is close to the surface of the molecule. The copper ion is coordinated to a surface exposed histidinyl imidazole as well as a cysteinyl thiolate, a methioninyl thioether and another histidinyl imidazole (Redinbo et al., 1993).

The geometry of the active site is an irregular tetrahedron. This distortion is imposed by the folding of the protein and is responsible for stabilizing the high midpoint potential

(+370 mV) of the protein. As mentioned in the previous section, plastocyanin and

cytochrome c6 are interchangeable in function in many cyanobacteria and algae, even though the structures of the individual protein are very different from one another.

Plastocyanin is mostly comprised of b-sheet secondary structure and cytochrome c6 is mostly alpha-helical in structure (Kerfeld et al., 1995). Ho and Krogmann (1984) noted

that the isoelectric points of plastocyanin and cytochrome c6 from the same organism are more similar to one another than they are to the pIs of plastocyanin or cytochrome c6 proteins from different organisms. This observation suggests that the similarity of isoelectric points within the same organism is the result of convergent evolution of 17 plastocyanin and cytochrome c6 within each organism in response to changes in a common, interacting reaction partners.

1.5 Terminal oxidases

All aerobic bacterial species examined to date have multiple respiratory oxidases that allow them to modify their respiratory systems according to their environmental challenges. These respiratory oxidases may fall into either the heme-copper respiratory oxidase super-family or into the unrelated cytochrome bd oxidase family. Heme-copper respiratory oxidases can be further divided into two subgroups: (I) heme-copper oxidases that are reduced by cytochrome c, which includes the mitochondrial cytochrome c oxidase, as well as the bacterial oxidases of the aa3, ba3, caa3, cao3, and bo3 type; and

(II) those heme-copper oxidases that are directly reduced by quinols (Garcia-Horsman et al., 1994a).

Membership in the heme-copper oxidase superfamily is determined by the presence of a homolog to subunit I of the mammalian cytochrome c oxidase. Subunit I is the largest subunit of the cytochrome c oxidase, and it contains the unique bimetallic or

binuclear center where O2 binds and is reduced to water (Garcia-Horsman et al., 1994a).

This bimetallic center consists of a heme and a copper ion, CuB. Subunit I has a second heme group in addition to the one at the binuclear center. This secondary heme facilitates electron transfer to the binuclear center. The mitochondrial heme-copper oxidase is comprised of 13 subunits, of which 3 (subunits I, II, and III) are encoded by the 18 mitochondrial genome, whereas most bacterial heme-copper respiratory oxidases have 3 to 4 subunits. Three of these bacterial subunits are homologues of subunit I, subunit II, and subunit III of mitochondria, and the fourth bacterial subunit, when present, is unrelated to any mitochondrial subunit. Figure 2 shows a scheme with the main catalytic subunits of the different types of heme-copper oxidases from the heme-copper oxidase superfamily. The variation observed in the heme-copper oxidases derives from the fact that individual oxidases use different combinations of the hemes a, o, and b in association with subunit I. Hemes a, o, and b can reside at the binuclear center and either heme b or heme a is found at the low-spin site. The proton-conducting channel is also associated with subunit I (Figure 2).

Subunit II is responsible for shuttling electrons from the redox partner of the oxidase to the binuclear center. The binding site for cytochrome c is located on subunit

II. Subunit II may also contain a second copper-containing redox center, denoted CuA

(Figure 2). This CuA is the initial electron acceptor from reduced cytochrome c in the case of cytochrome c oxidase. Amino acid residues in subunit II that are conserved in all cytochrome c oxidases have been implicated in either the binding of cytochrome c or in

liganding the CuA center (Saraste, 1990). These conserved amino acids are missing in subunit II of quinol-type oxidases, which additionally do not have a CuA center

(Abramson et al., 2000; Fukaya et al., 1993; Lauraeus et al., 1991; Minghetti et al., 1992).

Under changing oxidative conditions, the cells must balance different needs in order to optimize their respiratory electron transfer chains. Terminal oxidases may be used to generate a maximal H+/e- gradient (Puustinen et al., 1991), to remove excess 19

Figure 2. Schematic illustration of the similarities and differences of four subclasses of the heme-copper oxidase superfamily adapted from (Garcia-Horsman et al., 1994a).

Models indicate the major subunits (I and II) of the heme-copper oxidases as well as electron donors to the enzyme. Predicted electron flow follows the path of the arrows.

Panels A, B, and C depict three types of cytochrome c oxidases. Panel D depicts the generic model for a quinol oxidase. 20 Cyt c oxidases Quinol oxidases A D

Cyt c aa3, ba3 aa3, ba3, bb3, bo3

H+ H+ CuA e-

- e- - Fe e Fe e Fe CuB QH2 Fe CuB

II I II I

H+ H+ B

Cyt c e- caa3, cao3

Fe c

e- H+ CuA e-

e- Fe Fe CuB

II I

H+ C Cyt c

e- cbb3

Fe Fe H+ Fe

e- - Fe e Fe CuB

H+ 21 reducing equivalents, to consume oxygen to maintain anaerobicity or to lower the oxygen concentration for the cell (Kelly et al., 1990). R. sphaeroides represents a model organism that expresses a diverse number of terminal oxidases that may be used to regulate its metabolism, since the organism can grow aerobically, anaerobically, heterotrophically or photosynthetically (Garcia-Horsman et al., 1994a; Garcia-Horsman et al., 1994b). R. sphaeroides has three distinct respiratory oxidases, two of which utilize cytochrome c as an electron donor, whereas one terminal oxidase utilizes quinol as a

substrate. The predominant terminal oxidase is of the aa3 type when the cells are grown aerobically with high O2 tension (Hosler et al., 1992). The alternate cytochrome c oxidase is a cbb3 type oxidase and is present under microaerophilic conditions and when the cells are grown under photosynthetic conditions (Garcia-Horsman et al., 1994b). The

quinol oxidase is able to support aerobic growth in R. sphaeroides strains that lack the bc1 complex (Yun et al., 1990).

The majority of studies performed thus far regarding the function of cytochrome oxidases in cyanobacteria have been directed at examining cytochrome oxidase protein biochemistry or the function of the enzyme during respiration (Alge and Peschek, 1993b;

Howitt and Vermaas, 1998; Obinger et al., 1990; Peschek et al., 1989; Sone et al., 1993;

Tano et al., 1991; Trnka and Peschek, 1986; Wastyn et al., 1987). Previous biochemical studies examining P700+ reduction kinetics in cyanobacteria such as Synechococcus sp.

PCC 7002 (Yu et al., 1993) and Fremyella diplosiphon indicate that these organisms may use cytochrome oxidases as a sink for removing excess electrons not accounted for by

PSI activity (Schubert et al., 1995). 22

The first cyanobacterial ctaCIDIEI operon encoding the primary aa3-type cytochrome heme-copper oxidase was cloned from Synechocystis sp. PCC 6803 (Alge and Peschek, 1993a; Alge and Peschek, 1993b) and a similar gene cluster has been cloned from Synechococcus vulcanus (Sone et al., 1993). Two other respiratory oxidases were identified and characterized in Synechocystis sp. PCC 6803 by (Howitt and

Vermaas, 1998). One is a quinol oxidase of the bd-type and the second oxidase appears to be an oxidase of the bo-type. After characterization of mutants lacking all combinations of the oxidases, it was concluded that ctaCIDIEI and cydAB encode functional oxidases in Synechocystis sp. PCC 6803. However, these oxidases contributed little to the normal growth characteristics of the organism under the conditions that were studied (Howitt and Vermaas, 1998).

1.2 Purpose of the Present Work

The current project sought to identify similarities and differences between the freshwater, unicellular cyanobacterium Synechocystis sp. PCC 6803 and the marine cyanobacterium Synechococcus sp. PCC 7002 with regards to genes potentially involved in either photosynthetic or respiratory electron transport. The purpose of this project was to clone and sequence the genes for the type I NADH dehydrogenase, type II NADH dehydrogenases, hydrogenase, mobile electron transport proteins and terminal oxidases from the marine cyanobacterium Synechococcus sp. PCC 7002 for comparison of amino acid sequences and gene organizations with other cyanobacteria. 23

Another purpose of this work was to initiate the characterization of some of these genes and to examine the effects of mutations in specific genes that encode electron transport proteins on the physiology of Synechococcus sp. PCC 7002. Results from attempts to inactivate the ndhB and petJ1 genes are described. The initial characterization of mutations in the following genes are also described: the hydrogenase gene cluster, two putative mobile electron carriers, petJ2 and bcpA, and the ctaDI and ctaDII genes, that purportedly encode the large subunits of heme-copper oxidase complexes. The initial characterization of these genes is an important first step in understanding the roles that these electron transport proteins play within cyanobacteria, under different growth conditions. Chapter 2

MATERIALS AND METHODS

2.1 Bacterial strains and culture conditions

2.1.1 Synechococcus sp. PCC 7002

Synechococcus sp. PCC 7002 is a unicellular or filamentous, naturally transformable, marine cyanobacterium that was isolated by Van Baalen (Van Baalen,

1962). Synechococcus sp. strain PCC 7002 can be grown as a facultative photoheterotroph in the presence of glycerol (Rippka, 1972). It has a well-defined natural DNA uptake system that makes the organism attractive for genetic manipulation

(Stevens and Porter, 1980). The laboratory wild-type strain Synechococcus sp. PCC 7002

(formerly Agmenellum quadruplicatum strain PR6) was originally obtained from the

Pasteur Culture Collection, Unité de Physiologie Microbienne, Institut Pasteur, Paris,

France.

Synechococcus sp. PCC 7002 was grown in medium A (Stevens and van Baalen,

1973) supplemented with 1 g l-1 NaNO3 (referred to as A+ medium) in liquid culture and on 1.5% (w/v) agar plates. Medium A consists of: 18 g l-1 NaCl, 5 g l-1 MgSO4•7H2O, 1 g l-1 Tris-HCl pH 8.2, 600 mg l-1 KCl, 270 mg l-1 CaCl2, 50 mg l-1 KH2PO4, 30 mg l-1 25 tetrasodium EDTA, 34.3 mg l-1 H3BO3, 4.32 mg l-1 MnCl2 •4H2O, 3.89 mg l-1 FeCl3

•6H2O, 315 mg l-1 ZnCl2, 3 mg l-1 CuSO4•5H2O, 30 mg l-1 MoO3, 12.2 mg l-1 CoCl2

•6H2O, and 4 mg l-1 vitamin B12. Antibiotic concentrations used to select or maintain mutant strains were 100 mg ml-1 kanamycin, 100 mg ml-1 streptomycin, 100 mg ml-1 spectinomycin, 50 mg ml-1 chloramphenicol, and 100 mg ml-1 erythromycin.

Stock cultures were maintained on 1.5% (w/v) agar in A+ media, containing the appropriate antibiotic(s) for mutant strains of Synechococcus sp. PCC 7002, as necessary, in Petri dishes at approximately 28˚C under continuous illumination at an approximate light intensity of 60 mE m-2 s-1. Individual colonies for each strain were re-streaked on fresh plates once every three weeks.

Small-scale liquid cultures (25 ml) were grown in 22 ´ 175 mm culture tubes at

38˚C in aquarium water baths to maintain constant temperature. For large-scale cultures, cells were grown in sterile flasks at room temperature and were constantly bubbled with

CO2 in air with continuous stirring. Constant illumination was provided with

F72T12/CW fluorescent bulbs. The standard illumination conditions for liquid cultures were approximately 200-300 mE m-2 s-1. For lower light intensities, paper was used to shade the aquariums from light until the appropriate light level was achieved. For higher light intensities, 150 W halogen bulbs were added at appropriate distances to obtain the surface light intensity desired. Light intensities were measured using a model QSL-100 quantum scalar irradiance meter (Biospherical Instruments, Inc., San Diego, CA). For growth curve measurements, all strains were grown under standard conditions (250 µE

-2 -1 m s at 38°C with 1-5% (v/v) CO2/air constantly bubbling) into exponential phase and 26

were diluted to an OD550nm = 0.05 prior to a shift to a different light intensity. Once the cells were shifted to a new light intensity, the cell growth was monitored at that new light intensity over a 24-hour period.

Growth was determined by monitoring the turbidity of cells at 550 nm in a

Bausch and Lomb (now Milton Roy, Rochester, NY) Spectronic 20 spectrophotometer.

To determine the doubling time, the absorbance of cells in various growth phases was measured at various time points, minimally in triplicate, and the results were plotted on a semi-log scale.

2.1.2 Synechocystis sp. PCC 6803

Synechocystis sp. PCC 6803 is a unicellular, naturally transformable freshwater cyanobacterium (Grigorieva and Shestakov, 1982). Synechocystis sp. PCC 6803 cells were grown at 32˚C in liquid medium BG-11 (Stanier et al., 1971) buffered with 5 mM

HEPES in 22 x 175 mm culture tubes bubbled with 1.5% CO2 in air and on 1.5% (w/v) agar plates in B-HEPES medium. B-HEPES medium consists of: 1.5g l-1 NaNO3 , 50 mg

-1 l-1 KH2PO4, 75 mg l MgSO4•7H2O, 272 mg l-1 CaCl2, 6.56 mg l-1 citric acid•H2O, 12 mg l-1 ferric ammonium citrate, 2 mg l-1 tetrasodium EDTA, 2.86 mg l-1 H3BO3, 20 mg l-1 NaCO3, 1.81 mg l-1 MnCl2 •4H2O, 3.89 mg l-1 FeCl3 •6H2O, 222 mg l-1 ZnCl2, 390 mg l-1 NaMoO4•2H2O 79 mg l-1 CuSO4•5H2O, 49.4 mg l-1 Co(NO3)2•6H2O, 1.1 g l-1

HEPES pH 8.0 (titrated with 2 M KOH). Cells in liquid culture were bubbled constantly 27

-2 -1 with 1.5% CO2. Light intensities were 100-150 mE m s for growth of liquid cultures and 60 mE m-2 s-1 for stock cultures on plates.

2.1.3 Escherichia coli

E. coli DH5a (genotype: F-, endA, hsdR17, supE44, recA1, gyrA96, relA1, argF)

(Bethesda Research Laboratories, Gaithersburg, MD) was used for all recombinant DNA manipulations. This strain is a recombination-deficient, phage-suppressing strain that is capable of a -complementation with the amino-terminal, a- fragment of beta- galactosidase that is encoded by pUC and pBluescript vectors. The cells were grown in liquid cultures or on 1.5% (w/v) agar plates of LB (Luria-Bertani) medium at 37˚C. LB medium contains 1% (w/v) bacto-tryptone, 0.5% (w/v) yeast extract, and 1% (w/v) NaCl, pH 7.5 (adjusted with NaOH). Antibiotics (ampicillin 100 mg ml-1, kanamycin 40 mg ml-

1; chloramphenicol 25 mg ml-1; and erythromycin 50 mg ml-1) were added when required.

E. coli BL21(DE3) (genotype: F-, ompT, rB-, mB- (DE3)) and E. coli

BL21(DE3)(pLysS) (Novagen, Madison, WI) were used for overproduction of proteins.

The cells were grown in NZCYM medium (pH 7.0) containing 1% (w/v) bacto-tryptone,

0.5% (w/v) yeast extract, 0.5% (w/v) NaCl, 0.1% (w/v) casamino acids, and 0.2 % (w/v)

MgCl2•7H2O. 28

2.2 Standard laboratory methods

Standard recombinant DNA procedures were performed according to the recommendations of the manufacturer of the respective kit or enzyme and by consulting current laboratory manuals (Ausubel et al., 1987; Sambrook et al., 1989). Restriction endonucleases and other DNA modifying enzymes were purchased from New England

BioLabs (Beverly, MA), Promega Corporation (Madison, WI), Bethesda Research

Laboratories (Gaithersburg, MD), and Boehringer Mannheim Biochemicals

(Indianapolis, IN). Radiolabelled [a -32P] dATP and [a -35S] dATP were purchased from

New England Nuclear Products, Dupont de Nemours & Co. (Boston, MA).

DNA fragments generated by restriction digests were separated by electrophoresis on agarose gels and purified using GENECLEAN® DNA isolation kit (Bio101, La Jolla,

CA), or isolated from the excised gel slice with Sigma (formerly Supelco)-spin columns

(Sigma, Bellefonte, PA). DNA fragments were labeled with [a- 32P] dATP, using a

Random Primed DNA Labeling kit (Boehringer Mannheim Biochemicals, Indianapolis,

IN).

Polymerase chain reaction (PCR), using Taq polymerase from Boehringer

Mannheim Biochemicals (Indianapolis, IN), was performed with one of the following thermocyclers: a Barnstead/Thermolyne Corporation Temp•Tronic® Series 669 thermocycler (Dubuque, IA), a Techne Progene thermocycler (PGC Scientific, Frederick,

MD), or an Eppendorf® Mastercycler Gradient Thermal Cycler (Fisher Scientific, 29

Pittsburgh, PA). Annealing temperatures were varied ± 5˚C from the calculated Tm of the synthetic primers (as calculated in the MacVector version 6.5 software package,

Oxford Molecular Group). Reactions of 30-35 cycles were generally carried out with a denaturation temperature of 94˚C and an extension reaction temperature of 72˚C.

Template quantities per reaction for plasmid DNA and chromosomal DNA were generally 1-10 ng and 100-500 ng, respectively. 50 pmoles of each primer was added to the reactions.

Radioactive signals from Southern blots, Northern blots, colony filters, and sequencing gels were exposed to Kodak X-OMAT AR, BIOMAX MR, or BIOMAX MS

X-ray film using intensifying screens (Lightening Plus, DuPont, Wilmington, DE) at -

80˚C or to a Molecular Dynamics PhosporImager screen when appropriate.

2.3 Isolation and manipulation of DNA

2.3.1 Plasmid and cosmid isolation from Escherichia coli

An alkaline lysis method was used for small-scale plasmid and cosmid DNA preparations from E. coli. The cells were resuspended in 200 ml of solution 1 (0.05 M

Tris-HCl (pH 7.5), 0.01 M EDTA (pH 8.0), 0.1 mg ml-1 RNaseA). An equal amount of solution 2 was added (0.2 M NaOH, 1% SDS) and as soon as the suspension cleared, 200 ml of solution 3 (3M NaOAc at pH 5.0) was added. Cell debris was pelleted by centrifugation and the cleared supernatant was transferred to a new tube. The plasmid 30

DNA was precipitated by the addition of an equal volume of isopropanol to the collected aqueous solution.

An alkaline lysis method was also used for large-scale plasmid isolations

(Birnboim and Doly, 1979). Plasmid DNA was separated from chromosomal DNA by

CsCl-ethidium bromide, equilibrium density-gradient ultracentrifugation (Sambrook et al., 1989). The super-coiled plasmid DNA fraction was collected from the gradient, extracted with NaCl-saturated isopropanol (to remove ethidium bromide), and dialyzed against TE buffer (10mM Tris-HCl pH 8.0, 1 mM EDTA).

2.3.2 Total DNA isolation from Synechococcus sp. PCC 7002 and

Synechocystis sp. PCC 6803

For large-scale preparations of total DNA from Synechococcus sp. PCC 7002 or

Synechocystis sp. PCC 6803, cells were isolated by first collecting 1 L of exponentially growing cells by centrifugation at 5000´ g. The resulting cell pellet was resuspended in 4 ml of 5 mM Tris-HCl (pH 7.5), 5 mM EDTA, 50 mM NaCl with 3 mg ml-1 lysozyme.

The cells were then incubated for one hour with agitation to keep the cells in suspension at 37˚C. The cells were subsequently lysed by adding 400 µl of 10% (w/v) sarkosyl and an equal amount of Tris-buffered phenol. This mixture was agitated by vortexing for 15 min. The organic and aqueous phases were separated by centrifugation at 8000´ g for 10 min. The aqueous layer was collected and 1 ml of 5.0 M NaCl and 1 ml of 10% (w/v) cetyltrimethylamine bromide (CTAB)-700 mM NaCl were added to remove 31 polysaccharides. An equal volume of chloroform was added to the mixture and the suspension was agitated by vortexing or shaking and subsequently centrifuged at 8000Xg for 5 min to separate the organic and aqueous phases. The aqueous phase was collected and the DNA was precipitated by the addition of an equal amount of isopropanol to the aqueous phase.

Small-scale chromosomal DNA extractions were performed by harvesting exponentially growing cyanobacterial cells (25 ml) by centrifugation at 5000´ g for 5 min, resuspending the cell pellet in 500 ml of lysis buffer (10% (w/v) sucrose, 50 mM

Tris-HCl pH 8.0, 10 mM EDTA), and subjecting the suspension to a freeze/thaw cycle.

Lysozyme was added (5 mg) and the mixture was incubated at 37˚C for 30 min. SDS (to

1% w/v) was added and the resulting solution incubated at 37˚C for one hour before phenol:chloroform extractions were performed. The aqueous phase was removed and combined with NaCl (to 1 M), one-fifth volume CTAB/NaCl solution (10% (w/v) CTAB,

700 mM NaCl), and an equal volume of chloroform to separate polysaccharides from the

DNA. DNA was precipitated with an equal volume of isopropanol.

2.4 Transformation procedures

2.4.1 E. coli transformation procedures

E. coli cells were prepared for transformation using the following methods. To prepare cells for transformation by electroporation, E. coli of the desired strain (BL21R, 32

BL21(DE3), DH5-a, or DH10-B) were streaked onto an LB plate with no antibiotics from an axenic freezer stock and allowed to grow overnight at 37°C. A single colony was selected from the plate and transferred to 5 ml of LB liquid media and grown overnight at

37°C with vigorous shaking. This 5 ml starter culture was transferred to 1L of LB and

grown for 2-3 hours at 37°C to an OD500 of 0.5-1.0. Cells were harvested by centrifugation in sterile tubes by at 4000´ g at 4°C for 15 min. The cell pellet was resuspended in 1L of sterile, cold water and collected by centrifugation (4000´ g, 4°C) for

10 min. The cells were washed three more times with 500 ml of sterile cold water, and were resuspended in 3 ml of filter-sterilized cold 10% (v/v) glycerol, and aliquots (40 µl) of this solution was transferred into Eppendorf tubes in. Plasmid DNA (10-50 ng) was added to E. coli cells that had been prepared for transformation. E. coli cells were transformed by electroporation (Transporator Plus, BXT, San Diego, CA, USA) at 1.5V using ice-cold transformation cuvettes with a 1.5 mm gap space. The cells were incubated in 1 ml of SOC or LB for 30-60 min at 37°C. Aliquots of the cells incubated in

SOC were transferred to LB plates containing the appropriate antibiotic(s).

Transformants were usually selected by blue-white screening for initial clones on LB ampicillin (100 µg ml-1) and X-gal (40 µg ml-1) agar plates.

2.4.2 Cyanobacterial transformation procedures

Synechococcus sp. PCC 7002 cells are naturally transformable (Stevens and

Porter, 1980) and take up either circular or linear DNA. Insertional inactivation of genes 33 was performed by interposon mutagenesis. A DNA fragment containing a gene conferring resistance to an antibiotic was ligated into a restriction site within the coding region of the gene selected for mutation. Replacement of the wild-type gene in the

Synechococcus sp. PCC 7002 genome occurred via double crossover homologous recombination by transforming cells with linear DNA fragments containing an insertionally inactivated gene. Generally, at least 200 bp of homologous sequence were flanking each side of the drug resistance cartridge. Approximately 1-2 mg of linear DNA was added to 0.9 ml of cells that were grown to a transmittance of 20% at 550 nm. The culture was exposed to full light and bubbled with 1.5% CO2 in air for 1.5 doubling times. Aliquots of this transformation mixture were spread on A+ plates and incubated at low light intensity for 2-3 days. Plates were then overlaid with the appropriate antibiotic in 0.8% top agar and incubated under standard light intensity. Transformants were visible

4-10 days after being challenged with the appropriate antibiotic. Single colonies were inoculated into liquid culture (10-20 ml), and the resulting cultures were, again, diluted 4-

6 times with media containing the appropriate antibiotic in order to allow segregation of mutant and wild-type alleles. Southern blot hybridization analyses and PCR amplifications of chromosomal DNA from the transformants were performed to verify the gene interruption. Amplification of the DNA from whole cells was performed as described (Howitt et al., 1996). Synechococcus sp. PCC 7002 cells were resuspended in sterile water at an optical density at 550 nm of 15 ml-1. One ml of this suspension was used in a standard PCR reaction. 34

2.5 Southern blot transfer and hybridization

Agarose gels containing restricted DNA fragments that had been separated by electrophoresis were photographed alongside a fluorescent ruler under UV light using the

Biophotonics (Ann Arbor, MI) Gel Print 2000i video imaging system. The DNA was denatured by soaking the gel in denaturation solution (0.5 M NaOH, 1.5 M NaCl) for 1 hour and subsequently neutralized by soaking in neutralizing solution (0.5 M Tris pH 8.0,

1.5 M NaCl) for 1 hour. The DNA fragments were transferred to nitrocellulose membranes or nylon membranes (Schleicher & Schuell, Keene, NH) by capillary action in 10X SSC (0.15 M sodium citrate pH 7.0, 1.5 M NaCl) overnight. Nitrocellulose and nylon membranes were baked for 1 hour under vacuum at 80˚C before use in hybridization experiments. Pre-hybridization of the nitrocellulose filters or nylon filters was performed in a modified hybridization buffer described by Church and Gilbert

(1984). The modified version of the hybridization buffer consists of 5% (w/v) SDS,

0.5M NaPO4, and 1% (w/v) Bovine Serum Albumin (fraction V from Sigma, St. Louis).

The temperature of incubation determined the stringency of the hybridization and was determined empirically (Bryant and Tandeau de Marsac, 1988). Typically, low- stringency hybridizations were carried out between 50-55˚C, whereas high-stringency conditions were 60˚C - 70˚C. Following hybridization for at least 12 hours, blots were washed with wash buffer (Church and Gilbert, 1984) at the hybridization temperature six times or until there were no radioactive counts in the wash eluent. The wash buffer

consisted of 1% (w/v) SDS, 40 mM NaPO4, pH 7.2. 35

2.6 Cloning and sequencing

2.6.1 General cloning strategy

The following procedures were adapted for gene cloning from Ausubel et al. (1987) and Maniatis et al. (1982). First, a Southern blot hybridization experiment was carried out with chromosomal DNA, which had been digested with various combinations of restriction enzymes. The respective hybridization probe was chosen to maximize the signal intensity of the targeted gene to be cloned. In some cases several hybridization experiments were necessary to determine this. After analysis of the results of the hybridization experiment(s), a convenient restriction fragment was chosen for cloning based on size and restriction enzyme cutting efficiency. In the case of Synechococcus sp.

PCC 7002 a partial cosmid library was available, and if genes from this organism were to be cloned, this library was first screened to determine if the gene of interest was present.

Otherwise, size-fractionated libraries of fragments were created by restriction digestion of chromosomal DNA, by gel-purifying the desired range of fragments, and by ligating the fragments into a plasmid vector. The usual vector of choice was the high-copy- number vector pUC19 (Yanisch-Perron et al., 1985) or pBluescript SK+ (Stratagene, La

Jolla, CA). The fragment library was transformed into competent E. coli DH5a cells, and colonies were screened to be able to identify the targeted gene. Approximately 1000 colonies were generally analyzed by colony hybridization with the same probe that was 36 used for the initial Southern hybridization. Positive clones were selected and confirmed by further hybridization experiments. Final confirmation was obtained by DNA sequence analysis.

2.6.2 Construction of a Synechococcus sp. PCC 7002 genomic library

A total of 701 E. coli DH5-a clones made up the genomic library. Each clone harbors a cosmid containing a Synechococcus sp. PCC 7002 DNA fragment insert ranging in size from 20-kb to 45-kb. 576 clones originated from genomic DNA partially digested with EcoRI and ligated into cosmid vector pHC79. The remaining 125 clones (a gift from Dr. William R. Widger, University of Houston, USA) were made by partially digesting genomic DNA with Sau3A and with subsequent insertion of the resulting fragments into the cosmid vector SuperCos (Stratagene, La Jolla, CA, USA). The library was maintained on eight 96-well master plates; each clone was grown in 100 µL LB medium containing ampicillin, and stored at -80˚C after the addition of sterile glycerol to a final concentration of 10 % (v/v).

2.6.3 Probes

Genes putatively involved in electron transport were identified by sequence homology from the fully sequenced genome of the cyanobacterium Synechocystis sp.

PCC 6803 (Kaneko et al., 1996) and primers were made to amplify these genes (Table 2) 37

Table 2. Synechocystis sp. PCC 6803 electron transfer protein gene PCR primers

Primer name Nucleotide sequence (5’-3’) ORF number gene name slr1379R TTA CTC CTT AAC AAC AGC slr1379 cydA slr1379F ATG CAG GAT TTT TTG AGT AAC slr1379 cydA slr1185R TCA AAC CCA CCA GGG slr1185 petC slr1185F ATG GAT AAC ACA CAG GCG slr1185 petC slr1281F GTG GCT GAG GAA GTG AAC slr1281 ndhJ slr1281R CTA ATA GGC ATC CTG GAG slr1281 ndhJ slr1280R TCA GCC ACG GTT TAA TTG slr1280 ndhK slr1280F ATG AGT CCC AAC CCT GCT slr1280 ndhK sll0823F ATG GAA ATT GTT TGC AAA ATA sll0823 sdhB sll0823R TTA AAG GGG ATC CAT CAA GTC sll0823 sdhB sll0629F ATG ACG GCG ATC GCC AGG sll0629 psaK sll0629R CTA GAG AAT GCC ACT ACT AGC sll0629 psaK slr2009F ATG AAT TTT CCC ACG GCG slr2009 ndhF slr2009R TCA TAT TAA TAC CGA GCT slr2009 ndhF slr0261F ATG ACC AAG ATT GAA ACC slr0261 ndhH slr0261R CTA GCG GTC CAC CGA TCC slr0261 ndhH slr2007F ATG ACC CTA TTT GCA GTT slr2007 ndhD slr2007R TCA TAC TAA AAT CCA GGC slr2007 ndhD sll0027F ATG CTC AGT GCT CTC ATC sll0027 ndhD sll0027R CTA CAC TTC CCC CAA TTC sll0027 ndhD slr0851R CTA GGG GCC CAC TAG TTC slr0851 ndbA slr0851F ATG AAT TCC CCC ACC TC slr0851 ndbA slr1743F ATG ACG GAC GCT CGA CC slr1743 ndbB slr1743R GGA AGG TTC ATT TTT GAG slr1743 ndbB slr1484R CTA CCG ATT CAT ACC GG slr1484 ndbC slr1484F GTG GGG TTC CAG TTT CCA slr1484 ndbC 5'1226 ATG TCT AAA ACC ATT GTT ATC sll1226 hoxH 3' 1226 TTA ATC CCG CTG GAT GGA sll1226 hoxH 5'ndhB ATG GAC TTT TCT AGT AAC sll0223 ndhB 3'ndhB CTA GGG TAA ATC ATG GGA sll0223 ndhB slr1743.1 ATG ACG GAC GCT CGA CCA slr1743 ndbB slr1743.2 GAG CCA ATC CCC CAG GGG slr1743 ndbB slr1279 GTG TTT GTT TTA ACC GGT TAC slr1279 ndhC slr1279R CTA GGA CCA CTC CAG AGC slr1279 ndhC sll0026 250F CGG TGG AGA TTT CCC CGG sll0026 ndhF slr1380R CTA GTC GGT GAC AAT TTT GCC slr1380 cydB slr1380F ATG GAA CCG TTA GAA CCG slr1380 cydB 38 by PCR. Amplified PCR products were electrophoresed on standard agarose gels and purified as described previously (see 2.2). The purified Synechocystis sp. strain PCC

6803 PCR products were labeled with (a 32P)-dATP by use of a Random Primed DNA

Labeling Kit (Boehringer Mannheim, Germany) and used as probes to screen cosmid libraries, partial genomic libraries and genomic DNA blots. Hybridization probes made from Synechococcus sp. PCC 7002 DNA that had been isolated from clones or PCR products, were made in the same way as those for Synechocystis sp. PCC 6803.

2.6.4 Colony hybridization

Colony hybridizations were carried out as described by Sambrook et al. (1989).

Usually 100 individual transformants were transferred to a fresh LB agar plate containing the appropriate antibiotic(s) with a sterile loop. The cells were scraped into a grid template and the plate was marked on a single side as a frame of orientation for the colonies. The plate was replicated as described in Sambrook et al. (1989), and one plate served as the reference plate. The colonies were lifted from one plate onto circular nitrocellulose membranes (Schleicher & Schuell, Keene, NH), or in the case of the rectangular plates containing the cosmid library onto rectangular-cut Nytran-Plus membranes (Schleicher & Schuell, Keene, NH). After the colony transfer, the membranes were placed on filter paper soaked with denaturation solution to lyse the cells and denature the DNA. The membranes were then transferred to a filter paper soaked in neutralization solution to neutralize the denaturation solution. The membranes were air- 39 dried between the two steps. These are the same solutions as described above for

Southern transfers. The following steps are also similar to the steps leading to hybridization described in the section on Southern transfers. To lower the background hybridization of colony filters, cell debris was removed by gently rubbing the pre- hybridized filters, fresh pre-hybridization solution was added, and the filters were pre- hybridized for at least another hour prior to probe addition.

2.6.5 DNA sequencing and analysis

The sequences of all cloned genes were completely determined on both strands.

Manual sequencing was carried out by the dideoxy chain-termination method (Hattori and Sakaki, 1986; Tabor and Richardson, 1987a) following the protocol supplied with the

SEQUENASE® Version 2.0 DNA sequencing kit (U. S. Biochemical Company, Inc.,

Cleveland, OH). The templates were base-denatured (Hattori and Sakaki, 1986) prior to sequencing. Sequencing reactions were electrophoresed on 5% (w/v) acrylamide

(acrylamide:bis-acrylamide, 19:1 (w/w)) gels (40 cm x 33 cm x 0.4 mm) containing 8 M urea and TBE buffer (89 mM Tris pH 8.3, 89 mM boric acid, 2.75 mM EDTA) at a constant power of 52-60 W. Gels were soaked in 10% (v/v) acetic acid, 10% (v/v) methanol for 15-30 min before being transferred to Whatman paper (3MM) and dried under heat and vacuum. Dried gels were exposed to Kodak X-Omat AR or Biomax AR film. Other sequencing reactions were performed at the Nucleic Acid Facility at The

Pennsylvania State University with an ABI PRISM 377 DNA sequencer (Perkin-Elmer 40

ABI, Foster City, CA, USA) by using the method of 3' BigDye-labeled dideoxynucleotide triphosphates. Sequences were assembled in MacDNASIS Pro V2.5

(Hitachi Software Engineering Co., Ltd., Japan). Other sequence manipulations were performed with MacVector 6.5 (Oxford Molecular Co., Ltd., UK) software. Amino-acid sequences obtained in this work were compared with sequences obtained from the

National Center for Biotechnology Information (NCBI) databases and aligned with the

ClustalW multiple sequence alignment program (Thompson et al., 1994a) by using default parameters within the MacVector 6.5 program.

2.7 RNA isolation, Northern-blot hybridization, and RT-PCR analysis

2.7.1 RNA isolation

All glassware was baked at 400˚C for 4 hours, aqueous solutions were treated with

0.1% (v/v) DEPC (diethylpyrocarbonate) for 12 hours, and gloves were worn throughout the entire procedure in order to prevent contamination by RNases. Total RNA from

Synechococcus sp. PCC 7002 was isolated using a modified mechanical breakage protocol (Golden et al., 1987). Cultures (150-200 ml) were harvested by centrifugation and the cells were resuspended in 5 ml 50/100 TE (50 mM Tris-HCl pH 8.0, 100 mM

EDTA) and transferred to glass centrifuge tubes. Chloroform was added and incubated with the cell mixture for 5 min on ice with occasional shaking. After centrifugation at

5000´ g for 10 min at 4˚C, the cell pellet was resuspended in 5 ml of breakage buffer (30 41 ml 50/100 TE, 0.15 ml Triton X-100, 0.6 ml 25% (w/v) sarkosyl, 0.6 ml 20% (w/v)

SDS). Acid-washed, baked 0.45-mm glass beads (4.0 g) and a 1:1 mixture of phenol/chloroform (5 ml) were added. This suspension was vortexed in three bursts of 3 min each, with incubation on ice for 2 min between each burst. After centrifugation, the aqueous phase was transferred to a new tube and extracted twice with phenol/chloroform and twice with chloroform. The RNA and DNA was precipitated with ethanol, pelleted by centrifugation, dried at room temperature, and resuspended in 50/100 TE. The RNA was separated from DNA by precipitation with 2.5 M LiCl (Sakamoto and Bryant, 1997).

RNA concentrations were quantified by a fluorometric assay using the dye SYBR

Green II (Molecular Probes, Inc., Eugene, OR) as described by Schmidt and Ernst (1995) and/or by inspection of a UV spectrum for the RNA sample. A standard curve was made by measuring the fluorescence of known quantities of RNA, which had been determined by measuring the absorbance of a stock solution at 260 nm with a Cary 14

spectrophotometer (Olis, Bogart, GA); An absorbance of A260nm = 1.0 was assumed to contain 40 mg ml-1 RNA.

2.7.2 Northern-blot hybridization analyses

RNAs were size-fractionated by electrophoresis through 1.5 % (w/v) agarose gels made with running buffer (50 mM HEPES pH 7.8, 1 mM EDTA including 16% (v/v) formaldehyde). An equal volume of loading buffer (50% (v/v) formamide, 16% (v/v) formaldehyde, HEPES/EDTA buffer, 10% (w/v) glycerol, 0.025% (w/v) xylene cyanol, 42

0.025% (w/v) bromphenol blue, 0.025% (w/v) ethidium bromide) was added to each

RNA sample. The samples were then incubated in a 68˚C water bath for 5 min prior to loading on the gel. Gels were typically run at 25 volts for 16 hours in the dark at room temperature. The ribosomal RNA bands were visualized under UV light and photographed with a Biophotonics (Ann Arbor, MI) Gel Print 2000i video imaging system. The formaldehyde-agarose gels containing RNA were soaked in distilled water five times for 30 min. This was followed by a 30-minute soak in 20´ SSC. The filter papers were also soaked in 20´ SSC. The membrane was soaked in 10´ SSC prior to transfer of the RNA by capillary action to Nytran Plus nylon membranes (Schleicher &

Schuell, Keene, NH) using 20´ SSC (1´ SSC = 0.015 M Na citrate pH 7.0, 0.15 M NaCl) for 16-24 hours. After the transfer, the RNA was fixed to the nylon membrane for two min by UV crosslinking. A slightly modified set of hybridization and washing conditions were used as described in Church and Gilbert (1984). The membranes were incubated in

hybridization buffer (5% (w/v) SDS, 0.5 M NaPO4, pH 7.2, 1% (w/v) Bovine Serum

Albumin, Fraction V) for a minimum of 2 hours. After this time, the hybridization buffer was replaced and a denatured gene-specific probe was added to the membranes. The membranes and probe were incubated overnight at 55°C. The membranes were washed

with wash buffer (1% (w/v) SDS, 40 mM NaPO4, pH 7.2) six times for 15 min each until no counts were detectable with a pancake radiation monitor. 43

2.7.3 Reverse transcriptase-polymerase chain reaction (RT-PCR)

The following primers were used for the RT-PCR reactions with the

Synechococcus sp. PCC 7002 total RNA or cDNA templates. For ctaCII, primer ctaCII.1

(5'-CAC TTT CGG CGA TCG CCC TAC TTT TGG GGG-3') and primer ctaCII.2 (5'-

GGG ATG ATA ATT CAC CAC-3') were used. For ctaDII, primer ctaDII.1 (5'-CCA

TGA CCC AAG CTC CC-3') and primer ctaDII.2 (5'-CGG GAA CCA GTG GTA CAC

CGC-3') were used. For ctaEII, primer ctaEII.1 (5'-GAC TGC CAT CAA TGA AAC C-

3') and primer ctaEII.2 (5'-ATT GCC AGA GAT AAA TCA GCC-3') were used. 1 µg of total RNA isolated from Synechococcus sp. PCC 7002 was diluted to a final volume of 20

µl with 20 µM of primer (either primer 683, primer 1407, or primer 1288) and incubated at 70˚C for 5 min to denature secondary structure. Each mixture was briefly incubated on ice. 5 µl of 5´ M-MLV Reaction buffer (5 µl of 5´ M-MLV Reaction buffer = 250 mM

Tris-HCl, pH 8.3, 375 mM KCl, 15 mM MgCl2, 50 mM DTT), 1µl of 25 mM dNTPs,

0.625 µl RNAsin, and 1.5 µl of M-MLV reverse transcriptase was added to the mixture, and the mixture was incubated at 42˚C for 1 hour. Individual reaction mixtures were incubated at 70˚C for 5 min to stop the extension reaction and treated with 5U of RNAse

H and 5U of RNAse A for 20 min at 37˚C. Each reaction mixture was brought up to a volume of 200 µl with TE and concentrated using Nanosep 100 spin columns. The three different cDNAs were collected from the membrane by washing with 20 µl of TE.

Aliquots (4 µl) of this mixture were used for second strand synthesis by PCR. 44

2.8 Overproduction of the rBcpA protein in E. coli

2.8.1 Generation of E. coli expression plasmids

Plasmids for the overproduction of proteins in E. coli were constructed using the pET plasmid system (Studier et al., 1990). Appropriate restriction sites were engineered by

PCR into genes destined for overexpression. An NcoI or NdeI site was introduced at the start codon of the gene via the design of the 5' oligonucleotide primer, and a BlpI site was introduced downstream of the stop codon in the 3' oligonucleotide primer. The modified and amplified PCR product was digested with appropriate restriction enzymes and ligated into the equivalent sites of the chosen pET vectors, pET30C+. or pET16 The pET vectors were purchased from Novagen (Madison, WI), and the conditions for overexpression were according to those recommended by the manufacturer. Briefly, E. coli DH5-a cells were transformed with the pETBcpA plasmids as described in 2.4.1 and kanamycin resistant colonies were selected. Selected individual colonies picked were transferred to

5 ml of LB-kanamycin or LB-ampicillin liquid medium and grown overnight. These isolated pETBcpA plasmids were used to transform either BL21R or BL21(DE3) E. coli strains for recombinant protein overproduction. 45

2.8.2 Protein overproduction in E. coli and isolation of inclusion bodies

A frozen stock of pET30C+/BCP or pET16BCP was used to inoculate an LB- kanamycin or LB-ampicillin plate to grow individual colonies harboring the pET30C+/BCP or pET16BCP plasmid. The plates were allowed to grow overnight at

37˚C. An individual colony was picked from the plate and grown in either 5 ml of LB- ampicillin or 5 ml of LB-kanamycin media (depending on the plasmid) at 37˚C to an

OD600 of 0.5. These 5 ml starter cultures were used to inoculate 500 ml or 1 L of LB- kanamycin or LB-ampicillin liquid media and the cultures were incubated with rigorous shaking at 37˚C until the OD600 was between 0.4 and 1.0. This usually took between 2-3 hours, after which, IPTG from 100 mM stock was added to a final concentration of 0.4 mM and the incubation was continued for 2-3 hours. The flasks were placed on ice for 5 min and the cells were harvested by centrifugation at 5000´ g for 5 min at 4˚C. The cell pellet was resuspended in 0.25´ culture volume (125 ml) of TS (50 mM Tris, pH 8.0, 100 mM NaCl) and centrifuged again to wash the cells. The pellet was resuspended in 30 ml of TS, and the cells were broken by using a French Pressure cell operated at 20,000 psi and maintained at 4˚C. The broken cell mixture was centrifuged at 10,000´ g in a

Beckman JA14 rotor to separate the inclusion bodies/membrane fraction from soluble proteins for 5 min at 4˚C. The inclusion body pellet was resuspended in 3 ml of TS buffer and centrifuged to wash the pellet. The pellet was washed twice with 0.1% (v/v)

Triton X-100 and washed again with TS buffer to remove Triton X-100. Next, the 46 inclusion body pellet was resuspended in 10 M urea (TS, 4 mM 2-mercaptoethanol) using a minimal amount of buffer. This mixture was allowed to sit at room temperature for 1 hour and then centrifuged at 10,000´ g in a Beckman JA14 rotor for 10 min to remove insoluble material. The protein concentration was determined with the Pierce Coomassie protein concentration assay. The solubilized pellet was then diluted with buffers as described in the Results section to remove the chaotrope.

2.8.3 Purification of rBcpA from E. coli

The solubilized inclusion bodies were purified either by size exclusion chromatography using a Sephadex G-75 resin (Amersham Pharmacia, Piscataway, NJ) or a carboxymethyl (CM) sepharose resin (Amersham Pharmacia, Piscataway, NJ). For size exclusion chromatography, the rBcpA protein was diluted in 0.8 M Urea TS buffer and was loaded onto a pre-equilibrated G75 column. The running buffer was TS that had been filter-sterilized and degassed. 2-mercaptoethanol was added to final concentration of 4 mM to reduce disulfide bridges formed during the overproduction of the recombinant protein in E. coli. The column was kept at 4˚C throughout the experiment.

Fractions were collected according to the 280 nm absorbance of the protein. The fractions were dialyzed against TS after the column chromatography to remove the 2- mercaptoethanol. For the purification of rBcpA using an ion exchange column, the sample was diluted in 5PEM buffer (5 mM sodium phosphate, 1 mM EDTA, 2 mM 2- mercaptoethanol) until the urea concentration was 0.8 M. The sample was loaded onto a 47

CM-sepharose column and the protein was eluted from the column at room temperature by using a NaCl gradient (0-500 mM). The fractions collected were tested for protein content with the Pierce BCA Protein Assay and were electrophoresed on SDS polyacrylamide gels to determine the purity of the rBcpA protein.

2.9 Cytochrome c6 purification from Synechococcus sp. PCC 7002 and amino

terminal sequencing

20 L of Synechococcus sp. PCC 7002 cells were grown in A+ medium. Cells were collected by centrifugation at 10,000´ g, resuspended in 100 ml of Tris, pH 7.5 and broken by passage through a French Press. The soluble fraction of the crude extract was separated from the insoluble fraction by ultracentrifugation at 45000 ´ g for 1 hour. The supernatant was collected and precipitated overnight at 4˚C with NH4SO4 (65% saturation). The soluble fraction was separated by centrifugation at 8000 ´ g for 10 min and the supernatant was collected. The soluble supernatant was fractionated again by an overnight NH4SO4 (95% saturation) precipitation at 4˚C. The proteins were collected by centrifugation at 10000 ´ g for 10 min. The pellet was resuspended in a minimal amount of 20 mM Tris, pH 7.8 and deionized by dialysis in 20 mM Tris, pH 7.8 overnight at 4˚C.

Cytochrome c6 was separated from other proteins by FPLC using a linear gradient of 1M

NaCl in 50 mM Tris, pH 8.0 on a mono-Q column. Fractions from the mono-Q

chromatography were collected and cytochrome c6 was identified by its absorbance properties. Fractions containing cytochrome c6 were pooled and precipitated by 48

trichloroacetic acid. The precipitated samples of cytochrome c6 were resuspended in sodium dodecyl sulfate (SDS) sample buffer (60 mM Tris-HCl, pH 6.8, 2% (w/v) SDS,

10% (v/v) glycerol, 0.1 mg bromphenol blue per ml, 5% (v/v) 2-mercaptoethanol), and loaded onto SDS polyacrylamide gradient gels (10-20%) and electrophoresed as

described below. Cytochrome c6 was identified by TMBZ staining (Thomas et al., 1976) and Coomassie staining (see below). The protein band corresponding to cytochrome c6 was excised from the gel and electroeluted to PVDF membrane for N-terminal sequencing at the University of Nebraska Protein Core Facility (Department of

Biochemistry, The Beadle Center, P.O. Box 880664, Lincoln, NE 68588-0664).

2.10 SDS-polyacrylamide gel electrophoresis, TMBZ staining and immunoblot

analysis

2.10.1 SDS-PAGE

Polyacrylamide gel electrophoresis in the presence of SDS was performed as described by (Laemmli, 1970). Gels used in immunoblot studies and in protein expression studies were 10%, 15 %, or 10-20% (w/v) linear gradient polyacrylamide slab gels (30:0.8 (w/w) acrylamide:bisacrylamide). Stacking gels were typically 4% (w/v) acrylamide. Protein samples were heated to 65˚C in SDS-sample buffer (60 mM Tris-

HCl, pH 6.8, 2% (w/v) SDS, 10% (v/v) glycerol, 0.1 mg bromphenol blue ml-1, 5% (v/v)

2-mercaptoethanol) for 5 min prior to loading. Typically the gels were electrophoresed 49 for 12-16 hours under constant amperage in a buffer containing 25 mM Tris, 0.2 M glycine, 0.1% (w/v) SDS for 16 cm gels. Proteins separated by electrophoresis were stained with Coomassie blue stain (0.125% (w/v) Coomassie Brilliant Blue, 50% (v/v) methanol, 10% (v/v) acetic acid) and destained to visualize the protein by washing the gel with destaining solution (50% (w/v) methanol, 10% (v/v) acetic acid) until the protein bands were clearly visible.

2.10.2 3, 3', 5, 5'-tetramethylbenzidine (TMBZ) staining

Proteins with c-heme groups (such as cytochrome c6) can be specifically

visualized by 3, 3', 5, 5'-tetramethylbenzidine (TMBZ)-H2O2 staining (Thomas et al.,

1976). A 6.3 mM TMBZ (Sigma-Aldrich Chemical Co., St. Louis, MO) solution was freshly prepared in methanol. Immediately before use, 3 parts of the TMBZ solution were mixed with 7 parts of 0.25 M sodium acetate, pH 5.0. The gels were immersed in this mixture at room temperature in the dark. After 1-2 hours with occasional mixing

(every 10-15 min), H2O2 was added to a final concentration of 30 mM. The staining was visible within 3-30 min. The gels were washed with a (3:7) isopropanol:0.25 M sodium acetate, pH 5.0 solution that was replaced once or twice to remove any precipitated

TMBZ. 50

2.10.3 Immunoblot analysis

The nitrocellulose membrane, filter papers, and SDS-polyacrylamide gel were pre-soaked in transfer buffer (25mM Tris, 192 mM glycine (20% (v/v) methanol), pH

8.3) for 30 min. Proteins separated by SDS-PAGE were electrophoretically transferred onto the nitrocellulose membranes (Schleicher & Schuell, Keene, NH) for 10-45 min at

10-15V using a BioRad Trans-Blot SD Semi-dry transfer cell (Richmond, CA). The membrane was stained with 0.5% (w/v) Ponceau-S in 1.0% (v/v) acetic acid for 30 seconds to determine the transfer efficiency. The Ponceau-S stain was removed by washing the membrane with several changes of distilled water. The membrane was then placed in 5% (w/v) nonfat milk in TBS buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl) for 1 hour on a shaking platform at room temperature. Next, the membrane was incubated in a 3% nonfat milk/TBS solution with the primary antibody at a dilution of

1:5000 to 1:10000 for 1-4 hours at room temperature. After incubation with the primary antibody, the membrane was washed with TBST (10 mM Tris-HCl, pH 8.0, 150 mM

NaCl, 0.05% (v/v) Tween-20) buffer for 15 min, followed by two more washes with

TBST buffer of 5 min each. The membrane was then incubated with the secondary antibody (goat, anti-rabbit IgG-alkaline phosphatase conjugate, Sigma, A-8025, St.

Louis, MO) diluted 1:3000 in 1% (w/v) nonfat milk/TBS for 2-3 hours. The membrane was rinsed briefly with TBS buffer and then washed with TBST buffer once for 15 min, followed by 4 washes in TBST buffer of 5 min each. The membrane was finally washed 51 with TBS buffer to remove the Tween-20. The color of the blot was developed in 50 ml

of alkaline phosphatase buffer (100 mM Tris-HCl, pH 9.5, 100 mM NaCl, 5 mM MgCl2) containing 0.33 ml of nitroblue tetrazolium (50 mg ml-1 in 70% (v/v) dimethyl

-1 formamide) and 0.165 ml of bromochlorindolyl phosphate (50 mg ml in ddH2O). The membrane was incubated in this solution at room temperature with agitation until bands

were suitably dark. The blot was rinsed with ddH2O to stop the reaction and allowed to air dry. After color development was complete, the blots were rinsed in distilled water and stored in the dark.

2.11 Determination of chlorophyll a and carotenoid contents

Chlorophyll a and total carotenoid concentrations were determined as described by Sakamoto and Bryant (1998). Cyanobacterial cells (1.0 ml) in exponential growth phase were harvested by centrifugation in 15 ml COREX tubes at 10,000´ g at room temperature for 5 min. The supernatant was removed and chlorophyll a and carotenoids were extracted with dimethylformamide for 15 min at room temperature. The concentrations of chlorophyll a and carotenoids in the dimethylformamide extract were calculated from the following equations (A750nm was subtracted to correct for light scattering):

-1 [Chlorophyll a (µg ml )] = (A664nm-A750nm) ´ 11.92

-1 [Carotenoids (µg ml )] = {(A461nm-A750nm) - 0.046 ´ (A664nm-A750nm)} ´ 4 52

2.12 Oxygen evolution and consumption rates

Oxygen evolution and consumption rates were determined using a Clark-type electrode from Hansatech, Inc. (UK). The cyanobacterial cells were harvested by centrifugation, washed with fresh A+ media, and centrifuged again. The cyanobacterial cell pellets were resuspended to a final chlorophyll concentration of 3-5 µg chlorophyll a per ml for oxygen evolution measurements. The cyanobacterial cell pellets were resuspended to a final concentration of 50-100 µg of chlorophyll a per ml for oxygen consumption measurements. The cells were kept in the dark 10-15 min and then agitated to saturate the samples with air levels of oxygen prior to addition to the electrode chamber. A total of 1 ml of cells was added to the electrode chamber for each individual experiment. The electrode chamber temperature was maintained at 38˚C with a circulating water bath, and the chamber contents were continuously stirred.

Measurements of oxygen evolution were determined after two min in the dark by stimulation with saturating amounts of light (2.5 mE m-2 s-1). Measurements of oxygen consumption were determined in complete darkness.

2.13 P700+ kinetic measurements

The photoinduced absorption change attributable to P700 can be monitored with a single beam spectrophotometer (Maxwell and Biggins, 1976). Thus, the reduction 53 kinetics of P700+ in whole cells of Synechococcus sp. PCC 7002 were measured using a detection system similar to the one described by (Yu et al., 1993) and originally by

(Maxwell and Biggins, 1976). Cyanobacterial cells were grown under standard growth

-2 -1 conditions (38˚C, 250 µE m s , 1.5% (v/v) CO2/air) and harvested by centrifugation.

The cells were resuspended in 1 ml Ficoll saturated (200 mg ml-1) 25 mM Tris, pH 8.3 and adjusted to a chlorophyll a content of 40 µg ml-1. Modulated light was provided with an oscillation frequency of 22 kHz, and the measuring beam was passed through a 5-mm-

wide sample cuvette with a 1-cm pathlength. A colored glass bandpass filter (l cuton > 800 nm) was inserted between the cuvette and the detector to minimize scattered actinic illumination and fluorescence artifacts. The modulated near-IR measuring beam was detected with a photodiode (RCA 30810), and the signal was demodulated with a lock-in amplifier (model 5101; EG&G, Sunnyvale, CA). The instrument was operated with a time constant of 1 ms. The sample was excited with white light generated from a 150-W quartz-tungsten lamp that was focused on the cuvette (Yu et al., 1993). The cells were kept in the dark prior to the experiment when they were subjected to a flash of saturating light lasting 22 seconds to fully oxidize P700, followed by a dark incubation for 15 seconds to reduce P700+. The signal-to-noise ratio was improved by averaging 128 light- dark cycles with a Nicolet 4095A digital oscilloscope (12-bit resolution, 500 µs point-1).

The first light dark cycle was recorded separately and compared with the average; no changes in kinetics were found during the course of any measurement. The data were transferred to and analyzed with IGORPRO v. 3.5 as described in (Yu et al., 1993). In all cases, a single-exponential curve provided the best fit for the P700+ reduction kinetics. 54

2.14 77K fluorescence emission measurements

Exponentially growing cyanobacterial cells (10 ml) grown under various light intensities (see Results) were harvested by centrifugation at room temperature at 8000´ g for 5 min and resuspended in 200 µl of 60% (v/v) glycerol, 50 mM HEPES, pH 7.0.

Samples were adjusted to a final OD730 nm of 1.0 in 1.0 ml of 60% glycerol, 50 mM

HEPES, pH 7.0. Measurement of 77K fluorescence was determined with an SLM-

Aminco 8000C spectrofluorometer (Spectronic Instruments Inc, Rochester, NY, USA); the excitation wavelength was 440 nm for chlorophyll excitation. Absorption spectra were obtained with a Cary-14R spectrophotometer modified for computerized operation, data collection and data analysis by On-Line Instruments Systems (Bogart, GA, USA) as described by (Sakamoto and Bryant, 1998).

2.15 Pulse Amplitude Modulated (PAM) fluorescence measurements

In PAM fluorescence measurements, a constant, weak modulated light source is combined with a system that selectively detects the fluorescence emitted at the same frequency and phase as the modulated source (Schreiber et al., 1986). Because the modulated source is of constant irradiance, one can measure the modulated fluorescence signal once the unmodulated background irradiance is high enough to close all of the reaction centers (Geider and Osborne, 1992). Therefore, one can use PAM fluorescence 55 to determine the number of functional PSII complexes in whole cells. Exponentially growing cyanobacterial cells (25 ml) grown under standard growth conditions (38°C, 250

-2 -1 µE m s , 1.5% (v/v) CO2) were harvested by centrifugation at room temperature at

8000´ g for 5 min. The cell pellet was resuspended and washed with 25 mM HEPES-

NaOH buffer, pH 7.0. The cells were collected again by centrifugation and resuspended

-1 to a final OD550 nm = 1.0. Cells (1-3 ml) at a chlorophyll concentration of 5 µg ml in

25mM HEPES-NaOH buffer, pH 7.0 were added to the cuvette. Sodium bicarbonate (1.0 mM final concentration) was added to the cuvette as well. The cells were incubated for 2

min in complete darkness before establishing F0, or the ground state. FM’, or the maximal fluorescence, was obtained by exposing the cyanobacterial cells to a saturating light pulse for 1 second intervals. The fluorometer settings were changed to 100 kHz from 1,600

kHz to obtain FV’, or variable fluorescence. After establishing Fm’, DCMU was added to

1 µM in order to obtain FM. by closing all PSII reaction centers completely. By examining the ratio of FV’/FM’ (variable fluorescence to maximal fluorescence) one can determine the total number of active PSII clusters compared to total chlorophyll content.

2.16 Superoxide dismutase (SOD) activity measurements

SOD inhibits the reduction of nitro-blue tetrazolium by superoxide by dismutation of superoxide to hydrogen peroxide and oxygen (Winterbourn et al., 1975). This allows one to measure the amount of SOD activity in soluble and membrane fractions of

Synechococcus sp. PCC 7002. Cyanobacterial cells (150 ml) were collected by 56 centrifugation and washed with 50 mM potassium phosphate buffer, pH 7.8. The recollected cells were resuspended in 20 ml of 50 mM potassium phosphate buffer and broken by passage through an SLM-AMINCO French Press. The broken cells were centrifuged at 4°C, 5000´ g for 5 min to collect unbroken cells. The supernatant was collected and subjected to a high-speed centrifugation (45,000´ g for 1 h) at 4°C to separate the soluble and membrane fractions. The soluble fraction was collected and immediately used in SOD assays. The membrane fraction was collected and resuspended in a minimal amount of 50 mM potassium phosphate buffer, pH 7.8 and used for SOD assays. The protein concentration for each fraction was determined by Coomassie assay.

SOD activity assays were performed according to the protocol of (Winterbourn et al.,

1975).

2.17 Detection of hydroperoxides

A modified ferrous oxidation/xylenol orange assay was used to determine the levels of hydroperoxides in cyanobacterial cells (Sakamoto and Bryant, 1998).

Exponentially growing cyanobacterial cells were first diluted to an OD550nm of 0.2 and were incubated under standard growth conditions (250 µE m-2s-1 at 38°C) or in darkness for 4 hours in the presence or absence of 50 µM methyl viologen. The cells were harvested by centrifugation at 5000´ g for 10 min. The cell pellets were resuspended in

0.8 ml of methanol/0.01% (w/v) BHT, 0.1 ml of Reagent A (2.5 mM ammonium iron (II) sulfate/0.25M sulfuric acid) and 0.1 ml of Reagent B (40 mM BHT/1.25 mM xylenol 57

orange in methanol) to an OD550nm of 1.0 per ml. The mixture was incubated for 30 min at room temperature and then centrifuged at 10,000´ g to remove any cell debris. The absorbance at 560 nm was determined for the samples and the concentration of

4 -1 hydroperoxides was determined by using the extinction coefficient (E560nm = 4.3X10 M cm-1) (Sakamoto and Bryant, 1998).

2.18 Catalase activity

To measure catalase activity in Synechococcus sp. PCC 7002, the method of

(Beers and Sizer, 1952) was followed using whole cells. Exponentially growing cyanobacterial cells (25 ml) grown under standard growth conditions (38°C, 250 µE m-2

-1 s , 1.5% (v/v) CO2) were harvested by centrifugation at room temperature at 8000´ g for

5 min. The cell pellet was resuspended and washed with 50 mM potassium phosphate buffer, pH 7.0. The washed cells were again collected by centrifugation and resuspended

-1 to give a final OD550nm = 1.0 ml in a 3 ml sample. Catalase activity was measured by monitoring the rate of H2O2 decomposition at 240 nm by using the extinction coefficient

-1 -1 for H2O2 of 43.6 M cm (Beers and Sizer, 1952).

2.19 Peroxidase activity

Peroxidase activity can be measured by the following reaction: H2O2 + peroxidase

+ oxygen acceptor (colorless)àH2O + oxidized acceptor (colored) (Trinder, 1966). In 58 this case the oxygen acceptor is 4-aminoantipyrine in the presence of phenol.

Exponentially growing cyanobacterial cells (25 ml) grown under standard growth

-2 -1 conditions (38°C, 250 µE m s , 1.5% CO2) were harvested by centrifugation at room temperature at 8000´ g for 5 min. The cell pellet was resuspended and washed with 20 mM potassium phosphate buffer, pH 7.0. The washed cells were again collected by

centrifugation and resuspended to give a final OD550nm of 2.0 per ml in a 3.0 ml sample.

Peroxidase activity was measured by monitoring the change in color of phenol in the

presence of 4-aminoantipyrene and H2O2 at 510 nm (Trinder, 1966).

2.20 Cell viability

- - Exponentially growing cells (OD550nm = 0.25) from the wild-type, ctaDI , ctaDII , and ctaDI- ctaDII- strains of Synechococcus sp. strain PCC 7002 were divided into four culture tubes. Two tubes of the cultures of each strain were incubated under standard growth conditions with or without the addition of 50 µM MV. The other two tubes of the cultures of each strain were incubated in the dark at 37°C with or without the addition of

50 µM MV for 4 hours. The cells were harvested by centrifugation and washed with fresh A+ liquid media to remove the methyl viologen. The cells were resuspended in A+

to give a final OD550nm of 0.1 per ml. This culture was diluted 1000-fold and 10 µl of each dilute, resuspended cell culture was spread on an appropriate A+ plate. The cells were grown on plates for seven days prior to counting of colony forming units by visual inspection. Under normal growth conditions at 38°C, 1 ml of Synechococcus sp. PCC 59

7 7002 culture with an OD550nm = 1.0, is equivalent to 4.7 + 0.6 ´ 10 CFU (Sakamoto and

Bryant, 1998). 60

Chapter 3

RESULTS

Cloning of electron transport protein genes

In order to determine the number and type of electron transport protein encoding genes in Synechococcus sp. PCC 7002, forty open reading frames, putatively encoding proteins involved in electron transport based on sequence similarities to known electron transport proteins, were cloned and sequenced from the marine cyanobacterium

Synechococcus sp. PCC 7002. The ndh and ndb genes were cloned by Søren Persson, a lab technician who worked under my direct supervision. All of the clones were used to compare the number of electron transport genes and organization of electron transport genes to those of other cyanobacteria. The genes identified in this study can be divided into five major groups according to their presumed products: (1) the type I NADH dehydrogenase, (2) the type II NADH dehydrogenases, (3) the hydrogenase and its assembly proteins, (4) mobile electron transport proteins, and (5) cytochrome oxidases.

Table 3 shows the genes and some predicted properties of the proteins encoded by those genes identified or used in this study. The percent similarity and identity of the

Synechococcus sp. PCC 7002 electron transport proteins to their respective homologs from other bacteria or chloroplasts are shown in Table 4. Restriction maps of all of the 61

Synechococcus sp. PCC 7002 electron transport genes identified in this study are shown in APPENDIX A. ClustalW protein alignments of the electron transfer proteins from this study with their respective homologs from other bacteria and chloroplasts not shown in the RESULTS are located in APPENDIX B. 62

Table 3. Genes that encode electron transport proteins from Synechococcus sp. PCC

7002 and some predicted properties of those proteins. 63

Gene / gene cluster Synechocystis Source Amino MW Predicted Synechococcus sp. PCC7002 sp. PCC6803 acids (kDa) pI homolog Type I NADH dehydrogenase ndhB sll0223 this study 527 56.1 5.28 ndhC slr1279 this study 121 13.5 6.54 ndhK/psbG slr1280 this study 242 26.9 5.71 ndhJ slr1281 this study 174 19.9 4.22 ndhD1 slr0331 this study 527 57.6 6.83 ndhD2 slr1291 this study 532 58.0 9.12 ndhF3 sll1732 Klughammer et al., 1999 617 67.2 6.35 ndhD3 sll1733 Klughammer et al., 1999 499 53.9 8.16 ndhF4 sll0026 this study 633 69.3 6.25 ndhD4 sll0027 this study 494 53.5 7.43 ndhF1 slr0844 Schluchter et al., 1993 665 72.9 5.27 ndhF5 slr2007 this study 479 52.2 9.25 ndhF2 slr2009 this study 479 52.1 9.24 ndhH slr0261 this study 395 45.6 5.66 ndhA sll0519 this study 372 40.5 4.82 ndhI sll0520 this study 203 23.4 5.65 ndhG sll0521 this study 206 21.9 4.92 ndhE sll0522 this study 104 11.4 4.95 ndhL ssr1386 this study 77 9.2 9.42 Type II NADH dehydrogenase ndbA slr0851 this study 460 50.6 5.82 ndbB slr1743 this study 391 43.3 5.61 Cytochrome oxidase ctaCI slr1136 this study 362 39.6 4.55 ctaDI slr1137 this study 590 60.9 5.62 ctaEI slr1138 this study 197 22.0 5.96 ctaCII sll0813 this study 298 33.8 6.50 ctaDII sll2082 this study 551 61.0 6.37 ctaEII sll2083 this study 198 22.4 4.89 Hydrogenase hypE sll1464 this study 348 36.9 4.80 hoxE sll1220 this study 171 18.4 6.34 hoxF sll1221 this study 536 57.9 4.78 hoxU sll1223 this study 238 26.1 5.73 hoxY sll1224 this study 189 20.9 4.66 hyp3 NA this study 209 23.3 5.69 hoxH sll1226 this study 476 52.9 6.62 hoxW slr1876 this study 143 16.2 4.59 hypA slr1675 this study 114 12.0 4.40 hypB sll1079 this study 274 30.4 6.21 hypF sll0322 this study 766 86.0 7.18 hypC ssl3580 this study 80 8.2 4.04 hypD slr1498 this study 362 39.3 6.71 Mobile electron transport proteins bcpA NA this study 106 10.6 6.06 petJ1 sll1796 Nomura and Bryant 1997 112 9.4 4.89 petJ2 NA this study 88 9.4 9.29 cytM sll1245 this study 98 10.5 8.53 NA: not available, since no homolog occurs in Synechocystis sp. PCC 6803 64

Table 4. Percent identity and similarity of Synechococcus sp. PCC 7002 electron transport proteins with homologs from other bacteria and the spinach chloroplast.

X/Y in table represents the % identity / % similarity of the predicted protein from

Synechococcus sp. PCC 7002 compared to Synechocystis sp. PCC 6803, Anabaena sp. PCC 7120, E. coli, Spinacia oleracea, and R. eutropha.

ND: not determined.

(*) compared to sll1796 (PetJ) from Synechocystis sp. PCC 6803.

N-317 refers to the 317 amino acids at the amino terminus of the E. coli NuoF protein.

C-338 refers to the 338 amino acids at the carboxy-terminus of the R. eutropha HypF protein. 65

Predicted Protein Synechocystis Anabaena sp. E. coli S. oleracea R. eutropha Synechococcus sp. sp. PCC6803 PCC7120 PCC7002 Type I NADH dehydrogenase NdhB 73/85 72/83 27/45 46/64 ND NdhC 91/97 87/95 27/46 65/81 ND NdhK/PsbG 80/86 73/85 34/52 50/64 ND NdhJ 74/84 75/84 23/43 48/61 ND NdhD1 80/87 80/88 33/51 45/62 ND NdhD2 57/70 55/69 31/48 42/58 ND NdhD3 65/78 59/76 30/51 27/38 ND NdhD4 64/78 33/52 33/53 29/49 ND NdhF1 76/85 73/83 33/50 44/68 ND NdhF2 49/68 33/52 14/30 15/28 ND NdhF3 64/78 57/76 24/43 23/39 ND NdhF4 64/81 58/74 33/53 17/31 ND NdhF5 54/70 62/78 15/31 16/33 ND NdhH 92/95 85/92 37/55 71/83 ND NdhA 84/84 75/87 33/54 58/78 ND NdhI 81/88 81/87 21/35 54/64 ND NdhG 66/81 62/74 22/42 36/53 ND NdhE 80/92 81/88 29/56 58/80 ND NdhL 58/71 51/65 ND ND ND Type II NADH dehydrogenase NdbA 61/74 57/69 19/32 ND ND NdbB 53//74 50/68 20/31 ND ND Cytochrome oxidase CtaCI 61/74 47/65 ND ND ND CtaDI 80/90 63/78 ND ND ND CtaEI 67/78 55/74 ND ND ND CtaCII 40/57 51/70 ND ND ND CtaDII 64/76 72/86 ND ND ND CtaEII 56/69 61/71 ND ND ND Hydrogenase HypE 57/73 ND 42/56 ND 43/61 HoxE 67/82 56/73 24/44 ND ND HoxF 69/83 65/79 30/47 ND 28/45 HoxU 75/86 68/85 24/36(N-317) ND 32/49 HoxY 60/75 48/64 13/23 ND 30/50 Hyp3 ND 46/65 ND ND ND HoxH 66/89 64/80 18/33 ND 39/57 HoxW 36/51 30/46 ND ND 13/26 HypA 50/70 30/51 29/52 ND ND HypB 51/64 36/57 37/57 ND 30/43 HypF 48/63 ND 39/56 ND 30/44 (C-388) HypC 52/72 ND 32/47 ND 34/50 HypD 61/75 49/68 45/65 ND 43/62 Mobile electron transport proteins BcpA ND 62/75 ND ND ND PetJ1 46/64 46/56 ND ND ND PetJ2 37/51* 47/59 ND ND ND CytM 49/65 ND ND ND ND 66

3.1 Type I NADH Dehydrogenase

A full complement of genes that are predicted to encode subunits of the type I

NADH dehydrogenase and that exhibit strong sequence similarity to the genes found in the freshwater cyanobacterium Synechocystis sp. PCC 6803 were isolated from the marine cyanobacterium Synechococcus sp. PCC 7002. These include the ndhA, ndhI, ndhG, ndhE, ndhB, ndhC, ndhK, ndhJ, ndhD1, ndhD2, ndhD3, ndhD4, ndhF1, ndhF2, ndhF3, ndhF4, ndhF5, ndhH, and ndhL genes. The NADH dehydrogenase genes were cloned and the predicted protein sequences were used in comparisons with other cyanobacterial subunits of the NADH dehydrogenase as well as with their respective counterparts found in the chloroplast of Spinacia oleracea and in the gram-negative bacterium E. coli. As in chloroplasts and other cyanobacteria, Synechococcus sp. PCC

7002 is missing the homologs to the nuoE, nuoF and nuoG genes of E. coli and R. sphaeroides. Restriction maps for all Synechococcus sp. PCC 7002 ndh genes are shown in APPENDIX A. 67

3.1.1 ndhAIGE

The ndhAIGE gene cluster was isolated on a 3.8-kb EcoRI fragment from the

Synechococcus sp. PCC 7002 EcoRI cosmid library. Figure 3 reveals that the ndhAIGE gene cluster arrangement is nearly identical to that of the freshwater cyanobacterium,

Synechocystis sp. PCC 6803 and the gene cluster arrangement of the filamentous, nitrogen-fixing cyanobacterium, Anabaena sp. PCC 7120. This ndhAIGE gene organization may be conserved throughout all cyanobacteria. However, the genes flanking the ndhAIGE gene cluster are very different from one another. In

Synechococcus sp. PCC 7002, the ndhAIGE gene cluster is flanked by a homolog to the fraH gene, which would encode a putative zinc-finger protein related to the slr0298 open reading frame of Synechocystis sp. PCC 6803 at the 5’ end of the cluster. Examination of the 3’ end of the cluster revealed that it is homologous to the slr1536 open reading frame from Synechocystis sp. PCC 6803. Both of these reading frames are in the same transcriptional orientation as the ndhAIGE gene cluster. In Synechocystis sp. PCC 6803 the genes flanking the ndhAIGE gene cluster are transcribed divergently and consist of a valyl-tRNA and a gene encoding a putative dihydroorotate dehydrogenase (Kaneko et al.,

1996). The Anabaena sp. PCC 7120 ndhAIGE gene cluster is flanked by a predicted citrate synthase gene as well as a homolog to the hypothetical protein slr1415 (Kazusa

DNA Research Institute, 2000; Kaneko et al., 1996). The similarities of the ndhAIGE gene clusters and differences in the genes flanking the ndhAIGE gene cluster illustrate 68

Figure 3. Gene organization of the ndhAIGE gene cluster in cyanobacteria. Arrow directions indicate the direction of transcription. Scale is as indicated in bp for each individual map. (A) The gene organization of the ndhAIGE gene cluster of

Synechococcus sp. PCC 7002. (B) The gene organization of the ndhAIGE gene cluster of

Synechocystis sp. PCC 6803. (C) The gene organization of the ndhAIGE gene cluster of

Anabaena sp. PCC 7120.

ndhAIGE gene organization in cyanobacteria

Synechococcus sp. PCC7002 fraH (slr0298) ndhA ndhI ndhG ndhE slr1536

1K 2K 3K

Synechocystis sp. PCC6803

Dihydroorotate slr1419 dehydrogenase (slr1418) ndhA ndhI ndhG ndhE Valyl-T-RNA synthetase

1K 2K 3K 4K 5K 6K 7K

Anabaena sp. PCC7120

citrate synthase ndhA ndhI ndhG ndhE sll1415

1K 2K 3K 4K 5K 69

both the conservation of gene organization as well as the diversity in gene organization among these three cyanobacterial species.

The predicted proteins encoded by the Synechococcus sp. PCC 7002 ndhAIGE gene cluster are most similar to their homologs from the freshwater cyanobacterium

Synechocystis sp. PCC 6803 (Table 4), and have the least amount of similarity with the homologs from E. coli.

Sequence analysis indicates that the ndhA gene is predicted to encode a protein of

372 amino acids in Synechococcus sp. PCC 7002 with a predicted pI of 4.82. In E. coli, the NdhA homolog NuoH contains the ubiquinone binding site (Yagi et al., 1998). Based on the amino acid sequence similarity of the Synechococcus sp. PCC 7002 NdhA protein to other NdhA and ND1 proteins and the similarity of the NdhA and ND1 hydrophobicity profiles, the NdhA subunit may also contain a quinone-binding site (Ellersiek and

Steinmüller, 1992; Friedrich et al., 1990; Matsubayashi et al., 1987).

The ndhI gene is predicted to encode a protein of 203 amino acids with a predicted pI of 5.65. NdhI is the only protein in the predicted membrane arm of the

NDH-1 complex that would have iron-sulfur clusters. These clusters are bound to the

NdhI protein in a similar manner to the 4Fe-4S clusters of bacterial ferredoxins. The consensus-binding motif CysXXCysXXCysXXXCysPro, which provides ligands for tetranuclear iron-sulfur centers in ferredoxins, occurs twice in the predicted NdhI sequence of Synechococcus sp. PCC 7002. The first occurrence of the motif begins at

Cys-92 for the amino acid sequence CIACEVCVRVCP and the second begins at Cys-137 70 for the amino acid sequence CIFCGNCVEYCP. The NdhI protein is homologous to the E. coli NuoI protein that most likely contains two 4Fe-4S clusters, as well.

The ndhG gene putatively encodes a protein of 206 amino acids and a pI of 4.92 in Synechococcus sp. PCC 7002. The NdhG protein is homologous to the ND6 protein from the mitochondrial complex (Walker et al., 1992) and the NuoJ protein of E. coli

(Weidner et al., 1993). Analyses of NdhG predict a protein with 5 transmembrane helices and suggest that this gene encodes part of the membrane bound arm of the NDH-1 complex.

The ndhE gene of Synechococcus sp. PCC 7002 is the last gene found in this gene cluster. Analysis predicts a protein of 104 amino acids with a pI of 4.95. The NdhE protein is homologous to the NuoK protein from E. coli and the ND4L protein from the mitochondrial NDH-1 complex (Friedrich et al., 1995).

3.1.2 ndhB

A portion of the ndhB gene from Synechococcus sp. PCC 7002 was initially isolated on a 0.9-kb BamHI-EcoRI fragment, and the gene was completely sequenced by adding sequence from a cosmid 4A-B3 isolated from the EcoRI cosmid library. The ndhB gene of Synechococcus sp. PCC 7002 is predicted to encode a protein of 527 amino acids with a pI of 5.28. The NdhB protein is homologous to the NuoN protein in E. coli and the ND2 protein from the mitochondrial NDH-1 complex. 71

The gene organization of the Synechococcus sp. PCC 7002 ndhB gene is similar to that of Synechocystis sp. PCC 6803 since the gene in both organisms is not associated with any other ndh gene. Figure 4 shows that the ndhB gene from

Synechococcus sp. PCC 7002 is adjacent to an open reading frame predicted to encode a

2Fe-2S ferredoxin-like protein similar to one found in Clostridium pasteurianum

(Fujinaga and Meyer, 1993). These two genes are upstream of an open reading frame predicted to encode an adenine phosphoribosyltransferase related to the sll1430 open reading frame from Synechocystis sp. PCC 6803 (Kaneko et al., 1996). 72

Figure 4. Gene organization around the ndhB gene in cyanobacteria. Gene maps of three different cyanobacteria: Synechococcus sp. PCC 7002, Synechocystis sp. PCC

6803, and Anabaena sp. PCC 7120. Arrows represent transcriptional direction. Maps are individually scaled in bp.

7002 ndhB

ndhB Ferredoxin sll1430

300 600 900 1200 1500 1800 2100 2400 2700

6803 ndhB

phoA (sll0222) ndhB (sll0223) ssl0410 psaK (ssr0390)

300 600 900 1200 1500 1800 2100 2400 2700 3000 3300

7120 ndhB

hstK ndhB PAB1763

300 600 900 1200 1500 1800 2100 2400 2700 3000 73

3.1.2.1 Attempted mutagenesis of the Synechococcus sp. PCC 7002 ndhB gene by interposon mutagenesis

The inactivation of the ndhB gene in Synechococcus sp. PCC 7002 was attempted for two reasons: (1) The ndhB genes in Synechocystis sp. PCC 6803 (Ogawa, 1991) and

Synechococcus sp. PCC 7942 (Marco et al., 1993) were previously insertionally inactivated. Analysis of these ndhB mutants revealed strains that required much higher

concentrations of CO2 for growth. However, in tobacco, mutagenesis of the ndhB gene revealed impairment of cyclic electron flow around PSI (Shikanai et al., 1998). In order to determine whether the ndhB gene product from Synechococcus sp. PCC 7002 was involved in carbon uptake, cyclic electron flow or both, inactivation of the ndhB gene was attempted. (2) It had been previously demonstrated by Schluchter et al. (1993) that inactivation of ndhF1 of Synechococcus sp. PCC 7002 had an effect on cyclic electron flow. It had also been demonstrated by Klughammer et al. (1998) that inactivation of the ndhF3 and ndhD3 genes in Synechococcus sp. PCC 7002 lead to phenotypes affecting carbon assimilation. Both of these studies were done with genes (ndhF1, ndhF3 and ndhD3) that are present in multiple copies in the genome of Synechococcus sp. PCC 7002

(Table 3). On the other hand, ndhB is present as a single copy gene in Synechococcus sp.

PCC 7002 and its inactivation may lead to phenotypes different than those observed for genes present in multiple copies. Therefore, to examine the effects of inactivation of a 74 single copy ndh gene in Synechococcus sp. PCC 7002, mutagenesis of the ndhB gene was attempted.

The ndhB gene was interrupted by the insertion of a Klenow-filled-in HincII-

EcoRV 1.3-kb fragment containing the chloramphenicol acetyltransferase (cat) gene isolated from the plasmid pRL425 (Elhai and Wolk, 1988) into a unique BglII site within the ndhB coding sequence which was made blunt by Klenow treatment (see Figures 4 and 5). Several hundred independently isolated chloramphenicol/ampicillin E. coli colonies were screened to isolate the mutant plasmid. Two plasmids were isolated for both the parallel and anti-parallel orientation of the antibiotic resistance cartridge relative to the reading frame orientation of the cloned ndhB sequence. The resultant plasmids, pNDHB1 (parallel transcriptional orientation of the cat insert relative to ndhB) and pNDHB2 (antiparallel transcriptional orientation of the cat insert relative to ndhB) were used to transform the wild-type strain of Synechococcus sp. PCC 7002 (Figure 5A).

Transformants were selected for as described in Materials and Methods.

Chloramphenicol-resistant transformants were selected and grown in liquid culture. The segregation of the ndhB::cat locus in the chloramphenicol resistant transformants was checked by Southern Blot analysis. All of the transformants were merodiploid for the ndhB::cat and the wild-type copy of the ndhB gene (Figure 5B). The merodiploid ndhB::cat/wild-type ndhB strains of Synechococcus sp. PCC 7002 were grown on

+ selective medium A plates under high CO2 concentrations (5% CO2, 95% air) to try and force full segregation of the alleles, since ndhB- strains of Synechocystis sp. PCC 6803

(Ogawa, 1991) and Synechococcus sp. PCC 7942 (Marco et al., 1993) require higher 75

levels of CO2 for viability. This treatment failed to cause segregation of the alleles.

All subsequent attempts to segregate the mutant (increasing the antibiotic concentration

or increasing of CO2 levels) also failed to segregate the ndhB loci. These results imply that the ndhB gene is essential to Synechococcus sp. PCC 7002 under all of the conditions that were tested for segregation. The inability to segregate the ndhB mutant and wild- type alleles may be indicative of the central role of the type I NADH dehydrogenase in

Synechococcus sp. PCC 7002 compared to the enzymes function in other cyanobacterial species. 76

Figure 5. The ndhB gene of Synechococcus sp. PCC 7002 is required for cell viability.

(A) The ndhB gene was interrupted by insertion of the chloramphenicol acetyltransferase

(cat) gene into a unique BglII site within the coding sequence. The cat gene was digested with HincII and EcoRV. The ndhB plasmid was digested with BglII. This site was made blunt via a Klenow fill-in reaction and the cat gene was ligated into the now blunt BglII site. Transformants containing both orientations of the cat gene were isolated, and the new plasmids pNDHB(1.1), corresponding to the parallel orientation of insert and pNDHB(2.2), corresponding to the antiparallel orientation of insert. These plasmids were used to transform the wild-type strain of Synechococcus sp. PCC 7002. (B) Southern blot hybridization analysis of the ndhB loci. Genomic DNA was digested with EcoRI, transferred to a nylon membrane, and probed with a 32P-labeled PCR product for ndhB.

The sizes of the hybridizing bands are indicated on the left. Lane 1 represents genomic

DNA isolated from the merodiploid wild-type ndhB/ndhB::cat strain grown under normal growth conditions. Lane 2 represents genomic DNA isolated from the merodiploid wild-

type ndhB/ndhB::cat strain grown under 5% CO2/95% air. Lane 3 represents genomic

DNA isolated from the merodiploid wild-type ndhB/ndhB::cat strain grown in the presence of 10 mM glycerol under normal growth conditions. Lane 4 represents genomic

DNA isolated from the merodiploid wild-type ndhB/ndhB::cat strain grown under 5%

CO2/95% air and 10 mM glycerol. Lane 5 represents genomic DNA isolated from the wild-type strain of Synechococcus sp. PCC 7002. 77

A. HI HI RI RI II II Bam Eco Bam Bgl Bgl Eco

ndhB ndhB

CAT CAT RI RI cII cII dIII dIII RV RV Hin Hin Eco Eco Hin Hin Eco Eco

B. 1 2 3 4 5

2.1 kb

0.89 kb 78

3.1.3 ndhD1, ndhD2, ndhD3, ndhD4

Synechococcus sp. PCC 7002 has 4 genes that putatively encode proteins with strong sequence similarity to NdhD. There are an equivalent number of ndhD genes found in the genome of Synechocystis sp. PCC 6803 (Kaneko et al., 1996). NdhD is homologous to the NuoM protein of E. coli and to the ND4 protein from the mitochondrial NDH-1 complex (Friedrich et al., 1995). Non-cyanobacteria have only one gene copy of the ndhD-like genes; thus they lack the diversity displayed by the multiple copies of the ndhD-type genes seen in cyanobacteria (Kaneko et al., 1996). All of the ndhD genes are predicted to encode highly hydrophobic subunits that are most likely membrane bound. Restriction maps of all Synechococcus sp. PCC 7002 ndhD genes are shown in APPENDIX A.

The ndhD1 gene (slr0331 homolog) was cloned on 2.2-kb BamHI-HincII fragment and sequence analysis is predicted to encode a protein of 521 amino acids with a pI of 6.83. NdhD1 from Synechococcus sp. PCC 7002 is most similar to NdhD1 gene from Synechocystis sp. PCC 6803.

The ndhD2 gene (slr1291 homolog) was isolated on a 3.5-kb-XbaI fragment. The analysis of the ndhD2 nucleotide sequence is predicted to encode a protein of 532 amino acids with a pI of 9.12. NdhD2 from Synechococcus sp. PCC 7002 is most similar to

NdhD2 gene from Synechocystis sp. PCC 6803. 79

The ndhD3 gene (sll1773 homolog, accession number U97516) was cloned by

Klughammer et al. (Klughammer et al., 1999), and the gene is predicted to encode a protein of 499 amino acids with a pI of 8.16. NdhD3 from Synechococcus sp. PCC 7002 is most similar to NdhD3 gene from Synechocystis sp. PCC 6803.

The ndhD4 gene (sll0027 homolog) from Synechococcus sp. PCC 7002 was cloned and sequenced from two independently isolated clones. The first plasmid consists of a 2.6-kb EcoRV-EcoRI fragment, which contains the 3' end of the ndhF4 gene and the

5' end of the ndhD4 gene. A second clone consisting of a 2.5-kb HincII-HindIII fragment contains the 3' end of the ndhD4 gene. The ndhD4 gene is predicted to encode a protein of 494 amino acids with a pI of 7.43.

Figure 6 shows the ClustalW amino acid alignment of the four Synechococcus sp.

PCC 7002 NdhD proteins. Table 5 shows percent identity and similarity of the four

Synechococcus sp. PCC 7002 NdhD proteins to each other. It is clear from these results that, although the NdhD proteins from Synechococcus sp. PCC 7002 are related to one another, they are very diverse in size, homology, and pI. The differences observed in the

NdhD subunits may be responsible for the diversity in the number and types of NDH-1 complexes in cyanobacteria. 80

Figure 6. ClustalW alignment of the four NdhD amino acid sequences from

Synechococcus sp. PCC 7002. The Synechococcus sp. PCC 7002 NdhD amino acid sequences were aligned using the ClustalW alignment program from MacVector v. 6.5.

The consensus amino acid sequence is underneath the alignments. Gray high-lighted regions indicate identical amino acids and lighter shading indicates conserved amino acids. Dashes represent insertions/deletions included to maximize the sequence similarity. Percent identity and percent conserved amino acids were determined with the

ClustalW program (Thompson et al., 1994b). 81

7002 NdhD ClustalW Formatted Alignments

10 20 30 40 50 NdhD1-7002(slr0331) M N F A N F P WL S T I I L F P I I A A L F L P L I P D K D G K T V R WY A L T I G L I D F V I I V NdhD2-7002(slr1291) M D S L Q I P WL T T A I A F P L L A A L V I P L I P D K E G K T I R WY T L WR C P H R F C L L V NdhD3-7002(slr1773) M L S F L L F L P L V G I G A I A L F P R - - - - P L T R I V A T V F T V V T L A I S NdhD4 -7002(sll0027) M L S A L I WL P L A G A L L V A I L P Q G E K N Q F S R T M A L G A A A L V F V WT L S . I P L . . A L . . L . P ......

60 70 80 90 100 NdhD1-7002(slr0331) T A F Y T G Y D F G N P N L Q L V E S Y T WV E A I D L R WS V G A D G L S M P L I L L T G F I T T NdhD2-7002(slr1291) T A F WQ N Y D F G R T E F Q L T K N F A WI P Q L G L N WS L G V D G L S M P L I I L A T L I T T NdhD3-7002(slr1773) S G L L I N L N L Q D A G M Q Y T E F H N WL S I L G L N Y N L G V D G L S L P L I V L N S L L T L NdhD4 -7002(sll0027) A WL G F H Y D V A I A G L Q F V E H Y L WI E WL G L N Y D L G V D G L S L P L L A L N A L L T L . . Y D . Q E . W. L G L N . L G V D G L S P L I . L L . T

110 120 130 140 150 NdhD1-7002(slr0331) L A I L A A WP V S F K P K L F Y F L M L L M Y G G Q I A V F A V Q D M L L F F F T WE L E L V P V NdhD2-7002(slr1291) L A T L A A WN V T K K P K L F A G L I L V M L S A Q I G V F A V Q D L L L F F I M WE L E L V P V NdhD3-7002(slr1773) V A I Y S I G E S N H R P K L Y Y S L I L L I N S G I T G A L I A N N L L L F F L F Y E I E L I P F NdhD4 -7002(sll0027) V A L WI S P K D L H R P R F Y Y A L F L L L Q A S V N G A F L A Q D V L L F F L F Y E I E I I P L . A . . . . . P K L . Y L L L . G . F . . Q D . L L F F . . E . E L . P .

160 170 180 190 200 NdhD1-7002(slr0331) Y L I L S I WG G K K R L Y A A T K F I L Y T A G G S L F I L I A A L T M A F Y G D T V T F D M T A NdhD2-7002(slr1291) Y L L I S I WG G K K R L Y A A T K F I L Y T A L G S V F I L A F T L A L A F Y G G D V T F D M Q A NdhD3-7002(slr1773) Y L L I A I WG G E K K G Y A S T K F L I Y T A I S G L C V L A A F L G I V WL S Q S S N F D F E N NdhD4 -7002(sll0027) Y F L I A I WG G K K R G Y A A I K F L L Y T A V S G I L I L A S F L G L A F L T E S N T F A Y S A Y L L I I WG G K K R . Y A A T K F . L Y T A . . I L A L . . A F . T F D A

210 220 230 240 250 NdhD1-7002(slr0331) I A Q K D F G I N L Q L L L Y G G L L I A Y G V K L P I F P L H T WL P D A H G E A T A P A H M L L NdhD2-7002(slr1291) L G L K D Y P L A L E L L A Y A G F L I G F G V K L P I F P L H S WL P D A H S E A S A P V S M I L NdhD3-7002(slr1773) L T L E N L E F N T K V I L L T I L L I G F G I K I P L V P L H T WL P D A Y V E A N P A V T V L L NdhD4 -7002(sll0027) L H S D L L P L T T Q L I L L G G I L V G F G I K I P F L P F H T WL P D A H V E A S T P V S V I L L . L . L . G . L I G F G . K . P . P L H T WL P D A H . E A . P V . . L

260 270 280 290 300 NdhD1-7002(slr0331) A G I L L K M G G Y A L L R M N A G M L P D A H A L F G P V L V I L G V V N I V Y A A L T S F A Q R NdhD2-7002(slr1291) A G V L L K M G G Y G L I R L N M E M L P D A H I R F A P L L I V L G I V N I V Y G A L T A F G Q T NdhD3-7002(slr1773) G G V F A K L G T Y G L V R F G L Q L F P D V WS T V S P A L A V I G T V S V M Y G S L A A I A Q R NdhD4 -7002(sll0027) A G V L L K V G T Y G L L K F G I G L F P L A WA V V A P WL A I WA A I S A L Y G A S C A I A Q K A G V L L K G Y G L . R . P D A . . P . L . . . G . V . . Y G A L A A Q .

310 320 330 340 350 NdhD1-7002(slr0331) N L K R K I A Y S S I S H M G F V L I G M A S F T D L G T S G A M L Q M I S H G L I G A S L F F M V NdhD2-7002(slr1291) N L K R R L A S S S I F P H G L S S L G L L S F T D L G M N G A V L Q M L S H G F I A A A L F F L S NdhD3-7002(slr1773) D L K R M V A Y S S I G H M G Y I L V S T A A G T E L S L L G A V A Q M I S H S L I L A L L F H L V NdhD4 -7002(sll0027) D M K K V V A Y S S I A H M A F I L L A A A A A T P L S L A A A E I Q M V S H G L I S G L L F L L V L K R . A Y S S I H M G . . L . . A T . L G A . Q M . S H G L I . A . L F L V 82

360 370 380 390 400 NdhD1-7002(slr0331) G A T Y D R T H T L M L D E M G G V G - - - K K M K K I F A M WT T C S M A S L A L P G M S G F V A NdhD2-7002(slr1291) G V T Y E R T H T L M M D E M S G I A - - - R L M P K T F A M F T A A A M A S L A L P G M S G F V S NdhD3-7002(slr1773) G I I E R K V G T R D L D V L N G L M N P V R G L P L T S S L L I L A G M A S A G I P G L V G F V A NdhD4 -7002(sll0027) G I V Y K K T G S R D V D Y L R G L L T P E R G L P L T G S L M I L G V M A S A G L P G M A G F I A G . Y . T T . D G . . R . P T . . . M A S . . L P G M G F V A

410 420 430 440 450 NdhD1-7002(slr0331) E L M V F V G F A T S D A Y S P T F R V I I V F L A A V G V I L T P I Y L L S M L R E I L Y G P E N NdhD2-7002(slr1291) E L T V F L G L S N S D A Y S Y G F K P I A I F L T A V G V I L T P I T C F Q C C G V F Y G - - K G NdhD3-7002(slr1773) E F L V F Q G ------S F S R F P I P T L F C I I A S G L T A V Y F V I L L N R T C F G R L D NdhD4 -7002(sll0027) E F L I F R G ------S F P V Y P V A T L L C M V G T G L T A V Y F L L M I N K V F F G R L T E V F G S . P I . L . V G . L T . Y . . . . G

460 470 480 490 500 NdhD1-7002(slr0331) K E L V A H E K L I D A E P R E V F V I A C L L I P I I G I G L Y P K A V T Q I Y A S T T E N L T A NdhD2-7002(slr1291) S Q A P P R C G G E D A K P R E I F V A V C L L A P I I A I G L Y P K L A T T T Y D L K T V E V A S NdhD3-7002(slr1773) S H T A Y Y P K V F A S - - - E K I P A I A L T V I I L F L G L Q P A WL T R WI E P T T S Q F I A NdhD4 -7002(sll0027) P E L I N M S P V N WA - - - D Q F P A V M L V I L L F V F G L Q P Q WL V R WS E I D T A A L V A . . . A E F A . . L . . I . . . G L P . T . T . . A

510 520 530 540 550 NdhD1-7002(slr0331) I L R Q S V P S L Q Q T A Q A P ------S L D V A V L R A P E I R NdhD2-7002(slr1291) K V R A A L P L Y A E Q L P Q N G D R Q A Q M G L S S Q M P A L I A P R F NdhD3-7002(slr1773) A I P T V Q T I A L T P A E L S ------K A P NdhD4 -7002(sll0027) S P T A I E I S L K N . . . . A P 83

Table 5. Percent identity and similarity (identity/similarity) of the four NdhD proteins from Synechococcus sp. PCC 7002 compared to one another as determined by a ClustalW alignment (Thompson et al., 1994a).

% identity/similarity to Synechococcus sp. PCC 7002 protein

NdhD1 NdhD2 NdhD3 NdhD4

Synechococcus sp. PCC 7002 100/100 56/70 30/48 34/55 NdhD1 Synechococcus sp. PCC 7002 56/70 100/100 30/47 33/51 NdhD2 Synechococcus sp. PCC 7002 30/48 30/47 100/100 49/69 NdhD3 Synechococcus sp. PCC 7002 34/55 33/51 49/69 100/100 NdhD4

3.1.4 ndhF1, ndhF2, ndhF3, ndhF4, ndhF5

Cyanobacteria display great genetic diversity at the ndhF loci. Synechococcus sp.

PCC 7002 has five divergent copies of the ndhF gene. NdhF proteins are homologs to the NuoL protein from E. coli and the ND5 protein from the mitochondrial complex I

(Friedrich et al., 1995). All of the ndhF genes from Synechococcus sp. PCC 7002 are predicted to encode hydrophobic proteins, likely associated with the membrane. 84

The ndhF1 (slr0844 homolog, accession number AAA27311) gene from

Synechococcus sp. PCC 7002 was originally identified and characterized by Schluchter et al. (1993) and analysis of the gene sequence is predicted to encode a protein of 665 amino acids with a pI of 5.27.

The ndhF2 (slr2009 homolog) gene was originally cloned on a 1.5-kb BamHI-

HindIII fragment adjacent to the ndhF5 (slr2007 homolog) gene. The remaining sequence to obtain the entire sequence of both the ndhF2 and ndF5 genes was acquired from a cosmid from the cosmid library. The ndhF2 (slr2009 homolog) gene is predicted to encode a protein of 479 amino acids and a pI of 9.24.

The ndhF3 (sll1773 homolog) gene was originally identified by Klughammer et al. (1999) and the gene sequence is predicted to encode a protein of 617 amino acids with a pI of 6.35.

The ndhF4 (sll0026 homolog) gene was cloned with the ndhD4 gene on a 2.6-kb

EcoRV-EcoRI fragment. The 5' end of the ndhF4 gene was cloned on a 3.15-kb XbaI-

EcoRI fragment. The ndhF4 gene is predicted to encode a protein of 633 amino acids and a pI of 6.25.

The ndhF5 (slr2007 homolog) was partially isolated on a 1.5-kb BamHI-HindIII fragment. The remaining nucleotide sequence was obtained from cosmid AG-4. The ndhF5 gene is predicted to encode a protein of 479 amino acids and a pI of 9.25.

Figure 7 shows the ClustalW alignment of the five Synechococcus sp. PCC 7002

NdhF proteins. Table 6 shows the percent identity and similarity of the five

Synechococcus sp. PCC 7002 NdhF proteins to each other. Again, the diversity observed 85

Figure 7. ClustalW alignment of the five NdhF amino acid sequences from

Synechococcus sp. PCC 7002. The Synechococcus sp. PCC 7002 NdhD amino acid sequences were aligned using the ClustalW alignment program from MacVector v. 6.5.

ClustalW program (Thompson et al., 1994b). 86

7002 NdhF ClustlW Amino Acid Alignments

10 20 30 40 50 NdhF1-7002(slr0844) M E P L Y Q Y A WL I P V L P L L G A M V I G I G L I S L N K F T N K L R Q L Y A V F V L S L I G NdhF2-7002(slr2009) M N E L T I G WV I ------F P F V V G F S I Y L L P K I D R Y L A I F V S I C S NdhF3-7002(sll1732) M N T F F S Q S V WL V P C Y P L L G M G L S A L WM P S I T R K T G P R P A G Y V N M L L T F M A NdhF4-7002(sll0026) M S E F L L Q S V WL V P V Y G I T G A L L T L P WS L G L I R R T G P R P A A Y L N L I M T F L G NdhF5-7002(slr2007) M I D D I T I I WI L ------L P F V V G F S I Y L L P R WN R Y F A L A I A A L S . Q . WL . P . G . . P . . . G . . . . T . P R . Y ......

60 70 80 90 100 NdhF1-7002(slr0844) T S M A L S - F G L L WS Q I Q G H E A F T Y T L E WA A A G D F H L Q M G Y T V D H L S A L M S V NdhF2-7002(slr2009) L I F G - - - F V Q I F Q P E P ------Y S L K L L G M Y G V D L L V D - D Q S G Y F I L T N A NdhF3-7002(sll1732) L V H S C L A F I E R WE Q P A L - - - - K P S L T WL Q A A D L T L S I D L D I S S I T I G A L I NdhF4-7002(sll0026) L L H G S F A F A S L WN M P P Q - - - - Q L S L E WL Q V A D L N L S L V I E I S P V N L G A M E NdhF5-7002(slr2007) V V Y S - - - I G L L WS L E P ------F T L E L L D S F G V T L M F D - E L S G Y F I L M N G L . . F . L W P . S L E WL . . D . L . D . . S . I L .

110 120 130 140 150 NdhF1-7002(slr0844) I V T T V A L L V M I Y T D G Y M A H D P G Y V R F Y A Y L S I F S S S M L G L V F S P N L V Q V Y NdhF2-7002(slr2009) A V A I A ------V T V Y C WK S A K S A F F F T Q L V V L Q G A L N A V F V C A D L I S L Y NdhF3-7002(sll1732) L I A G I N L L A Q L Y A V A Y L E M D WG WA R F F A T M S L F E A G M C A L V L C N S L F F S Y NdhF4-7002(sll0026) L V T G I C F M A Q L Y G L G Y L E K D WS I A R F Y G L M G F F E A A L S G L A I S D S L L L S Y NdhF5-7002(slr2007) L V T G A ------V L L Y C F D K Q K S P F F Y T Q L V I L H G A V N A T F C C A D L I S L Y L V T G . . . Y . . . Y . . D A R F Y . L . . F . A . A L . . C L . . Y

160 170 180 190 200 NdhF1-7002(slr0844) I F WE L V G M C S Y L L I G F WY D R K A A A D A C Q K A F V T N R V G D F G L L L G M L G L Y W NdhF2-7002(slr2009) V A L E A I S I A A F L L M T Y Q R T D R S I WI G L R Y L F L S N - T A M L F Y L I G A V L V Y Q NdhF3-7002(sll1732) V V L E I L T L G T Y L L I G Y WF N Q S L V V T G A R D A F L T K R V G D L F L L M G V V A L L P NdhF4-7002(sll0026) G L L E V L T L S T Y L L V G F WY A Q P L V V T A A R D A F L T K R V G D I L L L M G I V A L S S NdhF5-7002(slr2007) V A L E C I G I A A F L L I T Y S R S D R S L WV G L R Y L F I S N - T A M L F Y L I G A V L V Y Q V . L E ...... Y L L I G Y W. . . . . G . R A F L T N R V G D L F L L . G . V . L Y

210 220 230 240 250 NdhF1-7002(slr0844) A T G S F E F D L M G D R L M D L V S T G Q I S S L L A I V F A V L V F L G P V A K S A Q F P L H V NdhF2-7002(slr2009) A T K S F A F V G L ------A E A P S D A I A L I F L G L L T K G G ------V F V S G L NdhF3-7002(sll1732) L A G T WN F D G L A E - - - - WA A T A E L D P T L A T L L C L A L I A G P L G K C A Q F P L H L NdhF4-7002(sll0026) Y G T G L T F S E L E T - - - - WA A N P P L P P WE A S L V G L A L I S G P I G K C A Q F P L N L NdhF5-7002(slr2007) A S N S F A F S G L ------A V A P K E A I A L I F L G L L T K G G ------I F V S G L A . S F F G L . A A P . . . . A I . L G L L . . G P . . K A Q F P L L

260 270 280 290 300 NdhF1-7002(slr0844) WL P D A M - E G P T P I S A L I H A A T M V A A G V F L V A R M Y P V F E P I P E A M N V I A WT NdhF2-7002(slr2009) WL P L T H S E A E T P V S A M L - S G V V V K A G I F P L L R C G - - - I L V P D L D L WL R L F NdhF3-7002(sll1732) WL D E A M - E S P V P A T V V R - N S L V V G T G A WV L I K L Q P I F A L S D F A S T F M I A I NdhF4-7002(sll0026) WL D E A M - E G P N P A G I I R - N S V V V S A G A Y V L L K M E P V F T I T P I T S D A L I I I NdhF5-7002(slr2007) WL P L T H G E S E T P V S A L L - S G V V V K A G V F P L A R C A - - - L L V P E L D P V V R L F WL P . A M E . P T P . S A . . . V V V A G . F . L . R . P . F . L . P ......

310 320 330 340 350 NdhF1-7002(slr0844) G A T T A F L G A T I A L T Q N D I K K G L A Y S T M S Q L G Y M V M A M G I G G Y T A G L F H L M NdhF2-7002(slr2009) G L A T A L L G I I F A I L E T D A K R L L A F S T I S K L G L L L S A P - - - - - A V A G L A A L NdhF3-7002(sll1732) G A T T A L G A A M V A I A Q I D I K R S L S Y S V S A Y M G M V F M A V G S Q Q D Q T T L V L L L NdhF4-7002(sll0026) G T V T T V G A S L V A L A Q I D I K R A L S H S T S A Y L G L V F I A V G L N Q V D I A L L L L L NdhF5-7002(slr2007) G V G T A L L G V G Y A V F E K D T K R M L A F H T V S Q L G F V L A A P - - - - - A V G G F Y A L G . . T A L L G . . . A . . Q D I K R . L A . S T S L G V . A G . . L . . L L

360 370 380 390 400 NdhF1-7002(slr0844) T H A Y F K A M L F L G S G S V I H G M E E V V G H N A V L A Q D M R L M G G L R K Y M P I T A T T NdhF2-7002(slr2009) S H G L V K S S L F L M A G ------Q L P - - - T R ------NdhF3-7002(sll1732) T Y G V A M A I L V M A I G G V V L ------V N I S Q D L T Q Y G G L WS R R P I T G I C NdhF4-7002(sll0026) T H A I A K A L L F M S I G A V I L ------N T H G Q N I T E M G G L WS R M P A T T S A NdhF5-7002(slr2007) T H G L V K G A L F L T A G ------Q L S - - - S R ------T H G . . K A . L F L . G V . Q Q G G L S R P . T 87

410 420 430 440 450 NdhF1-7002(slr0844) F L I G T L A I C G I P P F A G F WS K D E - - I L G L A F E A N P V L WF I G WA T A G M T A F Y NdhF2-7002(slr2009) ------N F Q E L R Q T K I A S S L WL P L A I A C L S M V G M P L L V G F S NdhF3-7002(sll1732) Y L V G A A S L V A L P P F G G F WS L A Q - - L T T N F WK T S P I L A V I L I T V N A L T S F S NdhF4-7002(sll0026) F V V G S A G L V C L F P L G T F WT M R R - - WV D G F WD T P P WL V L L L V G V N F C S S F N NdhF5-7002(slr2007) ------N F K V L R E Q S I P R A Y WWV L V L A C A S I S G L P F L A G Y S . . . G . . . P . F W. L R I . . . W P . L . . . . G . . L . . F S

460 470 480 490 500 NdhF1-7002(slr0844) M F R M Y F L T F E G E F R G T D Q Q L Q E K L L T A A G Q A P E E G H H G S K P H E S P L T M T F NdhF2-7002(slr2009) S K A L L L K N I A ------P ------NdhF3-7002(sll1732) I M R E F G L I F G G K ------P K Q M T V R S P E NdhF4-7002(sll0026) L T R V F R S V F L G A ------P K P K T R R S P E NdhF5-7002(slr2007) S K I L T M K N I L ------P ------R . . F . G P .

510 520 530 540 550 NdhF1-7002(slr0844) P L M A L A V P S V L I G L L G V P WG N R F E A F V F S P N E - - - - A A E A A E H G F E L T E F NdhF2-7002(slr2009) - - WQ A M G L N I A A V G T A L A - - - - F A K F L F I P ------H D A A T K NdhF3-7002(sll1732) G L WA L V L P M V I L A G F A L H S P F I L A K L N F L P ------D WH Q L N L P L A A V NdhF4-7002(sll0026) V V WQ M A V P M V S L I L M T L M V P F F L H Q WQ L L F N P S L P T L V E R P L I V T L A I P A NdhF5-7002(slr2007) - - WQ S F A L N V A A V G T A I S - - - - F A K F I F L P ------K K T D P S . WQ . . . P V . . . G A L F A K F . F L P . . .

560 570 580 590 600 NdhF1-7002(slr0844) L I M G G N S V G I A L I G I T I A S L M Y L Q Q R I D P A R L A E K F P V L Y Q L S L N K WY F D NdhF2-7002(slr2009) F T A K S T T F WG A I A F L F S G V I L G N ------G F Y L E NdhF3-7002(sll1732) L I I S T M V G G G T A M Y L Y L N E K I S K P I H I F S D P V R E F F ------A K D L Y T A NdhF4-7002(sll0026) L M I T G G L G L V A G L T I T L N P S L S R P R Q L Y L R F L Q D L L ------A Y D F Y I D NdhF5-7002(slr2007) L K I L P N - F Y G A I V L L L G G L F V T N ------S F Y L E L I . . G A . . L ...... F Y . .

610 620 630 640 650 NdhF1-7002(slr0844) D I Y N N V F V M G T R R L A R Q I L E V D Y R V V D G A V N L T G I A T L L S G E G L K Y I E N G NdhF2-7002(slr2009) A Y Q L D N I P K A L I K I A - - I G WA L Y WL I M K R I E F K - L P R I F ------NdhF3-7002(sll1732) E L Y K N T V I F A V A L I S K I I D WL D R Y F V D G V I N F L G L A T L F G G Q S L K Y N N S G NdhF4-7002(sll0026) R I Y N V T V V WL V T T L S K L A A WF D R Y V V D G F V N L T G L A T L F S G S A L R Y N V S G NdhF5-7002(slr2007) A Y Q P S N I L K A L V T I G - - I G WL A Y G L I F Q R I T V K - L P R V V ------. Y . . A . . I . . I . W. D Y . . V D G I N . G L A T L F G L . Y G

660 670 680 690 700 NdhF1-7002(slr0844) R V Q F Y A L I V F G A V L G - - - - F V I F F S V A NdhF2-7002(slr2009) - E A F E Q L I G A M S V V L - - - - - T G L F WM V T L N NdhF3-7002(sll1732) Q S Q S Y A L S I V A G I L L - - - - F I A A L S Y P L L K H WQ F NdhF4-7002(sll0026) Q S Q F Y V L T I V L G M I L G L V WF M A T G Q WT M I T D F WS N Q L A NdhF5-7002(slr2007) - E Q F E H L V G V M S L V L - - - - - T G L F WL V L A N Q F Y . L . . V . . . . L F . . F . . 88

Table 6. Percent identity and similarity (identity/similarity) of the four NdhD proteins from Synechococcus sp. PCC 7002 compared to one another as determined by a ClustalW alignment (Thompson et al., 1994a).

% identity/similarity to Synechococcus sp. PCC 7002 protein

NdhF1 NdhF2 NdhF3 NdhF4 NdhF5

Synechococcus sp. PCC 100/100 13/28 25/46 25/25 15/29 7002 NdhF1 Synechococcus sp. PCC 13/28 100/100 14/28 14/29 60/76 7002 NdhF2 Synechococcus sp. PCC 25/46 14/28 100/100 45/64 62/75 7002 NdhF3 Synechococcus sp. PCC 25/25 14/29 22/40 100/100 13/27 7002 NdhF4 Synechococcus sp. PCC 15/29 60/76 24/44 13/27 100/100 7002 NdhF5 89

among NdhF subunits may be responsible for multiple forms of the NDH-1 complex in cyanobacteria.

3.1.5 Gene organization and phylogenetic analysis of the ndhD and ndhF

genes in cyanobacteria

The gene organization of several of the ndhD-ndhF gene clusters of cyanobacteria is shown in Figure 8. The ndhF1 and ndhD1 genes from Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803 are not found in close proximity to other ndh genes.

However, in Anabaena sp. PCC 7120, the ndhF1 gene and the ndhD1 gene are arranged in a cluster with thioredoxin and an open reading frame homologous to the slr1621 gene from Synechocystis sp. PCC 6803. The ndhF3 and ndhD3 genes are oriented in the same transcriptional direction and arranged as clusters in all three cyanobacterial species. The ndhF4 and ndhD4 genes are also arranged in similar clusters for all three cyanobacterial species. The ndhF5 and ndhF2 genes are found nearly adjacent to one another in both

Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803. However, Anabaena sp.

PCC 7120 appears to have only one homolog of the ndhF5 gene. In all three cyanobacterial species, the ndhD2 gene is not found to be associated with other ndh genes.

Sequence analysis shows that the NdhF and NdhD proteins are derived from a common ancestor. Phylogenetic analysis reveals that the original nomenclature 90

Figure 8. Gene organization of the ndhF and ndhD genes in cyanobacteria. The ndhD and ndhF genes and gene clusters in

Synechococcus sp. PCC 7002, Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120 are shown. NADH dehydrogenase genes shown in dark gray, genes not related to the NADH dehydrogenase shown in light gray. HP: hypothetical protein. h: homolog. 91

Synechococcus sp. PCC7002 Synechocystis sp. PCC6803 Anabaena sp. PCC7120

1kb 0.5 kb

PetH ndhF1 HP, sll0175 ycf34h. , ssr1425 ndhF1 HP, ssl1533

1 kb

HP, ssl3451 h.

0.5 kb thioredoxin, ndhF1… ndhD1 HP, slr1612 h.

P1174 (1492<-1512) 0.5 kb PsaC HP, slr0333 ndhD1 ndhD1

1 kb 1 kb 1 kb

HP, sll1735 HP,h. sll1730 rbcR ndhF3 ndhD3 ndhF3 ndhD3 HP, sll1734 HP, slr1429 h.ndhF3 ndhD3 HP, sll1734 h.

HP, ORF427, sll1734 h.

1 kb 1 kb 1 kb

ndhF4 ndhD4 HP, sll0518 ndhF4 ndhD4 NrtD, slr0044 ccmk-2, sll… ndhF4 ndhD4HP, slr1302 h./sll1734 h.

HP, slr1302 h.

1 kb 1 kb 1 kb

HP, slr2006 HP, slr2008 HP, ssr3409 Na+/H+ antiporterndhF5 ndhF2 HP, ssr3410 h. ndhF5 ndhF2 HP, sll0528HP, h.slr2006ndhF5 h.HP, slr2010HP, slr2011h. h.

HP, slr2006 h. HP, slr2010 h. HP, slr2010

1 kb 1 kb 1 kb

SigGHP, slr1546 h. ndhD2 HP, sll1201 ndhD2 HP, sll1200 ExbB, sll1404 h. ndhD2 HP, slr1083 h. and classification of the cyanobacterial NdhF and NdhD proteins was incorrect. Figure 9 shows a representative phylogenetic tree with the evolutionary relationships among the predicted NdhF and NdhD proteins from Synechococcus sp. PCC 7002, Synechocystis sp.

PCC 6803, Anabaena sp. PCC 7120, as well as the Ndh4 protein (accession number

NP_039348) and Ndh5 protein (accession number NP_039343) from the liverwort

Marchantia polymorpha (Ohyama et al., 1986), the NuoM and NuoL proteins from E. coli (Weidner et al., 1993), and the NuoM protein (accession number AAC25004) and

NuoL (accession number AAC25003) proteins from R. capsulatus (Dupuis et al., 1995).

The Ndh protein sequences were aligned with the ClustalW program (Thompson et al.,

1994a) in MacVector v. 7.0 using MD (Dayhoff and National Biomedical Research

Foundation., 1965), PAM (Dayhoff and National Biomedical Research Foundation.,

1965), and BLOSUM (Henikoff and Henikoff, 1992) matrices to obtain the best alignments of the protein sequences. The phylogenetic trees were made using the neighbor-joining program in MacVector v. 7.0. Trees calculated from each of the matrices were subjected to bootstrapping over 1000 cycles to assure that the relationships displayed in the tree were those most likely to occur. The cyanobacterial NdhF proteins cluster with the NuoL proteins E. coli and R. capsulatus as well as the liverwort Ndh5 protein from M. polymorpha. The cyanobacterial NdhD proteins group with the NuoM proteins from E. coli and R. capsulatus as well as the liverwort Ndh4 protein from M. polymorpha. The cyanobacterial Ndh proteins are most closely related to their identified paralog in the other cyanobacteria except in the case of the NdhF5 and NdhF2 proteins.

NdhF5 was originally classified as an NdhD protein (Kaneko et al., 1996). However, 93

Figure 9. Phylogenetic analysis of NdhF and NdhD proteins. NdhF and NdhD proteins Synechococcus sp. PCC7002, Synechocystis sp. PCC6803 and Anabaena sp.

PCC7120 and homologs from E. coli, R. capsulatus and M. polymorpha were subjected to phylogenetic analysis with MacVector 7.0. A BLOSUM series matrix was used to calculate the alignment between individual sequences with an Open/Extended gap penalty of 10/0.05, Delay divergent: 60%. This is a representative tree after bootstrapping for 1000 iterations. Numbers on branches represent percentages of times that the proteins group together after analysis. 94

Method: Neighbor Joining; Bootstrap (1000 reps); tie breaking = Systematic Distance: Uncorrected (“p”) Gaps distributed proportionally

NuoM R. capsulatus

99 NdhF2 7002 NdhF5 7120 100 NdhF5 7002

NdhF5 6803 NdhF2 6803

100 100 NdhF4 7120

99 NdhF4 7002 100 NdhF4 6803

100 NdhF3 7120 100 99 NdhF3 7002 NdhF3 6803

100 NuoL E. coli NuoL R. capsulatus

100 Ndh5 M. polymorpha

100 NdhF1 7120

95 NdhF1 7002 NdhF1 6803

100 NdhD3 7120

98 NdhD3 7002 100 NdhD3 6803

99 NdhD4 7120

94 NdhD4 7002 NdhD4 6803

Ndh4 M. polymorpha

100 NdhD1 7120

100 NdhD1 7002 NdhD1 6803

72 NdhD2 7002

100 NdhD2 7120 NdhD2 6803 NuoM E. coli 95

phylogenetic analysis reveals that NdhF5 (slr2007 homolog) should be classified as an

NdhF protein and is actually most closely related to the NdhF2 protein from the same

organism. The ndhF5 and ndhF2 genes lie adjacent to each other in both the

Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803 genomes; the high degree

of sequence similarity between the NdhF5 and NdhF2 proteins may be indicative of a

relatively recent gene duplication of either ndhF5 or ndhF2.

3.1.6 ndhCKJ

The Synechococcus sp. PCC 7002 ndhCKJ gene cluster was cloned on a 3.5-kb

BamHI fragment. The organization of the ndhCKJ gene cluster organization is shown

and compared to the other cyanobacterial ndhCKJ gene clusters in Figure 10. This

figure reveals that the Synechococcus sp. PCC 7002 ndhCKJ gene cluster is organized in

a manner very similar to the ndhCKJ gene clusters from both Synechocystis sp. PCC

6803 and Anabaena sp. PCC 7120, although the genes flanking the ndhCKJ cluster are, as in the case of the cyanobacterial ndhAIGE gene cluster, very different from species to species.

The ndhC gene from Synechococcus sp. PCC 7002 is predicted to encode a protein of 121 amino acids and a pI of 6.54. Based on sequence similarity, NdhC is a homolog to the NuoA protein from E. coli as well as the ND3 protein from the mitochondrial complex I. 96

Figure 10. Gene organization of ndhCKJ gene clusters. Arrows indicate the direction of transcription and size of the gene. (A) Synechococcus sp. PCC 7002 ndhCKJ gene cluster. (B) Synechocystis sp. PCC 6803 ndhCKJ gene cluster. (C) Anabaena sp. PCC

7120 ndhCKJ gene cluster. Gene names or predicted protein names are indicated above each gene. Individual scales for each map are in bp.

Synechococcus sp PCC7002

hypothetical sll0606 protein ndhC ndhK ndhJ sll0997

1K 2K 3K

Synechosystis sp. PCC6803 ndhCKJ-6803

Threonine synthase ndhC ndhK ndhJ transposase (slr1282) transposase (slr1283)

1K 2K 3K 4K

Anabaena sp. PCC7120 rubredoxin ndhC ndhK ndhJ kinesin light chain

1K 2K 3K 4K 5K 97

Sequence analysis predicts that the ndhK gene encodes a protein of 242 amino acids and a pI of 5.71. NdhK is a homolog to the NuoB protein from E. coli and the

PSST protein from the mitochondrial complex I of B. taurus (Friedrich et al., 1995). The

Synechococcus sp. PCC 7002 NdhK protein contains the putative binding motifs (C57,

C58, C122, and C153) for a 4Fe-4S cluster. These cysteines are conserved in all NdhK proteins from cyanobacteria (Kazusa DNA Research Institute, 2000; Kaneko et al.,

1996), the NdhK from spinach chloroplast (Schmitz-Linneweber et al., 2000), and the homologous NuoB protein from E. coli (Weidner et al., 1993).

The ndhJ gene is predicted to encode a protein of 174 amino acids and a pI of

4.22. Based on sequence similarity, NdhJ is a homolog to the NuoC protein from E. coli and the 30(IP) protein from B. taurus (Friedrich et al., 1995).

3.1.7 ndhH

The ndhH gene from Synechococcus sp. PCC 7002 was isolated on a 2.4-kb

BamHI fragment and is predicted to encode a protein of 395 amino acids and a pI of 5.66.

NdhH is a homolog of the NuoD protein from E. coli. It is also related to the 49(IP) protein of mitochondrial complex I from B. taurus (Friedrich et al., 1995). The ndhH gene is not associated with other ndh-genes, similar to the gene organization of the

Synechocystis sp. PCC 6803 ndhH gene (Figure 11). The genes downstream of the ndhH gene are different in each species of cyanobacteria. In Synechococcus sp. PCC 7002, there is a putative serine esterase gene downstream of the ndhH gene, followed by a 98

Figure 11. Gene organization around the ndhH genes of Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803. Arrows indicate the direction of transcription and size of the gene. (A) Gene organization around the Synechococcus sp. PCC 7002 ndhH gene.

(B) Gene organization around the Synechocystis sp. PCC 6803 ndhH gene. Maps are individually scaled and numbers below each represent bp. Gene names and predicted product names are indicated above genes (arrows). Restriction sites are as indicated on maps. 99

A. Synechococcus sp. PCC 7002 ndhH

PvuI PvuI

StyIPvuI PvuI

BamHI EcoRV EcoRI HincII sll0355 BamHI ndhH serine esterase

300 600 900 1200 1500 1800 2100

SspI StyI

B. Synechocystis sp. PCC 6803 ndhH

HincII NcoI SspI

HincII SspI SphI EcoRI NcoI StyI HindIII slr0262 slr0263 NdeI ndhH StyI

300 600 900 1200 1500 1800 2100

StyI StyI SspI PstIPvuI PvuI

StyI 100

homolog of the Synechocystis sp. PCC 6803 hypothetical protein encoding sll0355.

In Synechocystis sp. PCC 6803 the gene organization around the ndhH gene is different.

It has two ORFs 3’ to ndhH (slr0262 and slr0263) which encode genes of unknown

function. These results reinforce that there are organizational differences for the genes

immediately surrounding the ndh genes amongst cyanobacteria.

3.1.8 ndhL

The ndhL gene from Synechococcus sp. PCC 7002 was isolated on a 1.75-kb

HindIII fragment from a partial genomic library. Sequence analysis of the

Synechococcus sp. PCC 7002 ndhL gene is predicted to encode a protein of 77 amino

acids and a pI of 9.42. A comparison of the gene organization around the ndhL genes of

Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803 is shown in Figure 12.

The genes immediately downstream from the ndhL genes in both Synechococcus sp. PCC

7002 and Synechocystis sp. PCC 6803 are the same (slr0815). Based on proximity and

gene organization, these ORFs may be involved with the assembly or the function of the

type I NADH dehydrogenase. The ndhL gene was originally isolated as a mutant deficient in its ability to uptake inorganic carbon and was called ictA (for inorganic

carbon transport A) (Ogawa, 1990) and was later renamed when found to be associated

with the type I NADH dehydrogenase complex (Ogawa, 1991). The predicted NdhL

proteins from cyanobacteria are very similar, and inactivation of the ndhL gene in 101

Figure 12. Gene organization around the ndhL genes of Synechococcus sp. PCC

7002 and Synechocystis sp. PCC 6803. Arrows indicate the direction of transcription and size of the gene. (A) Synechococcus sp. PCC 7002 ndhL gene. (B) Synechocystis sp.

PCC 6803 ndhL gene. Maps are individually scaled and numbers below each represent bp. Gene names and predicted product names are indicated above genes (arrows).

Restriction sites are as indicated on maps. 102

A. Synechococcus sp. PCC 7002 ndhL

StyI PvuI PvuI

StyI BamHI NcoI slr0815 HindIII TrpA (slr0966) PvuIHindIII ndhL

300 600 900 1200 1500

EcoRV PstI NcoI StyI StyI

PvuI StyI

B. Synechocystis sp. PCC 6803 ndhL

StyI

StyI StyI StyI

PstI NcoI sll0812 ndhL slr0815 SspI slr0816

300 600 900 1200 1500 1800

PvuI StuI PvuI StyI

NcoI 103

Synechococcus sp. PCC 7002 may lead to a phenotype similar to that observed by

Ogawa (Ogawa, 1990).

3.2 Type II NADH dehydrogenases

The ndb genes encode proteins of the type II NADH dehydrogenase family.

There are three ndb genes in Synechocystis sp. PCC 6803 (Howitt et al., 1999), but only two of the three ndb genes could be found in Synechococcus sp. PCC 7002: ndbA and ndbB. The Synechococcus sp. PCC 7002 ndbA open reading frame was isolated on a 2.1- kb HindIII fragment from a partial genomic library. The ndbA gene is predicted to encode a protein of 460 amino acids and a pI of 5.82. A comparison of the gene organization around the ndbA genes from Synechococcus sp. PCC 7002 and

Synechocystis sp. PCC 6803 is shown in Figure 13. The genes flanking the ndbA genes are very different in the two cyanobacteria.

The Synechococcus sp. PCC 7002 ndbB gene was isolated on a 2.0-kb BamHI-

HindIII fragment from a partial genomic library. Sequence analysis of the ndbB gene from Synechococcus sp. PCC 7002 is predicted to encode a protein of 391 amino acids with a pI of 5.61. A comparison of the gene organization around the ndbB genes from

Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803 is shown in Figure 14.

As in Synechocystis sp. PCC 6803, the ndbA and ndbB proteins from Synechococcus sp.

PCC 7002 are predicted to be hydrophilic proteins. 104

Figure 13. Gene organization around the ndbA genes of Synechococcus sp. PCC

7002 and Synechocystis sp. PCC 6803. Restriction sites are as indicated. Arrows indicate the direction of transcription and size of the gene. (A) Synechococcus sp. PCC

7002 ndbA gene. (B) Synechocystis sp. PCC 6803 ndbA gene. Maps are individually scaled and numbers below each represent bp. Gene names and predicted product names are indicated above genes (arrows). Restriction sites are as indicated on maps.

A. Synechococcus sp. PCC 7002 ndbA

EcoRV SspI

DraI SspI EcoRV StyI

DraI DraI HincII PvuI SspI PstI HincII HindIII PvuI PvuI StyI

PvuIPvuI StuI PvuIBglII EcoRIStyI HincII NcoI StyIStyI NcoI PvuI StyI hypothetical protein slr0722 hypothetical protein StuI ndbA (slr0851) slr1938 PvuIPvuI

500 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 6000 6500 7000 7500 8000 8500

HincII SspI SspI StyI BamHI SspI BglII BglII StyI EcoRV HincII StyI DraI SspI StyI PvuI PvuI

HindIII DraI BamHI HindIII PvuI PvuI PvuI NcoI SspI

SspI HindIII EcoRI EcoRI

B. Synechocystis sp. PCC 6803 ndbA

slr0852 EcoRI HincII StyI HincII StyI NcoI

EcoRV PvuISspI DraIPvuI PvuI EcoRI ribosomal-protein-alanine N-acetyltransferase DraI StyI ndbA

300 600 900 1200 1500 1800 2100 2400

SspI HincII StyI HincII StyI AvaI XbaI NcoI SacI

HindIII EcoRI StyI 105

Figure 14. Gene organization around the ndbB genes of Synechococcus sp. PCC

7002 and Synechocystis sp. PCC 6803. Arrows indicate the direction of transcription and size of the gene. (A) Synechococcus sp. PCC 7002 ndbB gene. (B) Synechocystis sp.

PCC 6803 ndbB gene. Maps are individually scaled and numbers below each represent bp. Gene names and predicted product names are indicated above genes (arrows).

Restriction sites are as indicated on maps. 106

A. Synechococcus sp. PCC 7002 ndbB

HincII BglII SspI SphI

StyI NcoI DraI PvuI hypothetical Zn-finger protein (CAB07242) KpnI ndbB

300 600 900 1200 1500 1800 2100

StyI PvuI StyI DraI StyI PvuI XbaI SacI

PvuI StyI BamHI StyI

B. Synechocystis sp. PCC 6803 ndbB

HincII

StyI NcoI

BamHI StyI StyI StyI StyI PvuI AvaI HincII

PvuI PvuI HincII StyIStyI slr1742 ndbB N-acetylmuramoyl-L-alanine amidase

300 600 900 1200 1500 1800 2100 2400 2700 3000

EcoRV NcoI HincII HincII PvuI StyI StyI AvaI SmaI SacI

NcoI HincII HincII

StyI 107

The results presented here suggest that there are only two Ndb-type II NADH dehydrogenases in Synechococcus sp. PCC 7002 that are most similar to the NdbA and

NdbB proteins from Synechocystis sp. PCC 6803.

3.3 Bi-directional Hydrogenase

The bi-directional hydrogenase apparent operon spans a 15-kb region of DNA in

Synechococcus sp. PCC 7002. Initially, the Synechococcus sp. PCC 7002 hoxH gene was identified by Southern blot hybridization with a Synechocystis sp. PCC 6803 hoxH- specific probe on a 4.5-kb EcoRV fragment. A Synechocystis sp. PCC 6803 hoxF- specific probe was similarly used to identify cosmid 3C8 containing the Synechococcus sp. PCC 7002 hoxF gene. The hydrogenase gene cluster was identified on two other overlapping cosmids, cosmid 1C6 and cosmid 1E6, by using the hoxH and hoxF clones isolated from Synechococcus sp. PCC 7002 as probes to screen the cosmid library. The entire hydrogenase gene cluster was also identified on cosmid 5D12. The genes encoding the bi-directional hydrogenase as well as the genes encoding the hydrogenase assembly factors are tightly clustered in Synechococcus sp. PCC 7002 compared to other cyanobacterial species (Figure 15). There are a total of 13 genes that have been identified in this 15-kb nucleotide sequence that may encode subunits of the hydrogenase enzyme or proteins involved in the assembly and maturation of the functional hydrogenase. The 15-kb of nucleotide sequence has the following open reading frames: hypE, hoxE, hoxF, hoxU, hoxY, hyp3, hoxH, hoxW, hypA, hypB, hypF, hypC, and hypD. 108

Figure 15. Hydrogenase gene cluster organization in cyanobacteria. Arrows indicate the direction of transcription and size of the gene. (A) The hydrogenase gene cluster of

Synechococcus sp. PCC 7002. (B) The hydrogenase gene cluster of Synechocystis sp.

PCC 6803. (C) The hydrogenase gene cluster of Anabaena sp. PCC 7120. Maps are individually scaled and numbers below each represent bp. Gene names and predicted product names are indicated above genes (arrows).

Cyanobacterial hydrogenase gene organization

Synechococcus sp. PCC7002 hoxW hypB hoxU UreC hypE hoxE hoxF hoxY hyp3 hoxH hypA hypF hypC hypD

1K 2K 3K 4K 5K 6K 7K 8K 9K 10K 11K 12K 13K 14K

Synechocystis sp. PCC6803

ssl2420 sll1222 slr1233 hoxH sll1225 hoxY hoxU hoxF hoxE transposase slr1334

1K 2K 3K 4K 5K 6K 7K 8K 9K 10K 11K

Anabaena sp. PCC7120

sll1388 sll0525 hyp8 slr0090 hoxE hoxF sll0163 sll0163 slr0143 hoxU hoxY hoxH ORF acetyl CoA synthase hoxW

1K 2K 3K 4K 5K 6K 7K 8K 9K 10K 11K 12K 13K 14K 15K 16K 17K 18K 19K 20K 21K 22K 23K 24K 25K 109

3.3.1 hypE

The hypE gene of Synechococcus sp. PCC 7002 is predicted to encode a protein of 348 amino acids with a pI of 4.8. This protein is presumably involved in assembly of the hydrogenase enzyme in other bacteria (Colbeau et al., 1993; Drapal and Bock, 1998;

Jacobi et al., 1992), and is predicted to serve a similar purpose in Synechococcus sp. PCC

7002.

3.3.2 hoxE

The hoxE gene of Synechococcus sp. PCC 7002 is predicted to encode a protein of 171 amino acids with a pI of 6.34. There is a putative 2Fe-2S binding motif

(CX4CX35CXGXC) beginning at Cys-86 and ending at Cys-131 of the predicted

Synechococcus sp. PCC 7002 HoxE protein; this motif is similar to the that found in the

Synechococcus sp. PCC 6301 HoxE protein (Boison et al., 1998). The HoxE protein also has some sequence similarity to the NuoE subunit of the E. coli (Blattner et al., 1997;

Weidner et al., 1993).

3.3.3 hoxF

The hoxF gene of Synechococcus sp. PCC 7002 is predicted to encode a protein of 536 amino acids and a pI of 4.78. This large subunit of the predicted diaphorase 110 heterodimer has sequence similarity to the NuoF subunit of the E. coli Type I NADH dehydrogenase and is predicted to contain a flavin mononucleotide binding site for transfer of electrons to and from NAD(P)H/NAD(P)+ (Appel and Schulz, 1996).

3.3.4 hoxU

The hoxU gene of Synechococcus sp. PCC 7002 is predicted to encode a protein of 238 amino acids and a pI of 5.73. The HoxU protein from Synechococcus sp. PCC

7002 is predicted to have 15 cysteines and has a conserved 2Fe-2S cluster binding motif

(PXLCX10-15CRXCXVX11-16C) near the amino terminus, starting at Pro-33 and ending at

Cys-64. This motif is found in all other HoxU proteins to date (Appel and Schulz, 1996).

Another conserved region of amino acids found in the Synechococcus sp. PCC 7002

HoxU protein which begins at Asp-95, is the sequence (EGNHXC). This sequence

overlaps the conserved sequence HXCX2CX5C and corresponds either to a 4Fe-4S cluster similar to the 4Fe-4S cluster found in the small, crystallized subunit from D. gigas

(Volbeda et al., 1995) or 3Fe-4S cluster binding site (Appel and Schulz, 1996). There is

also a ferredoxin-like binding motif (CX2CX2CX3CXnCX2CX2CX3CP) located near the carboxy terminus of the Synechococcus sp. PCC 7002 HoxU protein that begins at Cys-

148 and ends at Pro-203. These putative Fe-S clusters may be involved in distributing electrons to either NAD+ or other electron acceptors from hydrogen to transfer electrons through the respiratory electron transport chains. 111

3.3.5 hoxY

The hoxY gene of Synechococcus sp. PCC 7002 is predicted to encode a protein of

189 amino acids with five cysteines and a pI of 4.66. This proteins also has the Ni-Fe

hydrogenase consensus sequence CXGCXnGXCX3GXmGCPP, which is predicted to

ligand the proximal 4Fe-4S cluster in the hydrogenase moiety of the enzyme (Appel and

Schulz, 1996).

3.3.6 hyp3

The hyp3 gene of Synechococcus sp. PCC 7002 is predicted to encode a protein of

209 amino acids and a pI of 5.6. Synechocystis sp. PCC 6803 has no homolog of this protein, but there are copies of the gene in Anabaena variabilis (Boison et al., 1998) and

Anabaena sp. PCC 7120. The function of this protein is unknown.

3.3.7 hoxH

The hoxH gene of Synechococcus sp. PCC 7002 is predicted to encode a protein of 476 amino acids and a pI of 6.62. This large subunit of the hydrogenase moiety is predicted to have binding ligands for the Ni cofactor. There is a putative protease- cleavage site located at His-448 (His-475 in the numbering scheme in Figure 40 because of insertions to maximize the sequence similarity) in the Synechococcus sp. PCC 7002 112

HoxH protein. The 26 amino acids of the carboxy terminus may have to be cleaved by HoxW in order for the maturation of the protein similar to the HoxH proteins of R. eutropha (Massanz et al., 1997).

3.3.8 hoxW

The hoxW gene of Synechococcus sp. PCC 7002 is predicted to encode a protein of 143 amino acids and a pI of 4.59. The predicted HoxW protein has the consensus

hydrogenase-protease specific motif GXGNX4RDD/EGXG (Boison et al., 1998) and is responsible for cleavage of HoxH for the maturation of the hydrogenase enzyme in other bacteria (Massanz et al., 1997). It is likely that the HoxW protein serves the same purpose in Synechococcus sp. PCC 7002.

3.3.9 hypA

The hypA gene of Synechococcus sp. PCC 7002 is predicted to encode a protein of 114 amino acids and a pI of 4.40. HypA is a predicted metal-binding assembly protein containing five cysteines near the carboxy terminus of the protein. The HypA protein

from Synechococcus sp. PCC 7002 contains the CX2CX12-13CX2C motif that is found in all HypA proteins and is characteristic of non-heme iron proteins like rubredoxins (Berg and Holm, 1982) and certain zinc-binding regulatory domains (South and Summers,

1990), which suggests that HypA is a metalloprotein. 113

3.3.10 hypB

The hypB gene of Synechococcus sp. PCC 7002 is predicted to encode a protein of 274 amino acids and a pI of 6.21. This protein has 20 histidines in the amino terminus that may be involved in Ni binding (Fu and Maier, 1994; Olson et al., 1997). Based on the similarity that the Synechococcus sp. PCC 7002 HypB protein has with the HypB proteins from other bacterial species, it also contains four regions that may be involved in

GTP binding (Fu and Maier, 1994; Olson et al., 1997).

3.3.11 hypF

The hypF gene of Synechococcus sp. PCC 7002 is predicted to encode a protein of 766 amino acids and a pI of 7.18. This protein has a Zn-finger binding motif

CX2X18CX24CX2CX18CX2C (Rey et al., 1993) beginning at Cys-110 and ending at Cys-

184. It has been postulated that HypF is required for the assembly of the mature hydrogenase as well as hydrogen inducibility of the hydrogenase operon in R. capsulatus

(Colbeau et al., 1998). It has been postulated recently that the HypF proteins also have an acylphosphatase (ACP) motif near the N-termini (Wolf et al., 1998). The biological function of ACPs is unknown in prokaryotes. In eukaryotes, it is known that ACPs are small (100 amino acids) proteins that catalyze the hydrolysis of the carboxyl-phosphate bond in acylphosphates (Wolf et al., 1998). 114

Alignment of eukaryotic and prokaryotic ACPs with the Synechococcus sp. PCC

7002 HypF reveals that this regional similarity does exist in the HypF protein (Figure

16). Of note is the conserved Arg residue that corresponds to R23 in the well-defined

ACP from bovine testis. This Arg residue is conserved in all HypF proteins and ACPs to date (Wolf et al., 1998). This residue is presumed to bind the phosphate moiety at the active-site which abstracts a proton from a nucleophilic water molecule liganded to a conserved Asn (N41). This residue is also conserved in all HypF sequences reported to date. These observations point to the existence of an acylphosphatase domain in the

Synechococcus sp. PCC 7002 HypF protein. 115

Figure 16. ClustalW alignment of HypF proteins and acylphosphatases. The N-termini of several HypF proteins have been aligned to the acylphosphatase proteins from three major acylphosphatase groups (Wolf et al., 1998). Groups are represented as I, II, or III. Group I are the organ-common acylphosphatases, group II are the muscular acylphosphatases, and group III are the prokaryotic homologs of acylphosphatases.

HypF and ACP ClustalW Formatted Alignments

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 chicken I PO7032 A G S E G L M S V D Y E V S G R V Q G V F F R K Y T Q S E A K R L G L V G WV R N T S H G T V Q G Q A Q G - P A A R V R E L Q E WL R K I G S P - Q S R I S R A E F T N E K E I A A L E H T D F Q I R K pig I P24540 S M A E G D T L I S V D Y E V F G K V Q G V F F R K Y T Q A E G K K L G L V G WV Q N T D Q G T V Q G Q L Q G - P T S K V R H M Q E WL E T R G S P - K S H I D R A S F N N E K V I S K L D Y S D F Q I V K human I P07311 M A E G N T L I S V D Y E I F G K V Q G V F F R K H T Q A E G K K L G L V G WV Q N T D R G T V Q G Q L Q G - P I S K V R H M Q E WL E T R G S P - K S H I D K A N F N N E K V I L K L D Y S D F Q I V K chicken II P07031 S A L T K A S G S L K S V D Y E V F G R V Q G V C F R M Y T E E E A R K L G V V G WV K N T S Q G T V T G Q V Q G - P E D K V N A M K S WL S K V G S P - S S R I D R T K F S N E K E I S K L D F S G F S T R Y Pig II P00819 S T A R P L K S V D Y E V F G R V Q G V C F R M Y T E D E A R K I G V V G WV K N T S K G T V T G Q V Q G - P E E K V N S M K S WL S K I G S P - S S R I D R T N F S N E K T I S K L E Y S N F S I R Y human II P14621 M S T A Q S L K S V D Y E V F G R V Q G V C F R M Y T E D E A R K I G V V G WV K N T S K G T V T G Q V Q G - P E D K V N S M K S WL S K V G S P - S S R I D R T N F S N E K T I S K L E Y S N F S I R Y E. coli III P75877 M S K V C I I A WV Y G R V Q G V G F R Y T T Q Y E A K R L G L T G Y A K N L D D G S V E V V A C G - E E G Q V E K L M Q WL K S - G G P R S A R V E R V L S - - E P H H P S G E L T D F R I R A. fulgidus III O29440 M I A L E I Y V S G N V Q G V G F R Y F T R R V A R E L G I K G Y V K N L P D G R V Y I Y A V G - E E L T L D K F L S A V K S - G P P - - - - L A T V R G V E V K K A E I E N Y E S F E V A Y B. subtilis III O35031 M L Q Y R I I V D G R V Q G V G F R Y F V Q M E A D K R K L A G WV K N R D D G R V E I L A E G - P E N A L Q S F V E A V K N - G S P - F S K V T D I S V T E S R S L E G - - H H R F S I V Y 7002 HypF N-term 150 L L V K R R L R L E I Q G T V Q G V G F R P F V Y Q L A T A L N L F G WV N N S T A G - V T I E V E G - G R S P L N L F L E K L Q A E L P P - - - - N A K I D A L K Y Q Y L E L I G Y N N F E I H A 6803 HypF N-term 150 M L K T V A I Q V Q G R V Q G V G F R P F V Y T L A Q E M G L N G WV N N S T Q G A T V V I T A D - - E K A I A D F T E R L T K T L P P - - - - P G L I E Q L A V E Q L P L E S F T N F T I R P R. eutropha HypF2 N-term 150 M L M P R R P R N P R T V R I R I R V R G V V Q G V G F R P F V Y R L A R E L G L A G WV R N D G A G - V D I E A Q G S A A A L V D S R L R R L R R D A P P - L A R V D E I G E - - E R C A A Q V D A D G F A I L E E. coli HypF N-term 150 M A K N T S C G V Q L R I R G K V Q G V G F R P F V WQ L A Q Q L N L H G D V C N D G D G - V E V R L R E - - - - D P E T F L V Q L Y Q H C P P - L A R I D S V E R - - E P F I WS Q L P T E F T I R Q R. capsulatus HypF N-term 150 M Q A WR I R V R G Q V Q G V G F R P F V WQ L A R A R G L R G V V L N D A E G - V L I R V A G - - - - D L G D F A A A L R D Q A P P - L A R V D A V E V T - - A A V C D D L P E G F Q I A A V . V G . V Q G V G F R . T E A . . L G L . G WV . N . G V . . G V L G P R . D . . E ...... F I . 116

3.3.12 hypC

The HypC protein is required for hydrogenase maturation in R. eutropha (Dernedde et al.,

1993), E. coli (Lutz et al., 1991), B. japonicum (Olson and Maier, 1997), and may also be required for the maturation of the enzyme in cyanobacteria. The hypC gene of

Synechococcus sp. PCC 7002 is predicted to encode a protein of 80 amino acids and a pI of 4.04 and has a high degree of similarity to other bacterial HypC proteins.

3.3.13 hypD

The hypD gene of Synechococcus sp. PCC 7002 is predicted to encode a protein of 362 amino acids and a pI of 6.71. The predicted protein is very similar to other bacterial HypD proteins. HypD has been shown to play a role in hydrogenase maturation in E. coli (Jacobi et al., 1992) and in R. eutropha (Dernedde et al., 1993) and may play a similar role in Synechococcus sp. PCC 7002. 117

3.3.14 Interposon mutagenesis of the hoxH and hoxF genes in

Synechococcus sp. PCC 7002

The physiological role of the hydrogenase in cyanobacteria is unknown. In order to investigate its possible role in electron transport, the hoxH gene was chosen for interposon mutagenesis. The hoxH gene is predicted to encode the large Ni-containing subunit of the hydrogenase. It was hoped that the absence of the large subunit would lead to an inactivation of the entire hydrogenase. The hoxH gene was interrupted by the insertion of a 1.3-kb BamHI DNA fragment containing the aphII gene and conferring kanamycin resistance into a unique BglII site in the coding sequence of hoxH (Figure

17). This plasmid, pHOXH, was used to transform the wild-type strain of Synechococcus sp. PCC 7002. Segregation of alleles in the transformants hoxH::aphII of Synechococcus sp. PCC 7002 was verified by PCR analysis using primer 311 (5'-G CCC CAT CAT

CAG AAC G-3') and primer 312 (5'-GAT TTA GCA CGG GGT GG-3') (Figure 17).

Results of the PCR analysis reveal that the hoxH::aphII locus successfully replaced all wild-type copies of the hoxH gene. Therefore, the hoxH gene is not essential for cell viability, under normal photoautotrophic growth conditions.

As mentioned in 1.1.2, although cyanobacteria have ndh genes, which putatively encode a type I NADH dehydrogenase, genes encoding subunits for the diaphorase activity of type I NADH dehydrogenase are missing. It may be possible that some of the diaphorase active subunits from the hydrogenase of Synechococcus sp. PCC 7002 are able to substitute for these missing type I NADH dehydrogenase subunits. In order to 118

Figure 17. Interposon mutagenesis of hoxH of Synechococcus sp. PCC 7002. (A)

Physical map of the Synechococcus sp. PCC 7002 hoxH gene. Arrows indicate the direction of transcription and size of the gene. A 1.3-kb BamHI fragment containing the aphII gene conferring kanamycin resistance was inserted into a unique BglII site within the coding sequence of the hoxH gene. The resultant plasmid, pHOXH was used to transform the wild-type strain of Synechococcus sp. PCC 7002. (B) PCR analysis of the hoxH locus. Total DNA was isolated from the wild-type and kanamycin-resistant strains of Synechococcus sp. PCC 7002. This DNA was used as a template for PCR reactions. 119 A RV RV dIII cII cII dIII II Bgl Eco HIn HIn Eco HIn HIn

hoxH hoxW hypA hypB

aphII I Sph

hoxH::aphII B ∆ wild type HE ladder

2.4 kb

1.1 kb 120 assess the possible role of the bi-directional hydrogenase as a component of the type I

NADH dehydrogenase in Synechococcus sp. PCC 7002, interposon mutagenesis was performed on the hoxF gene. The hoxF gene was interrupted by inserting a 2-kb BamHI

DNA fragment containing the aadA gene from the Ω fragment (Prentki and Krisch, 1984) conferring spectinomycin and streptomycin resistance into a unique BglII site within the predicted coding sequence of hoxF (Figure 18). The resulting plasmid, pHOXF, was used to transform both the wild-type and hoxH::aphII strains of Synechococcus sp. PCC

7002. The segregation of the hoxF::Ω allele was verified by Southern blot analysis

(Figure 18). Results indicate that the all wild-type copies of the hoxF gene were successfully displaced by the hoxF::Ω allele in three independently isolated spectinomycin transformants and a kanamycin/spectinomycin resistant transformant.

These results indicate that the hoxF gene product is not essential for cell viability under normal growth conditions. The absence of a clear phenotype similar to those associated with either the NdhF1-deficient (Schluchter et al., 1993), or NdhF3-deficient and NdhD3- deficient (Klughammer et al., 1999) strains of Synechococcus sp. PCC 7002 suggests that the HoxF gene product is not important in complexes involving these Ndh proteins. 121

Figure 18. Interposon mutagenesis of hoxF of Synechococcus sp. PCC 7002. (A)

Physical map of the Synechococcus sp. PCC 7002 hoxF gene. Arrows indicate the direction of transcription and size of the gene. A BamHI fragment containing the Ω fragment conferring streptomycin and spectinomycin resistance was inserted into a unique BglII site within the coding sequence of the hoxF gene. (B) Southern blot analysis of the hoxF locus. Total DNA was isolated from the wild-type and spectinomycin resistant strains of Synechococcus sp. PCC 7002. This DNA was digested with HindIII and BamHI and transferred to a nylon membrane. A 32 P-labeled hoxF specific probe was hybridized to the blot. Lanes 1 and 6, wild-type Synechococcus sp.

PCC 7002; lane 2, hoxF/hoxF::Ω merodiploid; lane 3, hoxF::Ω; lane 4 and 5, hoxH::aphII hoxF::Ω. 122

A HI RI cII dIII II HIn Bam Hin Eco Bgl

hoxE hoxF

Ω I HI HI Sph Bam Bam B 123 456

3.2 kb

1.2 kb 123

3.3.15 Chlorophyll and carotenoid contents of the hoxH- and hoxH- hoxF-

strains

Although no immediate growth phenotypes were obvious, inactivation of the hox genes in Synechococcus sp. PCC 7002 may have had other effects on the cells and their contents. Physiological parameters that can be readily measured are the chlorophyll and carotenoid contents. The chlorophyll and carotenoid contents of the hoxH- and hoxH- hoxF- mutant strains were compared to those of the wild-type strain grown under standard conditions. The wild-type strain had a chlorophyll concentration of 3.6 ± 0.1 µg ml-1, compared to 3.3 ± 0.1 µg ml-1 for the hoxH- strain and 3.5 ± 0.1 µg ml-1 for the hoxH- hoxF- double mutant strain. The carotenoid contents were 1.0 ± 0.1 µg ml -1 for the wild- type strain, 1.0 ± 0.1 µg ml-1 for the hoxH- strain, and 1.1 ± 0.1 µg ml-1 for the hoxH- hoxF- double mutant strain. These results reveal that under standard growth conditions, there is no significant difference in chlorophyll and carotenoid contents between the hoxH- and hoxH- hoxF- mutant strains compared to the wild-type strain of Synechococcus sp. PCC 7002. 124

3.3.16 Growth analysis of hoxH- and hoxH- hoxF- double mutants

The doubling times of both the hoxH- mutant and hoxH- hoxF- double mutant are

nearly identical to those of the wild-type strain (3.8 ± 0.1 hours) under standard growth

conditions. The doubling time of the hoxH- strain is 3.9 ± 0.3 hours and the doubling time of the hoxH- hoxF- strain is 3.8 ± 0.2 hours. These results indicate that the doubling

times of the hoxH- and hoxH- hoxF- double mutant are identical to the wild-type under

° -2 -1 normal growth conditions (38 C, 250 µE m s , 1.5% CO2/98.5% air). Temperature

shifts from normal growth temperatures to lower temperatures have been shown to lead

to photoinhibition in other cyanobacterial strains with lesions in the electron transport

chain (Clarke and Campbell, 1996). Therefore, a temperature shift experiment was

conducted to determine whether or not there would be an effect on the growth rates from

the inactivation of the hydrogenase genes. The effects of a temperature-shift from 38°C

to 22°C on the growth rates of hoxH- and wild-type strains of Synechococcus sp. PCC

7002 are shown in Figure 19. These results indicate that the hoxH gene is not involved

in low temperature adaptation in Synechococcus sp. PCC 7002. Moreover, the wild-type

and mutant strains do not grow differently under conditions of nickel limitation at 22°C

when nitrate is the nitrogen source (T. Sakamoto, personal communication). These

results prove the fact that neither the hoxH nor the hoxF gene products are necessary for

the cell under these conditions. 125

Figure 19. Temperature-shift effects on wild-type and hoxH::aphII. Exponentially growing cells were either maintained at 38°C or transferred to a 22°C water bath. (A)

Temperature-shift effects on the hoxH::aphII strain of Synechococcus sp. PCC 7002.

The hoxH::aphII cells were grown at 38°C () and 22°C (). (B) Temperature-shift effects on the wild-type strain of Synechococcus sp. PCC 7002. Wild-type cells grown at

38°C () and 22°C (). The data shown are from a single experiment. The experiment was repeated three times, and the results were consistent and reproducible. 126 A

10 ∆hoxH::aphII (38ûC) ∆hoxH::aphII (22ûC)

1 550

OD 0.1

0.01 0 1020304050 Time (hours) B 10 WT(38ûC) WT (22ûC) 1 550

OD 0.1

0.01 0 1020304050 Time (hours) 127

3.4 Mobile Electron Carriers

Four potential mobile electron carriers were identified in this study. Three genes

(petJ1, petJ2, cytM) are predicted to encode cytochrome c proteins, and the fourth gene, bcpA, is predicted to encode a putative type I blue-copper protein. Plastocyanin is encoded by the petE gene in higher plants (Scawen et al., 1975), algae (Merchant et al.,

1990), and other cyanobacteria (Briggs et al., 1990; Clarke and Campbell, 1996).

Although Synechococcus sp. PCC 7002 genomic DNA blots were probed with heterologous petE specific probes, no cross-hybridizing signals could be found for the petE gene. Likewise, no plastocyanin-like sequences have yet been identified in the

Synechococcus sp. PCC 7002 genome sequencing project.

3.4.1 Cloning and sequence analysis of the Synechococcus sp. strain

PCC 7002 cytochrome c6 gene

Initial Southern blot hybridization screens using heterologous PCR product

probes (cytochrome c6 genes from Synechocystis sp. PCC 6803 or Synechococcus sp.

PCC 7942) at low stringency did not produce positive hybridization signals. Thus, the

Synechococcus sp. strain PCC 7002 protein was purified as described in Materials and

Methods. Cytochrome c6 was purified to apparent electrophoretic homogeneity based upon its characteristic absorbance maxima at 416 nm and 553 nm. Purity was assessed by electrophoresis on a polyacrylamide gel in the presence of SDS and was followed by 128

Coomassie staining of the proteins and TMBZ detection of heme groups (Figure 20).

The N-terminal sequence from the purified cytochrome c6 was determined to be

ADAAAGAQVFAANCA. Based upon this sequence, the degenerate oligonucleotide: 5'-

GCT GA(T/C) GCT GCT GCT GGT GCT CA(A/G) GTT TT(T/C) GCT GCT AA(T/C)

TG(T/C) GC-3' was synthesized for use as a forward primer for PCR. This primer was used with the degenerate oligonucleotide: 5'-CCA CGC TTT TTC TGC TTG GTC GAG

TAC GTA GCG TGC NAC (A/G)TC-3' derived from the conserved cytochrome c6 sequence (DVAAYVLDQAEKGW) of other cytochrome c6 proteins as the reverse primer to amplify by PCR an internal region of the Synechococcus sp. strain PCC 7002

cytochrome c6 gene sequence from total Synechococcus sp. strain PCC 7002 genomic

DNA for use as a probe. An internal degenerate PCR primer: 5'-AA(T/C) TG(T/C)

GC(T/C) GC(T/C) TG(C/T) C-3' was also designed from the conserved heme attachment

site amino acid sequence (NCAACH) as determined by cytochrome c6 sequence alignments using the BLAST search tool (Altschul et al., 1990). The resultant PCR products were used as probes for Southern blot analysis of Synechococcus sp. strain PCC

7002 chromosomal DNA digested with multiple restriction enzymes. A hybridizing

HindIII-XbaI fragment of approximately 1.7-kb was chosen to clone into pUC19 (Figure

21). The nucleotide sequence of this clone has been deposited into GenBank (accession number: AF020306). The petJ coding region contains a single complete open reading frame of 351 bp that can encode to a preprotein of 117 amino acids, this ORF was positioned between the sequences of two other partial open reading frames (Figure 22).

Sequence analysis of the partially sequenced open reading frame 5' 129

Figure 20. SDS-PAGE analysis of cytochrome c6 from Synechococcus sp. PCC

7002. Soluble protein fractions from Synechococcus sp. PCC 7002 isolated by ammonium sulfate precipitation and mono-Q ion-exchange chromatography were electrophoresed on two SDS polyacrylamide gels. (A) TMBZ stained polyacrylamide

gel. Lane 1, fraction 1 from mono-Q column chromatography of purified cytochrome c6 from Synechococcus sp. PCC 7002. Lane 2, fraction 2, mono-Q column chromatography, Lane 3, fraction 3, mono-Q column chromatography, Lane 4, 95% ammonium sulfate cut supernatant, Lane 5, E. coli protein extract, Lane 6, horse heart cytochrome c. (B) Coomassie stained polyacrylamide gel. Lanes are loaded identically as for Panel A. A total of 5 µg of protein was loaded per lane. 130 A. 1 2 3 4 5 6

Cyt c6

B. 1 2 3 4 5 6

Cyt c6 131

Figure 21. Probe design and Southern blot hybridization of petJ from Synechococcus sp. strain PCC 7002. (A) Degenerate oligonucleotide sequences for PCR primers.

Primer 1 was a degenerate oligonucleotide derived from the N-terminal sequence of the

Synechococcus sp. strain PCC 7002 purified cytochrome c6. Primer 2 was a degenerate oligonucleotide derived from the conserved amino acid sequence of the heme attachment

motif (XCXXCH) of several cytochrome c6 proteins. Primer 3 was a degenerate oligonucleotide derived from a semi-conserved amino acid sequence at the carboxy-

terminus of several cytochrome c6 proteins. The predicted amino acid sequences for the degenerate oligonucleotides are shown above the nucleotide sequences. Two different probes were made using these degenerate oligonucleotides, probe 1 and probe 2. The predicted PCR probe product sizes are beneath the primer sequences. (B) Autoradiogram of genomic digests of Synechococcus sp. strain PCC 7002 DNA probed with probe 2 (32P- radiolabelled PCR product of primer 1 and primer 3 of (A)). Genomic DNA from 7002 was digested with various restriction enzymes and hybridized at 55¡C. Lane 1: BglII,

Lane 2: BamHI, Lane 3: EcoRI, Lane 4: HindIII, Lane 5: XbaI, Lane 6: BglII/BamHI,

Lane 7: BglII/EcoRI, Lane 8: BglII/HindIII, Lane 9: BamHI/EcoRI, Lane 10:

BamHI/XbaI, Lane 11: BamHI/HindIII, Lane 12: EcoRI/HindIII, Lane 13: EcoRI/XbaI,

Lane 14: HindIII/XbaI. Only one copy of the petJ gene is detectable at a hybridization temperature of 55ûC. The smallest hybridizing DNA fragment (1.74-kb HindIII-XbaI) was chosen to clone into pUC19. 132 A. Primer 1 Primer 3

A D A A A G A Q V F A A N C A D V A A Y V L D Q A E K G W 5' GCT GAT/C GCT GCT GCT GGT GCT CAA/G GTT TTT/C GCT GCT AAT/C TGT/C GC 3' 3' CTG/A CAN CGT GCG ATG CAT GAG CTG GTT CGT CTT TTT CGC ACC 5'

Primer 2

N C A A C H 5' AAT/C TGT/C GCT/C GCT/C TGC/T C 3'

Probe 1 238 bp Probe 2 275 bp

B. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 kb

21.7

5.00 4.27 3.48

1.98 1.90 1.60 1.37

0.94 133

Figure 22. Gene organization around petJ1 in cyanobacteria. Arrows indicate direction of transcription. (A) Gene organization around the petJ1 gene of Synechococcus sp. PCC 7002. (B) Gene organization around the petJ gene of Synechocystis sp. PCC

6803. Maps are individually scaled in bp. Gene names and predicted product names are indicated above genes (arrows). Restriction sites are as indicated on maps. 134

A. Synechococcus sp. PCC 7002 petJ1

HindIII

StyI SspI PvuI BamHI

PvuI SacI AvaI PvuI

AvaI sll0185 homolog petJ1 sll0415 ABC transporter PvuI

300 600 900 1200 1500 1800 2100 2400 2700 3000 3300

EcoRV PvuI KpnI HincII PvuI StyI StyI HindIII

SspI XbaI KpnI

B. Synechocystis sp. PCC 6803 petJ

HincII HincII

NcoI StyI

PvuI PvuI slr1896 petJ srrA (slr1897)

300 600 900 1200

StyI NcoI

StyI StyI 135 to the petJ gene indicates that it has high sequence homology to an ABC transporter protein (sll0415 in the Synechocystis sp. PCC 6803) (Kaneko et al., 1996). The partial open reading frame 3' to the petJ gene is predicted to encode a hypothetical protein of unknown function (sll0815 in the Synechocystis sp. PCC 6803) (Kaneko et al., 1996).

Both of these hypothetical reading frames would be transcribed in the opposite direction of Synechococcus sp. strain PCC 7002 petJ gene.

The petJ nucleotide sequence is predicted to encode a pre-protein transit peptide sequence of 24 amino acids (Figure 23) that displays many characteristics (a basic N- terminal region with two lysines (n-region), a central hydrophobic region (h-region) and a

C-terminal region that follows the '-3, -1 rule' where the amino acids at the -3 and -1 positions are alanines) common for prokaryotic signal peptides (Gierasch, 1989; von

Heijne, 1986). The mature protein would begin at amino acid 25 (Ala) and would thus contain 93 amino acids. As noted above, this was confirmed by N-terminal amino acid

c Synechococcus sequencing of the gel purified cytochrome 6 protein from sp. strain PCC

7002. PetJ has a molecular mass of 9.4 kDa and analysis of the amino acid sequence predicts a pI of 4.89. There is a potential transcription terminator (-18 kcal mol-1) located between nucleotide positions 970 and 990 about 150 bases downstream from the stop codon of the petJ1 gene.

A comparison of mature cytochrome c6 proteins is shown in Figure 23. The cytochrome c6 proteins were aligned using the ClustalW tool (Thompson et al., 1994b).

Cytochrome c6 from Synechococcus sp. strain PCC 7002 has 24 amino acids that are

absolutely conserved and overall is 46%-54% identical to other cytochrome c6 proteins. 136

Figure 23. ClustalW alignment of PetJ amino acid sequences. (A) Deduced cyt c6 amino acid sequences were aligned by the ClustalW multiple protein sequence alignment function in the MacVector 6.5 program. Dark gray shading indicates identical or a majority identical amino acid, the lighter gray shading indicates conserved amino acids, and no shading indicates non-conserved amino acids. Dashes indicate gaps introduced to

maximize the alignment. The consensus cyt c6 sequence is indicated below. Organisms are indicated as follows: 7002, Synechococcus sp. PCC 7002; S. maxima, Spirulina maxima; C. reinhardtii, Chlamydomonas reinhardtii; 7937, Anabaena sp. PCC 7937;

7942, Synechococcus sp. PCC 7942; 7120, Anabaena sp. PCC 7120; 6803, Synechocystis

sp. PCC 6803. (B) Deduced cyanobacterial signal transit peptide sequences for cyt c6 and plastocyanin from various organisms were aligned as described above. Dark gray shading indicates identical or majority identical amino acids, while the lighter gray shading indicates conserved amino acids, and no shading indicates non-conserved amino acids. Hyphens indicate gaps introduced to maximize the alignment. Conserved or identical signal sequence amino acids are indicated below. Organisms are as described

above. The signal peptide of cyt c6 from Synechococcus sp. PCC 7002 shares many of the traits of typical prokaryotic signal peptide sequences (see text). 137

A. ClustalW alignment of cytochrome c6 proteins ClustalW Formatted Alignments

10 20 30 40 50 7002 ADAAAGAQVFAANCAACHAGGNNAVMPTKTLKADALKTYLAGYKDGSKSL 6803 ADLAHGKAI FAGNCAACHNGGLNAINPSKTLKMADLEANGK------NS S. maxima GDVAAGASVFSANCAACHMGGRNVIVANKTLSKSDLAKYLKGFDD- - -DA 7942 ADLAHGGQVFSANCAACHLGGRNVVNPAKTLQKADLDQYGM------AS C. reinhardtii ADLALGAQVFNGNCAACHMGGRNSVMPEKTLDKAALEQYLDGG- - - - - FK 7120 ADSVNGAK I FSANCASCHAGGKNLVQAQKTL KKADL EKYGM------YS 7937 ADVANGAKI FSANCASCHAGGKNLVQAQKTLKKEDLEKFGM------YS AD.A. GA. .F.ANCAACH GG.N. . .P KTLKKADLE.YG S

60 70 80 90 7002 EEAVAYQVTNGQGAMPAFGGRLSDAD I ANVAAYI ADQAENNKW 6803 VAA I VAQI TNGNGAMPGFKGRI SDSDMEDVAAYVLDQAEKG-W S. maxima VAAVAYQVTNGKNAMPGFNGRLSPKQI EDVAAYVVDQAEKG-W 7942 I EA I T TQV TN GKGAMPA FGSKL SADD I ADVA SYV LDQSEKG-WQG C. reinhardtii VESI I YQVENGKGAMPAWADRLSEEEI QAVAEYVFKQATDAAWKY 7120 A EA I I AQV TNGKNAMPA FKGRL KPEQ I EDVAA YV LGKADAD -WK 7937 A EA I I AQV TNGKNAMPA FKGRL KPDQ I EDVAA YV LGQADKS -WK .EAI..Q.TNGKGAMPAF GR.S ...EDVAAYVLDQAEK. W.

B. ClustalW alignment of cyanobacterial signal sequences forClustalW cyt c6 and Formatted plastocyanin Alignments

10 20 30 7002 C6 signal sequence L KKL LA I A LT -V LA TV FA FG- - - TPAFA 7120 C6 signal sequence MKKIFSLVLLGIALFTFAFS- - -SPALA 7937 C6 signal sequence MKKIFSLVLLGIALFTFAFS- - -SPALA 7942 C6 signal sequence MKRILGTAIA-ALVVLLAFI - - -APAQA 7942 PC signal sequence MKVLASFARRLSFAVA-AVLCVGSFFLSAAPASA 6803 PC signal sequence MSKKFLTI LAGLLLVVSSFFLSVSPAAAA 6803 C6 signal sequence MFKLFNQASRIFFGIALPCLIFLGGIFSLGNTALA MKK I F . . L . . L L . A F . PA A 138

It is interesting to note that there appears to be an insertion of 7 amino acids starting

at glycine 42 and ending at lysine 48. The most similar cytochrome c6 protein sequence is that from Spirulina maxima, which also has extra inserted amino acids when compared

to cytochrome c6 proteins from other cyanobacteria.

3.4.2 Attempted deletion of the petJ1 gene by interposon mutagenesis.

The aphII gene, which confers kanamycin resistance, was inserted into a unique BstXI site within the coding region of the Synechococcus sp. PCC 7002 petJ1 gene. This plasmid was used to transform the wild-type strain of Synechococcus sp. PCC 7002 and kanamycin resistant transformants from A+ plates were selected for growth in liquid media. Unlike the previously described deletion of the petJ gene from Synechocystis sp. strain PCC 6803 (Zhang et al., 1994) or the interposon interruption of the cytA gene from Synechococcus sp. strain PCC 7942 (Laudenbach et al., 1990) where the functional copies of the gene encoding cytochrome c6 were replaced by the mutant alleles, attempts to segregate the petJ1::aphII and petJ1 alleles in Synechococcus sp. strain PCC 7002 failed. This indicates the importance of a wild-type copy of the gene for cell viability

(Figure 24). Northern blot analysis of the Synechococcus sp. strain PCC 7002 petJ1 transcript reveals that the petJ1 gene is accumulated as a monocistronic transcript (Figure 25). The results of Northern blot analysis and the fact that the genes adjacent to petJ1 would be transcribed in the opposite direction (Figure 22) reveal that a polar effect in an essential gene is unlikely. Attempts to segregate the Synechococcus sp. strain PCC 7002 gene under different light intensities petJ were also unsuccessful. These results support the idea that the petJ1 gene is essential to Synechococcus sp. PCC 7002. 139

Figure 24. Attempted interposon mutagenesis of the petJ1 gene. (A) Physical map of the petJ1 gene and surrounding partial reading frames. The aphII gene, which confers kanamycin resistance, was digested with the blunt cutting enzyme HincII. This HincII fragment was inserted into a unique, blunt BstXI site within the coding region of petJ1. The plasmid, pPETJ1 was used to transform the wild-type strain of Synechococcus sp. PCC 7002. (B) DNA gel blot hybridization suggests that the gene encoding cytochrome c6 may be an essential gene in Synechococcus sp. strain PCC 7002. Lanes 1 and 2 represent genomic DNA from two different Synechococcus sp. strain PCC 7002 transformants digested with Hind III. The two hybridizing bands represent the wild-type petJ1 gene (2.0 kb) and the petJ1 gene interrupted with the aphII gene encoding kanamycin resistance inserted at the BstXI site (petJ1::aphII, 3.3 kb) indicative of the merodiploid status of these alleles. Lane 3 represents genomic DNA digested with HindIII and wild-type petJ1 gene (2.0 kb). 140

A 100 bp B 123 I

I X

d III Xba Bst

Hin

21.7

bif A petJ hyp I 5.00 4.27 3.3 kb I

X 1.98 2.0 kb Bst 1.90 1.60 ∆ petJ::aphII 1.37

0.94 0.83

aphII I I II II HI HI I I I II Xba Xba Hinc Hinc Bam Bam Bcl Pst Bgl Sph 141

Figure 25. Northern blot analysis of the petJ1 gene. The petJ1 gene is transcribed as a monocistronic transcript. Total RNA was isolated from wild-type Synechococcus sp.

PCC 7002 under standard conditions. The RNA was denatured, electrophoresed, and transferred to a nylon membrane. The blot was probed with a petJ1 specific probe generated by the primers in Figure 21. The size of the petJ1 transcript (450 bp) was estimated based on migration of the ribosomal RNA. Sizes of the ribosomal RNA species are given on the left.

(knt)

2.8 2.3 1.5

0.5 petJ1 mRNA transcript 142

3.4.3 Attempted functional substitution of the Synechococcus sp. strain PCC 7002 petJ1 gene with the petE and petJ genes from Synechocystis sp. strain PCC 6803

Because attempts to segregate the petJ1::aphII allele in Synechococcus sp. PCC

7002 failed under the conditions tested, plasmids were made to determine whether the

functional analog of cytochrome c6, plastocyanin, from Synechocystis sp. strain PCC

6803, could replace the Synechococcus sp. strain PCC 7002 cytochrome c6, and thus

induce complete segregation of the petJ1::aphII allele. The petE gene, which encodes

plastocyanin, was amplified by PCR from Synechocystis sp. strain PCC 6803 genomic

DNA using the primers (5' petE): 5'-ATC GCC AAG AAA CAT GTC TAA AAA G-3'

and (3' petE): 5'-ATA GGC CTT GCC ATT GCG AGA AGC-3'. The PCR product was

isolated and digested with AflIII and StuI. This AflIII-StuI DNA fragment was inserted

into the pSE280 vector at the AflIII compatible NcoI site so that the gene would be in the

correct reading frame for expression and under the control of the E. coli trc (trp-lac) promoter (Brosius, 1989). It has been demonstrated previously that introduced heterologous genes can be expressed with the trc promoter in cyanobacteria (Geerts et al.,

1995). The 3' end of the PCR product was engineered to have a StuI site for insertion into pSE280. This plasmid containing the Synechocystis sp. PCC 6803 petE gene was inserted into the platform vector pLAT2. The pLAT2 vector contains a marker for erythromycin resistance and a polylinker between Synechococcus sp. PCC 7002 argE3 gene flanking sequences. The argE3 gene has been determined to be a neutral site within the Synechococcus sp. PCC 7002 genome (Muñiz, 1993). The pLAT2-petEtrc plasmid 143 was then transformed into the petJ1/petJ1::aphII merodiploid strain of

Synechococcus sp. PCC 7002 (Figure 26). Erythromycin/kanamycin resistant transformants were selected and grown in liquid media with the appropriate antibiotics.

Chromosomal DNA was isolated from the cyanobacterial transformants as described in the Materials and Methods and segregation of the petJ1::aphII allele was assayed by

Southern blot hybridization. Results indicate that the pLAT2-petEtrc plasmid had been successfully inserted into the Synechococcus sp. PCC 7002 neutral site in all strains tested (Figure 26). The inserted Synechocystis sp. PCC 6803 petE plasmids failed to induce full segregation in the petJ1/petJ1::aphII merodiploid strain of Synechococcus sp.

PCC 7002. Even after transformation of this merodiploid strain with the pLAT2-petEtrc, the wild-type copy of the petJ1 gene could not be segregated under either photoautotrophic or photoheterotrophic growth conditions (Figure 27).

A similar strategy was used to incorporate the petJ gene from Synechocystis sp.

PCC 6803 into the Synechococcus sp. PCC 7002 petJ1/petJ1::aphII strain. The primer

6803 c6.1 (5'-CCT TGA AAG GAG AAC ACA TGT TTA AAT TAAT TC-3') containing the NcoI-compatible AflIII site (underlined) and the primer 6803 c6.2 (5'-CTC

CCC AGC CCC AGA GGA TCC CTG ATC AAA-3') containing a BamHI site

(underlined) were used to amplify the petJ gene from Synechocystis sp. PCC 6803. The resulting PCR product was digested and cloned into the pSE280 vector as described for the Synechocystis sp. PCC 6803 petE/pSE280 plasmid. The Synechocystis sp. PCC 6803 petJ/pSE280 plasmid was digested with SspI and inserted into the platform vector pLAT3. (Figure 28A). 144

Figure 26. (A) Schematic of the petJ1::aphII rescue strategy. This is an outline of the strategy to inactivate the petJ1 gene from the Synechococcus sp. PCC 7002 merodiploid

strain by functionally replacing the Synechococcus sp. PCC 7002 PetJ1 (cyt c6) with the

Synechocystis sp. PCC 6803 PetE (plastocyanin) protein. The Synechocystis sp. PCC

6803 petE gene was cloned into the expression vector pSE280 so that petE expression was regulated by the E. coli trc promoter. The plasmid pSE280-petEtrc was digested with SspI and BamHI, the SspI-BamHI petEtrc-containing fragment was inserted into the platform vector pLAT2. The pLAT2-petEtrc plasmid was used transform the petJ1/petJ1::aphII merodiploid strain of Synechococcus sp. PCC 7002. (B) Southern blot analysis of the petEtrc integration into the Synechococcus sp. PCC 7002 chromosome..

The blot was hybridized with a 32-P-labelled Synechocystis sp. PCC 6803 petE PCR product. Lanes 1 and 2: the total DNA isolated from kanamycin/erythromycin resistant strain transformed with the pLAT2-petEtrc plasmid digested with EcoRI. Lanes 3 and 4: total DNA isolated from kanamycin/erythromycin resistant strain transformed with the pLAT2 plasmid digested with EcoRI. Lane 5: pSE280-petEtrc digested with SspI. Lane

6: petE PCR product. Arrows adjacent to the autoradiogram indicate the Synechocystis sp. PCC 6803 petEÐspecific cross-hybridizing DNA fragments. 145 II Hinc I/ Acc HI I I I d III I

I/ Hin Kpn Sph Sst Pst I Sal Bam Xba

Ssp I Hinc II Hind III Ssp I

Ptrc petE Eco RI Em r Eco RI digest pSE280 with Ssp I 7002 ARG pLAT2 8500 bp

7002 ARG digest pLAT2 with Hind III ORI Ap r Not I

Klenow Apr Not I fill-in II

Hinc Ligate with T4 ligase I/ Acc HI I I I I/ I I Sal Sst Pst Kpn Sph Xba Bam

petE Eco RI Ptrc Em r 7002 ARG pLAT2/petE trc Eco RI 9697 bp II Hinc

ApORI r I/ Acc HI I Not I I I I I/ 7002 ARG I Sph Kpn Pst Bam r Sal Sst Ap Not I Xba

Eco RI petE P 7002 chromosome 7002 ARG trc Em r 7002 ARG 7002 chromosome

123456

3.1 kb

1.0 kb

0.4 kb 146

Figure 27. Attempted displacement of the petJ1 gene in a Synechococcus sp. PCC

7002 petJ1/petJ1::aphII merodiploid strain with the pLAT2-petEtrc platform vector.

The petJ1::aphII/petJ1 merodiploid strain of Synechococcus sp. PCC 7002 was transformed with the pLAT2-petEtrc plasmid. Kanamycin/erythromycin resistant cyanobacterial colonies were selected and grown in liquid culture in the presence or absence of 5µM Cu+ and in the presence or absence of 10 mM glycerol. Genomic DNA

was isolated from these cultures, digested with HindIII and transferred to a nylon membrane and probed with a 32P-labelled Synechococcus sp. PCC 7002 petJ1-specific

probe. Genetic backgrounds of isolated DNA are as follows: Lanes 1 and 2:

petJ1/petJ1::aphII/pLAT2-petEtrc . Lanes 3 and 4: petJ1/petJ::aphII, not transformed

with pLAT2-petEtrc . Lane 5: wild-type. Arrows point out Synechococcus sp. PCC

7002 petJ1-specific hybridization with the radiolabelled probe. 147 12345 10 mM glycerol + -++ - 5 µM Cu+ +++ - -

petJ1::aphII

petJ1 148

Figure 28. Insertion of the Synechocystis sp. PCC 6803 petJ gene into the petJ1::aphII merodiploid strain of Synechococcus sp. PCC 7002. (A) The pLAT3/Synechocystis sp.

PCC 6803 petJ plasmid. The petJ gene from Synechocystis sp. PCC 6803 was placed into the multi-cloning site interrupting the amtA gene in the pLAT3 plasmid. This plasmid was used to transform the petJ1/petJ1::aphII merodiploid strain of

Synechococcus sp. PCC 7002. Restriction sites in brackets ( ) represent sites that were eliminated when the plasmid was made. (B) Southern blot analysis of the insertion of the

Synechocystis sp. PCC 6803 petJ gene into the petJ1/petJ1::aphII merodiploid strain of

Synechococcus sp. PCC 7002. Total DNA isolated from several independent kanamycin/spectinomycin resistant transformants was digested with PstI and transferred to a nylon membrane. The blot was probed with a 32P-labelled pLAT3/Synechocystis sp.

PCC 6803 petJ plasmid. Lanes 1-4, Genomic DNA isolated from the petJ1/petJ1::aphII

merodiploid strain of Synechococcus sp. PCC 7002 transformed with

pLAT3/Synechocystis sp. PCC 6803. Lane 5, genomic DNA isolated from the

Synechococcus sp. PCC 7002 wild-type strain. Lane 6, pLAT3 plasmid. Lane 7,

pLAT3/Synechocystis sp. PCC 6803 petJ plasmid. 149 A. )

I I I I I I I

I I

I ) I I I I I I I I

I c

I I I H t

a d n

d s a h i e t d b n a m o P m n i l s p p H i n a m X c a ( i S P C S ( H S H B E B H amtA amtA I

p p s s S / S petJ / V V R R o o c I I I c I E

H E

o c c n m i N a H B B. 1 2 3 4 5 6 7

WT ∆amtA

6803 petJ 1.7 kb 1.1 kb 150

The pLAT3::petJtrc plasmid was used to transform the petJ1/petJ1::aphII

merodiploid strain of Synechococcus sp. PCC 7002 (Figure 28B). Figure 29 shows that,

even with the Synechocystis sp. PCC 6803 petJ gene, the Synechococcus sp. PCC 7002

petJ1/petJ1::aphII merodiploid strain did not segregate. Because there was no

segregation in either attempts at functional substitution of the petJ1 gene from

Synechococcus sp. PCC 7002 with the petE or petJ gene from Synechocystis sp. PCC

6803, the transcription of the Synechocystis sp. PCC 6803 petE and petJ genes was assayed by RNA blot analysis (Figure 30 and Figure 31). The results indicated that both the Synechocystis sp. PCC 6803 petE and petJ genes are transcribed in

Synechococcus sp. PCC 7002.

To insure that a protein was being translated from the petE mRNA transcript, the

production of the Synechocystis sp. PCC 6803 plastocyanin in the Synechococcus sp.

PCC 7002 petJ1/petJ1::aphII merodiploid strain was assayed by immunoblot analysis

with anti-Synechocystis sp. PCC 6803 plastocyanin antiserum (a gift from Dr. John

Whitmarsh, University of Chicago-Urbana). Figure 32 shows that the Synechocystis sp.

PCC 6803 plastocyanin is produced and processed to the correct mature size in

Synechococcus sp. PCC 7002. These results indicate that Synechococcus sp. PCC 7002 is

capable of producing and processing foreign proteins but that neither the Synechocystis

sp. PCC 6803 plastocyanin or cytochrome c6 can functionally substitute for the native

Synechococcus sp. PCC 7002 cytochrome c6 protein. 151

Figure 29. The petJ gene from Synechocystis sp. PCC 6803 cannot functionally

replace the petJ gene from Synechococcus sp. PCC 7002. The petJ1/petJ1::aphII

merodiploid/pLAT3 6803 petJ strain of Synechococcus sp. PCC 7002 was grown under various light and photoheterotrophic conditions and total DNA was isolated from the cells. DNA isolated from all strains was digested with HindIII. A Synechococcus sp.

PCC 7002 gene specific probe for petJ1 was used to check the segregation of the petJ1::aphII and the wild-type petJ1 alleles. Lanes 1-3: genomic DNA isolated from the petJ1/petJ1::aphII merodiploid/pLAT3 6803 petJ strain of Synechococcus sp. PCC 7002 grown under the different conditions listed. Lane 4: genomic DNA isolated from the wild-type Synechococcus sp. PCC 7002 strain. Arrows point out Synechococcus sp. PCC

7002 petJ1-specific hybridization with the 32P-radiolabelled probe.

1234

250 µE m-2 s-1 + - + + 10 mM glycerol + + - -

petJ1::aphII (3.4 kb)

petJ1 (2.0 kb) 152

Figure 30. RNA blot hybridization analysis of the Synechocystis sp. PCC 6803 petE gene in the petJ1/petJ1::aphII merodiploid strain of Synechococcus sp. PCC 7002. Total

RNA was isolated from merodiploid and wild-type strains. The RNA was electrophoresed and transferred to a nylon membrane. (A) The blot was probed first with a 32P-labelled Synechocystis sp. PCC 6803 petE gene specific probe. (B) As a control, the membrane was stripped of radioactivity and then hybridized with a 32P-labelled

Synechococcus sp. PCC 7002 petJ1 gene specific probe. Sizes of the ribosomal RNA bands are shown on the right. 153 A. B.

petE petJ petE petJ

68036803 68036803

petJ1::aphII,petJ1::aphII, petJ1,wild-type petJ1, petJ1::aphII,petJ1::aphII, petJ1,wild-type petJ1, 2.8 2.3 1.5

0.5 154

Figure 31. RNA blot hybridization analysis of the Synechocystis sp. PCC 6803 petJ in the petJ1::aphII merodiploid strain of Synechococcus sp. PCC 7002. Total RNA was isolated from mutant and wild-type strains. The RNA was electrophoresed and transferred to a nylon membrane. (A) The blot was probed first with a 32P-labelled

Synechocystis sp. PCC 6803 petJ gene specific probe. (B) As a control, the membrane was stripped of radioactivity and then hybridized with a 32P-labelled Synechococcus sp.

PCC 7002 petJ1 gene specific probe. Sizes of the ribosomal RNA markers are shown on the right. 155 A. B.

petE petJ petE petJ

6803 6803 68036803

wild-type petJ1::aphII,petJ1::aphII, petJ1,wild-type petJ1, wild-type petJ1::aphII,petJ1::aphII, petJ1,wild-type petJ1,

2.8 2.3 1.5

0.5 156

Figure 32. Immunoblot analysis of the Synechocystis sp. PCC 6803 PetE

(plastocyanin) protein produced in the petJ1::aphII/petJ1 merodiploid strain of

Synechococcus sp. PCC 7002. Immunoblot analysis of crude protein extracts from various cyanobacterial strains was performed. 40 µg of total protein was isolated from whole cells of the following strains and electrophoresed on a SDS polyacrylamide gel:

Lane 1, wild-type Synechococcus sp. PCC 7002, Lane 2, wild-type Synechocystis sp.

PCC 6803, and Lane 3, Synechococcus sp. PCC 7002 petJ::aphII/petJ1, pLAT2-petEtrc

,. The crude extracts were subjected to SDS-PAGE and blotted onto a nitrocellulose membrane. The membrane was probed with polyclonal rabbit antiserum against

Synechocystis sp. PCC 6803 plastocyanin. Plastocyanin was detected in the

Synechococcus sp. PCC 7002 petJ1::aphII/petJ1/pLAT2-petEtrc merodiploid strain and in the whole-cell extracts from the Synechocystis sp. PCC 6803 wild-type strain.

123

PC 157

3.4.4 petJ-2

The petJ2 gene was identified on a 3.7-kb BamHI-HincII fragment downstream of the ccmK-1 gene, which encodes a protein involved in carbon uptake. Sequence analysis of the Synechococcus sp. PCC 7002 petJ2 predicts a mature protein of 88 amino acids and a pI of 9.29.

The Synechococcus sp. PCC 7002 PetJ2 amino acid sequence was subjected to analysis with the SignalP server (http://www.cbs.dtu.dk/services/SignalP/index.html) to determine if there were a predicted signal sequence (Nielsen et al., 1997). Analysis of the sequence using the algorithms for the prediction of gram-negative and gram-positive signal sequences was used to identify a potential signal sequence AFG-AD, where (-) is the predicted cleavage site. The potential signal peptide is 27 amino acids

(MNKRLVQVIVFVMIVLLLVPLLATPAFG) at the N-terminus of the predicted protein.

A ClustalW alignment of PetJ2 with cytochrome c6 proteins is shown in Figure

33. The PetJ2 protein from Synechococcus sp. PCC 7002 is 40% identical to and 55% similar to the PetJ1 of Synechococcus sp. PCC 7002, 37% identical and 51% similar to the PetJ protein from Synechocystis sp. PCC 6803 (accession number BAA17354)

(Kaneko et al., 1996), 47% identical and 59% similar to the PetJ protein from Anabaena sp. PCC 7120 (accession number I39601) (Ghassemian et al., 1994), 48% identical to and

63% similar to the PetJ1 protein from Synechococcus sp. PCC 7942 (accession number 158

Figure 33. ClustalW alignment of the Synechococcus sp. PCC 7002 PetJ2 amino acid sequence with PetJ amino acid sequences from other cyanobacteria. Gray high- lighted regions indicate identical amino acids while lighter shading indicates conserved amino acids. Dashes represent insertions/deletions included to maximize the sequence similarity. The consensus sequence is shown beneath all other sequences. Percent identity and percent conserved amino acids determined by the ClustalW alignment tool

(Thompson et al., 1994b).

10 20 30 40 50 7002 PetJ2 MNKR L VQV I VF VM I VL L L VPL L AT PAFGADLDQGAQ I F EAHC A 7002 PetJ1 MKKLLAI ALTVLATVFAFGTPAFAADAAAGAQVFAANCA 6803 PetJ MFKLFNQASRIFFGIALPCLIFLGGIFSLGNTALAADLAHGKAIFAGNCA 7120 PetJ MKKI FSLVLLGI ALFTFAFSSPALAADSVNGAKI FSANCA S. maxima PetJ GDVAAGASVFSANCA . . L . L . F . . PA AAD . A GA . I F . ANC A

60 70 80 90 100 7002 PetJ2 GCHLNGGNI VRRGKNLKKRAMAKNGYTS------VEAI ANLVTQGKGNM 7002 PetJ1 ACHAGGNNAVMPTKTLKADAL KTYL AGYKDGSKSL EEAVAYQVTNGQGAM 6803 PetJ ACHNGGLN A I N PSKT L KMADL EANGKNS ------VAA I VAQ I TN - GNGAM 7120 PetJ SCHAGGKN L VQAQKT L KKADL EKYGMYS ------AEA I I AQVTNGKNAM S. maxima PetJ ACHMGGRNVI VANKTLSKSDLAKYLKGFDD- - -DAVAAVAYQVTNGKNAM ACH GG N . V KTLKK. DL KYG . S EAVA QVTNGKGAM 110 120 130 140 150 7002 PetJ2 SAYGDKL SSEQ I QAVSQYVLQQSQTD -WKS 7002 PetJ1 PAFGGRLSDADI ANVAAYI ADQAENNKW 6803 PetJ PGFKGRI SDSDMEDVAAYVLDQAEKG-W 7120 PetJ PAFKGRLKPEQI EDVAAYVLGKADAD-WK S. maxima PetJ PGFNGRLSPKQI EDVAAYVVDQAEKG-W PAF. GRLS QI EDVAAYVLDQAE . W 159

P25935) (Laudenbach et al., 1990), and is 46% identical and 59% similar to the PetJ protein from Anabaena sp. PCC 7937 (accession number P28597) (Bovy et al., 1992).

3.4.5 Interposon mutagenesis of the petJ2 gene in Synechococcus sp. PCC

7002

The petJ2 gene was interrupted by the insertion of a BamHI fragment containing the aphII gene, which confers kanamycin resistance, into a unique BglII site within the coding sequence of the petJ2 open reading frame. The segregation of the petJ2::aphII and petJ2 alleles was checked by PCR analysis (Figure 34). A 0.4-kb PCR, which corresponds to the wild-type copy of petJ2, product was produced when wild-type

Synechococcus sp. PCC 7002 chromosomal DNA was used as the template. In strains transformed with the pPETJ2 plasmid, a PCR product of 1.7-kb is produced that corresponds to the size expected for the insertionally inactivated petJ2::aphII allele and no product from the wild-type copy of the petJ2 gene is observed (Figure 34). This analysis reveals that the wild-type copy was fully replaced by the petJ2::aphII mutant allele. Therefore, the petJ2 gene does not have an essential function in electron transport under normal, photoautotrophic growth conditions in Synechococcus sp. PCC 7002. 160

Figure 34. Interposon mutagenesis of the petJ2 gene. (A) Physical map of the

Synechococcus sp. PCC 7002 petJ2 gene. The petJ2 gene was interrupted by inserting a

BamHI fragment containing the aphII gene into a unique BglII site within the coding sequence of petJ2. The plasmid pPETJ2 was used to transform the wild-type strain of

Synechococcus sp. PCC 7002. (B) PCR analysis of the petJ2 locus. Primers 5C62 (5’-

TCC CTG CCC GAA TTA CCG A-3’) and 3C62 (5’-TTA AGA TTT CCA GTC GGT

TTG GGA TTG C-3’) were used to amplify the petJ2 gene from genomic DNA isolated from wild-type and kanamycin-resistant strains of Synechococcus sp. PCC 7002. The position of the primers is indicated in (A). Two PCR products were produced, a 0.4 kb product corresponding to the wild-type petJ2 allele and a 1.7 kb product corresponding to the petJ2::aphII allele. 161

A. B.

100 bp

cII

II Hin

Bgl λ Ssp wild type petJ2::aphII HE

petJ-2

Bcl Pst

Sph Bgl

aphII 1.7 kb

cII

Ssp Hin

0.4 kb petJ-2 162

3.4.6 Growth analysis of petJ2::aphII

The doubling time of the petJ2::aphII strain of Synechococcus sp. PCC 7002 under standard growth conditions was 4.0 ± 0.6 hours, which is roughly the same doubling time as for the wild-type strain. In the related species, Synechococcus sp. PCC

7942, a deletion in the petE gene exacerbates a chilling effect on photoinhibition (Clarke and Campbell, 1996). By comparing the growth rates of wild-type and mutant strains of

Synechococcus sp. PCC 7002 when exposed to chilling stress, it was hoped that any exacerbations of photoinhibition in mutant strains would translate to a growth-limiting phenotype. Therefore, attempts were made to characterize the mutant strain by exposing the strain to a temperature-shift from 38°C to 22°C. However, the results of the temperature-shift from 38°C to 22°C (Figure 35) reveal that there is no significant difference between the growth rates after a temperature-shift from 38°C to 22°C of the wild-type strain and petJ2::aphII strain of Synechococcus sp. PCC 7002. 163

Figure 35. Temperature-shift effects on wild-type and petJ2::aphII strains of

Synechococcus sp. PCC 7002. Exponentially growing cells were either maintained at

38°C or transferred to a 22°C water bath. (A) Temperature-shift effects on the

petJ2::aphII strain of Synechococcus sp. PCC 7002. The petJ2::aphII cells were grown

at 38°C () and at 22°C (). (B) Temperature-shift effects on the wild-type strain of

Synechococcus sp. PCC 7002. Wild-type cells grown at 38°C () and at 22°C (). The data shown are from a single experiment, which was repeated three times, and the results were consistent and reproducible. 164 A

10 ∆petJ2 (38ûC) ∆petJ2 (22ûC) 1 550

OD 0.1

0.01 0 1020304050 B Time (hours)

10 WT(38ûC) WT (22ûC) 1 550

OD 0.1

0.01 0 1020304050 Time (hours) 165

3.4.7 cytM

The cytM gene, encoding cytochrome cM was originally identified in

Synechocystis sp. PCC 6803 by Malakhov et al. (1994). The function of this cytochrome

is unknown, but it has been proposed to be a substitute for PetE or PetJ in cells grown

under high-light or chilling-stress conditions in Synechocystis sp. PCC 6803 (Malakhov

et al., 1999). The cytM gene from Synechococcus sp. PCC 7002 was identified on contig

638 from the ongoing Synechococcus sp. PCC 7002 genome project. A comparison of

the gene organization around the cytM genes from Synechococcus sp. PCC 7002 and

Synechocystis sp. PCC 6803 is shown in Figure 36. The genes flanking the cytM genes are very different between Synechococcus sp. PCC 7002 and Synechocystis sp. PCC

6803, and the surrounding genes do not give any hints as to the function of the cytM gene

or its gene product. A ClustalW alignment of cytochrome cM proteins is shown in Figure

37. The results reveal that the CytM proteins have a high degree of sequence similarity

among cyanobacterial species.

The Synechococcus sp. PCC 7002 CytM amino acid sequence was subjected to

analysis with the SignalP server (http://www.cbs.dtu.dk/services/SignalP/index.html) to

determine if there were a predicted signal sequence (Nielsen et al., 1997). Analysis of the

sequence using the algorithms for the prediction of gram-negative and gram-positive

signal sequences was used to identify a potential signal sequence TQA-SD, where (-) is

the cleavage site. The potential signal peptide is 38 amino acids 166

Figure 36. Gene organization around the cytM gene of Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803. Arrows represent direction of transcription and size of genes. Maps are individually scaled and numbers below each represent bp. Gene names and predicted product names are indicated above genes (arrows). Restriction sites are as indicated on maps. 167

Synechococcus sp. PCC 7002 cytM restriction map

PvuI

StyI PvuI slr1215 BglII

PvuI StyI sll1434-penicillin binding pr… cytM sll1704 csgA (cell-cell signaling factor C)

300 600 900 1200 1500 1800 2100 2400 2700

NcoI PvuI DraI StyIStyI PvuI StyI XbaIPvuI StyI

PvuI PvuI StyI

PvuI

Synechococcus sp. PCC 6803 cytM restriction map

EcoRV

StuI AvaI StyI DraI cytM HincIIPvuI StyI StyI

SspI StyI DraI NcoI rpl9 (50S ribosomal protein L9) slr1351 (murF) NcoI slr1353

300 600 900 1200 1500 1800 2100 2400 2700 3000 3300 3600

BglII PvuI PvuI PvuI StyI NcoIStyI KpnI DraI NcoI PvuI PvuI StyI

PvuI StyI StyI EcoRV PvuI 168

Figure 37. ClustalW alignment of CytM proteins from cyanobacteria. Gray high- lighted regions indicate identical amino acids while lighter shading indicates conserved amino acids. Dashes represent insertions/deletions included to maximize the sequence similarity. The alignment of the CytM proteins was determined by the ClustalW alignment tool (Thompson et al., 1994b).

CytM ClustalW Amino Acid Alignment

10 20 30 40 50 7002 CytM VANSSELSVPNFSKLLFVI I LVLAI AALG A. variabilis CytM MDNQI TKPEI LI QRI ALVALVI LLAI PLG 7942 CytM MI I LRHVLQTTDSLSPAI ASAVEQSLSKSI ESRAAQTLGWLLATAVMVAI 6803 CytM MAPVI EKSPTVATVNASPTGI WI MAGI VSLVI L ...S ......

60 70 80 90 100 7002 CytM I FGVYSTQASDPYI QQVLALQGDELRGNAI FQI NCAGCHGPQADGNVGPS A. variabilis CytM FFGVQLVKASDPYVKSVLAMKGDPI QGHAI FQI NCAGCHGLEADGRVGPS 7942 CytM GL VVT L I RPADPYVST VL NL PGNAERGQAI F QI NCAGCHGPEGRGL VGPD 6803 CytM AVALFSFMNFDPYVSQVLALKGDADRGRAI FQANCAVCHGI QADGYI GPS . . V DPYV VL AL GD RG AI FQI NCAGCHG ADG VGPS

110 120 130 140 150 7002 CytM LRAVAQRKSDVRLI QQVI SGKTPPMPKFQPAPQEMADLLSYLRTLN A. variabilis CytM LQAVSKRKSKYGLI HQVI SGDTPPMPKFQPNTQEMADLLSFLETL 7942 CytM LANVSNRKSRKDLI RQVTTGETPPMPKFQPSPETMADLLRYLETL 6803 CytM LWGVSQRRSQSHI I HQVVSGQTPPMPQFEPNPQEMADLLNYLKTLN L . VS. RKS LI . QV. SG TPPMPKFQP PQEMADLL YL TL 169

(VANSSELSVPNFSKLLFVIILVLAIAALGIFGVYSTQA) at the N-terminus of the predicted protein. Interestingly, neither the CytM protein from Synechocystis sp. PCC

6803 nor the CytM protein from Synechococcus sp. PCC 7942 are predicted to have a signal sequence. However, analysis of the CytM protein from A. variabilis predicts that the protein is processed at the VKA-SD motif, where (-) represents the processing site.

3.4.8 bcpA

The bcpA gene was identified as an open reading frame on a 2.5-kb EcoRI fragment in close proximity to the glnB gene. Sequence analysis of the Synechococcus sp. PCC 7002 bcpA gene predicts a protein of 106 amino acids with a pI of 6.06. A

ClustalW alignment of BcpA with the Anabaena sp. PCC 7120 BcpA sequence is shown in Figure 38A. The predicted BcpA protein from Synechococcus sp. PCC 7002 is 62% identical and 75% similar to the BcpA protein from Anabaena sp. PCC 7120 (Kazusa

DNA Research Institute, 2000). Figure 38B shows a ClustalW alignment of cyanobacterial plastocyanin proteins with the BcpA proteins. The BcpA protein from

Synechococcus sp. PCC 7002 has low similarity to the plastocyanins from cyanobacteria, with the most significant similarity occurring near the putative Cu-binding ligands in the carboxy-terminus.

In order to assess whether the bcpA gene was being transcribed, RNA blot hybridization was performed using RNA isolated from wild-type Synechococcus sp. PCC

7002 grown under standard conditions with petJ1, petJ2, and bcpA gene specific probes 170

Figure 38. (A) BcpA ClustalW alignment. The predicted BcpA amino acid sequences of Synechococcus sp. PCC 7002 and Anabaena sp. PCC 7120 were aligned using the ClustalW alignment program from MacVector v. 6.5. The consensus amino acid sequence is located underneath. Gray high-lighted regions are identical amino acids and lighter shading indicates a conserved amino acid. A dash is equivalent to a gap in the sequence. Percent identity and percent conserved amino acids was determined by the

ClustalW program (Thompson et al., 1994a) and is shown in Table 4.

(B) BcpA/PetE ClustalW amino acid alignment. PetE and BcpA proteins were aligned using the ClustalW alignment program from Mac Vector v. 6.5. The consensus amino acid sequence is located underneath. Gray high-lighted regions are identical amino acids and lighter shading indicates a conserved amino acid. A dash is equivalent to a gap in the sequence. 171

A. BcpA ClustalW Amino Acid Alignments

10 20 30 40 50 7002-BcpA MVFHKYFI HGCRAL VL LGLI WFCL TPAAI ALPVPAQQP------LQEMQ 7120-BcpA MI SL LSPI VRQI CVVFTLLL CFTFTNTNSVLAAKESSDLLKQPVSEI T .R.... CT .A .E

60 70 80 90 100 7002-BcpA I HL GTTSGALRFVPDQLEFVAGQRYKL LLDNPSNQKHYFTAKDFADTSWT 7120-BcpA VSL GNSANELKFEPNNLEL VAGKRYLL HLNNPSQLKHYFTAKDFADGI WT . L G . L. F P . LE VAG RY L L NPS. KHYFTAKDFAD WT

110 120 130 140 150 7002-BcpA QKVEAGQGGSKRGDPRTRTQARAI AEWI LI P- QKTGKFEL HCSVPGHAAA 7120-BcpA QKVEAGKVEI KGAI HELEL KPGAEAEWVLVAI K- PGKYGLRCPI PGHTEA QKVEAG. K. AAEW.L. GK. L.C.PGH A

160 170 180 190 200 7002-BcpA GMVGT I QVI ADS 7120-BcpA GMTGEI VI NP GM G I .

B. BcpA/PetE ClustalW Amino Acid Alignments

10 20 30 40 50 7002-BcpA MVFHKYFIHGCRALVLLGLIWFCLTPAAIALPVPAQQP------LQEMQ 7120-BcpA1 MI SLL SPI VRQI CVVFTL LLCFTFTNTNSVLAAKESSDL LKQPVSEI T PetE-6803 MSKKFLTILAGLLLVVSSFFLSVSPAAAAN------AT PetE-7942 MKVLASFARRLSLFAVAAVLCVGSFFLSAAPASAQT------VA PetE-7120 MKL I AASL RRL SL AVL TVLLVVSSFAVFTPSASAET------YT PetE-Spinacia oleracea VE .R .....L.. . A .

60 70 80 90 100 7002-BcpA I HLGTTSGALRFVPDQLEFVAGQRYKLLLDNPSNQKHYFTAKDFADTSWT 7120-BcpA1 VSLGNSANEL KFEPNNLELVAGKRYL LHLNNPSQLKHYFTAKDFADGI WT PetE-6803 VKMGSDSGALVFEPSTVTI KAGE- EVKWVNN- KL SPHNI VFAADG- - - - - PetE-7942 I KMGADNGMLAFEPSTI EI QAGD- TVQWVNN- KLAPHNVVVEGQ------PetE-7120 VKLGSDKGLLVFEPAKLTI KPGD- TVEFLNN- KVPPHNVVFDAALN- - - - PetE-Spinacia oleracea VL LGGDDGSLAFL PGDFSVASGE- EI VFKNN- AGFPHNVVFDEDEI - - - - V.LGDGL.FEP . . AG. . ..NN . PHN.V .

110 120 130 140 150 7002-BcpA QKVEAGQGGSKRGDPRTRTQARAI AEWI LI P- QKTGKFELHCSVPGHAAA 7120-BcpA1 QKVEAGKVEI KGAI HELELKPGAEAEWVLVAI K- PGKYGLRCPI PGHTEA PetE-6803 - - - VDADTAAKLSHKGLAFAAGESFTSTFTE- - - PGTYTYYCEP- - HRGA PetE-7942 ------PELSHKDLAFSPGETFEATFSE---PGTYTYYCEP--HRGA PetE-7120 - PAKSADLAKSLSHKQLL MSPGQSTSTTFPADAPAGEYTFYCEP- - HRGA PetE-Spinacia oleracea - PSGVDAAKI SMSEEDLLNAPGETYKVTLTE- - - KGTYKFYCSP- - HQGA .. .S.L.PG. T GY.YCP HGA

160 170 180 190 200 7002-BcpA GMVGT I QVI A DS 7120-BcpA1 GMTGEI VI NP PetE-6803 GMVGKVVVE PetE-7942 GMVGKI VVQ PetE-7120 GMVGKI TVAG PetE-Spinacia oleracea GMVGK VT VN GMVGK I V 172

(Figure 39). Results reveal that the petJ1 gene transcript is present at the highest levels, petJ2 has a very low level of transcript, and the bcpA transcript is present at an intermediate level compared to petJ1 and petJ2. Although bcpA is expressed in

Synechococcus sp. PCC 7002 (Figure 39), it is clear that the bcpA gene product cannot functionally substitute for PetJ1 under normal growth conditions since attempts to segregate the petJ1 and petJ1::aphII alleles were not successful. It is not surprising that petJ2 also cannot functionally substitute for PetJ1 based its low transcript levels in

Synechococcus sp. PCC 7002 cells grown under normal conditions.

3.4.9 Interposon mutagenesis of the bcpA gene from Synechococcus

sp. PCC 7002

The bcpA gene was interrupted by the insertion of a 1.3-kb SmaI fragment containing the aphII gene into a unique Klenow filled-in NcoI site within the coding sequence of the bcpA gene. The resulting plasmid, pBCPA was used to transform the wild-type strain of Synechococcus sp. PCC 7002 (Figure 40A). Segregation of the bcpA allele was verified by PCR analysis (Figure 40B). This result indicates that the bcpA::aphII locus has been fully segregated and that the bcpA gene is not essential for the viability of Synechococcus sp. PCC 7002 under the conditions tested. 173

Figure 39. RNA transcript analysis of petJ1, petJ2, and bcpA. Total RNA was isolated from wild-type Synechococcus sp. PCC 7002. Total RNA (20 µg) was loaded per lane in a formaldehyde-agarose gel and electrophoresed as described in Materials and Methods, transferred to a nylon membrane and probed with either a petJ1-specific, petJ2-specific, or bcpA-specific 32P-labelled probe. Results shown are for three, identically treated, separately probed membranes. Ribosomal RNA marker sizes (kb) are indicated at right.

petJ1 petJ2 bcpA

2.8 2.3 1.5

0.5 174

Figure 40. Interposon mutagenesis of the Synechococcus sp. PCC 7002 bcpA gene. (A)

Physical map of the bcpA gene. The bcpA gene was interrupted by insertion of a 1.3-kb

SmaI fragment containing the aphII gene into a Klenow-treated, blunt NcoI site within the bcpA coding sequence. Primers (5BcpA 5’-ATG GAG AAA ATC CAT GGT ATT

TCA TAA ATA-3’ and 3BcpA 5’-TTG AGG GAG CTT AGC TTA CGA ATC AGC

AAT-3’) were used to amplify the bcpA gene for PCR analysis. Primer positions are indicated below the bcpA gene. (B) PCR analysis of the bcpA locus. PCR products are as indicated on gel. The sizes of the PCR products are given on the left and right of the figure. 175

I I

cII

Bam

Eco

Hin Nco Nco

bcpA

aphII

HE λ wild type bcpA::aphIIbcpA::aphII

1.8 kbp

464 bp 176

3.4.10 Growth rate analysis of bcpA- strain of Synechococcus sp.

PCC 7002

Under normal growth conditions, the bcpA- strain of Synechococcus sp. PCC 7002

has a doubling time of 4.0 ± 0.8 hours, which is nearly identical to that of the wild-type.

Therefore, interruption of the bcpA locus has no effect on the growth of Synechococcus

sp. PCC 7002 under standard conditions. In a previous study by (Clarke and Campbell,

1996), it was shown that inactivation of the petE gene in Synechococcus sp. PCC 7942

experienced photoinhibition at low temperatures. In order to determine whether or not

inactivation of bcpA would have a similar effect in Synechococcus sp. PCC 7002, growth of the wild-type and bcpA- strains of Synechococcus sp. PCC 7002 was monitored

following a temperature shift-down. Figure 41 shows the temperature-shift analysis of

the wild-type and bcpA- strains of Synechococcus sp. PCC 7002. These results indicate

that there is no difference in growth rate before and after a temperature-shift from 38°C

to 22°C in either the bcpA- or wild-type strains of Synechococcus sp. PCC 7002.

Therefore, the bcpA locus plays no role in low-temperature adaptation for Synechococcus sp. PCC 7002.

3.4.11 Overproduction of the BcpA protein in E. coli.

The bcpA gene of Synechococcus sp. PCC 7002 predicts a novel blue-copper protein that could have unique structural and biochemical traits. To investigate these 177

Figure 41. Temperature-shift effects on wild-type and bcpA::aphII strains of

Synechococcus sp. 7002. Exponentially growing cells were either maintained at 38°C or

transferred to a 22°C water bath. The bcpA::aphII cells were grown at 38°C () and

22°C () . (B) Temperature-shift effects on the wild-type strain of Synechococcus sp.

PCC 7002. Wild-type cells grown at 38°C () and 22°C (). The data shown are from

a single experiment, which was repeated three times, and the results were consistent and

reproducible. 178 A 10 ∆bcpA (38ûC) ∆bcpA (22ûC) 1 550

OD 0.1

0.01 0 1020304050 Time (hours) B 10 WT(38ûC) WT (22ûC) 1 550

OD 0.1

0.01 0 1020304050 Time (hours) 179

possibilities, two plasmids were made to overproduce the recombinant BcpA protein.

The first rBcpA plasmid was made using the following primers to amplify the bcpA gene

from Synechococcus sp. PCC 7002: BcpNcoI5’: 5’-CCC AGC GGC CAT GGC CTT

ACC CGT-3’ and BcpBlpI3': 5’- TTG AGG GAG CTT AGC TTA CGA ATC AGC

AAT –3’. The restriction sites for NcoI in the BcpNcoI5’ primer and BlpI for the

BcpBlpI3’ primer are underlined. These primers were used to amplify a PCR product

that eliminates the first 30 amino acids of the protein that encode a potential prokaryotic

signal sequence according to the rules of von Heijne (von Heijne, 1986). The PCR

product was digested with NcoI and BlpI and inserted into the pET16 vector for protein overproduction.

The following primers were used to amplify the bcpA gene from Synechococcus sp. PCC 7002: BcpNde5': 5'-CCT GCC CAA CAG CAT ATG CAG GAA ATG CAG-3'

(NdeI restriction site underlined) and the previously defined BcpBlpI3' primer for the

second rBcpA plasmid. The BcpNde5' primer was designed to truncate the amino

terminus of the BcpA at amino acid 39, and the primer also changed the leucine at

position 39 to a methionine. This portion of the protein was eliminated since the

remaining nine N-terminal amino acids were hydrophobic and possibly causing the

misfolding observed from the recombinant protein generated by the first plasmid. The

primers were used to amplify a product encoding a truncated bcpA gene, which was

digested with NdeI and BlpI. This DNA fragment was cloned into the NdeI and BlpI site

of the E. coli expression vector pET30C+. Overproduction of the protein led to the

formation of inclusion bodies. The rBcpA inclusion bodies were purified as described in 180 the Materials and Methods section. After purification, the proteins were checked by

SDS-PAGE (Figure 42). Attempts to refold the recombinant protein isolated from inclusion bodies produced by either pET plasmid were unsuccessful under a number of conditions (Table 7).

3.4.12 Immunoblot analysis of rBcpA

The rBcpA protein was purified as described in Materials and Methods and was sent to the antibody facility at The Pennsylvania State University for the production of rBcpA antisera. The anti-rBcpA antisera from two rabbits were assayed by immunoblot analysis (Figure 43). Results indicate that the antisera cross-reacts with the rBcpA protein and with a protein of similar size in the soluble extract from wild-type

Synechococcus sp. PCC 7002. The cross-reacting protein from Synechococcus sp. PCC

7002 is present in very low quantities and may represent the native BcpA protein. The

BcpA immunoblot results along with the result of the RNA blot analysis suggest that the bcpA gene is transcribed and translated at low levels in wild-type Synechococcus sp. PCC

7002 grown under standard growth conditions. 181

Figure 42. Overproduction of the rBcpA protein. The plasmid pET30C+BCP was

used to transform E. coli BLR. Four transformants were picked to start cultures in 5 ml

LB kanamycin. These cultures were grown overnight and three cultures (15 ml) was

transferred to a flask containing LB medium (1L) containing 30 µg ml-1 kanamycin and

grown for 3 hours at 37°C and BcpA expression was induced by addition of 0.5 mM

IPTG at 3 hours and the cells were grown for another 3 hours at which time they were

harvested in 250 ml centrifuge bottles in a GSA rotor (4100×g) for 10 min at 4°C. The cell pellets were washed with 50 mM Tris, pH 8.0 and re-centrifuged at 5000×g in a total volume of 25 ml. The pellet was collected and resuspended in 25 ml 50 mM Tris, pH

8.0. These cells were broken by passage through an SLM-AMINCO French Pressure cell as described in the Materials and Methods. The eluent was centrifuged at 3000×g for 10 min and an off-white pellet was isolated from the supernatant. The inclusion body pellet was washed with 20 ml of 50 mM Tris, pH 8.0 and the soluble fraction was saved. The inclusion body pellet was resuspended in 5 ml of 50 mM Tris, pH 8.0 and 25 µl was added to 25 µl of 4×SDS loading buffer. The same volume was used for the soluble fraction. Samples were heated to 70°C for 5 min and electrophoresed on an SDS- polyacrylamide. Lanes 1 and 2: 5 µl of resuspended BCP inclusion body pellet. Lane 3:

5 µl of soluble E. coli BLR pET30C+BCP fraction. Lane 4: low MW ladder from

BioRAD. Lanes 5 and 6: 10 µl of resuspended BCP inclusion body pellet. Lanes 7 and

8: 10 µl of soluble E. coli BLR pET30C+BCP fraction. The rBcpA protein is indicated with an arrow. 182

kDa 12 345678 107

76 52

36.8

27.2 19 13 183

Table 7. Protein refolding conditions for rBcpA. Attempts to refold the recombinant

BcpA proteins were made in the following buffers: 50 mM Tris, pH 8.0; 50 mM Tris, pH

8.0, 100 mM NaCl; 50 mM Tris, pH 8.0, 150 mM NaCl, 50 mM MES, pH 6.0; 50 mM

MES, pH 6.0, 100 mM NaCl; and 5 mM phosphate, pH 7.0, 2 mM EDTA. Attempts were done under the conditions described in the table. Fast dilution indicates that the chaotrope-solubilized protein was diluted with buffer rapidly (50:1-100:1 (v/v)) and then concentrated with an Amicon filter. Slow dilution indicates that the chaotrope- solubilized protein was diluted slowly with buffer (0.1 ml h-1). Dialysis was carried out overnight against 4000-fold dilution of buffer.

Chaotrope dilution method aerobic copper result

urea fast dilution yes yes precipitate urea fast dilution yes no precipitate urea fast dilution no yes precipitate urea fast dilution no no precipitate urea dialysis yes yes precipitate urea dialysis yes no precipitate urea dialysis no yes precipitate urea dialysis no no precipitate urea slow dilution yes yes precipitate urea slow dilution yes no precipitate guanidine-HCl fast dilution yes yes precipitate guanidine-HCl fast dilution yes no precipitate guanidine-HCl fast dilution no yes precipitate guanidine-HCl fast dilution no no precipitate guanidine-HCl dialysis yes yes precipitate guanidine-HCl dialysis yes no precipitate guanidine-HCl slow dilution yes yes precipitate guanidine-HCl slow dilution yes no precipitate 184

Figure 43. Immunoblot analysis of using rBcpA antisera. Immunoblot analysis was performed as described in Materials and Methods. rBcpA antisera from two rabbits (E and F) were used for the experiment. The primary antibodies were diluted as described above each blot. All gels were loaded in the same manner as follows: lane 1: 100 ng of rBcpA, lane 2: 50 ng of rBcpA, lane 3: 100 µg protein from the membrane fraction of

Synechococcus sp. PCC 7002, lane 4: 100 µg protein from the soluble fraction of

Synechococcus sp. PCC 7002. The cross-hybridizing signal from rBcpA/BcpA is indicated at right by the arrow (12.6 kDa). 185

Antisera from Rabbit F

1:1000 1:10,000

1234 12 34

12.6 kDa

Antisera from Rabbit E

1:1000 1:10,000

12 3 4 12 3 4

12.6 kDa 186

3.5 Heme-Copper Oxidases in Synechococcus sp. PCC 7002

Two separate gene clusters encoding putative heme-copper oxidases in

Synechococcus sp. PCC 7002 were identified in this study. One apparent operon

(ctaCIDIEI) represents the primary heme-copper cytochrome oxidase. The second apparent operon (ctaCIIDIIEII) represents a secondary heme-copper quinol oxidase.

Unlike Synechocystis sp. PCC 6803, no evidence for the occurrence of a cydAB-type quinol oxidase in Synechococcus sp. PCC 7002 was obtained.

3.5.1 Screening and Cloning of the ctaI and ctaII gene clusters from

Synechococcus sp. PCC 7002

A BamHI fragment of 4.5-kb was isolated from a partial genomic library of

Synechococcus sp. PCC 7002 DNA by cross-hybridization with a ctaDI PCR probe amplified from Synechocystis sp. PCC 6803 genomic DNA. This clone contained the sequence of the ctaDI gene. The Synechococcus sp. PCC 7002 ctaDII gene cluster was found by screening a cosmid genomic library with a PCR product probe derived from the slr2082 (Kaneko et al., 1996) open reading frame encoding the ctaDII gene in

Synechocystis sp. PCC 6803. The cosmid 2B1 was identified as a positive clone and a

3.5-kb HincII fragment was subcloned. Nucleotide sequence analysis of this 3.5-kb

HincII fragment revealed two open reading frames with homology to ctaCII and ctaDII from Synechocystis sp. PCC 6803 as well as the open reading frames sll1485 and sll1486. 187

Primers were designed to sequence cosmid 2B1 outside of the 3.5-kb HincII fragment. Sequencing downstream from ctaDII, it was possible to identify and sequence the ctaEII gene. Primers were also made to generate a PCR product corresponding to the

Synechocystis sp. PCC 6803 cydA gene, which encodes one subunit of the cytochrome bd-quinol oxidase, and the resulting PCR product was used to screen the Synechococcus sp. PCC 7002 genomic library for the presence of a quinol oxidase. No cross-hybridizing

DNA fragments corresponding to the cydA gene from Synechocystis sp. PCC 6803 were found in Synechococcus sp. PCC 7002 genomic DNA Southern blot hybridizations.

Moreover, no genes with similarity to the cydABDC genes have been found in the genome sequencing project from Synechococcus sp. PCC 7002. These results indicate that Synechococcus sp. PCC 7002 has two terminal oxidase operons, ctaCIDIEI and ctaCIIDIIEII, but does not have appear to an operon encoding a CydAB-like quinol oxidase.

A comparison of the gene organization for the ctaCIDIEI operon from

Synechococcus sp. PCC 7002 with the ctaCIDIEI operons from Synechocystis sp. PCC

6803 and Anabaena sp. PCC 7120 is shown in Figure 44. These data show that the gene organization for the ctaCIDIEI operon is identical for all three cyanobacterial strains.

Figure 45 shows a comparison of the gene organization of the ctaCIIDIIEII gene clusters from Synechococcus sp. PCC 7002, Synechocystis sp. PCC 6803, and Anabaena sp. PCC

7120. This figure reveals that the organization of the ctaCIIDIIEII gene cluster is differs for the three cyanobacterial strains. In Synechococcus sp. PCC 7002, the ctaCIIDIIEII genes occur in a single operon. However, arrangements of the ctaCII, ctaDII, and ctaEII 188

Figure 44. Gene organization of the ctaCIDIEI genes from cyanobacteria. The gene organization of the ctaCIDIEI operons of Synechococcus sp. PCC 7002, Synechocystis sp. PCC 6803, and Anabaena sp. PCC 7120 are depicted in this figure. Maps are individually scaled and numbers below each represent bp. Gene names and predicted product names are indicated above genes (arrows).

Cyanobacterial ctaI gene organization

Synechococcus sp. PCC7002 sll1898 ctaCI ctaDI ctaEI slr1542

700 1400 2100 2800 3500 4200 4900

Synechocystis sp. PCC6803

trxA (slr1139)

sll1084 ctaCI ctaDI ctaEI slr1140 nrtD (sll1082)

600 1200 1800 2400 3000 3600 4200 4800 5400 6000 6600 7200 7800 8400

Anabaena sp. PCC7120

ctaCI ctaDI ctaEI ORF8

400 800 1200 1600 2000 2400 2800 3200 3600 4000 4400 4800 189

Figure 45. Gene organization of the ctaCIIDIIEII genes from cyanobacteria. The gene organization of the ctaCIIDIIEII apparent operon of Synechococcus sp. PCC 7002, and the organization of the ctaCIIDIIEII genes from Synechocystis sp. PCC 6803, and

Anabaena sp. PCC 7120 are depicted in this figure. Maps are individually scaled and numbers below each represent bp. Gene names and predicted product names are indicated above genes (arrows).

Gene organization of cyanobacterial ctaII gene clusters

Synechococcus sp. PCC7002

sll1485 sll1486 ctaCII ctaDII ctaEII sldI

500 1000 1500 2000 2500 3000 3500 4000 4500 5000

Synechocystis sp. PCC6803

ssr3532 sll0236 slr2079 slr2080 tyrA ctaDII ctaEII sll1988 sll1987 (katG)

500 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 6000 6500 7000 7500 8000 8500 9000 9500 10000 10500

slr1080 sll0818 ssl1520 sll0817 sll0816 (trpX) sll0815 sll0814 sll0813 (ctaCII) slr0812

500 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 6000 6500 7000 7500

Anabaena sp. PCC7120 uvrC (sll0865) ORF sll1485 sll1486 ctaCII ctaDII

500 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 6000

ptxB rpsD ctaEII

500 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 6000 190

genes are different in the other cyanobacteria. The ctaDII and ctaEII are clustered together in Synechocystis sp. PCC 6803, whereas the ctaCII gene is located elsewhere in the genome of this organism. Preliminary analysis of the nucleotide sequence of contigs from the Anabaena sp. PCC 7120 genome sequencing project (Kazusa DNA Research

Institute, 2000) reveals that the ctaCII and ctaDII genes are clustered together, whereas the ctaEII gene is found elsewhere in the genome in Anabaena sp. PCC 7120.

3.5.2 Insertional Mutagenesis of ctaDI and ctaDII from

Synechococcus sp. PCC 7002

To characterize the role of the two cytochrome oxidases in electron transport pathways, interposon mutagenesis of the ctaDI and ctaDII genes was performed. A unique BglII site in ctaDI was used to introduce a 1.3-kb BamHI fragment containing the aphII gene, which confers kanamycin resistance. Two plasmids were made with the aphII gene interrupting ctaDI. The first plasmid, pctaDI 1.1 had ctaDI interrupted with the aphII gene in the same transcriptional orientation. The second plasmid, pctaDI 2.2 had the ctaDI interrupted with aphII in the opposite transcriptional orientation (Figure

46). Both pctaDI 1.1 and pctaDI 2.2 were independently used to transform wild-type

Synechococcus sp. PCC 7002 cells and ctaDII- strains (see below). Figure 46 shows that

all transformants were homozygous for the ctaDI::aphII locus. The ctaDII gene was

interrupted by insertion of a 2-kb SmaI fragment that had the aadA gene which confers

spectinomycin resistance (Prentki and Krisch, 1984) into a unique EcoRV site within the 191

Figure 46. Interposon mutagenesis of the ctaDI gene from Synechococcus sp. PCC

7002. (A) Physical map of the 4.5-kb BamHI fragment encoding the ctaCIDIEI gene cluster of Synechococcus sp. strain PCC 7002 and disruption of the ctaDI locus by interposon mutagenesis. Arrows indicate the direction of transcription and size of the gene. The aphII gene, which encodes aminoglycoside 3'-phosphotransferase II and confers kanamycin resistance, was inserted into a BglII site within the coding region of ctaDI in both orientations to create the plasmids pctaDI 1.1 and pctaDI 2.2. These plasmids were used to transform the wild-type and ctaDII- strains of Synechococcus sp.

PCC 7002. (B) PCR analysis of the ctaDI locus. PCR analysis was performed on genomic DNA isolated from the wild-type and mutant strains of Synechococcus sp. PCC

7002 using the primers ctaDI.I1 and ctaDI.I2 as indicated in (A). The sizes of the PCR products are indicated on the left. The 1.3-kb increase in size of the PCR product in the insertion mutants corresponds to the size of the aphII insert. Lane 1 represents the 2.0-kb wild-type ctaDI PCR product. Lane 2: Total DNA isolated from a kanamycin resistant strain that had been transformed with pctaDI 1.1 was used as the template for PCR. The

3.3-kb ctaDI::aphII PCR product and no wild-type product are indicative of successful segregation of the mutant and wild-type alleles. Lane 3: As lane 2 except that the wild- type strain was originally transformed with pctaDI 2.2. Lanes 4 and 5: Total DNA isolated from a kanamycin-spectinomycin strain had been transformed with either pctaDI

1.1 or pctaDI 2.2 were used as templates for PCR. This was done to check for reversion to a wild-type ctaDI after transformation with the pctaDII Ω plasmid (Figure 48). The results indicate that the double-mutant strains are still homozygous for ctaDI::aphII. 192

A. 500 bp

cII cII

cII

Bam

Bgl

Stu Stu Hin Hin

Hin Bam Eco slr1999 ctaCI ctaDI ctaEI sll1898 ctaDI.I1 ctaDI.I2 slr1542

aphII 1.1

Pst I

I Pst aphII 2.2

B. Ω Ω

ctaDI(1.1)::aphIIctaDI(2.2)::aphII wild type ctaDI(1.1)::aphIIctaDI(2.2)::aphIIctaDII:: ctaDII::

3.3 kb 2.0 kb 193

ctaDII coding sequence. The resulting plasmid, pctaDII Ω was used to transform

wild-type Synechococcus sp. PCC 7002 as well as the ctaDI- strains of Synechococcus sp.

PCC 7002 (Figure 47A). Attempts to PCR amplify the ctaDII::Ω locus were

unsuccessful, thus the segregation of the ctaDII and ctaDII::Ω alleles was determined by

Southern blot hybridization analysis (Figure 47B). The results of the Southern blot

analysis indicate that the ctaDII::Ω locus was also completely segregated in all genetic

backgrounds that were transformed with pctaDII. These results indicate that the ctaDI

and ctaDII genes have been successfully interrupted and are not essential for viability

under standard growth conditions for Synechococcus sp. PCC 7002.

3.5.3 Expression of the cta gene clusters in Synechococcus sp. PCC

7002

To assess whether or not the cta genes were expressed in Synechococcus sp. PCC

7002, the level of mRNA and the size of the transcripts of the ctaI and ctaII apparent operons were examined by Northern blot hybridization analysis. Specific DNA probes were made for all cloned and sequenced cta genes (Table 8). A 3.5-kb RNA species was detected for ctaCI, ctaDI, and ctaEI on the RNA blot (Figure 48). This indicates that this gene cluster is transcribed as a polycistronic mRNA encoding all three genes. No hybridization signal was detected by the ctaII-specific probes on RNA blots from wild- type Synechococcus sp. PCC 7002, indicating that the level of mRNA transcript 194

Figure 47. Interposon mutagenesis of the ctaDII gene from Synechococcus sp. PCC

7002. (A) Physical map of the 3.5-kb HincII fragment encoding ctaCIIDII and disruption

of the ctaDII locus by interposon mutagenesis. Arrows indicate the direction of

transcription and size of the gene. The Ω fragment, conferring spectinomycin resistance,

was inserted into an EcoRV site within the coding region of ctaDII. The plasmid pctaDII

Ω was used to transform the wild-type and ctaDI- strains of Synechococcus sp. PCC

7002. Transformants were selected for spectinomycin resistance as described in

Materials and Methods. (B) Southern blot analysis of the ctaDII locus. Genomic DNA

was digested with HincII and probed with a 32P-labeled PCR product for ctaDII. The

sizes of the hybridizing DNA fragments are indicated on the left. Lane 1: Hybridizing

fragment of 3.5-kb corresponds to the size of the wild-type copy of ctaDII. Lane 2:

Total DNA isolated from a spectinomycin resistant transformant was digested with

HincII. The 2.0 kb increase in size of the hybridizing ctaDII specific fragment corresponds to the insertion of the 2.0-kb Ω fragment which confers spectinomycin resistance. The absence of a 3.5-kb hybridizing fragment is indicative of the full segregation of the ctaDII and ctaDII::Ω alleles. Lanes 4 and 5: The ctaDI- strains made

previously (Figure 46) were transformed with pctaDII Ω. Total DNA isolated from the

kanamycin spectinomycin resistant transformants was digested with HincII. Results indicate that the strains were homozygous for ctaDII::Ω. 195

. 00 bp

I I

cII cII

Eco

Nco Nco

Hin Hin Nde

ctaDII ctaCII sll1486

Ω

HI HI

d III d

Sph

Hin Hin

Bam Bam Bam Bam Ω Ω Ω

ctaDI(1.1)::aphIIctaDI(2.2)::aphII . wild type ctaDII:: ctaDII:: ctaDII::

.5 kb .5 kb 196

Table 8. Cytochrome oxidase oligonucleotide list

Gene primer name nucleotide sequence (5’-3’) PCR product size (bp) ctaCI ctaCI.1 GTG AAT ATT CCC AAT AGC ATC 929 ctaCI.2 CCC CAT CTC CTC GGC GTA GGC ctaDI ctaDI.1 ATG AGT GAC GCG ACA ATA CAC 1133 ctaDI.2 TAG GTG TCA TGG ACA TGG ctaEI ctaEI.1 TAC GGC GAT CGC CAC CGA 566 ctaEI.1 CAA GGC CGA ACA GGA TAA TTC ctaCII ctaCII.1 CAC TTT CGG CGA TCG CCC TAC TTT TGG GGG 868 ctaCII.2 GGG ATG ATA ATT CAC CAC ctaDII ctaDII.1 CCA TGA CCC AAG CTC CC 1257 ctaDII.2 CGG GAA CCA GTG GTA CAC CGC ctaEII ctaEII.1 GAC TGC CAT CAA TGA AAC C 588 ctaEII.2 ATT GCC AGA GAT AAA TCA GCC 197

Figure 48. RNA blot hybridization analysis of the ctaCIDIEI gene cluster of

Synechococcus sp. PCC 7002. PCR products specific for the ctaEI, ctaDI and ctaCI genes were made and used as probes for RNA blot hybridization analysis. Total RNA

(20µg) was electrophoresed per lane of the gel. The size of the major hybridizing RNA species is indicated on the left. This 3.5-kb hybridizing fragment corresponds to the size of ctaCIDIEI operon. Smaller, non-specific hybridizing species may represent breakdown products of the main transcript. Sizes of the ribosomal RNAs are indicated on the right. 198

ctaEI ctaCI ctaDI markers kb kb 3.5 2.8 2.3 1.5

0.5 199 accumulation for ctaII was below the detection limit of Northern blot hybridization analysis.

Because the transcripts of the ctaCII, ctaDII, and ctaEII genes could not be detected by Northern blot hybridization, attempts to detect the ctaII transcripts were made using reverse transcriptase (RT)-PCR. ctaII gene specific primers and template

RNA isolated from wild-type Synechococcus sp. PCC 7002 cells were used for RT reactions. Three different cDNA templates were synthesized from total RNA isolated from wild-type Synechococcus sp. PCC 7002 using the following primers: ctaEII.2, ctaDII.2, and ctaCII.2. Primer ctaEII.2 corresponds to the 3' end of the ctaEII gene.

Primer ctaDII.2 corresponds to a region approximately 300 nucleotides downstream from the 3' end of the ctaDII gene. Primer ctaCII.2 corresponds to the 3' end of the ctaCII gene. The RT reaction with primer ctaEII.2 produced the cDNA template ctaEII.2T. The

RT reaction with primer ctaDII.2 produced the cDNA template ctaDII.2T. The RT reaction with primer ctaCII.2 produced the cDNA template ctaCII.2T. Primers corresponding to the 5' ends of the ctaCIIDIIEII genes are labeled as follows: ctaEII.1 (5' end of the ctaEII gene), ctaDII.1 (5' end of the ctaDII gene), and ctaCII.1 (5' end of the ctaCII gene). These 5’ primers were used in conjunction with the 3' primers, ctaEII.2, ctaDII.2, and ctaEII.2 for PCR amplifications of the cDNA templates. As a control, total

RNA used for the RT cDNA synthesis was used as a template for all PCR reactions. No

PCR products were produced when the total RNA was used as a template with any of the primers . This indicates that all of the PCR products from the reactions with the cDNA templates are the result of amplification of the cDNA template and not genomic DNA. 200

When the cDNA ctaEII.2T was used as a template and primers ctaEII.1 and ctaEII.2 were used for the subsequent PCR reaction a 589-bp product was detected by agarose gel electrophoresis (Figure 49). When the cDNAs ctaEII.2T and ctaDII.2T were used as templates and primers ctaDII.1 and ctaDII.2 were used for the subsequent PCR reaction, a 1655-bp product which corresponds to the ctaDII gene, was detected by agarose gel electrophoresis (Figure 49). When the cDNAs ctaEII.2T, ctaDII.2T and ctaCII.2T were used as templates and primers ctaCII.1 and ctaCII.1 were used for the subsequent PCR to amplify the RT-PCR products, an 868-bp product was detected by standard agarose gel electrophoresis, which corresponds to the ctaCII gene (Figure 49). These results indicate that the mRNA species covers the entire region of the ctaII gene cluster and that the three genes are co-transcribed. However, the inability to detect this mRNA species by RNA blot analysis indicates that the transcript abundance of the ctaII genes was low under standard growth conditions for Synechococcus sp. PCC 7002. 201

Figure 49. RT-PCR analysis of the ctaII gene cluster of Synechococcus sp. PCC

7002. (A). Gene arrangement of the ctaII gene cluster. The small numbered arrows below the physical map represent primers used for PCR amplification. Primer sequences are given in Table 8. (B). cDNA products from the reverse transcriptase reactions.

Primer ctaEII.2 was used to make the ctaEII.2T cDNA product. Primer ctaDII.2 was used to make the ctaDII.2T cDNA product. Primer ctaCII.2 was used to make the ctaCII.2T cDNA product. These cDNA products were used as templates for PCR. (C)

Electrophoretic analysis of PCR products amplified from templates listed above the lanes.

Primers ctaCII.1 and ctaCII.2 were used for lanes 1 to 4. Primers ctaDII.1 and ctaDII.2 were used for lanes 5 to 7. Primers ctaEII.1 and ctaEII.2 were used for lanes 8 and 9. 202 500 bp sll1485 ll1486 taCII ctaDII taEII sldI

868 bp 1655 bp 589 bp ctaCII.1 taCII.2 taDII.1 ctaDII.2 ctaEII.1 taEII.2

ctaEII.2T cDNA ctaDII.2T ctaCII.2T

p ctaEII.2TctaDII.2TctaCII.2T7002 DNActaEII.2TctaDII.2T7002 DNActaEII.2T7002 DNA bp 655 1655 68 868 589 589 203

3.5.4 Respiratory activity and oxygen evolution activity in Synechococcus

sp. PCC 7002 wild-type, ctaDI-, and ctaDII- strains

Table 9 shows respiratory rates of whole cells of the wild-type and various mutant strains grown under standard growth conditions. Strains in which the ctaDI gene was insertionally inactivated displayed a 3-fold decrease in oxygen uptake compared to the wild-type strain, while the CtaDII-deficient strain had the same rate of oxygen uptake as the wild-type strain rate. The ctaDI- ctaDII- double mutant exhibited a decrease in respiratory rate that was essentially identical to the ctaDI- mutant strain when compared to wild-type cells. Although no genetic evidence could be found for a Cyd AB quinol- type oxidase, the enzyme could still be present in Synechococcus sp. PCC 7002. To determine the existence of a possible third oxidase activity, a known inhibitor of terminal oxidase activity, KCN was added. All strains were sensitive to the addition of KCN to a final concentration of 1 mM, and only low rates of oxygen uptake were seen in the presence of KCN. This level of oxygen uptake can be attributed to the amount of oxygen consumed by the electrode itself, since this rate was observed even in the absence of live cells during the assay. The oxygen uptake activity observed in the ctaDI- and ctaDI- ctaDII- strains is within the range of error for oxygen consumed by the electrode. These results indicate that under the conditions tested, ctaDI encodes a subunit of a functional 204 terminal oxidase responsible for the respiratory activity of the cell and the CtaII enzyme has no significant oxygen uptake activity.

Table 9. Oxygen Consumption Rates of Photoautotrophically Grown Synechococcus sp.

PCC 7002 Strains in Darkness. Values shown represent the averages ± the standard deviation of five independent experiments.

Strain oxygen uptake oxygen uptake oxygen uptake -1 -1 -1 -1 -1 -1 (µmol of O2 (mg of chl) h ) (µmol of O2 (mg of chl) h ) (µmol of O2 (mg of chl) h )

no additions + 1mM KCN KCN sensitive activity wild-type 24 ± 6 2.5 ± 0.2 21 ± 6 ctaDI- 8 ± 2 2.5 ± 0.4 5 ± 2 ctaDII- 21 ± 6 2.4 ± 0.2 18 ± 6 ctaDI- ctaDII- 7 ± 1 2.5 ± 0.1 4 ± 1

Differences in oxygen evolution activities were not significant between mutant and wild-type strains (Table 10). For cells of all strains grown under 150 to 250 µE m-2 s-1 constant illumination, the oxygen evolving activities were virtually identical. There was a marked decrease in oxygen evolving capacity in all strains when they were grown under high to extremely high light intensities (700 to 4500 µE m-2 s-1) compared to cells grown under lower to standard constant illumination (Table 10). The oxygen evolving activity was not determined for the ctaDI- strain at 4.5 mE m-2 s-1 because the strain did not grow under these conditions. The differences in oxygen evolving activities observed 205

- Table 10. Doubling times, oxygen evolution rates and Fv’/Fm’ for wild-type, ctaDI , ctaDII-, and ctaDI- ctaDII- strains of Synechococcus sp. PCC 7002 grown under various light intensities. All values represent the averages ± standard deviations of 5 independent experiments. inviable: cells were unable to grow under these conditions.

Strain Light doubling oxygen evolution Fv’/Fm’ Intensity time -1 -1 (µmol of O2 (OD550nm) h ) (µE m-2 s-1) (hours) wild-type 150 4.0 ± 0.4 1394 ± 70 ND ctaDI- 150 4.2 ± 0.4 1360 ± 40 ND ctaDII- 150 4.1 ± 0.4 1460 ± 70 ND ctaDI- ctaDII- 150 3.9 ± 0.4 1460 ± 90 ND wild-type 250 3.6 ± 0.4 1428 ± 70 0.47 ± 0.1 ctaDI- 250 4.0 ± 0.4 1410 ± 90 0.38 ± 0.1 ctaDII- 250 3.8 ± 0.4 1440 ± 90 0.46 ± 0.1 ctaDI- ctaDII- 250 4.1 ± 0.4 1392 ± 90 0.33 ± 0.1 wild-type 700 4.0 ± 0.4 360 ± 15 ND ctaDI- 700 4.0 ± 0.4 351 ± 15 ND ctaDII- 700 4.0 ± 0.4 375 ± 30 ND ctaDI- ctaDII- 700 4.0 ± 0.4 294 ± 60 ND wild-type 4500 4.5 ± 0.4 340 ± 50 ND ctaDI- 4500 inviable inviable ND ctaDII- 4500 5.0 ± 0.6 339 ± 70 ND ctaDI- ctaDII- 4500 5.0 ± 0.4 320 ± 50 ND 206

between cells grown under standard constant illumination versus the oxygen evolving activities of cells grown under constant light stress correspond to an environmentally stimulated response within the cyanobacterial cells that causes a change in ratio of the photosystems as well as a change in the structural and functional activities of the photosystems (see results for 77K fluorescence).

3.5.5 Growth analysis of Synechococcus sp. PCC 7002 wild-type,

ctaDI-, ctaDII-, and ctaDI- ctaDII- strains

The growth rates of the mutant and wild-type strains of Synechococcus sp. PCC

7002 were analyzed for cells grown under several different continuous light intensities

(150 µE m -2 s-1, 250 µE m-2 s-1, 700 µE m-2 s-1, and 4.5 mE m-2 s-1). Under moderately low and normal light intensities (150 µE m-2 s-1 and 250 µE m-2 s -1) and under moderate light stress (700 µE m -2 s-1), the mutant strains could grow exponentially and the doubling times were nearly indistinguishable from the doubling time of the wild-type strain (Table

10). This indicates that under these conditions, cytochrome oxidase activity does not significantly contribute to the growth physiology and energetics of the cells.

In order to examine the effects of extremely high light intensity on the

Synechococcus sp. PCC 7002 strains, the cells were grown under a very high light

-2 -1 intensity (4.5 mE m s ) while the temperature and CO2 concentration were held constant. The growth yield of the ctaDI- strain was severely reduced compared to the 207 wild-type strain under these conditions (Figure 50). Additionally, although the growth rates are slightly slowed for the wild-type, ctaDII- and ctaDI- ctaDII- strains, the growth rate of the ctaDI- single mutant was drastically slowed when compared to the wild-type strain (Figure 51B). These results indicate that, although cytochrome oxidase I is dispensable for growth at light intensities from 150 µE m-2 s-1 to 700 µE m-2 s-1, it is important when the cells are grown under very high light stress (4.5 mE m-2 s-1).

However, under the same light intensity stress (4.5 mE m-2 s-1), the ctaDII- and ctaDI- ctaDII- strains had growth rates similar to the wild-type strain of Synechococcus sp. PCC

7002 (Figure 51ACD). These results suggest two things. First, the wild-type strain of

Synechococcus sp. PCC 7002 is able to tolerate extremely high light intensities as long as

the temperature is carefully maintained and high CO2 levels are provided. The second conclusion is that the absence of ctaDII allows the cells to grow at 4.5 mE m-2 s-1 even in the absence of ctaDI. Thus, the absence of ctaDII suppresses the phenotype observed when ctaDI is inactivated alone. 208

Figure 50. Effect of extreme high light intensity on growth of Synechococcus sp.

PCC 7002 wild-type (PR6000) and ctaDI- cells. Cells were grown under standard conditions until they reached exponential growth phase. The cells were then diluted to an

-2 -1 OD550 = 0.05 and grown under either standard light conditions (250 µE m s ) or extreme high light conditions (4.5 mE m-2 s-1) for 24 hours. (S) standard light conditions; (H) extreme high light conditions.

WT ctaDI- SH SH 209

Figure 51. Effect of extreme high light intensity on growth rates of wild-type, ctaDI-, ctaDII- and ctaDI- ctaDII- strains of Synechococcus sp. PCC 7002. All cells were grown under standard conditions until they reached the exponential phase of growth. The cells

-2 -1 -2 -1 were then diluted to OD550 = 0.05 and grown at either 250 µE m s or 4.5 mE m s continuous illumination. Open circles () represent cells grown under a continuous illumination at 250 µE m-2 s-1. Closed circles () represent cells grown under a continuous illumination at 4.5 mE m-2 s-1. Typical results of one of seven independent growth experiments are shown. Panel (A) wild-type. Panel (B) ctaDI-. Panel (C) ctaDII-. Panel (D) ctaDI- ctaDII-. 210

10 10 A. B.

1 1 550

OD 0.1 0.1

0.01 0.01 0 5 10 15 20 25 0 5 10 15 20 25 Time (h) Time (h) 10 10 C. D.

1 1 550 0.1 0.1 OD

0.01 0.01 0 5 10 15 20 25 0 5 10 15 20 25 Time (h) Time (h) 211

3.5.6 Chlorophyll and carotenoid contents of wild-type, ctaDI-,

ctaDII- and ctaDI ctaDII- strains

Table 11 shows chlorophyll a and carotenoid contents of wild-type and cta mutant strains grown under different light intensities. For cells grown under low to standard constant illumination (150 to 250 µE m -2 s-1) there was virtually no difference in the chlorophyll and carotenoid content between the mutant and wild-type strains. For cells grown under moderate to very high light stress (400 to 4500 µE m-2 s-1), the chlorophyll contents decrease in all strains compared to the chlorophyll concentrations from cells grown under normal light intensities. The exception is for the ctaDI- mutant strain, which fails to grow under extreme high light stress. These results suggest that in all strains there is a change in the structure of the photosynthetic apparatus when cells are grown under constant light stress, denoted by a decrease in the chlorophyll and carotenoid contents. There is, however, no significant difference between the wild-type and mutant strains in overall chlorophyll or carotenoid content under any of the light intensities. The exception to this pattern is the ctaDI- strain. The ctaDI- strain is unable to grow under conditions with constant extreme light stress (4.5 mE m-2 s-1) and thus chlorophyll and carotenoid concentrations are impossible to determine under these conditions. These observations are consistent with both oxygen evolution activity data 212

Table 11. Chlorophyll a and carotenoid contents for wild-type, ctaDI-, ctaDII-, and ctaDI- ctaDII- strains of Synechococcus sp. PCC 7002 grown under various light intensities. All values represent the averages of at five independent experiments ± standard deviations. inviable: cells were unable to grow under these conditions.

Strain Light Intensity Chlorophyll a Carotenoids -2 -1 (µE m s ) (µg ml-1 OD550 nm-1) (µg ml-1 OD550 nm-1) wild-type 150 3.7 ± 0.4 0.8 ± 0.2 ctaDI- 150 3.7 ± 0.3 0.8 ± 0.1 ctaDII- 150 3.6 ± 0.6 0.8 ± 0.1 ctaDI- ctaDII- 150 3.3 ± 0.3 0.8 ± 0.1 wild-type 250 3.4 ± 0.6 0.8 ± 0.1 ctaDI- 250 3.0 ± 0.5 0.8 ± 0.1 ctaDII- 250 3.4 ± 0.5 0.8 ± 0.2 ctaDI- ctaDII- 250 3.2 ± 0.7 0.9 ± 0.1 wild-type 400 3.4 ± 0.6 0.8 ± 0.1 ctaDI- 400 3.2 ± 0.6 0.8 ± 0.1 ctaDII- 400 3.4 ± 0.5 0.8 ± 0.2 ctaDI- ctaDII- 400 3.1 ± 0.5 0.8 ± 0.1 wild-type 700 1.5 ± 0.1 0.7 ± 0.3 ctaDI- 700 1.3 ± 0.1 0.7 ± 0.1 ctaDII- 700 1.5 ± 0.4 0.7 ± 0.3 ctaDI- ctaDII- 700 1.4 ± 0.6 0.7 ± 0.2 wild-type 1500 1.3 ± 0.1 0.7 ± 0.3 ctaDI- 1500 1.2 ± 0.1 0.7 ± 0.1 ctaDII- 1500 1.4 ± 0.4 0.7 ± 0.3 ctaDI- ctaDII- 1500 1.3 ± 0.2 0.7 ± 0.2 wild-type 4500 1.5 ± 0.1 0.7 ± 0.3 ctaDI- 4500 inviable inviable ctaDII- 4500 0.6 ± 0.4 0.6 ± 0.2 ctaDI- ctaDII- 4500 0.7 ± 0.3 0.6 ± 0.2 213

that show a decrease in the oxygen evolving activity with an increase in light induced stress in Synechococcus sp. PCC 7002 and the 77K fluorescence data (see below).

3.5.7 Photoinhibition of the whole chain electron transport activity

in Synechococcus sp. PCC 7002 wild-type, ctaDI-, ctaDII-, and

ctaDI- ctaDII- strains

The photoinhibition of the whole chain electron transport activity of wild-type and the Cta-deficient strains of Synechococcus sp. PCC 7002 grown under standard conditions was examined by incubation of the different strains under standard conditions

-2 -1 ° -2 -1 (250 µE m s , 38 C, 10 mM NaHCO3), or under high light conditions (4.5 mE m s ,

° 38 C, 10 mM NaHCO3) for fixed time periods after which the oxygen evolving activities of each of the samples were assayed. All strains were unaffected in their ability to evolve oxygen when incubated under standard light intensity conditions for any amount of time up to an hour (Figure 52). Differences were observed when the cells were incubated for various times under very high light intensity conditions. For the first 40 min of incubation under high light conditions, there was little effect on the oxygen evolving capacity the wild-type strain of Synechococcus sp. PCC 7002 compared to the first 40 min of incubation of the wild-type strain under standard conditions. However, after 1 hour of high light treatment the wild-type cells had an oxygen evolution rate that was 214

Figure 52. Effects of high light intensity on whole chain electron transport in wild- type, ctaDI- and ctaDII- strains of Synechococcus sp. PCC 7002. Cells were grown under standard conditions until they reached the exponential phase of growth. Cells were

° supplemented with 10 mM NaHCO3 and were treated at 38 C with either high light of 4.5 mE m-2 s-1 (wild-type ( ),ctaDI- (), ctaDII- (), ctaDI- ctaDII- (∇)), or standard light intensity of 250 µE m-2 s-1 for all strains (). Photosynthetic oxygen evolution from whole cells was measured at 38°C. The data shown are the averages ± standard deviation

(error bars) of five independent experiments.

100 80 60

40 20 % Oxygen evolution 0 0204060 Time (minutes) 215

40% that of the wild-type cells incubated under standard conditions. After 1 hour of

high light incubation, the ctaDII- strain had nearly the same level of oxygen evolving

activity as the ctaDII- strain incubated under standard conditions. The ctaDI-/ctaDII-

strain also showed little variance between incubations under standard light conditions and

high light conditions for the first 40 min. The ctaDI- ctaDII- strain exhibited a 30%

decrease in oxygen evolving activity after incubation for 1 hour under high light

conditions. The ctaDI- strain displayed a roughly exponential decrease after exposure to

high light (Figure 52). These results suggest that the CtaDI-deficient strain is much

more sensitive to photoinhibition than the wild-type strain when the cells are exposed to

high light stress. However, the absence of the ctaDII gene product can somehow compensate for the absence of the ctaDI gene product (see data for the ctaDI- ctaDII-

strain) and cause the cells to be less sensitive to photoinhibition.

3.5.8 Fluorescence emission at 77K of wild-type, ctaDI-, ctaDII- and

ctaDI- ctaDII- strains of Synechococcus sp. PCC 7002

Fluorescence emission spectra at 77K were collected for mutant and wild-type

strains in order to examine possible differences in PSI and PSII levels. Figure 53 shows

the 77K fluorescence emission spectra of whole cells of the wild-type strain, ctaDI-,

ctaDII-, and ctaDI- ctaDII- strains for cells grown at 100 µE m-2 s-1, 400 µE m-2 s-1, and

1500 µE m-2 s-1. Cells grown under moderately low light (100 µE m-2 s-1) exhibit little or

no difference in fluorescence emission maxima (Figure 53A). This implies that the 216 structure and ratio of both photosystems remains unchanged between the mutant strains and the wild-type strain with regards to the reaction-center-associated chlorophylls. When cells from both the wild-type and mutant backgrounds are grown under moderate light stress (400 µE m -2 s-1), all strains show a shift in the ratio of PSI to

PSII, with a relative decrease in PSI and increase in PSII associated chlorophylls (Figure

53B). There is a decreased PSII content in the ctaDI- and ctaDII- single mutant strains and a more pronounced reduction in fluorescence in the ctaDI ctaDII double mutant strain when compared to the wild-type strain. This is easily visualized by examining the decrease in fluorescence emission at 685 nm and 695 nm respectively of the mutant strains compared to the wild-type strains (Figure 53B). Although both emission peaks at

685 nm and 695 nm are indicative of relative PSII levels, because of the light-activated chlorophyll binding proteins fluorescence at 685 nm, the emission peak at 695 nm is more indicative of the PSII content (Dr. Gaozhong Shen, personal communication).

When all the strains are grown under high light intensities (1500 µE m-2 s-1) there is a relative decrease in PSII emission levels for all of the strains, but this is especially evident in the mutant strains (Figure 53C). These results indicate that Synechococcus sp.

PCC 7002 changes the ratio of PSI to PSII during changes in growth light intensity.

These effects are more drastic in the mutant strains for which especially the PSI and PSII emission levels are greatly decreased relative to the wild-type strain. Therefore, the structural integrity of the photosystem complexes may be compromised at higher light intensities and the lack of cytochrome oxidases further exacerbates this effect. 217

Figure 53. Fluorescence emission spectra at 77K of whole cells of Synechococcus sp. PCC 7002 wild-type and cytochrome oxidase mutant strains. Samples were

normalized to an OD730nm = 1.0 for all strains and were excited at a wavelength of 446 nm.

Strains used are indicated in the figure. (A) Cells grown at 100 µE m-2 s-1. (B) Cells grown at 400 µE m-2 s-1. (C) Cells grown at 1500 µE m-2 s-1. 218 A 3 30x10 Wild type ctaDI- 25 ctaDII- ctaDI- ctaDII- 20

Fluorescence Amplitude 5 Cells grown under 100 µE m-2 s-1

600 650 700 750 800 B Wavelength (nm) Wild type 3 - 80x10 ctaDI ctaDII- ctaDI- ctaDII- 60

20 Fluorescence Amplitude

-2 -1 0 Cells grown under 400 µE m s 600 650 700 750 800 C Wavelength (nm) 3 12x10 Wild type ctaDI- - 10 ctaDII ctaDI- ctaDII- 8

Fluorescence Amplitude 2 Cells grown under 1500 µE m-2 s-1

600 650 700 750 800 Wavelength (nm) 219

3.5.9 P700+ Reduction Kinetics

To determine the effect that the cytochrome oxidases have on photosynthetic electron transport, P700+ reduction kinetics were examined. Light-induced oxidation and dark-induced reduction of P700 are readily monitored by a single-beam spectrophotometer using an 830 nm measuring beam because of the complete absence of additional components undergoing absorption changes in the same spectral region (Yu et al., 1993). In order to obtain full reduction of P700+ in this experiment, 15-second dark incubation times were used. This was followed by a 22-second light exposure to induce the complete oxidation of P700. These light-dark cycles were repeated a total of 128 times per sample and the results were averaged using a Nicolet 4095 digital oscilloscope as described in the Materials and Methods. These data were transferred to a Macintosh

G3 computer, with which IGORPRO v 3.5 was used to determine a best-curve fit, from which the half-times for the reduction of P700+ were determined. The experiments were repeated a minimum of three times and differences between the half-times determined were less than 10%. The half-times of P700+ for the wild-type and mutant strains are tabulated in Table 12. 220

Table 12. Half-times (ms) for P700 reduction in Synechococcus sp. PCC 7002.

Values represent averages ± standard deviations for three independent experiments.

Strain no inhibitors 10 mM DCMU 10 mM DCMU, 1 mM KCN wild-type 120 ± 8 330 ± 10 237 ± 10 ctaDI- 70 ± 5 130 ± 10 146 ± 10 ctaDII- 87 ± 7 262 ± 10 203 ± 10 ctaDI- ctaDII- 53 ± 4 127 ± 10 160 ± 10 DCMU: 3-(3,4-dichlorophenyl)-1,1-dimethylurea, KCN: potassium cyanide

In the absence of any inhibitors in the ctaDI- strain the reduction of P700+ is 1.7- fold faster than in the wild-type strain. In the ctaDII- strain, P700+ reduction is 1.4-fold faster than the wild-type strain. For the ctaDI- ctaDII- strain, P700+ reduction is 2.2-fold faster than in the wild-type strain. These results indicate that in the absence of terminal

oxidases, there is an increase in the flow of electrons from the cytochrome b6f complex for P700+ reduction.

The inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) inhibits electron flow from PSII; therefore in the presence of DCMU the electron flow must be derived predominantly from the NADH dehydrogenase and from cyclic electron flow around PSI

(Yu et al., 1993). In the case of the wild-type strain, the addition of DCMU to a final concentration of 10 mM increased the half-time of P700+ reduction to 330 ms as opposed to 120 ms in the absence of DCMU. The cta mutant strains all have faster half-times as compared to the wild-type strain when DCMU was added (Table 12). In the presence of 221

10 mM DCMU, ctaDI- had a 2.5-fold faster half-time compared to the wild-type strain under the same conditions. The ctaDII- strain had a 1.7-fold faster half-time compared to the wild-type strain in the presence of 10 mM DCMU and the ctaDI- ctaDII- strain has a 2.6-fold faster reduction of P700+ compared to the wild-type strain. These results suggest that in the absence of Cta complexes, the majority of cyclic photosynthetic electron transport goes through PSI, but when Cta complexes are present, some of the electron flow is output through the terminal oxidases, resulting in a slower reduction of

P700+.

As a control, the cytochrome oxidase inhibitor KCN was added to all strains and the P700+ reduction kinetics were measured. It had been noted previously that the addition of KCN alone had no appreciable affect on the rate of P700+ reduction (Yu et al.,

1993). Therefore, these experiments were carried out in the presence of both KCN and

DCMU as described by (Yu et al., 1993). It was expected that in the presence of KCN, the half-time for P700+ reduction would decrease for the wild-type and ctaDII- strains relative to the same strains treated with DCMU alone. The half-time of P700+ reduction was not expected to change significantly in the ctaDI- strains upon the addition of KCN.

This is what was observed (Table 12). The addition of KCN to a final concentration of 1 mM in addition to the 10 mM DCMU caused a 1.4-fold decrease in the half-time in the wild-type strain compared to DCMU alone. The half-times for the ctaDI- and ctaDI- ctaDII- mutant strains did not decrease, but the half-time for P700+ reduction in the ctaDII- strain decreased 1.3-fold. 222

The faster half-times of P700+ reduction observed in the ctaDI- strains are

indicative of a bifurcation in electron flow from cytochrome c6 to PSI and cytochrome oxidase. The faster half-times observed in the ctaDII- strain are likely due to the changes in PSI stoichiometry discovered in the 77K fluorescence experiments (see section 3.5.8).

3.5.10 Pulse-Amplitude Modulated Fluorescence Measurements

Pulse-amplitude modulated (PAM) fluorescence measurements allow one to measure the number of functional photosystem II complexes in whole cells. In order to examine the electron flow around PSII, PAM fluorescence studies were performed at

room temperature. By examining the ratio of FV’/FM’ one can determine the total number of active PSII sites compared to total chlorophyll content. Results indicate that there is a decrease in this ratio for the strains harboring the ctaDI- mutation for cells grown at 250

µE m-2 s-1 (Table 10). Therefore, the ctaDI- and ctaDI- ctaDII- strains have a reduced number of active PSII complexes, which correlates with the decreased fluorescence emission at 685 and 695 nm at 77K (Figure 53). 223

3.5.11 Oxidative Stress and Cell Viability of Synechococcus sp.

PCC 7002 wild-type and ctaD mutant strains

Methyl viologen (MV) is a herbicide that can be reduced through a single-electron transfer from reduced Fe-S clusters associated with PSI. This results in the formation of a stable cation radical causing the subsequent reduction of oxygen, which forms the toxic superoxide radical anion. In order to examine the effects of oxidative stress on

Synechococcus sp. PCC 7002, wild-type and cytochrome oxidase mutant strains were grown in the presence and absence of MV and their growth rates were compared.

Tolerance to 50 µM of MV was observed for the ctaDII- and ctaDI- ctaDII- strains of

Synechococcus sp. PCC 7002 (Figure 54), which were able to grow in the presence of 50

µM MV unlike the wild-type and ctaDI- strains. The ctaDII- and ctaDI- ctaDII- strains grown in the presence of MV had slightly lower chlorophyll contents but similar

- ± -1 Ð1 ± -1 Ð1 carotenoid contents (ctaDII , 2.9 0.6 µg chl ml OD550 , 0.8 0.3 µg car ml OD550 , n

- - ± -1 Ð1 ± -1 Ð1 = 4 and ctaDI ctaDII , 3.0 0.7 µg chl ml OD550 , 0.8 0.4 µg car ml OD550 , n = 4) compared to cells grown without MV (see Table 11).

Figure 55 shows the growth curves of the Synechococcus sp. PCC 7002 wild-type and cta mutant strains grown in the presence and absence of 50µM MV. These growth curves indicate that the strains harboring the ctaDII- mutation are able to grow in the presence of 50 µM MV at rates nearly equal to cultures grown without MV (Figure 55C and 55D). This is in stark contrast to the growth rates of the wild-type strain or the ctaDI- strain in the presence of 50 µM MV (Figure 55A and 55B). These 224

Figure 54. Effects of methyl viologen on cell death of Synechococcus sp. PCC 7002 wild-type and ctaDII- strains. Cells were grown at standard growth conditions until they

reached exponential growth phase. These cells were then diluted to an OD550nm = 0.05.

The cells were then grown in the presence (+) or absence (-) of 50 µM methyl viologen for 24 hours. 225

Figure 55. Effects of MV on growth rates of wild-type, ctaDI- and ctaDII- strains of

Synechococcus sp. PCC 7002. Cells were grown under standard growth conditions until

they reached exponential growth phase. These cells were then diluted to an OD550 = 0.05 and grown in the presence or absence of 50 µM MV under otherwise identical conditions.

Open circles () represent cells grown without 50 µM MV, and closed circles () represent cells grown in the presence of 50 µM MV. Results represented are typical of one of seven independent growth experiments. Panel (A): wild type. Panel (B): ctaDI-.

Panel (C): ctaDII-. Panel (D): ctaDI- ctaDII-. 226

10 10 A. B. 550 1 1 OD

0.1 0.1 0 5 10 15 20 25 0 5 10 15 20 25 Time (h) Time (h) 10 10 C. D.

550 1 1 OD

0.1 0.1 0 5 10 15 20 25 0 5 10 15 20 25 Time (h) Time (h) 227 results indicate that strains harboring the ctaDII- mutation must have a mechanism to counteract the increase in superoxide generated by methyl viologen.

The tolerance of the ctaDII- and ctaDI- ctaDII- strains of Synechococcus sp. PCC

7002 toward MV was further verified by cell viability counts (Table 13). Viability counts reveal that there was a 45% decrease in the number of wild-type colony forming

-2 - units (CFU) per OD550 after a 4-hour incubation under standard conditions (250 µE m s

1 ° , 38 C, 1.5% CO2/air) in the presence of 50 µM MV. Similarly, there was a 40%

- decrease in the number of ctaDI CFUs per OD550 after a 4 hour incubation under standard

-2 -1 ° growth conditions (250 µE m s , 38 C, 1.5% CO2/air) in the presence of 50 µM MV.

However, there was no discernable difference in the number of CFUs for ctaDII- strains incubated with or without 50 µM MV. The total number of CFUs decreases for all strains when they were incubated in the dark compared to any of the strains incubated under standard conditions without MV (Table 13). However, there were no discernable differences in the number of CFUs for any of the strains incubated with or without 50 µM

MV in the dark when that particular strain was compared to itself. These results indicate that MV most likely forms the cation radical as described by (Epel and Neumann, 1973) during photosynthesis, but these results also indicate that MV is not very toxic when the cells are incubated in the dark. 228

Table 13. Cell viability after incubation with or without 50 µM MV, in either light or darkness. Conditions are described in Materials and Methods. Results are given as

number of colony forming units per OD550. Values presented are the average of three independent experiments ± standard deviation.

Strains of Synechococcus sp. PCC 7002 treatment wild-type ctaDI- ctaDII- ctaDI- ctaDII- none 8.0 ± 0.3 × 107 5.0 ± 0.3 × 107 7.4 ± 0.3 × 107 5.8 ± 0.5 × 107 50 µM MV 3.6 ± 0.1 × 107 2.0 ± 0.1 × 107 7.7 ± 0.4 × 107 6.0 ± 0.4 × 107 dark 4.8 ± 0.4 × 107 4.6 ± 0.3 × 107 6.6 ± 0.2 × 107 4.0 ± 0.3 × 107 dark + 50 µM MV 4.4 ± 0.4 × 107 4.6 ± 0.3 × 107 6.8 ± 0.3 × 107 3.7 ± 0.4 × 107 229

3.5.12 Superoxide Dismutase (SOD) Activity Assays

One possible explanation for the tolerance toward MV observed in the ctaDII- and ctaDI- ctaDII- strains is that these strains have increased levels of enzymes (superoxide dismutase (SOD), peroxidase, and catalase) related to the alleviation of oxidative stress.

In order to test this hypothesis, the wild-type and ctaDII- strains were assayed for differences in various enzyme levels. Both soluble and membrane fractions derived from the ctaDII- strain of Synechococcus sp. PCC 7002 have an increased level of SOD activity compared to the fractions from the wild type strains (see Table 14). The SOD activity of the membrane fractions for the ctaDII-strain was significantly higher than for the wild-type (>2-fold difference). These results suggest that ctaDII gene product is somehow involved in regulating the amount of membrane bound superoxide dismutase activity and in the absence of ctaDII, there are greater amounts of functional, membrane- bound superoxide dismutase. Thus, the higher SOD activity observed in the ctaDII- and ctaDI- ctaDII- strains may be responsible for the tolerance these strains exhibit towards

MV. 230

Table 14. Superoxide dismutase activity in cell extracts of Synechococcus sp. PCC

7002. Values shown represent the averages of five separate experiments ± standard deviation.

Strain WT ctaDI- ctaDII- ctaDI- ctaDII- soluble proteins (mg) 5 ± 15 ± 16 ± 15 ± 1 membrane-associated 5 ± 15 ± 16 ± 15 ± 1 proteins (mg) total proteins (mg) 10 ± 2 10 ± 1 12 ± 2 10 ± 1 soluble SOD units 173 ± 27 176 ± 20 220 ± 37 196 ± 16 membrane-associated 64 ± 12 67 ± 15 148 ± 30 134 ± 6 SOD units total SOD units 237 ± 33 243 ± 18 370 ± 63 330 ± 15 % of total activity 73% 72% 60% 59% (soluble) % of total activity 27% 28% 40% 39% (membrane) fold-increase in 1.0* 1.02 1.27 1.13 activity compared to WT (soluble) fold-increase in 1.0* 1.04 2.31 1.84 activity compared to WT (membrane) fold-increase in 1.0* 1.03 1.56 1.39 activity compared to WT (total) *normalized SOD activity, WT=1.0 231

3.5.13 Hydroperoxide levels in Synechococcus sp. PCC 7002

Another indicator of oxidative stress in cells is the concentration of hydroperoxides in cells. Hydrogen peroxide is the by-product of SOD dismutation of superoxide. In order to examine the effects of the mutations in the ctaDI and ctaDII loci, the total level of hydroperoxides were measured in both wild-type and mutant strains grown under standard conditions in the presence and absence of 50 µM MV. The results are summarized in Table 15. The results indicate that the wild-type and ctaDI- strains have similar levels of hydroperoxides both in the presence and absence of methyl viologen. The ctaDII- strain has a 31% higher level of hydroperoxides compared to the wild-type strain in the absence of methyl viologen and a 24% higher level of hydroperoxides when treated with methyl viologen. The ctaDI- ctaDII- double mutant

- had similarly increased levels of hydroperoxides as the ctaDII strain. H2O2 is a direct by product of superoxide dismutase activity (Fridovich and Hassan, 1979) and the presence of higher concentrations of hydroperoxides in the ctaDII- mutant strains implies that there is an increase in the level of activity of superoxide dismutase. However, this does not correspond to an increase in the activity of catalases or peroxidases in the ctaDII- strains

(see below). 232

Table 15. Hydroperoxide levels in Synechococcus sp. PCC 7002 and cta mutant strains.

Values shown represent the averages ± standard deviations of at least three separate experiments.

Strain 50 µM methyl Hydroperoxides % peroxides viologen treatment (nmol ml-1 OD550 nm-1 ) relative to wild-type wild-type no additions 58 ± 3 100 wild-type 50 µM MV 54 ± 9 100 treatment ctaDI- no additions 60 ± 9 103 ctaDI- 50 µM MV 54 ± 6 100 treatment ctaDII- no additions 76 ± 9 131 ctaDII- 50 µM MV 67 ± 2 124 treatment ctaDI- ctaDII- no additions 74 ± 1 128 ctaDI- ctaDII- 50 µM MV 70 ± 4 130 treatment 233

3.5.14 Catalase and peroxidase activity in Synechococcus sp. PCC

7002

To determine the whether or not the cta mutations had an effect on oxidative stress enzymes other than SOD, catalase and peroxidase activities for the wild-type and cta- strains were assayed. The results shown in Table 16 indicate that there is no appreciable difference in catalase activity between the wild-type, ctaDI-, ctaDII-, or ctaDI- ctaDII- strains. The results in Table 16 further reveal that there is no difference in peroxidase activity between the wild-type and mutant strains. These results support the previous observation from 3.5.13 where the levels of hydroperoxides were higher in the ctaDII- and ctaDI- ctaDII- strains compared to the wild-type and ctaDI- strains.

Therefore, although interruption of ctaDII has an effect on the levels of SOD activity, inactivation of the ctaDII gene has no effect on the levels of catalase or peroxidase activity compared to the wild-type strain. These results imply that the interruption of ctaDII only affects the SOD activity in these cells. 234

Table 16. Catalase and peroxidase activity in Synechococcus sp. PCC 7002 and cta mutant strains. Values shown represent the averages ± standard deviations of at five separate experiments. One unit results in the decomposition of one micromole of hydrogen peroxide per minute at 25°C and pH 7.0 per OD550 nm.

Strain units of catalase ml-1 units of peroxidase ml-1 OD550 nm-1 OD550 nm-1 wild-type 0.05 ± 0.01 1.7 ± 0.4 ctaDI- 0.05 ± 0.01 1.7 ± 0.4 ctaDII- 0.06 ± 0.02 1.7 ± 0.4 ctaDI- ctaDII- 0.06 ± 0.02 1.7 ± 0.4 Chapter 4

DISCUSSION AND CONCLUSIONS

4.1 Genes and gene organization in cyanobacteria

The completely sequenced genome of Synechocystis sp. PCC 6803 revealed 16 genes that encode subunits for the type I NADH dehydrogenase complex (Kaneko et al.,

1996). Synechococcus sp. PCC 7002 has homologs to all of these subunits. There were no cross-hybridizing fragments for the cydAB genes, which encode the quinol oxidase of

Synechocystis sp. PCC 6803, and these genes have not been detected in the

Synechococcus sp. PCC 7002 genome sequencing project. Synechococcus sp. PCC 7002 also lacks the hupLS genes, which encode the uptake hydrogenase in Anabaena sp. PCC

7120 (Carrasco et al., 1995).

This study reveals that most of the electron transfer genes have the highest sequence similarity to their respective counterparts in the freshwater cyanobacterial strain

Synechocystis sp. PCC 6803 (Kaneko et al., 1996) or the filamentous, nitrogen fixing cyanobacteria, Anabaena sp. PCC 7120 (Kazusa DNA Research Institute, 2000). The comparisons with Anabaena sp. PCC 7120 are still incomplete since a final, complete genomic sequence of that organism is still unavailable. However, the completely 236

sequenced genome of Synechocystis sp. PCC 6803 shows that there are many similarities

between the gene organization of electron transfer genes between Synechocystis sp. PCC

6803 and Synechococcus sp. PCC 7002. Examples of apparent operon organization

throughout cyanobacteria include the ndhAIGE cluster, the ndhF5 and ndhF2 genes, the

ndhCKJ cluster, and the ctaCIDIEI operon. However, there are some genes for which the

retention of operon structure appears to be more important in Synechococcus sp. PCC

7002 than in Synechocystis sp. PCC 6803. Examples of these apparent operons found in

Synechococcus sp. PCC 7002 but not found in Synechocystis sp. PCC 6803 or Anabaena sp. PCC 7120 include the hydrogenase gene cluster and the ctaCIIDIIEII operon.

In Synechococcus sp. PCC 7002, the genes for both the structural components of the hydrogenase enzyme, as well as the genes encoding hydrogenase assembly proteins, occur in a single operon. However, in the other cyanobacterial species examined in this study (Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120), although the genes for the hydrogenase subunits are in relatively close proximity to one another, the genes for the hydrogenase-assembly proteins are scattered throughout their respective genomes. In the case of the ctaCIIDIIEII genes, Synechococcus sp. PCC 7002 appears to be unique in that these genes form an operon; In contrast, although the ctaCII and ctaDII genes are in close physical proximity to one in another in Synechocystis sp. PCC 6803, the ctaEII gene is located elsewhere in the genome.

Why is there such diversity of gene organization in cyanobacteria? One possible explanation is the varied transposon activity within different cyanobacterial species.

Many (106) transposon-like elements have been identified in the genome of 237

Synechocystis sp. PCC 6803 (Kaneko et al., 1996). This number is considerably higher than the number of transposon-like elements identified in Synechococcus sp. PCC 7002

(5) thus far. Transposase activity may have been responsible for many of the gene rearrangements in Synechocystis sp. PCC 6803. Conversely, the lack of these transposon-like elements may explain the extensive retention of apparent operons in

Synechococcus sp. PCC 7002.

4.1.1 Future directions of the genome sequencing project in Synechococcus

sp. PCC 7002

Currently, the entire genome of Synechococcus sp. PCC 7002 is being sequenced and assembled by the Bryant lab in collaboration with the lab of Dr. Jindong Zhao at

Beijing University and The Institute for Genetics, Beijing, China. The sequence data gathered in this study are being used to orient and assemble contigs for the complete sequence determination of Synechococcus sp. PCC 7002. The nucleotide sequences from this study have also been used to fill in gaps from the sequencing project when they overlap with other contigs. 238

4.2 Type I NADH Dehydrogenase

There are 14 subunits that constitute NDH-1 complex in E. coli, R. capsulatus, P. denitrificans, and T. thermophilus (Yagi et al., 1998). These 14 subunits are homologous to subunits of the mitochondrial type I NADH dehydrogenase of B. taurus and are considered the minimal number of subunits necessary for the NADH-quinone oxidoreductase activity. In contrast to these bacteria, plant chloroplasts and cyanobacteria have homologs to only 11 of the 14 subunits found in other bacteria.

These subunits have been collectively termed the “alien complex” by (Friedrich et al.,

1995). An observation from this and another study (Friedrich et al., 1995) and is that the chloroplast and cyanobacterial genes encoding subunits for the type I NADH dehydrogenase are more closely related to one another than are mitochondrial and chloroplast genes for type I NADH dehydrogenases, even if mitochondrial and plastic

NDH-1 gene sequences are derived from the same plant. The cyanobacterial type I

NADH dehydrogenase genes are also less similar to the bacterial genes encoding type I

NADH dehydrogenases than they are to chloroplast encoded type I NADH dehydrogenase genes. These observations suggest a break in lineage or a separate lineage in the origins of the NDH-1 genes from cyanobacteria and the chloroplast from those of bacteria and the mitochondria. Noticeably absent in chloroplast and cyanobacteria are the genes nuoF and nuoE. These genes encode the subunits harboring the FMN cofactor and at least 4 Fe-S clusters. Furthermore, these subunits comprise the NADH dehydrogenase (diaphorase) portion of the enzyme in purple bacteria (Friedrich et al., 239

1995; Friedrich et al., 1990). This suggests that another donor/acceptor besides NADH is utilized by the type I NADH dehydrogenase in chloroplasts and cyanobacteria. A plausible electron donor/acceptor would be ferredoxin because of the predicted close proximity of the type I NADH dehydrogenase to PSI (Berger et al., 1993).

The names of the genes and proteins for the NDH-1 subunits in cyanobacteria have been assigned according to the nomenclature used for the chloroplast homologs and a similar system of nomenclature has been used in this study. The Synechococcus sp.

PCC 7002 NDH-1 genes are found in several locations throughout its genome and their organization is similar to the gene arrangements found in Synechocystis sp. PCC 6803.

The following NDH-1 genes were found in this study: ndhAIGE, ndhB, ndhD1, ndhD2, ndhD3, ndhD4, ndhF1, ndhF2, ndhF3, ndhF4, ndhF5, ndhCKJ, ndhH, and ndhL. The genes for two of the type I NADH dehydrogenase subunits (the ndhF1 gene (slr0844 homolog) (Schluchter et al., 1993) and the ndhD3 (slr1732 homolog) and ndhF3 (slr1733 homolog) genes (Klughammer et al., 1999) were characterized in previous studies.

Single-copy genes encoding putative subunits of the type I NADH dehydrogenase in Synechococcus sp. PCC 7002 are represented by the ndhA, ndhI, ndhG, ndhE, ndhB, ndhC, ndhK, ndhJ, ndhH, and ndhL genes. The phenotypes of mutations at several of these loci have been investigated previously in Synechocystis sp. PCC 6803 (Ogawa,

1990; Ogawa, 1991). The ndhL open reading frame identified in Synechocystis sp. PCC

6803 is predicted to encode a hydrophobic protein of 80 amino acids. Deletion of the ndhL gene in Synechocystis sp. PCC 6803 revealed that this strain is limited in its ability

- to transport CO2 and HCO3 (Ogawa, 1990). Thus far, inactivation of the single copy ndh 240

genes in cyanobacteria consistently leads to defects in their abilities to uptake CO2 and

- HCO3 . Therefore, similar phenotypes would be expected for such mutations in

Synechococcus sp. PCC 7002. Under the conditions presented in 3.1.1, the ndhB::cat allele could not be segregated from the wild-type copy in Synechococcus sp. PCC 7002.

This may seem surprising given that these genes could be inactivated in Synechocystis sp.

PCC 6803 (Ogawa, 1991) and Synechococcus sp. PCC 7942 (Marco et al., 1993).

However, the results of (Ogawa, 1991) have recently come into question, since attempts to repeat Ogawa's inactivation of the ndhB gene in Synechocystis sp. PCC 6803 by other groups have also been unsuccessful (Dr. Wim Vermaas, Arizona State University, personal communication). Thus, the initial observations made by Ogawa may have been due to a secondary mutation that rescues the phenotype of an essential single-copy gene.

Mutational analysis of the ndhD3 and the ndhF3 genes in Synechococcus sp. PCC

7002 has revealed that these mutants, like the ndhB- mutants of Synechocystis sp. PCC

6803 (Ogawa, 1991) and Synechococcus sp. PCC 7942 (Marco et al., 1993), also require

high levels of CO2 (Klughammer et al., 1999). A similar phenotype was observed in ndhD3- Synechocystis sp. PCC6803 strains (Price et al., 1998). However, mutations in

other ndhD genes are able to concentrate Ci at levels similar to the wild-type strain of

Synechocystis sp. PCC 6803 (Ohkawa et al., 2000). However, mutation analysis of the ndhF1gene in Synechococcus sp. PCC 7002 revealed that the gene product of ndhF1 is involved in cyclic electron transport and respiratory function (Schluchter et al., 1993) and not in inorganic carbon transport (Sültemeyer et al., 1997b). Therefore, it has been 241 proposed that the different ndhD and ndhF gene products are involved with multiple, functionally distinct, NDH-1-like complexes in cyanobacteria.

The phylogenetic analysis of the NdhD and NdhF protein sequences shows that certain genes were probably misnamed upon their initial discovery (i.e. NdhF2 and

NdhF5). The names for the NdhD and NdhF proteins from Synechocystis sp. PCC 6803 cyanobacteria have been changed in this report to reflect the results of this phylogenetic analysis. The close physical proximity and high degree of sequence similarity (>60%) observed with the ndhF2 and ndhF5 genes throughout all of the cyanobacteria examined in this study may be the result of a recent gene duplication at one of these loci. The function of these genes and their gene products is still unknown.

4.2.1 Future directions for research of the type I NADH dehydrogenase

genes in Synechococcus sp. PCC 7002

To further investigate the function of the type I NADH dehydrogenase in cyanobacteria, attempts should be made to inactivate the ndh genes discovered in this study by interposon mutagenesis. Restriction maps have been provided in the

APPENDIX so designing these experiments would be relatively straightforward. Since a plasmid with an interrupted ndhB gene has been constructed, and further attempts could be made to isolate an NdhB-deficient strain under different growth conditions in order to find out more about its relationship with the type I NADH dehydrogenase in

Synechococcus sp. PCC 7002. 242

4.3 Type II NADH dehydrogenases

There are two genes (ndbA and ndbB) that are predicted to encode type II NADH dehydrogenases in Synechococcus sp. PCC 7002. However, the function of these gene products has not been examined. Attempts to identify a homolog to ndbC of

Synechocystis sp. PCC 6803 by heterologous gene hybridization and examination of the

Synechococcus sp. PCC 7002 genome sequence data have thus far been unsuccessful.

The ndbA and ndbB genes have not yet been inactivated in Synechococcus sp. PCC 7002, and thus their function is unknown.

Analyses of the predicted amino acid sequences of the NDH-2 proteins from

Synechococcus sp. PCC 7002 revealed that they all have putative NADH and FAD binding motifs. These motifs consist of a β-sheet-α-helix-β-sheet structure, a glycine- rich phosphate binding consensus sequence (GXGXG), a conserved negatively charged residue (D or E) at the end of the second β-sheet, and six positions typically occupied by small hydrophobic residues. Four of these hydrophobic residues are present in the

NADH binding motif, while the other two precede the conserved D or E residue. In

NADPH binding proteins, the third G in the GXGXXG motif is replaced by S, A, or P and the negative charge at the end of the second β-sheet is missing (Howitt et al., 1999).

Since these features are absent in the predicted NDH-2 proteins from Synechococcus sp.

PCC 7002, it is likely that these enzymes bind NADH and not NADPH. 243

Both of the NDH-2 proteins predicted from the Synechococcus sp. PCC 7002 sequence also have the conserved nucleotide-binding domain for FAD and a secondary motif downstream that is characteristic of FAD binding motifs (Howitt et al., 1999). This motif contains the conserved Asp that forms a hydrogen bond with FAD. These characteristics, along with the potential ability to bind NADH, are hallmarks of NDH-2 proteins.

4.3.1 Future directions for research of the type II NADH dehydrogenase

genes in Synechococcus sp. PCC 7002

(Howitt et al., 1999) proposed that the NDH-2 proteins act as redox sensors within cyanobacteria. Now that the nucleotide sequence for the ndbA and ndbB genes is available from Synechococcus sp. PCC 7002, it would be interesting to inactivate the ndbA and ndbB genes in Synechococcus sp. PCC 7002 strains in which the PSI genes have also been inactivated. If the high-light tolerance found in strains of Synechocystis sp. PCC 6803 where both ndb genes and PSI genes have been inactivated (Howitt et al.,

1999) could be reproduced in Synechococcus sp. PCC 7002, it would be attractive to see if the inactivation of these genes contributes to generate higher levels of proteins designed to deal with oxidative stress (i.e. SOD, peroxidase, or catalase) similar to the ctaII mutants. Additionally, it would be interesting to determine if the ndb- strains of

Synechococcus sp. PCC 7002 display the same tolerance to MV as the ctaDII- strains of

Synechococcus sp. PCC 7002. The Synechococcus sp. PCC 7002 NdbA and NdbB 244

proteins are predicted to be hydrophilic and it may be of interest to generate recombinant

versions of these proteins in E. coli for the generation of NdbA-specific and NdbB-

specific antibodies for use in physiological characterization of the Ndb proteins in

Synechococcus sp. PCC 7002. These recombinant Ndb proteins could also be used for

biochemical and biophysical characterization.

4.4 Bi-directional hydrogenase

An interesting finding in this study is that the genes for the bi-directional

hydrogenase are organized in a unique manner in Synechococcus sp. PCC 7002.

Compared to the organization of hydrogenase genes in other cyanobacterial species, all of

the hydrogenase genes are in close physical proximity to each other, possibly forming an

operon. There are many assembly proteins associated with the maturation of the

hydrogenase enzyme in bacteria (Jacobi et al., 1992). These assembly proteins may be

necessary for proper maturation of the hydrogenase enzyme. Synechococcus sp. PCC

7002 is also unique because the genes predicted to encode hydrogenase assembly proteins

(hyp genes) are clustered with the genes predicted to encode the hydrogenase itself. The

other cyanobacterial species examined in this study (Synechocystis sp. PCC 6803 and

Anabaena sp. PCC 7120) have the hyp genes located elsewhere in their respective genomes.

Mutants of Synechococcus sp. PCC 7002 in which either the hoxH gene or both the hoxH and hoxF genes have been inactivated show no apparent growth phenotypes 245 under normal growth conditions. These results are similar to results in Synechocystis sp.

PCC 6803, in which the hoxF gene was inactivated (Howitt and Vermaas, 1999). A popular proposal was that the hoxF, hoxE, and hoxU subunits were the missing cyanobacterial homologs of the type I NADH dehydrogenase genes nuoF, nuoE, and nuoG (Appel and Schulz, 1996). However, this hypothesis seems unlikely, since the mutant strains in both Synechocystis sp. PCC 6803 (Howitt and Vermaas, 1999) and

Synechococcus sp. PCC 7002 lack distinguishing phenotypes that could be attributed to the absence of a functional type I NADH dehydrogenase. If the hoxF, hoxE, and hoxU gene products were indeed functionally substituting for the nuoF, nuoE and nuoG gene products, one would predict that the absence of a functional HoxF subunit would exhibit a phenotype similar to that of strains in which a gene encoding a unique subunit of the type I NADH dehydrogenase had been inactivated. However, the hoxF mutant strain has no significant phenotypes when grown under standard growth conditions. It remains possible that HoxF functions in conjunction with the type I NADH dehydrogenase under alternative growth conditions (e. g. anaerobiosis).

4.4.1 Future directions for research of the bi-directional hydrogenase and

hyp genes in Synechococcus sp. PCC 7002

Although there has been no phenotype associated with the inactivation of the

HoxH and HoxF subunits of the hydrogenase in Synechococcus sp. PCC 7002, there may be conditions that have not been assayed that will elucidate a function for the 246 hydrogenase in cyanobacteria. Hydrogenase expression in lithoautotrophic bacteria is dependent on the level of hydrogen present. In R. eutropha, hydrogenases are produced

in the presence of H2 as well as during growth on poor carbon sources. Hydrogenase synthesis is blocked during growth on preferentially utilized carbon sources, such as succinate and pyruvate (Schwartz et al., 1998). In Bradyrhizobium japonicum, the expression of hydrogenase is repressed by oxygen and carbon substrates and hydrogenase expression is stimulated by hydrogen and carbon dioxide (Merberg et al., 1983). In cyanobacteria, hydrogenase activity levels in heterocysts and vegetative cells of A. variabilis and in the unicellular cyanobacteria, A. nidulans, were increased by incubation

of the cultures under anaerobic conditions and the addition of molecular H2 (Houchins and Burris, 1981; Papen et al., 1986). Growth of Synechococcus sp. PCC 7002 under anaerobic or microaerophilic conditions may also increase expression of the hydrogenase enzyme and give some insight as to the physiological function of the bi-directional hydrogenase in cyanobacteria. A simple way to conduct this experiment would be to

bubble cultures in liquid media with a 5% CO2 95% N2 gas mixture which would

effectively lower the O2 levels.

The Hyp proteins have been shown to be necessary for proper assembly and function of the mature hydrogenase in other bacterial species (Jacobi et al., 1992;

Magalon and Bock, 2000; Olson et al., 1997; Olson and Maier, 1997). The hydrogenase assembly factors have also been implicated in the proper assembly of other Ni-containing enzymes in bacteria (Olson et al., 2001). These assembly factors may be necessary for the assembly of other Ni containing enzymes in Synechococcus sp. PCC 7002. The hypB 247 gene may be a good target for inactivation to test the hypothesis of whether or not the corresponding polypeptide is necessary for the assembly of Ni-containing enzymes in

Synechococcus sp. PCC 7002. It is known that Synechococcus sp. PCC 7002 has a urease which utilizes Ni for its catalytic activity (Sakamoto et al., 1998). Inactivation of the hypB gene could lead to the inability of Synechococcus sp. PCC 7002 to grow on urea.

Although the structures for other members of the hydrogenase family have been solved, no structural data exist for the cyanobacterial bi-directional hydrogenases. The subunits of bi-directional hydrogenase from Synechococcus sp. PCC 7002 are predicted to be hydrophilic and it may be beneficial to try and produce recombinant forms of the proteins for structural studies. Production of recombinant forms for the hydrogenase subunits could also be used for antibody production. Hox-specific antibodies would be useful to determine the location of the hydrogenase in the cell as well as for physiological studies.

4.5 Mobile electron carriers

It is clear from this study that Synechococcus sp. PCC 7002 has some unique genes compared to other cyanobacteria. Synechococcus sp. PCC 7002 has the genetic potential to encode two soluble c-type cytochromes, petJ1 and petJ2. The petJ2 gene is

predicted to encode a second cytochrome c6 protein that would be different in primary amino acid sequence from the cytochrome cm proteins found in Synechocystis sp. PCC

6803 and Synechococcus sp. PCC 7002. At this point, there is evidence that petJ1 is the 248

essential cytochrome c6 and there is no evidence that the petJ2 gene product can functionally replace the petJ1 gene product. Synechococcus sp. PCC 7002 also has the potential to synthesize a blue-copper protein encoded by the bcpA gene. The bcpA gene is also found in Anabaena sp. PCC 7120, but it is not present in the genome of

Synechocystis sp. PCC 6803.

The failure to segregate the petJ1 gene in Synechococcus sp. strain PCC 7002 is quite surprising, since inactivation of the petJ gene was achieved in both Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7942 (Laudenbach et al., 1990; Zhang et al.,

1994). However, both Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7942 have a petE gene, encoding a plastocyanin. No evidence for the presence of the petE gene in Synechococcus sp. strain PCC 7002 was obtained by Southern hybridization using heterologous probes, and thus far, examination of the genome sequence of

Synechococcus sp. PCC 7002 has failed to reveal the presence of a petE gene.

Plastocyanin has never been isolated from nor detected in Synechococcus sp. strain PCC

7002. Caution must be exercised when making an argument for the absence of plastocyanin, since heterologous hybridization analysis for the petE gene in

Synechococcus sp. strain PCC 7942 had also failed to reveal the presence of the gene sequence or the gene product even though it was present (Clarke and Campbell, 1996).

However, the failure to obtain segregation of petJ1/petJ1::aphII under various growth conditions points towards the absence of either plastocyanin or another mobile electron carrier that could substitute for PetJ1, in Synechococcus sp. strain PCC 7002. The absence of alternative electron carriers that could substitute for the PetJ1 gene product is 249 supported by an observation from (Manna and Vermaas, 1997). They observed that in

Synechocystis sp. strain PCC 6803, if the petE gene is deleted, the petJ gene cannot be deleted and vice-versa. It is presumed, therefore, that these two mobile electron carriers can substitute for one another, but a functional copy of one of these genes must always be present in cyanobacteria for viability. Therefore, if there were only one gene for a functional mobile electron carrier (petJ1) in Synechococcus sp. PCC 7002, attempts to inactivate the petJ1 gene in Synechococcus sp. PCC 7002 would be similar to attempts to inactivate both the petE gene and petJ gene in other cyanobacteria. This observation supports the idea that Synechococcus sp. PCC 7002 has only one mobile electron carrier,

namely cytochrome c6, and that there is no plastocyanin in this species.

An alternative explanation for the failure to obtain a petJ1- strain would be that, if plastocyanin or another mobile electron carrier is present in Synechococcus sp. strain

PCC 7002, it is unable to functionally substitute for cytochrome c6 under the conditions tested. It is clear that Synechococcus sp. PCC 7002 has a copy of the cytM gene,

encoding cytochrome cM, at least one other gene predicted to encode a c-type cytochrome, petJ2, as well as the gene encoding the potential blue-copper protein BcpA.

It is also clear that none of these genes can functionally substitute for the petJ1 gene product. The attempted substitution of the Synechococcus sp. PCC 7002 petJ1 gene with either the Synechocystis sp. PCC 6803 petE or petJ gene was also unsuccessful. These heterologous genes were expressed in Synechococcus sp. PCC 7002 and a processed

Synechocystis sp. PCC 6803 petE gene product was detected by immunoblot analysis.

Since neither of these gene products could substitute for the Synechococcus sp. PCC 7002 250

PetJ1, it must be concluded that the interaction of PetJ1 with its redox partners is very

specific. Whether it is the reductive (cytochrome b6f) or oxidative (PSI or cytochrome oxidase) side, or both that require such apparent specificity is unknown at this point. Of special interest on the oxidative side of this electron transfer reaction is the psaF gene

product. The psaF gene product has a function as a docking protein for cytochrome c6 or plastocyanin, allowing electron transfer between cytochrome c6 and P700 of PSI. In a mutant strain of the cyanobacterium Synechococcus elongatus, Hippler et al. (1999) generated a strain that expressed a hybrid Chlamydomonas reinhardtii/S. elongatus version of the PSI subunit, PsaF. This hybrid PsaF was incorporated in vivo into the cyanobacterial PSI and showed an increase by 2-3 orders of magnitude in the ability for the C. reinhardtii mobile electron carriers to reduce P700+ (Hippler et al., 1999). Thus, although the PsaF proteins from Synechococcus sp. PCC 7002 and Synechocystis sp. PCC

6803 are very similar (60% identical, 73% similar), the interactions of the PsaF proteins with their mobile electron transport proteins are very specific, enough so that the

Synechocystis sp. PCC 6803 mobile electron carriers cannot functionally substitute for

PetJ1 in Synechococcus sp. PCC 7002.

Specificity of interaction of electron donors and acceptors is a reasonable hypothesis as to why neither of the putative secondary electron carrier gene products,

BcpA or PetJ2, or for that matter the heterologous gene products from Synechocystis sp.

PCC 6803 cannot functionally substitute for PetJ1 in Synechococcus sp. PCC 7002. A similar situation has been observed in the purple non-sulfur bacteria R. sphaeroides and

R. capsulatus. In R. capsulatus, there are two electron carriers between the cytochrome 251 bc1 complex and the reaction center, cytochrome c2 and cytochrome c y that can functionally substitute for one another (Jenney and Daldal, 1993; Jenney et al., 1996). If

cytochrome c2 is deleted in R. capsulatus, this bacterium can still grow photosynthetically using cytochrome cy (Jenney et al., 1996). The case for R. sphaeroides is different even though the two organisms are closely related. Deletion of the cytochrome c2 gene results in a non-photosynthetic mutant in R. sphaeroides (Hochkoeppler et al., 1995). This is in

spite of the fact that R. sphaeroides has a cytochrome cy gene. However, if the R. capsulatus cytochrome cy gene is expressed in the R. sphaeroides cytochrome c2-deficient strain, the cells again become photosynthetic (Jenney et al., 1996). Perhaps

Synechococcus sp. strain PCC 7002 does have a plastocyanin as R. sphaeroides has a

copy of the gene encoding cytochrome cy but this gene product is unable to substitute for the main mobile electron carrier, which in Synechococcus sp. strain PCC 7002 is

cytochrome c6. This may point to a functional difference based upon specific structural

interactions between cytochrome c6 and any of its putative redox partners within the

b f c thylakoid membrane (cytochrome 6 , PSI, or cytochrome oxidase). Cytochrome 6 in

Synechococcus sp. strain PCC 7002 may also be serving an alternative and unique function that is different than that described in other cyanobacterial species. 252

4.5.1 Future directions for research of the mobile electron carrier genes in

Synechococcus sp. PCC 7002

Thus far, here have not been any phenotypes associated with the inactivation of either the petJ2 or bcpA genes in Synechococcus sp. PCC 7002. Further physiological characterization of the petJ2- and bcpA- strains under different growth conditions may uncover a phenotype for these deficiencies. These genes may be involved in anaerobic or microaerophilic metabolism, and therefore, no phenotype would have been observed under the growth conditions tested. In Synechococcus sp. PCC 7002, the unique genes present possibilities for new electron transfer pathways. An sqr gene encoding sulfide- quinone oxidoreductase has been found on contig 450 from the Synechococcus sp. PCC

7002 genome sequencing project and presents the possibility that Synechococcus sp. PCC

7002 may be able to use hydrogen sulfide as an electron source. Sulfide-quinone oxidoreductases have also recently been found in the filamentous cyanobacteria

Oscillatoria limnetica and Anabaena sp. PCC 7120 (Bronstein et al., 2000). Thus, it is possible that the mobile electron-transport gene products may be involved with electron transfer to and from the sulfide-quinone oxidoreductase.

Attempts to refold the rBcpA protein produced in E. coli have been unsuccessful.

The initial design of this plasmid was made with the belief that the mature protein would be processed at a signal-sequence site in the amino terminus based on the ideas of ((von

Heijne, 1986). Since these truncated plasmids have not worked, perhaps a new rBcpA/pET plasmid should be made with the full-length, unprocessed predicted protein, 253 if any further attempts will be made to overproduce this recombinant protein for biochemical characterization.

4.6 Terminal Oxidases Present in Synechococcus sp. PCC 7002

It has been demonstrated previously that there are three gene clusters that encode

ORFs similar to known quinol and cytochrome oxidases in Synechocystis sp. PCC 6803

(Alge and Peschek, 1993a; Alge and Peschek, 1993b; Howitt and Vermaas, 1998). This study reports the identification of two cta gene clusters similar to the two heme-copper oxidase operons found in the related cyanobacterium, Synechocystis sp. PCC 6803

(Howitt and Vermaas, 1998). The CtaD protein is the largest subunit of the bacterial cytochrome oxidase. Synechococcus sp. PCC 7002 has two genes that potentially encode CtaD subunits, and these genes have been named ctaDI and ctaDII. The ctaDI gene is predicted to encode a 549 amino acid polypeptide that would have a molecular mass of 60.9 kDa and an estimated pI of 5.62. This polypeptide also contains the

2+ putative Mg binding site. The ctaDII gene is predicted to encode a 551 amino acid protein with a molecular mass of 61 kDa and an estimated pI of 6.37. Alignments in the

2+ region of the Mg binding site show a high degree of identity with their counterparts from Synechocystis sp. PCC 6803 (Figure 56). As in Synechocystis sp. PCC 6803, the

2+ CtaDII subunit of Synechococcus sp. PCC 7002 is divergent in the region of the Mg binding site. 254

The CtaC subunit of the bacterial cytochrome oxidase contains the putative CuA binding motif. Synechococcus sp. PCC 7002 has two genes that potentially encode CtaC subunits, and these genes have been named ctaCI and ctaCII. The ctaCI gene is predicted to encode a 362 amino acid polypeptide that would have a molecular mass of

39.6 kDa and an estimated pI of 4.55. The ctaCII gene is predicted to encode a 298 amino acid protein with a molecular mass of 33.8 kDa and an estimated pI of 6.50.

Figure 56 also shows an alignment of the CuA binding motif for the Synechococcus sp.

PCC 7002 and Synechocystis sp. PCC 6803 CtaCI proteins. The CuA binding motifs of the CtaCII proteins from both Synechococcus sp. PCC 7002 and Synechocystis sp. PCC

6803 are similarly divergent in this binding motif. 255

2+ Figure 56. Oxidase Mg and CuA binding motif alignments. A. Alignments of the heme-copper oxidase Mg2+ binding region in the cyanobacterial CtaD subunit with the large subunits of other cytochrome oxidases. Putative binding ligands are in bold. In the cyanobacterial CtaDII subunits, the conserved histidine and aspartic acid have been replaced by asparagines. B. Alignments of the heme copper oxidase CuA binding region in the cyanobacterial CtaC subunit with subunit II from other cytochrome oxidases. Putative binding ligands are in bold. In the cyanobacterial CtaCII subunits, the conserved cysteine is replaced by aspartic acid. The conserved glutamic acid is replaced by glutamine. The conserved histidine is replaced by phenylalanine. A single methionine is conserved in all CtaC subunits of heme copper oxidases.

A. Alignment of the Mg2+ binding motif from CtaD

CtaDI 7002 368 APFDIHVHDTYFVVGHFHYV CtaDI 6803 371 VPFDIHVHDTYFVVGHFHYV CtaDII 7002 382 VPVDIHVNNTYFVVGHFHYV CtaDII 6803 361 APFDLHVNNTYFVVGHFHYV Bos taurus 361 SSLDIVLHDTYYVVAHFHYV S. cerevisiae 361 ASLDVAFHDTYYVVGHFHYV P. denitrificans 396 GSLDRVYHDTYYIVAHFHYV B. subtilis 368 AAADYQFHDTYFVVAHFHYV

B. Alignment of the CuA binding motif from CtaC

CtaCI 7002 274 PVICAELCGSYHGGMKTTMTVETAEGYDQW CtaCI 6803 245 PVICAELCGAYHGGMKSVFYAHTPEEYDDW CtaCII 7002 211 RIRDSQFSGTYFAAMQADVVVESQEDYQTW CtaCII 6803 230 KLHDSQFSGTYFAVMTAPVVVQSLSDYQAW Bos taurus 193 YGQCSEICGSNHSFMPIVLELVPLKYFEKW S. cerevisiae 221 YGACSELCGTGHANMPIKIEAVSLPKFLEW P. denitrificans 213 FGQCSELCGINHAYMPIVVKAVSQEKYEAW B. subtilis 214 FGKCAELCGPSHALMDFKVKTMSAKEFQGW 256

From these observations, it may be concluded that, as in Synechocystis sp. PCC

6803, the CtaDII and CtaCII subunits are similar to the bo-type quinol oxidase subunits,

whereas the counterparts CtaDI and CtaCI are similar to the subunits of the aa3 type cytochrome oxidase (Howitt and Vermaas, 1998).

Both the ctaDI and ctaDII genes of Synechococcus sp. PCC 7002 are dispensable for growth under normal to moderately high light conditions. As in Synechocystis sp.

PCC 6803, the primary cytochrome oxidase is encoded by the ctaCIDIEI gene cluster in

Synechococcus sp. PCC 7002, and the secondary cytochrome oxidase, encoded by the ctaCIIDIIEII gene cluster, seems to contribute little to respiratory processes under the conditions tested. However, unlike Synechocystis sp. PCC 6803, which has a functional quinol oxidase encoded by the cydAB operon, no cydAB-like genes were detected in

Synechococcus sp. PCC 7002 by heterologous Southern blot hybridization using

Synechocystis sp. PCC 6803 cydA or cydB specific probes. The absence of the cydAB genes in Synechococcus sp. PCC 7002 is not surprising based upon the oxygen-uptake data in this study. In Synechocystis sp. PCC 6803, mutagenesis of either cydAB or ctaDIEI singly had no effect on respiratory rates when compared to Synechocystis sp.

PCC 6803 wild type respiratory rates, while a double mutation in these loci had a dramatic effect on oxygen consumption (Howitt and Vermaas, 1998). In marked contrast, the single mutant, ctaDI- in Synechococcus sp. PCC 7002 drastically reduces the rates of oxygen consumption when compared to the Synechococcus sp. PCC 7002 wild- 257 type strain. This is further functional evidence that Synechococcus sp. PCC 7002 lacks a cydAB-type quinol oxidase.

4.6.1 Effects of Cytochrome Oxidase on Electron Flow Around PSII

It is clear from the change in ratios of FV’/FM’ between the wild-type and the ctaDI- and ctaDI- ctaDII- mutant strains of Synechococcus sp. PCC 7002 that electron flow around PSII has been modified. However, this ratio is virtually unchanged in the

- strain with the ctaDII single mutation. FV’/FM’ reflects the photochemical efficiency of open PSII centers in higher plants under a given light acclimation status and represents the combined regulation of PSII via both reversible non-photochemical quenching and photoinhibitory inactivation of PSII (Campbell et al., 1998). However, in cyanobacteria,

changes in FV’/FM’ also combine non-photochemical influences on PSII function as well as photoinhibitory inactivation of PSII (Campbell et al., 1998). It has been documented that cyanobacteria have a high capacity to remove electrons from PSII with oxygen as a terminal acceptor for electron flow from water (Campbell et al., 1998). This electron flow is low under low light conditions, variable but significant at the standard growth light intensity, and large under conditions of excess light (Campbell et al., 1998). This activity is sensitive to cyanide in other cyanobacteria, which also suggests that there is a contribution from the activities from cytochrome oxidase (Schubert et al., 1995). Thus, with the evidence presented here, it is logical to conclude that the primary oxidase,

CtaCIDIEI serves as a buffer from excess excitation of PSII by removing electrons as 258 necessary in Synechococcus sp. PCC 7002. These conclusions are in complete agreement with our physiological results with the ctaDI- mutants and are reflected in the ratio of

FV’/FM’.

4.6.2 Cytochrome Oxidase and its Role in High Light Stress

Under low to moderate light stress conditions, the mutations in the cta loci cause little or no physiological duress to Synechococcus sp. PCC 7002. However, the P700+ reduction kinetics of the mutants were strongly affected, with the half-times of P700+ reduction decreasing dramatically in the ctaDI- strain. This suggests that cytochrome oxidase plays a role within cyanobacterial cells to process the electrons generated by PSII and competes with PSI for electrons. There are no significant differences in the total amount of chlorophyll from the cells of the Cta-deficient mutants when compared to the chlorophyll levels in wild type cells under many different light conditions. An exception to this pattern was observed in the ctaDI- single mutant strain, which failed to grow under

-2 -1 ° extremely high-light stress (4.5 mE m s , 38 C, 1.5% CO2/air). The absence of the primary cytochrome oxidase from these cells places an increased amount of redox stress on the other proteins in the electron transport chain. The resulting increased amount of reductant may have a deleterious effect and cause an overall reduction in the number of functional PSII complexes in the ctaDI- strain at higher light intensities because of oxidative damage. The inability of the ctaDI- cells to manage an increased amount of light stress becomes more evident when the cells are exposed to increasing light 259

intensities. As the light intensities increased, the cells had a gradual loss of PSII as

reflected by a decrease in the 77K fluorescence emission peaks at 685 nm and 695 nm.

These observations lead to the conclusion that the CtaI complex acts as an electron sink,

removing excess electrons to insure the fidelity of the other proteins in the electron

transport chain.

4.6.3 Tolerance of Methyl Viologen and High Light Stress in ctaDII- and

ctaDI- ctaDII- strains of Synechococcus sp. PCC 7002

Unlike the ctaDI- strain of Synechococcus sp. PCC 7002, the ctaDII- strain is able

to tolerate growth at 4.5 mE m-2 s-1. This suggests that the CtaII complex does not play

the same role as the CtaI complex in Synechococcus sp. PCC 7002. Interestingly, the

tolerance to extreme light intensities is observed even in the ctaDI- ctaDII- double mutant

strain. This observation is unusual because ctaDI- strain is sensitive to high light.

Therefore, the ctaDII- strain suppresses one defect associated with the ctaDI mutant. The ctaDI- ctaDII- strain, similar to the ctaDI mutant strain, is severely affected in its ability

to uptake oxygen. Therefore, it is not simply an increased level of respiratory activity

that accounts for the increased tolerance to light stress observed in the ctaDII- mutant

strains, but rather, some unknown mechanism.

An additional observation was that the ctaDII- strains showed a high tolerance

towards oxidative stress based on the tolerance these cells exhibit towards the addition of

methyl viologen to the growth medium. Methyl viologen (Paraquat) is a herbicide that 260

- causes oxidative stress by increasing the amount of superoxide (O2 ) in cells. Mutations in the ctaDII locus are resistant to high levels (50 µM) of MV in the growth medium, whereas wild-type and ctaDI- cells are both sensitive to the addition of MV to the growth medium.

Superoxide dismutase (SOD) is an enzyme that catalyzes the destruction of the

- superoxide (O2 ) radical. SOD has been widely implicated in protecting cells against the harmful effects of active oxygen (Asada, 1999; Storz and Imlay, 1999). There are three types of SOD enzymes that can be distinguished by their metal cofactors at the active site: iron (Fe-SOD), manganese (Mn-SOD), and copper/zinc (Cu/Zn-SOD) (Asada,

1999). Two of the enzymes have been found in cyanobacteria: the iron (Fe-SOD) enzyme and the manganese (Mn-SOD) enzyme (Herbert et al., 1992; Okada et al., 1979).

The SOD activity can be divided into a soluble activity that most likely represents the constitutively expressed Fe-SOD enzyme activity and a membrane fraction containing the

Mn-SOD enzyme (Okada et al., 1979). The genes for both the Fe-SOD (contig 344) and

Mn-SOD (contig 384) have been identified in the sequencing project for the

Synechococcus sp. PCC 7002 genome. The SOD activity assays indicate that there are significantly higher levels of superoxide dismutase activity in both of the ctaDII- strains of Synechococcus sp. PCC 7002. Higher SOD activity would allow cells to tolerate the excess generation of superoxide anion from the MV added to the media. Thus, higher levels of SOD activity partially explain the tolerance of the ctaDII- strains exhibit in the presence of MV. 261

Examination of the activity of oxidative stress enzymes (SOD, catalase, and peroxidase) as well as the accumulation of total hydroperoxides has indicated that only

SOD activity is significantly increased in the ctaDII- strains. Therefore, it can be implied that although the other enzymes linked to oxidative stress may be important to the cells, they are nonetheless not the major determinants for the tolerance to prolonged exposure to high-light stress or to a sudden oxidative stress, specifically the generation of superoxide by MV.

The nucleotide sequence surrounding the ctaDII gene cluster reveals two open reading frames immediately upstream of the ctaCII gene that are homologs of the sll1485 and sll1485 open reading frames in Synechocystis sp. PCC 6803. The function of these open reading frames is unknown at this time. Immediately downstream of the ctaEII gene, there is a partially sequenced open reading frame with similarity to the

Gluconobacter oxydans sldI gene. The sldI gene encodes the large subunit of the sorbitol dehydrogenase. The ctaDII gene was interrupted with the Ω fragment (Prentki and

Krisch, 1984), which has two transcriptional terminators flanking the aadA gene sequence. Thus, substantial increase in the transcription of the open reading frames immediately upstream or downstream of the ctaDII loci is unlikely.

The unusual phenotype of oxidative stress tolerance observed in the ctaDII- strains may be due to an interruption in a signaling pathway in which the secondary cytochrome oxidase is involved. If the CtaDII enzyme is somehow involved in the down-regulation of superoxide dismutase, the absence of CtaDII in the ctaDII- strains could result in cells that contain higher basal levels of the SOD. These higher levels of 262

SOD could be responsible for the tolerance of oxidative stress. It has been shown here that the ctaDII- strains have increased levels of membrane bound SOD activity, but that the levels of other enzymes associated with oxidative stress (catalase and peroxidase) are not affected by this mutation. The use of an oxidase as a signaling transducer is not unprecedented in prokaryotes. An aerotaxis transducer has been found in archaebacteria

(Brooun et al., 1998). In addition, R. sphaeroides contains a cbb3-type cytochrome that is responsible for the repressing expression of photosynthesis gene in the presence of oxygen (O'gara and Kaplan, 1997) and is believed to play a role in aerotaxis signaling

(Armitage et al., 1985). It remains to be determined whether there are other enzymes involved in regulating the levels of membrane bound SOD activity in Synechococcus sp.

PCC 7002.

4.6.4 Future research directions for cta- mutants in Synechococcus sp. PCC

7002

Further characterization of the heme-copper oxidase mutant strains may reveal additional insight into the functions of these enzymes in vivo. If the CtaCIIDIIEII enzyme is acting as a redox response regulator, it is important to identify its redox partners and to determine how it regulates the expression of other genes within cyanobacteria. Now that the genes encoding subunits from three large electron transport enzymes have been cloned, interposon mutagenesis may be performed to help elucidate the physiological function of these enzymes in cyanobacteria. 263

It has been recently found that the ratios of specific carotenoids are altered in response to oxidative stress in plants (Carol and Kuntz, 2001) and R. sphaeroides (O'gara and Kaplan, 1997; O'gara et al., 1998). In both plants and bacteria, these physiological changes are mediated by specific oxidases. In R. sphaeroides, this event is controlled by

the cbb3 oxidase (O’gara and Kaplan, 1997; O’gara et al., 1998) and in Arabidopsis thaliana carotenoid contents are controlled by the plastidal terminal oxidases (PTOX)

(Carol and Kuntz, 2001). Although the overall carotenoid content did not change in the wild-type and cta mutant strains of Synechococcus sp. PCC 7002, the ratios of the different species of carotenoids may have changed. In addition to the production of oxidative stress related enzymes (SOD, peroxidase, catalase), changes in the ratios of carotenoids are also important for adaptation to oxidative stress (Carol and Kuntz, 2001).

The future addition of a photodiode array detector to the Bryant lab will facilitate the determination of differences in the carotenoid contents of the wild-type and cta mutant strains by HPLC analysis.

Figure 57 shows an alternative explanation to the toxic effects observed when ctaDI is inactivated. In the wild-type strain, the genes encoding both CtaCIDIEI and

CtaCIIDIIEII are transcribed and the proteins are presumably produced. When the wild- type cells are grown under 4.5 mE m-2 s-1 constant illumination, no toxicity effect is observed (Figure 57A). Similarly, no toxicity effects are observed in the ctaDII- and ctaDI- ctaDII- strains (Figure 57C and Figure 57D). However, the absence of CtaDI 264

Figure 57. Model for toxicity in ctaD mutant strains grown under 4.5 mE m-2 s-1 constant illumination. (A) The wild-type strain of Synechococcus sp. PCC 7002 is able to produce complete ctaCIDIEI and ctaCIIDIIEII transcripts and products. It is possible that the presence of the CtaCIDIEI complex counteracts any toxic effects from a CtaCIIDIIEII complex. (B) Inactivation of the ctaDI gene still allows the possibility for the expression of the ctaCI gene and production of a hybrid CtaCIDIIEII complex as well as the native

CtaCIIDIIEII complex. Either of these could be toxic to the cell. (C) Inactivation of the ctaDII gene still allows the possibility for the expression of the ctaCII gene and production of a hybrid CtaCIIDIEI complex as well as the native CtaCIDIEI complex.

Either the CtaCIIDIEI complex is not toxic to the cells or the CtaCIDIEI enzyme is able to compensate for toxic effects generated by the hybrid CtaCIIDIEI enzyme. (D)

Inactivation of both the ctaDI and ctaDII genes eliminates the possibility of making either complex, therefore no toxic effect is observed. 265 possible possible toxic transcripts products

A. WT CI DI EI CI DI EI

CII DII EII CII DII EII B. ctaDI- CI DI EI CI DII EII

CII DII EII CII DII EII

C. ctaDII- CI DI EI CI DI EI

CII DII EII CII DI EI

D. ctaDI- ctaDII- CI DI EI no products? CII DII EII 266 singularly is toxic to cells grown under 4.5 mE m-2 s-1 constant illumination. Two possible explanations of these phenomena are: (1) the inactivation of ctaDI eliminates the production of the entire CtaCIDIEI enzyme which, when present, counteracts a toxic effect generated by the CtaCIIDIIEII enzyme or (2) the inactivation of the ctaDI gene eliminates only the CtaDI subunit, but the capacity to produce the CtaCI subunit still exists (Figure 57B).

It is clear from this study that the levels of detectable transcript for ctaCIDIEI are much higher than those of ctaCIIDIIEII. Thus, one could assume, if ctaCI were expressed, that the CtaCI protein could be produced in higher concentrations than the

CtaCII subunit. Based on the similarity between the Cta subunits, one might imagine that the CtaCI subunit could interact and form complexes with the CtaDII and CtaEII subunits. If the CtaCI subunit were functional in these hybrid complexes, it would

presumably be able to accept electrons from cytochrome c6. This could possibly place a higher level of reducing equivalents through what is normally a low activity enzyme

(CtaCIIDIIEII). Perhaps it is the higher levels of reducing equivalents through a hybrid

CtaCIDIIEII enzyme that causes the toxicity observed in the ctaDII- strain.

In the ctaDII- strain, it is impossible to produce either the CtaCIIDIIEII enzyme or the CtaCIDIIEII hybrid enzyme, thus no toxic effects are observed (Figure 57C) and in the ctaDI- ctaDII- strain it is impossible to produce either of the CtaCIIDIIEII or the

CtaCIDIIEII enzyme, therefore, no toxic effects are observed in this strain either (Figure

57D). An experiment where the ctaCI and ctaCII genes were inactivated could answer whether the proposed hybrid CtaCIDIIEII enzyme was toxic to the cells. Inactivation of 267 the ctaCI gene would eliminate the possibility of a CtaCI protein interacting with the

CtaDII and CtaEII subunits, therefore preventing the formation of the hybrid CtaCIDIIEII enzyme. If growth under 4.5 mE m-2 s-1 constant illumination no longer proved fatal to a ctaCI- strain, it would lend credence to the idea that presence of a CtaCIDIIEII enzyme causes toxicity within the cells. However, if growth under constant extreme light intensity were still fatal to the ctaCI- strain, it would lend support to the idea that the

CtaCIIDIIEII enzyme or the product of its reaction is toxic to the cell. It would also suggest that the CtaCIDIEI enzyme is able to counteract the toxicity caused by the

CtaCIIDIIEII enzyme.

This study has established protocols to perform experiments where cyanobacterial cells could be exposed a constant level of either extremely high light stress (growth under

4.5 mE m-2 s-1) or oxidative stress in the presence of methyl viologen. It will be interesting to determine the effects of these stresses on other electron transport mutants, which may now be created based on genomic sequence information. Experiments such as these will allow us to better characterize the stress responses of cyanobacterial cells.

4.7 Concluding Remarks

Information provided by the sequencing of genomes from different organisms allows us to compare the types of genes, number of genes as well as the gene organization of different species. The value of comparative analysis of both gene sequences and organization is evident from this study. Comparing the sequences of 268 predicted gene products allows us to make predictions about the functions of the gene products. Examination of gene organization may also provide a means to identify the function of the genes, especially if these genes are organized in apparent operons. This information provides an invaluable starting point for experimentation.

Although many (40) genes predicted to encode electron transport proteins were identified in this study, it likely does not represent the total number of electron transport proteins within the cell. There are still many open reading frames with unassigned function that have been identified in Synechocystis sp. PCC 6803 and homologs to many of these open reading frames have also been found in Synechococcus sp. PCC 7002.

Some of these unassigned open reading frames may encode electron transport proteins.

Further studies of electron transport proteins will allow the opportunity to discover new functions for these proteins. In this study, phenotypes discovered from the inactivation of the ctaD genes of Synechococcus sp. PCC 7002 have given us new insights to the function of heme-copper oxidases in cyanobacteria. It will be exciting to find out what other electron proteins may be acting in oxidative stress response pathways.

This study has shown that it is dangerous to hold a single organism as the model for all others simply because genomic information is readily available. Two genes characterized in this study (petJ1, ndhB) were found to be essential for viability in

Synechococcus sp. PCC 7002 whereas they were non-essential in other cyanobacteria. It is important to recognize that these lab ‘workhorses’ were once isolated from different environments, and the adaptation to these environments has allowed these organisms to acquire some unique proteins. In studying the differences and similarities of the proteins 269 between organisms, we will better understand how nature uses similar scaffolding to create proteins which fulfill different needs in the cell. 270

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APPENDIX A: RESTRICTION MAPS OF ELECTRON TRANSFER GENES

Restriction maps of Synechococcus sp. PCC 7002 electron transport genes

The restriction enzyme maps for all Synechococcus sp. PCC 7002 genes/clones reported for this study are shown in this appendix. For details, please refer to RESULTS. 296

Type I NADH dehydrogenase genes from Synechococcus sp. PCC 7002

A1. Restriction map of the Synechococcus sp. PCC 7002 ndhAIGE gene cluster

AvaI StyI StyI AvaI

PvuI DraI PvuI StyI StyI DraI EcoRI StyI StyI HincII SacI DraI SspINdeI

StyI HincIIStyI HindIII EcoRI PvuI StyI SspI PvuI HindIII fraH (slr0298 homol.) ndhA ndhI ndhG ndhE hypo. prot. (slr1536 homol.)

200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600

StyI DraI SspI HindIII StyI DraI StyI SmaI PvuI 297

A2. Restriction map of the Synechococcus sp. PCC 7002 ndhB gene

BglII AvaII EcoRI AflIII AflII SapI BamHIBglI SspI EcoRI ndhB Ferredoxin sll1430

300 600 900 1200 1500 1800 2100 2400 2700

XbaI 298

A3. Restriction map of the Synechococcus sp. PCC 7002 ndhD1 gene

PvuI P1174 (1492<-1512) SspI HindIII NcoI

HincII ndhD1 PvuI AvaI SspI PvuI BamHI

200 400 600 800 1000 1200 1400 1600 1800 2000 2200

StyI BglII

A4. Restriction map of the Synechococcus sp. PCC 7002 ndhD2 gene

HindIII

AvaI PvuI

AvaI NcoI PvuI HincII StyI NcoI PvuI PvuI

XbaI StyI sigG slr1546 StyI ndhD2 StyIXbaI

300 600 900 1200 1500 1800 2100 2400 2700 3000 3300

SmaI HincII StyI StyI HincII StyI

SmaI 299

A4. Restriction map of the Synechococcus sp. PCC 7002 ndhF3 ndhD3 genes. Data from (Klughammer et al., 1999).

XbaI PvuI

SspI PvuI KpnI SspI

HincII StyI NcoI NcoI SspI KpnI EcoRI EcoRI EcoRV

PvuI StyI PvuI HincIIStyI PvuI StyI DraI PvuI HindIII StyIStyI DraI sll1735 EcoRI rbcR (sll1594) ndhF3 ndhD3 sll1734 XbaI

400 800 1200 1600 2000 2400 2800 3200 3600 4000 4400 4800 5200 5600 6000 6400

StyI PstI StyIPvuI NdeI StyI PstI HincII EcoRI PvuI PvuI DraIHindIII PvuI HincII

StyI StyI StyI BglII

EcoRV EcoRV 300

A5. Restriction map of the Synechococcus sp. PCC 7002 ndhF4 ndhD4 genes.

KpnI StyI StyI PvuI PvuI

StyIHincII EcoRV SspI PvuI HincII StyI HincII NcoI EcoRI DraI SspI SspI

XbaI ndhF4 ndhD4 slr1302 HindIII

300 600 900 1200 1500 1800 2100 2400 2700 3000 3300 3600 3900 4200 4500

AvaI StyI KpnI PvuI NcoI SacI StyI StyI StyI StyI PvuIPvuIPvuI

KpnI 301

A6. Restriction map of the Synechococcus sp. PCC 7002 ndhF1 gene. Data from (Schluchter et al., 1993).

StyI StyI HincII

NcoI StyI HindIII StyI NcoI HincII

HincII PstI PvuI NcoI BamHI NcoI KpnI AvaI PvuI SmaI NcoI DraI petH ssl3451 StyI PvuI StyI ndhF1 StyIPvuI sll0175 EcoRI

300 600 900 1200 1500 1800 2100 2400 2700 3000 3300 3600 3900 4200 4500

StyI StyI NcoI StyI EcoRV PvuI AvaI SspI StyI

StyI NcoI DraI StyI 302

A7. Restriction map of the Synechococcus sp. PCC 7002 ndhF5 and ndhF2 genes

BamHI SmaI

SmaI HincII DraI NcoI XbaI PvuI NcoI DraI HindIII PvuI PvuI PvuI StyI StyI

PvuI PvuI NcoI StyI NcoI SspI AvaI HincII PvuI NcoI SspI StuI Na+/H+ antiporter (sll0689) slr2010 ssr3410 StuI ndhF5 ndhF2

300 600 900 1200 1500 1800 2100 2400 2700 3000 3300 3600 3900 4200 4500 4800 slr2006

AvaI StyIAvaI EcoRI StyI PvuIStyI StyI StyI SmaI BglII StyI StyI SspI StyI SspI NcoI SspI StyI

SmaI StyI StyI AvaI StyI StuI DraI

HindIII 303

A8. Restriction map of the Synechococcus sp. PCC 7002 ndhCKJ gene cluster

StyI

PvuINcoI StyI StyI

KpnI StyI HP Vng2274c (Halobacterium sp. NRC-1) BamHI SspIHincII ndhC sll0997 HincII ndhK ndhJ HP, sll0606 homol.

500 1000 1500 2000 2500 3000 3500

PvuI StyI DraI SspI PvuIStyI NcoI BglII StyI DraIStuI KpnI XbaI BamHI

HindIII StyI PvuI SspIStyI PvuI BglII PvuI

StyIStyI NcoI 304

A9. Restriction map of the Synechococcus sp. PCC 7002 ndhH gene

PvuI StyI PvuI PvuI StyI BamHI

BamHI EcoRV EcoRI HincII SspI sll0355 ndhH serine esterase

200 400 600 800 1000 1200 1400 1600 1800 2000 2200

PvuI 305

A10. Restriction map of the Synechococcus sp. PCC 7002 ndhL gene

PvuI PvuI

EcoRV PstI NcoI StyI BamHI PvuI NcoI

HindIII StyI slr0815 TrpA (slr0966) HindIII ndhL

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700

StyI PvuI StyI

StyI 306

Type II NADH dehydrogenase genes from Synechococcus sp. PCC 7002

A11. Restriction maps of the Synechococcus sp. PCC 7002 ndbA and ndbB genes 307

StyI

NcoI PvuI AflIII

EcoNI AvaII BstXI XmnI ndbA (slr0851 homolog)

200 400 600 800 1000 1200 1400 1600 1800

HindIII - HindIII construct (pUC19), 2111bp

HincII BglII

HaeII RsaI SpeI XcmI

HindIII AclI NcoI BglI BamHI ndbB (slr1743 homolog)

200 400 600 800 1000 1200 1400 1600 1800

HindIII - BamHI construct (pBluescript II SK+) 308

Hydrogenase gene cluster genes from Synechococcus sp. PCC 7002

A12. Restriction map of the hydrogenase gene cluster from Synechococcus sp. PCC 7002.

NcoI

EcoRV SmaI EcoRI

DraI EcoRV DraI SspI HindIII

HincII PvuI StyI StyI NcoI SspI HincII DraI DraI StyI

NcoI NcoI PvuI NcoIBglII StyI PvuI EcoRI StyI PvuI DraI PvuI DraI PvuI PvuI PvuI StyI PvuI PvuI

PvuI StyI PvuI PvuI NcoI SspI HindIIIHincII BglII DraIEcoRV HincII DraIBglII DraI StyI PvuI SspI SspIPvuI PvuI SmaI hoxW XbaI NcoI KpnI StyI PvuI PvuI StyI PvuI EcoRV XbaI EcoRV StyI DraI SspI BglII DraI

UreC hypE hoxE hoxF hoxU hoxY hyp3 hoxH hypA hypB hypF hypC hypD

1K 2K 3K 4K 5K 6K 7K 8K 9K 10K 11K 12K 13K 14K

DraI PvuI DraIHincIIStyIPvuI EcoRI BamHI StyI PvuI SspI PvuISspI PvuI PvuISspI HincII PvuI StyI NcoI StyI AvaI

StyI StyI PvuI NcoI PvuI EcoRV StyI HindIII DraI EcoRI SspI SspI SspI PvuI StyI

SspI StyI StyI HindIIIHindIII PvuI HindIII

PvuI AvaI HincII StyI

DraI

StyI 309

Mobile electron transport genes from genes from Synechococcus sp. PCC 7002

A13. Restriction map of petJ1 gene from Synechococcus sp. PCC 7002

HindIII

StyI SspI PvuI BamHI

PvuI SacI AvaI PvuI

AvaI sll0185 homolog petJ1 sll0415 ABC transporter PvuI

300 600 900 1200 1500 1800 2100 2400 2700 3000 3300

EcoRV PvuI KpnI HincII PvuI StyI StyI HindIII

SspI XbaI KpnI 310

A14. Restriction map of the petJ2 gene from Synechococcus sp. PCC 7002

PvuI PvuI

BglII XbaI DraI AvaI NcoI

PvuI StyI PvuI SspI sll0102 algI (alginate-o-acetyltransferase) slr0436 ccmK-1 StyI petJ2 HincII

300 600 900 1200 1500 1800 2100 2400 2700 3000

BamHI PstI SacI HindIII SspI SmaI BglII

StyI StyI 311

A15. Restriction map of the cytM gene from Synechococcus sp. PCC 7002

PvuI

StyI PvuI slr1215 BglII

PvuI StyI sll1434-penicillin binding pr… cyt M sll1704 csGA (cell-cell signaling factor C)

300 600 900 1200 1500 1800 2100 2400 2700

NcoI PvuI DraI StyI StyI PvuI StyI XbaIPvuI StyI

PvuI PvuI StyI

PvuI 312

A16. Restriction map of the bcpA gene from Synechococcus sp. PCC 7002

BglII HindIII SspI NcoI EcoRI StyI

StyI HincII XbaI PvuI SspI DraI PvuI PvuI PvuI BamHI StyI

PvuI StyI PvuI SmaI StyI KpnI PvuI StyI KpnI DraI HincII EcoRV StyI StyIHincII pepM-slr0918 hypothetical protein StyI HincII HincII HincIIEcoRI AvaI StyI PvuI StyI StuI HincIIDraI bioF gene-slr0917 blue-copper protein EcoRV trigger factor-sll0533 ilvC gene-sll1363 DNA polymerase I-slr0707 hypothetical-sll1858 mfd gene-sll0377 StuI SspI PvuI

800 1600 2400 3200 4000 4800 5600 6400 7200 8000 8800 9600 10400 11200 12000 12800 13600

PvuI XbaI DraI NcoI PvuI HincII BamHI DraI AvaI XbaISspIXbaI SspI SmaI EcoRI hypothetical protein hypothetical-slr0204glnB gene PvuI SspI

StuI EcoRI StyI NcoI SspI PvuI SspISmaI AvaI NcoI StuI PvuI PvuIStyI DraI PvuI DraI PvuIBamHI

HincIIKpnI StyI SspI XbaI XbaIStyI HindIII DraI StyI BamHI HincII PvuI NcoIStyI

StyI PstI HincII DraI PvuI HincII NcoI

StyI EcoRV PvuI 313

Heme-copper oxidase genes from Synechococcus sp. PCC 7002

A17. Restriction map of the ctaCIDIEI operon from Synechococcus sp. PCC 7002

PvuI

StyI

BamHI StyI SspI

KpnI StyI StyI HincII PvuI StyI XbaI

SacI PvuI HincII NcoI PvuI NcoI StuI PvuI BglIIEcoRV NcoI PvuI NcoI PstI

EcoRI sll1898 DraI ctaCI ctaDI ctaEI PvuIDraI slr1542 SphI

300 600 900 1200 1500 1800 2100 2400 2700 3000 3300 3600 3900 4200 4500 4800

AvaI SspI NcoI StyI PvuI StyI StyI StyI HincII

SmaI NcoI BamHI

NcoI HincII

StyI 314

A18. Restriction map of the ctaCIIDIIEII operon from Synechococcus sp. PCC 7002

PvuI

XbaI NcoI

SmaI PvuI PvuI SspI PvuI PvuI PvuI PvuI

AvaI EcoRI DraI PvuI PvuI NcoI PvuI SspI NdeI HincII PvuI PvuI DraI sll1485 sll1486 ctaCII ctaDII ctaEII sldI

300 600 900 1200 1500 1800 2100 2400 2700 3000 3300 3600 3900 4200 4500 4800 5100

BamHI NdeI StyI EcoRV StyI

HincII StyI 315

APPENDIX B: CLUSTALW ALIGNMENTS OF ELECTRON TRANSPORT PROTEINS

The ClustalW amino acid alignments for the Synechococcus sp. PCC 7002 electron transport proteins are presented in this appendix. For details, please see

RESULTS.

Figure B1. ClustalW alignment of NdhA amino acid sequences. NdhA amino acid sequences of the following organisms were aligned using the ClustalW alignment program from MacVector v. 6.5: Synechococcus sp. PCC 7002, Synechocystis sp. PCC

6803, Anabaena sp. PCC 7120, Spinacia oleracea, and E. coli. The consensus amino acid sequence is shown underneath. Gray high-lighted regions indicate identical amino acids while lighter shading indicates conserved amino acids. Dashes represent insertions/deletions included to maximize the sequence similarity. The percent identity and percent conserved amino acids were determined with the ClustalW alignment tool.

(Thompson et al., 1994b). 316

NdhA ClustalW Amino Acid Alignment

10 20 30 40 50 NdhA-7002 MNSGIDLQGSFIETLQSFGLSHEIAKTIWLPLPLLLMIIGATVGVLVVV NdhA-6803 MTSGIDLQNSFLQSLQGFGLPPGLAKLFWIPLPSILMIIGATVGVLVVV NdhA-7120 MNSGIDLQGTFIKSLIDLGIPPGTAKAIWMPLPMILMLIGATVGVLVCV NdhA-Spinacia oleracea MIIDTTTTKVQAINSFSRLEFLKEVYETIWMLFPILILVLGITIGVLVIV NuoH-E. coli MSWISPELIEILLTILKAVVILLVVVTCGAFMS M SGIDLQ..FI SL G. PE.AK.IW PLP..LM.IGATVGVLV.V

60 70 80 90 100 NdhA-7002 WLERKISAAAQQRVGPEYAGPLGVLQPVADGLKLVFKEDVVPAKTDPWLF NdhA-6803 WLERKISAAAQQRIGPEYAGPLGVLQPVADGIKLVFKEDVVPAKADPWLF NdhA-7120 WLERKISAAAQQRIGPEYIGPLGLLAPVADGLKLVFKEDIVPAQADPWLF NdhA-Spinacia oleracea WLEREISASIQQRIGPEYAGPLGILQALADGTKLLFKENLLPSRGDTYLF NuoH-E. coli FGERRLLGLFQNRYGPNRVGWGGSLQLVADMIKMFFKEDWIPKFSDRVIF WLERKISAAAQQRIGPEYAGPLG.LQPVADG.KLVFKED.VPA..DPWLF

110 120 130 140 150 NdhA-7002 TLGPALVVIPVFLSYLIVPFGQNLVITDLNVGIFLWISLSSIAPIGLLMS NdhA-6803 TLGPVLVVLPVFLSYLIVPFGQNLVITDINVGIFLWIALSSIAPIGLLMS NdhA-7120 TLGPILVVLPVFLSYLIVPFGQNIVITNVGTGIFLWIALSSIQPIGLLMA NdhA-Spinacia oleracea SIGPSIAVISILLGYLIIPFGSRLVLADLSIGVFLWIAVSSIAPIGLLMS NuoH-E. coli TLAPMIAFTSLLLAFAIVPVSPGWVVADLNIGILFFLMMAGLAVYAVLFA TLGP.LVV.PVFLSYLIVPFGQNLVITDLN.GIFLWIALSSIAPIGLLMS

160 170 180 190 200 NdhA-7002 GYSSNNKYALLGGLRAAAQSISYEIPLALAVLAIAMMSNSLSTIDIVEQQ NdhA-6803 GYASNNKYSLLGGLRAAAQSISYEIPLSLAVLAIVMMSNSLSTIDIVDQQ NdhA-7120 GYSSNNKYSLLGGLRAAAQSISYEIPLALSVLAIVMMSNSLSTVDIVNQQ NdhA-Spinacia oleracea GYGSNNKYSFLGGLRAAAQSISYEIPLTLCVLSISLLSNSSSTVDIVEAQ NuoH-E. coli GWSSNNKYSLLGAMRASAQTLSYEVFLGLSLMGVVAQAGSFNMTDIVNSQ GYSSNNKYSLLGGLRAAAQSISYEIPL.L VLAIVMMSNSLST.DIV.QQ

210 220 230 240 250 NdhA-7002 SGYGILGWNIWRQPVGFLIFWIAALAECERLPFDLPEAEEELVAGYQTEY NdhA-6803 SGYGILGWNIWRQPVGFLIFWIAALAECERLPFDLPEAEEELVAGYQTEY NdhA-7120 SGYGILGWNIWRQPLGFMIFWIAALAECERLPFDLPEAEEELVAGYQTEY NdhA-Spinacia oleracea SKYGFWGWNLWRQPIGFIVFIISSLAECERLPFDLPEAEEELVAGYQTEY NuoH-E. coli A----HVWNVIPQFFGFITFAIAGVAVCHRHPFDQPEAEQELADGYHIEY SGYGILGWNIWRQP.GF.IFWIAALAECERLPFDLPEAEEELVAGYQTEY

260 270 280 290 300 NdhA-7002 AGMKFGLFYVGSYVNLVLSALIVSILYLGGWEFPIPLDKLAGWLNVAPST NdhA-6803 AGMKFALFYLGSYVNLVLSALVFSVLYLGGWDFPIPLENVANWLGVAPTT NdhA-7120 SGMKFALFYLSSYVNLILSALLVAVLYLGGWDFPIPINVLANLVGVSEAN NdhA-Spinacia oleracea SGIKFGLFYVASYLNLLISSLFVTVLYLGGWNLSIPYIFIS---EFFEIN NuoH-E. coli SGMKFGLFFVGEYIGIVTISALMVTLFFGGWQGPLLPPFIW------SGMKFGLFYVGSYVNLVLSAL.V.VLYLGGW.FPIP. .A . V

310 320 330 340 350 NdhA-7002 PWLQVITASLGIIMTLVKTYALVFIAVLLRWTLPRVRIDQLLNFGWKFLL NdhA-6803 SWLQVLMAALGITMTVLKSYFLIFIAILLRWTVPRVRIDQLLNLGWKFLL NdhA-7120 PVLQVVSAALGITMTLVKAYFLVFIAILLRWTVPRVRIDQLLDLGWKFCY NdhA-Spinacia oleracea KIDGVFGTTIGIFITLAKTFLFLFIPITTRWTLPRLRMDQLLNLGWKFLL NuoH-E. coli ------FALKTAFFMMMFILIRASLPRPRYDQVMSFGWKICL LQV. A LGI MTL.KTYFL.FIAILLRWTLPRVRIDQLLNLGWKFLL

360 370 380 390 400 NdhA-7002 PVALVNLLLTAALKLAFPIAFGG NdhA-6803 PVALANLLITAALKLTFPMAFGG NdhA-7120 QLVSEPTFNRQPLKLAFPRPSAG NdhA-Spinacia oleracea PISLGNLLLTTSSQLFSL NuoH-E. coli PLTLINLLVTAAVILWQAQ P..L.NLL.TAALKL FP .G 317

Figure B2. ClustalW alignment of NdhI amino acid sequences. NdhI amino acid sequences of the following organisms were aligned using the ClustalW alignment program from MacVector v. 6.5: Synechococcus sp. PCC 7002, Synechocystis sp. PCC

(Thompson et al., 1994b). 318

NdhI ClustalW Amino Acid Alignment

10 20 30 40 50 NdhI-7002 MFKILKQVGDYAKDAAQAAK------YIGQGLSV NdhI-6803 MFNNILKQVGDYAKESLQAAK------YIGQGLAV NdhI-7120 MLKFLKQVGDYAKEAVQAGR------YIGQGLSV NdhI-Spinacia oleracea MFPMVTGFINYGQQTIRAAR------YIGQSFMI NuoI-E. coli PGNVLRLENLPAADADQLAGNGGCHSLAGAIRGNKTMTLKELLVGFGTQV M .LKQVGDYAK...QAA. YIGQGL V

60 70 80 90 100 NdhI-7002 -----TFDHMRRRPVTVQYPYEKLIPSERFRGRIHFEFDK-----CIACE NdhI-6803 -----TFDHMSRRPITVQYPYEKLIPSERFRGRIHFEFDK-----CIACE NdhI-7120 -----TFDHMRRRPVTVQYPYEKLIPGERFRGRIHYEFDK-----CIACE NdhI-Spinacia oleracea -----TLSHANRLPVTIQYPYEKLITSERFRGRIHFEFDK-----CIACE NuoI-E. coli RSIWMIGLHAFAKRETRMYPEEPVYLPPRYRGRIVLTRDPDGEERCVACN TFDHM RRPVTVQYPYEKLIPSERFRGRIHFEFDK CIACE

110 120 130 140 150 NdhI-7002 VCVRVCPINLPVVDWEFDKSIKKKTLKHYSIDFGVCIFCGNCVEYCPTNC NdhI-6803 VCVRVCPINLPVVDWEFNKAVKKKELKHYSIDFGVCIFCGNCVEYCPTNC NdhI-7120 VCVRVCPINLPVVDWEFDKATKKKKLNHYSIDFGVCIFCGNCVEYCPTNC NdhI-Spinacia oleracea VCVRACPIDLPVVDWKLETDIRKKRLLNYSIDFGICIFCGNCVEYCPTNC NuoI-E. coli LCAVACPVGCISLQKAETKDGR-WYPEFFRINFSRCIFCGLCEEACPTTA VCVRVCPINLPVVDWEF.K .KKK L HYSIDFGVCIFCGNCVEYCPTNC

160 170 180 190 200 NdhI-7002 LSMTEEYELATYDRHELNYDN-VALGRLPYKVTQDPMVTPLRELAYLPQG NdhI-6803 LSMTEEYELAAYDRHDLNYDN-VALGRLPYKVTEDPMVTPLRELGYLPKG NdhI-7120 LSMTEEYELATYDRHELNYDS-VALGRLPYKVTDDPMVTPLRELVYLPKG NdhI-Spinacia oleracea LSMTEEYELSTYDRHELNYNQ-IALGRLPISITDD------YTIRT NuoI-E. coli IQLTPDFEMGEYKRQDLVYEKEDLLISGPGKYPEYN------FYRMAGM LSMTEEYELATYDRHELNYD. VALGRLPYKVT.DPMVTPLREL.YLP.G

210 220 230 240 250 NdhI-7002 VIDPHDLPQGSQRAGEHPEDILERLQAEKQQETADQ NdhI-6803 VIEPHNLPKGSQRAGQHPEDLVKAE NdhI-7120 VLDPHDLPANAPRPGARPEDLVEKTEA NdhI-Spinacia oleracea ILN---SPQTKEKACD NuoI-E. coli AID--GKDKGEAENEAKPIDVKSLLP VIDPH LP G RAG .PED.. 319

Figure B3. ClustalW alignment of NdhG amino acid sequences. NdhG amino acid

sequences of the following organisms were aligned using the ClustalW alignment

program from MacVector v. 6.5: Synechococcus sp. PCC 7002, Synechocystis sp. PCC

(Thompson et al., 1994b). 320

NdhG ClustalW Amino Acid Alignment

10 20 30 40 50 NdhG-7002 VNLAEGVQI VSFVI LTVMMLGSALGVVLFENI VYSAFLLGGVFI SI SGFY NdhG-6803 MNLAEGVQYI SFLI LAFLVI GAALGVVLLSNI VYSAFLLGGVFLSI SGI Y NdhG-7120 MNLAEGVQVVPFGI LATMLI GPALGVVLATSI VYSAFLLGGVFI SI AGMY NdhG-Spinacia oleracea MDLPGPI HDFLLVFLGSGLI LGALGVVLFTNPI FSAFSLGLVLVCI SLFY NuoJ-E. coli MEFAFYICGLIAILATLRVITHTNPVHALLYLIISLLAISGVF MNLAEGVQ . F.IL. ..IG.ALGVVL TNIVYSAFLLGGVF. SI SG Y

60 70 80 90 100 NdhG-7002 I LLNADFVAAAQVLI YVGAI NVLI LFAI MLVNKKEDFSQMPGRLLRQGAT NdhG-6803 I LLNADFVAAAQVLVYVGAVSVLI LFAI MLVNKREDFSKI PGRWLRNVST NdhG-7120 LLLNGDFVAAAQVLVYVGAVNVLI LFAI MLVNKRQDFTPYPSAGI RKVLT NdhG-Spinacia oleracea I LANSHFVASAQLLI YVGAI NVLI I FSVMFMSGPEYDKKFQLWTVGDGVT NuoJ-E. coli FSLGAYFAGALEI I VYAGAI MVLFVFVVMMLN----LGGSEIEQERQWLK I LLNADFVAAAQVLVYVGAI NVLI LFAI MLVNK. EDF. P. . R...T

110 120 130 140 150 NdhG-7002 ALVCLGLFALLGTMVLVTPWE------LSTISPAAVGNSVVAI AKHFFS NdhG-6803 ALVCTGIFALLSTMVLITPWQ------INETGPFVEN-TLVTIGKHFFS NdhG-7120 AI VSVGLFALLSTMVLATPWA------YSTTPKVGDG- SI I VI GEHFFS NdhG-Spinacia oleracea SLVCI SLFVSLI STI LNTSWYGI I WTTKSNQI LEQDLI NASQQI GI HLST NuoJ-E. coli PQVWI GPAI LSAI MLVVI VYAI LG----VNDQGIDGTPISAKAVGI TLFG ALVC.GLFALL.TMVL.TPW .N . . . S...IG HFFS

160 170 180 190 200 NdhG-7002 DFLLPFELASVLLLI AMVGAI I LARRDI I PEI TTTEEGSTGLQLPERPRE NdhG-6803 DYLLPFELASVLLLMAMVGAI I LARRDL I PEL SEENKTATALTLPERPRE NdhG-7120 DFLLPFELASVLLLMAMVGAI I LARREYLPEVTPI WLTSTVLTLPERPRE NdhG-Spinacia oleracea DFFLPFELI SI I LLVSLI GAI AVARQ NuoJ-E. coli PYVLAVELASMLLLAGLVVAFHVGREERAGEVLSNRKDDSAKRKTEEHA DFLLPFELASVLLL. AMVGAI I LARR. . PE. . T. L LPERPRE

210 220 230 240 250 NdhG-7002 LAGANSPKSKQN NdhG-6803 LTSASK NdhG-7120 L VGAGSET QE NdhG-Spinacia oleracea NuoJ-E. coli LA 321

Figure B4. ClustalW alignment of NdhE amino acid sequences. NdhE amino acid

sequences of the following organisms were aligned using the ClustalW alignment

program from MacVector v. 6.5: Synechococcus sp. PCC 7002, Synechocystis sp. PCC

(Thompson et al., 1994b).

NdhE ClustalW Amino Acid Alignment

10 20 30 40 50 NdhE-7002 MQIQLEYI LVLAAALFCI GIYGLVTSRNAVRVLMSI ELL NdhE-6803 MHLQLQYCLI LAAALFCI GIYGLITSRNAVRVLMSI ELL NdhE-7120 MQLRYFLLLAAALFCI GIYGLITSRNAVRVLMSI ELL NdhE-Spinacia oleracea MILEHVLVLSAFLFSI GIYGLVTSRNLVRALMCLELI NuoK-E. coli RRQREKKNGGARMI PLQHGLI LAAI LFVLGLTGLVI RRNLLFMLI GLEI M . QL Y. L. LAAALFCI GI YGLVTSRNAVRVLMSI ELL

60 70 80 90 100 NdhE-7002 LNSVNLNFMGFSNFLDPGEI KGQVFTVFVLTVAAAEAAVGLAI I LAI YRN NdhE-6803 LNAVNLNLMGFSNYLDPSNI RGQVFAI FVI TI AAAEAAVGLAI VLAI YRN NdhE-7120 LNAVNLNLMAFSNYLDSTLI KGQVFTVFVI TVAAAEAAVGLAI VLAI YRN NdhE-Spinacia oleracea LNAVNLNFVTFSDFFDSRQLKGNI FSI FVI AI AAAEAAI GPAI VSSI YRN NuoK-E. coli I NASALAFVVAGSYWG- - QTDGQVMYI LAI SLAAAEASI GLALLLQLHRR LNAVNLNFM. FSNYLD . I KGQVF. I FVI T. AAAEAAVGLAI VLAI YRN

110 120 130 140 150 NdhE-7002 RNTI DMEQFNL L KN NdhE-6803 RETTDMEQFNL L KW NdhE-7120 RDTVDMEQFNL L KW NdhE-Spinacia oleracea RKSI RI NQSNL L NK NuoK-E. coli RQNL NI DSVSEMRG RT.DMEQFNLLK 322

Figure B4. ClustalW alignment of NdhB amino acid sequences. NdhB amino acid sequences of the following organisms were aligned using the ClustalW alignment program from MacVector v. 6.5: Synechococcus sp. PCC 7002, Synechocystis sp. PCC

6803, Synechococcus sp. PCC 7942, Anabaena sp. PCC Spinacia oleracea, and E. coli.

The consensus amino acid sequence is underneath. Gray high-lighted regions are identical amino acids while lighter shading indicates a conserved amino acid. A dash is equivalent to a gap in the sequence. Percent identity and percent conserved amino acids determined by the ClustalW search program (Thompson et al., 1994b). 323

NdhB ClustalW Amino Acid Alignment

10 20 30 40 50 NdhB-7002 MDFSSTVASQLYTGAI LPESI VI LTLI VVLVGDL NdhB-6803 MDFSSNVAAQLNAGTI LPEGI VI VTLLLVLI VDL NdhB-7942 MDFLT- LAGQLNAGVI LPEGI VI VTLLTVLVTDL NdhB-7120 MDFAN- LAAQLNAGTI LPEGI VI VTLMGVLI VDL NdhB-Spinacia oleracea MIWHVQNENFILDSTRIFMKAFHLLLFDGSLIFPECILIFGLILLLMIDS NuoN-E. coli MDF .A.QL .G ILPE IVI.TL..VL..DL

60 70 80 90 100 NdhB-7002 I VGRARSGWI PYAAI AGLLGSVFALYLGWDNPHPVAFLGAFNSDNLSI LF NdhB-6803 I GGRKVALALPYLAI AGLLVSVGLLVTSWSMADPI GFI GAFNGDNLSI I F NdhB-7942 I LGRQSLRLTPALAI TGLSAAI AVLTLQWNTSQNLAFLGGFNGDNLSI VF NdhB-7120 I L GRT SSRWI GYL AI AGL L AAI VAL YF QWDAT NPI SF T GAF I GDDL SI I F NdhB-Spinacia oleracea TSDQKDI PWLYFI SSTSLVMSI TALLFRWREEPMI SFSGNFQTNNFNEI F NuoN-E. coli MDVTPLMRVDGFAMLY I.GR ...AIGL....L W .FG.F..DNLSI.F

110 120 130 140 150 NdhB-7002 RGI I VLSTAFTI MMSVRYVERSGTALSEFI CI LLTATLGGMFLSGANELV NdhB-6803 RAI I ALSTVVTI LMSVRYVQQTGTSLAEFI AI LLTATLGGMFLSAANELV NdhB-7942 RGI VLLSAAVTI LLSI RYVEQSGTSLGEFI TI LLTASLGGMFLSGANELV NdhB-7120 RGI I ALSAVVTI LMSI RYVEQSGTALAEFI AI LLTATLGGMFVSGASELV NdhB-Spinacia oleracea QFLI LLCSTLCI PLSVEYI ECTEMALTEFLLFI LTATLGGMFLCGANDLI NuoN-E. coli TGLVLLASLATCTFAYPWLEGYNDNKDEFYLLVLI AALGGILLANANHLA RGI I . LS. . . TI S. RYVE . GT L EFI . I LLTATLGGMFLSGANELV

160 170 180 190 200 NdhB-7002 MI FVSLEMLSI SSYLLTGYMKRDPRSNEAALKYLLI GAASSAI FLYGVSL NdhB-6803 MVFI SLEMLSI SSYLMTGYMKRDPRSNEAALKYLLI GASSSAI FLYGLSL NdhB-7942 TI FVSLETLSI SSYLLTGYMKRDPRSNEAALKYLLI GAASSAI FLYGVSL NdhB-7120 MI FI SLETLSI SSYLLTGYTKRDPRSNEAALKYLLI GASSTAVFLYGVSL NdhB-Spinacia oleracea TI FVAPECFSLCSYLLSGYTKKDVRSNEATTKYLLMGGASSSI LVHGFSW NuoN-E. coli SLFLGI ELI SLPLFGLVGYAFRQKRSLEASI KYTI LSAAASSFLLFGMAL I F. SLE LSI SSYLLTGY KRDPRSNEAALKYLLI GAASSAI FLYG. SL

210 220 230 240 250 NdhB-7002 LYGLSGGKTI LSEI ALGFTDPQ- GGQSLALAI ALVFAI AGI AFKI SAVPF NdhB-6803 LYGLSGGETQLVLI AEKLVNADTVGQSLGLAI ALVFVI AGI AFKI SAVPF NdhB-7942 LYGLAGGETQLPAI AEKLGEAQ- - - - PLALLI SLI FVI AGI AFKI SAVPF NdhB-7120 LYGLSGGQTELNAI ANGI I TAN- VGQSLGAVI ALVFVI AGI GFKI SAAPF NdhB-Spinacia oleracea LYGSSGGEI ELQEI VNGLI NTQMYN- SPGI SI ALI FI TVGI GFKLSPAPS NuoN-E. coli VYAQSG-- - DLSFVALGKNLGDGMLNEPLLLAGFGLMIVGLGFKLSLVPF LYGLSGGT L IA G...... SL.L.IAL.F.IAGI.FKISAVPF

260 270 280 290 300 NdhB-7002 HQWTPDVYEGSPTPVVAFLSVGSKAAGFALAI RLLVTVFNPVSEEWHFI F NdhB-6803 HQWTPDVYEGSPTPVVAFLSVGSKAAGFAVAI RLLVTAFGGI TDEWHVI F NdhB-7942 HQWTPDVYEGSPTPI VAFLSVGSKAAGFALAI RLLVTAYPALTEQWHFVF NdhB-7120 HQWTPDVYEGAPTPVI AFLSVGSKAAGFALAI RLLTTVFPFVAEEWKFVF NdhB-Spinacia oleracea HQWTPDVYEGSPTPVVAFLSVTSKVAASASATRI FDI PFYFSSNEWHLLL NuoN-E. coli HLWTPDVYQGAPAPVSTFLATASKI AI FGVVMRLFLYAPVGDSEAI RVVL HQWTPDVYEGSPTPVVAFLSVGSKAAGFA. AI RLL. T. F . . EEWH . F 324

310 320 330 340 350 NdhB-7002 TALAI LSMVLGNVVALAQTSMKRMLAYSSI AQAGFVMI GLVAG- - TDAGY NdhB-6803 TALAVLSMVLGNVVALAQTSMKRMLAYSSI GQAGFVMI GLVAG- - SEDGY NdhB-7942 TALAI LSLVLGNVVALAQTSMKRLLAYSSI AQAGFVMI GLI AG- - TEAGY NdhB-7120 TALAVLSMI LGNVVALAQTSMKRMLAYSSI AQAGFVMI GLI AG- - TDAGY NdhB-Spinacia oleracea EI LAI LSMI LGNLI AI TQTSMKRMLAYSSI GQI GYVI I GI I VGD- SNDGY NuoN-E. coli AI I AFASI I FGNLMALSQTNI KRLLGYSSI SHLGYLLVALI ALQTGEMSM TALA. LSM. LGNVVALAQTSMKRMLAYSSI . QAGFVMI GLI AG . . GY

360 370 380 390 400 NdhB-7002 SSVI FYLLVYLFMNLGAFTCVI LFSLRT- G- - TDQI AEYAGLYQKDPLLT NdhB-6803 ASMVFYMLI YLFMNLGAFSCI I LFTLRT- G- - SDQI SDYAGLYHKDPLLT NdhB-7942 SSMVYYLLI YLFMNLGGFACVI LFSLRT- G- - TDQI SEYAGLYQKDPLVT NdhB-7120 ASMI FYLLVYLFMNLCGFTCI I LFSLRT- G- - TDQI AEYSGLYQKDPLLT NdhB-Spinacia oleracea ASMI TYMLFYI SMNLGTFACI VLFGLRT- G- - TDNI RDYAGLYTKDPFLA NuoN-E. coli EAVGVYLAGYLFSSLGAFGVVSLMSSPYRGPDADSLFSYRGLFWHRPI LA SM. . YLL. YLFMNLG. F C. I LFSLRT G TDQI . YAGLY KDPLLT

410 420 430 440 450 NdhB-7002 LGLSVCLLSLGGIPPLAGFFGKI YLFWAGWQAGLYGLVLLGLI TSVI SI Y NdhB-6803 LGLSI CLLSLGGI PPLAGFFGKI YI FWAGWQSGLYGLVLLGLVTSVVSI Y NdhB-7942 LGLSLCLLSLGGIPPLAGFFGKLYLFWAGWQAGLYGLVLLALI TSVI SI Y NdhB-7120 LGLSI SLLSLGGIPPLAGFFGKI YLFWAGWQAGLYWLVLLGLVTSVI SI Y NdhB-Spinacia oleracea LSLALCLLSLGGLPPLAGFFGKI YLFWCGWQAGLYFLVLI GLLTSVLSI Y NuoN-E. coli AVMTVMMLSLAGI PMTLGFI GKFYVLAVGVQAHLWWLVGAVVVGSAI GLY L GL S. CL L SL GGI PPL AGF F GKI YL F WAGWQAGL Y L VL L GL . T SVI SI Y

460 470 480 490 500 NdhB-7002 YYI RVI KMMVVKEPQEMSDSVRNYPAVTWTAVGMKPLQVGLVLSVI I TSL NdhB-6803 YYI RVVKMMVVKEPQEMSEVI KNYPAI KWNLPGMRPLQVGI VATLVATSL NdhB-7942 YYI RVI KMMVVKEPQEMSESVRNYPETNWNLPGMQPLRAGLVVCVI ATAV NdhB-7120 YYI RVVKMMVVKEPQEMSDVVKNYPEI RWNLPGFRPLQVGLVLTLI ATSV NdhB-Spinacia oleracea YYLKI I KLLMTGRNQEI TPHVRNYRRS- - PLRSKNSI EFSMI VCVI ASTI NuoN-E. coli YYLRVAVSLYLHAPE- - - QPGRDAPSN- WQYSAGG- - - I VVLI SALLVLV YYI RV. KMMVVKEPQEMS. VRNYP W. L G PL . G. V. . . I AT. .

510 520 530 540 550 NdhB-7002 AGI LSNPLFVI ADQSVTS- - - TPMLQVANHPTEQVVAQVDSELVGVAI AD NdhB-6803 AGI LANPLFNLATDSVVS- - - TKMLQTALQQTGETPAI AI SHDLP NdhB-7942 AGI LSNPLFNLASASVSGSSFLGLAPAAEVVTTTATPVALSEPPAAS NdhB-7120 AGI LSNPLFTLANNSVAN- - - TAI LQATKVVSTQVSAI PAEKPDGLN NdhB-Spinacia oleracea PGISMNPIITIAQDTLF NuoN-E. coli LGVWPQPLI SI VRLAMPLM AGIL NPLF .A SV . .

560 570 580 590 600 NdhB-7002 H NdhB-6803 NdhB-7942 NdhB-7120 NdhB-Spinacia oleracea NuoN-E. coli 325

Figure B5. ClustalW alignment of NdhD1 amino acid sequences. NdhD1 amino acid sequences of the following organisms were aligned using the ClustalW alignment program from MacVector v. 6.5: Synechococcus sp. PCC 7002, Synechocystis sp. PCC

(Thompson et al., 1994b). 326

NdhD1 ClustalW Amino Acid Alignment

10 20 30 40 50 NdhD1-7002(slr0331) MNFANFPWLSTI I LFPI I AALFLPLI PDKDGKTVRWYALTI GLI DFVI I V NdhD1-7120(slr0331) MNTANFPWLTTI I LLPI AASLLI PI I PDKDGKTI RWYALTVGLI DFALI V NdhD1-6803(slr0331) MNTFPWLTTI I LLPI VAALFI PI I PDKDGKTVRWYSLAVGLVDFALI V NdhD-Spinacia oleracea MIELLLMT NuoM-E. coli MLLPWL---ILIPFIGGFLCWQTERFGVKVPRWI ALI TMGLTLALSL FPWL.TIIL.PI.A.L .P.IPDKDGKT.RWYAL .GLIDFALIV

60 70 80 90 100 NdhD1-7002(slr0331) TAFYTG-YDFGN----PNLQLVESYTWVEAI DLRWSVGADGLSMPLI LLT NdhD1-7120(slr0331) YAFYTS-YDFAN----PDLQLVESYPWVPQLDLNWSVGADGLSMPLI I LT NdhD1-6803(slr0331) YAFYSG-FDLSE----PGLQLVESYTWLPQI DLKWSVGADGLSMPLI I LT NdhD-Spinacia oleracea YVFFYH-FQPDD----PLIQLVEDYKWINFFDFHWRLGIDGLSIGPILLT NuoM-E. coli QLWLQGGYSLTQSAGI PQWQSEFDMPWI PRFGI SI HLAI DGLSLLMVVLT YAFY. G YD . P LQLVESY W. P . DL. WSVGADGLSMPLI . LT

110 120 130 140 150 NdhD1-7002(slr0331) GFI TTLAI LAAWPVSFKPK- LFYFLMLLMYGGQI AVF AVQDML L FFFTWE NdhD1-7120(slr0331) GFI TTLATLAAWPVTLKPR-LFYFLLLAMYGGQI AVF AVQDML L F F L VWE NdhD1-6803(slr0331) GFI TTLATMAAWPVTLKPK- LFYFLMLLMYGGQI AVF AVQDI L L F F L VWE NdhD-Spinacia oleracea GFI TTLATLAAWPVTRNSQ- LFHFLMLAMYSAQI GLFSSRDLLLFFI MWE NuoM-E. coli GLLGVLAVLCSWKEI EKYQGFFHLNLMWILGGVI GVFLAI DMFLFFFFWE GF I T T L AT L AAWPVT KP. L F YF L ML . MYGGQI AVF AVQDML L F F . WE

160 170 180 190 200 NdhD1-7002(slr0331) LELVPVYLI LSI WG-----GKKRLYAATKFI LYTAGGSLFI LI AALTMAF NdhD1-7120(slr0331) LELI PVYLLLAI WG-----GKKRQYAATKFI LYTAGGSLFI LLASLTMAF NdhD1-6803(slr0331) LELVPVYLI LSI WG-----GKKRLYAATKFI LYTAGGSLFI LLAGLTLAF NdhD-Spinacia oleracea LELI PVYLLLSMWG-----GKKRLYSATKFI LYTAGGSI FLLMGVLGVGL NuoM-E. coli MMLVPMYFLI ALWGHKASDGKTRI TAATKFFI YTQASGLVMLI AI LALVF LELVPVYLLLSI WG GKKRLYAATKFI LYTAGGSLFI L. A. LT. AF

210 220 230 240 250 NdhD1-7002(slr0331) YG----DTVTFDMTAIAQKDFGINLQLLLYGGLLIAYGVKLPI FPLHTWL NdhD1-7120(slr0331) YG----DTVTFDMRSLALKDYALNFQLLLYAGFLI AYAI KLPI I PLHTWL NdhD1-6803(slr0331) YG----DVNTFDMSAIAAKDI PVNLQLLLYAGFLI AYGVKLPI FPLHTWL NdhD-Spinacia oleracea YGS---NEPTLNLETLVNQSYPVALEI I FYI GFFI AFAVKLPI I PLHTWL NuoM-E. coli VHYNATGVWTFNYEELLNTPMSSGVEYLLMLGFFI AFAVKMPVVPLHGWL YG D TFDM LA. KD. . NLQLLLY. GFLI AYAVKLPI . PLHTWL

260 270 280 290 300 NdhD1-7002(slr0331) PDAHGEATAPAHMLLAGI LLKMGGYALLRMNAGMLPDAHALFGPVLVI LG NdhD1-7120(slr0331) PDAHGEATAPAHMLLAGI LLKMGGYALI RMNAGI LPDAHAYFAPVLVVLG NdhD1-6803(slr0331) PDAHGEATAPAHMLLAGI LLKMGGYALLRMNVGMLPDAHAVFAPVLVI LG NdhD-Spinacia oleracea PDTHGEAHYSTCMLLAGI LLKMGAYGLVRI NMELLPHAHSI FSPWLMI I G NuoM-E. coli PDAHSQAPTAGSVDLAGI LLKTAAYGLLRFSLPLFPNASAEFAPI AMWLG PDAHGEATAPAHMLLAGI LLKMGGYALLRMN. G. LPDAHA. FAPVLVI LG 327

310 320 330 340 350 NdhD1-7002(slr0331) VVNI VYAALTSFAQRNLKRKI AYSSI SHMGFVLI GMASFTDLGTSGAMLQ NdhD1-7120(slr0331) VVNI I YAALTSFAQRNLKRKI AYSSI SHMGFVI I GFASFTDLGLSGAVLQ NdhD1-6803(slr0331) VVNI I YAAFTSFAQRNLKRKI AYSSI SHMGFVLI GLASFTDLGMSGAMLQ NdhD-Spinacia oleracea TMQI I YAASTSPGQRNLKKRI AYSSVSHMGFIIIGISSITDTGLNGAILQ NuoM-E. coli VI GI FYGAWMAFAQTDI KRLI AYTSVSHMGFVLI AI YTGSQLAYQGAVI Q VVNI I YAA TSFAQRNLKRKI AYSSI SHMGFVLI G. ASFTDLG SGA. LQ

360 370 380 390 400 NdhD1-7002(slr0331) MI SHGLI GASLFFMVGATYDRTHTLMLDEMGGVGKKMKKI FAMWTTCSMA NdhD1-7120(slr0331) MVSHGLI GASLFFLVGATYDRTHTLMLDEMGGVGKRMPKI FAMFTACSMA NdhD1-6803(slr0331) MI SHGL I GASL F F MVGAT YDRT HT L ML DEMGGI GQKMKKGF AMWTACSL A NdhD-Spinacia oleracea I I SHGFI GAALFFLAGTSYDRI RLVYLDEMGGI AI PMPKI FTLFSSFSMA NuoM-E. coli MI AHGLSAAGLFI LCGQLYERI HTRDMRMMGGLWSKMKWLPALSLFFAVA MI SHGLI GASLFFLVGATYDRTHTLMLDEMGG. G KMKKI FAM. T CSMA

410 420 430 440 450 NdhD1-7002(slr0331) SLALPGMSGFVAELMVFVGFATSDAYSPTFRVI I VFLAAVGVI LTPI YLL NdhD1-7120(slr0331) SLALPGMSGFVAELMVFVGFATSDAYSSTFKVI VVFLMAVGVI LTPI YLL NdhD1-6803(slr0331) SLALPGMSGFVAELMVFVGFATSDAYNLVFRTI VVVLMGVGVI LTPI YLL NdhD-Spinacia oleracea SLALPGMSGFI AELI VFFGLI TSQKYLLI PKLLI TFGMAI GMI LTPI YLL NuoM-E. coli TLGMPGTGNFVGEFMILFG------SFQVVPVI TVI STFGLVFASVYSL SLALPGMSGFVAELMVFVGFATSDAYS F..IIVFLMAVGVILTPIYLL

460 470 480 490 500 NdhD1-7002(slr0331) SMLREI LYGPENKELVAHEKLI DAEPREVFVI ACLLI PI I GI GLYPKAVT NdhD1-7120(slr0331) SMLREI FYGKENEELVSHQQLI DAEPREVFVI ACLLVPI I GI GFYPKLLT NdhD1-6803(slr0331) SMLREMLYGPENEELVNHTNLVDVEPREVFI I GCLLVPI I GI GFYPKLI T NdhD-Spinacia oleracea SMSRQMFYG- YKLFNI SNSSFFDSGPRELFVSTSI FLPVI GI GVYPDLVL NuoM-E. coli AMLHRAYFG- KAKSQI ASQELPGMSLRDVFMI LLLVVLLVLLGFYPQPI L SMLRE. . YG EN ELV H L. D. EPREVFVI . CLLVPI I GI GFYPKL. T

510 520 530 540 550 NdhD1-7002(slr0331) QI YASTTENLTAI LRQSVPSLQQTAQAPSLDVAVLRAPEI R NdhD1-7120(slr0331) QMYDATTVQLTARLRDSVPTLAQEKQE- - VARVSLSAPVI GN NdhD1-6803(slr0331) QI YDPTI NQLVQTARRSVPSLVQQANLSPLEVTALRPPTI GF NdhD-Spinacia oleracea SLSVEKVEAI LSNYFYR NuoM-E. coli DTSHSAI GNI QQWFVNSVTTTRP Q. Y T. . L . R SVP. L Q . . L P I 328

Figure B6. ClustalW alignment of NdhD2 amino acid sequences. NdhD2 amino acid sequences of the following organisms were aligned using the ClustalW alignment program from MacVector v. 6.5: Synechococcus sp. PCC 7002, Synechocystis sp. PCC

(Thompson et al., 1994b). 329

NdhD2 ClustalW Amino Acid Alignment

10 20 30 40 50 NdhD2-7002 (slr1291) MDSLQI PWLTTAI AFPLLAALVI PLI PDKEGKTI RWYTLWRCPHRFCLLV NdhD2-6803(slr1291) MLEHFPWLTTMI ALPLVAALFI PLI PDKDGKQVRWYALGVGLADFVLMS NdhD2-7120(slr1291) MADGFPWLTAI I LLPLVASAFI PLLPDKEGKLVRWYALGVGI ADFVLMC NdhD-Spinacia oleracea MI E L L L MT NuoM-E. coli MLLPWL- - - I LI PFI GGFLCWQTERFGVKVPRWI ALI T- MGLTLALS PWLT I . . PL. A. . I PL. PDK. GK . RWYAL. . . F. LM.

60 70 80 90 100 NdhD2-7002 (slr1291) T AF WQN- - YDF GR- - - - T EF QL T KNF AWI PQL GL NWSL GVDGL SMPL I I L NdhD2-6803(slr1291) YVFWTN- - YDI SS- - - - TGFQLQEKFSWI PQFGLSWSVSVDGI SMPLVLL NdhD2-7120(slr1291) YTFWHH- - YDTSS- - - - ATFQLVEKYDWLPQI GFSWAVSVDGI SMPLVLL NdhD-Spinacia oleracea YVFFYH- - FQPDD- - - - PL I QL VEDYKWI NFFDFHWRLGI DGL SI GPI L L NuoM-E. coli LQLWLQGGYSLTQSAGI PQWQSEFDMPWI PRFGI SI HLAI DGLSLLMVVL Y. FW . YD . FQL E . WI PQFG. SW L. VDGLSMPLVLL

110 120 130 140 150 NdhD2-7002 (slr1291) ATLI TTLATLAAWNVTKKPK- LFAGLI LVMLSAQI GVFAVQDLLLFFI MW NdhD2-6803(slr1291) AGLVTTLSI FAAWQVDHKPR- LFYFLMLVLYAAQI GVFVAQDMLLLFI MW NdhD2-7120(slr1291) AGFVTTLSMLAAWQVNLKPR- LFYFLMLVLYSAQI GVFVAQDLLLFFI MW NdhD-Spinacia oleracea TGFI TTLATLAAWPVTRNSQ- LFHFLMLAMYSAQI GLFSSRDLLLFFI MW NuoM-E. coli TGLLGVLAVLCSWKEI EKYQGFFHLNLMWILGGVI GVFLAI DMFLFFFFW AGL. TTLA LAAW. V . KP. LF FLMLV. YSAQI GVF. AQDLLLFFI MW

160 170 180 190 200 NdhD2-7002 (slr1291) ELELVPVYLLI SI WG- - - - - GKKRLYAATKFI LYTALGSVFI LAFTLALA NdhD2-6803(slr1291) ELELVPVYLLVCI WG- - - - - GQKRQYAAMKFLLYTAAASVFI LVAALGLA NdhD2-7120(slr1291) ELELVPVYLLVSI WG- - - - - GQKRRYAATKFLLYTAAASI FI LI AGLAMA NdhD-Spinacia oleracea ELELI PVYLLLSMWG- - - - - GKKRLYSATKFI LYTAGGSI FLLMGVLGVG NuoM-E. coli EMMLVPMYFLI ALWGHKASDGKTRI TAATKFFI YTQASGLVMLI AI LALV ELELVPVYLL. SI WG GKKR. YAATKF. LYTAA. S. FI L. A. LALA

210 220 230 240 250 NdhD2-7002 (slr1291) FYG- GDV- - - TFDMQALGLKDYPLALELLAYAGFLI GFGVKLPI FPLHSW NdhD2-6803(slr1291) FYG- DVT- - - TFDI AELGLKDYPI ALELFLYAGLLI AFGVKLAI FPFHTW NdhD2-7120(slr1291) LYG- DNT- - - TFDI VELGAKNYPLALELLLYAGLLI AFGVKLAI FPLHTW NdhD-Spinacia oleracea LYGSNEP- - - TLNLETLVNQSYPVALEI I FYI GFFI AFAVKLPI I PLHTW NuoM-E. coli FVHYNATGVWTFNYEELLNTPMSSGVEYLLMLGFFI AFAVKMPVVPLHGW FYG T TFD. ELG. K YP. ALELLLYAGFLI AFGVKLPI FPLHTW

260 270 280 290 300 NdhD2-7002 (slr1291) LPDAHSEASAPVSMI LAGVLLKMGGYGLI RLNMEMLPDAHI RFAPLLI VL NdhD2-6803(slr1291) LPDAHGEASAPVSMI LAGVLLKMGGYGLI RLNLGLLEDAHVYFAPI LVI L NdhD2-7120(slr1291) LPDAHGEASAPVSMI LAGVLLKMGGYGLI RLNLELLPDAHI YFAPVLATL NdhD-Spinacia oleracea LPDTHGEAHYSTCMLLAGI LLKMGAYGLVRI NMELLPHAHSI FSPWLMI I NuoM-E. coli LPDAHSQAPTAGSVDLAGI LLKTAAYGLLRFSLPLFPNASAEFAPI AMWL LPDAHGEASAPVSMI LAGVLLKMGGYGLI RLNLELLPDAH. FAP. L. . L

310 320 330 340 350 NdhD2-7002 (slr1291) GI VNI VYGALTAFGQTNLKRRLASSSI FPHGLSSLGLLSFTDLGMNGAVL NdhD2-6803(slr1291) GVVNI I YGGFSSFAQDNMKRRLAYSSVSHMGFVLLGI ASFTDLGI SGAML NdhD2-7120(slr1291) GVI NI I YGGLNSFAQTHMKRRLAYSSVSHMGFVLLGI ASFTDVGVSGAML NdhD-Spinacia oleracea GTMQI I YAASTSPGQRNLKKRI AYSSVSHMGFI I I GI SSI TDTGLNGAI L NuoM-E. coli GVI GI FYGAWMAFAQTDI KRLI AYTSVSHMGFVLI AI YTGSQLAYQGAVI GV. NI I YGA . SFAQTN. KRRLAYSSVSHMGFVLLGI . SFTDLG. . GA. L

360 370 380 390 400 NdhD2-7002 (slr1291) QMLSHGFI AAALFFLSGVTYERTHTLMMDEMSGI ARLMPKTFAMFTAAAM NdhD2-6803(slr1291) QMLSHGLI AAVLFFLAGVTYDRTHTLSLAQMGNI GKVMPTVFALFTMGAM NdhD2-7120(slr1291) QMLSHGLI AAVLFFLAGVTYDRTHTMAMDNLGGI GQAMPKVFALFTAGTM NdhD-Spinacia oleracea QI I SHGFI GAALFFLAGTSYDRI RLVYLDEMGGI AI PMPKI FTLFSSFSM NuoM-E. coli QMI AHGLSAAGLFI LCGQLYERI HTRDMRMMGGLWSKMKWLPALSLFFAV QMLSHGLI AA. LFFLAGVTYDRTHT. MD MGGI . . MPK. FALFT . AM 330

NdhD2-7002 (slr1291) ASLALPGMSGFVSELTVFLGLSNSDAYSYGFKPI AI FLTAVGVI LTPI TC NdhD2-6803(slr1291) ASLALPGMSGFVSELAVFVGVSSSDI YSTPFKTVTVFLAAVGLVLTPI YL NdhD2-7120(slr1291) ASLALPGMSGFVSELKVFI GVTTSDI YSPTFCTVMVFLAAVGVI LTPI YL NdhD-Spinacia oleracea ASLALPGMSGFI AELI VFFGLI TSQKYLLI PKLLI TFGMAI GMI LTPI YL NuoM-E. coli ATLGMPGTGNFVGEFMILFGS------FQVVPVI TVI STFGLVFASVYS ASLALPGMSGFVSEL VF.G...SD.YS FK V..FL AVG.ILTPIYL

460 470 480 490 500 NdhD2-7002 (slr1291) FQCCGVFYGKGSQAPPRCGGE------D NdhD2-6803(slr1291) LSMLRQLFY- GNNI PPSCNLEQDNLSANSDQEAVCFGTSCVLPGNAI YDD NdhD2-7120(slr1291) LSMLRQVFY- GTGAELSCNI NNGAYQNQEDEGTACFGTDCLLPGEAVYRD NdhD-Spinacia oleracea LSMSRQMFY-GYKLFNISNSS------FFD NuoM-E. coli LAMLHRAYF-GKAKSQIASQELP------G L SMLRQ. FY G . CN. E . D

510 520 530 540 550 NdhD2-7002 (slr1291) AKPREI FVAVCLLAPI I AI GLYPKLATTTYDLKTVEVASKVRAALPLYAE NdhD2-6803(slr1291) ARPREVFI AACFLLPI I AVGLYPKLATQTYDATTVAVNSQVRQSYVQI AE NdhD2-7120(slr1291) ASVREVFI AVSFLVLI I GVGVYPKI ATQLYDVKTVAVNTQVRQSYTQI AQ NdhD-Spinacia oleracea SGPRELFVSTSI FLPVI GI GVYPDLVLSLSVEKVEAI LSNYFYR NuoM-E. coli MSLRDVFMILLLVVLLVLLGFYPQPI LDTSHSAI GNI QQWFVNS---VTT A PREVF.A. .L.PII..G.YPKLAT TYD. KTVAV. S. VR. S . A

560 570 580 590 600 NdhD2-7002 (slr1291) QLP---QNGDRQAQMGLSSQMPALIAPRF NdhD2-6803(slr1291) TNPRVYAEALTAPHI PTTDFATVKVQP NdhD2-7120(slr1291) SNPQI YAKGFFTPQI VEPEVMAVSGVI K NdhD-Spinacia oleracea NuoM-E. coli TRP .P . .. 331

Figure B7. ClustalW alignment of NdhD3 amino acid sequences. NdhD3 amino acid sequences of the following organisms were aligned using the ClustalW alignment program from MacVector v. 6.5: Synechococcus sp. PCC 7002, Synechocystis sp. PCC

(Thompson et al., 1994b). Data for NdhD3 is taken from Klughammer et al.

(Klughammer et al., 1999). 332

NdhD3 ClustalW Amino Acid Alignment

10 20 30 40 50 NdhD3-7002(sll1733) MLSFLLFLPLVGIGAIALFP-----RPLTRI VATVFTVVTLAISSGLLI NdhD3-7120(sll1733) ML S V LI WIPI LSAI VI GFWPSNPNQSSRI RLVALTV AA I V L I WNL F I L F NdhD3-6803(sll1733) MLSLLLI LPVIGALII GFFPGNI-PAKQLRQITEVFAVLTLVWSLLVLF NdhD-Spinacia oleracea MI E L L L M T Y V F F Y H F NuoM-E. coli ML L P WLILIPFIGGFLCWQTERFG--VKVPRWIALITMGLTLALSLQLWL MLS L. .P..G...I. .P R .A . .LTL..SL .LF

60 70 80 90 100 NdhD3-7002(sll1733) N- -L NL QD - - - - A GMQY TEFHNWLSI LGLNYNLGVDGLSLPLI VLNSLLT NdhD3-7120(sll1733) K--FDISN----PGMQFQEYLPWNETLGLSYQLGVDGLSILMLVLNSLLT NdhD3-6803(sll1733) K--FDVTD----PQFQFQEYLPWIPQLGLNYSLAIDGLSLPLVI LNNLLT NdhD-Spinacia oleracea Q--P--DD----PLI QLVEDYKWINFFDFHWRLGI DGLS I GPI L LT GFI T NuoM-E. coli QGGYSLTQSAGI PQWQSEF DMPWIPRFGI SI HLAI DGLSLLMVVLT GLLG ....DP.Q.E.PWILGLYLGIDGLSL..VLNLLT

110 120 130 140 150 NdhD3-7002(sll1733) LVAI Y SI GESNHRPK-L YYSLI LL I NSGI T GALI ANNLL LFF LF YEI ELI NdhD3-7120(sll1733) WI A I Y SSSNKTERPR-LFYSMILLVSGGVAGAFLSENLLLFFLFYELELI NdhD3-6803(sll1733) GV AI Y SI GPNVNRSR-L YYGLI LL I NAGI SGALLAQNLL LFI VF YELELI NdhD-Spinacia oleracea TLATLAAWPVTRNSQ-LFHFLMLAMYSAQI GLFSSRDLLLFFI MWELELI NuoM-E. coli VLAVLCSWKEI EKYQGFFHLNLMWILGGVI GVFLAI DMFLFFFFWEMMLV .. AIY S. R . LFY LI LLI . G.. GAFLA NLLLFF.FYELELI

160 170 180 190 200 NdhD3-7002(sll1733) PFYLLI AIWG-----GEKKGYASTKFLI YTAISGLCV LAAFL GI VWLSQS NdhD3-7120(sll1733) PFYLLI SI WG-----GEKRAYAGIKFLI YTAVSGAFI LATFLGMVWLTGS NdhD3-6803(sll1733) PFYLMIAIWG-----GEKRGYASMKFLLYTAFSGLLV LAAFL GMSLLSGS NdhD-Spinacia oleracea PVYLLLSMWG-----GKKRLYSATKFI LYTAGGSI FLLMGVLGVGLYGSN NuoM-E. coli PMYFLI ALWGHKASDGKTRI T AAT KFFI YT QASGLVMLI AI L ALVFV HYN PFYLLI AI WG GEKR. YA. T KFLI YTA. SGL . LAAFLG. V. L. S

210 220 230 240 250 NdhD3-7002(sll1733) SN----FDFENLTLENLEFNTKVI LLTI LLI GFGI KI PLVPLHTWLPDAY NdhD3-7120(sll1733) TS----FAFDAI STQGLSAGMQFI LLVGI I LGFGI KI PLVPFHTWLPDAY NdhD3-6803(sll1733) HS----FDYNPEITQTFTESAQTILLILILLGFGIKIPLVPLHTWLPDAY NdhD-Spinacia oleracea EP---TLNLETLVNQSY PVALEII FYIGFFI AFAVKLPI IPLHTWLPDTH NuoM-E. coli AT GV WT FNYEELLNT PMSSGVEYL LMLGFFI AFAVKMPVVPL HGWLPDAH . F .E L. Q . .. ILL.G..IGFGIKIPLVPLHTWLPDAY

260 270 280 290 300 NdhD3-7002(sll1733) VEANPAVT VLLGGVFAKLGTYGLVRFGLQLFPDVWST VSPALAVI GTVSV NdhD3-7120(sll1733) VEASAPIAILLGGVLAKLGTYGLLRFGLGMFPQTWSTVAPTLAIWGAVSA NdhD3-6803(sll1733) TEASPATAILLGGILAKLGTYGII RFGLQLFPQTWAQFAPVLAIIGTVTV NdhD-Spinacia oleracea GE AHY STCMLLAGI LLKMGAYGLVRI NMELLPHAHSI FSPWLMI IGTMQI NuoM-E. coli SQAPTAGSVDLAGILLKTAAYGLLRFSLPLFPNASAEFAPIAMWLGV IGI . EA A. . LLGGILAKLGTYGL. RFGL LFP. . WS FAP. LAIIGTV. .

310 320 330 340 350 NdhD3-7002(sll1733) MYGS LAAI AQR DL KR MV AYSSI GHMGYI LVSTAAGTELSLLGAVAQMISH NdhD3-7120(sll1733) IYGAV IAIAQKDIKRMV AYSSI GHMGYVLLASAASTSLALVGAVSQMFSH NdhD3-6803(sll1733) LY GALS AI AQKDI KR MV AYSSI GHMGYI LVAAAAGTELSVLGAVAQMVSH NdhD-Spinacia oleracea I YAAST SPGQRNLKKRI AYSSVSHMGFI I I GI SSI TDTGLNGAI LQI I SH NuoM-E. coli FYGAWMAFAQTDI KRLI AYTSVSHMGFVLI AI YTGSQLAYQGAVI QMIAH .YGA. AIAQ.DIKRMV AYSSI GHMGYI L. A. AAGT. L. L. GAV. QMISH

360 370 380 390 400 NdhD3-7002(sll1733) SLI LALLFHLVGI I ERKVGTRDLDVLNGLMNPVRGLPLT SSLLI LAGMAS NdhD3-7120(sll1733) GLI LAI LFHLVGI I EEKVGTRELDKLNGLMSPI RGLPLI SALLVLSGMAS NdhD3-6803(sll1733) GLI LALLFHLVGI VERKAGTRDLDVLNGLMNPI RGLPLT SALLI TGGMAS NdhD-Spinacia oleracea GFIGAALFFLAGTSYDRIRLVYLDEMGGIAI PMPKIF---TLFSSFSMAS NuoM-E. coli GL SAAGLF I L C GQLYER I HTR DMRMMGGLWSKMKWLP -- -AL SL FF AV AT GLI LA. LFHLVGI . E. K. GTRDLD LNGLM P. RGLPL SAL L. GMAS 333

410 420 430 440 450 NdhD3-7002(sll1733) AG I P GL V GFVAEFLVFQG-----SFSRFP--IPTLFCII ASGLTAVYFVI NdhD3-7120(sll1733) AGIPGLTGFI AEFIVFQG-----SFTAFP--LSTLLCVASSGLTAVYFVI NdhD3-6803(sll1733) AG I P GL V GFAAEFI VFQG-----S FPTFP--I PTLLCI LASGLT AVYFVI NdhD-Spinacia oleracea LALPGMSGFI AELI VFFGLI T SQKYLLI PKL LI TFGMAI GMI LT PI YLLS NuoM-E. coli LGMPGTGNFVGEFMILFG-----SFQVVP--VITVISTFGLVFASVYSLA AGIPGL.GF.AEFIVFQG SF .FP . TL.C...SGLTAVYFVI

460 470 480 490 500 NdhD3-7002(sll1733) LL NRTCFGRLDSHTAYYPKVF ASE KI PAI AL --TVI I LFLGLQPAWLTRW NdhD3-7120(sll1733) LLNRTCFGRLDNNQAYYAKVLWSEKTPALIL--AALIIFLGVQPTWLVRW NdhD3-6803(sll1733) LLNRTCFGKLDNQRAYYPKVLASEMIPALVL--TAII FFLGVQPNYLVHW NdhD-Spinacia oleracea MSRQMFYGYKLFNIS---NSSFFDSGPRELF-- V STSIFLPVIG--IGVY NuoM-E. coli MLHRAYFGKAKSQ------I ASQELPGMSLRDV FMILLLV VLLVLLGFY LLNRTCFG.LD . AYY KV.ASE .PA..L ...I.FLGVQP .L..W

510 520 530 540 550 NdhD3-7002(sll1733) IEPTTSQFIAAIPTVQTIALTPAELSKAP NdhD3-7120(sll1733) SE PT TT AMVAAI PPVEKTV ISQVV LK NdhD3-6803(sll1733) TQTT TNEMIAQLPHETSAI EQLAQGVTLP NdhD-Spinacia oleracea PDLVLS LS VEKV EAI LSNYFYR NuoM-E. coli PQPI LDTS HS AI GNI QQWFVNS VT TT RP .PTT..AAIP...... 334

Figure B8. ClustalW alignment of NdhD4 amino acid sequences. NdhD4 amino acid sequences of the following organisms were aligned using the ClustalW alignment program from MacVector v. 6.5: Synechococcus sp. PCC 7002, Synechocystis sp. PCC

(Thompson et al., 1994b). 335

NdhD4 ClustalW Amino Acid Alignment

10 20 30 40 50 NdhD4-7002(sll0027) MLSALI WLPLAGALLVAI LPQGEKNQFSRTMALGAAALVFVWTAWLGFH NdhD4-7120(sll0027) MLSVLI FAPLLGALLI GLLPSGI NGRSSRNVALI FASVTFLWSVI LASQ NdhD4-6803(sll0027) MLSAL I WGPI FGAI L I AI I PNPDHDCYSRKI AL GI MVAMAGL SVL L AGQ NdhD-Spinacia oleracea MIELLLMTYVFFYH NuoM-E. coli MLLPWLI LI PFI GGFLCWQTER- FGVKVPRWI ALI TMGLTLALSLQLWLQ MLS.LI..P..GA.L....P SR .AL. M.. .. S..L. Q

60 70 80 90 100 NdhD4-7002(sll0027) YDV------AIAGLQFVEHYLWIEWLGLNYDLGVDGLSLPLLALNALLTL NdhD4-7120(sll0027) FQP------GEVNQQFSEFLPWIDTLGLSYNLGVDGLSLPLLVLNGLLTG NdhD4-6803(sll0027) FNI------SDPQMQFVEYYPWLPSLGLNYHLGVDGLSLPLLLLNSALVV NdhD-Spinacia oleracea FQP------DDPLIQLVEDYKWINFFDFHWRLGIDGLSIGPILLTGFITT NuoM-E. coli GGYSLTQSAGI PQWQSEFDMPWI PRFGI SI HLAI DGLSLLMVVLTGLLGV F. . . P. QFVE YPWI LGL Y. LGVDGLSLPLL. LNGLLT.

110 120 130 140 150 NdhD4-7002(sll0027) VALWI SPKDLHRPR- FYYALFLLLQASVNGAFLAQDVLLFFLFYEI EI I P NdhD4-7120(sll0027) I AI YSSDESLQRPK- FYYSLI LVLSAGVSGAFLAQDLLLFFLFYELELI P NdhD4-6803(sll0027) I AI FSTNTEI ERPR-FYYALLLLLSGGVAGAFLAQDLLLFFLFFELEI I P NdhD-Spinacia oleracea LATLAAWPVTRNSQ- LFHFLMLAMYSAQI GLFSSRDLLLFFI MWELELI P NuoM-E. coli LAVLCSWKEI EKYQGFFHLNLMWILGGVI GVFLAI DMFLFFFFWEMMLVP . A. . S . . RP. FYY. L. L. L . GV. GAFLAQDLLLFFLF. ELELI P

160 170 180 190 200 NdhD4-7002(sll0027) LYFLIAIWG-----GKKRGYAAI KFLLYTAVSGI LI LASFLGLAFLTESN NdhD4-7120(sll0027) LYLLIAIWG-----GARRGYAATKFLI YTAFSGI LI LASFLGLVWLSGSG NdhD4-6803(sll0027) LYFLIAIWG-----GQRRGYAAMKFLLYTALSGFLVLVSFLGWFWLTKAP NdhD-Spinacia oleracea VYLLLSMWG-----GKKRLYSATKFI LYTAGGSI FLLMGVLGVGLYGSNE NuoM-E. coli MYFLI ALWGHKASDGKTRI TAATKFFI YTQASGLVMLI AI LALVFVHYNA LYFLI AI WG GK. RGYAATKFLLYTA. SGI L. L. SFLGL. . L.

210 220 230 240 250 NdhD4-7002(sll0027) T----FAYSALHSDLLPLTTQLI LLGGI LVGFGI KI PFLPFHTWLPDAHV NdhD4-7120(sll0027) S----FALSTLNAQSLPLATQLLLLAGILVGFGIKMPLVPFHTWLPDAHV NdhD4-6803(sll0027) N----FDYNPSLADALPVKTQMLLLLPLLLGLGIKI PI FPFHTWLPDAHV NdhD-Spinacia oleracea P---TLNLETLVNQSYPVALEI I FYI GFFI AFAVKLPI I PLHTWLPDTHG NuoM-E. coli TGVWTFNYEELLNTPMSSGVEYLLMLGFFI AFAVKMPVVPLHGWLPDAHS . F Y L. LP. . TQ.LLL. G.L. GFGIK. P. . PFHTWLPDAHV

260 270 280 290 300 NdhD4-7002(sll0027) EASTPVSVI LAGVLLKVGTYGLLKFGI GLFPLAWAVVAPWLAI WAAI SAL NdhD4-7120(sll0027) EASTPI SVLLAGVLLKLGTYGLLRFGMNLLPAAWNYLAPWLAAWAVVSVL NdhD4-6803(sll0027) EASTPVSVLLAGVLLKLGTYGLLRFGLGLYLEAWVEFAPYLATLAAI SAL NdhD-Spinacia oleracea EAHYSTCMLLAGI LLKMGAYGLVRI NMELLPHAHSI FSPWLMI I GTMQI I NuoM-E. coli QAPTAGSVDLAGI LLKTAAYGLLRFSLPLFPNASAEFAPI AMWLGVI GI F EASTP. SVLLAGVLLK. GTYGLLRFG. L. P AW. FAPWLA. . A. I S. L

310 320 330 340 350 NdhD4-7002(sll0027) YGASCAI AQKDMKKVVAYSSI AHMAFI LLAAAAATPLSLAAAEI QMVSHG NdhD4-7120(sll0027) YGSSCAI AQTDMKKMVAYSSI GHMGYVLLAAAAATPLSTLGAVMQMI SHG NdhD4-6803(sll0027) YGASCAI AQKDMKKVVAYSSI AHMGYI LLAAAAATRLSVTAASAQMVSHG NdhD-Spinacia oleracea YAASTSPGQRNLKKRI AYSSVSHMGFIIIGISSITDTGLNGAILQIISHG NuoM-E. coli YGAWMAFAQTDI KRLI AYTSVSHMGFVLI AI YTGSQLAYQGAVI QMI AHG YGASCAI AQ. DMKK. VAYSSI . HMGFI LLAAAAAT LS. GA. . QMI SHG 336

360 370 380 390 400 NdhD4-7002(sll0027) LI SGLLFLLVGIVYKKTGSRDVDYLRGLLTPERGLPLTGSLMILGVMASA NdhD4-7120(sll0027) LI SALLFLLVGVVYKKAGSRDLDVI RGLLNPERGMPVI GTLMI VGVMASA NdhD4-6803(sll0027) I I SALLFLLVGVVYKKTGSRDVDKLQGLLTPERGLPI TGSLMILGVMASA NdhD-Spinacia oleracea FI GAALFFLAGTSYDRI RLVYLDEMGGI AI P- - - MPKI FTLFSSFSMASL NuoM-E. coli LSAAGLFI LCGQLYERI HTRDMRMMGGLWSKMKWLP- - - ALSLFFAVATL LI SALLFLLVG. VYKK. GSRD. D . GLL. PERGLP. G. LMI . GVMASA

410 420 430 440 450 NdhD4-7002(sll0027) GLPGMAGFI AEFLI FRG- - - - - SFPVYP- - VATLLCMVGTGLTAVYFLLM NdhD4-7120(sll0027) GTPGMVGFI SEFI I FRG- - - - - SFAVFP- - VQTLLSMI GTGLTAVYFLI L NdhD4-6803(sll0027) GI PGMVGFI AEFLVFRG- - - - - SFPI FP- - TQTLLCLI GSGLTAVYFLLM NdhD-Spinacia oleracea ALPGMSGFI AELI VFFGLI TSQKYLLI PKLLI TFGMAI GMI LTPI YLLSM NuoM-E. coli GMPGTGNFVGEFMI LFG- - - - - SFQVVP- - VI TVI STFGLVFASVYSLAM G.PGM.GFIAEF.IFRG SF V.P V.TLL. IG.GLTAVYFL.M

460 470 480 490 500 NdhD4-7002(sll0027) I NKVFFGRLTPELI N- - MSPVNWADQFPAVMLVI LLFVFGLQPQWLVRWS NdhD4-7120(sll0027) VNKAFFGRLSAQVMN- - LPRI YWSDRAPAFI LAVLI VI FGI QPSWLARWT NdhD4-6803(sll0027) I NRVFFGRLTMELSH- - LPKVRWQEQI PAI ALAVVI I ALGI QPHWLTQWS NdhD-Spinacia oleracea SRQMFYGYKLFNI SNSSFFDSGPRELFVSTSI FLPVI GI GVYPDLVLSLS NuoM-E. coli LHRAYFGKAKSQI ASQELPGMSLRDVFMI LLLVVLLVLLGFYPQPI LDTS .N..FFGRL. .. N LP . W D FPA..L.VL....G.QP WL. WS

510 520 530 540 550 NdhD4-7002(sll0027) EIDTAALVA------SPTAIEISLKN NdhD4-7120(sll0027) EPTI TAMFS------VESTVATVSLNKVKEKS NdhD4-6803(sll0027) EPQTAVLLTGHPDLVSVPAPPRI EI EVKELGEV NdhD-Spinacia oleracea VEKVEAI LS------NYFYR NuoM-E. coli HSAIGNIQQ------WFVNSVTTTRP E ..A...... 337

Figure B9. ClustalW alignment of NdhD amino acid sequences from Synechococcus sp.

PCC 7002 against each other. The Synechococcus sp. PCC 7002 NdhD amino acid sequences were aligned using the ClustalW alignment program from MacVector v. 6.5.

The consensus amino acid sequence is underneath the alignments. Gray high-lighted regions indicate identical amino acids while lighter shading indicates conserved amino acids. Dashes represent insertions/deletions included to maximize the sequence similarity. Percent identity and percent conserved amino acids determined with the

ClustalW program (Thompson et al., 1994b). 338

NdhD ClustalW Amino Acid Alignment

10 20 30 40 NdhD1-7002(slr0331) MNFANFPWLSTI ILFPI IAALFLPLIPDKDGKTVRWYALT NdhD2-7002 (slr1291) MDSLQIPWLTTAIAFPLLAAL V IPLIPDKEGKTIRWYTLW NdhD3-7002(sll1773) ML S F L L F L PLV GIGAIALFPR----PLTR IVAT NdhD4-7002(sll0027) MLSALIWLPLAGALLVAILPQGEKNQFSRTMAL LS .I PL..AL.. L.P . . .

50 60 70 80 NdhD1-7002(slr0331) IGL IDFV I IVTAFYTGYDFGNPNLQLVESYTWVEA IDLRW NdhD2-7002 (slr1291) RC PHRFC LLVTAFWQNYDFGRTEFQLTKNFAWI PQLGLNW NdhD3-7002(sll1773) V FTVVT LA I SSGLL INLNLQDAGMQYT EFHNWLS I LGLNY NdhD4-7002(sll0027) GAAALV FVWTAWLGFHYDVA IAGLQFV EHYLWI EWLGLNY ...... YD . Q E . W. LGLN .

90 100 110 120 NdhD1-7002(slr0331) SVGADGLSMPLILLTGF ITTLAILAAW PV SFK PKL FYF LM NdhD2-7002 (slr1291) SLGVDGLSMPLI ILATLITTLATLAAW NV TKK PKL FAGL I NdhD3-7002(sll1773) NLGVDGLSLPLIVLNSLLTLVAIYSIGESNHRPKLYYSL I NdhD4-7002(sll0027) DLGVDGLSLPLLALNALLTLVALWISPKDLHRPRFYYALF LGVDGLS PLI.L L.T .A. .. ..PKL.Y L

130 140 150 160 NdhD1-7002(slr0331) LLMYGGQIAVFAVQDMLLFFFTW ELELVPVYLI LS IWGGK NdhD2-7002 (slr1291) LVMLSAQ IGV FAVQDLLLFF IMW ELELVPVYLLI S IWGGK NdhD3-7002(sll1773) LLINSGITGALIANNLLLFFLFYEIELIPFYLLIAIWGGE NdhD4-7002(sll0027) LLLQASVNGAFLAQDVLLFFLFYEIEI IPLYFLIAIWGGK LL . G.F..QD.LLFF. .E.EL.P.YLLI IWGGK

170 180 190 200 NdhD1-7002(slr0331) KRLYAATKF I LYTAGGSLF I L IAALTMAFYGDTVTFDMTA NdhD2-7002 (slr1291) KR LYAATKF I LYTALGSV F I LAFTLALAFYGGDVT FDMQA NdhD3-7002(sll1773) KKGYASTKFL IYTA I SGLCVLAAFLG IVWLSQSSNFDFEN NdhD4-7002(sll0027) KRGYAA IKFLLYTAV SG I L I LASFLGLAFLT ESNT FAYSA KR . YAATKF . LYTA . . I LA L . .AF . TFD A

210 220 230 240 NdhD1-7002(slr0331) IAQKDFG INLQLLLYGGLL IAYGVKLP I FPLHTWLPDAHG NdhD2-7002 (slr1291) LGLKDYPLALELLAYAGFL IGFGVKLP I FPLHSWLPDAHS NdhD3-7002(sll1773) LTLENLEFNTKVILLTILLIGFGIKIPLVPLHTWLPDAYV NdhD4-7002(sll0027) LHSDLLPLT TQL I LLGG I LVGFG IK I PFLPFHTWLPDAHV L . L.L .G.LIGFG.K.P. PLHTWLPDAH.

250 260 270 280 NdhD1-7002(slr0331) EATAPAHMLLAG I LLKMGGYALLRMNAGMLPDAHALFGPV NdhD2-7002 (slr1291) EASAPV SMI LAGVLLKMGGYGL IRLNMEMLPDAH IR FAPL NdhD3-7002(sll1773) EANPAVTVLLGGVFAKLGTYGLVRFGLQLFPDVWSTVSPA NdhD4-7002(sll0027) EASTPV SV I LAGVLLKVGTYGLLKFG IGLFPLAWAVVAPW EA. PV. .LAGVLLK G YGL.R . PDA . .P.

290 300 310 320 NdhD1-7002(slr0331) LV I LGVVNIVYAALTSFAQRNLKRKIAYSSISHMGFVL IG NdhD2-7002 (slr1291) L IVLGIVNIVYGALTAFGQTNLKRRLASSS IFPHGLSSLG NdhD3-7002(sll1773) LAV IGTV SVMYGSLAA IAQRDLKRMV AYSS IGHMGYI LV S NdhD4-7002(sll0027) LAIWAAISALYGASCAIAQKDMKKVV AYSS IAHMAF I LLA L...G.V ..YGAL A AQ. LKR .AYSSI HMG..L.. 339

330 340 350 360 NdhD1-7002(slr0331) MASFTDLGT SGAMLQMI SHGL IGASLFFMVGATYDR THT L NdhD2-7002 (slr1291) LLSFTDLGMNGAVLQMLSHGFIAAALFFLSGVTYERTHTL NdhD3-7002(sll1773) TAAGTELSLLGAVAQMISHSLILALLFHLVGI IERKVGTR NdhD4-7002(sll0027) AAAAT PLSLAAAE IQMVSHGL I SGLLFLLVG IVYKKTGSR AT.LGA.QM.SHGLI.A.LFLVG.Y.TT

370 380 390 400 NdhD1-7002(slr0331) MLDEMGGVG ---KKMKK I FAMWTTCSMASLALPGMSGFVA NdhD2-7002 (slr1291) MMD EMSG I A - --R LMPK T FAMF TAAAMA S LA L PGMSG FV S NdhD3-7002(sll1773) DLDVLNGLMNPVRGLPLTSSLLILAGMASAGIPGLVGFVA NdhD4-7002(sll0027) DVDYLRGLLT PERGLPLTGSLMI LGV MA S AG L PGMA G F I A .D G. . R. P T . . .MAS. .LPGM GFVA

410 420 430 440 NdhD1-7002(slr0331) ELMVFVGFATSDAYSPTFRV I IVFLAAVGVILTPIYLLSM NdhD2-7002 (slr1291) ELTV F LGLSNSDAY SYGFKP IA I FLTAVGV I LT P I TC FQC NdhD3-7002(sll1773) EFLVFQG ------SFSR FP I PT LFC I IASGLTAVYFV I L NdhD4-7002(sll0027) EFLIFRG------SFPV YPVATLLCMVGTGLTAVYFLLM EVFG S.PI.L.VG.LT.Y..

450 460 470 480 NdhD1-7002(slr0331) LR E I LYGPENKELVAHEKL IDAEPREV FV IAC LL I P I IG I NdhD2-7002 (slr1291) CGVFYG --KGSQAPPRCGGEDAKPREIFVAVCLLAPI IA I NdhD3-7002(sll1773) LNRTCFGRLDSHTAYYPKVFAS---EKIPAIALTV I ILFL NdhD4-7002(sll0027) INKVFFGRLTPEL INMSPVNWA---DQFPAVMLV ILLFVF ..G...AEFA..L..I...

490 500 510 520 NdhD1-7002(slr0331) G LY PKAV TQ IYAST T ENL TA I LRQSV PSLQQTAQAP---- NdhD2-7002 (slr1291) GLYPKLATTTYDLKTVEV ASKV RAALPLYAEQLPQNGDRQ NdhD3-7002(sll1773) GLQPAWLTRWIEPTTSQF IAAIPTVQT IALTPAELS---- NdhD4-7002(sll0027) GLQPQWLVRWSEIDTAALVASPTAI E I SLKN GL P . T . T . .A . . . .

530 540 550 560 NdhD1-7002(slr0331) -----SLDVAVLRAPEIRN NdhD2-7002 (slr1291) AQMGLSSQMPAL IAPRF NdhD3-7002(sll1773) ------KAP NdhD4-7002(sll0027) AP 340

Figure B10. ClustalW alignment of NdhF1 amino acid sequences. NdhF1 amino acid sequences of the following organisms were aligned using the ClustalW alignment program from MacVector v. 6.5: Synechococcus sp. PCC 7002, Synechocystis sp. PCC

(Thompson et al., 1994b). Data for NdhF1 was acquired from Schluchter et al.

(Schluchter et al., 1993). 341

NdhF1 ClustalW Amino Acid Alignment

10 20 30 40 50 NdhF1-7002(slr0844) MEPLYQYAWLIPVLPLLGAMVIGIGLISLNKFTNKLRQLYAVFVLSLIGT NdhF1-6803(slr0844) MELLYQLAWLI PVLPLFGATVVGI GLI SFNQATNKLRQI NAVFI I SCLGA NdhF1-7120(slr0844) MEVI YQYAWLI PVLPLLGAMLVGLGLI SFNQTTNRLRQLNAVLI I SLMGA NdhF-Spinacia oleracea MEHIYQYAWII PFLPLPVPLLI GAGLLFFPTATKNLRRI WAFSSI SLLSI NuoL-E. coli MNMLAL T I I LPLI GFVLLAFSRGRWSE NVSAI VGVGS VAL AAL VT A ME . YQYAWLI PVLPL. GA L. G. GLI SFN TN. LRQ. AV . I SL. GA

60 70 80 90 100 NdhF1-7002(slr0844) SMALS FGLLWSQI QGHEAFT YT L EWAAAGDF HL QMGYT VDHLSALMSVI V NdhF1-6803(slr0844) ALVMSGALLWDQI QGHASYAQMIEWASAGSFHLEMGYVI DHLSALMLVI V NdhF1-7120(slr0844) ALGLSSALLWSQLQGHPTYLRTLEWAAAGNFHLTMGYTI DNLTSLMLVI A NdhF-Spinacia oleracea VMIFSMKLAIQQINSNSIYQYLWSWTINNDFSLEFGYLMDPLTSIMSMLI NuoL-E. coli LS ALI SS LT ASKT YS ----QPLWTWMSVGDF NI GFNLVL DGLSL T MLSVV . . LS . LLWSQI QGH Y . EWA. AGDFHL MGY. . D LS. LMLVI V

110 120 130 140 150 NdhF1-7002(slr0844) TTVALLVMIYTDGYMAHDPGYVRFYAYLSI FSSSMLGLVFSPNLVQVYI F NdhF1-6803(slr0844) TSVALLVMIYTDGYMAHDPGYVRFYAYLSLFASSMLGLVI SPNLVQVYI F NdhF1-7120(slr0844) TSVAVLVMVYTDGYMAHDPGYVRFYAYLSLFGSSMLGLVVSPNLVQI YI F NdhF-Spinacia oleracea TTVAI LVLI YSDNYMSHDQGYLRFFAYMSFFNTSMLGLVTSSNLI QI YI F NuoL-E. coli TGVGFLI HMYASWYMRGEEGYSRFFAYT NLFI ASMVVLVLADNLLLMYLG T. VA. LVMIYTDGYMAHDPGYVRFYAYLSLF. SSMLGLV. SPNLVQ. YI F

160 170 180 190 200 NdhF1-7002(slr0844) WELVGMCSYLLI GFWYDRKAAADACQKAFVT NRVGDFGLLLGMLGLYWAT NdhF1-6803(slr0844) WELVGMCSYL LI GFWYDRKAAADACQKAFVT NRVGDFGL LL GI L GLYWAT NdhF1-7120(slr0844) WELVGMCSYL LVGFWYDRKS AADAAQKAFVT NRVGDFGL LL GI L GLF WAT NdhF-Spinacia oleracea WELVGMCSYLLI GFWFTRPI AANACQKAFVTNRVGDFGLLLGI LGLYWIT NuoL-E. coli WEGVGLCSYL LI GFYYT DPKNGAAAMKAFVVT RVGDVFL RF ALF I LYNE L WELVGMCSYL LI GFWYDRK. AADACQKAFVT NRVGDFGL LL GI L GLYWAT

210 220 230 240 250 NdhF1-7002(slr0844) GS FEF DL MGDRL MDL VST GQI SS LLAI VFAVLVFLGPVAKS AQF PLHVWL NdhF1-6803(slr0844) GS FDF GT I GE RL EGL VSS GVLSGAI AAI LAI LVFLGPVAKS AQF PLHVWL NdhF1-7120(slr0844) GS FDF QI MGDRL AEL VQT GS I SNFLAVL FAI LVFLGPVAKS AQF PLHVWL NdhF-Spinacia oleracea GSFEFRDLFEI FNNLI KNNEVNSLFCI LCAFLLFAGAVAKSAQFPLHVWL NuoL-E. coli GTLNFREMVELAPAHLADG---NNMLMWATLMLLGGAVGKSAQLPLQTWL GSF.F MGERL LV .G .S ..A.. A.LVFLGPVAKSAQFPLHVWL

260 270 280 290 300 NdhF1-7002(slr0844) PDAMEGPT PI SALI HAAT MVAAGVFL VARMYPVFEPI PE AMNVI AWTGAT NdhF1-6803(slr0844) PDAMEGPTPI SALI HAATMVAAGVFLVARMYPVFEPI PVVMNTI AFTGCF NdhF1-7120(slr0844) PDAMEVPT PI SALI HAAT MVAAGVFL VARMYPVFEHVPAAMNVI AFT GAF NdhF-Spinacia oleracea PDAMEGPTPI SALI HAATMVAAGI FLVARLLPLFVVI PYI MYVI SFI GI I NuoL-E. coli ADAMAGPT PVSALI HAAT MVT AGVYL I ART HGL FLMTPE VL HLVGI VGAV PDAMEGPT PI SALI HAAT MVAAGVFL VARMYPVFE I P . MNVI AFT GA

310 320 330 340 350 NdhF1-7002(slr0844) TAFLGATI ALTQNDIKKGLAYSTMSQLGYMVMAMGIGGYTAGLFHLMTHA NdhF1-6803(slr0844) TAFLGATI ALTQNDI KKGLAYSTI SQLGYMVMAMGI GAYSAGLFHLMTHA NdhF1-7120(slr0844) TAFLGATI AI TQNDI KKGLAYSTI SQLGYMVMAMGVGAYSAGLFHLMTHA NdhF-Spinacia oleracea TVLLGATLALAQKDI KRSLAYYTMSQLGYMMLALGMGSYRTALFHLI THA NuoL-E. coli T L LLAGF AAL VQT DI KRVLAYST MSQI GYMFLALGVQAWDAAI F HLMTHA TAFLGATI ALTQNDI KKGLAYSTMSQLGYMVMAMG. GAY. AGLFHLMTHA

360 370 380 390 400 NdhF1-7002(slr0844) YF KAMLF LGS GS VI HGMEEVVGHNAVLAQDMRL MGGLRKYMPI T AT T FL I NdhF1-6803(slr0844) YF KAMLF LCS GS VI HGMEGVVGHDPI LAQDMRI MGGLRKYMPI T AT CFL I NdhF1-7120(slr0844) YF KAMLF LGS GS VI HGMEAVVGHDPALAQDMRL MGGLRKYMPAT GLT FL I NdhF-Spinacia oleracea YSKALLFLASGSLI HSMGTI VGYSPDKSQNMVLMGGLTKHVPI TKTSFLI NuoL-E. coli FFKALLFLASGSVI LACH----H----EQNI FKMGGLRKSI PLVYLCFLV YF KAMLF L. S GS VI HGME VVGH P. LAQDMRL MGGLRKYMPI T . T . FL I 342

410 420 430 440 450 NdhF1-7002(slr0844) GT LAI CGI PPF-AGFWSKDEI LGLAFEANPV-LWFI GWATAGMTAFYMFR NdhF1-6803(slr0844) GT LAI CGI PPF-AGFWSKDEI LGLAFQANPL-LWFVGWATAGMTAFYMFR NdhF1-7120(slr0844) GCLAI SGI PPF-AGFWSKDEI LGAAYASNPL-LWFI GWMTAGI T AFYMFR NdhF-Spinacia oleracea GTLSLCGI PPL-ACFWSKDEI LNDSWVYSPI -FAI I AYFTAGLTAFYMFR NuoL-E. coli GGAALSALPLVT AGFFSKDEI LAGAMANGHI NLMVAGLVGAFMTSLYT FR GTLAICGIPPF AGFWSKDEILG. A. . NP. LWFIGW. TAGMTAFYMFR

460 470 480 490 500 NdhF1-7002(slr0844) MYFLTFEG------EFRGTDQQLQEKLLTA NdhF1-6803(slr0844) MYFMTFEG------GFRGNDQEAKDGVLQF NdhF1-7120(slr0844) MYFSTFEG------KFRGNDEKI KDKLLKA NdhF-Spinacia oleracea I YLLT FEGHLNFFCKNYSGKKSSSFYSI SLWGKKELKT I NQKI SLLNLLT NuoL-E. coli MIFI VFHG------K---EQ------I HA MYF.TFEG FRGDQ...LA

510 520 530 540 550 NdhF1-7002(slr0844) AGQAP------EEGHHGSKPHESPLTM NdhF1-6803(slr0844) YGLLPNFGPGAMNVKELDHEAGH------DDHGHSSEPHESPLTM NdhF1-7120(slr0844) KT I LLELESAEPT PVFGPGAMKKGELAAT GGHHDGHGHHSSSPHESPWTM NdhF-Spinacia oleracea MNNKERASFFSKKPYEINVKLTKLLRSFITITYFENKNISLYPYESDNTM NuoL-E. coli HAVKG------VTH ...... HSS PHESP.TM

560 570 580 590 600 NdhF1-7002(slr0844) TFPLMALAVPSVLI GLLGVPWGN-----RFEAFVFSPNEAAEAAEHG--- NdhF1-6803(slr0844) TFPLMALAVPSVLI GLLGRPWAN-----QFEAFI HAPGEVVEHAAE---- NdhF1-7120(slr0844) TLPLLI LAI PSMLIGLVGTPYNN-----YFERFI FSPTESLAEVLEKAAE NdhF-Spinacia oleracea LFPLI I LI MFTLFVGFI GI PFNQEGMDLDI LTKWLTPSI NLLHSNSEN-F NuoL-E. coli SLPLI VLLI LSTFVGALI VPPLQG------VLPQTTELAHG------TFPL..LA.PS.LIGLLG.P..N FE F. .P.E L.H.

610 620 630 640 650 NdhF1-7002(slr0844) FELTEFLI MGGNSVGI ALI GI TI ASLMYLQQRI DPARLAEK------NdhF1-6803(slr0844) FEWGEFYVMAGNSI GI ALI GI TVASLMYWQHKFDPKVLAEK------NdhF1-7120(slr0844) FDPNEFYI MAGGSVGVSLI GI TLASLMYLQRKI DPAAI AAK------NdhF-Spinacia oleracea VDWYEFVINAIFSISIAFFGIFIAFFFYKPIYSSLKNFDLINSFDKRGQK NuoL-E. coli S----MLTLEI TSGVVAVVGI LLAAWLWLGKRTLVTSI ANSAP------F. EF. I MAG S. GI ALI GI T. ASLMYLQ. . DP . A K

660 670 680 690 700 NdhF1-7002(slr0844) ------FPVLYQLSLNKWYFDDI YNNVFVMGTRRLARQI LEVDYRVVDG NdhF1-6803(slr0844) ------FPSLYQLSLNKWYFDDLYDKLFVQGSRRVARQI MEVDYKVI DG NdhF1-7120(slr0844) ------I KPLYELSLNKWYFDDI YHRVFVLGLRRLARQVMEVDFRVVDG NdhF-Spinacia oleracea RILGDNIITIIYNWSANRGYIDAFYSTFLIKGIRSLSELVSFFDRRIIDG NuoL-E. coli ------GRLLGTWWYNAWGFDWLYDKVFVKPFLGI AWLLK---RDPLNS . . LY. LSLNKWYFDD. Y . VFV G RRLARQ. EVD. RV. DG

710 720 730 740 750 NdhF1-7002(slr0844) AVNLTGIATLLSGEGLKYIENGRVQFYAL-IVFGAVLGFVIFFSVA NdhF1-6803(slr0844) AVNLTGLVTLVSGEGLKYLENGRAQFYAL-I VFGAVLGFVI VFSLT NdhF1-7120(slr0844) AVNLTGFFTLVSGEGLKYLENGRVQFYAL-I VFGAVLGLVI VFGVT NdhF-Spinacia oleracea I PNGFGVTSFFVGEGIKYVGGGRI SSYLF-WYLLYVSI FLFI FTFT NuoL-E. coli MMNIPAVLSRFAGKGLLLSENGYLRWYVASMSI GAVVVLALLMVLR AVNLTG..TL.SGEGLKY.ENGR.QFYAL IVFGAVLGFVI.F..T 343

Figure B11. ClustalW alignment of NdhF2 amino acid sequences. NdhF2 amino acid sequences of the following organisms were aligned using the ClustalW alignment program from MacVector v. 6.5: Synechococcus sp. PCC 7002, Synechocystis sp. PCC

(Thompson et al., 1994b). 344

NdhF2 ClustalW Amino Acid Alignment

10 20 30 40 50 NdhF2-7002(slr2009) MNELTI GWVI FPFVVGFSI YLLPKIDR------YLAI NdhF2-6803(slr2009) MNFPTAI APNNLLI AI VALLVLALMGAFGGYLFRPLVR------PSAL NdhF-Spinacia oleracea MEHI YQYAWII PFLPLPVPLLI GAGLLFFPTATKNLRRI WAFSSI S NuoL-E. coli MNMLALT I I LPLI GFVLLAFSRGRWSENVSAI VGVGSVALAA ...... L...... F. . .. A.

60 70 80 90 100 NdhF2-7002(slr2009) FVSI CSLI FGFVQI FQPEP------YSLKLLGMYGVDL--LVDDQSGYF NdhF2-6803(slr2009) LMTLGTI LLAAVGFALPNA------QQWQLMDRFGI LL--QLDNLGSYF NdhF-Spinacia oleracea LLSI VMIFSMKLAI QQI NSNSI YQYLWSWTI NNDFSLEFGYLMDPLTSI M NuoL-E. coli LVTALSALI SSLTASKTYS----QPLWTWMSVGDFNI GFNLVLDGLSLTM L...... W . F . ..D L.

110 120 130 140 150 NdhF2-7002(slr2009) I LTNAAVAI AVTVY--CWKSAKS--AFFFTQLVVLQGALNAVFVCADLI S NdhF2-6803(slr2009) LLTNGLVTLAVLLY--CWASPRT--TFFYVQLMVLHVSLNAAFLSTDLI S NdhF-Spinacia oleracea SMLI TTVAI LVLI YSDNYMSHDQGYLRFFAYMSFFNTSMLGLVTSSNLI Q NuoL-E. coli LSVVTGVGFLI HMYASWYMRGEEGYSRFFAYTNLFI ASMVVLVLADNLLL ..V...V.Y.SFF...S...LI

160 170 180 190 200 NdhF2-7002(slr2009) LYVALEAI SI AAFLLMTYQRTDRSI WIGLRYLFLSNTAMLF-YLI GAVLV NdhF2-6803(slr2009) LYVCLEVVGLSSFLLI I YPRQAASSWIGLRYLFVTNTALLF-YLI GVMLV NdhF-Spinacia oleracea I YI FWELVGMCSYLLI GFWFTRPI AANACQKAFVTNRVGDFGLLLGI LGL NuoL-E. coli MYLGWEGVGLCSYLLI GFYYT DPKNGAAAMKAFVVT RVGDVFLRFALFI L .Y. E.VG. S.LLI.. T ... .FV.N .. F L.G. ..

210 220 230 240 250 NdhF2-7002(slr2009) YQATKSFAFVGLAEAPSDAI A------LI FLGLLTKGGVFVS NdhF2-6803(slr2009) YQATNSLDFQGLATAPYEAI A------LI FLGLLI KGEI FLS NdhF-Spinacia oleracea YWITGSFEFRDLFEI FNNLI KNNEVNSLFCI LCAFLLFAGAVAKSAQFPL NuoL-E. coli YNELGT LNFREMVELAPAHLADG---NNMLMWATLMLLGGAVGKSAQLPL Y..T S F L.E. .IA L.F.G...K . F

260 270 280 290 300 NdhF2-7002(slr2009) GLWLPLTHSEAETPVSAMLSG-VVVKAGI FPLLRCG---I LVPDLDLWLR NdhF2-6803(slr2009) GLWSPQT SSI ASAPVAALLSG-I VVKAGI LPLLRFA---SLSERLAMMVW NdhF-Spinacia oleracea HVWLPDAM-EGPT PI SALI HAAT MVAAGI FLVARLLPLFVVI PYI MYVI S NuoL-E. coli QT WLADAM-AGPT PVSALI HAAT MVT AGVYLI ART HGLFLMTPEVLHLVG . WLP . TPVSAL. . V AGI . . . R . . . P . .

310 320 330 340 350 NdhF2-7002(slr2009) LFGLATALLGI I FAI LETDAKRLLAFSTI SKLGLLLSAPAVAG-----LA NdhF2-6803(slr2009) GLAI AT ALLGMGLGMFARDSRRI LAYST I SQMGFI LVAPAVGG-----LY NdhF-Spinacia oleracea FI GI I TVLLGATLALAQKDI KRSLAYYTMSQLGYMMLALGMGSYRTALFH NuoL-E. coli I VGAVTLLLAGFAALVQTDI KRVLAYSTMSQI GYMFLALGVQAWDAAI FH ..G..T.LLG. .A.. D.KR.LAYST SQ.G. .A .V..

360 370 380 390 400 NdhF2-7002(slr2009) ALSHGLVKSSLFLMAGQLPT-----RNFQELRQT------KI AS NdhF2-6803(slr2009) ALTHGLAKACLFLLVGSLPE-----RDLDKLQAQ------PI SY NdhF-Spinacia oleracea LI THAYSKALLFLASGSLI HSMGTI VGYSPDKSQNMVLMGGLTKHVPI TK NuoL-E. coli LMTHAFFKALLFLASGSVI LACHHEQNI FKMGGLR------KSI PLVY ..TH. KA LFL. GSL PI 345

410 420 430 440 450 NdhF2-7002(slr2009) S------LWLP------LAI ACLSMVGMPL NdhF2-6803(slr2009) K------LWLP------MVLASSSI I GLPI NdhF-Spinacia oleracea TSFLI GTLSLCGI PPLAC-FWSKDEI LNDSWVYSPI -FAI I AYFTAGLTA NuoL-E. coli LCFLVGGAAL SALPL VT AGF FSKDEI LAGAMANGHI NLMVAGLVGAF MTS W..A.G.

460 470 480 490 500 NdhF2-7002(slr2009) LVGFSSKALLLK------NI APWQA------NdhF2-6803(slr2009) LAGFEAKTLTLETLS------LNELPWTG------NdhF-Spinacia oleracea FYMFRI YLLTFEGHLNFFCKNYSGKKSSSFYSI SLWGKKELKTI NQKI SL NuoL-E. coli LYTFRMIFI VFHGKEQ------I HAHAVKG------LF L . W.

510 520 530 540 550 NdhF2-7002(slr2009) ---MGLN------NdhF2-6803(slr2009) ---I LMN------NdhF-Spinacia oleracea LNLLTMNNKERASFFSKKPYEI NVKLTKLLRSFI TI TYFENKNI SLYPYE NuoL-E. coli ---VTHS------.N

560 570 580 590 600 NdhF2-7002(slr2009) ------I AAVGTALAFAKFLFI P------HDAATKFTAKSTTFWG-- NdhF2-6803(slr2009) ------LAGVGTAI I LAKFI FLI PS-----FDKNVDLDKSPWGLFL-- NdhF-Spinacia oleracea SDNTMLFPLIILIMFTLFVGFIGIPFNQEGMDLDILTKWLTPSINLLHSN NuoL-E. coli ------LPLI VLLI LSTFVGALI VPPLQG--VLPQTTELAHGSMLTLEI T L...... F.F..P D T S

610 620 630 640 650 NdhF2-7002(slr2009) ------AI AFLFSGVI LGNGFYLEAYQLDNI PKALI KI A- NdhF2-6803(slr2009) ------AVLLLLGALTLGNVI YPEAFSMENGI KATASFL- NdhF-Spinacia oleracea SENFVDWYEFVI NAI FSI SI AFFGI FI AFFFYKPI YSSLKNFDLI NSFDK NuoL-E. coli ------SGVVAVVGI LLAAWLWLGKRTLVTSI ANSAPGR- .... G. L. Y ... . .

660 670 680 690 700 NdhF2-7002(slr2009) ------I GWALY------WLI MK------NdhF2-6803(slr2009) ------LGSAI Y------WWGLR------NdhF-Spinacia oleracea RGQKRILGDNIITIIYN-----WSANRGYIDAFYSTFLIKGIRSLSELVS NuoL-E. coli ------LLGTWWYNAWGFDWLYDKVFVKPFLGI AWLLK------.G...Y W . .

710 720 730 740 750 NdhF2-7002(slr2009) ----RI EFKLPRI FEAFEQLI GAMSVVLTG------LFWMVTL NdhF2-6803(slr2009) ----KI PWQPPDWGERLDHLI GTMAI MLML------LFSSVLI NdhF-Spinacia oleracea FF DRRI I DGI PNGFGVT S FF VGE GI KYVGGGRI SSYLFWYL LYVSI F LF I NuoL-E. coli ---RDPLNSMMNI PAVLSRFAGKGLLLSENG----YLRWYVASMSI GAVV .I P . . .G .. . . LFWV.

760 770 780 790 800 NdhF2-7002(slr2009) NdhF2-6803(slr2009) NdhF-Spinacia oleracea FTFT NuoL-E. coli VLALLMVLR 346

Figure B12. ClustalW alignment of NdhF3 amino acid sequences. NdhF3 amino acid sequences of the following organisms were aligned using the ClustalW alignment program from MacVector v. 6.5: Synechococcus sp. PCC 7002, Synechocystis sp. PCC

6803, Anabaena sp. PCC 7120, Spinacia oleracea, and E. coli. The consensus amino acid sequence is shown underneath. Gray high-lighted regions indicate identical amino acids while lighter shading indicates conserved amino acids. Dashes represent insertions/deletions included to maximize the sequence similarity. Dashes represent insertions/deletions included to maximize the sequence similarity. The percent identity and percent conserved amino acids were determined with the ClustalW alignment tool.

(Thompson et al., 1994b). 347

NdhF3 ClustalW Amino Acid Alignment

10 20 30 40 50 NdhF3-7002(sll1732) MNTFFSQSVWLVPCYPL---LGMGLSALWMPSI TRKTGPRPAGYVNMLLT NdhF3-6803(sll1732) MLESLSRI I WLVPCYAL---LGALLAVPWSPGLTRQTGPRPAGYI STLMT NdhF3-7120(sll1732) MAQFLLETVWLVPLYAL---I GGLLAI PWSPGI I RKTGPRPAGYVNLI MT NdhF-Spinacia oleracea MEHI YQYAWII PFLPLPVPLLI GAGLLFFPTATKNLRRI WAFSSI SLLS NuoL-E. coli MNMLALT I I LPLI GF--VLLAFSRGRWSENVSAI VG-VGSVALAALVT M . . WLVP Y. L LG. . L. . WSP . TR TGPRPAGY. L. T

60 70 80 90 100 NdhF3-7002(sll1732) FMALVHSCLAFI ERWEQPALKPSLTWLQAADLTLSI DLDI SSI TI GALI L NdhF3-6803(sll1732) FVAFLHSLLVLI HI WQQPAI DLSFPWLHAADLEI NFDLKI STVNIAALVL NdhF3-7120(sll1732) FLALVHSAI ALQAT WSHPPQEVFLPWLST AGLDLT I AI EI SSI SVGAMVV NdhF-Spinacia oleracea I VMIFSMKLAI QQI NSNSI YQYLWSWTI NNDFSLEFGYLMDPLTSI MSML NuoL-E. coli ALSALI SSLTASKTYSQP----LWTWMSVGDFNIGFNLVLDGLSLTMLSV F.A..HS LA. WSQP. ..WL .ADL L F L IS.....AL.L

110 120 130 140 150 NdhF3-7002(sll1732) I AGI NLLAQLYAVAYLEMDWGWARFFATMSLFEAGMCALVLCNSLFFSYV NdhF3-6803(sll1732) I T GLNLGAQI YAI GYLERDWGWARFFSLMALFEAGLCT LVLCNSLFFSYV NdhF3-7120(sll1732) I TGLNFLAQI FAI GYMEMDWGLGRFYSLLGLFEAGLCALVLCNNLFFSYV NdhF-Spinacia oleracea I TTVAI LVLI YSDNYMSHDQGYLRFFAYMSFFNTSMLGLVTSSNLIQI YI NuoL-E. coli VTGVGFLI HMYASWYMRGEEGYSRFFAYTNLFI ASMVVLVLADNLLLMYL I TG. N. LAQIYA. . YME DWG. . RFFA M LFEAGMC. LVLCNNLFFSYV

160 170 180 190 200 NdhF3-7002(sll1732) VLEI LTLGTYLLI GYWFNQSLVVTGARDAFLTKRVGDLFLLMGVVALLPL NdhF3-6803(sll1732) VLEI LTLGTYLLI GYWFNQSLVVTGARDAFLTKRVGDLI LLMGVVALLPL NdhF3-7120(sll1732) I LEI LTLGTYLLVGLWFSQPLVVSGARDAFLTKRVGDLFLLMGVLGLWPL NdhF-Spinacia oleracea FWELVGMCSYLLI GFWFTRPI AANACQKAFVTNRVGDFGLLLGI LGLYWI NuoL-E. coli GWEGVGLCSYLLI GFYYT DPKNGAAAMKAFVVT RVGDVFLRFALFI LYNE . LEI LTLGTYLLI G. WF. QPLVV. GARDAFLTKRVGDLFLLMGV. . L. PL

210 220 230 240 250 NdhF3-7002(sll1732) AGT WNFDGLAEWAATAELDPT LAT LLCLA----LI AGPLGKCAQFPLHLW NdhF3-6803(sll1732) AGSWNYDDLAQWAASADLNPT AAT LLCLA----LI AGPLAKCAQFPLHLW NdhF3-7120(sll1732) AGT WNYPELAQWAQTANVDPT I I T LVGLA----LVAGPMGKCAQFPLHLW NdhF-Spinacia oleracea TGSFEFRDLFEI FNNLI KNNEVNSLFCI LCAFLLFAGAVAKSAQFPLHVW NuoL-E. coli LGTLNFREMVELAPAHLADGNNMLMWATL---MLLGGAVGKSAQLPLQTW AGTWNF .LAEWA.A .DPT..TL.CLA L.AGP.GKCAQFPLHLW

260 270 280 290 300 NdhF3-7002(sll1732) LDEAMESPVPA-TVVRNSLVVGTGAWVLI KLQPI FALSDFASTFMIAI GA NdhF3-6803(sll1732) LDEAMEGPI PA-TI LRNTLVVATGAWVLVKVQPI LALSPVALTVMIAI GS NdhF3-7120(sll1732) LDEAMEGPMPS-TI LRNSVVVASGAWVLI KLQPVLTLSPVVSSFI VAI GA NdhF-Spinacia oleracea LPDAMEGPTPISALIHAATMVAAGIFLVARLLPLFVVIPYIMYVISFIGI NuoL-E. coli LADAMAGPTPVSALI HAATMVTAGVYLI ARTHGLFLMTPEVLHLVGI VGA LDEAMEGP P. T..RN..VVA.GAWVL.KLQP.F.LSP . ....AIGA

310 320 330 340 350 NdhF3-7002(sll1732) TTALGAAMVAI AQI DI KRSLSYSVSAYMGMVFMAVGSQQDQTTLVLLLTY NdhF3-6803(sll1732) VTAI GASLI AI AQI DI KRFLSYVVSAYMGLVFI AVGTGQGETALQLI FTY NdhF3-7120(sll1732) VTAI GGSLI AI AQI DVKRCLSYSVSAYMGLVFI AVGARQDEAALLLVLTH NdhF-Spinacia oleracea I TVLLGATLALAQKDI KRSLAYYTMSQLGYMMLALGMGSYRTALFHLI TH NuoL-E. coli VTLLLAGFAALVQTDI KRVLAYSTMSQI GYMFLALGVQAWDAAI FHLMTH VTALGA. . AI AQI DI KR LSYSVSAYMG VF. AVG Q . TAL LL. TH

360 370 380 390 400 NdhF3-7002(sll1732) GVAMAI LVMAI GGVVLVN------I SQDLTQYGGLWSRRPI TGI CYL NdhF3-6803(sll1732) TFAMAI LVMCVGGI I LNN------VTQDLTQYGGLWSRRPI SGLSYL NdhF3-7120(sll1732) AVSAALLVMSTGGI I WNS------I TQDVTQLGGLWSRRPI SGLAFI NdhF-Spinacia oleracea AYSKALLFLASGSLI HSMGTI VGYSPDKSQNMVLMGGLTKHVPI TKTSFL NuoL-E. coli AFFKALLFLASGSVI LACH------HEQNI FKMGGLRKSI PLVYLCFL A. ALLVMA. GG. I L . . QD. T Q GGLWSRRPI . GL FL 348

410 420 430 440 450 NdhF3-7002(sll1732) VGAASLVALPPFGG-FWSLAQLTTNFWKTSPI LAVI LI TV-NALTSFSI M NdhF3-6803(sll1732) VGVASLI ALPPFGT-FWAWLKLAENLSATSPLLVGVLLVV-NLLTAFNVT NdhF3-7120(sll1732) VGT LGLI GFPPLGG-FWALLKLADGLWGT HPWLVGI VI AV-NALT AFSLV NdhF-Spinacia oleracea I GTLSLCGI PPLAC-FWSKDEI LNDSWVYSPI FAI I AYFT-AGLTAFYMF NuoL-E. coli VGGAALSALPLVT AGFFSKDEI LAGAMANGHI NLMVAGLVGAFMTSLYT F VG.ASL.ALPP.G. FWS. L. .W.TSPIL..I...V N.LTAF .

460 470 480 490 500 NdhF3-7002(sll1732) REFGLI FGGKP------KQMTVRSPEGLWA------NdhF3-6803(sll1732) RGFCLI FGGEA------KPMTVRSPEGLWA------NdhF3-7120(sll1732) REFGLI FGGKA------KQMTERSPEVHWP------NdhF-Spinacia oleracea RI YLLTFEGHLNFFCKNYSGKKSSSFYSI SLWGKKELKTI NQKI SLLNLL NuoL-E. coli RMIFI VFHGKE------QI HAHAVKGVTH------RF.LIFGGK. KQMT.RSPEGLW.

510 520 530 540 550 NdhF3-7002(sll1732) ------NdhF3-6803(sll1732) ------NdhF3-7120(sll1732) ------NdhF-Spinacia oleracea TMNNKERASFFSKKPYEINVKLTKLLRSFI TI TYFENKNISLYPYESDNT NuoL-E. coli ------

560 570 580 590 600 NdhF3-7002(sll1732) LVLPMVI LAGFALHSP-FI LAKLN------FLPDWHQLNLPLAA----- NdhF3-6803(sll1732) LVLPMVVTVGFALHLS-LI LKQGN------LLPDFADI NWGLSS----- NdhF3-7120(sll1732) MVLPMMILFGFVLHLP-LI LQALS------LLPDWAI LNKDVVL----- NdhF-Spinacia oleracea MLFPLIILIMFTLFVG-FIGIPFNQEGMDLDILTKWLTPSINLLHSNSEN NuoL-E. coli -SLPLI VLLI LSTFVGALI VPPLQGVLPQTTELAHGSMLTLEI TSG---- VLPM.IL.GF.LH. LIL LN .LPDW. LN. L.

610 620 630 640 650 NdhF3-7002(sll1732) -----VLI I STMVGGGTAMY-LYLNEKI SKPI HI FSDPVREFFAKD---- NdhF3-6803(sll1732) -----VLI ASSLLGVGSSAF-I YLNPKI TKPI DLPLPVVQNFFAYD---- NdhF3-7120(sll1732) -----LLI WSTI FGCSI SSV-I YLSNI I PKPI RLPWKGLQDLLAYD---- NdhF-Spinacia oleracea FVDWYEFVI NAIFSI SI AFFGIFI AFFFYKPI YSSLKNFDLI NSFDKRGQ NuoL-E. coli ----VVAVVGI LLAAWLWLGKRTLVTSI ANSAPGRLLGTWWYNAWG---- VLI.S...G. . . IYL I KPI . L .. . A.D

660 670 680 690 700 NdhF3-7002(sll1732) ------LYTAELYKNTVI FAVALI SKI I DWLDRYFVD NdhF3-6803(sll1732) ------LYTDKFYKLTI VAVI DSI SRLI NWFDKTFVD NdhF3-7120(sll1732) ------FYTPNLYRI TI I FSVAQLSKFADMIDRFVVD NdhF-Spinacia oleracea KRILGDNIITIIYNWSANRGYIDAFYSTFLIKGIRSLSELVSFFDRRIID NuoL-E. coli ------FDWLYDKVFVKPFLGI AWLLK---RDPLN .YTDLY.T.I...IS.L..DRVD

710 720 730 740 750 NdhF3-7002(sll1732) GVI NFLGLAT LFGGQSLKYNNSGQSQSYALSI VAGI LLFI AALSYPLLKH NdhF3-6803(sll1732) GVI NLI GI VT I FSGQSLKYNVSGQT QFYVLSI VLGLT LI GAFLSYSLLGQ NdhF3-7120(sll1732) GI VNFVGLFSLLGGEGLKYST SGQT QFYALT VLLGVGVLGAWVT WPFWGF NdhF-Spinacia oleracea GI PNGFGVT SFFVGEGI KYVGGGRI SSYLFWYLLYVSI FLFI FT FT NuoL-E. coli SMMNIPAVLSRFAGKGLLLSENGYLRWYVASMSI GAVVVLALLMVLR G..N..G..S.F.G GLKY SGQ.Q.Y.LS..LG.....A.L..

760 770 780 790 800 NdhF3-7002(sll1732) WQF NdhF3-6803(sll1732) AF NdhF3-7120(sll1732) QFLDLMF NdhF-Spinacia oleracea NuoL-E. coli 349

Figure B13. ClustalW alignment of NdhF4 amino acid sequences. NdhF4 amino acid sequences of the following organisms were aligned using the ClustalW alignment program from MacVector v. 6.5: Synechococcus sp. PCC 7002, Synechocystis sp. PCC

(Thompson et al., 1994b). 350

NdhF4 ClustalW Amino Acid Alignment

10 20 30 40 50 NdhF4-7002(sll0026) MSEFLLQSVWLVPVYGI TGALLT-LPWSLGLI RRTGPRPAAYLNLI MTFL NdhF4-6803(sll0026) MSDFLLQSSWFIPFYGLI GSI LS-LPWSFRLI KQTGPRPAAYFNVFMTLV NdhF4-7120(sll0026) MNQFLFATSWCVPFYSLLGALLT-LPWGI GI VRRTGPRPAAYFNLLTTI V NdhF-Spinacia oleracea MEHI YQYAWII PFLPLPVPLLI -GAGLLFFPTATKNLRRI WAFSSI SLL NuoL-E. coli MNMLALTI I LPLI GFVLLAFSRGRWSENVSAI VGVGSVALAALVT ALS M . FLLQ. . W.. PFYGL. G. LL. LPWS. . . . TGPRPAAY. NL. TL.

60 70 80 90 100 NdhF4-7002(sll0026) GLLHGSFAFASLWNMPPQ---QLSLEWLQVADLNLSLVI EI SPVNLGAME NdhF4-6803(sll0026) SAI HGMVALSAI WQTPSE---QI VFHWLQVADLDLTLAVEI SPVSLGALS NdhF4-7120(sll0026) GFAHSLWVFKDIWSREQE---NLVITWFQAADLNLSFALELSPVSMGATV NdhF-Spinacia oleracea SI VMI FS MKL AI QQI NSNSI YQYLWSWTI NNDF SLE FGYLMDPL T SI MS NuoL-E. coli ALI SSLTASKTYS------QPLWTWMSVGDFNI GFNLVLDGLSLTMLS ...H. A IW. Q....W QVADLNL.F..E.SPVSLGA

110 120 130 140 150 NdhF4-7002(sll0026) LVTGI CFMAQLYGLGYLEKDWSI ARFYGLMGFFEAALSGLAI SDSLLLSY NdhF4-6803(sll0026) VVTGI SFLVQI FGLGYMEKDWSLARFYGLLGFFEAALGGI ALSDSLFLSY NdhF4-7120(sll0026) LI TGLSLLAQI YALGYMEKDWSLARFFGLLGFFEAALSGLAI SDSLFLSY NdhF-Spinacia oleracea LI TTVAI LVLI YSDNYMSHDQGYLRFFAYMSFFNTSMLGLVTSSNLI QI Y NuoL-E. coli VVTGVGFLI HMYASWYMRGEEGYSRFFAYTNLFI ASMVVLVLADNLLLMY LVTG. FL. QI Y. LGYMEKDWS. ARFFGL GFFEAAL. GLA. SDSL. LSY

160 170 180 190 200 NdhF4-7002(sll0026) GLLEVLTLSTYLLVGFWYAQPLVVTAARDAFLTKRVGDI LLLMGI VALSS NdhF4-6803(sll0026) GLLEMLTLSTYLLVGFWYAQPLVVTAARDAFLTKRVGDI I LLMGVVALSS NdhF4-7120(sll0026) GLLEI LTLSTYLLVGFWYAQPLVVTAARDAFWTKRVGDLLLLMAVVTLST NdhF-Spinacia oleracea I FWELVGMCSYLLI GFWFTRPI AANACQKAFVTNRVGDFGLLLGI LGLY NuoL-E. coli LGWEGVGLCSYLLI GFYYTDPKNGAAAMKAFVVTRVGDVFLRFALFI LYN GLLE. LTLSTYLLVGFWYAQPLVVTAARDAF. TKRVGD. . LLMG. V. LS.

210 220 230 240 250 NdhF4-7002(sll0026) YGTGLTFSELETWAANPP----LPPWEASLVGLALI SGPI GKCAQFPLNL NdhF4-6803(sll0026) YGQGL T F SQL DNWAS T VP----VT GI T AT LL GL SLI AGPT GKCAQFPLNL NdhF4-7120(sll0026) LAGSL NF SDL YE WVQT AN----L DPVT AT LL CL GLI AGPAGKCAQFPLHL NdhF-Spinacia oleracea ITGSFEFRDLFEIFNNLIKNNEVNSLFCILCAFLLFAGAVAKSAQFPLHV NuoL-E. coli EL GT L NF REMVE LAPAHL AD---GNNMLMWAT L MLL GGAVGKSAQLPLQT .G.L FS.L EWA . . . A.L..L.LIAGP.GKCAQFPL.L

260 270 280 290 300 NdhF4-7002(sll0026) WLDEAMEGPNP-AGI I RNSVVVSAGAYVLLKMEPVFTI TPI TSDALI I I G NdhF4-6803(sll0026) WLDEAMEGPNP-AGI MRNSVVVSAGAYVLI KLQPVFTLSPI ASKTLI VLG NdhF4-7120(sll0026) WLDEAMEGPNP-ASVMRNSL VVAGGAYL LYKLQPI L I LS PVALNVLI I I G NdhF-Spinacia oleracea WLPDAMEGPTPI SALI HAATMVAAGI FLVARLLPLFVVI PYI MYVI SFI G NuoL-E. coli WLADAMAGPTPVSALI HAATMVTAGVYLI ARTHGLFLMTPEVLHLVGI VG WLDEAMEGPNP A..IRNS.VV.AGAYLL.KL P.F...P.. .LIIIG

310 320 330 340 350 NdhF4-7002(sll0026) TVTTVGASLVALAQI DI KRALSHSTSAYLGLVFI AVGLNQVDI ALLLLLT NdhF4-6803(sll0026) TLTVVMTSLI AIAQIDIKRTLSHSTSVYLGLVFI AVGLGQVDIAFLLLFA NdhF4-7120(sll0026) GVT AI GASLVSI AQT DI KRALSHST S AYMGL VF LAVGLE QGGVALMLLL T NdhF-Spinacia oleracea I I TVLLGATLALAQKDIKRSLAYYTMSQLGYMMLALGMGSYRTALFHLI T NuoL-E. coli AVT LL LAGFAAL VQT DI KRVLAYST MSQI GYMFLAL GVQAWDAAI FHLMT .VT...ASL.ALAQ DIKR.LSHSTS.YLGLVFLAVGL Q.D.AL LL.T

360 370 380 390 400 NdhF4-7002(sll0026) HAI AKALLFMSI GAVI LNTH------GQNI TEMGGLWSRMPATTSAF NdhF4-6803(sll0026) HAI AKALLFMSI GSI I FTTS------GQNI TEMGGLWNRMPVTTTSF NdhF4-7120(sll0026) HAI AKALLFMSSGSVI FTTH------SQDLTEMGGLWSRMPATTTAF NdhF-Spinacia oleracea HAYSKAL LFL AS GSL I HS MGT I VGYS PDKSQNMVLMGGLT KHVPI T KT S F NuoL-E. coli HAFFKALLFLASGSVI LACH------HEQNI FKMGGLRKSI PLVYLCF HAI AKAL LFMSS GSVI . T H QNI T EMGGLW RMP. T T T F 351

410 420 430 440 450 NdhF4-7002(sll0026) VVGSAGLVCLFPLGT-FWTMRRWVDGFWDTPPWLVLLLVGV-NFCSSFNL NdhF4-6803(sll0026) VVGSAGLLAVFPLGM-FWTWQKWFSGDWLVSWPLLALLI FV-NLFSALNL NdhF4-7120(sll0026) VVGSAGMVTLLPLGS-FWAMLSWADGLVRVSPWVI GVLI LV-NGLTALNL NdhF-Spinacia oleracea LI GTLSLCGI PPLAC-FWSKDEI LNDSWVYSPI FAI I AYFT-AGLTAFYM NuoL-E. coli LVGGAALSALPLVT AGFFSKDEI LAGAMANGHI NLMVAGLVGAFMTSLYT VVGSAGL..L PLG FW. W. G W. SP ....L..V N. TALNL

460 470 480 490 500 NdhF4-7002(sll0026) TRVFRSVFLGAP--KPKTRRSPE------VVW------NdhF4-6803(sll0026) TRVFRLVFLGKP--QPKTRRAPE------VPW------NdhF4-7120(sll0026) TRVFRLAFWGQP--QQKTRRAPE------VGW------NdhF-Spinacia oleracea FRI YLLT FEGHLNFFCKNYSGKKSSSFYSI SLWGKKELKT I NQKI SLLNL NuoL-E. coli FRMIFI VFHGKE--QI HAHAVKG------VTH------TRVFRLVF G. P Q KTRR. PE V. W

510 520 530 540 550 NdhF4-7002(sll0026) ------NdhF4-6803(sll0026) ------NdhF4-7120(sll0026) ------NdhF-Spinacia oleracea LTMNNKERASFFSKKPYEINVKLTKLLRSFITITYFENKNISLYPYESDN NuoL-E. coli ------

560 570 580 590 600 NdhF4-7002(sll0026) QMAVPMVSLI LMTLMVPFF------LHQWQLLFNPSLPTLVER NdhF4-6803(sll0026) PMAVPMVSLI I VTLLVPI A------PLQWSFWLSATYPLGLTS NdhF4-7120(sll0026) TMAFPMVTLI I LTLLLPLM------LQQWYLLP------AWES NdhF-Spinacia oleracea TMLFPLI I LI MFTLFVGFI GI PFNQEGMDLDI LTKWLTPSI NLLHSNSEN NuoL-E. coli --SLPLI VLLI LSTFVGAL------I ---VPPLQGVLPQTTEL MA.PMV.LII.TL VP.. L QW LP . E

610 620 630 640 650 NdhF4-7002(sll0026) PLI VTLAI PALMITGGLGLVAGLTI T---LNPSLS------RPRQL NdhF4-6803(sll0026) P-VTQWAMPLLMVAGI TGI LLGSLMP---LRRNLS------RSSRL NdhF4-7120(sll0026) I --DWYVVLVLVSSTVAGVVI GSTI H---LHKAWS------RSTVL NdhF-Spinacia oleracea F-VDWYEFVINAIFSISIAFFGIFIAFFFYKPIYSSLKNFDLINSFDKRG NuoL-E. coli A---HGSMLTLEI TSGVVAVVGI LLAAWLWLGKRT------LVTSI . .. ..L I....G.V.G. I L. S R . L

660 670 680 690 700 NdhF4-7002(sll0026) YLRFLQDLLAYDFY------I DRI YNVTVVWLVTTLSKLAAWFDRYVV NdhF4-6803(sll0026) PVRFLQDLFAYDVY------LDKI YGATVVAAVAAI AKI STWFDRYVI NdhF4-7120(sll0026) AWRFI QDLLGYDFY------I DRI YRLTVVSAVALLSRI SAWSDRYLV NdhF-Spinacia oleracea QKRILGDNIITIIYNWSANRGYIDAFYSTFLIKGIRSLSELVSFFDRRII NuoL-E. coli ANSAPGRLLGTWWYNAWG----FDWLYDKVFVKPFLGI AWLLK---RDPL RFLQDLL.YD.Y ID.IY .TVV .V..LS.L. WFDRY..

710 720 730 740 750 NdhF4-7002(sll0026) DGFVNLTGLATLFSGSALRYNVSGQSQFYVLTI VLGMILGLVWFMATGQW NdhF4-6803(sll0026) DGI VNLVSLVTI FSGSALKYNVTGQSQFYLLTI LVGVAL-LI WFSLSGQW NdhF4-7120(sll0026) DGLVNLVGFAT I FGGQGLKYSI SGQSQGYMLT I LAVVGALGFFI SWSLGL NdhF-Spinacia oleracea DGI PNGFGVT SFFVGEGI KYVGGGRI SSYLFWYLLYVSI FLFI FT FT NuoL-E. coli NSMMNIPAVLSRFAGKGLLLSENGYLRWYVASMSI GAVVVLALLMVLR DG.VNL G..T.F.G GLKY ..GQSQ.Y.LTIL.GV...L..F....

760 770 780 790 800 NdhF4-7002(sll0026) TMITDFWSNQLA NdhF4-6803(sll0026) MAI RQFWSSWLSLI LP NdhF4-7120(sll0026) LDKLPF NdhF-Spinacia oleracea NuoL-E. coli F 352

Figure B14. ClustalW alignment of NdhF5 amino acid sequences. NdhF5 amino acid sequences of the following organisms were aligned using the ClustalW alignment program from MacVector v. 6.5: Synechococcus sp. PCC 7002, Synechocystis sp. PCC

(Thompson et al., 1994b). 353

NdhF5 ClustalW Amino Acid Alignment

10 20 30 40 50 NdhF5-7002(slr2007) MIDDI TI I WILLPFVVGFSI YLLPRWNR------YFALA NdhF5-6803(slr2007) MTLFAVSAEFNAAPWATVVI CLALMAGFTGYLLPATI R------FLTLA NdhF5-7120(slr2007) MTTI TLTWITLPFLLGFI I YLVPKLDK------YLALG NdhF-Spinacia oleracea MEHI YQYAWII PFLPLPVPLLI GAGLLFFPTATKNLRRI WAFSSI S NuoL-E. coli MNMLALTI I LPLI GFVLLAFSRGRWSENVSAI VG------VGSVA . . I ...I LPF..GF .YL.P . . ...LA

60 70 80 90 100 NdhF5-7002(slr2007) I AALSVVYSI GLLWSLEP------FTLELLDSFGVTL--MFDELSGYF NdhF5-6803(slr2007) VCFGTGFLAYLGFSLPEA------QSWYLLDSFGVVF--QLDALSGYF NdhF5-7120(slr2007) AALASAGYAAQLFVAQSP------LELRLLDNFGVTL--TLDELTGYF NdhF-Spinacia oleracea LLSI VMIFSMKLAI QQI NSNSI YQYLWSWTI NNDFSLEFGYLMDPLTSI M NuoL-E. coli LAALVTALSALI SSLTAS-KTYSQPLWTWMSVGDFNI GFNLVLDGLSLTM .A...... S..L . ..W LLD FGV F LD LSGYF

110 120 130 140 150 NdhF5-7002(slr2007) I LMNGLVTGAVLLYCFDKQKSPFFYTQLVI LHGAVNATFC----CADLI S NdhF5-6803(slr2007) LLTNALVTLAVLVYCWNTGRSAFFYAQLI I LHASLNSAFL----CADFMS NdhF5-7120(slr2007) I LTNALVTI AVI LYCWQSDKTAFFYVQTMMLHGSVNAAFA----CTDFI S NdhF-Spinacia oleracea SMLITTVAI LVLI YSDNYMSHDQGYLRFFAYMSFFNTSMLGLVTSSNLIQ NuoL-E. coli LSVVTGVGFLI HMYASWYMRGEEGYSRFFAYTNLFI ASMVVLVLADNLLL .L N.LVT.AVL.YC.. .. FFY.Q .LH. .NA.F. C DLIS

160 170 180 190 200 NdhF5-7002(slr2007) LYVALECI GI AAFLLI TYSRSDRSLWVGLRYLFI SNTAMLF-YLI GAVLV NdhF5-6803(slr2007) LYVALEVVAI AAFCLMTYPREPRI I WLGLRYLLLSNTAMLF-YLI GVALV NdhF5-7120(slr2007) LYVALEVSGI AAFLLI AYPRTDRSI WVGLRYLFI SNVAMLF-YLVGAVLA NdhF-Spinacia oleracea I YI FWELVGMCSYLLI GFWFTRPI AANACQKAFVTNRVGDFGLLLGI LGL NuoL-E. coli MYLGWEGVGLCSYLLI GFYYT DPKNGAAAMKAFVVT RVGDVFLRFALFI L LYVALE. VGI AAFLLI . Y RTDR . W.GLRYLF. SN AMLF YL. G. . L.

210 220 230 240 250 NdhF5-7002(slr2007) YQASNSFAFSGLAVAPKEAI A------LI FLGLLTKGGI FVS NdhF5-6803(slr2007) YKTNQSFAFSGLTQAPPEAI A------LI FLGLLTKGGI FLA NdhF5-7120(slr2007) YQTNHSFAFSSLRGAPPEALA------LI FLGLLVKGGVFVS NdhF-Spinacia oleracea YWITGSFEFRDLFEI FNNLI KNNEVNSLFCI LCAFLLFAGAVAKSAQFPL NuoL-E. coli YNELGT LNFREMVELAPAHLADG---NNMLMWAT LMLLGGAVGKSAQLPL Y. SFAFS L APPEAIA LIFLGLL.KGG.F..

260 270 280 290 300 NdhF5-7002(slr2007) GLWLPLTHGESETPVSALLSG-VVVKAGVFPLARCA---LLVPELDPVVR NdhF5-6803(slr2007) GLWLPQTHGEAATPVSAMLSG-AVVKAGALPLLRCA---LLSDQLLLLVQ NdhF5-7120(slr2007) GLWLPLTHSESDTPVSALLSG-VVVKTGVYPLVRCA---LI LDEVDPVI R NdhF-Spinacia oleracea HVWLPDAM-EGPT PI SALI HAAT MVAAGI FLVARLLPLFVVI PYI MYVI S NuoL-E. coli QT WLADAM-AGPT PVSALI HAAT MVT AGVYLI ART HGLFLMTPEVLHLVG GLWLP TH E. TPVSALLSG . VVKAGV. PLARCA L. . PE. VV

310 320 330 340 350 NdhF5-7002(slr2007) LFGVGT ALLGVGYAVFEKDT KRMLAFHT VSQLGFVLAAPAVGG-----FY NdhF5-6803(slr2007) I LGVAT ALFGVVYAMLAKDSKRMLAFHT VSQMGFVLAAPI AGG-----FY NdhF5-7120(slr2007) ILGAGTALLGVSYAIFEKDTKRMLAWSTISQLGWIMSAPEVAG-----FY NdhF-Spinacia oleracea FI GI I TVLLGATLALAQKDI KRSLAYYTMSQLGYMMLALGMGSYRTALFH NuoL-E. coli I VGAVTLLLAGFAALVQTDI KRVLAYSTMSQI GYMFLALGVQAWDAAI FH I . G. . TALLGV YA. . KD. KRMLA. T. SQLG. . . AP. VGG FY

360 370 380 390 400 NdhF5-7002(slr2007) ALTHGLVKGALFLTAGQLS------SRN---FKVLRE-----QSI PR NdhF5-6803(slr2007) ALSHGLVKSSLFLLAGNLP------SRD---FKVLKK-----TPI AA NdhF5-7120(slr2007) ALTHGLVKSVLFLI AGSLP------SRN---FKELKN-----KPI NT NdhF-Spinacia oleracea LI T HAYSKALLFLASGSLI HSMGTI VGYSPDKSQNMVLMGGLT KHVPI T K NuoL-E. coli LMTHAFFKALLFLASGSVI LACH----HEQNI FKMGGLRKS----I PLVY ALTHGLVK. . LFL. AGSL SRN FKVL. PI 354

410 420 430 440 450 NdhF5-7002(slr2007) A------YW------WVLVLA----- NdhF5-6803(slr2007) G------FW------VPLLLA----- NdhF5-7120(slr2007) S------VW------I ALVI G----- NdhF-Spinacia oleracea TSFLI GTLSLCGI PPLAC-FWSKDEI LNDSWVYSPI FAI I AYFTAGLTAF NuoL-E. coli LCFLVGGAALSALPLVTAGFFSKDE------I LAGAMAN---- .FW.AL..A

460 470 480 490 500 NdhF5-7002(slr2007) ------CASI SG-----LPFLAGYSSK------NdhF5-6803(slr2007) ------SSSI AG-----FPLLAGFEAK------NdhF5-7120(slr2007) ------SLSI SG-----FPLFSGFGAK------NdhF-Spinacia oleracea YMFRI YLLTFEGHLNFFCKNYSGKKSSSFYSI SLWGKKELKTI NQKISLL NuoL-E. coli ------GHI NLMVAGLVG-----AFMTSLYTFR------M .SISG FP .SG. K

510 520 530 540 550 NdhF5-7002(slr2007) -I LTMKN------I LPWQS NdhF5-6803(slr2007) -TLTLKG------LPPWLA NdhF5-7120(slr2007) -LLTMKN------LLPWQV NdhF-Spinacia oleracea NLLTMNNKERASFFSKKPYEI NVKLTKLLRSFI TI TYFENKNI SLYPYES NuoL-E. coli I FI VFHGKEQ------I HAHAVK .LTMKN L. PW

560 570 580 590 600 NdhF5-7002(slr2007) FALNVAAVGTAI SFAKFI -FLPKKTDPS----LKI LPNFYGAI VLLLG-- NdhF5-6803(slr2007) I ALNI AAVGTAI SFSKFV-FLKPTFVG-----KTYPLGLGLALVVLLG-- NdhF5-7120(slr2007) I VMNVAALGTAI TFAKFI -FLPHGGK------REVKQGLWPGVI LLI S-- NdhF-Spinacia oleracea DNT MLFPL I I LI MFT LFVGFI GI PFNQEGMDLDI LT KWLT PS I NL LHS NS NuoL-E. coli GVTHSLPLI VLLI LSTFVGALI VPPLQG---VLPQTTELAHGSMLTLEI T .. N.AALGTAI.F.KFV FL . L ...LLL

610 620 630 640 650 NdhF5-7002(slr2007) ------GLFVTNSFYLEAYQ------NdhF5-6803(slr2007) ------GLAVGNVVYWQAFT------NdhF5-7120(slr2007) ------ALI VANI VYYDAYT------NdhF-Spinacia oleracea ENFVDWYEFVI NAIFSI SI AFFGI FI AFFFYKPIYSSLKNFDLI NSFDKR NuoL-E. coli S------GVVAVVGI LLAAWLWLG------GL.V.N..Y AY.

660 670 680 690 700 NdhF5-7002(slr2007) --PSNI LKALVTI GI GWLA------YGLI FQ NdhF5-6803(slr2007) --PSNLI KATLTCVVGAGL------YWVVVK NdhF5-7120(slr2007) --VDNI I KALAI I GI GWLV------YLFI VQ NdhF-Spinacia oleracea GQKRILGDNIITIIYNWSANRGYIDAFYSTFLIKGIRSLSEL--VSFFDR NuoL-E. coli --KRTLVTSI ANSAPGRLLGTWWYNAWGFDWLYDKVFVKPFLGIAWLLKR NL.KA..TI..GWL. Y .. .

710 720 730 740 750 NdhF5-7002(slr2007) RI TVKLPR------VVEQFEHLV-G------VMSLVLTGLFWLVLAN NdhF5-6803(slr2007) RLTLKLPD------EGEQVDHLL-G------MMSI SLTI LFAWILV NdhF5-7120(slr2007) RLTI KLPR------VLEQFEHLV-G------FMSLMLVLLFWMVLAN NdhF-Spinacia oleracea RI I DGI PNGFGVTSFFVGEGI KYVG-GGRI SSYLFWYLLYVSI FLFI FTF NuoL-E. coli DPLNSMMNI PAVLSRFAGKGLLLSENGYLRWYVASMSI GAVVVLALLMVL R. T. KLP VGEQ. . HL. G MSL. LV. LF. . L.

760 770 780 790 800 NdhF5-7002(slr2007) NdhF5-6803(slr2007) NdhF5-7120(slr2007) NdhF-Spinacia oleracea T NuoL-E. coli R 355 356

Figure B15. ClustalW alignment of NdhF amino acid sequences from Synechococcus sp.

PCC 7002 against each other. The Synechococcus sp. PCC 7002 NdhD amino acid sequences were aligned using the ClustalW alignment program from MacVector v. 6.5.

ClustalW program (Thompson et al., 1994b). 357

7002 NdhF ClustlW Amino Acid Alignments

460 470 480 490 500 NdhF1-7002(slr0844) MFRMYFLTFEGEFRGTDQQLQEKLLTAAGQAPEEGHHGSKPHESPLTMTF NdhF2-7002(slr2009) SKALLLKNIA------P------NdhF3-7002(sll1732) IMREFGLIFGGK------PKQMTVRSPE NdhF4-7002(sll0026) LTRVFRSVFLGA------PKPKTRRSPE NdhF5-7002(slr2007) SKILTMKNIL------P------R. . F. G P.

Figure B16. ClustalW alignment of NdhC amino acid sequences. NdhC amino acid sequences of the following organisms were aligned using the ClustalW alignment program from MacVector v. 6.5: Synechococcus sp. PCC 7002, Synechocystis sp. PCC

6803, Anabaena sp. PCC 7120, E. coli, and Spinacia oleracea . The consensus amino acid sequence is shown underneath. Gray high-lighted regions indicate identical amino acids while lighter shading indicates conserved amino acids. Dashes represent insertions/deletions included to maximize the sequence similarity. The percent identity and percent conserved amino acids were determined with the ClustalW alignment tool.

(Thompson et al., 1994b).

NdhC ClustalW Amino Acid Alignment

10 20 30 40 50 NdhC-7002 VFVLSGYEYFLGFLI VSSLVPI LALTASKLLRPKGGGPERKTTY NdhC-6803 MFVLTGYEYFLGFLFI CSLVPVLALTASKLLRPRDGGPERQTTY NdhC-7120 MFVLSGYEYLLGFLI I CSLVPALALSASKLLRPTGNSLERRTTY NdhC-Spinacia oleracea MFLLYEYDI FWAFLI I SSVI PI LAFLFSGI LAPI SKGPEKLSSY NuoA-E. coli MSMSTSTEVI AHHWAFAI FLI VAI GLCCLMLVGGWFLGGRARARSKNVPF MFVL. GYEYFLGFLI I SLVP. LAL. ASKLLRP. . GPER TTY

60 70 80 90 100 NdhC-7002 ESGMEPI GGAWI QFNI RYYMFALVFVVFDVETVFLYPWAVAFNQLGLLAF NdhC-6803 ESGMEPI GGAWI QFNI RYYMFALVFVVFDVETVFLYPWAVAFNQLGLLAF NdhC-7120 ESGMEPI GGAWI QFNI RYYMFALVFVVFDVETVFLYPWAVAFHRLGLLAF NdhC-Spinacia oleracea ESGI EPMGDAWLQFRI RYYMFALVFVVFDVETVFLYPWAMSFDI LGVSVF NuoA-E. coli ESGI DSVGSARLRLSAKFYLVAMFFVI FDVEALYLFAWSTSI RESGWVGF ESGMEPI GGAWI QFNI RYYMFALVFVVFDVETVFLYPWAVAF LGLLAF

110 120 130 140 150 NdhC-7002 VEALI FI AI LVI ALVYAWRKGALEWS NdhC-6803 VEALI FI AI LVVALVYAWRKGALEWS NdhC-7120 I EALI FI AI LVVALVYAWRKGALEWS NdhC-Spinacia oleracea I EALI FVLI LI VGLVYAWRKGALEWS NuoA-E. coli VEAAI FI FVLLAGLVYLVRI GALDWTPARSRRERMNPETNSI ANRQR VEALI FI AI LVVALVYAWRKGALEWS 360

Figure B17. ClustalW alignment of NdhK amino acid sequences. NdhK amino acid sequences of the following organisms were aligned using the ClustalW alignment program from MacVector v. 6.5: Synechococcus sp. PCC 7002, Synechocystis sp. PCC

6803, Anabaena sp. PCC 7120, E. coli, and Spinacia oleracea. The consensus amino acid sequence is shown underneath. Gray high-lighted regions indicate identical amino acids while lighter shading indicates conserved amino acids. Dashes represent insertions/deletions included to maximize the sequence similarity. The percent identity and percent conserved amino acids were determined with the ClustalW alignment tool.

(Thompson et al., 1994b). 361

NdhK ClustalW Amino Acid Alignment

10 20 30 40 50 NdhK-7002 MNP T S F E Q Q Q - NdhK-6803 MS P NP A NP T D L E R V A - NdhK-7120 M V LNSDLT TQD- NdhK Spinacia oleracea MGNEFRYIGCIRIYRSFNFRAYPNCWFSLCMAKRSIGMVLIPEDSDKKKK NuoB-E. coli MD Y T L T R I D P N G E N D R - ..P.D.E

60 70 80 90 100 NdhK-7002 TEKLLNPMGRS QVTQDLSENVI LT TVDDLYNWARL SS LWPLL YGTACCF I NdhK-6803 TAKI LNPASRS QVTQDLSENVI LT TVDDLYNWAKLSS LWPLL YGTACCF I NdhK-7120 KERI I NPI ERPTI TQDLSENVI LT TVDDLYNWARL SS LWPLL FGTACCF I NdhK Spinacia oleracea I ETVMNSI EFPLLDQIAQNSVISTTSNDLSNWSRLSSLWPLLYGTSCCFI NuoB-E. coli YP LQKQEI VT DPL EQEVNKNVF MGKL NDMV NWGRKNSI WPYNFGLSCCYV E. . . NPI R . TQDLSENVILTTVDDLYNWARLSSLWPLLYGTACCFI

110 120 130 140 150 NdhK-7002 EFAALLGSRFDFDRFG-LVPRSSPRQADLI LVAGTVTMKMAPALVRLYEE NdhK-6803 EFAALI GSRFDFDRFG-LVPRSSPRQADLI I TAGTI T MKMAPALVRLYEE NdhK-7120 EFAALI GSRFDFDRFG-LI PRSSPRQADLI I TAGTI T MKMAPQLVRLYEQ NdhK Spinacia oleracea EFASLI GSRFDFDRYG-LVPRASPRQADLI LTAGT VT MKMAPSL LRLYE Q NuoB-E. coli EMVT LF TAVHDVARFGAEVL RASPRQADLMVVAGT CF TKMAPVI QRLYDQ EFAALI GSRFDFDRFG LVPRSSPRQADLI . TAGT. TMKMAP.LVRLYEQ

160 170 180 190 200 NdhK-7002 MPEPKYVI AMGACTI TGGMFSSDSTTAVRGVDKLI PVDLYIPGCPPRPEA NdhK-6803 MPEPKYVIAMGACTI TGGMFSSDSTTAVRGVDKLI PVDVYIPGCPPRPEA NdhK-7120 MPEPKYVIAMGACTI TGGMFSVDSPTAVRGVDKLI PVDVYLPGCPPRPEA NdhK Spinacia oleracea MPEPKYVIAMGACTI TGGMFSTDSYSTVRGVDKLI PVDVYLPGCPPKPEA NuoB-E. coli MLEPKWVISMGACANSGGMY--DIYSVVQGVDKFI PVDVYIPGCPPRPEA MPEPKYVIAMGACTI TGGMFS. DS TAVRGVDKLI PVDVYIPGCPPRPEA

210 220 230 240 250 NdhK-7002 II DAII KLRKKVSNETI QERSLKTEQTHRYYSTAHSMKVV EP I L TGKYL G NdhK-6803 IFDAII KLRKKVANESI QER-AITQQTHRYYSTSHQMKVV AP I L DGKY L Q NdhK-7120 IIDAMIKLRKKIANDSMQER-SLIRQTHRFYSTTHNLKPV AE I L T GKY MQ NdhK Spinacia oleracea VI DAI T KL RKKI S RE I YEDR -I KS QP KNRC F T I NHKF RVGRS I HTGNYDQ NuoB-E. coli YMQALMLLQESIG----KER------RPLSWVVG---DQ IIDAIIKLRKKI.NE. QER . . QTHR.YST H KV V ILTGKY Q

260 270 280 290 300 NdhK-7002 MDTWNNPP KEL TE AMGMPVP PALL TAKQRE EA NdhK-6803 QGTRSAPPREL QEAMGMPVPPALT TSQQKEQLNRG NdhK-7120 SETRFNPPKELTEAI GLPVPPALLTSQTQKEEQKRG NdhK Spinacia oleracea AL L YKY KS PS T SE I P PE T F F KYKNAASS RE L VN NuoB-E. coli GVYRANMQSERERKRGE--RIAVTNLRTPDEI . TR NPP . EL . E A G PVP PAL T. . . E E. . 362

Figure B18. ClustalW alignment of NdhJ amino acid sequences. NdhJ amino acid sequences of the following organisms were aligned using the ClustalW alignment program from MacVector v. 6.5: Synechococcus sp. PCC 7002, Synechocystis sp. PCC

6803, Anabaena sp. PCC 7120, E. coli, and Spinacia oleracea. The consensus amino acid sequence is shown underneath. Gray high-lighted regions indicate identical amino acids while lighter shading indicates conserved amino acids. Dashes represent insertions/deletions included to maximize the sequence similarity. The percent identity and percent conserved amino acids were determined with the ClustalW alignment tool.

(Thompson et al., 1994b).

NdhJ ClustalW Amino Acid Alignment

10 20 30 40 50 7002-NdhJ MAEENQNP-----T PEEAAI VEAGAVS QLLT ENGFSHES LERDHSGI EI 6803-NdhJ MAEEVNSPNEAVNLQEETAI APVGPVSTWLTTNGFEHQSLT ADHLGVEM 7120-NdhJ MADEEL QPV -- -- -P AAE AAI VP SGPT S QWLT ENGFAHES LAADKNGVE I NdhJ-Spinacia oleracea MQGRLSAWLVKHGLVHRSLGFDYQGI ET NuoC-E. coli MVNNMTDLTAQE PAWQT RDHLDDP VI GEL RNRFGPDAFT VQATRTGVPV M.EE . E AI . G . S WLT NGF H SL AD. GVE.

60 70 80 90 100 7002-NdhJ I KVDAD------L LI PLCTALYAFGFNYL----QCQGAYDLGPGKEL VSF 6803-NdhJ VQVEAD------L LL PLCTALYAYGFNYL----QCQGAYDEGPGKSL VSF 7120-NdhJ I KVEAD------F LL PI ATALYAYGFNYL----QF QGGVDLGPGQDL VSV NdhJ-Spinacia oleracea L QI KPE------DWHSI AVI LYVYGYNYL----RS QCAYDVAPGGLL ASV NuoC-E. coli V WI KRE QL LEV GDFL KKL PKPYV MLFDL HGMDERL RT HRE GL PAADF SV F . V. AD LLP. . TALYAYGFNYL Q QGAYD. GPG . LVSF

110 120 130 140 150 7002-NdhJ YHLLKVGDNVTDPEEVRVKVFLPRENPVVPSVYWIWKGADWQERESYDMY 6803-NdhJ YHLVKLTEDTRNPEEVRLKVFLPRENPVVPSVYWIWKAADWQERECYDMF 7120-NdhJ YHLVKVSDNADKPEEIRVKVFLPRENPVVPSVYWIWKTADWQERESYDMF NdhJ-Spinacia oleracea YHLTRI EYGVDQPEEVCI KVFAPRRNPRIPSVFWVWKSADFQERESYDMF NuoC-E. coli YHLISI DRNRD----IMLKVALAENDLHVPTFTKLFPNANWYERETWDLF YHL. K. . N. D PEEVR. KVFLPRENPVVPSVYWIWK ADWQERESYDMF

160 170 180 190 200 7002-NdhJ GI V YEGHP NLKRI LMPEDWI GWPLR KDY VS PDFYE LQDAY 6803-NdhJ GI V YEGHP NLKRI LMPEDWVGWPLR KDY I S PDFYE LQDAY 7120-NdhJ GI I YEGHP NLKRI LMPEDWVGWPLR KDY I S PDFYE LQDAY NdhJ-Spinacia oleracea GI S YDNHP RLKRI LMPES WI GWPLR KDY I V PNFYE I QDAY NuoC-E. coli GI T FDGHP NLR RI MMPQT WKGHP LR KDY PR AR YRI LAV GI . YEGHP NLKRI LMPEDW. GWPLR KDY I S PDFYE LQDAY 363

Figure B19. ClustalW alignment of NdhL amino acid sequences. ClustalW alignment of NdhL amino acid sequences. NdhL amino acid sequences of the following organisms were aligned using the ClustalW alignment program from MacVector v. 6.5:

Synechococcus sp. PCC 7002, Synechocystis sp. PCC 6803, Anabaena sp. PCC 7120, and

Synechococcus sp. PCC 7942. The consensus amino acid sequence is shown underneath.

Gray high-lighted regions indicate identical amino acids while lighter shading indicates conserved amino acids. Dashes represent insertions/deletions included to maximize the sequence similarity. The percent identity and percent conserved amino acids were determined with the ClustalW alignment tool. (Thompson et al., 1994b).

NdhL ClustalW Amino Acid Alignment

10 20 30 40 7002-NdhL MPFDI PVETLLI ATLYLSLSVTYLLVLPAGLYFYLNN 6803-NdhL MEDLLGLLLSETGLLAI I YLGLSLAYLLVFPALLYWYLQK 6301-NdhL MTVTLI I AALYLALAGAYLLVVPAALYLYLQK 7120-NdhL MIVPLLYLALAGAYLLVVPVALLFYLKL T. . . A. LYL. L . AYLLV. PA. LY. YL.

50 60 70 80 7002-NdhL RWYVASSI ERLVMYFFVFFLFPGMLLLSPFLNFRPRRREV 6803-NdhL RWYVASSVERLVMYFLVFLFFPGLLVLSPVLNLRPR-RQA 6301-NdhL RWYVASSWERAFMYFLVFFFFPGLLLLAPLLNFRPRSRQI 7120-NdhL RWYVVSSI ERTFMYFLVFLFFPGLLVLSPFVNLRPRPRKI RWYVASS.ER.MYFLVFFFPGLL.LSPLNRPRR.

90 100 110 120 7002-NdhL 6803-NdhL A 6301-NdhL PA 7120-NdhL EV 364

Figure B20. ClustalW alignment of NdbA amino acid sequences. sequences of the following organisms were aligned using the ClustalW alignment program from

MacVector v. 6.5: Synechococcus sp. PCC 7002, Synechocystis sp. PCC 6803, Calothrix vigueri, Corneybacterium glutamicum, and Mycobacterium smegmatis. The consensus amino acid sequence is shown underneath. Gray high-lighted regions indicate identical amino acids while lighter shading indicates conserved amino acids. Dashes represent insertions/deletions included to maximize the sequence similarity. The percent identity and percent conserved amino acids were determined with the ClustalW alignment tool.

(Thompson et al., 1994b). 365

NdbA ClustalW Amino Acid Alignment

10 20 30 40 50 NdbA-7002 MVNTQLPNLTPQTDKHKVVI I GGGFGGLYAAKTLGKYEA- AVDVTLI DKR NdbA-6803 MNSPTSPRRPHVVI VGGGFAGLYTAKNLRRSP- - - VDI TLI DKR Ndb(1)-7120 LYTAKTLATAN- - - VSVTLI DKR Ndb(2)-7120 LYAAKALAKTN- - - VNVTLI DKR Ndb1-E. coli MSRPRI VI VGAGFAGYRTARTLSRLTRHQADI TLLNPT . ..VI.G.GF.GLYTAKTL.. VDVTLIDKR

60 70 80 90 100 NdbA-7002 NFHLFQPLLYQVATGTLSPADI ASPLRGVLSGNKNTHVLLDEVVDI DPDS NdbA-6803 NFHLFQPLLYQVATGSLSPADI ASPLRGVLKGQKNI RVLMDKVI DI DPDK Ndb(1)-7120 NFHLFQPLLYQVATGTLSPGDI SSPLRAVFSKSKNTQVLLGEVKDI NPKA Ndb(2)-7120 NFHLFQPLLYQVATGTLSPADI SAPLRSVLSKSKNTKVLLGEVNDI DPNL Ndb1-E. coli DYFLYLPLLPQVAAGI LEPRRVSVSLSGTLP- - - HVRLVLGEADGI DLDG NFHLFQPLLYQVATGTLSPADI SSPLRGVLS KNT. VLLGEV DI DPD.

110 120 130 140 150 NdbA-7002 KTVVMNEGI-----VNYDSLIVATGVSHHYFGNDHWKPYAPGLKTVEDAL NdbA-6803 QKVVLEDHAP----IAYDWLVVATGVSHHYFGNDHWAALAPGLKTIEDAL Ndb(1)-7120 QQVILDDKV-----VPYDTLIVATGANHSYFGKDHWKDVAPGLKTVEDAI Ndb(2)-7120 QQIIVGDKV-----VPYDTLIVATGAKHSYFGKDNWQELAPGLKTVEDAI Ndb1-E. coli RTVHYTGPEGGEGTLAYDRLVLAAGSVNKLLPI PGVAEHAHGFRGLPEAL Q V. . D. . V YD. LI VATG. H. YFG DHW . . APGLKTVEDAL

160 170 180 190 200 NdbA-7002 EI RHRI FMAFEAAEKETDPALQQAWLTFVI VGGGPTGVELAGAI AEI AYS NdbA-6803 TI RQRI FAAFEAAEKESNPERQQAWLTFVI VGAGPTGVELAGAI AEI AHS Ndb(1)-7120 EMRRRI FGAFEAAESETDPEKRRAWLTFVI VGGGPTGVELAGAI AELAYK Ndb(2)-7120 EMRRRI FGAFEAAEKETDLEKRKALLTFVI VGGGPTGVELAGAI AELAYK Ndb1-E. coli YLRDHVTRQVELAAAADDRAECAARCTFVVVGAGYTGTEVAAHGAMYTDA E. R. RI F. AFEAAEKETDPE. AWLTFVI VGGGPTGVELAGAI AE. AY

210 220 230 240 250 NdbA-7002 VLKKDFRKI DTTRARI I LLEGMDRVLPPYDPSLSAKAQKSLENLGVQVQT NdbA-6803 SLKDNFHRI DTRQAKI LLI EGVDRVLPPYKPQLSARAQRDLEDLGVTVLT Ndb(1)-7120 TLKEDFRSI DTSETKI LLLQGGDRI LPHI APELSQVAAESLQKLGAI I QT Ndb(2)-7120 TLQEDFRNI NTSETRI LLLQGGDRI LPHI APELSQAATTSLREFGVVVQT Ndb1-E. coli QVRRHPMRTG- MRPRWMLLDVAPRVMPEMDERLSRTAERVLRQRGVDVRM .LK.DFR.IDT. RILLL.G.DRVLP P LS A .SL LGV VQT

260 270 280 290 300 NdbA-7002 KSLVTNI EDHLVTFKQGDDQCEI AAKTI VWAAGVKASGMSKVLEDRLSAT NdbA-6803 ERMVTDI NPEQVTVHNNGQTETI VTKTVLWGAGVRASSLGKI I GDRTGAE Ndb(1)-7120 KTRVTNI ENDI VTFKKGDEVKEI PSKTI LWAAGVKASPMGQVLAERTGVE Ndb(2)-7120 KTRVTSI ENDI VTFKQGDELQTI TSKTI LWAAGVKASPMGKVLAERTGVE Ndb1-E. coli GTSVKEATHDGVVLTDG- - - STVDTRTLVWCVGVRPDP- - - - LVESLGLP KT VT I E D. VTFK. GD. TI . KTI LWAAGVKASPMGKVL. ERTG. E

310 320 330 340 350 NdbA-7002 LDRAGRVI VEPNLSVAGYPDVFVI GDLANFPHQN- - ERPLPGVAPVAMQE NdbA-6803 LDRAGRVVVNPDLSVASFDNI FVLGDLANYSHQG- - DQPLPGVAPVAMQE Ndb(1)-7120 CDHAGRVI VEPDL TI RDYKNI FVVGDL GNFSHQN- - GKPL PGVAPVATQQ Ndb(2)-7120 CDRAGRAI VEPDL SI KGHQNI FVVGDL ANFSHQN- - GQPL PSVAPVAI QE Ndb1-E. coli MER- GRLLVDPHLQVPGRPELFACGDVAAVPDLNQPGQYTPMTAQHAWRH . DRAGRVI VEPDLSV G. NI FV. GDLANFSHQN GQPLPGVAPVA QE 366

360 370 380 390 400 NdbA-7002 GEYVAKLI KQRVNGQE- - MAPFRYMELGSLAVI GQNAAVVDLGFVKFSGF NdbA-6803 AAYLSKLI PARLAEKEQI MVPFRYI DYGSLAVI GQNKAVVDL GFAQFTGL Ndb(1)-7120 GEYVAKLI KKRLKGQT- - LPQFRYNDVGSLAMIGQNLAVVDLGLI KLQGF Ndb(2)-7120 GEYVAKLI KKRLQGKT- - LPAFKYNDHGSLAMI GQNAAVVDL GLLKL KGF Ndb1-E. coli GKVCAHNVVASLGRGQ- - RRAYRHRDMGFVVDLGGAKAAANPLGLPMSGP GEYVAKLI K RL G FRY D GSLA I GQN. AVVDLG. . K . GF

410 420 430 440 450 NdbA-7002 LAWLI------WIFAHVY------YLIEF NdbA-6803 VAWMI------WVWAHVY------YLIEF Ndb(1)-7120 IAWVF------WLLIHIY------FLIEF Ndb(2)-7120 SAWAF------WLLIHIY------FLIEF Ndb1-E. coli AAGAVTRGYHL AAMPGNRVRVAADWLLDAVL PRQAVQLGLVRSWSVPLES .AW.. WLL.HVY .LIEF

460 470 480 490 500 NdbA-7002 DNKMVVML------NdbA-6803 DNKLIVML------Ndb(1)-7120 DTKLLVVF------Ndb(2)-7120 DSKLLVMI------Ndb1-E. coli SSPEVARVPGRPEQSGKDAGTAGSAGTAGKHADGDEGKKQPGGEPAKGQP D. KL . VM.

510 520 530 540 550 NdbA-7002 ------QWGWNYFTRG------NdbA-6803 ------QWGWNYFTRG------Ndb(1)-7120 ------QWAWNYITRN------Ndb(2)-7120 ------QWAWNYITRK------Ndb1-E. coli GGEPGKNQPGGEPGKNQPGGEPGKNQPGGEPAKNQPGGEPAERGPDASAG QWGWNY. T RG

560 570 580 590 600 NdbA-7002 ------RGARLITGEKDLVS----- NdbA-6803 ------RGARLITDTPNPQS----- Ndb(1)-7120 ------RRSRLITGREAFVE----- Ndb(2)-7120 ------RGSRLITGKESLAF----- Ndb1-E. coli SRSHGERAKRSQSGSPRASGKPRRAVEAAGPRSARGGSGKPGKSSRASGT RGARL I TG. . S

610 620 630 640 650 NdbA-7002 ------GFKLYQEAADKETKVNIKVEA NdbA-6803 ------TSTVQKELVRP Ndb(1)-7120 ------PKTVNQQN Ndb(2)-7120 ------ANNFDDSNDTNNYSAANNRQPLNV Ndb1-E. coli GSAGKRPTAPSGPSRSAGQPADPGPEPPAHQPPPGPDI APGPVRRTDGRA .. Q .

660 670 680 690 700 NdbA-7002 NdbA-6803 Ndb(1)-7120 Ndb(2)-7120 Ndb1-E. coli VEGDS 367

Figure B22. ClustalW alignment of NdbB amino acid sequences. ClustalW alignment of NdbB amino acid sequences. NdbB amino acid sequences of the following organisms were aligned using the ClustalW alignment program from MacVector v. 6.5:

Synechococcus sp. PCC 7002, Synechocystis sp. PCC 6803, Anabaena sp. PCC 7120, and

E. coli. The consensus amino acid sequence is shown underneath. Gray high-lighted regions indicate identical amino acids while lighter shading indicates conserved amino acids. Dashes represent insertions/deletions included to maximize the sequence similarity. The percent identity and percent conserved amino acids were determined with the ClustalW alignment tool. (Thompson et al., 1994b). 368

NdbB ClustalW Amino Acid Alignment

10 20 30 40 50 NdbB-7002 MSQPHI VI I GGGF AGL YT AL RL L QF PWET SQRPDI T L I DRQNHF VF SP NdbB-6803 MT DARPRI CI L GGGF GGL YT AL RL GQL SWEGHT PPEI VL VDQRDRF L F AP NdbB-7120 MT EQT KRI VI L GGGF GGL YT AL RVSQL PWET QQKPEI VL VDQSDRF L F SP Ndb-E. coli MSRPRI VI VGAGFAGYRTARTLSRLTRHQA- - - DI TLLNPTDYFLYLP PRIVI.GGGF.GLYTALRL QL WE P.I L.D D.FLF P

60 70 80 90 100 NdbB-7002 LLYELI TEEMQPWEVAPTYTELLRHGPVKFVQTQVQTVDPEQKNVVCGDR NdbB-6803 FLYELVTEEMQTWEI APPFVELLAESGVI FRQAEVTAI DFDHQKVLLNDQ NdbB-7120 LLYELLTGELQSWEI APPFI ELLEGTGI RFYQAVVSGI DI DQQRVHLQDG Ndb-E. coli L L PQVAAGI L EPRRVSVSL SGTL PH- - VRL VL GEADGI DL DGRTVHYTGP LLYEL. T E Q WE. AP . ELL V. F Q. V . I D D V D

110 120 130 140 150 NdbB-7002 Q- - - - - I TYDYL VI AAGGTTKFVNL PGI KEYAL PFKTL NDAL HL- KEKL R NdbB-6803 DKGTESLAFDQLVI ALGGQTPLPNLPGLKDYGLGFRTLEDAYKL- KQKLK NdbB-7120 PE- - - - I PYDRLVLTLGGETPLDLVPGAI SYAYPFRTI ADTYRL- EERLR Ndb-E. coli EGGEGTLAYDRLVLAAGSVNKLLPI PGVAEHAHGFRGLPEALYLRDHVTR . YD LV.A.GGT L .PG. .YA FRTL DA .L .LR

160 170 180 190 200 NdbB-7002 ALETSVAEKIR------IAIVGGGYSGVELACK------LADR NdbB-6803 SLEQADAEKIR------IAIVGGGYSGVELAAK------LGDR NdbB-7120 VLEESDAEKIR------VAIVGAGYSGVELACK------LADR Ndb-E. coli QVELAAAADDRAECAARCTFVVVGAGYTGTEVAAHGAMYTDAQVRRHPMR L E AEKI R . AI VG. GYSGVEL A K L . DR

210 220 230 240 250 NdbB-7002 LGDRGRLRI I DRGDEI LKNAPKFNQLAAKEALEARGI WVDYATEVTEVTA NdbB-6803 L GERGRI RI I ERGKEI L AMSPEFNRQQAQASL SAKGI WVDTETTVTAI TA NdbB-7120 L GERGRFRL VEI SDQI L RTSPDFNREAAKKAL DAKGVFI DL ETKVESI GQ Ndb-E. coli TGMRPRWMLLDVAPRVMPEMDERLSRTAERVLRQRGVDVRMGTSVKEATH LG.RGRR... . IL P.FN A .L A.G..VD T V .T

260 270 280 290 300 NdbB-7002 DSLSL RYKGEVDTI PADL VL WTGGTAI APWVKDL AL PHAGNGKLDVNAQL NdbB-6803 TDVTL QFREQEDVI PVDL VL WTVGTTVSPL I RNL AL PHNDQGQLRTNAQL NdbB-7120 NTI SL EYKNQVDTI PVDL VI WTVGTRVTNVVKSL PFKQNQRGQI TNTPTL Ndb-E. coli DGVVL TDG- - - STVDTRTL VWCVGVRPDPL VESL GL PME- RGRL L VDPHL . . L . . DTI P. DL V. WTVGT . P. V. L . L P G L L

310 320 330 340 350 NdbB-7002 QIQNHPNIFALGDVAQAEDN------LPMTAQVAIQQADVCAWNLRGLIT NdbB-6803 QVEGKT NI F AL GDGAEGRDAS- - GQL I PT T AQGAF QQT DYCAWNI WANL T NdbB-7120 QVL DHPDI FAL GDL ADCI DAE- - GQQVPATAQAAFQQADYAAWNI WASL T Ndb-E. coli QVPGRPEL FACGDVAAVPDL NQPGQYTPMTAQHAWRHGKVCAHNVVASL G QV .P IFALGD.A . D. GQ.P TAQ.A.QQ.D CAWN. A LT 369

360 370 380 390 400 NdbB-7002 NKPLLPFKFFNLGEMLTLGENNATLSGLGLELEGNLAHVARRLVYLYRLP NdbB-6803 GRPLLPCRYQPLGEMLALGTDGAVLSGLGI KLSGPAALLARRLVYLYRFP NdbB-7120 QRPLLPFRYQQLGEMMALGTDNATLTGLGVKLDGSLAYVARRLAYLYRLP Ndb-E. coli RGQRRAYRHRDMGFVVDLGGAKAAANPLGLPMSGPAAGAVTRGYHLAAMP . PLLP. R. LGEM. LG A L. GLG. L G . A . ARRL. YLYR P

410 420 430 440 450 NdbB-7002 ------TWEHQVQVGLN--WLV------NdbB-6803 ------TWQHQLTVGLN--WLT------NdbB-7120 ------TLDHQLKVGFN--WLV------Ndb-E. coli GNRVRVAADWLLDAVLPRQAVQLGLVRSWSVPLESSSPEVARVPGRPEQS THQ.VGLNWLV

460 470 480 490 500 NdbB-7002 ------QPLTKLLAQ NdbB-6803 ------RPLGDWLKNEPS NdbB-7120 ------RPIIETIYSAVDAVNEK Ndb-E. coli GKDAGT AGSAGT AGKHADGDEGKKQPGGEPAKGQPGGEPGKNQPGGEPGK P. . . . .

510 520 530 540 550 NdbB-7002 NdbB-6803 NdbB-7120 GT RY Ndb-E. coli NQPGGEPGKNQPGGEPAKNQPGGEPAERGPDASAGSRSHGERAKRSQSGS

560 570 580 590 600 NdbB-7002 NdbB-6803 NdbB-7120 Ndb-E. coli PRASGKPRRAVEAAGPRSARGGSGKPGKSSRASGTGSAGKRPTAPSGPSR

610 620 630 640 650 NdbB-7002 NdbB-6803 NdbB-7120 Ndb-E. coli SAGQPADPGPEPPAHQPPPGPDI APGPVRRTDGRAVEGDS 370

Figure B23. ClustalW alignment of HypE amino acid sequences. HypE amino acid sequences of the following organisms were aligned using the ClustalW alignment program from MacVector v. 6.5: Synechococcus sp. PCC 7002, Anabaena sp. PCC 7120,

Synechocystis sp. PCC 6803, and E. coli. The consensus amino acid sequence is shown underneath. Gray high-lighted regions indicate identical amino acids while lighter shading indicates conserved amino acids. Dashes represent insertions/deletions included to maximize the sequence similarity. The percent identity and percent conserved amino acids were determined with the ClustalW alignment tool. (Thompson et al., 1994b). 371

HypE ClustalW Amino Acid Alignment

10 20 30 40 50 HypE-7002 MDLTCPL LQN---- TSHVLLA GGGGKL M DL I DKMF T AF GEPC - - HypE-7120 MKYVDEF EPE------KAEAL REI EKLS QLDKHI K MEVCGGH - - HypE-6803 MNLVCPV LDR---- YPQVLLA GGGGKL S QL L KQI F PAF GASE - - HypE-E. coli M QLI NSLF EAFANPW AE HypE-R. eutropha MSGTVKL YQRPLNI SGRI DMG GAGGRAA QLI QELF AAFDNEW RQ M ...... G. GGKL QL I . F AF .

60 70 80 90 100 HypE-7002 ------HDAAALP STEKLAF TDSYVVQ ----LFF GG---DI KL HypE-7120 ------HSIFK GIEEILP NIELIHG GCPVCVM KGRLDDA AI HypE-6803 ------G HDAAVFT NQSSLAF TDSYVI N ----LFF GG---DI SL HypE-E. coli QEDQAR- LDLAQLV EGDRLAF TDSYVI D ----LFF GG---NI KL HypE-R. eutropha GND---- -QAAFAM AGARMVM TDAHVVS ----LFF GG---DI SL DAA.. ...LAF TDSYV. LFF GG DI L

110 120 130 140 150 HypE-7002 A-----I GTVNDLA A-GARPL LSV---G ILEEGL- LETLWQV VS HypE-7120 SQNPNVI TTFGDTM VPGSKTT LQAKAQG DI RMVYS LDSL- QI RN HypE-6803 A-----V GTVNDLA A-GATPR ISV---G ILEEGL- METLWRV QS HypE-E. coli A-----I GTANDVA S-GAIPR LSC---G ILEEGL- METLKAV TS HypE-R. eutropha S-----V GTINDVA A-GAKPL LAA---S ILEEGF- LADLKRI ES A I GT.ND.A A GA.P LS. G ILEEGL LETL V S

160 170 180 190 200 HypE-7002 MQQ---A QVVGVQI TGDTKV- ERGKGDG --FINTS -IGIIES LN HypE-7120 HPDKEI V FALGFET APSTALT LQAASEN TNFSMFS HVLVI PA QA HypE-6803 LGQ---A QNCGVEI TGDTKV- DRGKGDG --FINTS -IGSLDH QT HypE-E. coli MAE---T RAAGIAI TGDTKV- QRGAVDK --FINTA -MGAIPA IH HypE-R. eutropha MAG---A REAGVPI TGDTKV- EQGKGDG --FITTT -VGVVPA I L M. A ..GV I TGDTKV .RGKGDG FINTS .G.IPA .

210 220 230 240 250 HypE-7002 I QPKAI A GDTI LVS ----DLG HGIAIMA R--EGLA ESPI ESD - A HypE-7120 LLNNPDL LDGFI GP HVSMVI G EPYEFI A QYHKPI V SGFEPLD FQ HypE-6803 IHPNQVQ GDRLILS ----DLG HGMAIMA R--QGLE ETTIESD -A HypE-E. coli WGAQTLT GDVLLVS ----TLG HGATILN R--EQLG DGELVSD -A HypE-R. eutropha IDGAGAR GDAILLS ----TMGHGVAILS R--ESLE DTEIRSD -A I . . GD..L.S LG HG.AI.A R E L. .. I SD A

260 270 280 290 300 HypE-7002 PLVEPVQ LLKAGI E H- CL- RD TRGG- - L AVLNELA AAGYQFK H- HypE-7120 SI WMLL Q L VENRCE ENQYNRL QKGGNQI L AAMHKV AVREKFA RG HypE-6803 PVHREVQ LLSAGI P H- CL- RD TRGG- - L SAVNEI A TSGVTMA R- HypE-E. coli VLTPLI - TLRDI PG K- AL- RD TRGG- - V AVVHEFA ACGCGI E S- HypE-R. eutropha ALHDLVA MLAVVPG R- VL- RD TRGG- - L TTLNEI S QSGVGMV D- L LVQ LL .. . L RD TRGG L ...NE.A A G . .

310 320 330 340 350 HypE-7002 GNQI PVI AVRGACE FGFD- PV I ANEGRF AI LPEAR AEGLA- L QH HypE-7120 LDEI P- D GLKI REE AQFDAEL FTI PNLK ADHKACK GEI LKGV KP HypE-6803 ETLI PVE EVQAACE LGFD- PL VANEGRF AI VPPEA QKTVE- I QT HypE-E. coli EAALPVK AVRGVCE LGLD- AL FANEGKL I AVERNA EQVLA- A HS HypE-R. eutropha EAAI PVL QVDAACE LGLD- PL VANEGKL AI CAAAD DALLA- A RG E .IPV .V..ACE LGFD PL .ANEG. AI. .LA . .

360 370 380 390 400 HypE-7002 YNA------NARQ GLVTTQN GEQHLAI PVVVEND GVTRI LE LS HypE-7120 WQCKVFG ACTPETP GTCMVS------SE ACAAYYK GRFSTTL KQ HypE-6803 FHP------QATA GTVT--- GKSAQTL LVSLESS GAPRLLD I S HypE-E. coli HPLGK-- ---DAAL GEVVE------R GVRLAGL GVKRTLD PH HypE-R. eutropha HPLGR-- ---EARR GEVIED------GR FVQMRTK GGMRVVD LS .. A GV .V. G.R.LD.S

410 420 430 440 450 HypE-7002 GEQL PRI HypE-7120 AAEKPKV I SS HypE-6803 GEQL PRI HypE-E. coli AEPLPRI HypE-R. eutropha GEQL PRI GEQL PRI 372

Figure B24. ClustalW alignment of HoxE amino acid sequences. HoxE amino acid sequences of the following organisms were aligned using the ClustalW alignment program from MacVector v. 6.5: Synechococcus sp. PCC 7002, Synechocystis sp. PCC

6803, Synechococcus sp. PCC 6301, Anabaena sp. PCC 7120 and E. coli. The consensus amino acid sequence is shown underneath. Gray high-lighted regions indicate identical amino acids while lighter shading indicates conserved amino acids. Dashes represent insertions/deletions included to maximize the sequence similarity. Percent identity and percent conserved amino acids determined by the ClustalW search program (Thompson et al., 1994b).

HoxE ClustalW Amino Acid Alignment

10 20 30 40 50 HoxE-7002 LSFETI TMAI SHAVP- ADKRFRVL EVAMKRNQYRQDTLI EI L HKAQEV HoxE-6803 MTVATDRQTVPPSAAHPSGDKRFKVL DATMKRNQFNQDALI EI L HKAQEI HoxE-6301 MAT SET TPSVDPRRRRL EL AI KRQAAQADAL I EI L HEAQSL HoxE-7120 MTTTTPPHPHPSGDKRLKMLDAAI KRHQYQQDALI EI LHKAQEL NuoE-E. coli MHENQQPQTEAFELSAAEREAI EHEMHHYEDPRAASI EAL KI VQKQ T S A PS.DKR ..LE.AMKR.Q..QDALIEILHKAQE.

60 70 80 90 100 HoxE-7002 FGYLEDEVL EYVARGLKLPL SRVYGVATFYHLFSLKPKGKHTCVVCL GTA HoxE-6803 FGYLEEDVL LYVARGLKLPL SRVFGVATFYHLFSLKPSGKHTCVVCL GTA HoxE-6301 YGYLDRELL QWVAEQLALPRSKVYGVASFYHLFQLNPSGRHRCHVCL GTA HoxE-7120 FGYLENDLL LYI AHSLKLPPSRVYGVATFYHLFSLAPQGVHSCVVCTGTA NuoE-E. coli RGWVPDGAI HAI ADVLGI PASDVEGVATFYSQI FRQPVGRHVI RYCDSVV FGYLE. . . L YVA. . LKLP. SRVYGVATFYHLFSL P G. H. CVVCLGTA

110 120 130 140 150 HoxE-7002 CYVKGSQEL LDKI DETL HI KPGETTPDDQI SLVTARCI GACGI APAVVYD HoxE-6803 CYVKGAGDL LKTLDQEVHLKPGETTEDGQMSLVTARCI GACGI APAVVYD HoxE-6301 CYVKGSQAI LDCLI AEL GI REGETTNDGSVSLGTVRCVGACGI APVVVYD HoxE-7120 CYVKGSSAI LADLEKATRI HAGETTADGQLSLLTARCLGACGI APAVVFD NuoE-E. coli CHI NGYQGI QAALEKKLNI KPGQTTFDGRFTLLPTCCLGNCDKGPNMMI D CYVKGSQ. I L L. L . I KPGETT DGQ. SL. TARC. GACGI APAVVYD

160 170 180 190 200 HoxE-7002 DEVCGKQNADHL MARL RQLSEGS HoxE-6803 GKVL GKQNDEAVL AAI QPWLSNS HoxE-6301 GDI QGRQESEAVWQQVQAWQQEAH HoxE-7120 GKVL GNQT PESVNERVQGWL NuoE-E. coli EDTHAHL TPEAI PEL LERYK G. V G. Q EAV . Q W 373

Figure B25. ClustalW alignment of HoxF amino acid sequences. HoxF amino acid sequences of the following organisms were aligned using the ClustalW alignment program from MacVector v. 6.5: Synechococcus sp. PCC 7002, Synechocystis sp. PCC

6803, Anabaena sp. PCC 7120, Synechococcus sp. PCC 6301, Anabaena variabilis, E. coli, and R. eutropha. The consensus amino acid sequence is shown underneath. Gray high-lighted regions indicate identical amino acids while lighter shading indicates conserved amino acids. Dashes represent insertions/deletions included to maximize the sequence similarity. Percent identity and percent conserved amino acids determined by the ClustalW search program (Thompson et al., 1994). 374

HoxF ClustalW Amino Acid Alignment

10 20 30 40 50 HoxF-7002 MTDLAELFEIAEAEQDSH------HoxF-6803 MDIKELKEIATKSREKQ------HoxF-7120 MELTELLDIGRQERSQQ------HoxF-6301 MDWEDLGRLANEELTCQ------HoxF-A. variabilis MELNELLDIGRQERSQQ------NuoF-E. coli MMTVAAEIR------HoxF-Ralstonia eutropha M SRI TTI LERYRSDRTRLI DI LWDVQHEYGHI PDAVLPQLGAGLKLSPL MD. E L . . I A E R Q

60 70 80 90 100 HoxF-7002 ------TK---IQIRCCTAAGCMSSGSLAVKEELEKQIKEKN HoxF-6803 ------TK---IRIRCCSAAGCLSSEGETVKKNLTTAIAAAG HoxF-7120 ------KP---VQIRCCTAAGCLSANSQAVQQQLEQAVKAEG HoxF-6301 ------KP---IRLRCCTATGCRANGAEAVFKAVQQTIADQN HoxF-A. variabilis ------KP---VQIRCCTAAGCLSANSQAVKQQLEEAVKAEG NuoF-E. coli ------GSNPEKLLAPVLSAFWDE- HoxF-Ralstonia eutropha D RETASFYHFFLDKPSGKYRI YLCNSVI AKI NGYQAVREALERETGI RF KP . I RCCT AAGC S AV. L E A. .

110 120 130 140 150 HoxF-7002 L DR- - - - LEVVPVGCMKLCGFAPLVDVSDET------CFQQVMPEVAP HoxF-6803 L EK- - - - VEVCGVGCMKFCGRGPLVAVDDRNQ------LYEFVTPDQVG HoxF-7120 L GE- - - - VQVSGVGCMRLCCQGPLVEVEGSGEEKTKQRLYEKVTPEDAS HoxF-6301 L DR- - - - CEAVSVGCLGLCGAGPLVQCDPSDR------LYSDI RPDQAA HoxF-A. variabilis L DG- - - - VQVAGVGCMRL CCQGPL VEVEGSGEEETT QKL YGKVRSEDAS NuoF-E. coli ------HoxF-Ralstonia eutropha G TDPNGMFGLFDTPCI GLSDQEPAMLI DK------VVFTRLRPGKI T L . . V VGCM. LC GPLV V. LY V P. A

160 170 180 190 200 HoxF-7002 E VDVALG- - ETPSDKLEI CDRQAPFFTLQKPVVLENSGKI DPERI EAYI HoxF-6803 D VKKLQKPDAVAETGLI SGDPHHPFYALQRNI ALENSGRI DPESI DEYI HoxF-7120 A I GALKG- - - - KEAQLSVVNLEQPFFTYQAPI VLENSGKI DPERI QAYI HoxF-6301 D VAAAQG- - - - AAMDLPEVDQAQPFFSQQLKI VNRHSGLI NPDRLESYL HoxF-A. variabilis V VGTLRG- - - - KAAQLSVVDLKQPFFTYQAPI VLENSGKI DPERI QAYI NuoF-E. coli ------D------ESWTLDVYR HoxF-Ralstonia eutropha D I AQLKQ------GRSPAEI ANPAGLPSQDI AYVDAMVESNVRTKGPV . V. L G L .D .PFF. Q IVLENSG.IDPERI..YI

210 220 230 240 250 HoxF-7002 A GGYRSLHQVLE- - - DLTPMAVVEEI TQSGLRGRGGGGYPTGLKWATVA HoxF-6803 A GGYEQLHKVVY- - - EMTPEEVI VEMNKSGLRGRGGGGYPTGLKWATVA HoxF-7120 A QGYQGLYQVLR- - - EMTPAAVVDSVSRSGLRGRGGAGYPTGLKWATVA HoxF-6301 A GGYRALMHTI F- - - DLTPTEVVEI I RLSGLRGRGGGGYPTGLKWATVA HoxF-A. variabilis A QGYQAL YQVL R- - - EMT PAGVVDSVNRSGL RGRGGAGYPT GL KWAT VA NuoF-E. coli R EGYEGLRKAL- - - - AMAPDDLI AYVKESGLRGRGGAGFPTGMKWQFI P HoxF-Ralstonia eutropha F RGRTDLRSLLDQCLLLKPEQVI ETI VDSRLRGRGGAGFSTGLKWRLCR A GY . L. VL . MTP VV. . SGLRGRGGAGYPTGLKWATVA

260 270 280 290 300 HoxF-7002 K PGDQKYVVCNADEGDPGAFMDRAVLESDPHRVLEGMAI AGYAVGANHG HoxF-6803 K PGQQKYVI CNADEGDPGAFMDRSVLESDPHRI LEGMAI AAYAVGANHG HoxF-7120 K KGERKFVI CNADEGDPGAFMDRSVL ESDPHRVL EGMAI AAYAVGASQG HoxF-6301 K PSDRKFVVCNGDEGDPGAFMDRSVL ESDPHQVI EGMAI AAYAVGANFG HoxF-A. variabilis K KGERKFVI CNADEGDPGAFMDRSVL ESDPHRVL EGMAI AAYAVGASQG NuoF-E. coli Q DGKPHYLVVNADESEPGTCKDI PLLFANPHSLI EGI VI ACYAI RSSHA HoxF-Ralstonia eutropha D ESEQKYVI CNADEGEPGTFKDRVLLTRAPKKVFVGMVI AAYAI GCRKG K G. KYVI CNADEGDPGAFMDRSVLESDPHRVLEGMAI AAYAVGA . G

310 320 330 340 350 HoxF-7002 Y YI RAEYPLAI QRLEKAI KQAKSKGLLGSQI FN- SPFNFTI DI RI GAGA HoxF-6803 Y YVRAEYPLAI QRLQKAI QQAKRYGLMGTQI FD- SPI DFKI DI RVGAGA HoxF-7120 Y YVRAEYPI AI KRLHTAI HQAQRLGLLGSNI FE- SPFDFKI DI RI GAGA HoxF-6301 Y YVRAEYPLAI ARLNQAI RQARRRGLLGNSVLD- SRFSFDLEVRI GAGA HoxF-A. variabilis Y YVRAEYPI AI KRLQTAI HQAQRLGLLGSNI FE- SPFDFKI DI RI GAGA NuoF-E. coli F YLRGEVVPVLRRLHEAVREAYAAGFLGENI LG- SGLDLTLTVHAGAGA HoxF-Ralstonia eutropha I YLRGEYFYLKDYLERQLQELREDGLLGRAI GGRAGFDFDI RI QMGAGA Y YVRAEYP.AI RL AI.QA.R GLLG..IF. SPFDF IDIRIGAGA 375

360 370 380 390 400 HoxF-7002 FVCGEETALI ASI EGGRGTPRPRPPYPAQSGLWGHPTLI NNVETYANI VP HoxF-6803 FVCGEETALI ASVEGKRGTPRPRPPYPAQSGLWQSPTLI NNVETYANVVP HoxF-7120 YVCGEETALMASI EGKRGVPHPRPPYPAESGLWGYPTLI NNVETFANI AP HoxF-6301 FVCGEETALI HSI QGERGVPRVRPPYPAESGLWGHPTLI NNVETFANI AP HoxF-A. variabilis YVCGEETALMASI EGKRGVPHPRPPYPAESGLWGYPTLI NNVETFANI AP NuoF-E. coli YI CGEETALLDSLEGRRGQPRLRPPFPAVAGLYACPTVVNNVESI ASVPA HoxF-Ralstonia eutropha YI CGDESALI ESCEGKRGTPRVKPPFPVQQGYL GKPT SVNNVET F AAVSR YVCGEETALI ASI EGKRG PRPRPPYPA SGLWG PTLI NNVETFANI . P

410 420 430 440 450 HoxF-7002 I I REGADWFSSI GTEKSKGTKVFALTGKVKNNGLI EVPMGTPVRQI VEQM HoxF-6803 I I REGGDWYGSI GTEKSKGTKVFALTGKVENAGLI EVPMGTTVRQVVEEM HoxF-7120 I I RKGADWFASI GTAKSKGTKVFALAGKI RNTGLI EVPMGTSLRQI VAEM HoxF-6301 I VEQGADWFAAI GTPTSKGTKVFAL TGKL RNNGL I EVPMGI PLRSI VDGM HoxF-A. variabilis I I RKGADWFASI GTAKSKGTKVFALAGKI RNTGLI EVPMGTSLRQI VEQM NuoF-E. coli I LNKGKDWFRSMGSEKSPGFTLYSLSGHVAGPGQYEAPLGI TLRQLLDMS HoxF-Ralstonia eutropha I MEEGADWFRAMGTPDSAGTRLLSVAGDCSKPGI YEVEWGVTLNEVLAMV I I R GADWF. SI GT KSKGTKVFAL. GK. . N GLI EVPMGT. LRQI V. M

460 470 480 490 500 HoxF-7002 GGGI PDSGTVKSVQTGGPSGGCI PADYLDTPI EYDSLI KLGTMMGSGGMI HoxF-6803 GGGVPNGGQVKAVQTGGPSGGCI PADKLDTPI EYDTLLALGTMMGSGGMI HoxF-7120 GGGI PDGGVAKAVQTGGPSGGCI PASAFDTPVDYESLTNLGSMMGSGGMI HoxF-6301 G- - I PES- PVKAVQTGGPSGGCI PLAQLDTPVDYDSLI QLGSMMGSGGMV HoxF-A. variabilis GGGI PDGGVAKAVQTGGPSGGCI PASAFDTPVDYESLTNLGSMMGSGGMI NuoF-E. coli GG- MRPGHRLKFWTPGGSSTPMFTDEHLDVPLDYEGVGAAGSMLGTKALQ HoxF-Ralstonia eutropha G-----ARDARAVQISGPSGECVSVAK-----DGERKLAYEDLSCNGAFT GGGI P. GG . KAVQTGGPSGGCI PA LDTP. DYESL. LGSMMGSGGMI

510 520 530 540 550 HoxF-7002 VMDEATNMVDVAKFYMEFCQCESCGKCI PCRAGTVQMSGLLSKMLKGQAE HoxF-6803 VMDESTNMVDVAQFYMDFCKSESCGKCI PCRAGTVQLYDLLTRFLEGEAT HoxF-7120 VMDDTTNMVDVARFFMEFCMDESCGKCI PCRVGTVQLHGLLSKI REGKAS HoxF-6301 VMDENTDMVAI ARFYMEFCRSESCGKCI PCRAGTVQLHELLGKLSSGQGT HoxF-A. variabilis VMDDTTNMVDVARFFMEFCMDESCGKCI PCRVGTVQLHGLLSKI REGKAS NuoF-E. coli CFDETTCVVRAVTRWTEFYAHESCGKCTPCREGTYWLVQLLRDI EAGKGQ HoxF-Ralstonia eutropha I FNCKRDL L EI VRDHMQFFVEESCGI CVPCRAGNVDLHRKVEWVI AGKAC VMDE. TNMVDVARF. MEFC ESCGKCI PCRAGTVQLH LL. K. GKA.

560 570 580 590 600 HoxF-7002 PKDI ELLEQLCHMVKEASLCGLGQSAPNPI LSTLRYFRAEYDALVGSNAD HoxF-6803 QEDLI KLENLCHMVKETSLCGLGMSAPNPVI STLRYFRHEYEELLKV HoxF-7120 LADLELLEELCDMVKNTSLCGLGQSAPNPVFSTLHYFRDEYLALI AVGAV HoxF-6301 AI DLQQLEDLCYLVKDTSLCGLGMSAPNPI LSTLRWFRQEYESRLI PERA HoxF-A. variabilis FADLELLEELCDMVKNTSLCGLGQSAPNPVFSTLRYFRDEYLALI AE NuoF-E. coli MSDLDKLNDI ADNI NGKSFCALGDGAASPI FSSLKYFREEYEEHI TGRGC HoxF-Ralstonia eutropha QKDLDDMVSWGALVRRTSRCGLGATSPKPI LTTLEKFPEI YQNKLVRHEG DL. LE. LC MVK TSLCGLG SAPNPI . STLRYFR. EY. L. .

610 620 630 640 650 HoxF-7002 HoxF-6803 HoxF-7120 SLNSTD HoxF-6301 IALTH HoxF-A. variabilis NuoF-E. coli PFDPAKSTAWADRTEVKA HoxF-Ralstonia eutropha PLLPSFDLDTALGGYEKALKDLEEVTR 376

Figure B26. ClustalW alignment of HoxU amino acid sequences. The HoxU amino acid sequence from Synechococcus sp. PCC 7002 was aligned with HoxU proteins from the following organisms using the ClustalW alignment program from MacVector v. 6.5:

Synechococcus sp. PCC 7002, Synechocystis sp. PCC 6803, Synechococcus sp. PCC

6301, Anabaena variabilis, the N-terminus of the NuoG protein from E. coli and R. eutropha. The consensus amino acid sequence is shown underneath. Gray high-lighted regions indicate identical amino acids while lighter shading indicates conserved amino acids. Dashes represent insertions/deletions included to maximize the sequence similarity. Percent identity and percent conserved amino acids determined by the

ClustalW search program (Thompson et al., 1994). 377

HoxU ClustalW Amino Acid Alignment

10 20 30 40 50 HoxU-7002 MAVKT------LTINDQLISARAGETILEAARDAGIHI HoxU-6803 MSVVT------LTIDDKAIAIEEGASILQAAKEAGVPI HoxU-6301 MSVVT------LQIDDQELAANVGQTVLQVAREASIPI HoxU-7120 MSVKT------LTINDQLISAQEEETLLQAAQEAGIHI HoxU-A. variabilis MSVKT------LTINDQLISAQEEETLLQAAQEAGIHI NuoG-E. coli (371 N-TERM) MTVTTSTPSGGGAAAVPPEDLVTL TI DGAEI SVPKGTLVI RAAEQLGI EI HoxU-Ralstonia eutropha MSIQ------ITIDGKTLTTEEGRTLVDVAAENGVYI MSV T LTIDDQ.ISA EG T.LQAA EAGI I

60 70 80 90 100 HoxU-7002 PTLCHLEGVSDVGACRLCLVEI EGSNKL QPACVTEVMEGMVVQTH- - TEK HoxU-6803 PTLCHLEGI SEAAACRLCMVEVEGTNKL MPACVTAVSEEMVVHTN- - TEK HoxU-6301 PTLCHLQGVSDVGACRLCVVEVAGSPKL QPACLLTVSEGLVVQTR- - SPR HoxU-7120 PTLCHLEGVGDVGACRLCLVEI TGSNKL LPACVTKVAEGMEVRTN- - SDR HoxU-A. variabilis PTLCHLEGVGDVGACRLCLVEVAGSNKL LPACVTKVAEGMEVSTN- - SDR NuoG-E. coli (371 N-TERM) PRFCDHPL LDPAGACRQCI VEVEGQRKPMASCTI TCTDGMVVKTQLTSPV HoxU-Ralstonia eutropha PTLCYLKDKPCL GTCRVCSVKVNGN- - VAAACTVRVSKGLNVEVN- - DPE PTLCHLEGV DVGACRLC. VEV GSNKL PACVT V. EGMVV TN S. .

110 120 130 140 150 HoxU-7002 LEEYRRMTVELL FAEGNHVCAVCVANGNCELQDMAVEVGMDHSRFP- YQY HoxU-6803 L QNYRRMTVEL L FSEGNHVCAI CVANGNCEL QDMAI TVGMDHSRFK- YQF HoxU-6301 LERYRRQI VELFFAEGNHVCAI CVANGNCELQDAAI AVGMDHSRYP- YRF HoxU-7120 LQKYRRTI VEMLFAEGNHI CSVCVANNNCELQDLAI EMGMDHVRLE- YHF HoxU-A. variabilis L QRYRRTI VEML FAEGNHI CSVCVANNNCEL QDL AI EMGMDHVRL E- YHF NuoG-E. coli (371 N-TERM) AEKAQHGVMELL LI NHPLDCPVCDKGGECPLQNQAMSHGQSDSRFEGKKR HoxU-Ralstonia eutropha LVDMRKAL VEFL FAEGNHNCPSCEKSGRCQLQAVGYEVDMMVSRFP- YRF L . YRR . VELL FAEGNH. C VCVANGNCELQD. AI EVGMDHSRF Y. F

160 170 180 190 200 HoxU-7002 PKREVDI SHKQF GI DHNRCI L CTRCVR- VCDEI EGAHVWDVSNRGGESKI HoxU-6803 PKREVDL SHPMFGI DHNRCI L CTRCVR- VCDEI EGAHVWDVAYRGAECKI HoxU-6301 PKRDVDL SHRFF GL DHNRCI L CTRCVR- VCDEI EGAHVWDVAMRGEHCRI HoxU-7120 PNRKVDI SHDRF GVDHNRCVL CTRCI R- VCDEI EGAHTWDMAGRGTNSHV HoxU-A. variabilis PNRKVDI SHDRF GVDHNRCVL CTRCI R- VCDEI EGAHTWDMAGRGTNSHV NuoG-E. coli (371 N-TERM) TYEKPVPI STQVLLDRERCVLCARCTR- FSNQVAGDPMI ELI ERGALQQV HoxU-Ralstonia eutropha PVRVVDHASEKI WLERDRCI FCQRCVEFI RDKASGRKI FSI SHRGPES- - P R VD. SH F G. DHNRCI L CTRCVR VCDEI EGAH. WD. A RG S. .

210 220 230 240 250 HoxU-7002 VSGLNQPWG------AVDACTS------CGKCV HoxU-6803 VSGLNQPWG------TVDACTS------CGKCV HoxU-6301 VAGMDQPWG------AVDACTN------CGKCI HoxU-7120 ITDLSQPWG------TSDTCTS------CGKCV HoxU-A. variabilis ITDLSQPWG------TSDTCTS------CGKCV NuoG-E. coli (371 N-TERM) GTGEGDPFESYFSGNTI QI CPVGALTSAAYRFRSRPFDLI SSPSVCEHCS HoxU-Ralstonia eutropha RIEIDAELA------NAMPPEQ------VKEAV . . GL QPWG T . D. CT S CGK CV

260 270 280 290 300 HoxU-7002 DACPTGSIFRKG------ATVGSKLGDRQKLEFLITAR------HoxU-6803 DACPTGSIFHKG------ETTAEKIGDRRKVEFLATAR------HoxU-6301 DACPTGALFHKG------ETTGEI ERDRDKL AFLAEAR------HoxU-7120 NACPTGAIFYQG------SSVGEMKRDRAKLDFLVTAR------HoxU-A. variabilis NACPTGAIFYQG------SSVGEMKRDRAKLDFLVTAR------NuoG-E. coli (371 N-TERM) GGCATRTDHRRGKVMRRL AANEPEVNEEWI CDKGRF GFRYAQQRDRL T TP HoxU-Ralstonia eutropha AICPVGTILEKR------VGYDDPIGRRKYEIQSVRAR------ACP T G. I F . KG . GE . DR KL . F L . T AR

310 320 330 340 350 HoxU-7002 ------TKGEWTR HoxU-6803 ------KEKEWVR HoxU-6301 ------GQRRWTR HoxU-7120 ------EKQQWNL HoxU-A. variabilis ------EKQQWNL NuoG-E. coli (371 N-TERM) LVRNAEGELEPASWPE HoxU-Ralstonia eutropha ------ALEGEDK W. 378

Figure B27. ClustalW alignment of HoxY amino acid sequences. HoxY amino acid sequences of the following organisms were aligned using the ClustalW alignment program from MacVector v. 6.5: Synechococcus sp. PCC 7002, Synechocystis sp. PCC

6803, Synechococcus sp. PCC 6301, and R. eutropha. The consensus amino acid sequence is shown underneath. Gray high-lighted regions indicate identical amino acids while lighter shading indicates conserved amino acids. Dashes represent insertions/deletions included to maximize the sequence similarity. Percent identity and percent conserved amino acids determined by the ClustalW alignment tool (Thompson et al., 1994b). 379

HoxY ClustalW Amino Acid Alignment

10 20 30 40 50 HoxY-7002 MAQET QQKI RF AT I WLAGCSGCHMSF L D HoxY-6803 MAKI RFATVWLAGCSGCHMSFLD HoxY-6301 MDKI KLATVWLAGCSGCHMSFLD HoxY-Ralstonia eutropha MRAPHKDEI ASHELPATPMDPALAANREGKI KVATI GLCGCWGCTLSFLD KI . AT. WLAGCSGCHMSFLD

60 70 80 90 100 HoxY-7002 LDEFLI ELI KYVDVVFSPVGSDVKDYPKNVDVCLI EGAVANQENLELLEK HoxY-6803 MDEWLI DLAQKVDVVFSPVGSDLKEYPDNVDVCLVEGAI ANEENLELALE HoxY-6301 LDEWLI DLADGVEVVFSPVACDRKDYPEGVDL CL I EGAVANLDNLELLHQ HoxY-Ralstonia eutropha MDERLLPLLEKVTLLRS- SLTDI KRI PERCAI GFVEGGVSSEENI ETLEH DE. LI . L. V. VVFSPV. . D. K. YP. VD. CL. EGAVAN ENLELL

110 120 130 140 150 HoxY-7002 VRQNTKLLI AFGDCAVTTNVTGI RNQK- - GD- AQTI LERGYKELTEEH- R HoxY-6803 LRQKTKVVI SFGDCAVTANVPGMRNMLKGSD- - - PVLRRAYI ELGDGTPQ HoxY-6301 VRDRTRI L VSFGDCAI HANVPGMRNLW- GAESAAAVLERGYLELADTTPQ HoxY-Ralstonia eutropha FRENCDI L I SVGACAVWGGVPAMRNVFELKDCLAEAYVNSATAVPGAKAV .R T..LISFGDCAV .NVPGMRN . D . .L R.Y EL .

160 170 180 190 200 HoxY-7002 LPQQI TGGI LPPLLPRVLPI HEVVDI DLFLPGCPPDADRI KAAI APLLEG HoxY-6803 LPDEP- - GI VPPLLDKVI PLHEVI PVDI FMPGCPPDAHRI RATLEPLLNG HoxY-6301 LPNEP- - GI VPPLLNRVTPI HELVAI EHYLPGCPPPADRI RSLLQALLDQ HoxY-Ralstonia eutropha VPFHP---DIPRITTKVYPCHEVVKMDYFI PGCPPDGDAI FKVLDDLVNG LP P GI . PPLL . V P. HEVV . D F. PGCPPDADRI . . L LL G

210 220 230 240 250 HoxY-7002 KLPEMEGREMI KFG HoxY-6803 EHPLMEGRAMI KFG HoxY-6301 QTPPHSGRDLLKFG HoxY-Ralstonia eutropha R- PFDLPSSI NRYD PGR.KFG 380

Figure B28. ClustalW alignment of Hyp3 amino acid sequences. Hyp3 amino acid sequences of the following organisms were aligned using the ClustalW alignment program from MacVector v. 6.5: Synechococcus sp. PCC 7002, Anabaena variabilis, and

Prochlorothrix hollandica. The consensus amino acid sequence is shown underneath.

Gray high-lighted regions indicate identical amino acids while lighter shading indicates conserved amino acids. Dashes represent insertions/deletions included to maximize the sequence similarity. Percent identity and percent conserved amino acids determined by the ClustalW alignment tool (Thompson et al., 1994b). 381

Hyp3 Amino Acid Alignment

10 20 30 40 50 Hyp3-7002 ML T AADI MNPNVVTI KGL AT I ASATQCMRVNKTRVL I VDRRHVHDAYGI L Hyp3-A. variabilis MLKASDVMTKDVATI RSSATVAEAVKLMRARDWRALI VDRRHEQDAYGI I Hyp3-7120 MTKDVATI RSSATVAEAVKLMRARDWRALI VDRRHEQDAYGI I Hyp3-P. hollandica MTTPVVTI RRL RTI ADAVRL MRNKGAHAL MVERRNEADAYGI V MT V. TI R AT. A. AV. LMR. . RALI VDRRHE DAYGI .

60 70 80 90 100 Hyp3-7002 TATDI VSKVI AYGRDPRAI RVYEI MTKPCI FVSPDL AVEYVARL FSQWNL Hyp3-A. variabilis SESDI VYKVI AYGRDPYKI RVYEI MSKPCI AVNPDLGLEYVARLFADYGL Hyp3-7120 SESDI VYKVI AYGKDPNKI RVYEVMSKPCI AI NPDLGLEYVARLFADYGL Hyp3-P. hollandica TETDI VYQVTAHGKDPKTVRVFEVMTKPCI TVNPDL EVEYVARL FAMTGI . E. DI VYKVI AYG. DP I RVYE. M. KPCI VNPDL. . EYVARLFA . GL

110 120 130 140 150 Hyp3-7002 HSAPVMTDKLLGI I TVEDLI SKSDFLERPKELLFAAEMQAAI QKTKLI CQ Hyp3-A. variabilis HRAPVI QGELVGI I SLTDI LAQSDFLEQPYTI LLEQQLQDEI KKARAVCT Hyp3-7120 HRAPVI QGDLRGI I SLTDI L AQSDFL EQPYTI LL EQQLQDEI KKARAVCT Hyp3-P. hollandica RRAPVI QGQLL GMI STTDI L VKSNFVEEPKAQRL EQLI QEAI AAARQI CA HRAPVI QG L. GI I S. TDI L. SDFLE P . LLEQ . Q. I KAR. . C

160 170 180 190 200 Hyp3-7002 EKGHDSSDCI QAWAMVEELQAKTAYQQSTKVDKTAL EEYL EKNPEAI DHL Hyp3-A. variabilis QKGI NSEECAAAWDVI EEMQAEMAHQRAEKVSKI AFDDYCDEYPEALEA Hyp3-7120 QTGI NSEECAAAWDAVEEMQAEI AHQRAEKVSKTAFEDYCDEYPEALEAR Hyp3-P. hollandica DEGTTSPGCAAAWDVVEELQAEAAHQEAKGLI KTAFEEYL EENPEALEAR G S . CAAAWD. VEE QAE AHQ A KV KTAFE. Y . E PEALEA

210 220 230 240 250 Hyp3-7002 MV DNWCS G Hyp3-A. variabilis Hyp3-7120 LYEL Hyp3-P. hollandica VYDV . 382

Figure B29. ClustalW alignment of HoxH amino acid sequences. HoxH amino acid

sequences of the following organisms were aligned using the ClustalW alignment

program from MacVector v. 6.5: Synechococcus sp. PCC 7002, Synechocystis sp. PCC

6803, Synechococcus sp. PCC 6301, Anabaena variabilis, Anabaena sp. PCC 7120, and

R. eutropha. The consensus amino acid sequence is shown underneath. Gray high- lighted regions indicate identical amino acids while lighter shading indicates conserved amino acids. Dashes represent insertions/deletions included to maximize the sequence similarity. Percent identity and percent conserved amino acids determined by the

ClustalW alignment tool (Thompson et al., 1994b). 383

HoxH ClustalW Amino Acid Alignment

10 20 30 40 50 HoxH-7002 MSKTIIIDPMTRIEGHAKISIYLDDQGEVHEARFHVGEFRGFEKFCEGRP HoxH-6803 MSKTI VI DPVTRI EGHAKI SI FLNDQGNVDDVRFHVVEYRGFEKFCEGRP HoxH-6301 MTRTI VI DPVTRI EGHAKI SVFLDDQGNAEAARFHVVEYRGFEKFCEGRP HoxH A. variabilis MSKRI VI DPVTRI EGHAKI SI YLDDTGQVSDARFHVTEFRGFEKFCEGRP HoxH-7120 MSKRI VI DPVTRI EGHAKI SI YLDDTGQVNDARFHVTEFRGFEKFCEGRP HoxH-Ralstonia eutropha MSRKLVI DPVTRI EGHGKVVVHLDDDNKVVDAKL HVVEFRGFEKFVQGHP MSK I VI DPVTRI EGHAKI SI . LDD G. V DARFHV. EFRGFEKFCEGRP

60 70 80 90 100 HoxH-7002 FWEMPALTSRI CGI CPVSHLI ASSKTGDQLLAVKI P------VGAGKLRR HoxH-6803 MWEMAGI TARI CGI CPVSHLLCAAKTGDKLLAVQI P------PAGEKLRR HoxH-6301 FTEMAGI TARI CGI CPVSHLLAAAKTGDKI LAVQI P------PAAENLRR HoxH A. variabilis LWEMPGI TARI CGI CPVSHLLASAKAGDRI LSVTI P------PTATKLRR HoxH-7120 LWEMPGI TARI CGI CPVSHLLASAKAGDRI LSVTI P------PTATKLRR HoxH-Ralstonia eutropha FWEAPMFLQRI CGI CFVSHHLCGAKALDDMVGVGLKSGI HVTPTAEKMRR WEMPGI TARI CGI CPVSHLLA AK GD. . L. V I P P A KLRR

110 120 130 140 150 HoxH-7002 MI NLAQI TQSHALSFFHLSSPDLI FGWDSDPKTRNVFGLI AADPDLARGG HoxH-6803 LMNLGQI TQSHALSFFHLSSPDFLLGWDSDPATRNVFGLI AADPDLARAG HoxH-6301 LI NLAQI I QSHALSFFHLSSPDFLLGWDSDPARRNL FGL I AADPESARAG HoxH A. variabilis LMNLGQILQSHALSFFHLTAPDLLLGMDSDPQKRNI FGLIAAQPELARGG HoxH-7120 LMNLGQI LQSHALSFFHLSAPDFLLGMDSDPQKRNI FGLI AAQPELARGG HoxH-Ralstonia eutropha LGHYAQMLQSHTTAYFYLI VPEMLFGMDAPPAQRNVLGLI EANPDLVKRV L NL. QI . QSHALSFFHLS PD LLG DSDP RN. FGLI AA P. LAR. G

160 170 180 190 200 HoxH-7002 IRLRKFGQDIIKILGSQKVHPAWSIPGGVRSPLTKEGQTYIKEG------HoxH-6803 I RLRQFGQTVI ELLGAKKI HSAWSVPGGVRSPLSEEGRQWI VDR------HoxH-6301 I RL RQF GQQL I EWLGGRKI HAAWAVPGGVRSLLSQEGMVWIRDR------HoxH A. variabilis I RLRQFGQEI I EVLGGAKI HPAWAVPGGVREPLSVEGRTHI QER------HoxH-7120 I RLRQFGQEI I EVLGGAKI HPAWAVPGGVREPLSAEGRTHI QER------HoxH-Ralstonia eutropha VMLRKWGQEVI KAVFGKKMHGI NSVPGGVNNNLSI AERDRFL NGEEGL L S IRLRQFGQ..IE.LGGKIH AWVPGGVR PLS EGR I .R

210 220 230 240 250 HoxH-7002 LPEAKNTVKNALSLFKKI LDS- HQEEVTVFGNFPSLYLGLI GESGAWEHY HoxH-6803 LPEAKETVYLALNLFKNMLDR- FQTEVAEFGKFPSLFMGLVGKNNEWEHY HoxH-6301 LPAELATVRATLDRFKRLLDREFREETAVFGDFPSLFMGLVADNGDWEHY HoxH A. variabilis I PEARTI ALDALDRFKKLLKD- YEKEVQTFGNFPSLFMGLVTPDGLWETY HoxH-7120 I PEARTI ALDALDRFKKLLKD- YEKEAQTFGNFPSLFMGLVTPDGLWETY HoxH-Ralstonia eutropha VDQVI DYAQDGLRLFYDFHQK- HRAQVDSFADVPALSMCLVGDDDNVDYY . PEA. . AL FK. . L . EV FG FPSLFMGLV. G WE Y

260 270 280 290 300 HoxH-7002 GGI LRMI DSHGNI VGDRLDPNNYHEFLGEKLQEDSYLKSPYYKLFG---- HoxH-6803 GGSLRFTDSEGNI VADNLSEDNYADFI GESVEKWSYLKFPYYKSLGYP- - HoxH-6301 GGHLRFVDSQGRI I ADRLREDDYASFLGEAVEPWSYLKFPYYKPWGYP- - HoxH A. variabilis DGYI RFVDSAGNI I ADKLDPARYQEFI GEAVQPDSYLKSPYYRPLGYPDQ HoxH-7120 DGYI RFVDSAGNI I ADKLDPTRYQEFI GEAVQPDSYLKSPYYRPLGYPDQ HoxH-Ralstonia eutropha HGRL RI I DDD- KHI VREFDYHDYL DHFSEAVEEWSYMKFPYLKELGR- - - G LRF. DS GNI I AD. LD Y . F. GEAV SYLK PYYK LGYP 384

310 320 330 340 350 HoxH-7002 ------ETGVYRVGPLARLNICEHFGTEAADQELIEYRQRHGR-IVQASF HoxH-6803 ------DG-IYRVGPLARLNVCHHI GTPEADQEL EEYRQRAGG- VATSSF HoxH-6301 ------EG-MYRVGPLARLNVCDRI GTGRGGSRATGTARSRGG- TVTSSF HoxH A. variabilis HDQCRI DSGMYRVGPLARLNI CSHI GTTLADRELREFRELTSG- TAKSSF HoxH-7120 HDQCRI DSGMYRVGPLARLNI CSHI GTTLADRELREFRELTSG- TAKSSF HoxH-Ralstonia eutropha ------EQGSVRVGPLGRMNVTKSLPTPLAQEALERFHAYTKGRTNNMTL . G YRVGPLARLN. C HI GT . AD EL E. R G T. SSF

360 370 380 390 400 HoxH-7002 VYHHARLI EI LGSLERI ERMI DDPDLFSN- - RLQAEAGVNQTEAVGVSEA HoxH-6803 FYHYARLVEI LACLEAI ELLMADPDI LSK- - NCRAKAEI NCTEAVGVSEA HoxH-6301 LYHYARLVEI LASLEKI AEMVEDPNLQTG- - FLRSQAGVNCLEAI GVSEA HoxH A. variabilis FYHYARLI EI LACI EHI EMLLDDPDI LSN- - RLRSEAGVNQLEAVGVSEA HoxH-7120 FYHYARLI EI LACI EHI EI LLNDPDI LST- - RLRSEAGVNQLEGVGVSEA HoxH-Ralstonia eutropha HTNWARAI EI LHAAEVVKELLHDPDLQKDQLVLTPPPNAWTGEGVGVVEA YHYARLI EI LA . E. I E L. DPD. S LR AGVN . EAVGVSEA

410 420 430 440 450 HoxH-7002 PRGTLFHHYQVDENGLLKKI NLVI ATGQNNFAI NRTVTQI AKHYI HGE- T HoxH-6803 PRGTLFHHYKI DEDGLI KKVNLI I ATGNNNLAMNKTVAQI AKHYI RNH- D HoxH-6301 PRGTL FHHYRVDSHGKI ERVNL I I ATGQNNL AMNRTVTQI AQHYI RHG- E HoxH A. variabilis PRGTL FHHYQVDENGL L QKVNL I I ATGQNNL AMNRTVAQI ARHFI QGT- E HoxH-7120 PRGTL FHHYQVDENGL L QKVNL I I ATGQNNL AMNRTVAQI ARHFI QGT- E HoxH-Ralstonia eutropha PRGTLLHHYRADERGNI TFANLVVATTQNHQVMNRTVRSVAEDYLGGHGE PRGTLFHHY VDE GL. KVNLI I ATGQNNLAMNRTV QI A. HYI G E

460 470 480 490 500 HoxH-7002 VAEGI LNRVEAGVRNYDPCLSCSTHAAGQMPMI LQLI SPDGSI VKEI RRD HoxH-6803 VQEGFLNRVEAGI RCYDPCLSCSTHAAGQMPLMI DLVNPQGELI KSI QRD HoxH-6301 VQESFLNRVEAGI RCFDPCLSCSTHTAGQMPLKI EI FDSRGELYQCLCRD HoxH A. variabilis I PEGMLNRVEAGI RAFDPCLSCSTHAAGQMPLHI QLVAANGNI VNQVWRE HoxH-7120 I LEGMLNRVEAGI RAFDPCLSCSTHAAGQMPLHI QLVAADGNI VNQVWRE HoxH-Ralstonia eutropha I TEGMMNAI EVGI RAYDPCLSCATHALGQMPLVVSVFDAAGRLI DERAR . EG LNRVEAGI R . DPCLSCSTHAAGQMPL I L. G . . . R.

510 520 530 540 550 HoxH-7002 S HoxH-6803 HoxH-6301 L HoxH A. variabilis KLGV HoxH-7120 HoxH-Ralstonia eutropha 385

Figure B30. ClustalW alignment of HoxW amino acid sequences. HoxW amino acid sequences of the following organisms were aligned using the ClustalW alignment program from MacVector v. 6.5: Synechococcus sp. PCC 7002, Synechocystis sp. PCC

6803, Anabaena sp. PCC 7120, and R. eutropha. The consensus amino acid sequence is shown underneath. Gray high-lighted regions indicate identical amino acids while lighter shading indicates conserved amino acids. Dashes represent insertions/deletions included to maximize the sequence similarity. The percent identity and percent conserved amino acids were determined with the ClustalW alignment tool (Thompson et al., 1994b).

HoxW ClustalW Amino Acid Alignment

10 20 30 40 50 HoxW-7002 MKQTLMI GYGNTLRSDDGAGQKVAEAFFDQ- - ENI TAI ATHQLTP HoxW-6803 MPGQSTKSTLI I GYGNTLRGDDGVGRYLAEEI AQQNWPHCGVI STHQLTP HoxW-7120 MKKTVMVI GYGNDLRSDDGI GQRI ANEVASWRLPSVESLAVHQLTP HoxW-Ralstonia eutropha MKEQE- I DRI ATMI YEAPLGEYI GRDGAAI LAEHAAEARLLKGDE K IGYGNTLR DDG.G .A ..A . . HQLTP

60 70 80 90 100 HoxW-7002 -ELVEDLVQVEQVYFIDAAP------IETVTIKPIQRRDN- EHNFGHF HoxW-6803 - ELAEAI AAVDRVI FI DAQLQESANEPSVEVVALKTLEPNEL- SGDLGHR HoxW-7120 - DLADSLASVDLAI FI DACLPVHG-----FDVKVQPLFAAGD- I DSNVHT HoxW-Ralstonia eutropha FLYRRGDVTSSFYI VTDGRLALVREKTNERTAPI VHVLEKGDLVGELG- F .L.. .. V. .IFIDA L V . . GH

110 120 130 140 150 HoxW-7002 I D- - PKSL- - LNL- AQEI YHYAPDAYLVLI - - - PAQDFKLGEN- YSEI TQ HoxW-6803 GN- - PREL- - LTL- AKI LYGVEVKAWWVLI - - - PAFTFDYGEK- LSPLTA HoxW-7120 GD--PRSL--LAL-TKAIYGNCPTAWWVTI---PGANFEIGDR-FSRTAE HoxW-Ralstonia eutropha I DQTPHSLSVRALGDAAVLSFSAESI KPLI TEHPELI FNFMRAVI KRVHH . D P. SL L L . . Y A. VLI P. F G. S .

160 170 180 190 200 HoxW-7002 KAI ETAI HL- - LQERLTPCMK HoxW-6803 RAQAEALAQ- - I RPLVLGER HoxW-7120 TGKAI ALVK- - I I QI LDKVNNLWFEVGAVA HoxW-Ralstonia eutropha VVVTVGEHERELQEYI STGGRGR---G .A... 386

Figure B40. ClustalW alignment of HypA amino acid sequences. HypA amino acid sequences of the following organisms were aligned using the ClustalW alignment program from MacVector v. 6.5: Synechococcus sp. PCC 7002, Synechocystis sp. PCC

6803, Synechococcus sp. PCC 6301, Aquifex aeolicus and Bradyrhizobium japonicum.

The consensus amino acid sequence is shown underneath. Gray high-lighted regions indicate identical amino acids while lighter shading indicates conserved amino acids.

Dashes represent insertions/deletions included to maximize the sequence similarity.

Percent identity and percent conserved amino acids determined by the ClustalW alignment tool (Thompson et al., 1994b).

HypA ClustalW Amino Acid Alignment

10 20 30 40 50 HypA-7002 MHE AIMTETV IANAAAE QNATKI GLTMRIG ISGVVPE LSFAFEA HypA-6803 MHE SLMEQTL I AI AQAE HGASQI RLTLRVG QSGVVAD LRFAFEV HypA-6301 MHE SLATALV TALWQAV AEAQQI SLKLRLG WAGVDAE LRFAFSL HypA-Aquifex aeolicus MHE SI VQSLL LI EDYAR NNAKSV KVVVSI G LSGVEPH LEMAFNT HypA-Bradyrhizobium japonicum MHE ALCEGI I I VEEEAR RAFAKV VVCLEI G LSHVAPE LQFCFEA MHE SL. .. IA A A I L LRIG .SGV.PE L FAFE.

60 70 80 90 00 HypA-7002 VAG TLAEQAQ I I ETVPV CYCAQC RPFTPPD FYECPLC SLSQHI L HypA-6803 VRQ TMAAEAR EI EEI PV CRCQHC ENFQPED I YRCPHC QI SQTVM HypA-6301 VQQ TI AASAQ VI ESVPA FRCQTC QQTPPP- LAACSHC SDRWQLQ HypA-Aquifex aeolicus FKE TVAEKAE I MEI EKL I KCMDC KESEKEE NMLCPGC SLNTQI I HypA-Bradyrhizobium japonicum VAA TI AQGAK EI VETPG AWCMAC KSVEI KQ YEPCPSC GYQLQVT V T.A A .IE .P. .C C . P . CP C S. Q.

110 120 130 140 50 HypA-7002 SGK VELKSLE I HypA-6803 DGK LELASLE S HypA-6301 QGR L QL QSME V HypA-Aquifex aeolicus SGQ MFLKSLE EVD HypA-Bradyrhizobium japonicum GGE MRVREL E D G. . L . SL E 387

Figure B41. ClustalW alignment of HypB amino acid sequences. HypB amino acid sequences of the following organisms were aligned using the ClustalW alignment program from MacVector v. 6.5: Synechococcus sp. PCC 7002, Synechocystis sp. PCC

6803, Synechococcus sp. PCC 6301, Bradyrhizobium japonicum, and Azotobacter vinelandii. The consensus amino acid sequence is shown underneath. Gray high-lighted regions indicate identical amino acids while lighter shading indicates conserved amino acids. Dashes represent insertions/deletions included to maximize the sequence similarity. Percent identity and percent conserved amino acids determined by the

ClustalW alignment tool (Thompson et al., 1994b). 388

HypB ClustalW Formatted Alignments

10 20 30 40 50 HypB-7002 MCGNCGCN- - AVEKPVEI HTHAHDH------SHGHHDHHHPHDHEHHHH HypB-6803 MCQNCGCS- - AVGTVAHSHHHHGDG------NFAHSHDDHDQQEHHHHH HypB-6301 MCGVCGCQ--SPDPVVIATPEP------HypB-7120 MCVTCGCSDESEDKI TNL ETGEMVHN------HQCI QHTL ADGTVI THS HypB-B. japonicum MCTVCGCSD- GKASI EHAHDHHHDHGHDHDHGHDGHHHHHHGHDQDHHHH MC.CGCS.....HH DH .HHH HHHH

60 70 80 90 100 HypB-7002 HNHDG------NHS------HAIDINQSIFAKNDRLAERNRG HypB-6803 GNYSK------SPSQQTVTIEPDRQSIAIGQGILSKNDRLAERNRG HypB-6301 ------RQISLAEAILHRNDHAAAHNRE HypB-7120 HSHEQ------EPSQ--IPAKIHNTTISLEQEILGKNNLLAAQNRG HypB-B. japonicum HDHAHGDAGL L DCGANPAGQKI TGMSSDRI I QVERDI L GKNDRL AADNRA HH PS .. .I.QIL.KNDRLAA.NRG

110 120 130 140 150 HypB-7002 YFLAKDLFVI NVVSSPGSGKTALLERTI EQLKNKLNSAVI VGDLKTDNDA HypB-6803 YFQAKGL L VMNFL SSPGAGKT AL I EKMVGDRQKDHPTAVI VGDL AT DNDA HypB-6301 HFQRAGVLALNLLSSPGSGKTALLVRSFRELPPTLRPAAI VGDLATDRDA HypB-7120 WFKGRNI LSLNLMSSPGAGKTTLLTQTI NDLKHQLPI TVI EGDQETI NDA HypB-B. japonicum RFRADEVLAFNLVSSPGAGKTSLLVRAVSELKDSFAI GVI EGDQQTSNDA .F A. .L..NL.SSPGAGKTALL R.. .LK L AVIVGDL TDNDA

160 170 180 190 200 HypB-7002 QRLRKNNI PVAQI TTGTLCHLDAEMVLNAARKLALDGVHLLI I ENVGNLV HypB-6803 QRLRSAGAI AI QVTTGNI CHLEAEMVAKAAQKLDLDNI DQLI I ENVGNLV HypB-6301 QRLQATGAPADQI EPAELCHLEADLVHRACHQLDLSAI DI LFI ENVGNLV HypB-7120 EKI KETGCKVVQI NTGTGCHLDASMI ERGLQQLNPPI NSVLMI ENVGNLV HypB-B. japonicum ERI RATGVPAI QVNTGKGCHLDAAMVGEAYDRLPWLNGGLLFI ENVGNLV QRLR TG.PA.QI TG .CHLDA.MV..A. .L L .. .L IENVGNLV

210 220 230 240 250 HypB-7002 CPAAYDLGEHKRI VLLSTTEGEDKPLKYPTMFKSADVVI I NKI DI AEVVG HypB-6803 CPTTYDLGEDLRVVLFSVTEGEDKPLKYPATFKSAQVI LVTKQDI AAAVD HypB-6301 CPAAFDLGEERRVLLLSVTEGEDKPLKYPSAFSRADLVLI TKVDLAEAVE HypB-7120 CPALFDLGEQAKVVI LSVTEGEDKPI KYPHI FRASEI MI LTKI DLLPYVN HypB-B. japonicum CPAAFDLGEACKI VVFSTTEGEDKPLKYPDMFAASSLMLI NKI DLASVLD CPAAFDLGE RVVLLSVTEGEDKPLKYP F. A. . . LI TKI DLA . V.

260 270 280 290 300 HypB-7002 FDRDSALENVKKMCPQAQI FELSARTGEGMESWLNYLQSQYQDI AFQAVI HypB-6803 FDAEL AWQNLRQVAPQAQI FAVSARTGKGLQSWYEYLDQWQLQHYSPL VD HypB-6301 FDQAAAI ANLRAVNPTATI LPVSSRGGQGWSDWLDWLQVQRSHLLAPVKA HypB-7120 FDVQRCVKYAKQVNPNI QI FQV------FLQLRVLRV HypB-B. japonicum FDLARTI EYARRVNPKI EVLTLSARTGEGFAAFYAWI RKRMAATTPAAMT FD. A. N. R VNP. AQI F VSARTG G W . LQ . . .

310 320 330 340 350 HypB-7002 TV HypB-6803 PALA HypB-6301 HypB-7120 HypB-B. japonicum AAE . 389

Figure B42. ClustalW alignment of HypF amino acid sequences. HypF amino acid

sequences of the following organisms were aligned using the ClustalW alignment

program from MacVector v. 6.5: Synechococcus sp. PCC 7002, Synechocystis sp. PCC

6803, Aquifex aeolicus and R. sphaeroides. The consensus amino acid sequence is shown underneath. Gray high-lighted regions indicate identical amino acids while lighter shading indicates conserved amino acids. Dashes represent insertions/deletions included to maximize the sequence similarity. Percent identity and percent conserved amino acids determined by the ClustalW alignment tool (Thompson et al., 1994b). 390

HypF ClustalW Amino Acid Alignment

10 20 30 40 50 HypF-7002 L L VKRRL RL EI QGTVQGVGF RPF VYQL AT AL NL F GWVNNST AGVT I EVEG HypF-6803 ML KTVAI QVQGRVQGVGF RPF VYT L AQEMGL NGWVNNST QGAT VVI T A HypF-Aquifex aeolicus MPFKRLKLHLKGAVQGVGFRPFVYRI AKELGLKGFVI NDSKGVYI EVEG HypF-Rhodobacter capsulatus MQAWRI RVRGQVQGVGFRPFVWQL ARARGL RGVVL NDAEGVL I RVAG . . . . . G VQGVGF RPF VY L A GL G. V N . GV I V G

60 70 80 90 100 HypF-7002 GRSPLNLFLEKLQAELPPNAKI DALKYQYLELI GYNNFEI HASQT- GEKI HypF-6803 DEKAI ADFTERLTKTLPPPGLI EQLAVEQLPLESFTNFTI RPSSD- GPKT HypF-Aquifex aeolicus EEERLKKFLFKLNREKPPLARI YSQEI QFLEPVNYEDFVI RKSEEKGEKE HypF-Rhodobacter capsulatus D---LGDFAAALRDQAPPLARVDAVEVTAAVCDDL PEGFQI AASGAAGAE . L F. .L .PP A.I. . . L . F I. S G K

110 120 130 140 150 HypF-7002 AI VLPDLATCSECI AEI FDPQNRRYQYPFTNCTHCGPRYSI I ETLPYDRS HypF-6803 ASI LPDLSTCSACLTELFDPSDRRYLYPFI NCTHCGPRYTI I EALPYDRC HypF-Aquifex aeolicus VL VL PDI ATCEDCLREL FTPEDRRYMYPFI NCTNCGPRFTI I ERL PYDRK HypF-Rhodobacter capsulatus TRVTPDAATCPDCLAEI RG- EGRRRGYAFTNCTHCGPRFSI L QSL PYDRA . VLPD.ATC .CL E.F P RRY YPF NCTHCGPR..IIE LPYDR

160 170 180 190 200 HypF-7002 LTSMADFSMCADCQREYEDPGDRRFHAQPNACPI CGPKLEFLSHCQGQEN HypF-6803 RTTMARFRQCTDCEREYKQPGDRRFHAQPNACPRCGPQLAFWNRQGQVI A HypF-Aquifex aeolicus NTTMKVFEMCPECKREYENPLDRRFHAQPNACPKCGPWVSLYKD- GKLI A HypF-Rhodobacter capsulatus RTTMAPFAMCPACRAEYEDPADRRFHAQPI ACPDCGPRLWLEAGGAELPG TTMA F MC . C REYE P. DRRFHAQPNACP CGP L . . .

210 220 230 240 250 HypF-7002 TNQSPLEAAI QYI RDGKI VALKGLGGFQLLVDARNNKAVQQLRGRKQRPD HypF-6803 EANEALNFAVDNLKVGNI I AI KGLGGFHLCCDATDFEAVEKLRLRKHRPD HypF-Aquifex aeolicus EKNEALELLI EEI KI GKI VAVKGVGGFHLI CNATNEESVRTLRKRKRRSE HypF-Rhodobacter capsulatus ---DAIGLAAARLKAGEILAVKGLGGFHLACDATNADAVDLLRARKRRPA ..AL .A. .K.G I.A.KGLGGFHL.CDATN .AV LR.RK.RP.

260 270 280 290 300 HypF-7002 KPFAVMYPDL PSI KNDCFL SQLEEAFL TSQASPTVLLQKKKEFNLAENVA HypF-6803 KPLAVMYGNLGQI VEHYQPNNLEVELLQSAAAPI VLLNKKKQLI LVENI A HypF-Aquifex aeolicus KPFAVMFKSLEQVEAYANPTELEKALLI SPERPI VLI QKKK- - ELAPSVS HypF-Rhodobacter capsulatus KPFALMAR- EEDLARI VAVSPAALAALRDPAAPI VLMPARG- - SLPETLA KPFAVM. L . . LE A. L S A PI VL. . KKK L. E . A

310 320 330 340 350 HypF-7002 PHNPNLGVMLPYTPLHHLLLKSLNFPVI ATSGNRSDEPI CI DETEALERL HypF-6803 PGNPRVGVMLAYTPLHHLLLKKLKKPMVATSGNLAGEQICI DNI DALTRL HypF-Aquifex aeolicus PGLKRVGAFLPYSPLHHLI LNSLDFPVVATSANI SEEPI I KDNKEALEKL HypF-Rhodobacter capsulatus PGMAELGVMLPYTPLHHLLLDAFGGVLVMTSGNLSGAPQVI GNDEAREKL PG . GVMLPYTPLHHLLL L P. VATSGN. S EPI I DN EALE. L

360 370 380 390 400 HypF-7002 KNI ADGFLI HNRRI LRPVDDSVVRVMNNTPI I LRRSRGYAPEPLTLK- - R HypF-6803 QNI ADGFLVHDRPI VCPVDDSVVQI VAGKPLFLRRARGYAPQPI TLP- - K HypF-Aquifex aeolicus GEL ADLI L VHNRDI KRRCDDSVVKVVSGVPTPI RRSRGYAPL PVEVP- - F HypF-Rhodobacter capsulatus SAFADAFL MHDRAI ARRL DDSVVRVDP- - PMVL RRARGQVPGTL PL PPGF .AD.FL.H R I.R .DDSVV.V P LRR RGYAP P. LP

410 420 430 440 450 HypF-7002 TLSKNVLAMGAHLKNTVAI AQKNRLFLSQHI GDLSNQLTLQAMTKTLQKL HypF-6803 PTQKKLLAMGGHYKNTVAI AKQNQAYVSQHLGDLNSAPTYQNFEEAI AHL HypF-Aquifex aeolicus ELPKRVLAVGGMLKNTFALGFKNQVI LSQHI GDI ENLNTLKVFEESVFDL HypF-Rhodobacter capsulatus ETAPQI VAYGGQMKAALCLI KTGQALLGHHLGELDEALTWEAFLQADADY K . L A GG KNT . A. . NQ. L SQH. GDL . T . F . L 391

460 470 480 490 500 HypF-7002 SQI YDFQPDI I ACDLHPDYL STQYAKNL AQKL NI SLI SVQHHHAHI YACM HypF-6803 SQLYDFSPQEI VADLHPDYFSHQYAENQAL PVTF- - - - VQHHYAHI L AVM HypF-Aquifex aeolicus MELYEFEPDVVVCDMHPRYETTRWAEEFSRKRGI PLI KVQHHYAHMLSCM HypF-Rhodobacter capsulatus AALFDHRPQAVAVDLHPDFRASRHGAARAGRL GVPLI AVQHHHAHLAACL LYDF P . . . DLHPDY . . . A A . . . LI VQHH AH. . ACM

510 520 530 540 550 HypF-7002 AEH- QLESPL LG- - - - VAWDGTGYGEDGTI WGGEFFWVTKQSCERI AHFK HypF-6803 AE HGVMEE SV L G- - - - I A WDGT GY GMDGT I WGGEF L K I T QGT WQRI A HL Q HypF-Aquifex aeolicus AE N- GI KE KV L G- - - - I A WDGT GY GE DGT L WGGEF L V ADYT SY ERAF NF K HypF-Rhodobacter capsulatus GE N- - L WPKDGGKV AV I V L DGL GL GP DGT V WGGEL L L GDYK GF ERVA WL K AE . .LG IAWDGTGYG DGT.WGGEFL . ..ER.A K

560 570 580 590 600 HypF-7002 PFPL PGGDRASREPRRSALGLL SQLYDL QVLKKLNLPTI QAFSATEL EL L HypF-6803 PF HL L GNQQAI KYPHRI AL AL L WPTF GDDF SADS- L GNWLNFNNGFKNKI HypF-Aquifex aeolicus PVKL I GGEKAVKEPRRVALSLL FDI FGEEALNLD- LL PVKSFSEREL KNL HypF-Rhodobacter capsulatus PAPL I GGDRAQI EPWRNAL VRL DAAGL SDL ADRL ------FPAAPRDL A P L. GG. . A . EP. R AL. LL ...... L F .

610 620 630 640 650 HypF-7002 LSMLKKDIN------TPFTSSVGRLFDGVASLLDLR-QRESFEG HypF-6803 NSRL NQDL NNKNL RQL WQRGQAPL TSSMGRL F DGI AT L I GL I - NEVT FEG HypF-Aquifex aeolicus YLAWKKGIN------SPLSSSVGRLFDALASLLNLK-QILSYEG HypF-Rhodobacter capsulatus RQLAAKGIN------APLSSSAGRLFDAVAACLGICPMRQSYEG . K IN PL.SS.GRLFD..A.LL L . S.EG

660 670 680 690 700 HypF-7002 QAAMALEFSI DGLQI PDFYQFQYTKNDSI L EI DSRGI FQGI I QDL QNDL P HypF-6803 QAAI AL EAQI MPNL TEEYYPLTLNNKEKKL AVDWRPL I KAI TTEDR- - SK HypF-Aquifex aeolicus QGAMMVEDLYDP- L VKDNYPYEI RGKE- - - - VDLRKAFL EVLKEKD- - - K HypF-Rhodobacter capsulatus EAAMRL ESLAADTGPVPDLPCVGG------AI DPAPL FQLL AAGER- - - P QAAMLE. . .YP. . .DR.F....

710 720 730 740 750 HypF-7002 KNFI AAKFHNTLVEI I FDI YLHTLKLGFNSRKNI VLAGGCFQNKYLLEQT HypF-6803 TNLI ATKFHNSL VNLI I TI AQQ------QGI EKVALGGGCFQNCYLL AST HypF-Aquifex aeolicus S- LAASRFI NTL AKVCEDI ALM------VGI ERVCLSGGVMQNDPLVTKI HypF-Rhodobacter capsulatus D- RVAHAL HASL AQAFAAEARR- - LI EAGQAEAVALTGGCFQNSRLATMT .A .FHN.L. . IA .E V.L GGCFQN L. T

760 770 780 790 800 HypF-7002 I QKL ESSGANI YYPQKFPPNDGAI AL GQVMVVTGQAI HypF-6803 I TAL KKAGFSPL WPRELPPNDGAI CMGQLL AKI QARQYI C HypF-Aquifex aeolicus KELL EKEKFKVYTHQKVPPNDGGI AL GQAVFGLSL V HypF-Rhodobacter capsulatus RNFL ADQG- - I L TQGRI PANDGGLAL GQAL VAAAKLESN L G . ..PPNDG.IALGQ...... 392

Figure B43. ClustalW alignment of HypC amino acid sequences. HypC amino acid sequences of the following organisms were aligned using the ClustalW alignment program from MacVector v. 6.5: Synechococcus sp. PCC 7002, E. coli, Synechocystis sp.

PCC 6803, and R. eutropha. The consensus amino acid sequence is shown underneath.

Gray high-lighted regions indicate identical amino acids while lighter shading indicates conserved amino acids. Dashes represent insertions/deletions included to maximize the sequence similarity. Percent identity and percent conserved amino acids determined by the ClustalW alignment tool (Thompson et al., 1994b).

HypC ClustalW Amino Acid Alignment

10 20 30 40 50 HypC-7002 MCLAVPGKI LEI VGD- DPLFKMGRVSFSGVVREVSLAYVPEA------QV HypC-E. coli MCIGVPGQIRTIDGN------QAKVDVCGI QRDVDLTLVGSCDENGQPRV HypC-6803 MCLALPGQVVSLMPNSDPLLLTGKVSFGGI I KTI SLAYVPEV------KV HypC-Ralstonia eutropha MCLAIPARLVELQAD-----QQGVVDLSGVRKTISLALMADA------VV MCLA.PG .. . . G.V G. . .SLA V .. V

60 70 80 90 100 HypC-7002 GDYAVVHAGFALSVLDEVAATETLATLAEMESFAGG HypC-E. coli GQWVLVHVGFAMSVI NEAEARDTLDALQNMFDVEPDVGALLYGEEK HypC-6803 GDYVI VHVGFAI SI VDEEAAQETLI DLAEMGV HypC-Ralstonia eutropha GDYVI VHVGYAI GKI DPEEAERTLRLFAELERVQPPASEPMHGMNI HQEP GDYV. VHVGFA. S. . DE A . TL LAEM

110 120 130 140 150 HypC-7002 HypC-E. coli HypC-6803 HypC-Ralstonia eutropha A 393

Figure B44. ClustalW Alignment of HypD Amino Acid Sequences. HypD amino acid sequences of the following organisms were aligned using the ClustalW alignment program from MacVector v. 6.5: Synechococcus sp. PCC 7002, Synechocystis sp. PCC

6803, Anabaena sp. PCC 7120, E. coli, and R. eutropha. The consensus amino acid sequence is shown underneath. Gray high-lighted regions indicate identical amino acids while lighter shading indicates conserved amino acids. Dashes represent insertions/deletions included to maximize the sequence similarity. Percent identity and percent conserved amino acids determined by the ClustalW alignment tool (Thompson et al., 1994b). 394

HypD ClustalW Amino Acid Alignment

10 20 30 40 50 HypD-7002 MKFVDEFRDPAAVQKYVQAI AALVTRP----WT---IMEICGGQT HSI VK HypD-6803 MKYVDEYRDAQAVAHYRQAI AREI TKP----WT---LMEICGGQT HSI VK HypD-7120 MKYVDEFREPEKAEALRREI EKLSQQL----DKHIKIMEVCGGHTHSIFK HypD-E. coli MRFVDEYRAPEQVMQLI EHLRERASHLSYTAERPLRI MEVCGGHTHAI FK HypD-Ralstonia eutropha MKYI EEFRDGELAQRI AAHVRAEARPG----QR-YNFMEFCGGHTHAISR MKYVDEFRDPE. V . . . I .... . IME.CGGHTHSI K

60 70 80 90 100 HypD-7002 YGI DQLLPPEI TLI HGPGCPVCVTPAELI DQAI ALAQLPDVVLCSFGDML HypD-6803 YGLDALLPKNLTLI HGPGCPVCVTPMELI DQALWLAKQPEI I FCSFGDML HypD-7120 YGI EEI LPANI ELI HGPGCPVCVMPKGRLDDAI AI SQNPNVI LTTFGDTM HypD-E. coli FGLDQLLPENVEFI HGPGCPVCVLPMGRI DTCVEI ASHPEVI FCTFGDAM HypD-Ralstonia eutropha YGVTELLPENVRMI HGPGCPVCVLPI GRI DLALHLALERDAI VCTYGDTM YG. D LLP N. LI HGPGCPVCV P GRI D A. LA P. VI . CTFGD M

110 120 130 140 150 HypD-7002 RVPGTR- LDLLSVKAQGAAVKMVYSPLDALKMAQENPDKQVI FFAVGFET HypD-6803 RVPGSG- ADL L SI KAQGGDVRI VYSPLDCLAI ARENPNREVVFFGVGFET HypD-7120 RVPGSK- TTLLQAKAQGADI RMVYSPLDSLQI ARNHPDKEI VFFAL GFET HypD-E. coli RVPGKQ- GSLLQAKARGADVRI VYSPMDALKLAQENPTRKVVFFGLGFET HypD-Ralstonia eutropha RVPASGGMSLI RAKAHGADI RMVYSAADALKI AQRHPQREVVFLAI GFET RVPGS . . LL AKAQGADVRMVYSPLDALKI AQENP REVVFFA. GFET

160 170 180 190 200 HypD-7002 TAPTTAMAVYQAAKLGLTNFSLLVAHVSVPPAI EAI LSAP------DRTI HypD-6803 TAPATAMTLHQARAQGI SNFSLLCAHVLVPPAMEALLGNP------NSLV HypD-7120 TAPSTALTI LQAASENI TNFSMFSNHVLVI PALQALLNNP------DLQL HypD-E. coli TMPTTAITLQQAKARDVQNFYFFCQHITLIPTLRSLLEQP------DNGI HypD-Ralstonia eutropha TTPPTALI I REAKARQVDNFSVLCCHVL TPSAI THI LESPEVRDYGTVPI TAP. TA. T. QA. A . . NFS. LC HVLVPPA. ALL . P D I

210 220 230 240 250 HypD-7002 QGFLLAGHVCTVMGYQEYEAI AKNYQI PLI VTGFEPLDI VQGI YLCVKQL HypD-6803 QGFLAAGHVCTVTGERAYQHI AEKYQVPI VI TGFEPVDI MQGI FACVRQL HypD-7120 DGFI GPGHVSMVI GTEPYEFI AQQYHKPI VVSGFEPLDI FQSI WMLLQQL HypD-E. coli DAFLAPGHVSMVI GTDAYNFI ASDFHRPLVVAGFEPLDLLQGVVMLVQQK HypD-Ralstonia eutropha DGFVGPAHVSI VI GTRPYEHFSREYGKPVVI AGFEPLDVMQAI LMLVRQV DGFL. PGHVS VI GT YE I A Y . P. VV. GFEPLDI QGI . MLV. QL

260 270 280 290 300 HypD-7002 EEGRSHI ENQYRRVVQAAGNATAQQLVTEI FEI VPR- TWRGI GEI SQSGL HypD-6803 ESGQFTCNNQYRRSVQPQGNAHAQKI I DQVFEPVDR- HWRGLGLI PASGL HypD-7120 VENRCEVENQYNRLVQKGGNQI ALAAMHKVFAVREKFAWRGLDEI PDSGL HypD-E. coli I AAHSKVENQYRRVVPDAGNLLAQQAI ADVFCVNGDSEWRGLGVI ESSGV HypD-Ralstonia eutropha NSGRAEVENEFVRAVTRDGNESAQAMVSEVFELRPSFEWRGLGEVPYSAL GR VENQYRR. VQ . GN. AQ . . . VFE. . WRGLGEI P SGL

310 320 330 340 350 HypD-7002 GLREKYAVFDASRKFKLDLTHFQP- QASSSCI SGEI LRGRKKPKQCPAFG HypD-6803 GLRPAFAPWDAAVKFANLLQTMAPTMGETVCI SGEI LQGQRKPSDCPAFG HypD-7120 KI REEYAQFDAELKFTI PNLKVAD---HKACKCGEILKGVLKPWQCKVFG HypD-E. coli HLTPDYQRFDAEAHFRPAPQQVCD---DPRARCGEVLTGKCKPHQCPLFG HypD-Ralstonia eutropha RI RAQFARFDAEQRFDLRYRPVPD---NKACECGAILRGVKKPTDCKLFA .LR YA FDAE.KF . V D .C CGEIL.G .KP QCP.FG

360 370 380 390 400 HypD-7002 TTCTPERPLGAPMVSSEGACAAYYRYG HypD-6803 TI CTPEQPLGAPMVSSEGACAAYYRYRQQLPEPVGAARV HypD-7120 TACTPETPI GTCMVSSEGACAAYYKYGRFSTTLQKQAAEKPKVTI SS HypD-E. coli NTCNPQTAFGALMVSSEGACAAWYQYRQQESEA HypD-Ralstonia eutropha TVCTPENPMGSCMVSSEGACAAHYSYGRFKDI PLVAA T. CTPE P. GA MVSSEGACAAYY. YG A 395

Figure B45. ClustalW alignment of CtaCI amino acid sequences. CtaCI amino acid sequences of the following organisms were aligned using the ClustalW alignment program from MacVector v. 6.5: Synechococcus sp. PCC 7002, Synechocystis sp. PCC

6803, and Anabaena sp. PCC 7120. The consensus amino acid sequence is shown underneath. Gray high-lighted regions indicate identical amino acids while lighter shading indicates conserved amino acids. Dashes represent insertions/deletions included to maximize the sequence similarity. Percent identity and percent conserved amino acids determined by the ClustalW search program (Thompson et al., 1994b). 396

CtaCI ClustalW Amino Acid Alignment

10 20 30 40 50 CtaCI-7002 MKI VTNQRHFQTVCFNVVTSSYSKKAPTFPVNI PNSI TTLI AGI AI TLI S CtaCI-6803 MKI PGSVI TLLI GVVI TVVS CtaCI-7120 MQQI PVSLWTLI AGI VVGVI S . I P. S. TLI AGI VI TVI S

60 70 80 90 100 CtaCI-7002 LWYGQNHGLLPVAASADANDVDELFNLMMTI ATGLFI LI EGVLVI CLI RF CtaCI-6803 LWYGQNHGLMPVAASADAEKVDGI FNYMMTI ATGLFLLVEGVLVYCLI RF CtaCI-7120 LWI GQNHNLLPI QASEQAPLVDGFFNI MFTI AVALFLVVEGTI LI FLFKY LWYGQNHGLLPVAASADA VDG. FN. MMTI ATGLFLLVEGVLVI CLI RF

110 120 130 140 150 CtaCI-7002 RRKQGDLTDGPAI EGNVPLEI VWTAI PTVI VFI LAI YSFEI YNKMGGLDP CtaCI-6803 RRRKDDQTDGPPI EGNVPLEI LWTAI PTVI VFTLAVYSFEVYNNLGGLDP CtaCI-7120 RRRRGDNTDGVPVEGNVPLEI FWTAI PSI I VI CLGI YSVDVFNQMGGLEP RRR. GD. TDGPPI EGNVPLEI . WTAI PTVI VF LAI YSFEVYN. MGGLDP

160 170 180 190 200 CtaCI-7002 MV- - SGGGMTMAHHHQHNPNTMDNMVAMEPDSKI AI GI GKSMSAND- EDP CtaCI-6803 TI SRDNAGQQMAHNHMGHMGSMGNMVAMAGDGDVALGI GLDSEEQG- VNP CtaCI-7120 GT- HPHASAHVAHSSGTALAATLNDTSTSAI N- PGI GI GASPTTAGKTAD . . AG MAH H . . M. NMVAM . D . AI GI G. S . . G P

210 220 230 240 250 CtaCI-7002 LVVDVNGLQYAWI FTYPDTGI VSGDLHVPVDRPI QLNMKAADVI HAFWLP CtaCI-6803 LMVDVKGI QYAWI FTYPETGI I SGELHAPI DRPVQLNMEAGDVI HAFWI P CtaCI-7120 LVVNVTGMQFAWXFDYPDNGVSAGELHVPVGADVQLNLSAQDVI HSFWVP LVVDV G. QYAWI FTYPDTGI . SGELHVPVDRPVQLNM A. DVI HAFW. P

260 270 280 290 300 CtaCI-7002 EFRI KQDVMPGQVSQLSFVANREGTYPVI CAELCGSYHGGMKTTMTVETA CtaCI-6803 QLRLKQDVI PGRGSTLVFNASTPGQYPVI CAELCGAYHGGMKSVFYAHTP CtaCI-7120 QFRLKQDAI PGVPTELRFVATKPGTYPVVCAELCGGYHGSMRTQVI VHTP QFRLKQDVIPG .S L FVA..PGTYPVICAELCG.YHGGMKT VHTP

310 320 330 340 350 CtaCI-7002 EGYDQWVQSRTVALQDGEGQPLPVDSTALTDAEFLQAYAEEMGI I ENTLE CtaCI-6803 EEYDDWVAANAPAPTESMAMTLPKATTAMTPNEYLAPYAKEMGVQTEALA CtaCI-7120 EEFDSWLAENQVAQQQNLHQAVAVNPANLSTSEFLAPHTQDLGI SAATLE EEYD WVA N VA Q. . Q LPV . TALT EFLAPYA EMGI TLE

360 370 380 390 400 CtaCI-7002 QI PHHPLGMMSMAQ CtaCI-6803 QLKDQTSPVGDLL CtaCI-7120 TLHTTSVN QL . . . . 397

Figure B46. ClustalW alignment of CtaDI amino acid sequences. CtaDI amino acid sequences of the following organisms were aligned using the ClustalW alignment program from MacVector v. 6.5: Synechococcus sp. PCC 7002, Synechocystis sp. PCC

CtaDI ClustalW Amino Acid Alignment

10 20 30 40 50 CtaDI-7002 MSDATIHHAGD----R------KWTDYFTFCTDHKVIGI CtaDI-6803 MTIAAENLTANHPRR------KWTDYFTFCVDHKVIGI CtaDI-7120 MTRVEFPPHI PPDDNQPKNLAVGHGLTLPAWKWRDYFTFNVDHKVI GI . . T. HHP D . KWTDYFTFCVDHKVI GI

60 70 80 90 100 CtaDI-7002 QYLVTAFLFYFI GGALAEI VRTELATPDPDFVSPEVYNQMFTMHGTI MI F CtaDI-6803 QYLVTSFLFFFI GGSFAEAMRTELATPSPDFVQPEMYNQLMTLHGTI MI F CtaDI-7120 QYLVTAFLFYLI GGLMAI AI RTELATPDADFI DPNLYNAFMTNHGTI MI F QYLVTAFLFYFI GG. AEA. RTELATPDPDFV PE. YNQ MT HGTI MI F

110 120 130 140 150 CtaDI-7002 LWI VP- AGAAFANYLI PLMI GADDMAFPRLNAVAFWMQPVGGI LLI LSFF CtaDI-6803 LWI VP- AGAAFANYLI PLMVGTEDMAFPRLNAVAFWLTPPGGI LLI SSFF CtaDI-7120 LWI VPSAI GGFGNYLI PLMI GARDMAFPKLNAI AFWLNPPAGLLLLLSFI LWI VP AGAAFANYLI PLMI GA. DMAFPRLNAVAFWL. PPGGI LLI LSFF

160 170 180 190 200 CtaDI-7002 VGAPQAGWTSYPPLSLI SGKWGEELWI LCLLI LGTASI LGAI NFVTTI FK CtaDI-6803 VGAPQAGWTSYPPLSLLSGKWGEELWI LSLLLVGTSSI LGAI NFVTTI LK CtaDI-7120 FGGSQSGWTAYPPLSLVTAPTAQTLWILAI VLVGTSSI LGSVNFVVTI LM VGAPQAGWTSYPPLSL. SGKWGEELWI L LLLVGTSSI LGAI NFVTTI LK

210 220 230 240 250 CtaDI-7002 MRAPDMDI HSMPLFCWAMLATSALI LLSTPVLAAALI LLSFDLMAGTAFF CtaDI-6803 MRI KDMDLHSMPLFCWAMLATSSLI LLSTPVLASALI LLSFDLI AGTSFF CtaDI-7120 MKVPSMKWDQLPLFCWAI LATSVLALLSTPVLAAGLVLLLFDLNFGTSFF MR.PDMD.HSMPLFCWAMLATS.LILLSTPVLAAALILLSFDL AGTSFF

260 270 280 290 300 CtaDI-7002 NPTGGGDPI VYQHLFWFYSHPAVYI MVLPFFGVI SEI LPVHSRKPI FGYR CtaDI-6803 NPVGGGDPVVYQHLFWFYSHPAVYI MI LPFFGVI SEVI PVHARKPI FGYR CtaDI-7120 KPDAGGNVVI YQHLFWFYSHPAVYLMI LPI FGI MSEVI PVHARKPI FGYK NP GGGDPVVYQHLFWFYSHPAVYI MI LPFFGVI SEVI PVHARKPI FGYR

310 320 330 340 350 CtaDI-7002 AI AYSGLAI SFLGLI VWAHHMFTSGTPGWLRMFFMATTMLVAVPTGI KI F CtaDI-6803 AI AYSSLAI SFLGLI VWAHHMFTSGTPGWLRMFFMATTMLI AVPTGI KI F CtaDI-7120 AI AYSSVAI CVVGLFVWVHHMFTSGTPGWMRMFFTI STLI VAVPTGVKI F AI AYSSLAI SFLGLI VWAHHMFTSGTPGWLRMFFMATTMLVAVPTGI KI F 399

360 370 380 390 400 CtaDI-7002 SWCATLWGGKLQLNSALLFAI GFLSSFLI GGLTGVMLAAAPFDI HVHDTY CtaDI-6803 SWCGTLWGGKI QLNSAMLFAFGFLSSFMI GGLTGVMVASVPFDI HVHDTY CtaDI-7120 GWVATLWGGKI RFTSAMLFAI GLLSMFVMGGLSGVTMGTAPFDVHVHDTY SWCATLWGGKIQLNSAMLFAIGFLSSF.IGGLTGVM.A.APFDIHVHDTY

410 420 430 440 450 CtaDI-7002 FVVGHFHYVLFGGSVFALFGAVYHWFPKMTGKMYNETWGKI HFAMTFI GF CtaDI-6803 FVVGHFHYVLFGGSAFALFSGVYHWFPKMTGRMVNEPLGRLHFI LTFI GM CtaDI-7120 YVVAHFHYVLFGGSVFGI YAGI YHWFPKMTGRKLGEGWGRI HFALTLVGT FVVGHFHYVLFGGSVFALF. GVYHWFPKMTGRM. NE WGRI HFALTFI G

460 470 480 490 500 CtaDI-7002 NMTFLPMHYLGLQGMNRRI ALYDPQFQPLNQVCTLGSYI LALSTLPFLVS CtaDI-6803 NLTFMPMHELGLMGMNRRI ALYDVEFQPLNVLSTI GAYVLAASTI PFVI N CtaDI-7120 NLTFLPMHKLGLQGMPRRVAMYDPQFVDLNVLCTI GAFI LGLSVI PFAI N NLTFLPMH LGLQGMNRRI ALYDPQFQPLNVLCTI GAYI LALSTI PF. I N

510 520 530 540 550 CtaDI-7002 I VLGLVNGKAAGRNPWRALTLEWQTTSPPSI ENFDEPPVLWAGPYEYGI D CtaDI-6803 VFWSLFKGEKAARNPWRALTLEWQTASPPI I ENFEEEPVLWCGPYDFGI D CtaDI-7120 VI WSWSKGELAGDNPWEALSLEWTTSSPPLVENWEVLPVVTHGPYDYGHS V. WSL KGE. AGRNPWRALTLEWQT. SPP. I ENFEE PVLW GPYDYGI D

560 570 580 590 600 CtaDI-7002 GEPRD- EDSI EEMLAEVAEMS CtaDI-6803 T EL MDDEET VQT L I ADAAGS CtaDI-7120 LEAAP- EVSVST .E. D E.SV T .A..A 400

Figure B47. ClustalW alignment of CtaEI amino acid sequences. CtaEI amino acid sequences of the following organisms were aligned using the ClustalW alignment program from MacVector v. 6.5: Synechococcus sp. PCC 7002, Synechocystis sp. PCC

CtaEI ClustalW Amino Acid Alignment

10 20 30 40 50 CtaEI 7002 MQSSTTNTAIATDYQTPPT-----E CtaEI-6803 MTSPMGPTARDFWQNSRNSATI QRLI VSPMQTTALSSDLNSTYTPGEAHG CtaEI-7120 MTSVTAHEAHG------G MS T TA...D.. T G

60 70 80 90 100 CtaEI 7002 HHGHPDYRMFGLVMFLVAESMIFLGLFAAFLI YKATMPGF----TKELEL CtaEI-6803 HHGHPDLRMFGVVLFLVAESAI FLGLFTAYLI YRSVMPAWPPEGTPELEL CtaEI-7120 HEAHPDLRVWGLLTFLI SESLMFGGFFATYLFFKGTTEVWPPE-GTEVEL HHGHPDLRMFGLV FLVAES. I FLGLFAAYLI YK. TMP. WPPE T ELEL

110 120 130 140 150 CtaEI 7002 LVPT VNT I I LVSSSFVMHKGQSAI KNDDVKGLQLWFGI T ALMGAVFLGGQ CtaEI-6803 LLPGVNSI I LI SSSFVMHKGQAAI RNNDNAGLQKWFGI TAAMGI I FLAGQ CtaEI-7120 FVPAI NTAI LLSSSVVI HFGDMAI KKGNVWGMRI WYFLTAI MGAVFLAGQ LVP. VNTIIL. SSSFVMHKGQ AIKN DV GLQ. WFGITA. MGAVFLAGQ

160 170 180 190 200 CtaEI 7002 VYEYAHMEFGLTEHLFGSCFYVLTGFHGLHVTAGLLFTLAVLWRSREAGH CtaEI-6803 MYEYFHLEMGLTTNLFASCFYVLTGFHGLHVTFGLLLI LSVLWRSRQPGH CtaEI-7120 VYEYQNLGYGLTANVFANCFYI MTGFHGLHVFI GLLLI LGVLWRSRRSGH VYEY HLE. GLT NLFASCFYVLTGFHGLHVT. GLLLI L. VLWRSR GH

210 220 230 240 250 CtaEI 7002 YSGQAHFGVEAAELYWHFVDVVWNYPVRPCL CtaEI-6803 YSRTSHFGVEAAELYWHFVDVVWIVLFI LVYLL CtaEI-7120 YSETKHTGIEMAEIYWHFVDIIWIVLFTLVYLLNLL YS T HFGVEAAELYWHFVDVVWIVLF LVYLL 401

Figure B48. ClustalW alignment of CtaCII amino acid sequences. CtaCII amino acid sequences of the following organisms were aligned using the ClustalW alignment program from MacVector v. 6.5: Synechococcus sp. PCC 7002, Synechocystis sp. PCC

CtaCII ClustalW Amino Acid Alignment

10 20 30 40 50 CtaCII-7002 MKLKTI I SLSAI ALLL GVFSYWVGQWSYSWLPPQASVESQLVDRLFSTLV CtaCII-6803 MSRKNLI L LAVYI VFTVGASL WLGQRAYQWLPPAAAQEAQPVGDLFSFLV CtaCII-7120 NI VTLVI GAI AVTI TSFWI GKLAYTWLPPQAAAESI LI DDLFSFLV M KNI I . L. . . A. . . . . S. W. GQ AY. WLPPQAA. ESQLVDDLFSFLV

60 70 80 90 100 CtaCII-7002 AI GTFI LFGVAGTMTYSI LFHRAGRYDTSDGPPI EGNVTLEI VWTTI PFL CtaCII-6803 SL GSVVFL GVAGAMAYSVI FHRFSLQNPQ- GAPI RGNARLEI FWTVVPI I CtaCII-7120 TMGAFI FL GVTSTLFYSL LFHRAAENDLSDGPHI EGNVTLEVVWQAI PI L . . G. FI FL GVAGTM YS. LFHRA. . D SDGPPI EGNVTLEI VWT. I PI L

110 120 130 140 150 CtaCII-7002 IVIYLAYFSYQTYREMNIQAPG------HMHQEALVTNVSTGKTTMAMPV CtaCII-6803 LVTWI AWYSYVI YQRMNVLGPLPVVEVPQLL GEKAI AADAPAEL AMAQGS CtaCII-7120 LVVWI ATYSYQI YEQMGI QGRT- - - ALVHLHNPMEMESAYAATEDGLVEP LV.WIA.YSYQIY MNIQGP. . HLH.E .. . A MA

160 170 180 190 200 CtaCII-7002 TN- - - - VEVTAKQWAWI FHYPEKNVTSTELHLPVNQRAHFI L RSPDVI HG CtaCII-6803 NI NPERI GVEVKQWLWTFTYPNGGVTSHELHLPL DRRVTLNMTSKDVLHG CtaCII-7120 EE- - - KI DVI AKQWAWVFHYPEKNVTSTELHLPSDRRVKLAL HSEDVLHG . I . V AKQWAW. FHYPEKNVTSTELHLP. DRRV. L. L . S DVLHG

210 220 230 240 250 CtaCII-7002 FF I PAFRVKQDVI PFEDT DFEFT PI KTGKYRI RDSQF SGTYF AAMQADVV CtaCII-6803 FYVPNFRI KQDI VPNREI EFSFTPNRLGEYKLHDSQFSGTYFAVMTAPVV CtaCII-7120 FYI PAFRL KQDI I PNHNI DFEFTPI REGKYHLTDSQYSGTYFATMQANVV FYI PAFR. KQDI I PN. . I DFEFT PI R GKY. L . DSQF SGTYF A. MQA VV

260 270 280 290 300 CtaCII-7002 VESQEDYQTWLNQAARQPPTPAPNQAVSEYARRQQKEDRAAWKTVPPAPP CtaCII-6803 VQSLSDYQAWLESQKSLTPGELPNPALDEFKQTPTTPLKSGWPTVPPGTR CtaCII-7120 VESPEEYHKWLAKI ATHKPGTAYNQASAEYAQSI TQQVKTGWKTVAP VES EDYQ WL . A. PG APNQA. EYAQ. T . K. GWKTVPP.

310 320 330 340 350 CtaCII-7002 PVVNYHP CtaCII-6803 Q CtaCII-7120 403

Figure B49. ClustalW alignment of CtaDII amino acid sequences. CtaDII amino acid sequences of the following organisms were aligned using the ClustalW alignment program from MacVector v. 6.5: Synechococcus sp. PCC 7002, Synechocystis sp. PCC

CtaDII ClustalW Amino Acid Alignment

10 20 30 40 50 CtaDII-7002 MTQAPPEALTNEGQPLTNLP--NWRTFFSFSTDHKVI GLQYIVTSFFFFL CtaDII-6803 MTNQPTASVPFQRP------WHDYLKFSTDHKVI GI QYLLMSFCFFL CtaDII-7120 MTNI PI EGVQLPEGKPHHPSPGGWKEYFSFSHDHKVI GI QYLVTSFI FFL MTN. P E. V W.. YFSFSTDHKVI GI QYLVTSF FFL

60 70 80 90 100 CtaDII-7002 VAGI FAMIMRGELI TPEPDLVDRTVYNALFTMHGSI MLFGWTFPVLAGFA CtaDII-6803 VAGLLAMII RAELLTPQLDVVDRSLYNGLFTLHGTI MIFLWIFPANVGLA CtaDII-7120 VGGI FAMILRGELI TPESDLI DRTVYNGMFTMHGTVMLFLWTFPSLVGLA VAGI FAMI. RGELI TPE DLVDRTVYNGLFTMHGTI MLFLWTFP. LVGLA

110 120 130 140 150 CtaDII-7002 NYLVPLMIGAQDMAFPRLNAIAFWMVPVFGSLLVLSFLAPGGPAQAGWWS CtaDII-6803 NYLI PLMIGARDVAFPVLNAI AFWLMPVVGVLLI GSFFLPTGTAQAGWWS CtaDII-7120 NYLVPLMIGARDMAFPRLNAAAFWMVPVVGILLMTSFFVPGGPAQSGWWA NYLVPLMIGARDMAFPRLNAIAFWMVPVVG. LL. . SFF. PGGPAQAGWWS

160 170 180 190 200 CtaDII-7002 YPPVSI QNPAGVLLNGEFLWLVSVALSGISSI LGAVNIVTTI VQMRCPGM CtaDII-6803 YPPVSI QNPSGNFI NGEFLWLLAVATSGI SSI MGGVNFVTT VWKLRAPGL CtaDII-7120 YPPVSTQNPTGNLI NGQVI WLLAVAI SGVSSI MGAVNFVTT I VKMRAPGM YPPVSI QNP. GNLI NGEFLWLLAVA. SGI SSI MGAVNFVTT I VKMRAPGM

210 220 230 240 250 CtaDII-7002 GWFKTPAFVWTVLAAQII QLFGLPALTAGAVMLLFDLTVGTTFFDASQGG CtaDII-6803 TLLKMPVYVWTI LSAQILQLFCLPALTGGAVMLLFDLSFGTTFFDPFNQG CtaDII-7120 GFFRMPLFVWAVFSAQI I QLFGLPALTAGAVMLLLDI TVGTSFFDPSKGG G. FKMP. FVWTVLSAQII QLFGLPALTAGAVMLLFDLTVGTTFFDPS. GG

260 270 280 290 300 CtaDII-7002 SPVLYQHFFWFYSHPAVYVMVLPVFGFFSELFPVYARKPLFGYKVVVVSS CtaDII-6803 NPI I YQHLFWFYSHPAVYVMALPAFGVFSEI LPVFARKPLFGYKTVAI SS CtaDII-7120 NPVMFQHYFWFYSHPAVYVI I LPI FGI FSEI FPVYSRKPLFGYKVVAI SS NPV. YQH. FWFYSHPAVYVM.LP. FG. FSEI FPVYARKPLFGYKVVAI SS

310 320 330 340 350 CtaDII-7002 MII VVLSAVVWVHHMFASGTPPWMRMLFMFSTMLI SVPTGI KVFAWVATL CtaDII-6803 FLIAIQGTFVWVHHMFTSATPNWMRMFFMASTMLIAVPTGIKVLAWTATV CtaDII-7120 MLI AVVSAI VWVHHLYVSGTPAWMRMFFMLTTMLVSVPTGI KVFAWVATI MLI AV. SA. VWVHHMF. SGTP WMRMFFM. STMLI SVPTGI KVFAWVAT.

360 370 380 390 400 CtaDII-7002 WGGKL RL DT PMLF AMGGLI NF VF AGI T GI MLAS VPVDI HVNNT YF VVGHF CtaDII-6803 WRGSLRLKTPMLFCLGGILMFLFAGITGIMLASAPFDLHVNNTYFVVGHF CtaDII-7120 WGGKI RLNT PMLFALGGLI LFVFAGI VGI MLSSVPVDVHVNNT YFVVGHF WGGKLRL T PMLFALGGLI FVFAGI T GI MLASVPVD. HVNNT YFVVGHF

410 420 430 440 450 CtaDII-7002 HYVI YGAI VFGI YGAVYHWFPKMTGKMYYEGLGKLHFWLTMIGTT LNFLP CtaDII-6803 HYVVFGT VT MAIYGAI YFWFPKMTGRMYNEAWGKLHFALTFI GANLNFFP CtaDII-7120 HYVLFGT VT MGMYAAI YHWFPKMTGRMYYEGWGKLHFWLTFI GTNLNFFP HYV. FGT VT MGIYGAI YHWFPKMTGRMYYEGWGKLHFWLTFI GTNLNFFP

460 470 480 490 500 CtaDII-7002 MH P V G L MG MP R R V A S Y D P E F A F WN V I A S I G G F I L G MS T I P F L L N MI A S WI CtaDII-6803 MHPI GLQGMLRRI SSYDPEYT AWNVVASLGAFLLGMSTLPFI ANMVASAF CtaDII-7120 MHPLGLQGMLRRVSSYAPEYEGWNIVASLGAFLLGMSTLPFI FNMVVSWM MHP. GLQGMLRRVSSYDPEY . WNVVASLGAFLLGMSTLPFI . NMVASW

510 520 530 540 550 CtaDII-7002 NGDRAPANPWRAIGLEWLVPSPPEHENFEELPIVIAEPYGYGKSEPLTEN CtaDII-6803 QGRRVGNNPWNSLGLEWTTPSPPPEENFEVI PTI TVEPYGYDRPVDLTTE CtaDII-7120 HGEKAPDNPWRAI GLEWLVASPPPVENFEEI PVVI SEPYGYGKSEPLTAE .G.RAP NPWRAIGLEWLVPSPPP ENFEEIP.VI.EPYGYGKSEPLT E

560 570 580 590 600 CtaDII-7002 LGG CtaDII-6803 VTT CtaDII-7120 . 405

Figure B50. ClustalW alignment of CtaEII amino acid sequences. CtaEII amino acid sequences of the following organisms were aligned using the ClustalW alignment program from MacVector v. 6.5: Synechococcus sp. PCC 7002, Synechocystis sp. PCC

CtaEII ClustalW Amino Acid Alignment

10 20 30 40 50 CtaEII-7002 MTAI NETPI SATSHGHGEEDHRLFGFI VFLLSESVI FI SFFVGYI VYKLS CtaEII-6803 MESGNHLPHVEPTEEQ- EPDNLGFGFPVFLMSESVVFI SFFVTYTI LRLT CtaEII-7120 GHEI SHEHGHDEEGNKMFGFI VFL LSESVI FL SFFAGYI VYKTT M . N . P . . HGH EEDN. . FGFI VFL LSESVI FI SFFVGYI VYKL T

60 70 80 90 100 CtaEII-7002 PTDWLPPGVEGLEI HDPAI NTVVLVSSSGVI YL AERFL HKENLWGFRFFW CtaEII-6803 NKPWFPPGVDGLDVTRGAI NTMVLVTSSGAI I L AEKAL HRGEMKLFRLL W CtaEII-7120 TPNWLPVGVEGLEVRDPAI NTVVLVASSFVI YFAELAL KRQNLRLFRI FL WLPPGVEGLEV. DPAI NTVVLV. SSGVI YL AE. AL HR NL. LFR. FW

110 120 130 140 150 CtaEII-7002 LLTMAMGSYFLYGQAVEWQSLEFEFTSGVYGGI FYLLTGFHGLHVLTGVL CtaEII-6803 LATI SL GI VFL FGQAAEWAGMPFGLDAGSAGGTFFLLTGFHGLHVFTGVC CtaEII-7120 SATMAMGSYFLVGQAI EWSHLEFGFTSGVYGGMFYLLTGFHGLHVFTGI L LATMAMGSYFL . GQA. EW LEFGFTSGVYGG FYLLTGFHGLHVFTGVL

160 170 180 190 200 CtaEII-7002 LQGVMLGRSFL PNNYAGGQYGVEATSWFWHFVDVI WI I LFGLI YLWQ CtaEII-6803 LLL YMYWRSLQPHNFDRGHEGVTAI AL FWHFVDVI WI I LFI LLYLWPAN CtaEII-7120 LQFI I L VRSLI PGNYDTGHFGVNATSL FWHFVDV LQ. . ML. RSL. P NYD GH. GV ATSL FWHFVDVI WI I LF. L. YLW CHRISTOPHER T. NOMURA

EDUCATION 1988-1989 Laney Junior College, Oakland 1989-1994 B. A., Biology with Honors, University of California, Santa Cruz 1994-2001 Ph.D., Biochemistry and Molecular Biology, The Pennsylvania State University

HONORS/PROFESSIONAL TRAINING

1994-1996 NIH Biotechnology Predoctoral Fellow, Dept. of Biochemistry and Molecular Biology, The Pennsylvania State University

1994-1996 Graham Endowed Graduate Fellowship, Dept. of Biochemistry and Molecular Biology, The Pennsylvania State University

1995 Teaching Assistant, Dept. of Biochemistry and Molecular Biology, The Pennsylvania State University

1995 NSF Predoctoral Fellow, Honorable Mention

1994 MIRT Fellow, Laboratorio de Mamiferos Marinos (Dr. Claudino Campagna) and University of California-Santa Cruz (Dr. C. Leo Ortiz)

1993 MARC Undergraduate Fellow, University of California-Santa Cruz, (Dr. C. Leo Ortiz)

1992-1993 MBRS Fellow, University of California-Santa Cruz, (Dr. Barry Bowman)

PUBLICATIONS

1. Nomura, C. T. and D. A. Bryant. 1997. Characterization of cytochrome c6 from Synechococcus sp. PCC 7002, p. 269-274. In G.P. Peschek, W. Löffelhardt, and G. Schmetterer (ed.), The Phototrophic Prokaryotes. Kluwer Academic/Plenum Publishers, New York, New York. 2. Huckauf, J., Nomura, C. T., Forchhammer, K., and M. Hagemann. 2000. Stress responses of Synechocystis sp. strain PCC 6803 mutants impaired in genes encoding putative alternative sigma factors. Manuscript submitted to Microbiology. 3. Nomura, C. T., Persson, S., Inoue-Sakamoto, K., Sakamoto, T., Shen, G., and D. A. Bryant. 2001. A role for cytochrome oxidase in high-light and oxidative stress response in the cyanobacterium Synechococcus sp. PCC 7002. Manuscript in preparation. 4. Nomura, C. T., Persson, S., and D. A. Bryant. 2001. Cloning and sequence analysis of electron transport protein genes from Synechococcus sp. PCC 7002: A comparative study of electron transport proteins from cyanobacteria and chloroplasts. Manuscript in preparation.