The Pennsylvania State University
The Graduate School
Eberly College of Science
CYANOBACTERIAL QUINOMICS
STUDIES OF QUINONES IN CYANOBACTERIA
A Thesis in
Biochemistry, Microbiology, and Molecular Biology
by
Yumiko Sakuragi
Submitted in Partial Fulfillment of the Requirements for the Degree of
Doctor of Philosophy
August 2004
The thesis of Yumiko Sakuragi has been reviewed and approved* by the following:
Date of Signature
Donald A. Bryant Ernest C. Pollard Professor in Biotechnology Professor of Biochemistry and Molecular Biology Thesis Advisor Co-Chair of Committee
John H. Golbeck Professor of Biochemistry and Biophysics Professor of Chemistry Co-Chair of Committee
J. Martin Bollinger Jr. Associate Professor of Biochemistry and Molecular Biology
A. Daniel Jones Senior Scientist
B. Tracy Nixon Associate Professor of Biochemistry and Molecular Biology Special Signatory
Robert A. Schlegel Professor of Biochemistry and Molecular Biology Head of the Department of Biochemistry and Molecular Biology
*Signatures are on file in the Graduate School. iii ABSTRACT
Roles and functions of isoprenoid quinones (phylloquinone, plastoquinone-9) and
α-tocopherol were investigated in cyanobacteria. Comparative genome analyses of 14
cyanobacteria suggested that phylloquinone (PhyQ) biosynthesis in most but not all
cyanobacteria occurs similarly to menaquinone biosynthesis in Escherichia coli. This was
further supported by the discovery that two cyanobacteria, Synechococcus sp. PCC 7002
and Gloeobacter violaceus PCC 7421, synthesize menaquinone-4 (MQ-4). Targeted
inactivation of the menB, menF, and menG genes resulted in the incorporation of plastoquinone-9 (PQ-9) and demethyl-MQ or demethyl-PhyQ into Photosystem I (PS I)
complexes. In the PS I complexes containing demethyl-PhyQ, the rate of electron transfer
from A1 to the iron-sulfur clusters slowed by a factor of two, while the kinetics of the
+ - P700 [FA/FB] backreaction increased by a factor of 3 to 4. These results were explained
- - by a lowering of the equilibrium constant between Q /Q and FX /FX in the demethyl-PhyQ
containing PS I complexes by a factor of ~10.
Populations of α-tocopherol mutants of the cyanobacterium Synechocystis sp.
PCC 6803, previously isolated in the presence of glucose, were found to be
phenotypically and genotypically heterogeneous. Newly isolated, “authentic” tocopherol
mutants were unable to grow in the presence of glucose at pH 7.0; this was suggested to
be due to a significant reduction of the amounts of sigA and rbcL transcripts in cells
under these conditions. The slr2031 product, which has been previously shown to be
involved in sulfur, nitrogen, and carbon metabolism, and genes encoding inorganic
carbon uptake mechanisms, were found to be constitutively down-regulated in the iv “authentic” tocopherol mutants. The results indicate that α-tocopherol is involved in the
transcriptional regulation of these metabolic genes and plays an important role in the
coordination of nitrogen, sulfur, and carbon metabolism in Synechocystis sp. PCC 6803.
The PQ-9 biosynthesis pathway was predicted to be similar to that for ubiquinone biosynthesis based on comparative genome analyses of 14 cyanobacteria. However, targeted inactivation mutagenesis of eight genes encoding putative methyltransferase genes similar to UbiE/MenG in E. coli did not affect PQ-9 biosynthesis in Synechocystis sp. PCC 6803. Based on the results obtained, a possible PQ-9 biosynthesis pathway is proposed. v TABLE OF CONTENTS
Page List of Figures vii List of Tables xiii Acknowledgements xv
Chapter 1 General Introduction 1 Cyanobacterial oxygenic photosynthesis 3 Phylloquinone and Photosystem I 6 Plastoquinone and Photosystem II 9 Role of α-tocopherol 12 Cyanobacterial quinomics 13 References 16
Chapter 2 Comparative genome analysis and identification of menaquinone-4 32 biosynthetic pathways in cyanobacteria Abstract 33 Introduction 35 Results 38 Discussion 45 Summary 49 Materials and Methods 50 References 54
Chapter 3 Recruitment of a foreign quinone into the A1 site of Photosystem I. 72 Interruption of menG in the phylloquinone biosynthetic pathway of Synechocystis sp. PCC 6803 results in the incorporation of 2-phytyl-1,4-naphthoquinone and alteration of the equilibrium constant for electron transfer between A1 and FX. Abstract 73 Introduction 76 Results 80 Discussion 92 Summary 100 Materials and Methods 101 References 108
vi
Page Chapter 4 Physiological characterization of tocopherol biosynthesis mutants in 129 the cyanobacterium Synechocystis sp. PCC 6803: demonstration of a conditionally lethal phenotype in the presence of glucose at pH 7.0 Abstract 130 Introduction 131 Results 137 Discussion 153 Summary 159 Materials and Methods 160 References 167
Chapter 5 Transcriptional regulation of the metabolic and sigma factor genes in 194 the vitamin E-deficient mutant of Synechocystis sp. PCC 6803 Abstract 195 Introduction 196 Results 201 Discussion 216 Summary 220 Materials and Methods 221 References 225
Chapter 6 Comparative genome analysis and genetic study of plastoquinone 242 biosynthetic pathway in cyanobacteria Abstract 243 Introduction 245 Results 248 Discussion 258 Summary 262 Materials and Methods 263 References 266
Appendix 291 vii LIST OF FIGURES
Page
Figure 1.1 Structure of phylloquinone, plastoquinone-9, and α-tocopherol 27
Figure 1.2 Scheme of oxygenic photosynthesis in cyanobacteria 28
Figure 1.3 The spatial arrangement and the redox potentials of the electron 29 transfer cofactors in Photosystem I
Figure 1.4 The electron transfer cofactors in Photosystem II 30
Figure 1.5 Biosynthetic pathway of PQ-9 and α-tocopherol in higher plants 31
Figure 2.1 Menaquinone biosynthetic pathway in E. coli 62
Figure 2.2 The men gene arrangements 63
Figure 2.3 Phylogenetic trees based on the amino acid sequences of MenA, 64
MenB, and MenD
Figure 2.4 HPLC profiles of solvent extracts from PS I complexes and whole 65 cells
Figure 2.5 Absorption spectra of solvent-extracted components analyzed by 66
HPLC
Figure 2.6 LC-mass spectrometric analysis of the solvent extracts from 67
Synechococcus sp. PCC 7002
Figure 2.7 Restriction maps of the menB, menF, and menG coding regions in 68
Synechococcus sp. PCC 7002
Figure 2.8 PCR analyses on genomic DNAs isolated from the menB rubA, menF, 69 and menG mutants of Synechococcus sp. PCC 7002 viii Figure 2.9 Reverse-phase HPLC analyses of the solvent extracts from the PS I 70 complexes
Figure 2.10 Absorption spectra of eluting components analyzed for PS I 71 complexes isolated from Synechococcus sp. PCC 7002
Figure 3.1 Relationship between the molecular structure of 2-phytyl-1,4-NQ and 117 the axes of the electronic g-tensor
Figure 3.2 Construction and verification of the menG mutant strain of 118
Synechocystis sp. PCC 6803
Figure 3.3 Mass spectra recorded for pigments and quinone extracts from the PS I 119
complexes isolated from the wild-type and menG mutant strains of Synechocystis
sp. PCC 6803
Figure 3.4 77 K fluorescence emission spectra from whole cells of Synechocystis 120
sp. PCC 6803
Figure 3.5 SDS-PAGE analysis of PS I complexes isolated from the wild-type 121
and menG mutant strains of Synechocystis sp. PCC 6803
Figure 3.6 Photoaccumulated Q-band EPR spectra and simulations of PS I 122
complexes from Synechocystis sp. PCC 6803
Figure 3.7 Transient spin-polarized EPR spectra of the charge separated P700+ Q- 123
state in PS I complexes.
Figure 3.8 Simulations of the Q-band spectrum of the menG mutant carried out 124
for various angles between the largest principal A11 and the gxx axis.
Figure 3.9 Spin-polarized EPR transient of wild type and menG mutant 125 ix Figure 3.10 Decay-associated transient EPR spectra at ambient temperature 126
Figure 3.11 Flash-induced absorbance changes at 812 nm in PS I complexes 127 isolated from Synechocystis sp. PCC 6803
Figure 3.12 Steady-state rates of flavodoxin reduction in PS I complexes isolated 128 from the wild-type and menG mutant strains.
Figure 4.1 Biosynthetic pathway of α-tocopherol in Synechocystis sp. PCC 6803 176
Figure 4.2 Colony morphologies of three tocopherol mutants previously isolated 177
in the presence of glucose.
Figure 4.3 Growth curves of wild type and cell lines derived from small and large 178
colonies of tocopherol mutants selected in the presence of glucose
Figure 4.4 PCR analysis of the genomic DNA extracted from the newly isolated 179
tocopherol mutants selected in the presence of glucose
Figure 4.5 Growth curves of wild type and the “authentic” tocopherol mutants in 180
the presence and in the absence of glucose
Figure 4.6 Growth curves of wild type and the “authentic” slr1736 mutant in the 181
presence of glucose
Figure 4.7 Growth analyses of wild type and the “authentic” slr1736 mutant of 182
Synechocystis sp. PCC 6803 in the presence of various carbon sources
Figure 4.8 Chlorophyll a and quinone contents in wild type and the “authentic” 183
tocopherol mutants grown in the absence and in the presence of glucose
Figure 4.9 Oxygen-evolution activities of the whole-chain and the Photosystem 184
II-dependent electron transport chains in wild type and the “authentic” tocopherol x mutants of Synechocystis sp. PCC 6803
Figure 4.10 Immunoblotting analysis for the D1 and PsbO proteins in whole cells 185 of wild type and three “authentic” tocopherol mutants
Figure 4.11 Time-dependent RT-PCR analysis of the sodB and nblA transcripts in 186 wild type and the “authentic” slr1736 mutant of Synechocystis sp. PCC 6803
Figure 4.12 Relative phycobiliprotein contents in wild type and the “authentic” 187 tocopherol mutants of Synechocystis sp. PCC 6803
Figure 4.13 Growth curves of wild type and the “authentic” slr1736 mutant the 188 presence of glucose at 3% (v/v) CO2 and air
Figure 4.14 Cultures of wild type and the “authentic” tocopherol mutants of 189
Synechocystis sp. PCC 6803 grown at various pH values
Figure 4.15 Growth curves of wild type and the “authentic” slr1736 mutant of 190
Synechocystis sp. PCC 6803 at various pH values in the presence of glucose in air
and 1% (v/v) CO2
Figure 4.16 Cultures of wild type and the “authentic” slr1736 mutant of 191
Synechocystis sp. PCC 6803 grown under various conditions for 48 h at pH 7.0
Figure 4.17 PS II-dependent O2-evolution activities of wild type and the 192
“authentic” slr1736 mutant in the absence and in the presence of glucose
Figure 4.18 Phycobiliprotein contents in wild type and the tocopherol mutants of 193
Synechocystis sp. PCC 6803 grown at 1% (v/v) CO2.
Figure 5.1 Time-dependent RT-PCR analysis on the cpcA, nblA, and sbtA 233
transcripts in wild type and slr1736 mutant grown at pH 7.0 and 8.0 xi Figure 5.2 Time-dependent RT-PCR analysis of the alternative sigma factor 234 transcripts in wild-type and slr1736 mutant grown at pH 7.0 and 8.0
Figure 5.3 Time-dependent RT-PCR analysis of the sigA, sigG, and Ci genes in 235
wild-type and slr1736 mutant grown at pH 7.0 and 8.0
Figure 5.4 Thin-section micrographs of the wild-type cells grown in the absence 236 and in the presence of glucose for 24 h
Figure 5.5 Thin-section micrographs of the slr1736 mutant grown in the absence 237 and in the presence of glucose for 24 h
Figure 5.6 Time-dependent RT-PCR analysis of the slr2031 and CCM gene 238 transcripts in wild type and slr1736 mutant grown at pH 7.0 and 8.0
Figure 5.7 PCR analysis of the genomic DNAs extracted from the slr2031::aadA 239 and pmgA::aacC1 mutants
Figure 5.8 Cultures of wild type, the slr1736, slr2031, and pmgA mutants of 240
Synechocystis sp. PCC 6803 in the presence of glucose at pH 7.0 and 8.0
Figure 5.9 Summary and a model of the non-antioxidant role of α-tocopherol in 241
Synechocystis sp. PCC 6803
Figure 6.1 UQ biosynthesis pathway in Escherichia coli 280
Figure 6.2 Hypothetical PQ-9 biosynthesis pathway in cyanobacteria 281
Figure 6.3 An alignment of the Ycf21/UbiC homologs in cyanobacteria and 282 eukaryotic phototrophs
Figure 6.4 Phylogenetic analyses based on nucleotide sequences of 16S rRNA 283 and amino acid sequences of Ubi proteins in cyanobacteria and proteobacteria xii Figure 6.5 Phylogenetic trees based on amino acid sequences of UbiE homologs 285 in cyanobacteria
Figure 6.6 HPLC analysis of the sll1653 mutant of Synechocystis sp. PCC 6803 286 and the 2-phytyl-1,4-benzoquinone (MPBQ) standard.
Figure 6.7 PQ-content in whole cells of the wild-type and methyltransferase 287 mutant strains of Synechocystis sp. PCC 6803
Figure 6.8 Restriction maps of coding regions and their flanking sequences for the 288
UbiE homologs in Synechocystis sp. PCC 6803
Figure 6.9 PCR analyses of genomic DNA isolated from the single, double, and 289 triple methyltransferase (UbiE homolog) mutants of Synechocystis sp. PCC 6803
Figure 6.10 Aromatic amino acid biosynthesis and an alternative proposal for 4- 290 hydroxybenzoate synthesis in cyanobacteria
xiii LIST OF TABLES
Page
Table 2.1 Whole genome analyses for the MQ/PhyQ biosynthesis enzymes in 14
cyanobacteria, Escherichia coli, Bacillus subtilis, Chlorobium tepidum and 60
Halobacterium sp. NRC
Table 3.1 Doubling times of Synechocystis sp. PCC 6803 wild type and the 114 menG mutant strains
Table 3.2 Chlorophyll content in cells grown photoautotrophically at 32 °C 115 under various illumination conditions.
- Table 3.3 Magnetic parameters of PhyQ (A1 ) and 2-phytyl-1,4-NQ in the A1 116 binding site
Table 6.1 Amino acid sequence similarities between UbiA homologs in 14 272 cyanobacteria and E. coli
Table 6.2 Amino acid sequence similarities between UbiH homologs in 14 273 cyanobacteria and E. coli
Table 6.3 Amino acid sequence similarities between UbiD homologs in 14 274 cyanobacteria and E. coli
Table 6.4 Amino acid sequence similarities between UbiX homologs in 14 275 cyanobacteria and E. coli
Table 6.5 List of the UbiE homologs in 14 cyanobacteria 276
Table 6.6 Ycf21/UbiC homologs in cyanobacteria and eukaryotic phototrophs 277
Table 6.7 Sequences of primers used for PCR analyses 278 xiv
Table 6.8 Construction of methyltransferase mutants in Synechocystis sp. PCC 279
6803
xv ACKNOWLEDGEMENTS
I would like to thank my advisor Dr. Donald A. Bryant and my co-advisor Dr.
John H. Golbeck for their guidance and support. I would also like to acknowledge my
collaborators who have contributed to the work on phylloquinone: Dr. Gaozhong Shen,
Dr. Boris Zybailov, Dr. Art van der Est, Dr. Robert Bittl, Dr. Stephan Zech, and Dr.
Dietmar Stehlik; and my collaborators who have contributed to the work on plastoquinone and α-tocopherol: Dr. Dean DellaPenna, Hiroshi Maeda, and Dr. Zigang
Cheng.
My special thanks are also due to the current and former members of both the
Bryant and Golbeck laboratories. Lastly, I would like to thank my husband, Dr. Niels-
Ulrik Frigaard, for his support and advice throughout my studies. 1 Chapter 1
General Introduction
Cyanobacteria are a phylogenetically coherent group of organisms, yet morphologically, physiologically, and genetically they are very diverse (Bryant, 1994).
They are found in a variety of terrestrial and aqueous environments such as open oceans, coastal waters, fresh water lakes, rivers, municipal reservoirs, ponds, and even kitchen sinks in domestic households. Some perform N2 fixation, and some are capable of chemotaxis or phototaxis or both. While the majority are obligate photoautotrophs, a few are able to grow chemoheterotrophically. Genome sizes vary from 2 Mbp to ~15 Mbp.
Despite these differences, there is one thing that is common among all cyanobacteria: the unique ability to perform oxygenic photosynthesis. All cyanobacteria perform oxygenic photosynthesis, which uses CO2 as the source of carbon, water as the source electrons, and light as the source of energy, in order to produce biomass. As a result, molecular oxygen is produced as a byproduct (Barber and Anderson, 2002). Through the activity of oxygenic photosynthesis, cyanobacteria have affected, and are continuing to affect, our environment since their first appearance more than 3 billion years ago. This activity of cyanobacteria has transformed the atmosphere of our planet from an extremely reducing to a rather oxidizing environment, and this change has supported the evolution of diverse forms of life on Earth. In an era of global warming, these organisms attract more attention given that about half of the global CO2 fixation known to occur on Earth is carried out by cyanobacteria (Bryant, 2003). 2 Much has been learned about the molecular mechanisms of oxygenic
photosynthesis owing to decades of study, which have dissected and largely solved the
mystery of water oxidation and the detailed functions of the photosynthetic apparatus
primarily from the viewpoint of protein functions. Such a perspective, however, has
somewhat overlooked the biological importance of non-proteineous molecules that are
also involved in the process. A group of such molecules are the quinones. In
cyanobacteria these are phylloquinone (PhyQ, 2-methyl-3-phytyl-1,4-naphthoquinone,
also known as vitamin K1), plastoquinone (PQ-9, 2,3-dimethyl-5-solanyl-1,4-
benzoquinone). These two compounds are isoprenoid quinones, which are composed of a
quinonoid nucleus with a polyprenyl substituent (Fig 1.1). There are two types of quinone
substructures: 1,4-naphthoquinone (1,4-NQ), and 1,4-benzoquinone (1,4-BQ). PhyQ is a
methyl- and phytyl-substituted 1,4-NQ, while PQ-9 is a methyl- and solanyl-substituted
1,4-BQ. The phytyl substituent is a partially saturated tetra-isoprene unit (equivalent to
20 carbon atoms), while the solanyl substituent is a nona-isoprene unit (equivalent to 45
carbon atoms). α-Tocopherol (2,5,7,8-tetramethyl-6-chromanol, also known vitamin E)
is a tetramethyl-substituted chromanol, another type of quinone nucleus that is derived
from a phytyl-substituted 1,4-BQ after ring closure (Fig 1.1).
These quinones are synthesized only in oxygenic phototrophs including
cyanobacteria, algae and higher plants (Collins and Jones, 1981; Threlfall and Whistance,
1971), and their functions are tightly connected to oxygenic photosynthesis. PhyQ and
PQ-9 are bound cofactors of the Photosystem I (PS I) and Photosystem II (PS II) complexes, respectively, and in this context they mediate electron transfer as one-electron carriers (see below). PQ-9 also serves as a membrane-associated two-electron and two- 3 proton carrier, both in the photosynthetic electron transport and the respiratory electron
transport chains. α-Tocopherol, on the other hand, is thought to provide protection
against oxidative stress in animals and plants, although its role(s) in cyanobacteria has not
yet been demonstrated (see below).
Cyanobacterial oxygenic photosynthesis
Fig 1.2 illustrates the process of oxygenic photosynthesis in cyanobacteria. In
general, photosynthesis is described in the context of two processes known as the light
reactions and dark reactions. The light reactions are the processes in which the energy of
photons is transformed into an electrochemical membrane potential that drives ATP
synthesis and into a strong reducing power that ultimately generates NADPH. The dark
reactions are the processes in which CO2 is reduced or “fixed” into biomass at the
expense of NADPH and ATP as the reducing power and energy source, respectively.
The light reactions are catalyzed by the photosynthetic electron transport chain
that resides in the thylakoid membranes (Bryant, 1994; Hervás et al., 2003). This electron
transport chain involves 3 membrane integral protein complexes (PS I, PS II, cytochrome
b6f complex) and 2 water-soluble electron carriers (cytochrome c6 or plastocyanin, and
ferredoxin or flavodoxin), as well as the lipid-soluble electron carrier PQ-9 (Fig 1.2). X-
ray crystallographic structures of PS I, PS II, and cytochrome b6f complexes are now
available at resolutions of 2.5 Å (Jordan et al., 2001), 3.5 Å (Ferreira et al., 2004), and 3.0
Å (Kurisu et al., 2001), respectively. Upon illumination, photons absorbed by the
peripheral antenna complexes known as phycobilisomes are transferred predominantly to
the PS II complexes, in which they trigger photochemistry that drives the light-dependent 4 oxidation of water and the reduction of plastoquinone (Barry et al., 1994; Bricker and
Ghanotakis, 1996; Britt, 1996; Diner and Babcock, 1996). Water oxidation occurs in the
thylakoid lumen and results in the release of molecular oxygen and protons (2H2O Æ O2
+ 4H+ + 4e-), while the reduction and protonation of PQ-9 occurs on the stromal side of
the membrane and releases doubly reduced plastoquinone, or plastoquinol (PQH2), into
+ - the membrane matrix (2 PQ-9 + 4 H + 2 e Æ 2 PQH2). PQH2 shuttles electrons to the
quinol oxidation site (o) of the cytochrome b6 f complex in a diffusion-dependent fashion,
where it is reoxidized and becomes available for a new round of photochemical reduction
by the PS II complexes. At this step, the electron transfer bifurcates into two separate
pathways within the cytochrome b6f complex; one electron is transferred to the Reiske
iron-sulfur protein and subsequently to a soluble electron carrier such as cytochrome c6 or plastocyanin in the lumen with concomitant release of two protons into the lumen, while the other electron is transferred to heme bL and heme bH and reduces PQ-9 at the quinone
reduction site (r) near the stromal surface of the thylakoid membranes (Kallas, 1994;
Hauska et al., 1996). After two rounds of PQH2 oxidation, one PQ-9 at the quinone
reduction site becomes doubly reduced and is protonated by concomitant uptake of two
protons from the stroma (cytoplasm). The resulting PQH2 is released into the membrane
matrix where it joins the pool of PQH2 and participates in electron donation to the
+ + cytochrome b6f complex (2PQH2 (O) + PQ (r) + 2H Lumen Æ 2PQ(O) + PQH2(r) + 4H Stroma
+ 2e-). This process is known as the Q cycle, and the result is an amplification of the
proton gradient across the membranes, which is coupled to ATP synthesis by ATP synthase (Michell, 1976). The final steps of the electron transport chain involve the PS I complex. Essentially, this light-dependent cytochrome c6/plastocyanin- 5
ferredoxin/flavodoxin oxidoreductase shuttles an electron from cytochrome c6 or
plastocyanin in the lumen to ferredoxin or flavodoxin in the stroma (Golbeck, 1994;
Brettel and Leibl, 2001). The resulting strong reductant donates an electron to ferredoxin-
NADPH+ oxidoreductase, which catalyzes the reduction of NADP+ to NADPH (Morand et al., 1994).
It is noteworthy that the cyanobacterial photosynthetic electron transport chain shares the membrane-associated PQ-9 pool and cytochrome b6f complexes with the
respiratory electron transport chain (Fig 1.2). In this case, electrons enter the electron
transport chain through NADH dehydrogenase in the form of NAD(P)H that is generated
through glycolysis and the TCA cycle, and the oxidative pentose phosphate pathway. The
terminal electron acceptor is molecular oxygen, which is reduced and protonated by
cytochrome c oxidase resulting in generation of water (Schmetterer, 1994).
NADPH and ATP generated through the photosynthetic electron transport chain
are used to fuel the dark reactions. A CO2 molecule is first incorporated into the C5
backbone of ribulose-1,5-bisphosphate, which is then split into two molecules of 3-
phosphoglycerate (Tabita, 1987, 1994). This reaction is catalyzed by ribulose-1,5-
bisphosphate carboxylase/oxygenase (RuBisCO), which is probably the most abundant
protein on Earth (Garrett and Grisham, 1999). In cyanobacteria, RuBisCO is housed
within protein-encapsulated structures known as carboxysomes (Badger and Price, 2003),
which play a role in locally increasing the CO2 concentration. 3-Phosphoglycerate is
reduced to 3-phosphoglyceraldehyde, which is processed through the reductive pentose
phosphate pathway to regenerate ribulose-1,5-bisphosphate. The processes are known as
the Calvin cycle and cause the net production of organic molecules that constitute the 6 metabolic precursors for the biosynthesis of proteins, nucleic acids, sugars, pigments, and other metabolites. The net production of biomass in the Calvin cycle can be expressed as the following equation:
+ 6 CO2 + 12 NADPH + 18 ATP Æ C6H12O6 + 12 NADP + 18 ADP + 18 Pi
PhyQ and Photosystem I
The PS I complex consists of twelve protein subunits, 96 Chl a molecules, 22 β- carotene molecules, two PhyQ molecules, three iron-sulfur clusters, and some structural lipids and water molecules (Jordan et al., 2001). Tightly bound electron transfer cofactors are found within the core of the complex formed by PsaA and PsaB proteins. These included a chlorophyll a (Chl a) dimer (P700), two Chl a monomers (A0), two PhyQ molecules (A1), and three [4Fe-4S] clusters (denoted as FX, FA, FB). These cofactors are arranged in two branches related by C2 symmetry. The spatial arrangement of these cofactors and their estimated in situ redox potentials are shown in Fig. 1.3. Upon photoexcitation, the primary donor P700 reduces the primary acceptor, A0, resulting in
+ - the initial charge separated state [P700 A0 ]. Subsequent electron transfer to the
+ - secondary acceptor A1 stabilizes the charge-separated state [P700 A0 A1 ] and minimizes
+ - the rapid charge recombination that occurs between P700 and A0 . The electron is
+ - subsequently transferred to FX [P700 A0 A1 FX ], and then to the terminal acceptors FA
+ - + - [P700 A0 A1 FX FA ] and FB [P700 A0 A1 FX FA FB ], which are located in the peripheral
PsaC subunit. The reduction of ferredoxin or flavodoxin takes place upon direct protein- protein interaction between these acceptors and the stromal proteins: PsaC, PsaD, and
PsaE (Zhao et al., 1991, 1993; Li et al., 1991; Golbeck, 1994). On the lumenal side, the 7 + photooxidized P700 accepts an electron from reduced cytochrome c6 or plastocyanin
(Durán et al., 2004).
Since the first discovery of PhyQ in oxygenic phototrophs by Dam in 1941 (cited
in Hauska, 1988), the role of PhyQ in PS I as the secondary acceptor A1 was obscure for
more than two decades. After its initial discovery by Thornber and coworkers in the
1970’s, in the 1980s it was found to be exclusively associated with PS I (Thornber et al.,
1976; Takahashi et al., 1985; Shoeder and Locau, 1986) and to participate in electron
transfer as shown by spin-polarized EPR analysis (Petersen et al., 1987) and optical
kinetic analysis (Brettel et al., 1987; see also reviews by Golbeck, 1994, 2003 and
references therein). Further supporting evidence was derived from solvent extraction
studies, in which extraction of PhyQ from PS I with hexane or ether resulted in a rapid,
+ - nano-second-scale charge recombination between P700 and A0 . Stable charge
separation was restored by the addition of exogenous PhyQ (Biggins and Mathis, 1988;
Itoh and Iwaki, 1989).
This in vitro reconstitution method was subsequently used to study the structural
and thermodynamic requirements for A1 by introducing diverse quinones of abiotic origin
into the A1 site, including benzoquinones, naphthoquinones, and anthraquinones with
various ring substitutions (Iwaki and Itoh, 1989, 1991; Itoh and Iwaki, 1991; see also
review by Itoh 2001). These authors concluded that the successful restoration of stable charge separation occurs only when quinones with redox potentials between those of A0
and FX are used. They additionally found that neither the structure of the head group nor
the presence of an alkyl substituent is important. This conclusion contradicted earlier
studies, in which the presence of an alkyl substituent was shown to be essential when 8 hexane/hexane-methanol-extracted PS I complexes were studied (Biggins and Mathis,
1998). These contradictory interpretations imply that different solvent-extraction methods affect the architecture of PS I differently; hence, the inconsistency in conclusions may reflect artifacts of the methods employed. Such artifacts can be avoided if “biologically” or “physiologically” modified materials are used. Materials that have not gone through harsh extraction processes, which result in co-extraction of other important PS I constituents such as water, Chl a, carotenoid, and lipid molecules (Biggins and Mathis,
1988; Itoh and Iwaki, 1989), should produce more consistent and interpretable results.
Some of these molecules are located in the vicinity of the quinone-binding site; in particular, as revealed by 2.5Å-resolution crystallographic structure (Jordan et al., 2002),
β-carotenes and chlorophylls form hydrophobic contacts with the phytyl side chain of
PhyQ and seem to provide structural stability for this important electron carrier.
Genetic manipulation of the PhyQ biosynthetic pathway was first introduced by
Chitnis and colleagues (Johnson et al., 2000) and provided an alternative and very powerful experimental system, by which the selective elimination of PhyQ from PS I could be achieved in vivo. Based on the menaquinone biosynthetic pathway in
Escherichia coli (Meganathan, 2001), a part of the PhyQ biosynthetic pathway in
Synechocystis sp. PCC 6803 was first predicted. Targeted insertional mutagenesis of genes encoding dihydroxynaphthoate phytyltransferase (MenA) and dihydroxynaphthoate-CoA synthase (MenB) successfully interrupted the PhyQ biosynthesis in the mutant strains (Johnson et al., 2000). The PS I complexes isolated from these mutants completely lacked PhyQ and accumulated PQ-9 (Johnson et al., 2000;
Zybailov et al., 2000; Semenov et al., 2000). As confirmed by continuous-wave EPR, 9 electron spin-polarized transient EPR, and electron spin-echo modulation experiments
(Zybailov et al., 2000), the orientation of the carbonyl bonds relative to the membrane
normal and the distance between PQ-9 and P700+ were the same as with PhyQ in the
wild-type PS I complexes. In the PS I complexes isolated from the mutants, PQ-9 was
shown to participate in electron transfer from A0 to FX (Zybailov et al., 2000; Semenov et
al., 2000), although the rate of forward electron transfer from A1 to FX slowed at least by
a factor of 100. The estimated in situ redox potential of PQ-9 differs from that of PhyQ
by ca. 130 mV (Shinkarev et al., 2002), which is consistent with the faster rate of charge
recombination between P700+ and FeS- in PS I (Semenov et al., 2000). Despite this significant defect in the efficiency of forward electron transfer, the PS I complexes
containing PQ-9 supported nearly 85% of the wild-type activity of PS I, as measured by
the rate of cytochrome c6:flavodoxin oxidoreduction (Johnson et al., 2000). More recent
studies have shown that PS I complexes are functional when the A1 site is occupied by
PhyQ, PQ-9 and even anthraquinones with various substituents (Golbeck et al., 2001;
Zybailov, 2003). The combined results indicate that PS I has an innate capacity to accommodate and use a wide range of redox-active quinones with various structures and substituents.
PQ-9 and Photosystem II
Cyanobacterial Photosystem II is a light-driven, water:plastoquinone oxidoreductase and is composed of at least 19 protein subunits, seven carotenoid molecules, two pheophytin molecules, two PQ-9 molecules, two bicarbonate molecules, one none-heme Fe, one heme b, and one heme c (Zouni et al., 2001; Ferreira et al., 10 2004). Four manganese atoms and one calcium atom are bound to the oxygen-evolving complex that is attached to the lumenal surface of the PS II complex and is responsible for water oxidation. As shown in Fig 1.4, the electron transfer cofactors are arranged in two branches that are related by a pseudo-C2 symmetry axis within the core of the PS II complex that is formed by the D1 and D2 proteins. Aside from the differences in the specific types of the cofactors and their arrangement, the principle of the photochemistry is largely the same in PS II as in PS I. Upon photoexcitation of a Chl a monomer (PD1, the primary donor P680), an electron is transferred to a pheophytin molecule (PheoD1)
(Danielius et al., 1987). This initial charge separation [P680+ Pheo-] is further stabilized
+ - by electron transfer to a tightly bound PQ-9 molecule (QA) [P680 Pheo QA ], which is
+ - then followed by transfer to a second PQ-9 molecule (QB) [P680 Pheo QA QB ] (Renger,
1992; Barry et al., 1994). The second electron transfer doubly reduces QB, which, after protonation, dissociates from the complex into the thylakoid membrane matrix (Bouges-
Bocquet, 1973; Velthuys and Amesz, 1974; Govindjee and van Rensen, 1993). The
+ oxidized P680 is reduced by the redox-active tyrosine Z (TyrZ, Y161 in the D1 protein)
(Gerken et al., 1988; Diner and Babcock, 1996), which is then reduced by a tetra- manganese cluster that catalyzes the abstraction of electrons from water molecules
(Bricker and Ghanotakis, 1996). With the exception of QA, all redox-active cofactors that are involved in electron transfer are in principle bound by subunit D1 (PsbA) (Ferreira et al., 2004).
Prolonged exposure to high light intensity is known to cause irreversible damage to PS II, and this phenomenon is generally known as photoinhibition (see review by Aro et al., 1993, and references therein). Under such conditions, QA becomes doubly reduced 11 and leaves the binding pocket (Vass et al., 1992). This leads to rapid charge
recombination between P680+ and Pheo- and to formation of the triplet P680T. P680T can
1 readily react with molecular oxygen in its vicinity and generate singlet oxygen ( O2).
Singlet oxygen, and other reactive oxygen species generated from it, are responsible for the photodamage. Photoinhibition can also occur under normal light intensity, when the donation of electrons to P680 occurs slower than their removal by the acceptor side
(Chen et al., 1995; Aro et al, 1993; Anderson and Chow, 2002). In such cases, highly
+ + oxidizing Tyr Z and P680 cation radicals are formed, which can extract electrons from
the surrounding environment and also cause irreversible damage to PS II. In either case,
the D1 protein is rapidly degraded (Gong and Ohad, 1991; Philbrick et al., 1991; Aro et
al., 1993).
Genetic manipulation of the quinone species present in the PS II quinone-binding
sites has not been demonstrated. The PQ-9 biosynthesis pathway has been intensively studied, and the complete pathway is known in higher plants (Threlfall and Whistance,
1971). The synthesis occurs from the precursor homogentisate after two enzymatic steps involving homogentisate solanyltransferase and 2-methyl-6-solanyl-1,4-benzoquinone
(MSBQ) methyltransferase (Soll et al., 1980, 1985; Collakova and DellaPenna, 2001;
Cheng et al., 2003; also see review by Threlfall and Whistance, 1971) (Fig. 1.5). This pathway in plants overlaps with the α-tocopherol biosynthesis pathway, since both utilize homogentisate as a precursor, and both pathways share the step that introduces the second
methyl group, a reaction which is catalyzed by MPBQ/MSBQ methyltransferase (Cheng
et al. 2003) (Fig 1.5). Recent studies have shown that the cyanobacterial PQ-9
biosynthetic pathway involves neither homogentisate (Dänhardt et al., 2002) nor MPBQ 12 activity (Cheng et al., 2003). Therefore, PQ-9 seems to be synthesized by a completely different pathway in cyanobacteria.
Role of α-Tocopherol
α-Tocopherol was first discovered by Evans and Bishop (1922) as a factor
essential for reproduction in rats. Forty years later, its antioxidant activity was recognized
by Epstein and colleagues (Epstein et al., 1966). Because α-tocopherol is an essential
component of our diet, much work since then has been done on its significance in animal
systems. Studies in animals, animal cell cultures, and artificial membranes have shown
that tocopherols scavenge or quench various reactive oxygen species and lipid oxidation
by-products that would otherwise propagate lipid peroxidation chain reactions in
membranes (Kamal-Eldin and Appelqvist, 1996). Upon interaction with a prooxidant, α- tocopherol can undergo one-electron transfer and form a relatively stable tocopheroxyl cation radical, which is then recycled back to α-tocopherol by an antioxidant network consisting of ascorbate, glutathione, and NADPH/NADH (see review by Packer et al.,
2001). α-Tocopherol can also undergo two-electron transfer and form α- tocopherylquinone. In higher plants, this antioxidant function of α-tocopherol has been discussed in connection with protection against oxidative stress caused by various environmental factors (Munné-Bosch and Leonor, 2000). In the eukaryotic green algae
Chlamydomonas reinhardtii, the herbicide-mediated interruption of the α-tocopherol biosynthetic pathway rendered PS II more susceptible to oxidative stress induced by an 13 extreme high light illumination (Trebst et al., 2002). Under these conditions, the D1
protein was rapidly degraded.
In addition to these antioxidant functions, other “non-antioxidant” functions related to modulation of signaling and transcriptional regulation in mammals have also been reported (Chan et al., 2001; Azzi et al., 2002; Ricciarelli et al., 2002). For example,
α-tocopherol has been shown to bind to phospholipase A2 specifically at the substrate-
binding pocket and to act as a competitive inhibitor, which thereby decreased the release
of arachidonic acid for eicosanoid synthesis (Chandra et al., 2002). α-Tocopherol has
also been suggested to modulate the phosphorylation state of protein kinase Cα in rat smooth-muscle cells, possibly via phosphorylation of protein phosphatase 2A (Ricciarelli et al., 1998). α-Tocopherol is also directly involved in transcriptional regulation in animals, including the expression of the genes encoding liver collagen αI, α-tocopherol transfer protein, and α-tropomyosin collagenase (Yamaguchi et al., 2001; Azzi et al.,
2002). Whether α-tocopherol in cyanobacteria performs one or both of these roles has not yet been demonstrated.
Cyanobacterial Quinomics
The goal of this study was to develop experimental systems in which the type and amounts of the quinone species found in cyanobacteria could be manipulated in vivo.
Such systems would allow the functions of the complexes in which they occur to be
studied. This has been accomplished by using comparative genome analyses, combined
with a reverse genetic approach, to predict and verify the biosynthetic pathways of PhyQ, 14 α-tocopherol, and PQ-9 (Chapter 2, 3, 6). Consequences of altering the nature of these
quinone species are studied in the context of PS I function (Chapter 3), stress responses
(Chapter 4), and gene regulation (Chapter 5). To express the relatively wide scope of and
inclusiveness of these studies, I introduce the term “quinomics” to describe my investigations of the cyanobacterial “quinome”. 15 ABBREVIATIONS
BQ benzoquinone
Chl a chlorophyll a
DHNA 1,4-dihydroxy-2-naphthoate
MPBQ 2-methyl-6-phytyl-1,4-benzoquinone
MSBQ 2-methyl-6-solanyl-1,4-benzoquinone
NQ naphthoquinone
PhyQ phylloquinone
PQ-9 plastoquinone-9
PQH2 plastoquinol
PS I Photosystem I
PS II Photosystem II
RuBisCO ribulose-1,5-bisphosphate carboxylase/oxygenase
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Fig 1.1: Structures of phylloquinone (PhyQ), plastoquinone-9 (PQ-9), and
α-tocopherol. 28
Fig 1.2: Scheme for oxygenic photosynthesis in cyanobacteria.
ATPase, ATP synthase; COX, cytochrome c oxidase; Cyt b6f, the cytochrome b6 f + complex; Cyt c6, cytochrome c6; Fd, ferredoxin; Fl, flavodoxin; FNR, ferredoxin:NADP oxidoreductase; NDH, NADH dehydrogenase; PBS, phycobilisome; PC, plastocyanin; PS I, Photosystem I; PS II, Photosystem II. 29
Fig 1.3: The spatial arrangement and redox potentials of the electron transfer cofactors in Photosystem I.
The soluble electron donor cytochrome c6 (Cytc6) and electron acceptor ferredoxin (Fd) are also shown. 30
A
B
Fig 1.4: The electron transfer cofactors in Photosystem II.
(A) Illustration of electron transfer between the cofactors, (B) the spatial arrangement of the cofactors obtained from the 3.5-Å resolution X-ray crystal structure, adapted from Ferreira KN, Iverson TM, Maghlaoui K, Barber J, Iwata S (2004) Science 303: 1831- 1838.
31
Fig1.5: Biosynthetic pathway of PQ-9 and α-tocopherol in higher plants.
BQ, benzoquinone; HDDP, 4-hydroxyphenylpyruvate dioxygenase; HTP, homogentisate phytyltransferase; MSBQ/MPBQ MT, 2-methyl-6-solanyl-1,4-benzoquinone/2-methyl-6- phytyl-1,4-benzoquinone methyltransferase; SAM, S-adenosyl-L-methionine; TC, tocopherol cyclase; γ-TMT, γ-tocopherol methyltransferase.
32
Chapter 2
Comparative Genome Analysis and Identification of Menaquinone-4 Biosynthetic
Pathways in Cyanobacteria
Publications:
Yumiko Sakuragi, Boris Zybailov, Gaozhong Shen, Ramakrishnan Balasubramanian,
Bruce A. Diner, Irina Karygina, Yulia Pushkar, Dietmar Stehlik, Donald A. Bryant and
John H. Golbeck, Recruitment of a foreign quinone into the A1 site of Photosystem I.
Spectroscopic characterization of a menB rubA double deletion mutant in Synechococcus
sp. PCC 7002 containing plastoquinone-9 but devoid of FX, FB, FA, in preparation
Yumiko Sakuragi and Donald A. Bryant, a review article ‘Genetic Manipulation of the
Quinone Pathway in Photosystem I’ in Photosystem I: the Plastocyanin:Ferredoxin
Oxidoreductase in Photosynthesis’, Golbeck JH (ed), in preparation 33 ABSTRACT
Phylloquinone (PhyQ, 2-methyl-3-phytyl-1,4-naphthoquinone) is an electron transfer cofactor at the A1 site of Photosystem I and is synthesized only in oxygenic phototrophs such as cyanobacteria, algae, and higher plants. The biosynthetic pathway of PhyQ was investigated by means of comparative genomics using the menaquinone (MQ) biosynthesis genes of Escherichia coli as queries. The homologs of menA, menB, menC, menD, menE, menF, menG and menH were found to be conserved in most of the cyanobacteria. In some of these organisms, these genes formed clusters similar to those found in Escherichia coli, Bacillus subtilis, and Chlorobium tepidum, suggesting that the
PhyQ biosynthetic pathway has evolved from that for MQ. In support of this hypothesis it was demonstrated that Gloeobacter violaceus and Synechococcus sp. PCC 7002 wild- type strains synthesize MQ-4 (2-methyl-3-geranylgeranyl-1,4-naphthoquinone) that functions at the A1 site in Photosystem I complexes. Targeted insertional mutagenesis of menB, menF, and menG in Synechococcus sp. PCC 7002 results in the complete interruption of MQ-4 biosynthesis and demonstrates that the predicted pathway indeed functions in this cyanobacterium. Phylogenetic analysis of MenA (dihydroxynaphthoate phytyltransferase) showed that the MenA sequences of the plant Arabidopsis thaliana and Oryza sativa group with cyanobacterial sequences, whereas MenB
(dihydroxynaphthoate-CoA synthase) and MenD (2-succinyl-6-hydroxy-2,4- cyclohexadiene-1-carboxylate synthase) do not. This leads to the hypothesis that PhyQ biosynthesis is a result of mosaic evolution derived from a non-cyanobacterial MQ 34 pathway together with a cyanobacterial-type MenA, possibly derived from endosymbiosis.
ABBREVIATIONS
Chl a chlorophyll a
DHNA dihydroxynaphthoate
HPLC high-performance liquid chromatography
MQ-4 menaquinone-4
PCR polymerase chain reaction
PhyQ phylloquinone
PQ plastoquinone-9
PS I Photosystem I 35 INTRODUCTION
Phylloquinone (PhyQ, 2-methyl-3-phytyl-1,4-naphthoquinone), also known as vitamin K1, is synthesized only in oxygenic phototrophs such as cyanobacteria, algae, and
higher plants. PhyQ plays a crucial role in photosynthesis as a cofactor in Photosystem I
by mediating electron transfer between the primary acceptor A0 (Chl a) and the terminal
acceptor Fx (4Fe-4S cluster) after photoexcitation of the primary donor P700 (Golbeck,
1994, 2003; Brettel and Leibl, 2001). Two molecules of PhyQ are found within the protein environments of the PS I complex, which is composed of 12 protein subunits, 96
Chl a molecules, 22 β-carotenes, 3 [4Fe-4S] clusters, and 4-5 structural lipids (Jordan et al., 2001). PS I is a light-driven cytochrome c6/plastocyanin:ferredoxin/flavodoxin
oxidoreductase and is responsible for providing electrons that are ultimately used for the
generation of NADPH, the reducing power for cellular anabolism. The quantum
efficiency of this enzyme is largely dependent on the stability of a charge-separated state
+ - between P700 and FX , and the presence of PhyQ ensures an optimal rate of transfer
between these cofactors.
Biosynthesis of PhyQ has been studied by isotope tracer and direct enzymatic
activity assays in higher plants (see a review by Pennock and Threlfall, 1981). The results
have indicated that PhyQ biosynthesis resembles menaquinone (MQ) biosynthesis in E. coli (see reviews by Meganathan 1996, 2001). MQ biosynthesis occurs through eight enzymatic steps as summarized in Fig 2.1. The first committed step is the isomerization of chorismate to isochorismate by isochorismate synthase (MenF), followed by the 36 condensation of isochorismate and thiamine-pyrophosphate-succinic semialdehyde by 2-
succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate (SHCHC) synthase (MenD).
Dehydration of SHCHC is catalyzed by o-succinylbenzoate (OSB) synthase (MenC) and
the subsequent CoA thioesterification by OSB-CoA synthetase (MenE); the ring
cyclization reaction is catalyzed by 1,4-hydroxynaphthoyl-CoA synthase (MenB), and deesterification by 1,4-hydroxynaphthoyl-CoA thioesterase (MenH) results in 1,4- dihydroxy-2-naphthoate (DHNA). DHNA polyprenyltransferase (MenA) catalyzes the condensation of DHNA and polyprenyl diphosphate, yielding 2-polyprenyl-1,4- naphthoquinone (demethylmenaquinone). The final methylation at the C2 position is carried out by 2-polyprenyl-1,4-naphthoquinone methyltransferase (MenG) with S-
adenosyl-methinone as a methyl donor, resulting in the production of MQ. PhyQ and MQ
have the same general molecular structure except that PhyQ has a partially saturated, C20
phytyl substituent and MQ found in other bacteria has an unsaturated polyprenyl
substituent typically consisting of 30 to 50 carbon units (Collins and Jones, 1981).
The targeted inactivation of the menA, menB, menD, and menE homologs in
Synechocystis sp. PCC 6803 was shown to result in the complete interruption of PhyQ
biosynthesis. This demonstrated that a MQ-like pathway is responsible for PhyQ
biosynthesis in this organism (Johnson et al., 2000; Johnson et al., 2002). The following
questions remained to be answered: is this MQ-like PhyQ biosynthesis pathway
ubiquitous among cyanobacteria and higher plants; what is its evolutionary origin; how
has it evolved among the various oxygenic phototrophs? In this study, MQ biosynthesis
was investigated in 14 cyanobacteria whose genomes have been or are currently being 37 sequenced. The results indicate that the MQ-like pathway is conserved among most, although not all, of the cyanobacteria and higher plants. A detailed analysis of the gene arrangements and phylogenetic reconstruction lead to the hypothesis that the PhyQ biosynthetic pathway has evolved from the MQ pathway in both cyanobacteria and higher plants. 38 RESULTS
As of April 26, 2004, complete genomic sequences of 8 cyanobacteria are
available: Synechocystis sp. PCC 6803 (Kaneko et al., 1996), Nostoc PCC 7120 (Kaneko
et al., 2001), Thermosynechococcus elongatus BP-1 (Nakamura et al., 2002ab),
Gloeobacter violaceus (Nakamura Y et al., 2003), Prochlorococcus marinus MED4 and
Prochlorococcus marinus MIT9313 (Rocap et al., 2003), Prochlorococcus marinus
SS120 (Dufresne et al., 2003), Synechococcus sp. WH8102 (Palenik et al., 2003); and
incomplete sequences exist for Synechococcus sp. PCC 7002 (Jürgen Marquardt, Tao Li,
Jindong Zhao, and Donald A. Bryant, unpublished), Nostoc punctiforme (Gene Bank accession number: NZ_AAAY00000000), Trychodesmium erythraeum (Gene Bank
accession number: NZ_AABK00000000), Synechococcus elongatus sp. PCC 7942 (Gene
Bank accession number: NZ_AADZ01000001), Anabaena variabilis ATCC 29413 (Gene
Bank accession number: NZ_AAEA01000001), and Crocosphaera watsonii WH 8501
(Gene Bank accession number: NZ_AADV01000004). Similarity searches with the
MenA through MenH protein sequences of E. coli as queries were performed against each genome, and the results are summarized in Table 2.1. Of 14 cyanobacteria examined, 11 were shown to possess the complete set of 8 Men protein homologs.
Similarities based on amino acid sequences were typically in the range of 30-70%, although the MenB sequences were very highly conserved among a wide range of organisms including cyanobacteria, the γ-proteobacterium E. coli, the green sulfur bacterium Chlorobium tepidum, the Gram-positive bacterium Bacillus subtilis, and the archeon Halobacterium sp. NRC-1 (similarity >70%). In the genome of G. violaceus, 39 homologs of only MenA and MenG were detected. When the Synechocystis sp. PCC
6803 MenB sequence was used to search the G. violaceus genome database, the best hit
was Gll2549 with only 38 % similarity. Further database searching has revealed that
Gll2549 is highly similar to 6-oxo camphor hydrolase in Rhodococcus sp. NCIMB 9784
(69%). When Synechocystis sp. PCC 6803 MenC, MenD, MenE, and MenF sequences were used to search the G. violaceus database, the best hits were Gll3099 (35%), Gll2804
(44%), Glr1146 (45%), and Gll0757 (43%), respectively. These ORFs also showed higher similarities to other hypothetical proteins: Gll3099 is similar to chloromuconate cycloisomerase in Clostridium acetobutylicum (57%); Gll2804 is similar to acetolactate synthase in Synechocystis sp. PCC 6803 (Sll1981, 86%); Glr1146 is similar to a long-
chain fatty-acid CoA ligase in Bacillus cereus (54%); and Gll0757 is similar to anthranilate synthase component I in Synechocystis sp. PCC 6803 (Slr1979, 66%). No homologs of MenD (A. variabilis) or MenC and MenF (C. watsonii) have been identified to date, but these genomes are not yet completely sequenced.
Some of the men genes involved in PhyQ biosynthesis are found to form clusters in several cyanobacterial genomes. A cluster composed of menF, menA, menC, and menE is conserved in T. erythraeum, N. punctiforme, Nostoc sp. PCC 7120, Synechococcus sp. WH 8102, and all three Prochlorococcus species (Fig 2.2). In a separate region of the genome, menD and menB are also found to form a cluster. The observed gene arrangements in these cyanobacteria are similar to parts of the gene clusters found in E. coli, C. tepidum, B. subtilis and Halobacterium sp. NRC-1 (Fig 2.2). Combined with the conservation in the amino acid sequences among Men proteins, the conserved 40
arrangements of the men genes are strong indications that PhyQ biosynthesis in cyanobacteria evolved from the MQ pathway of other bacteria.
Given that oxygenic photosynthesis is believed to have originated in cyanobacteria and that PhyQ is associated exclusively with PS I, one may expect that the common ancestor of higher plants and algae acquired the entire PhyQ biosynthetic pathway from cyanobacteria upon endosymbiosis. Interestingly, the men gene arrangement in Arabidopsis thaliana does not resemble that in cyanobacteria (Fig 2.2). Lack of conserved gene arrangements does not necessarily indicate the absence of an evolutionary relationship; however, it is sufficient to cast doubt about it. Thus the evolutionary relatedness between cyanobacterial and higher plant PhyQ biosynthetic genes was evaluated further. As shown in Table 2.1, A. thaliana contains the whole set of men genes in its nuclear chromosomes, which indicates that PhyQ biosynthesis in A. thaliana is similar to that in cyanobacteria and to MQ synthesis in other prokaryotes. In a phylogenetic tree based on the MenA sequences, the two higher plants, A. thaliana and Oryza sativa, form a clade within a domain composed entirely of cyanobacteria, which suggests that MenA in higher plants is evolutionarily more related to MenA in cyanobacteria than to MenA of other eubacteria (Fig 2.3A). On the contrary, in trees based on the MenB and MenD sequences, these two higher plants form a clade outside the cyanobacterial domain. This suggests that MenB and MenD in higher plants are more closely related to their homologs in other eubacteria than to those in cyanobacteria (Fig 2.3B and C). Trees based on the MenC, MenE, and MenF sequences showed the topology similar to those observed for the MenB and MenD sequences; however, the detailed branching orders within the cyanobacterial domain were variable probably due to low sequence conservation of these proteins (< ~50%). The observed variation in the 41
phylogenetic relationships therefore suggests that not all men genes in higher plants originated from cyanobacteria.
By comparative genome analyses and phylogenetic reconstructions, it was so far hypothesized that PhyQ biosynthesis in cyanobacteria and higher plants evolved from that of MQ, which suggests that early cyanobacteria synthesized and utilized MQ for the PS I complexes. This hypothesis was supported by the discovery that G. violaceus and Synechococcus sp. PCC 7002 synthesize MQ-4 instead of PhyQ. Fig 2.4A shows the HPLC profile of the solvent extract obtained from the PS I complexes isolated from Synechocystis sp. PCC 6803 wild type. PhyQ, which typically elutes at 22.4 min, was shown to be present. In the HPLC profiles of the solvent extracts obtained from the PS I complexes isolated from Synechococcus sp. PCC 7002 wild type and from the G. violaceus whole cells, no such peak was present as judged by the absence of UV- absorbing compounds at this retention time (Fig 2.4B and C). Further examination of the chromatograms led to the discovery of a new peak at ca. 14 min that had an intense UV- absorption with maxima at 248, 263, 270, and 332 nm (Fig 2.5B and C), which coincide well with the absorption spectrum of phylloquinone obtained from Synechocystis sp. PCC 6803 (Fig 2.5A). This spectrum is characteristic of a 1,4-naphthoquinoid compound with
two alkyl substitutions at the C2 and C3 positions (Dunphy and Brodie, 1971). The absence of PhyQ in G. violaceus was further confirmed by the absence of the PhyQ absorption in the eluate at 22.5 min (Fig 2.5C). The same results were obtained for Synechococcus sp. PCC 7002. Mass spectrometric analysis of the solvent extracts of PS I complex isolated from Synechococcus sp. PCC 7002 showed that the compound eluting at ca. 14 min has a m/z of 444, as opposed to a m/z of 450 for PhyQ (Figure 2.6). This difference is best explained by the presence of a geranylgeranyl substituent with four unsaturated isoprenoid units. Thus, Synechococcus sp. PCC 7002 synthesizes MQ-4 42 instead of PhyQ. The absence of PhyQ was confirmed by the absence of any component with a m/z of 450 at 22.5 min (Fig 2.6). The solvent extracts from the PS I complexes isolated from Synechococcus sp. PCC 7002 revealed the presence of 2.3 MQ-4 molecules per 100 Chl a molecules, demonstrating that MQ-4 plays a role as the A1 cofactor in the PS I complexes in this cyanobacterium. Similar quantitative analysis for G. violaceus has not yet performed.
Targeted insertional inactivation of menB, menF, and menG in Synechococcus sp. PCC 7002 was performed to verify the involvement of these gene products in the MQ-4 biosynthesis (Fig 2.7). The menF and menG mutations were introduced into the wild type, whereas the menB mutation was introduced into the rubA mutant for the purpose of generating PS I complexes that contain plastoquinone-9 but lack iron-sulfur clusters for future studies concerning electron transfer kinetics and thermodynamics (Y. Sakuragi, B. Zybailov, G. Shen, R. Balasubramanian, B. A. Diner, I. Karygina, Y. Pushkar, D. Stehlik,
D. A. Bryant and J. H. Golbeck, manuscript in preparation). The full segregation of the mutated alleles from the respective wild-type alleles was analyzed by PCR as shown in Fig 2.8. In the rubA mutant, PCR using the designed primers that are targeted to the menB region resulted in a product of 1.0 kb, which is expected based on the restriction map, as shown in Fig 2.7. Likewise, in the wild type, PCR using the designed primers that are targeted to the menF and menG regions resulted in products of 1.7 kb, and 1.8 kb, respectively. No products with corresponding sizes were detected for the mutants; instead products with larger sizes were detected. In the menB rubA and the menF mutants, the size of the products were ca. 2.1 kb and 2.8 kb, respectively. The difference between the PCR products from the wild type and the mutants corresponds to the size of the 1.1-kb gentamicin-resistance cartridge derived from pMS266. In the menG mutant a product of 2.2 kb was observed. The difference between the PCR products from the wild type and 43 the menG mutant is 0.3 kb, which corresponds to the difference between the 1.1-kb gentamicin-resistance cartridge derived from pMS266 and the 0.8-kb deletion in the N- terminal region (Fig 2.7C). These results show that the menB rubA double mutant and menF and menG single mutants are free of the respective wild-type alleles and therefore homozygous in the menB::GmR, menF::GmR , and menGB::GmR alleles, respectively.
When the pigment extracts from the PS I complexes isolated from the menB rubA and the menG mutants were analyzed, no peak with UV-absorption was detected at ca. 14 min, demonstrating that the menB and menG homologs are indeed required for MQ-4 synthesis in Synechococcus sp. PCC 7002 (Fig 2.9). In extracts of PS I complexes from the menF mutant, a small peak was detected at this retention time; however, the absorption properties of the component eluted at this retention time were significantly different from those of MQ-4 with an absorption maximum at 292 nm (Fig 2.10B). This component is virtually absent in the wild type and its chemical nature has not yet been determined. These results confirm that the interruption of the menF gene also results in the complete loss of MQ-4 and that MenF is required for MQ-4 synthesis in Synechococcus sp. PCC 7002. In the menF and the menB rubA mutants, a peak at 35.1 min was detected, which is absent in the extracts of PS I complexes in the wild type (Fig 2.9). The component eluted at this retention time showed absorption properties with a single peak at 256 nm (Fig 2.10C), which is characteristic of PQ-9 (Crane and Dilley, 1963). Mass spectrometric analysis of the component has shown a m/z of 748, which is characteristic of PQ-9. These results are in good agreement with a previous study in which the interruption of PhQ biosynthesis in Synechocystis sp. PCC 6803 resulted in the incorporation of PQ-9 in PS I (Johnson et al., 2000). Likewise, no MQ was detectable in the chromatogram of PS I complexes isolated from the menG mutant (Fig 2.9D). A new peak was detected at 11.9 min, which showed a strong UV-absorption with a maximum at 44
248 nm and a shoulder at 265 nm. Because the loss of a methyl group is expected to lower the hydrophobicity and size of a molecule, it was expected that demethylphylloquinone would elute earlier than MQ-4 in the reverse phase HPLC analysis. Therefore, it is highly plausible that the component eluting at 11.9 min is due to demethylmenaquinone. Combined with the sequence similarities to E. coli counterparts, these results confirm that menB, menG, and menF encode dihydroxynaphthoic acid-CoA synthase, 2-geranylgeranyl-1,4-naphthoquinone methyltransferase, and isochorismate synthase, respectively, in Synechococcus sp. PCC 7002. Together with the previous identification of menA, menB, menD, menE, and menG in Synechocystis sp. PCC 6803, it is concluded that PhyQ/MQ-4 biosynthesis in cyanobacteria requires MenF, MenD, MenE, MenB, MenA, and MenG. 45 DISCUSSION
Comparative genome analyses were conducted on 14 cyanobacteria, E. coli, B. subtilis, Halobacterium sp. NRC-1 and A. thaliana. Based on the results, it was concluded that PhyQ biosynthesis in cyanobacteria is similar to the MQ biosynthesis pathway in E. coli. Supporting this hypothesis is the discovery that G. violaceus and
Synechococcus sp. PCC 7002 synthesize MQ-4 instead of PhyQ and that Synechococcus sp. PCC 7002 utilizes it as the A1 cofactor in PS I complexes. G. violaceus is thought to be one of the earliest branching lineages among the cyanobacteria (Nelissen et al., 1995).
The fact that this organism synthesizes MQ-4 is a strong indication that PhyQ biosynthesis probably evolved from MQ biosynthesis pathway in other bacteria. It is therefore highly plausible that the early cyanobacteria synthesized MQ-4, and PhyQ biosynthesis evolved from the MQ-4 biosynthesis in cyanobacteria. This is consistent with the fact that the occurrence of PhyQ biosynthesis is restricted to cyanobacteria and to algae and higher plants, which are evolutionarily related to cyanobacteria. A recent study has shown that the red alga Cyanidium caldarium also synthesizes MQ-4 and utilizes it in its PS I complexes (Yoshida et al., 2004). Therefore, MQ-4 synthesis may be a widely occurring phenomenon among oxygenic phototrophs.
Based on phylogenetic reconstructions, it was concluded that not all men genes in
higher plants are of cyanobacterial origin. It is believed that plastids, and hence the ability
to perform oxygenic photosynthesis, in eukaryotic phototrophs were acquired from a
cyanobacterium upon endosymbiosis (Margulis, 1970). Therefore, the presence of a gene 46 with strong similarity to the cyanobacterial menA in the plant nuclear chromosome is
probably a result of gene transfer from the plastids. This gene is presumably derived from
the endosymbiotic cyanobacterium and would have been transferred to a host nuclear
chromosome during endosymbiotic interaction (Douglas, 1998; Delwiche and Palmer,
1997; Martin et al., 1998). On the other hand, the presence of the non-cyanobacterial-type
menB and menD in the plant nuclear chromosome suggests they originated in other
organisms. One possibility is that menB and menD were already present in the eukaryotic
host cells before the endosymbiotic cyanobacterium was incorporated into them. It is
believed that mitochondria in eukaryotic cells originated from a proteobacterium,
possibly in the α-subdivision (Olsen et al., 1994; Andersson et al., 1998; Martin and
Müller, 1998; Emelyanov 2001; Horner et al., 2001; Emelyanov, 2003), through an
endosymbiosis that preceded the acquisition of plastids. Given that some, although not
all, proteobacteria synthesize both ubiquinone and MQ (Collins and Jones, 1981), it is
possible that the endosymbiotic proteobacterium brought the ability to synthesize MQ
into the eukaryotic host cells. Another possibility is that menB and menD genes were
acquired via horizontal gene transfer from bacteria to plant cells. Interestingly, the men
genes in the plastid genomes of the red algae Cyanidium caldarium and Cyanidioschyzon merolae form a cluster (menD-menF-menB-menA-menC-menE), which is very similar to the cluster found in a variety of prokaryotes (Fig 2.2). Phylogenetic analyses show that neither the MenA nor the MenB sequences of the red algae Cyanidioschzon merolae
forms a cluster with the cyanobacterial sequences (Fig 2.3). These observations therefore
support the occurrence of horizontal gene transfer from bacteria other than cyanobacteria
to these eukaryotic phototrophs. 47 Targeted mutagenesis of the menB, menF, and menG homologs in Synechococcus
sp. PCC 7002, encoding for DHNA synthase, isochorismate synthase, and 2-methyl-1.4-
naphthoquinone methyltransferase, respectively, resulted in the expected interruption of
PhyQ biosynthesis. This confirms that the pathway responsible for MQ-4 synthesis in
Synechococcus sp. PCC 7002 is similar to that in other bacteria.
PhyQ mediates electron transfer between A0 and FX of PS I during the charge separation process upon photoexcitation of P700. The optimal rate of electron transfer from A0 to FX is ensured by the thermodynamically downhill arrangement of these
cofactors. The molecular structures of PhyQ and MQ-4 are identical except for the level
of saturation of the C3 polyprenyl side chain, and thus the redox potentials of the two
quinones are also expected to be identical. Therefore, it is not surprising that MQ-4 can
function at the A1 site in the PS I complexes. The prenylation reaction of DHNA in
Synechococcus sp. PCC 7002, G. violaceus, and C. caldarium is likely to be different
from that in PhyQ-synthesizing species either by i) higher substrate specificity of the
MenA protein for geranylgeranyl diphosphate compared to phytyl diphosphate; and/or ii)
substantially higher intracellular concentration of geranylgeranyl diphosphate than phytyl
diphosphate in these cells.
The whole-genome analysis of G. violaceus seems to suggest that menB, menC,
menD, menE, and menF homologs are absent in this organism. ORFs that show similarity
to the proteins encoded by these men genes are more related to other proteins that do not
play roles in MQ-4 biosynthesis. One example is the MenB-like protein Gll2549, which
shows high similarity to 6-oxo camphor hydrolase. This enzyme is a member of the 48 crotonase superfamily that catalyzes the cleavage of carbon-carbon bonds. Therefore, it is possible that Gll2549 participates in the PhyQ pathway and catalyzes the ring cyclization reaction of o-succinylbenzoate. However, MenB is a very highly conserved enzyme among a wide variety of organisms (Table 2.1), and the absence of its homolog together with four other Men proteins in G. violaceus suggests the presence of an alternative pathway that can lead to the synthesis of DHNA. Alternatively, it was also possible that
G. violaceus does not synthesize PhyQ, MQ-4 or related compounds at all. Previous studies have shown that PhyQ-less mutants recruit PQ into the A1 site of PS I, and, despite a slightly lower catalytic efficiency, as measured by the flavodoxin reduction rate, the cells are able to grow photoautotrophically (Johnson et al., 2000). However, HPLC analyses performed in this study demonstrated that MQ-4 is present in the whole cell extracts of G. violaceus (see above). This result largely rules out the possibility of PQ-9 dependent PS I function in this organism. It also suggests that a part of the MQ-4 biosynthetic pathway in this organism is very different from those in the rest of the oxygenic phototrophs analyzed in this study.
In this study, a comparative genomic approach was shown to be useful in predicting the entire biosynthetic pathway for a secondary metabolite. Combined with reverse genetics, one can now genetically modify the quinone species in PS I in cyanobacteria. The advantage of this approach is that one can study the biochemical and biophysical properties of PS I, as well as the physiology of the cells, by using a biologically intact system. Further examples are provided in the following chapters.
49 SUMMARY
Synechococcus sp. PCC 7002 and G. violaceus were found to synthesize MQ-4 instead of PhyQ. Targeted inactivation of the menF, menB, and menG genes in
Synechococcus sp. PCC 7002 demonstrated that these genes are required for MQ-4 biosynthesis. The PS I complexes isolated from the menB rubA and menF mutants contained PQ-9, while the PS I complexes isolated from the menG mutant contained an
UV-absorbing compound, which may be demethyl-MQ-4. Comparative genome analyses of 14 cyanobacteria and four eukaryotic phototrophs suggested that PhyQ biosynthesis in cyanobacteria evolved from MQ biosynthesis in bacteria and that this pathway in higher plants may have developed as a result of mosaic evolution. 50
MATERIALS AND METHODS
Comparative Genome Analyses The Men protein sequences of E. coli were used as queries to detect the presence of homologs in cyanobacteria using the Blast-P search method. Retrieved sequences were named after their locus tags in the genomes. Sequence similarities between the homologs were obtained based on pairwise alignment by BLOSUM30 using ClustalW. Multiple amino acid sequence alignments were also generated by BLOSUM30 using ClustalW. For the phylogenetic reconstructions based on the MenA, MenB, and MenD sequences, unambiguous regions in the alignments were removed. The resulting “trimmed” multiple alignments were subjected to phylogenetic reconstruction by the Parsimony method using PAUP*4.0 beta. The bootstrap values are results of 10,000 repetitions.
Generation of Mutant and Verification of Segregation
Wild type and mutant strains of Synechococcus sp. PCC 7002 were grown as
described previously (Frigaard et al., 2004). A DNA fragment containing the menB
coding region was amplified from the isolated genomic DNA by PCR using the primer
B1 (5’-CGTCGGGACACATCTTTCAC-3’) and the primer B2 (5’-
CGGTATCTACTCTACCAAATTGGG-3’). The resulting fragment was digested with
HindIII and BamHI, and inserted into the HindIII-BamHI sites of pUC19 (pUC19menB).
A fragment containing the menF coding region was amplified by PCR using the primer
F1 (5’-TTTGGGCAGCATCCCGGGTAAGCTGG-3’) that contains the engineered KpnI
cleavage site and the primer F2 (5’-GGAACCTATGAAGTTAGTCTGCGTTTCG-3’).
The resulting fragment was digested with KpnI and HindIII, and inserted into the KpnI- 51 HindIII fragment of the pUC19 cloning vector (pUC19menF). A fragment containing the
menG coding region was amplified by PCR using the primer G3 (5’-
GATGGTATTGTCGTCAACACCGC-3’) and the primer G4 (5’-
CACCGCCGCTATGGTACCAGGCG-3’). The resulting fragment was digested
(pUC19menG). The accC1 gene, derived from the plasmid pMS266 and conferring gentamycin resistance, was inserted into the unique PstI site and SmaI cleavage sites in
the plasmids pUC19menB and pUC19menF, respectively. The accC1 gene was inserted
into the EcoRV sites of pUC19menG, resulting in a deletion of 740 bp encompassing the
N-terminal and the upstream region of the menG gene. The menB::accC1 construct was
used to transform the rubA mutant of Synechococcus sp. PCC 7002 as previously
described (Shen et al., 2002). The menF::accC1 construct was used to transform a psaB-
strain. After achieving complete segregation of the menF::accC1 locus, the wild type psaB gene was introduced into the menF::accC1 psaB mutant to generate the menF::accC1 single mutant by homologous recombination at the psaB locus. The menG::accC1 construct was introduced into the wild-type strain. The full segregation of each allele was tested by PCR using the primers B1 and B2 for the menB::accC1 transformants, the primers F1 and F2 for the menF::accC1 transformant, and the primers
G1 (5’- CGTTTCAATTTCCCAGGCAAAGTC-3’) and G2 (5’-
CCGAGGAAGTAAACCGCATATTC-3) for the menG::accC1 transformant. 52 Isolation of Thylakoid Membranes and PS I Particles
Thylakoid membranes were isolated from cells in the late-exponential growth
phase as described previously (Shen et al., 1993). Cells were broken at 4° C by three
passages through a French pressure cell at 124 MPa. The thylakoid membranes were
resuspended and solubilized for 2 h at 4° C in the presence of 1% (w/v) n-dodecyl-β-D-
maltoside. PS I complexes were separated from other membrane components by
centrifugation on 5-20% (w/v) sucrose gradients with 0.05% n-dodecyl-β-D-maltoside in
the buffer. Further purification was achieved by a second centrifugation on sucrose
gradients in the buffer in the absence of n-dodecyl-β-D-maltoside. Trimeric PS I
complexes were used in these studies (Golbeck, 1995).
Quinone and Chlorophyll Analysis
Isolated PS I was either lyophilized or dried under N2 gas before extraction with acetone:methanol (7:2 v/v). Pigments and quinones were extracted with cold
acetone:methanol (7:2 v/v) after brief ultrasonication. Alternatively, 20 µL of the PS I preparations were extracted with 400 µL of acetone:methanol. After centrifugation and filtration through a PTFE filter membrane with a 0.2-µm pore size (Whatman International Ltd., Maldstone, UK), the extract in the organic phase was directly injected into an Agilent Technology 1100 series HPLC system (Agilent Technology, Palo Alto, CA, USA) equipped with a SUPELCO (Sigma-Aldrich Corp., St. Louis, MO, USA)
® Discovery C18 column (25 cm x 4.6 mm, 5 µm). Analyses were carried out using the following protocol: 80%A/20%B from 0 to10 min, a linear change to 20%A/80%B in 40 min, and 20%A/80%B for 5 min, where solvent A and solvent B are 100% methanol and 100% isopropanol, respectively. The flow rate was 0.75 ml min-1. Detection of eluates was performed with a diode array detector (Agilent 1100 series). The chlorophyll a, 53
phylloquinone, and plastoquinone contents of each sample were determined based on
integrated peak areas and their molar absorption coefficients, which are 17.4 mM-1 cm-1 at 618 nm, 18.9 mM-1 cm-1 at 270 nm (Dunphy and Brodie, 1971), and 15.2 mM-1 cm-1 at 254 nm (Crane and Dilley, 1963), respectively. The absorption coefficient of chlorophyll a at 618 nm was calculated from the ratio of the absorption peaks at 618 nm and 666 nm in methanol-isopropanol (6:4, v/v) and from its absorption coefficient at 666 nm in methanol (MacKinney, 1941). The peak assignments were confirmed by mass spectrometry using atmospheric-pressure chemical ionization with a Quattro II time-of- flight mass spectrometer (Micromass, Beverly, MA, USA) operated in the negative-ion mode. 54
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Table 2.1: Whole genome analyses for the MQ/PhyQ biosynthesis enzymes in 14 cyanobacteria, E. coli, B. subtilis, C. tepidum, and Halobacterium sp. NRC-1. Similarity to Synechocystis sp. PCC 6803 proteins are shown in parentheses (%). The similarity is based pairwise alignment of the entire sequences constructed by BLOSUM30. (Table continues on next page)
S. 6803 S. 7002 Nostoc T. ery N. pun A. var C. wat S. elo T. elo G. vio
b Alr0033 Tery1070 Npun0604 Avar026348 aCwat143901 Selo020647 Tlr2196 Gll1578 MenA Slr1518 (67%) (70%) (73%) (70%) (68 %) (34%) (66%) (70%) (53%) b All2347 Tery1085 Npun2498 Avar413601 Cwat159701 Selo187301 Tll2458 MenB Sll1127 - (89%) (91%) (83%) (91%) (91%) (91%) (89%) (88%) b Alr0034 Tery1067 Npun0605 Avar026382 Selo020304 Tlr1174 MenC Sll0409 - - (52%) (54%) (54%) (52%) (54%) (50%) (55%) b Alr0312 Tery1075 Npun2496 Cwat131201 Selo02718 Tll0130 MenD Sll0603 - - (62%) (61%) (61%) (59%) (61%) (61%) (56%) b Alr0035 Tery1066 Npun0606 Avar026381 Cwat026026 Selo02305 Tll1221 MenE Slrl0492 - (44%) (56%) (56%) (54%) (52%) (52%) (45%) (50%) b All0032 Teryal71 Npun0603 Avar026437 Selo021474 Tll1213 MenF Slr0817 - - (54%) (58%) (57%) (60%) (58%) (53%) (54%) b Alr5252 Tery3321 Npun3758 aAvar287101 Cwat179201 Selo020383 Tll2373 Gll0127 MenG Slr1653 (70%) (72%) (63%) (65%) (48%) (68%) (66%) (63%) (53%) b All0111 Tery1986 Npun0061 Avar513701 Cwat024832 Selo020062 Tlr2066 MenH Sll1916 - (43%) (68%) (46%) (55%) (65%) (45%) (52%) (43%) S. 6803, Synechocystis sp. PCC 6803; S.7002, Synechococcus sp. PCC 7002; Nostoc, Nostoc sp. PCC 7120; N. pun, Nostoc punctiforme; T.elo, T. elongatus
BP-1; T. ery, Trichodesmium erythraeum; G.vio, G. violaceus PCC 7421; A.var, Anabaena variabilis ATCC 29413; S. elo, Synechococcus elongatus sp.
7942; C.wat, Crocosphaera watsonii WH8501. aN-terminal sequence was truncated. bNo locus tags are available. 60
Table 2.1 continued
S. 8102 P. MED4 P. MIT P.SS120 Arabidopsis E. coli C. tep B. sub
Synw2307 Pmm0176 Pmt2059 Pro0201 At1g60600 B3930 CT11511 Bsu38490 MenA (56%) (58%) (55%) (55%) (55%) (47%) (44%) (41%) Synw0998 Pmm0608 Pmt0405 Pro1053 At1g60550 B2262 CT1846 Bsu3075 MenB (80%) (77%) (79%) (79%) (78%) (78%) (77%) (81%) Synw2306 Pmm0175 Pmt2058 Pro0200 At1g68900 B2261 CT1847 Bsu30780 MenC (39%) (41%) (43%) 43%) (46%) (38%) (35%) (35%) Synw0997 Pmm0607 Pmt0406 Pro1054 At1g68890 B2264 CT1839 Bsu3077 MenD (46%) (46%) (47%) (46%) (45%) (43%) (44%) (49%) Synw2305 Pmm0174 Pomt2057 Pro0199 At3g48990 B2260 CT1848 Bsu3074 MenE (35%) (34%) (36%) (35%) (43%) (46%) (39%) (39%) Synw2308 Pmm0177 Pmt2060 Pro0202 At1g74710 B2265 CT1838 Bsu3078 MenF (42%) (48%) (47%) (47%) (51%) (44%) (44%) (47%) Synw1674 Pmm0431 Pmt0276 Pro0427 At1g23360 B3833 CT0462 Bsu22750 MenG (56%) (51%) (37%) (57%) (50%) (47%) (52%) (45%) Synw0681 Pmm1628 Pmt0128 Pro1790 At5g38520 B2263 CT1845 Bsu3076 MenH (38%) (35%) (38%) (40%) (43%) (35$) (31%) (34%)
S. 8102, Synechococcus sp. WH8102; P.MED4, Prochlorococcus marinus MED4; P.MIT, Prochlorococcus marinus MIT9313; P.SS120, Prochlorococcus marinus SS120; Arabidopsis, Arabidopsis thaliana; E. coli, Escherichia coli; C. tep, Chlorobium tepidum; B. sub, Bacillus subtilis. aN-terminal sequence was truncated. 61
62
Fig 2.1: Menaquinone biosynthetic pathway in E. coli.
DHNA, dihydroxynaphthoate; DMQ, demethylmenaquinone; OSB, o-succinylbenzoate; SHCHC, 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate; TPP-SS, thiamine- pyrophosphate-succinic semialdehyde. 63
Fig 2.2: The men gene arrangements.
Nostoc, Nostoc sp. PCC 7120; Bsu, Bacillus subtilis; Ctep, Chlorobium tepidum; Eco, Escherichia coli; Hal, Halobacterium sp. NRC-1; Arabidopsis, A. thaliana; Cyme, Cyanidiochyzon merolae; Cyca, Cyanidium caldarium; Npun, Nostoc punctiforme; Pmm, Prochlorococcus marinus MED4; Pmt, P.marinus MIT9313; Synw, Synechococcus sp. WH8102; Tery, Trichodesmium erythraeum. P. marinus SS220 shows the same arrangement as Pmm. 64
Fig 2.3:Phylogenetic trees based on the amino acid sequences of (A) MenA, (B) MenB, and (C) MenD.
The multiple alignments were generated by BLOSUM30 using Clustal W, and the trees were constructed by the Parsimony method using PAUP*4.0 beta. Cyanobacteria, red algae and higher plants are indicated with black circles, black diamonds, and stars, respectively. The locus tags used to indicate organisms are summarized in Table 2.1, except for the following: Cymecp095, Cymecp092, Cymecp094 for MenA, MenB, and MenD in C. merolae, respectively; Oj1217B09.18 and P0671D01.18 for MenA and MenB in Oryza sativa, respectively. AF2036 and AF0435 are hypothetical proteins in the archeon Archeoglobus fulgidis DSM4304. Ml2270 is a MenD homolog in Mycobacterium leprae. Bootstrap values were obtained after 10,000 repetitions and those larger than 50 are shown. 65
Fig2.4: HPLC profiles of solvent extracts from the PS I complexes and whole cells.
Chromatograms of reverse-phase HPLC analysis of solvent extracts obtained from (A) PS I complexes isolated from the wild-type Synechocystis sp. PCC 6803 (B) PS I complexes isolated from wild-type Synechococcus sp. PCC 7002, and (C) whole cells of the wild-type G. violaceus. Chromatograms were recorded at 270 nm. Chl a, chlorophyll a; β-car, β-carotene; MQ-4, menaquinone-4. 66
Fig 2.5: Absorption spectra of solvent-extracted components analyzed by HPLC.
Components eluting (A) at 22.5 min from the PS I complex of the wild-type Synechocystis sp. PCC 6803, (B) at 14.5 min from the PS I complex of the wild-type Synechococcus sp. PCC 7002, and (C) at 14.5 min from the whole cell of the wild-type G. violaceus (solid line) (see Fig 2.4). A component eluting at 22.5 min in G. violaceus is also shown by dotted line and does not appear to be a quinone. 67
Fig 2.6: LC-mass spectrometric analysis of the solvent extracts from Synechococcus sp. PCC 7002.
(A) Chromatograms at the m/z of 444.3 (top) and 450.3 (bottom), (B) mass spectra at the retention time at 30 min (top) and 12.4 min (bottom). PQ-9 elutes at 30 min. 68
Fig 2.7: Restriction maps of the menB (A), menF (B), and menG (C) coding regions in Synechococcus sp. PCC 7002.
Short black arrows show the primers used for verification of segregation by PCR. 69
Fig 2.8: PCR analyses on genomic DNAs isolated from the menB rubA, menF, and menG mutants of Synechococcus sp. PCC 7002.
Primers used for the analyses are shown by the black arrows in Fig 2.7. Control amplifications using template DNA from the rubA and wild-type (WT) strains are also shown. 70
Fig 2.9: Reverse-phase HPLC analyses of the solvent extracts from PS I complexes.
(A) wild type, (B) the menB rubA mutant, (C) the menF mutant, and (D) the menG mutant of Synechococcus sp. PCC 7002. The retention time for MQ-4 is indicated by the black arrows. 71
Fig 2.10: Absorption spectra of eluting components analyzed for PS I complexes isolated from Synechococcus sp. PCC 7002 using the reverse-phase HPLC analysis (see Fig 2.9).
(A) MQ-4 at 13.5 min in wild type, (B) unidentified component eluting at 13.5 min in the menF mutant, (C) PQ-9 at 35.1 min in the menB rubA mutant, (D) unidentified component (possibly demethyl-MQ-4) eluting at 11.9 min in the menG mutant. 72
Chapter 3
Recruitment of a Foreign Quinone into the A1 Site of Photosystem I. Interruption of menG in the Phylloquinone Biosynthetic Pathway of Synechocystis sp. PCC 6803 Results in the Incorporation of 2-Phytyl-1,4-Naphthoquinone and
Alteration of the Equilibrium Constant between A1 and FX
Publication:
Yumiko Sakuragi, Boris Zybailov, Gaozhong Shen, Parag R. Chitnis, Art van der Est,
Robert Bittl, Stephan Zech, Dietmar Stehlik, John H. Golbeck and Donald A. Bryant. (2002) Biochemistry 41: 394-405
73
ABSTRACT
A gene (menG), encoding a methyltransferase was identified in Synechocystis sp. PCC
6803 as responsible for transferring the methyl group to 2-phytyl-1,4-naphthoquinone
(demethylphylloquinone) in the biosynthetic pathway of phylloquinone, the secondary
electron acceptor in Photosystem I (PS I). Mass spectrometric measurements showed that
targeted inactivation of the menG gene prevents the synthesis of phylloquinone and leads
to the accumulation of 2-phytyl-1,4-naphthoquinone in PS I complexes. Growth rates of
the wild-type and the menG mutant strains under photoautotrophic and photomixotrophic
conditions were virtually identical. The chlorophyll a content in whole cells of the menG mutant was similar to that in the wild type when the cells were grown at a light intensity of
50 µE m-2 s-1 but slightly lower when grown at 150 µE m-2 s-1. Low temperature chlorophyll
fluorescence emission measurements showed a larger increase in the ratio of PS II to PS I
in the menG mutant strain relative to the wild type as the light intensity was elevated from
50 µE m-2 s-1 to 300 µE m-2 s-1. CW EPR studies at 34 GHz and transient EPR studies at multiple frequencies showed that the quinone radical in the menG mutant has the same
overall linewidth as in the wild type, but the pattern of hyperfine splittings showed two lines in the low-field region consistent with the presence of an aromatic proton at ring position 3. The spin polarization pattern indicated that 2-phytyl-1,4-naphthoquinone is in the same orientation as phylloquinone, and out-of-phase, spin-echo modulation spectroscopy showed the same P700+ to Q- center-to-center distance in the two samples.
- Transient EPR studies indicated that forward electron transfer from Q to FX is slowed from 74 290 ns in the wild-type to 600 ns in the menG mutant. The redox potential of
2-phytyl-1,4-naphthoquinone was estimated to be 50 to 60 mV more oxidizing than
phylloquinone in the A1 site, which translates to a lowering of the equilibrium constant
- - + - between Q /Q and FX /FX by a factor of ca. 10. The kinetics of the P700 [FA/FB] backreaction increased from 80 msec in the wild-type to 20 msec in the menG mutant
strain, thus supporting a thermally-activated uphill electron transfer through the quinone
- + rather than a direct route for charge recombination between [FA/FB] and P700 .
75
ABBREVIATIONS bp basepair(s) Chl chlorophyll CW continuous wave ENDOR electron nuclear double resonance EPR electron paramagnetic resonance HEPES 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid HPLC high performance liquid chromatography kb kilobase MS mass spectrometry NQ naphthoquinone ORF open reading frame PCR polymerase chain reaction PhyQ phylloquinone PQ-9 plastoquinone-9 PS photosystem Tricine N- [2-hydroxy-1,1-bis(hydroxymethylethyl)]glycine 76
INTRODUCTION
Photosystem I (PS I) in cyanobacteria is a membrane-integral pigment-protein complex consisting of 12 protein subunits (PsaA through PsaF, PsaI through PsaM, and
PsaX) and a variety of cofactors, including 96 molecules of chlorophyll a (Chl a), two molecules of 2-phytyl-3-methyl-1,4-naphthoquinone (phylloquinone, PhyQ), three
[4Fe-4S] clusters, and about 22 molecules of β-carotene (Jordan et al., 2001). After the absorption of a photon by one of the antenna molecules, charge separation takes place between a special pair of Chl a molecules (P700) and a Chl a monomer (A0). The charge-separated state is stabilized on the donor side by electron transfer from
+ plastocyanin or cytochrome c6 to P700 , and on the acceptor side by electron transfer to
PhyQ in the A1 site, through the iron-sulfur clusters at FX, FA and FB, and ultimately to ferredoxin or flavodoxin. This light-driven enzyme therefore functions as cytochrome c6/plastocyanin:ferredoxin/flavodoxin oxidoreductase that ultimately provides reducing power to the cell in the form of NADPH.
Two molecules of PhyQ are present per P700 in PS I complexes of Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7942 (Biggins and Mathis, 1988; Malkin, 1986;
Schoeder and Lockau, 1986). Both quinones have been located on the 2.5 Å-resolution electron density map of Synechococcus elongatus PS I complexes (Jordan et al., 2001), and there is a good agreement with EPR studies that provide distance (Bittl et al., 1997) and orientation (Kamlowski et al., 1998; Zech et al., 2000) information for the EPR-active quinone. For example, the same orientation of the carbonyl oxygen bonds of PhyQ, tilted 77 ca. 30 degrees from the membrane plane, is deduced from the orientation of the magnetic
gxx [(Zech et al., 2000) and earlier references therein] and the electron density map (Jordan et al., 2001). PhyQ is structurally related to menaquinone-9 (2- nonaprenyl-3-methyl-1,4-naphthoquinone). Both have 3-methyl-1,4-naphthoquinone core
structures with a poly-isoprenoid chain at the C2 ring position: menaquinone has an
unsaturated C45 isoprenoid chain, while PhyQ has a partially saturated C20 phytyl chain.
Because of their similarities in structure, it is likely that the PhyQ biosynthetic pathway is
similar to the menaquinone biosynthetic pathway in Escherichia coli (Johnson et al., 2000,
see also Chapter 2). The biosynthetic pathway of PhyQ is interesting for two reasons.
Firstly, the genes and enzymes of this pathway have not yet been completely characterized in cyanobacteria. Secondly, knowledge of this pathway should enable one to devise biological and chemical strategies to modify the quinone present in the A1 site.
A pathway of PhyQ biosynthesis in Synechocystis sp. PCC6803 was recently
proposed and included the genes menA, menB, menC, menD, menE, menF, and menG
(Johnson et al., 2000) (see Fig 2.1). Inactivation of the menA and menB genes, predicted to
encode 1,4-dihydroxy-2-naphthoic acid phytyltransferase and 1,4-dihydroxy-2-naphthoate
synthase, respectively, prevented the synthesis of PhyQ and led to the incorporation of
plastoquinone-9 (PQ-9) into the A1 site of PS I (Johnson et al., 2000). PQ-9 is the
secondary quinone acceptor in Photosystem II (PS II) and additionally functions as the
molecule that shuttles electrons to the cytochrome b6f complex. It was reported that PQ-9 is
present in the A1 site of PS I with the same orientation and asymmetric spin-density
distribution as PhyQ in wild type PS I complexes (Zybailov et al., 2000). PQ-9 is a 78 benzoquinone with a C45 alkyl tail and is structurally and functionally only distantly
related to PhyQ. The fact that PQ-9 could be inserted into the A1 site and could function in
lieu of PhyQ indicates that the binding site is not highly specific for PhyQ. Rather, the
redox properties of the quinone appear to be conferred largely by the protein environment
of the A1 site rather than by the identity of the quinone (Semenov et al., 2000).
The object of this study is the protein encoded by ORF sll1653, which was
originally annotated as gerC2, a spore germination protein in Bacillus subtilis. This ORF is
homologous to menG, which codes for a methyltransferase enzyme in E. coli and other
bacteria. In Synechocystis sp. PCC 6803, this gene is anticipated to encode a
2-phytyl-1,4-naphthoquinone (2-phytyl-1,4-NQ) methyltransferase enzyme (Johnson et
al., 2000). 2-phytyl-1,4-NQ differs from PhyQ only by the absence of the methyl group at
ring position C3 and by a higher redox potential (Depew and Wan, 1988). The relationship
between the molecular structure of this quinone and the axes of the molecular g-tensor is
depicted in Fig 3.1.
This study has two aims. The first is to confirm that sll1653 codes for the methyltransferase responsible for synthesizing 2-phytyl-3-methyl-1,4-NQ from
2-phytyl-1,4-NQ. Indeed, it was found that inactivation of ORF sll1653 resulted in the synthesis of PS I complexes containing 2-phytyl-1,4-NQ in the A1 site. The second is to
study the roles of the methyl group in quinone binding and electron transfer in PS I. Here, it
was shown that 2-phytyl-1,4-NQ is present in the A1 site with the same orientation and
asymmetric spin-density distribution as PhyQ in native PS I. It was also reported that 79 2-phytyl-1,4-NQ functions in forward electron transfer with some attendant alteration of the PS I activity and electron-transfer kinetics. 80
RESULTS
Analysis of the Genotype of the menG Mutant Strain
As shown in Fig 3.2A, the menG gene (ORF sll1653) was inactivated by insertion of a 1.3-kb DNA cassette, encoding the aphII gene and conferring resistance to kanamycin, in the unique KpnI site within the coding sequence. Full segregation of the menG and menG::aphII alleles was confirmed by PCR amplification and by Southern blot-hybridization analysis of the genomic DNA. For PCR analysis, the primers flanking the menG coding sequence (Fig 3.2A small arrows) were used to amplify the DNA fragments from the wild-type and the menG mutant genomic DNAs. As expected, a 1.2-kb fragment was amplified by PCR for wild type (Fig 3.2B). For the menG::aphII mutant strain a 2.5-kb fragment was amplified by PCR; no DNA fragment with a size equal to that expected from the wild-type menG allele was detected. This result indicates that the menG mutant is homozygous for the menG::aphII gene interruption. To verify the menG mutation further, the genomic DNA isolated from the wild-type and the menG mutant strains were digested with restriction enzymes BglII and HindIII (see in Fig 3.2A) for the
Southern-blot hybridization analysis. Hybridization was performed using the PCR product from the wild type genome as a probe. A single band with the size of 2.3 kb was observed for the wild type strain (Fig 3.2C). For the menG mutant, two bands with the sizes of 2.7 kb and 1.5 kb were observed due to the presence of the BglII site in the Kmr cartridge. All of these results confirmed that the menG::aphII allele has fully segregated from the wild-type gene.
81 High Performance Liquid Chromatography/Mass Spectrometry Analysis
To determine whether the menG gene is involved in PhyQ biosynthesis,
solvent-extracted quinone fractions from PS I complexes isolated from the wild-type and
the menG mutant strains were subject to orthogonal acceleration/time-of-flight mass
spectrometry. Pigments and quinones extracted from the isolated PS I trimers were
separated by reverse-phase HPLC, and the molecular mass of each eluate was determined
by mass spectrometry. In the wild-type complexes, PhyQ was detected by its characteristic
retention time, its UV absorption spectrum, and its m/z of 450 (Fig 3.3A). In PS I from the
menG mutant strain, no compound absorbing at 270 nm eluted at this retention time, nor
was a m/z of 450 detected. Rather, a UV-absorbing component eluted near the retention
time of Chl a that had a m/z of 436, which corresponds to the mass of 2-phytyl-1,4-NQ (Fig
3.3B).
Growth Rate under Photoautotrophic and Photomixotrophic Conditions
As indicated by HPLC and mass spectrometry, mutation of the menG gene
resulted in the replacement of PhyQ by 2-phytyl-1,4-NQ in PS I complexes. To measure
the effectiveness of 2-phytyl-1,4-NQ in supporting photosynthesis, growth rates of the
wild-type and menG mutant strains were compared. The results are summarized in Table
3.1. Growth of the wild-type and menG mutant strains were virtually the same under low
(50 µE m-2 s-1) and moderate (150 µE m-2 s-1) illumination conditions, in either the presence
or absence of glucose (Table 3.1). When cells were grown photoautotrophically, doubling
times of both the wild-type and the menG mutant strains were ca. 10 h under low light
intensity conditions and ca. 8 h under moderate light intensity conditions. When cells were 82 grown photomixotrophically, doubling times of the wild-type and the menG mutant strains were both ca. 8 h under low light intensity conditions, and ca. 6 h under the moderate light intensity conditions. The observed differences in the growth rates between the wild-type and the menG mutant strains were all within the range of error. These results suggest that under these illumination regimes, replacement of PhyQ with 2-phytyl-1,4-NQ has no significant effect on growth.
PS II/PS I Ratio Measured by 77 K Fluorescence Emission Spectra
The relative ratios of PS II to PS I were compared by measuring 77 K fluorescence emission spectra of wild-type and the menG mutant cells grown at various light intensities.
The results are summarized in Fig 3.4. When cells were grown under low-light conditions, which is typical for Synechocystis sp. PCC 6803 (Fig 3.4A), the emission amplitude from
PS I at 721 nm was much greater than that from PS II at 695 nm. When cells were grown at higher light intensities, an obvious decrease at 721 nm was found in the emission spectra of the menG mutant cells when compared to wild-type cells (Fig 3.4B and 3.4C). In wild-type cells the emission peak at 721 nm due to Chl a associated with PS I decreased approximately 10% in cells grown at a light intensity of 300 µE m-2 s-1 relative to cells grown at a light intensity of 50 µE m-2 s-1, while emission peaks at 685 and 695 nm due to
Chl a associated with PS II remained nearly constant. However, the peak amplitude at 721 nm decreased substantially in the menG mutant cells grown at higher light intensities. The amplitude of this peak in cells grown at a light intensity of 300 µE m-2 s-1 was less than
70% that for cells grown at 50 µE m-2 s-1. The PS II/PS I ratio is slightly higher in the menG mutant than in wild type when grown at 50 µE m-2 s-1 and significantly higher when 83 grown at 300 µE m-2 s-1. These results suggest that there may be either a defect in the assembly of PS I complexes or an accelerated degradation of PS I complexes in the menG mutant cells grown at high light intensities.
Chlorophyll Content of Whole Cells Grown at Various Light Intensities
The chlorophyll contents of wild-type and the menG mutant cells grown under various light intensities were also determined (Table 3.2). When cells were grown under low or moderate light intensities (50 or 150 µE m-2 s-1), the chlorophyll content of the
menG mutant cells was similar to that of the wild-type cells. However, when the light
intensity was increased to 300 µE m-2 s-1, the chlorophyll content in the wild-type cells
decreased to approximately 70% of the initial value (Table 3.2). A significantly lower
value, approximately 58% of the value obtained at lower light intensities, was observed for
menG mutant cells grown at a light intensity of 300 µE m-2 s-1. This value is only 80% that
of the wild-type strain under the same conditions, and this decrease can be correlated with
the loss of fluorescence emission from PS I observable in the decreased emission at 721 nm
in Fig 3.4. In combination, the results from Table 3.2 and Fig 3.4 suggest that content of PS
I complexes is lower in the menG mutant strain when cells were grown at high light intensity. This difference could be due to a defect either in biogenesis or in stability of the
PS I complexes in the mutant strain.
Polypeptide Composition of Isolated PS I Trimers
To analyze whether the interruption of the menG gene has any effect on the PS I
subunit composition, PS I trimers were analyzed by SDS-polyacrylamide gel
electrophoresis. Polypeptide bands were visualized by silver staining. All of the eleven 84 polypeptides detected in the wild-type, PsaA though F and PsaI, J, L and M, were found to
be present in the menG mutant strain (Fig 3.5). No additional polypeptides were observed
in PS I complexes isolated from the menG mutant strain. These results indicate that the
replacement of PhyQ by 2-phytyl-1,4-NQ does not affect the subunit composition of the
PS I complexes in the menG mutant strain.
EPR spectroscopy of Q- and P700+ Q-
Fig 3.6A shows continuous wave EPR spectra measured at 34 GHz (Q-band) of
the photoaccumulated semiquinone radical (Q-) in PS I complexes isolated from the
wild-type (solid line, top) and the menG mutant strains (solid line, bottom). The PhyQ
anion radical is characterized (Fig 3.6A, solid line) by principal g-tensor values (Zybailov
et al., 2000) and the principal hyperfine A-tensor for the three protons in the methyl group
at the C2 position (Rigby et al., 1996) as summarized in Table 3.3. The hyperfine splitting due to the remaining aromatic hydrogen atoms and the methylene group of the phytyl chain
at the C3 position remain unresolved and are included in the inhomogeneous line
broadening using linewidths of 3.0, 6.0, and 4.0 G for each of the three g-tensor
components (Fig 3.6B, dotted line). The deviations of the experimentally obtained
spectrum (Fig 3.6B, solid line) from the simulated spectrum (Fig 3.6A, dotted line) seen in
- the mid- and high field regions are due to contamination by A0 .
The Q-band spectrum of the photoaccumulated semiquinone radical in the PS I
complexes isolated from menG mutant strain is shown in Fig 3.6B (solid line). The
spectrum has about the same overall width as that of wild type, which indicates that the
g-tensor of the semiquinone radical is about the same as that of the PhyQ anion radical. The 85 pattern of hyperfine splitting, however, is different in the menG mutant sample. The usual
four hyperfine lines are missing and instead, only two lines are present in the low-field
region. Such properties are expected if the semiquinone radical in the A1 site is replaced
with a 2-phytyl-1,4-NQ that lacks the methyl group at ring position 3 (see below). Indeed,
a satisfactory simulation of the spectrum of the menG mutant strain was obtained by modifying the wild-type simulation, with the A-tensor of the methyl group replaced by that of an aromatic C-H fragment with the same tensor components evaluated from the spin polarized spectra described in the next section (Fig 3.6B, dotted line).
Transient EPR Spectroscopy
Fig 3.7 compares transient, spin-polarized EPR spectra (X-, Q-, W-band) of the transient charge separated P700+ Q- state in the PS I complexes isolated from the wild-type
(dashed line) and the menG mutant strains (solid line). The two sets of spectra coincide
quite well in the high-field region, which is dominated by the P700+ contribution, but are
quite different in the low-field region. The W-band spectrum indicates a slightly larger
g-anisotropy for the menG mutant than that of the wild type PS I complex. The best fit
yields gxx = 2.00633 (menG) versus 2.00622 (wild type) while gyy = 2.00507 and gzz =
2.00218 remain unchanged from the wild type (Zech et al., 2000) as summarized in Table
3.3.
Closer inspection reveals that the main difference in the low field region concerns the partially resolved hyperfine pattern. It is sufficiently resolved to permit an initial, qualitative analysis directly from the spectra. For wild-type, the characteristic hyperfine pattern due to the 1:3:3:1 quartet associated with a methyl group in ring position 2 is 86 particularly obvious in the Q-band spectrum. The fact that it is partially resolved results
from the increased spin density at the carbon ring position to which the methyl group is
attached (specific to the semiquinone radical ion in the A1 site). The components of the
axially symmetric hyperfine tensor have been determined by ENDOR to be A11 = 12.8
MHz and A⊥ = 9.0 MHz (see Rigby et al., 1996, for summary and further references; see
also Kamlowski et al., 1998; Zech et al., 2000). In the menG mutant, the CH3 group is
expected to be replaced by an aromatic C-H fragment. Indeed, the quartet hyperfine pattern
due to the CH3 group is missing in the menG mutant spectrum. Instead it is replaced by a
doublet (menG), the center position of which is shifted down field with respect to the
center of the quartet (wild type). Such a shift is expected with an altered orientation of the
dominant hyperfine principal axis with respect to the molecular axis frame. As concluded
from the observed shift direction, the dominant hyperfine axis will point more in the
direction of the gxx(Q) tensor axis in the menG mutant while it points more in the direction
of the gyy tensor axis in the wild type. Since the dominant hyperfine axis A11 of the CH3 hyperfine tensor is usually almost parallel to the C-CH3 bond axis, its direction is closer to
(about 30 degrees off) the gyy tensor axis. In contrast, the direction of the dominant
hyperfine axis of the aromatic C-H fragment (which is in-plane and perpendicular to the
C-H bond, see Carrington and MacLachlan, 1967) is closer to (about 30 degrees off) the
gxx(Q) tensor axis. The associated downfield shift of the center of the hyperfine doublet is
therefore in agreement with the experimental result.
To prepare for a quantitative simulation of the spectra, the following findings
were taken into account. The spin polarization pattern shows that 2-phytyl-1,4-NQ has the 87
same orientation in the A1 site as native PhyQ (Fig 3.7). The same P700 to A1 center-to-center distance has been verified by out-of-phase, spin-echo modulation spectroscopy (data not shown). An independent determination of the hyperfine tensor components of the C-H fragment has been obtained from pulsed ENDOR spectroscopy of the same P700+ Q- state in the menG mutant (data not shown). The principal hyperfine
tensor components are given in Table 3.3. While the two large components, A11 and A22 are clearly resolved, the third component is part of the line manifold in the center part of the
ENDOR spectrum and cannot be evaluated. With the reasonable assumption that the spin density of the relevant carbon ring position does not change with respect to wild type
(corresponding to Aiso = -10.3 MHz with the appropriate McConnell relation) one can
estimate the third hyperfine principal component to be A33 = -3.6 MHz.
Although the axis associated with the largest hyperfine component is expected to
be in-plane and perpendicular to the C-H bond, this direction can be influenced by
significant spin densities on the neighboring molecular atoms, such as the spin density that
is known to reside on the carbonyl groups of the semiquinone radical. Therefore,
simulations of the Q-band spectrum (Fig 3.8) were carried out for various angles between
the largest principal axis A11 and the gxx (Q) axis (in-plane along the carbonyl bond direction). The A11 axis is perpendicular to the C-H bond direction when this angle is 30°.
Indeed, of the simulations shown, the one for this value (Fig 3.8, bottom, long dashed curve) comes closest to the experimental spectrum (Fig 3.8, top). A slightly smaller angle than 30 degrees improves the simulation result to some degree. This indicates a weak influence from spin density on neighboring atoms. The only significant spin density is 88 expected for the oxygen atom of the carbonyl group in ring position 1, as concluded from the alternating spin density distribution due to asymmetric H-bonding to the opposite carbonyl group in position 4 (see Kamlowski et al., 1998; Zech et al., 2000, and references therein). However, the oxygen atom is already quite distant from the proton spin of the C-H fragment, and thus the deviation of the C-H fragment hyperfine tensor from the (3:2:1) ratio of the undisturbed C-H fragment is small, as indeed is observed here.
- Kinetics of Forward Electron Transfer from A1 to FX
Forward electron transfer kinetics from the quinone to the iron sulfur clusters in
PS I complexes isolated from the wild-type and the menG mutant are compared in Fig 3.9
and Fig 3.10, which show spin-polarized EPR transients and decay-associated spectra,
respectively. The transients in Fig 3.9 labeled a, b and c were taken at the corresponding
field positions indicated with arrows in Fig 3.10. In wild-type PS I complexes, two
- consecutive spectra are observed as electron transfer from A1 to FX occurs. At early times
+ - (< ~1 µs) an emission/absorption/emission (EAE) pattern due to P700 A1 is observed;
this pattern changes to an emissive spectrum due to P700+ in the state P700+ FeS- at later
times (> ~1 µs). The identity of the iron-sulfur cluster partner to P700+ in the state giving
the late spectrum requires some discussion. It has been shown that electron transfer
+ - proceeds via FX (van der Est et al., 1994), which suggests the spectrum is due to P700 FX .
- However, there is evidence (Leibl et al., 1995) that electron transfer from FX to FA/FB is
+ faster than the transfer from A1 to FX. In this case, the late spectrum would be due to P700
- + - FA and/or P700 FB . Calculations (Kandrashkin et al., 1998) show the polarization
+ - + - + - patterns corresponding to P700 FX , P700 FA , and P700 FB show only minor 89 differences. Thus, the state giving the late spectrum P700+ FeS- is labeled P700+ FeS- and the identity of the iron sulfur cluster(s) is left open. In the kinetic traces (Fig 3.9), the electron transfer is seen most clearly at field position b. The positive (absorptive) signal at early times (< ~50 ns) is due to P700+ Q-, while the negative (emissive) signal at late times
(< ~1 µs) is due to P700+ FeS-. The electron transfer dominates the decay of the early signal
while relaxation of the spin polarization causes the decay of the late signal. At positions a and c the intensity of the late signal is weak and thus the transients decay primarily with the electron transfer rate. As can be seen at all three field positions shown in Fig 3.10, the rate of electron transfer is considerably slower in the mutant than in the wild type sample. For the mutant PS I complexes, a fit of the entire data set as described in (van der Est, 1994) yields an average electron transfer lifetime of 600 ± 100 ns. This compares with a value of
290 ± 70 ns for the wild-type complexes.
Kinetics of Charge Recombination Measured by Optical Spectroscopy
The reduction of P700+ was monitored at 811 nm after a brief laser flash in PS I trimers isolated from the wild-type and the menG mutant strains (Fig 3.11). Because no
terminal electron acceptors such as ferredoxin or flavodoxin were present, the reduction
+ - + rate of P700 represents the rate of charge recombination between [FA/FB] to P700 . The kinetics of P700+ reduction in the wild-type PS I complexes were fitted by one stretched
exponent with (1/e) lifetimes of 0.70, 30.7, and 94.7 ms (Fig 3.11A). The latter two values
are similar to the two lifetime components found previously for the P700-FA/FB complexes
from Synechocystis sp. PCC 6803 and are attributed to charge recombination between
- + [FA/FB] and P700 (Vassiliev et al., 1997). The first value is probably due to charge 90 + - recombination between P700 and FX in damaged PS I complexes that lack an intact PsaC
subunit. The kinetics of P700+ reduction in PS I complexes from the menG mutant (Fig
3.11B) were also fitted by two exponentials with (1/e) lifetimes of 10.3 and 29.3 ms, These
- + values are attributed to charge recombination between [FA/FB] and P700 and are ca. 3
times faster than the wild-type rates. Even though the reason for the heterogeneity in P700+ reduction is not understood, it is noteworthy that the two kinetic phases attributed to charge
- + recombination between [FA/FB] and P700 are equally slowed in the menG mutant
samples.
Steady-State Rates of Flavodoxin Reduction
Steady-state rates of flavodoxin reduction as a function of light intensity were
measured for PS I complexes isolated form the wild-type and menG mutant strains to
assess the relative efficiencies of forward electron transfer. The rates at saturating light
intensity were determined by treating light as a substrate in a Michaelis-Menten kinetic analysis (Fig 3.12). The maximal rate of flavodoxin reduction was found to be 1527 ± 247
µmol (mg Chl a)-1 h-1 for the wild-type PS I complexes and 1845 ± 399 µmol (mg Chl a)-1 h-1 for the menG mutant PS I complexes. Assuming that 100 Chl a molecules are present per P700 in all PS I complexes, these maximal rates of electron transfer correspond to 37.9
± 6.2 e- PS I-1 s-1 in the wild-type PS I complexes and 45.8 ± 8.4 e- PS I-1 s-1 in the menG
mutant PS I complexes. Although the value obtained for the PS I complexes from the
menG mutant was slightly higher than that for the wild-type, this difference is within the
margin of error and therefore is not statistically significant (Fig 3.12). Hence, the steady-state rate of electron transfer for the PS I complexes of the menG mutant, which 91 contain 2-phytyl-1,4-NQ, was virtually identical to the steady-state rate of electron transfer for the PhyQ-containing PS I complexes of the wild-type.
92 DISCUSSION
The menG gene, encoding the 2-phytyl-1,4-NQ (demethylphylloquinone) methyltransferase, was identified by targeted inactivation of ORF sll1653 in Synechocystis sp. PCC 6803. This open reading frame was originally assigned as gerC2, which encodes the spore germination protein C2 in Bacillus species and other spore-former bacteria
(Kaneko et al., 1996). In fact, the deduced amino acid sequence for ORF sll1653 shows only 27% sequence identity to the menG gene product of E. coli. Targeted inactivation of
ORF sll1653 was accomplished by insertion of a kanamycin resistance cartridge in the coding region, which was confirmed by PCR amplification of the region containing the
ORF and by Southern-blot hybridization analysis. The presence of 2-phytyl-1,4-NQ in PS I complexes was confirmed by high performance liquid chromatography and mass spectrometry, and its function in electron transfer was demonstrated by CW and transient
EPR spectroscopy. The orientation and distance of 2-phytyl-1,4-NQ relative to P700 was confirmed to be the same as for PhyQ in wild-type PS I complexes by transient EPR, out-of-phase, spin-echo modulation, and pulsed ENDOR spectroscopy. It can be safely concluded that ORF sll1653 in Synechocystis sp. PCC 6803 is menG; that it encodes
2-phytyl-1,4-NQ methyltransferase, the enzyme required for the last step of the PhyQ biosynthetic pathway; and that 2-phytyl-1,4-NQ functionally replaces PhyQ at the A1 site in PS I complexes.
Despite the absence of PhyQ and the presence of 2-phytyl-1,4-NQ in PS I complexes, the growth rates of the wild-type and menG mutant cells were identical. As observed for many other cyanobacteria (Fujita, 1997), the ratio of PS II to PS I is known to 93 change in Synechocystis sp. PCC 6803 as a function of the light intensity during cell
growth (G. Shen, J. H. Golbeck, and D. A. Bryant, unpublished observation). An
interesting observation was that the change in the ratio of PS II to PS I was more
pronounced in the menG mutant strain than in the wild type as the light intensity was
increased from 50 µE m-2 s-1 to 300 µE m-2 s-1. In wild-type cells, the PS II to PS I ratio
increased by a factor of 1.1, while in the menG mutant cells the PS II to PS I ratio increased
by a factor of 2 as estimated from the amplitudes of the fluorescence emission spectra at 77
K. It is noteworthy that the overall chlorophyll content in the wild-type and menG mutant cells were similar at light intensities of 50 and 150 µE m-2 s-1 and differed only at 300 µE
m-2 s-1. Therefore, the difference in the PS II to PS I ratio between wild type and the menG
mutant strain can be primarily attributed to a change in the content of PS I, i.e. the number of PS I complexes declines in the menG mutants to a greater extent than in the wild-type
cells as the light intensity is raised. By way of comparison, the menA and menB mutant
strains, which contain plastoquinone-9 in the A1 site, have been observed to grow significantly more slowly and can be grown only under very low light intensity conditions
(< 20 µE m-2 s-1). The ratio of PS II to PS I is already greater than 2:1 at low light intensity,
and might be expected to be even greater as the light intensity is increased (the cells,
however, do not survive). Thus, it appears that Synechocystis sp. PCC 6803 must exceed ca. 2:1 ratio of PS II to PS I before light becomes toxic to the growing cells.
To understand further the behavior of 2-phytyl-1,4-NQ in electron transfer, one must consider the redox potential of this quinone in the A1 site. In aqueous solution, the
- E(Q/Q )7 of 2-methyl-1,4-NQ is –203 mV vs. NHE (normal hydrogen electrode) and the 94 - - E(Q/Q )7 of 2,3-dimethyl-1,4-NQ is –240 mV vs. NHE (Swallow, 1982). The E(Q/Q )7 of
2-phytyl-3-methyl-1,4-NQ (PhyQ) in aqueous solution (containing 5 M 2-propanol and
2M acetone) is reported to be –170 mV. The presence of the second alkyl group, ortho to the first, therefore lowers the redox potential of the quinone by ca. 37 mV. However, the A1 site is highly hydrophobic (Iwaki and Itoh, 1991, 1994), and reduction potentials of quinones in organic solvent are probably more relevant to their properties in PS I complexes. In dimethylformamide (DMF), the E1/2 value for 2-methyl-1,4-NQ has been
reported as –650 mV vs. SCE and the E1/2 value for 2,3-dimethyl-1,4-NQ has been reported
to be –746 mV (Prince et al., 1983) vs. SCE (standard calomel electrode). The lower redox
potential for the dimethyl quinone is logical chemically; a methyl group is an electron
donor and should therefore destabilize the semiquinone radical in either aqueous solution
or in organic solvent. A prenyl substituent on the quinone ring is not as effective as a
second alkyl group at electron donation and lowers the E1/2 value by only 50 to 60 mV
(Prince et al., 1983). For example the E1/2 of menaquinone-2
(2-all-trans-polyprenyl-3-methyl-1,4-NQ) is –746 mV vs. SCE (to the best of our
knowledge, the reduction potential of 2-phytyl-1,4-NQ in DMF is not available). Using the
redox potentials for 2-methyl-1,4-NQ and 2,3-dimethyl-1,4-NQ as models for
2-phytyl-1,4-NQ and PhyQ (both differ by the addition of one methyl group), the addition
of a second methyl group ortho to the first should lower the redox potential by ca. 96 mV.
If one further employs the concept of an ‘acceptor number’ that was utilized for PQ-9 in the
menA and menB mutants (Semenov et al., 2000), the redox potential of 2-phytyl-1,4-NQ in
the A1 site would be 61 mV more oxidizing than PhyQ. An acceptor number corrects the 95
E1/2 value for the degree of the nucleophilic (donor) or electrophilic (acceptor) properties of
the solvent (Gutmann, 1976) and was employed initially by Itoh (Iwaki and Itoh., 1994) to
estimate the redox potential of PhyQ in the A1 site of PS I. It is a dimensionless number that
expresses the acceptor properties of a solvent relative to SbCl5.
The estimated redox potential of 2-phytyl-1,4-NQ in the A1 site can now be
compared to estimates based upon the experimentally measured rate of electron transfer.
Forward electron transfer is ca. 3-times slower when 2-phytyl-1,4-NQ rather than PhyQ
occupies the A1 site. According to the Moser-Dutton formulation (Moser et al., 1992), the
rate of electron transfer in proteins depends on the Gibbs free energy between the
donor-acceptor pair, the edge-to-edge distance between the donor-acceptor pair, and the
reorganization energy. The distance between the 2-phytyl-1,4-NQ anion radical and P700+ was experimentally determined here to be the same as the center-to-center distance between the PhyQ anion radical and P700+. The orientation of 2-phytyl-1,4-NQ relative to the membrane plane was also shown to be identical to that of PhyQ. Hence, the edge-to-edge distance between 2-phytyl-1,4-NQ and FX is likely to be the same as the
distance between PhyQ and FX. It seems reasonable to assume similar reorganization
energies when 2-phytyl-1,4-NQ and PhyQ occupy the A1 site, given that the only
difference between the two quinones is the presence or absence of a single methyl group.
Using the values of 11.3 Å for the edge-to-edge distance between the quinone and FX
(Klukas et al., 1999), and a value of 0.7 eV for the reorganization energy, a (1/e) lifetime of ca. 290 ns for Q- in the wild-type translates to an 81 mV difference in Gibbs free energy
- - between Q /Q and FX /FX. Retaining the same values for the distance and reorganization 96 energy, a (1/e) lifetime of 600 ns for Q- in the menG mutant translates to a 28 mV
- - difference in the Gibbs free energy between Q /Q and FX /FX. According to this analysis,
the addition of methyl group to 2-phytyl-1,4-NQ lowers the redox potential of the quinone
in the A1 site by 53 mV. This value is only a rough estimate, especially given the
uncertainties in the edge-to-edge distance between the cofactors and given the assumed
(unchanged) reorganization energy of the site. Nevertheless, this value agrees quite well
with the difference in redox potentials of 2-methyl-1,4-NQ and 2,3-dimethyl-1,4-NQ in
organic solvents, especially after correction for the site’s “acceptor number”. The
replacement of PhyQ by 2-phytyl-1,4-NQ would therefore lower the equilibrium constant
- - between Q /Q and FX /FX from 27 to 3, a factor of ca. 10. Estimating the absolute redox potential of 2-phytyl-1,4-NQ in the A1 site is somewhat more difficult because the redox
potential of A1 is not known with certainty. Three values have been published for the redox
potentials of the Q-/Q couple [-810 mV vs. NHE (Vos and Gorkom, 1990), -800 mV vs.
NHE (Chamrovoskii and Cammack, 1983) and -754 mV vs. NHE (Iwaki and Itoh, 1994)].
If one uses the lower redox potentials, the replacement of PhyQ by 2-phytyl-1,4-NQ would
therefore raise the redox potential of the Q-/Q couple to ca. –750 mV, a value that is
- probably still lower than the redox potential of the FX /FX couple.
The kinetics of reduction of P700+ after a single flash represents dissipative
+ - charge recombination between P700 and terminal electron acceptors [FA/FB] in the
absence of an acceptor such as ferredoxin or flavodoxin. It is not yet clear whether electron
- + transfer occurs directly from [FA/FB] to P700 , or whether the electron travels backward 97 by a thermally activated, uphill electron transfer through the quinone. The latter
------presupposes that each redox pair (Q /Q ↔ FX /FX; FX /FX ↔ FB /FB; FB /FB ↔ FA /FA) is
described by an equilibrium constant that can be determined from the midpoint potentials
of the acceptors. The large distance between P700 and the terminal iron-sulfur clusters [46
Å center-to-center distance from P700 to FA (Schubert et al., 1998)] argues against the
direct recombination mechanism, although experimental data to support thermally
activated uphill electron transfer have been lacking. If the charge recombination between
- + [FA/FB] and P700 were to occur through backward electron transfer, then a change in the
- - equilibrium constant between Q /Q and FX /FX should logically affect the rate. The
- - equilibrium constant between Q /Q and FX /FX would be correspondingly higher as the
redox potential of Q-/Q were driven more negative, thereby resulting in a depopulation of
- - reduced quinone. Conversely, the equilibrium constant between Q /Q and FX /FX would be correspondingly lower as the redox potential of Q-/Q were driven more positive, thereby
resulting in a repopulation of reduced quinone. Consequently, the back-reaction kinetics in
the mutant would be faster than in wild-type PS I complexes.
The latter description is precisely the behavior observed. The forward
- electron-transfer kinetics from Q to FX becomes correspondingly slower as PhyQ [1/e
lifetime of 200 ns (van der Est et al., 1994)], 2-phytyl-1,4-NQ (1/e lifetime of 600 ns), and
PQ-9 [1/e lifetime of 15 µs (Semenov et al., 2000)] occupy the A1 site. Conversely, the
- + charge recombination kinetics between [FA/FB] and P700 become more rapid as PhyQ
[1/e lifetime of 80ms (Vassiliev et al., 1997)], 2-phytyl-1,4-NQ (1/e lifetime of 20 ms) and
PQ-9 [1/e lifetime of 3 ms (Semenov et al., 2000)] occupy the A1 site. The trend among the 98 wild type, menA or menB, and menG mutants therefore strongly supports the backward,
through-carrier route of electron transfer for charge recombination. Since the redox
potential of the quinone in the A1 site influences the rate of charge recombination from the
terminal electron acceptors to P700+ as well as the forward electron transfer between the quinone and the terminal iron-sulfur clusters, this suggests that charge recombination
occurs by thermally activated, uphill electron transfer through the secondary quinone
acceptor in PS I.
Finally, although the kinetics of electron transfer within PS I are altered when
PhyQ is replaced by 2-phytyl-1,4-NQ, these changes do not affect the growth rate of the
organism. An explanation for this apparent discrepancy is that the slower rate of forward
- electron transfer from A1 to FX is still not the rate-limiting step in the overall electron
transfer reaction to flavodoxin (or probably to ferredoxin). Indeed, the maximal rate of
flavodoxin reduction of the PS I complexes isolated from the menG mutant was virtually identical to that observed for wild-type PS I complexes. Analysis of the rate of electron donation from PS I to flavodoxin is complicated by the two-electron reduction using this acceptor. Since the potentials at pH 7 for the oxidized flavodoxin/flavodoxin semiquinone and the flavodoxin semiquinone/hydroquinone couple are –212 and –436 mV (Schubert et al., 1998), respectively, it is likely that only the latter couple is physiologically relevant.
Studies show that the reduction of flavodoxin semiquinone is biphasic; the fast, first-order phase corresponds to electron transfer within a preformed complex, but the slower phase is concentration-dependent, with a second-order rate constant of 1.7 x 108 M-1 s-1. Given the nearly wild-type electron-transfer rates from cytochrome c6 to flavodoxin in the menG 99 mutant, it is clear that forward electron transfer continues to out-compete the faster back
reaction rate in the mutant. Hence, PS I complexes are remarkably robust and are tolerant to changes caused by replacement of wild-type components with “suboptimal” ones. This robustness still allows for a remarkable level of functionality of the bound electron-transfer cofactors under suboptimal conditions.
100
SUMMARY
The menG gene, encoding the 2-phytyl-1,4-NQ (demethylphylloquinone)
methyltransferase, was identified in Synechocystis sp. PCC 6803. The PS I complexes
isolated from the menG mutant accumulated 2-phytyl-1,4-NQ, which functioned as the A1 cofactor in the electron transfer. The rate of forward electron transfer from A1 to the
iron-sulfur clusters was slowed by a factor of two, while the rates of the P700+ [FA/FB]-
backreaction increased by a factor of 3 to 4. These results were explained by the lowering
- - of the equilibrium constant between Q /Q and FX /FX by a factor of ~10. Despite the defect
in the forward electron transfer, the PS I complexes containing 2-phytyl-1,4-NQ showed
catalytic activity similar to those of PS I complexes containing phylloquinone, which
demonstrates that PS I complexes are remarkably robust and can function with presumably
“suboptimal” electron transfer cofactors. 101
MATERIALS AND METHODS
Generation of the menG Mutant Strain of Synechocystis sp. PCC 6803
To generate the construct for inactivation of the menG gene, a 1.2-kb DNA
fragment containing the sll1653 ORF was amplified through PCR from the genome of the
Synechocystis sp. PCC 6803. For cloning convenience, a new EcoRI site was created in the
3'-end primer with one nucleotide change. The PCR-amplified DNA fragment was cloned
into pUC19 vector and confirmed by sequencing. To inactivate the menG gene, a 1.3-kb
kanamycin-resistance cartridge encoded by the aphII gene was inserted into the KpnI site
in the middle of the menG gene. The resulting plasmid was linearized by digestion with
EcoRI and was used to transform the Synechocystis sp. PCC 6803 wild-type cells. The
transformation and the transformant screening were carried out as described (Shen et al.,
1993). After several rounds of restreaking to single colonies, full segregation of the
menG::aphII from the menG alleles was verified by PCR and Southern blot-hybridization analyses.
Growth of Synechocystis sp. PCC 6803
Wild-type Synechocystis sp. PCC 6803 was grown in medium B-HEPES. This medium is prepared by supplementing BG-11 medium (Stainer et al., 1971) with 4.6 mM of HEPES [4-(2-hydroxyethyl)piperazine-N’-(2-ethnesulfonic acid)]-KOH and 18 mg L-1 ferric ammonium citrate. The menG mutant cells were grown in medium B-HEPES supplemented with 50 µg mL-1 kanamycin. The growth temperature was maintained at 32o
C and the light intensity was adjusted to 50, 150, and 300 µE m-2 s-1 by adding fluorescent 102 lamps or by shielding with paper as required. For photomixotrophic growth, the medium
was supplemented with 5 mM glucose. Growth of the cells was monitored by measuring
the absorbance at 730 nm using a Cary-14 spectrophotometer that had been modified for
computerized data acquisition by On-Line Instruments, Inc (Bogart, GA). Cells from a
liquid starter culture in the exponential phase (OD730nm < 0.7) were adjusted to the same initial cell density (OD730nm = 0.05) for growth-curve measurements. The liquid cultures
were bubbled with 1% (v/v) CO2 balanced with air.
DNA Isolation, PCR, and Southern Blotting
Chromosomal DNA from Synechocystis sp. PCC 6803 wild-type and mutant
strains was isolated as previously described (Shen, et al., 1993). PCR primers used to
amplify the menG DNA fragment for evaluation of the menG alleles were positioned as
shown in the Fig 3.2. The sizes of the PCR products from the wild type and menG mutant
strains were determined by agarose gel electrophoresis. For Southern blot-hybridization
analysis, the chromosomal DNAs were subjected to restriction enzyme digestion, agarose
gel electrophoresis, and capillary transfer to nitrocellulose membranes. Hybridization
probes were generated by labeling the wild-type menG PCR fragment with the
random-primed DNA labeling kit (Boehringer-Mannheim, Indianapolis, IN).
Hybridization conditions and detection were as previously described (Shen and Bryant
1995)
103 Chlorophyll Extraction and Analysis
Chlorophyll was extracted from whole cells, thylakoid membranes and PS I
particles with 100% methanol, and its concentration was determined
spectrophotometrically as described (MacKinney, 1941).
Isolation of Thylakoid Membranes and PS I Particles
Thylakoid membranes were isolated from cells in the late exponential growth
phase as described previously (Shen et al., 1993). Cells were broken at 4°C by three
passages through a French pressure cell at 124 MPa. The thylakoid membranes were resuspended and solubilized for 2 h at 4°C in the presence of 1% (w/v)
n-dodecyl-β-D-maltoside. PS I complexes were separated from other membrane components by centrifugation on 5-20% (w/v) sucrose gradients with 0.05% n-dodecyl-β-D-maltoside in the buffer. Further purification was achieved by a second centrifugation on sucrose gradients in the buffer in the absence of n-dodecyl-β-D-maltoside. Trimeric PS I complexes were used in these studies (Golbeck,
1995).
SDS-Polyacrylamide Gel Electrophoresis Analysis
Methods used for SDS-polyacrylamide gel electrophoresis were identical to those previously described (Shen and Bryant, 1995). A Tricine/Tris discontinuous buffer system was used to resolve the polypeptide composition of the PS I complexes prepared from the wild type and the menG mutant strains of Synechocystis sp. PCC 6803 (Schägger and van 104 Jagow, 1987) The separating gel contained 16% acrylamide and 6 M urea, and the resolved
proteins were visualized by silver staining (Blum et al., 1987)
Analysis of Quinones by High Performance Liquid Chromatography and Mass Spectrometry
For quinone extraction, PS I particles isolated from the wild-type and the menG
mutant strains were exchanged into distilled water by dialysis for 4 h and lyophilized. The
pigments were extracted from the lyophilized samples with acetone:methanol, 1:1 (v/v) at
4 °C, vacuum dried at room temperature in the dark, and resuspended in 100% methanol.
The extracts were separated by reverse-phase high performance liquid chromatography
(HPLC) using a C18 column as previously described (Johnson et al., 2000). Eluates were
analyzed using atmospheric-pressure chemical ionization with a Micromass Quattro II
mass spectrometer operated in the negative-ion mode.
Flavodoxin Photoreduction
Steady-state rates of flavodoxin reduction were measured for the PS I trimers
isolated from the wild-type and the menG mutant strains as previously described (Jung et
al., 1995). Cyanobacterial flavodoxin was overexpressed in E. coli and purified as
described (Fillat et al., 1991). PS I trimers were resuspended to a final concentration of 5
-1 µg Chl mL in 25 mM Tris-HCl buffer, pH 8.3, containing 50 mM MgCl2, 15 µM
cytochrome c6, 15 µM flavodoxin, 6.0 mM sodium ascorbate, and 0.05 %
n-dodecyl-β-D-maltoside. Measurements were made by monitoring the rate of change in
the absorption at 467 nm using a modified Cary 219 spectrophotometer fitted with a 465
nm interference filter on the surface of the photomultiplier. The 4-sided clear cuvette was 105 illuminated from two sides using high intensity, red light-emitting diodes (LS1, Hansatech
Ltd., Norfolk, UK). Initial rates of the reaction were recorded under various light
intensities and plotted as a function of the light intensity to assess the relative efficiency of
forward electron transfer to flavodoxin.
77 K Fluorescence Emission Spectra
Fluorescence emission spectra were measured at 77 K using an SLM 8000C
spectrofluorometer as previously described (Shen and Bryant, 1995). Cells from the
exponential phase of growth were harvested and resuspended in 25 mM HEPES/NaOH,
pH 7.0, containing 60% glycerol. Samples were adjusted to equal cell density (OD730nm =
1.0) prior to freezing in liquid nitrogen. The excitation wavelength was 440 nm, the
excitation slit width was 4 nm, and the emission slit width was 2 nm. Each spectrum shown
is the average of four spectra.
Q-band EPR of Photoaccumulated PS I Complexes
Photoaccumulation experiments were performed using a Bruker ER300E
spectrometer and an ER 5106-QT resonator equipped with an opening for in-cavity
illumination similar to that described in (Yang et al., 1998). Low temperatures were maintained with an ER4118CV liquid nitrogen cryostat and an ER4121 temperature controller. The microwave frequency was measured with a Hewlett-Packard 5352B frequency counter and the magnetic field was measured with a Bruker ER035M NMR gaussmeter. The pH of the sample was adjusted to 10.0 with 1.0 M glycine buffer, and sodium dithionite was added to a final concentration of 50 µM. After incubation for 10 min in the dark, the sample was placed into the resonator and the temperature was adjusted to 106 205 K. The sample was illuminated for 40 min with a 20 mW He-Ne laser operating at 630 nm. A dark background spectrum was subtracted from the photoaccumulated spectrum.
EPR spectral simulations were carried out on a 466 MHz Power Macintosh G3 computer using a Windows 3.1 emulator (SoftWindows 3.0, Insignia Solutions, UK) and SimFonia software (Bruker Analytik GMBH, Germany).
Time-Resolved EPR Spectroscopy at Multiple Frequencies
Transient EPR spectra as well as pulsed EPR/ENDOR data were obtained using the same instrumentation described earlier (Zybailov et al., 2000; Bittl et al., 1997).
Flash-Induced Absorbance Changes at 811 nm
The transient absorbance changes were measured with a home-built, double-beam spectrophotometer described in (Vassiliev et al., 1997). The sample was prepared at a chlorophyll concentration of 7 µM in 50 mM Tris buffer, pH 8.3. Measurements were made at pH 8.3 in 50 mM Tris buffer in the presence of 2 mM sodium ascorbate and 5 µM
2,6-dichlorophenolindophenol. The samples were measured in a quartz cuvette with a
1-cm path for the measuring light. The excitation beam was provided by a frequency-doubled Nd-YAG laser (532 nm) with the flash energy attenuated to ca. 20 mJ using the Q-switch delay and neutral density filters. The measuring beam was provided by a 5 mW semiconductor laser operating at 811 nm. Kinetic analysis was performed using a non-linear regression algorithm in Igor Pro (WaveMetrics Inc., Lake Oswego, OR)
(Vassiliev et al., 1997). All kinetic constants are reported as 1/e lifetimes (τ). 107 X-band Transient EPR Spectroscopy at Room Temperature
Room-temperature, transient EPR experiments were carried out using a modified
Bruker ESP 200 equipped with a home-built broad-band amplifier (bandwidth >500 MHz) for direct detection experiments. Excitation was provided by a Continuum YAG/OPO laser system operating at 680 nm and 1 Hz. The EPR signals were digitized using a LeCroy
LT322 500 MHz digital oscilloscope and transferred to a PC for storage and analysis. The samples were measured using a flat cell and a Bruker rectangular resonator fitted with a piece of rough-surfaced glass in front of the window to provide optimal illumination. The response time of the system in direct detection mode is limited by the bandwidth of the resonator and is estimated as ~50 ns, and the decay of the spin polarization limits the accessible time range to times shorter than a few microseconds. The same set-up can also be used with field modulation and lock-in detection. In this mode the response time is ~50
µs but the sensitivity is much higher, and the charge recombination can be monitored in the millisecond time range. In both modes of operation, full time/field data sets are collected and analyzed to determine the lifetimes of the species and their decay-associated spectra as described in detail (van der Est et al., 1994; Semenov et al., 2000). 108
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Table 3.1: Doubling times of Synechocystis sp. PCC 6803 wild type and the menG mutant strains (hours).
Strains Growth conditions Wild type menG
Photoautotrophic, 50 µE m-2 s-1 (n = 4) 9.8 ± 0.0.85 9.8 ± 0.95
Photoautotrophic, 150 µE m-2 s-1 (n = 3) 8.1 ± 0.12 8.1 ± 0.25
Photomixotrophic, 50 µE m-2 s-1 (n = 4) 7.9 ± 0.90 7.5 ± 0.47
Photomixotrophic, 150 µE m-2 s-1 (n = 4) 5.6 ± 0.73 6.3 ± 0.56
115 Table 3.2: Chlorophyll content in cells grown photoautotrophically at 32° C under various illumination conditions.
Chlorophylls were extracted from whole cells with methanol. The absorbance coefficient of 82 (µg/ml) –1 at 666 nm was used to calculate the chlorophyll content in 1 ml of the cultures with an optical density of 1.0 at 730 nm.
µg Chlorophyll / OD730nm (n = 3) Light intensity -2 -1 (µE m s ) Wild type menG
50 3.55 ± 0.24 3.40 ± 0.14
150 3.65 ± 0.21 3.40 ± 0.29
300 2.48 ± 0.34 1.98 ± 0.15
116 - - Table 3.3: Magnetic parameters of PhyQ (A1 ) and 2-phytyl-1,4-NQ (Q ) in the A1 binding site.
g-tensors a
gxx gyy gzz
b A1- 2.00622 2.00507 2.00218
Q- 2.00633 2.00507 2.00218
Hyperfine coupling principal values (MHz)
A11 A22 A33
- b A1 12.8 9.0 9.0
Q- -15.5 ± 0.5 -11.8 ± 0.5 <5
a the principal axes of the g-tensors are shown in Fig 3.1. b Zech et al., (2000) J. Phys. Chem.
B 104, 9728-9739 and references therein. 117
Fig 3.1: Relationship between the molecular structure of 2-phytyl-1,4-NQ and the axes of the electronic g-tensor. The axes of the electronic g-tensor of 2-phtyl-1,4-NQ (indicated by arrows) are parallel to the molecular axes system. 118
Fig 3.2: Construction and verification of the menG mutant strain of Synechocystis sp. PCC 6803. (A) Restriction map of the genomic region surrounding the menG gene in the wild type (top) and the menG mutant strain (bottom). The small arrows indicate the position of the PCR primers used to amplified the menG coding region. (B) Electrophoretic analysis of the DNA fragments amplified from the genomic DNA of wild type and the menG mutant strain through PCR analysis. (C) Southern-blot analysis of the genomic DNA isolated from wild type and menG mutant strain. Chromosomal DNA was digested using restriction enzymes BglII and HindIII. DNA fragment obtained by PCR from the wild type genome was used as probe, as shown in top panel. 119
Fig 3.3: Mass spectra recorded for pigments and quinone extracts from the PS I complexes isolated from the wild-type and menG mutant strains of Synechocystis sp. PCC 6803. (A) Wild type, and (B) the menG mutant. 120
Fig 3.4: 77 K Fluorescence emission spectra from whole cells of Synechocystis sp. PCC 6803. Chlorophyll fluorescence emission spectra were measured for the whole cells of wild type (solid lines) and the menG mutant strain (dotted lines) grown under three different light intensities, (A) 50 µE m-2 s-1, (B) 150 µE m-2 s-1, (C) 300 µE m-2 s-1. Each spectrum was recorded at the same cell density and presented as the average of four measurements. 121
Fig 3.5: SDS-PAGE analysis of PS I complexes isolated from the wild-type and menG mutant strains of Synechocystis sp PCC 6803. The equal amount of Chl a (5 µg) is loaded for each lane. Polypeptides were visualized after silver staining. 122
Fig 3.6: Photoaccumulated Q-band EPR spectra and simulations of PS I complexes from Synechocystis sp. PCC 6803.
Wild type (solid line), and the simulated spectrum (dotted line). B, menG mutant strain (solid line), and the simulated spectrum (dotted line). See text for details of the parameters of the simulation. 123
Fig 3.7: Transient spin-polarized EPR spectra (X-, Q- and W-) of the charge separated P700+ Q- state in PS I trimers. Wild-type spectra (dashed lines) and the menG mutant (solid lines). The frequencies are from bottom to top: 9, 35, and 95 GHz. 124
Fig 3.8: Simulations of the Q-band spectrum of the menG mutant carried out for various angles between the largest principal axis A11 and the gxx axis. Top: experimental spectrum as in Fig 3.4 (middle). Bottom: simulations as described and with the parameters specified in the text. 125
MenG Wild Type a
b
c
0246 024 time/µs time/µs
Fig 3.9: Spin-polarized EPR transients of wild type and menG mutant.
The traces a, b, c were collected using direct detection at the field positions indicated by the similarly labeled arrows in Fig 3.10. The traces on the left are for PS I complexes isolated from the menG mutant, and those on the right are those from the wild type. 126
Wild Type
+ - P Q + - P (FeS) MenG
a b c
3460 3470 3480 3490 3500
B0 /Gauss
Fig 3.10: Decay-associated transient EPR spectra at ambient temperature.
Spectra of PS I complexes the wild-type and menG mutant strains extracted from the full time/field datasets as described in detail in Van der Est et al., (1994) Biochemistry 33, 11789-11797. The solid curves correspond to the state P700+ Q- while the dashed curves are the spectra of P700+ FeS-. The arrows labeled a, b, c indicate the field positions corresponding to the transients shown in Fig 3.9.
127
Fig 3.11: Flash-induced absorbance changes at 812 nm in PS I complexes isolated from Synechocystis sp. PCC 6803. The experimental data are depicted as dots, and the computer-generated exponential fits are shown as solid lines, with the lifetimes of the phase indicated. Top: P700+ reduction kinetics in wild-type PS I complexes. Bottom: P700+ reduction kinetics in the menG mutant PS I complexes. 128
Fig 3.12: Steady-state rates of flavodoxin reduction in PS I complexes isolated from the wild-type and menG mutant strains.
Seven independent measurements were performed for PS I complexes from each strain at each light intensity. The open and filled diamonds indicate the average values obtained for the complexes for the wild-type and menG mutant strains, respectively. The solid and dashed lines indicate fitted curves derived from the Michaelis-Menten equation for the rates obtained for PS I complexes from the wild-type and menG mutant strains, respectively. Bars on the data points indicate the standard deviation. 129
Chapter 4
Physiological Characterization of Tocopherol Biosynthesis Mutants in the
Cyanobacterium Synechocystis sp. PCC 6803: Demonstration of a Conditionally
Lethal Phenotype in the Presence of Glucose at pH 7.0
Publications:
Zigang Cheng, Scott Sattler, Hiroshi Maeda, Yumiko Sakuragi, Donald A. Bryant, Dean
DellaPenna (2003) Highly divergent methyltransferases catalyze a conserved reaction in tocopherol and plastoquinone synthesis in cyanobacteria and photosynthetic eukaryotes.
Plant Cell 15: 2343-2356
Yumiko Sakuragi, Hiroshi Maeda, Dean DellaPenna and Donald A. Bryant, manuscript in preparation. 130 ABSTRACT
The phenotypes of three tocopherol mutant populations, slr1736, sll0418 and slr0089,
previously selected in the presence of glucose in the cyanobacterium Synechocystis sp.
PCC 6803, have been reexamined. When cultured on solid media containing glucose, the
mutant populations showed extremely heterogeneous colony morphologies, which
suggested that the strains were genotypically inhomogeneous. Newly isolated tocopherol
mutant strains were unable to grow in the presence of glucose at pH values below 7.2.
Therefore, it was concluded that “authentic” slr1736, sll0418, and slr0089 mutants are
glucose-sensitive and are not able to grow in the presence of glucose. Further
characterization of the “authentic” mutants revealed that the chlorophyll a,
phylloquinone, and plastoquinone contents decreased when cells were grown in the
presence of glucose, and Photosystem II (PS II) activity was completely lost after 24 h.
However, levels of the D1 and PsbO proteins remained nearly constant, and the levels of the sodB transcripts (encoding the superoxide dismutase) were virtually identical between the wild-type and mutant strains before and after transfer to medium containing glucose.
These results indicate that the loss of the D1 and PsbO proteins is not responsible for the
loss of PS II activity and strongly support the conclusion that the glucose-induced
phenotype is not directly associated with oxidative stress. The authentic mutants
exhibited chlorosis when grown in the presence of glucose. The relative phycobiliprotein
contents decreased to nearly one-third of the wild-type level, and the concomitant
accumulation of the nblA (encoding a protein essential for the degradation of
phycobilisomes) transcript was detected. These results indicate that the authentic mutants 131 experience macronutrient starvation when grown in the presence of glucose. Further growth analyses revealed that the glucose-induced growth defects, PS II inactivation, and the macronutrient starvation responses occur at pH below 7.2. At pH 7.0 in the presence of glucose, the PS II activity was completely lost, and the growth ceased after 24 h. The results further demonstrate that α-tocopherol plays an essential role for the growth and survival of Synechocystis sp. PCC 6803 in the presence of glucose and suggest that α- tocopherol plays a role in the regulation of macronutrient metabolism in cyanobacteria. 132 ABBREVIATIONS
Chl a chlorophyll a
DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl-urea
HEPES 4-(2-hydroxyethyl)-piperazine-N’-(2-ethanesulfonic acid)
HGA homogentisic acid
HPP 4-hydroxyphenylpyruvate
HPPD 4-hydroxyphenylpyruvate dioxygenase
HPT homogentisate phytyltransferase
MPBQ 2-methyl-6-phytyl-1,4-benzoquinol
MPBQ MT 2-methyl-6-phytyl-1,4-benzoquinol methyltransferase
PBP phycobiliprotein
PCR polymerase chain reaction
PS II Photosystem II
RT-PCR reverse transcriptase-polymerase chain reaction
133 INTRODUCTION
α-Tocopherol (vitamin E) is a lipid-soluble, organic molecule that is synthesized
only by oxygen-evolving phototrophs, including some cyanobacteria and all green algae
and plants (Threlfall and Whistance, 1971; Collins and Jones, 1981). The conservation of
α-tocopherol synthesis during the evolution of photosynthetic organisms suggests that the
molecule performs one or more critical functions. In plants, α-tocopherol is synthesized
and localized in the plastids (Soll et al., 1980, 1985), and it is particularly abundant in the
thylakoid membranes (Fryer, 1992), in which it is thought to afford protection against various oxidative stresses (Munné-Bosch and Lenor, 2000). Because α-tocopherol is also
an essential component of animal diets, most of our knowledge of tocopherol function
has been obtained from studies in these systems. Studies in whole animals, animal cell
cultures and artificial membranes have shown that tocopherols scavenge and quench
various reactive oxygen species and lipid oxidation byproducts, which would otherwise
propagate lipid peroxidation chain reactions in membranes (Kamal-Eldin and Appelqvist,
1996). In addition to these antioxidant functions, other “non-antioxidant” functions
related to modulation of signaling and transcriptional regulation in mammals have also
been reported (Chan et al., 2001; Azzi et al., 2002; Ricciarelli et al., 2002). In
photosynthetic organisms, tocopherol functions have not yet been rigorously defined, but
it is believed that they are likely to include some or all of the functions reported in
animals, as well as other possible functions specific to photosynthetic organisms. 134 The biosynthetic pathway of α-tocopherol is summarized in Fig 4.1. It was elucidated in plants in the mid-1980’s and during the past five years, most of the genes encoding the enzymes of this pathway have been identified in both plants and cyanobacteria (Threlfall and Whistance, 1971; d’Harlingue and Camara, 1985; Norris et al., 1998; Shintani and DellaPenna, 1998; Collakova and DellaPenna, 2001; Schledz et al., 2001; Porfirova et al., 2002; Savidge et al., 2002; Shintani et al., 2002; Dähnhardt et al., 2002; Collakova and DellaPenna, 2003; Koch et al., 2003; Cheng et al., 2003).
Briefly, homogentisic acid (HGA) is the aromatic head-group precursor for all tocopherols, and HGA is produced from 4-hydroxyphenylpyruvate (HPP) by the enzyme
HPP dioxygenase (Norris et al., 1998; Dähnhardt et al., 2002). The committed step in tocopherol synthesis is the condensation of HGA and phytyl-pyrophosphate by the enzyme homogentisate phytyltransferase to produce 2-methyl-6-phytyl-1,4-benzoquinol
(MPBQ) (Collakova and DellaPenna, 2001; Schledz et al., 2001; Savidge et al., 2002;
Collakova and DellaPenna, 2003). The ring of MPBQ is methylated by MPBQ methyltransferase to yield 2,3-dimethyl-5-phytyl-1,4-benzoquinol (Soll et al., 1985;
Shintani et al., 2002; Cheng et al., 2003), which is cyclized by tocopherol cyclase to yield
γ-tocopherol (Soll et al., 1985; Porfirova et al., 2002; Sattler et al., 2003). Finally, a second ring methylation by γ-tocopherol methyltransferase yields α-tocopherol (Soll et al., 1980; d’Harlingue and Camara, 1985; Shintani and DellaPenna, 1998, Koch et al.,
2003). Genes encoding HPP dioxygenase (slr0090), homogentisate phytyltransferase
(slr1736), MPBQ methyltransferase (sll0418) and γ-tocopherol methyltransferase
(slr0089) have been identified in Synechocystis sp. PCC 6803 (Shintani and DellaPenna, 135 1998; Shintani et al., 2002; Collakova and DellaPenna, 2001; Dähnhardt et al., 2002,
Cheng et al., 2003). The remaining enzyme, tocopherol cyclase, has recently been shown to be the product of slr1737 in Synechocystis sp. PCC 6803 (Sattler et al., 2003).
In the cyanobacterium Synechocystis sp. PCC 6803, the tocopherol biosynthetic pathway has been studied genetically and homozygous mutants of the slr1736, sll0418, and slr0089 genes, encoding homogentisate phytyltransferase, MPBQ methyltransferase, and γ-tocopherol methyltransferase, respectively, have been obtained (Shintani and
DellaPenna, 1998; Shintani et al., 2002; Cheng et al., 2003; Collakova and DellaPenna,
2001). As expected from the biosynthetic pathway (Fig 4.1), the slr0089 mutant accumulates only γ-tocopherol (Shintani and DellaPenna, 1998), the sll0418 mutant accumulates 30% of the wild-type level of α-tocopherol and a small amount of β- tocopherol (Cheng et al., 2003), and the slr1736 mutant does not accumulate any tocopherols (Collakova and DellaPenna, 2001). In light of the established antioxidant activity of α-tocopherol in animal systems, one would expect that the loss or reduction of
α-tocopherol would lead to a detectable phenotypic difference between the wild-type and
mutant strains. Intriguingly, however, the tocopherol-deficient slr1736 mutant grew similarly to the wild-type strain both in the absence and in the presence of glucose at a moderate light intensity (110 µE m-2 s-1) (Collakova and DellaPenna, 2001). A limited
comparative analysis of the photosynthetic activity of the wild-type and slr1736 mutant strains failed to reveal any differences. This led the authors to conclude that α-tocopherol is dispensable for the survival of Synechocystis sp. PCC6803 under the conditions tested
(Collakova and DellaPenna, 2001). 136 In this study, it is demonstrated that the “authentic” tocopherol mutants are
glucose-sensitive and that they cannot maintain growth in the presence of glucose at pH
7.2 or lower. Mutant populations obtained in previous studies probably contained mixtures of secondary suppressor mutations that accumulate during cultivation in the presence of glucose, although their exact nature has yet to be identified. The glucose- sensitive phenotype of the “authentic” mutants was characterized, and the data indicate that α-tocopherol is essential for the survival of Synechocystis sp. PCC 6803 in the
presence of glucose at pH 7.2 or lower.
137
RESULTS
Extremely Heterogeneous Colony Morphologies in Tocopherol Mutants Previously
Isolated in the Presence of Glucose
When maintained on solid media containing 5 mM glucose, three tocopherol mutants, slr1736::aphII, sll0418::aphII, and slr0089::aphII that had been isolated in previous studies in the presence of glucose, showed much larger variations in colony size and pigmentation than what is typically observed for the wild-type strain (Fig 4.2 E-H).
The larger colonies exhibited more intense blue-green pigmentation, which is characteristic of wild-type colonies, while the smaller colonies appeared more chlorotic and were yellow-green in color. Generally such non-homogenous colony morphology is indicative of heterogeneous genotypes within a given population. However, PCR analysis of genomic DNA isolated from three mutant strains showed that the targeted, mutated loci were homozygous and that no corresponding wild-type allele could be detected in any of the three mutant strains. These results eliminate the possibility that the observed colony heterogeneity (Fig 4.2) was due to incomplete segregation of the mutant loci or to contamination of the cultures with wild-type cells. When the tocopherol mutants were maintained in the absence of glucose, more uniform colony morphologies were observed
(Fig 4.2 A-D). Although the size of colonies showed somewhat larger variation as compared to wild type, all colonies showed intense blue-green pigmentation. Therefore it was concluded that the heterogeneity of colony morphologies observed for the tocopherol mutants is a specific response to growth in the presence of glucose. 138 The growth characteristics of two cell lines, one derived from a small colony and
the other derived from a large colony of the slr0089 mutant selected in the presence of
glucose, were analyzed in the absence and in the presence of glucose. In the absence of
glucose, the growth behavior of both cell lines was virtually indistinguishable from that
of wild type (Fig 4.3 A and B). Upon transfer to media containing glucose, however, the
cell line derived from the small colony ceased growth within 24 h and did not recover
during the time course of measurements (Fig 4.3 C). The cell line derived from the large colony grew somewhat more slowly than the wild-type strain, but it was able to maintain
growth during the course of the experiment (Fig 4.3 D). The dramatic phenotypic
difference between the two cell lines strongly suggested that they were genotypically
distinct, and that the mutant populations previously obtained by selection in the presence
of glucose (Shintani and DellaPenna, 1998; Collakova and DellaPenna, 2001; Shintani et
al., 2002) were genotypically inhomogeneous.
Tocopherol Mutants Are Glucose Intolerant
“Authentic” tocopherol mutant strains were constructed by transforming an
immotile, glucose-tolerant wild-type strain of Synechocystis sp. PCC 6803 with genomic
DNAs isolated from the slr1736, sll0418, and slr0089 mutants selected in the presence of
glucose. Because the presence of glucose was found to be unfavorable for some
populations of these tocopherol mutants, kanamycin-resistant transformants were selected
in the absence of glucose. Segregation of each mutant allele from the corresponding wild- type allele was tested by PCR analysis using specifically designed oligonucleotide primers (Fig 4.4). Products anticipated for the wild-type slr1736, sll0418, and slr0089 139 genes were 1.3 kbp, 1.0 kbp, and 1.2 kbp, respectively. No such product was observed for any of the mutant strains; instead, a product ~1.3 kbp larger was detected in each mutant line. This difference in size corresponds to the aphII-containing, kanamycin-resistance cartridge originally used for the insertional inactivation of each gene. It was therefore concluded that each mutant strain is homozygous and that the slr1736::aphII, sll0418::aphII, and slr0089::aphII alleles in each mutant line segregated completely in the absence of glucose.
The newly isolated slr1736, sll0418, and slr0089 mutants produced uniform colonies and grew similarly to the wild type in the absence of glucose under moderate light intensity (< 50 µE m-2 s-1; Fig 4.5 A). In the presence of glucose, however, all three mutants showed severe growth defects and were incapable of maintaining growth after 24 h (Fig 4.5 B and C). This growth phenotype of the mutant strains selected in the absence of glucose is very similar to that observed for the cell line derived from the small colonies of the mutants selected in the presence of glucose (see Fig 4.3 C), but sharply contrasts with the growth behavior of the cell lines derived from the large colonies (Fig 4.3 D).
These results clearly demonstrate that “authentic” slr1736, sll0418, and slr0089 mutants are glucose sensitive. The data also suggest that α-tocopherol plays an essential role for the survival of Synechocystis sp. PCC 6803 in the presence of glucose.
When cells from these “authentic” mutants grown in the presence of glucose for 72 h were transferred to fresh medium containing no glucose, no growth was detected even after 3 weeks of incubation. These results indicated that these “authentic” mutants lost viability during cultivation in the presence of 140 glucose for 72 h. The pH of the cultures grown in the presence of glucose typically shifted from the original value (pH 8.0) to a value in the range of 7.0 to
7.3. Preconditioned media prepared from cultures of the “authentic” mutants grown in the presence of glucose supported the normal growth of the wild-type cells. These results show that the glucose toxicity of the mutants is not the result of products excreted from the cells in the presence of glucose.
To test whether the growth light intensity was responsible for the observed failure of the “authentic” tocopherol mutants to grow in the presence of glucose, the “authentic” slr1736 mutant was grown in the presence of glucose at various light intensities. Under both weak (< 5 µE m-2 s-1) and strong (300 µE m-2 s-1) illumination conditions, however, the presence of glucose in the growth medium resulted in the death of the “authentic” slr1736 mutant (Fig 4.6 A and B). This indicates that light intensity is unlikely to be a contributing factor in the observed glucose toxicity of the tocopherol mutants. The effects of inhibitors of the electron transport chain on the glucose toxicity of the slr1736 mutant were also studied. Addition of a sublethal concentration of DCMU (3-(3,4- dichlorophenyl)-1,1-dimethyl-urea) to the growth medium led to a small recovery of growth for the slr1736 mutant in the presence of glucose (Fig 4.6 C). However, increasing the DCMU concentration further did not improve the growth of the slr1736 mutant. Addition of potassium cyanide in various concentrations did not rescue the slr1736 mutant from the glucose toxicity (Fig 4.6 D).
The effects of other organic carbon sources were analyzed on the growth of the
“authentic” slr1736 mutant. The presence of 5 mM pyruvate, succinate, and fumarate, 141 which are intermediates of the TCA cycle, did not cause growth defects in the slr1736
mutant. (Fig 4.7 A-D). The presence of 15 mM glutamate did not affect the growth of the
slr1736 mutant (Fig 4.7 E). The presence of ornithine, however, resulted in growth defects similar to those caused by glucose (Fig 4.7 F). The results suggested that the toxicity induced by glucose is caused by one or more metabolic intermediate that accumulate during glucose and ornithine catabolism. Ornithine is an intermediate in the citrulline cycle, in which as much as 20 % of CO2 fixation can take place (Tabita, 1987).
On the other hand, glucose is metabolized through the glycolytic pathway or oxidative
pentose phosphate pathway. Intermediates of these pathways are also shared with the
Calvin-Benson cycle (reductive pentose phosphate pathway), in which the major CO2
fixation occurs (Tabita, 1987). The fact that both glucose and ornithine caused lethal
effects on the tocopherol mutants implies that α-tocopherol is perhaps involved in carbon
metabolism by coordinating inorganic and organic carbon metabolic balance in the cells.
Chlorophyll a, Phylloquinone and Plastoquinone Content
The complete loss of, or a decrease in, tocopherol content led to small changes in
the pigmentation of the “authentic” tocopherol mutants grown in the absence of glucose.
The chlorophyll a (Chl a), phylloquinone, and plastoquinone contents were
approximately 10%, 10-20 %, and 3-30% lower, respectively, in these tocopherol
mutants than in the wild type (Fig 4.8). The slr1736 mutant cells contained nearly the
same amount of Chl a and quinones as wild type, while the sll0418 and slr0089 mutant
cells contained somewhat lower amounts. These differences, however, did not seem to
affect the photosynthetic electron transport activity; the rates oxygen evolution observed 142 for both whole-chain electron transport and the Photosystem II (PS II)-dependent electron transport for the three tocopherol mutants were very similar to, or perhaps somewhat higher than, those of wild type (Fig 4.9, see below).
When grown in the presence of glucose, however, the mutants exhibited more dramatic changes in the contents of Chl a, phylloquinone, and plastoquinone. The Chl a and phylloquinone contents were largely unchanged by 24 h after transfer from medium without glucose to medium containing glucose and decreased significantly after 48 to 72 h to as low as 50% of the corresponding wild-type level. Under these conditions, the Chl a as well as phylloquinone level of wild type was largely unaffected during the course of measurements (Fig 4.8 A and B). Because phylloquinone is associated exclusively with the Photosystem I complexes in cells, the parallel decreases in the Chl a and phylloquinone contents may imply a decrease in the Photosystem I content in the cells.
In contrast, following transfer from a medium without glucose to a medium containing glucose, the plastoquinone content gradually decreased in the wild-type strain
(Fig 4.8 C); by 48 h, the plastoquinone content of wild type was only half of that found in the absence of glucose. An even more severe decrease was observed in the mutants, in which the plastoquinone content decreased to ~50 % of the wild-type level by 24 h and as low as 15 % by 72 h after transfer to medium containing glucose (Fig 4.8 C). Recent studies have shown that the redox state of the plastoquinone pool in the thylakoid membranes modulates the expression levels of photosynthetic genes and affects the cellular contents of components of the photosynthetic apparatus, including the phycobilisomes and Photosystem I (Alfonso et al., 2000; Li and Sherman, 2000). This 143 raises the question as to whether the dramatic decrease in the plastoquinone content of the
“authentic” mutants could lead to a change in its redox state, which in turn could be responsible for the decrease in the phycobiliprotein content and PS II activity (see below), and ultimately cell death. The redox state of the plastoquinone pool can be modified by various growth conditions including the intensity of illumination applied during cell growth. Low light intensity (< 5 µE m-2 s-1) typically causes reduction of the plastoquinone pool while high light intensity (300 µE m-2 s-1) causes its oxidation
(Cooley and Vermaas, 2001). However, incubation under either of these light conditions did not lead to recovery of growth in the “authentic” tocopherol mutants in the presence of glucose (Fig 4.6 A and B). Similarly, addition of various concentrations of potassium cyanide, which promotes reduction of the plastoquinone pool, did not alleviate the glucose sensitivity of these mutants (Fig 4.6 D), and addition of DCMU, which causes oxidation of the plastoquinone pool, did so only to a small extent (Fig 4.6 C). These combined results therefore suggest that the changes in the plastoquinone pool size, and its possibly altered redox state, are not the primary cause of the glucose toxicity observed for the authentic tocopherol mutants in the presence of glucose.
Photosystem II Activity
Given the significant reduction in the Chl a, phylloquinone, and plastoquinone contents in response to the presence of glucose, whole-chain and PS II-dependent electron transport was assessed in the mutants (Fig 4.9). In cells grown in the absence of glucose, the whole-chain and the PS II-dependent oxygen evolution rates of all three mutants were similar to, or even slightly higher, than that of the wild type. Likewise, 144 somewhat higher O2-evolving activity (the whole-chain or the PS II-dependent) was detected for the wild type when grown in the presence of glucose. In contrast, no O2- evolving activity (the whole-chain and the PS II-dependent) was detected for the tocopherol mutants when grown in the presence of glucose.
Loss of PS II activity is often seen when cells are exposed to oxidative stress caused by exposure to extremely high light, to low temperature, or to an environment containing a high salt concentration (Allakhverdiev et al., 1999; Hideg et al., 2000; see a review by Aro et al., 1993, and references therein), and this phenomenon is known as photoinhibition. Under such conditions, the D1 protein, a subunit that forms a part of the
PS II core structure (Ferreira et al., 2004), rapidly degrades, and, as a result, the activity of PS II is lost. The possibility that the observed PS II inactivation is related to photoinhibition in the tocopherol mutant in response to glucose was tested by immunological assay. When cells were grown in the absence of glucose, the amount of immunologically detectable D1 protein was virtually indistinguishable between the wild- type and the mutant strains (Fig 4.10). After transfer to medium containing glucose, the amount of immunologically detectable D1 protein was only slightly reduced in the mutants compared to wild type, even though by 24 h a complete loss of PS II-mediated oxygen evolution activity was observed (Fig 4.9). PsbO, or the 33 kDa protein, is a subunit that forms a peripheral domain of PS II known as the oxygen-evolving complex
(Ferreira et al., 2004). This complex is responsible for the catalysis of water splitting and the production of oxygen. The immunologically detectable PsbO protein was also largely unaffected in response to glucose in both wild-type and three tocopherol mutant strains. 145 These results demonstrate that the loss of PS II activity in the tocopherol mutants in the
presence of glucose is not associated with structural damage that leads to a loss of the D1
protein and PsbO, and they suggest that oxidative stress is not the cause of the PS II
inactivation. The expression of the sodB gene, encoding superoxide dismutase, which is
known to accumulate several-fold in response to various reactive oxygen species
(Ushimaru et al., 2002), was analyzed by RT-PCR (Fig 4.11). The amounts of the sodB transcript detected for the wild type and the slr1736 mutant were very similar when cells were grown in the absence of glucose (Fig 4.11, shown at 0 h) and in the presence of glucose (Fig 4.11, at 4, 8, and 24 h). The combined results therefore strongly suggest that the loss of α-tocopherol does not cause oxidative stress in response to glucose and that the inactivation of PS II is unrelated to oxidative stress.
Relative Phycobiliprotein Content
The relative content of phycobiliproteins (PBPs), the primary protein components of the light-harvesting antennae known as phycobilisomes, was determined in response to the transfer of the wild type and the “authentic” tocopherol mutants to medium containing glucose (Fig 4.12 A). In the absence of glucose, all three tocopherol mutants contained 20-25% less PBP than the wild type. After 24 h of growth in the presence of glucose, the PBP content of wild-type cells decreased to ~60% of the level found in the absence of glucose, while the PBP content of the three tocopherol mutants decreased even more sharply to 18-26% of the level in their respective controls obtained for the cells grown in the absence of glucose. Time-course analyses revealed that the reduction of PBPs initiates by 4 h after transfer to medium containing glucose (Fig 4.12 B). In 146 comparing the decreased PBP levels of the wild type and the three tocopherol mutants during growth in the presence of glucose, it is obvious that the loss of PBPs is much more severe in the three mutants than in the wild type.
These data are consistent with the pale, chlorotic (yellow-green) coloration of the mutant colonies in the presence of glucose. The mutants had significantly lower
absorption in the orange-red region of their visible spectra, and as a result were less blue-
green in coloration than control cells. This bleaching, due to a decrease in the cellular
content of PBPs, is well known to be associated with nutrient starvation, and can be
caused by carbon (Miller and Holt, 1977), nitrogen (Allen and Smith, 1969; Foulds and
Carr, 1977; Wood and Haselkorn, 1980; Yamanaka and Glazer, 1980; Stevens et al.,
1981; Elmorjani and Herdman, 1987), phosphorous (Ihlenfeldt and Gibson, 1975), sulfur
(Schmidt et al., 1982; Jensen and Rachlin, 1984; Warner et al., 1986), or iron limitation
(Sherman and Sherman, 1983). The nblA gene, encoding a factor essential for the controlled degradation of phycobilisomes, is known to accumulate in Synechocystis sp.
PCC 6803 cells in response to nitrogen deprivation (Richaud et al., 2001). RT-PCR analyses showed that, in the wild-type samples, the nblA transcript was barely detectable
when cells were grown in the absence of glucose (Fig 4.11, shown at 0 h) and that a small
induction occurred by 4 h after transfer to medium containing glucose. In the slr1736
mutant sample, the wild-type level of the nblA transcript was detected in the absence of glucose and a dramatic increase followed by 4 h after transfer to medium containing
glucose. This correlates well with the observed behavior of PBP contents in the
tocopherol mutants (Fig 4.12, also see above) and suggests that the slr1736 mutant is 147 experiencing nutrient limitation in response to glucose. Because the BHEPES medium
used in culturing these mutants contains an appropriate mixture of all micronutrients and macronutrients to support the growth of Synechocystis sp. PCC 6803 (Stainer et al., 1971;
see Materials and Methods), the observed macronutrient starvation response of the mutants in the presence of glucose is most likely to be due to an impairment in an essential biosynthetic or regulatory process, which involves carbon, nitrogen, and glucose.
Involvement of CO2 in the Glucose Toxicity
The observed glucose-sensitive phenotype of the “authentic” tocopherol mutants
resembled that of the icfG mutant reported in a previous study (Beuf et al., 1994). The icfG gene encodes a putative protein phosphatase similar to RsbU (anti anti-sigma factor
B) in Bacillus subtilis (Price, 2000; Woodbury et al., 2004). This mutant showed a glucose sensitive-phenotype only at low CO2 [air level, 0.035% (v/v)] levels. The
presence of succinate, fumarate, pyruvate, and glutamate did not cause lethality at low
CO2, while the presence of ornithine caused a lethal effect. The striking behavioral
resemblance between the icfG mutant and the “authentic” tocopherol mutants in their
response to various carbon sources suggested a possible involvement of CO2 in the
glucose toxicity observed for the tocopherol mutants. Fig 4.13 shows the growth curves
of wild type and the “authentic” slr1736 mutant in the presence of glucose in air and 3 %
(v/v) CO2. The slr1736 mutant grew similarly to the wild type in air even in the presence
of glucose, and in fact, the growth of the two strains was virtually indistinguishable under these conditions. When cells were grown in the presence of glucose at 3% (v/v) CO2, 148 however, the slr1736 mutant exhibited severe growth defects as described above.
Therefore, these results demonstrated that the glucose toxicity occurs only at high CO2
levels. This led to the hypothesis that α-tocopherol is involved in the global regulation of
organic and inorganic carbon metabolism in Synechocystis sp. PCC 6803, possibly
through a signal transduction cascade involving IcfG.
Involvement of pH in Glucose Toxicity
The observed dependence of the glucose toxicity on the CO2 concentration was, however, questioned after discussion with Prof. Aaron Kaplan of the Hebrew University,
Israel. In the presence of 3% (v/v) CO2, the growth medium was found to acidify from
the initial pH 8.0 to about pH 7 (see above). Acidification of cultures can be caused by a
high concentration of CO2, such as 3% (v/v), since the in organic carbon (Ci ) supplied
- into medium as dissolved CO2 is rapidly hydrated and reaches equilibrium with HCO3 .
Thus, the possibility that the high-CO2 requirement of the glucose toxicity was actually
because of medium acidification was tested directly. The wild-type and the “authentic”
tocopherol mutants were grown at various pH values at 1% CO2 (v/v) in medium
BHEPES40, which is a modified BHEPES medium supplemented with 40 mM HEPES
for higher pH buffering capacity (see Materials and Methods). After culturing for 2 days
in this medium at 1% (v/v) CO2, the pH remained near the original value within a deviation of ≤ 0.05 pH unit. In the absence of glucose, both the wild type and the
tocopherol mutant strains grew well at all pH values tested between 6.8 and 8.0, and no
significant difference was observed among the four strains (Fig 4.14). This establishes
that the loss of α-tocopherol does not affect the growth rates of the cells at all pH values 149 between 6.8 and 8.0 in the absence of glucose. When the cells were transferred to media
containing glucose, the wild type grew faster as compared to the growth in the absence of
glucose (Fig 4.14), which confirms that the glucose is taken up and metabolized in the
wild-type cells. The tocopherol mutants grew similarly to wild type at pH 8.0 and 7.6
(Fig 4.14). However, they grew very poorly or not at all at pH 7.2, 7.0, and 6.8. In
comparing the pH-independent growth phenotype in the absence of glucose and the pH-
dependent growth in the presence of glucose, it is obvious that the observed glucose
toxicity of the tocopherol mutants occurs at pH 7.2 or lower. Therefore, the glucose-
sensitive phenotype characterized in detail in the earlier section of this chapter is also due
to the lowering of the pH caused by the acidification of the growth media during growth
in the presence of 3% (v/v) CO2.
Fig 4.15 shows that growth curves of the wild-type and the “authentic” slr1736
mutant cultures at various pH values in air and 1% (v/v) CO2. The slr1736 mutant grew
similarly to wild type at pH 8.0 and 7.6 both in air and 1% (v/v) CO2 (Fig 4.15 A and B).
At pH 7.2, the slr1736 mutant grew slightly slower than wild type during the initial 20 h
and grew even more slowly after 20 h (Fig 4.15 C). At pH 7.0 and 6.8, the mutant grew
substantially slower than wild type during the first 20 h and then stopped growing after
20 h (Fig 5.2 D and E). The growth behavior of the slr1736 mutant was virtually
indistinguishable either when grown with air or with 1% (v/v) CO2 (Fig 4.15 A-E). These results demonstrate it is the pH of the medium and not the extracellular CO2 concentration that causes the glucose-sensitive growth phenotype of the slr1736 mutant. 150 It is noteworthy that the pH-dependent glucose toxicity occurs after 20 h, which suggests that the cells were accumulating a toxic substance during the initial 20 h. 3-O- methyl-glucose is known to be transported into the cells through the glucose transporter; however, it is not phosphorylated and therefore is not metabolized further (Beuf et al.,
1994). Both the wild type and the slr1736 mutant grew well in the presence of this glucose analogue in the medium at pH 7.0 (Fig 4.16, top). The growth rates were very similar to those in the absence of glucose (based on visual inspection). The fact that the slr1736 mutant was able to grow similarly to wild type in the presence of this glucose analog suggests that the cause of the glucose toxicity is not the presence of glucose per se that accumulates inside the cells. The presence of 5 mM pyruvate, succinate, or fumarate in growth medium at pH 7.0 caused the tocopherol mutant to grow slightly slower yet very similarly to wild type (Fig 4.16, top). These results strongly suggest that the causative agent for the glucose toxicity at pH 7.0 in the tocopherol mutants is likely to be metabolites accumulated either during glucose metabolism through the glycolytic pathway or the pentose phosphate pathway. The glucose toxicity was observed when cells were grown at RT (ca. 25 °C). The growth experiments described so far were conducted under constant pH values; in other words, the cells were grown at pH 7.0 in the absence of glucose before being transfer to medium containing glucose at pH 7.0 in order to assess the effect of glucose on the growth of these cells. Likewise, the cells were grown at pH 8.0 in the absence of glucose before transferred to medium containing glucose at pH 8.0. Fig 4.16 (bottom) shows a similar, yet somewhat different experiment.
In this experiment, the cells were initially grown at pH 8.0 in the absence of glucose and then were transferred to medium containing glucose at pH 7.0. The purpose of this 151 experiment was to test whether the incubation at pH 7.0 (in the absence of glucose)
“sensitizes” the tocopherol mutant cells to glucose or its by-product. The results showed that the tocopherol mutants were unable to grow after transfer from medium at pH 8.0
(containing no glucose) to medium containing glucose at pH 7.0. This suggested that the pH value of the medium before transfer to medium containing glucose has nothing to do with the observed glucose toxicity in the tocopherol mutant cells.
Photosynthetic Activity
Fig 4.17 shows the activities of PS II measured for cells grown at various pH values in the absence and the presence of glucose. In the absence of glucose, PS II activities of the slr1736 cells (filled squares) were higher those that of wild type (filled circles) at all pH values. Although the reason for this higher activity for the slr1736 mutant is not known, this result is reproducible, as can be also seen in Fig 4.9. In the presence of glucose, the PS II activities of the wild-type cells were higher than those in the absence of glucose at all pH values (open circles). On the contrary, no PS II activity was detectable for the slr1736 mutant at pH 6.8 and 7.0. Higher activity was observed as the pH value was increased, and at pH 8.0 the PS II activity was virtually indistinguishable from that of wild type. Therefore, the inactivation of the PS II is also dependent on the presence of glucose, the pH of the surrounding environment, and the absence of tocopherols.
The PBP contents
When the cells were grown at pH 7.0 in the absence of glucose and with 1% CO2
(v/v), the PBP contents of the wild type and the “authentic” tocopherol mutants were 152 virtually indistinguishable (Fig 4.18A). When cells were grown in the presence of
glucose at pH 7.0, however, the PBP contents in the tocopherol mutants decreased to
about one-third of the wild type level (Fig 4.18A). This is consistent with the results
described in Fig 4.12, and demonstrates that the tocopherol mutants are experiencing
macronutrient starvation in response to glucose at pH 7. The effect of pH on the PBP
content was analyzed for the wild-type and the slr1736 mutant cells grown in the
presence of glucose (Fig 4.18B). At all pH values tested, the PBP content in the wild-type
cells remained constant. The PBP content in the slr1736 mutant cells was significantly lower, less than 40 % of the wild-type level at pH 7.0 and below, which confirms the reproducibility of the pH-dependent macronutrient starvation response in the tocopherol mutant as described above. As the pH of the medium increased, higher PBP contents were detected. At pH 8.0, the PBP content of the slr1736 mutant was 70% of the wild- type level. The observed reduction of the PBP content over a wide range of pH values strongly indicates that the presence of glucose alone induces a macronutrient starvation response in the tocopherol mutants and that the macronutrient starvation response is severely exacerbated at pH values below 7.2.
153 DISCUSSION
In this study, it was determined that the previously reported Synechocystis sp.
PCC 6803 mutants of genes slr1736, sll0418, and slr0089, encoding homogentisate phytyltransferase, MPBQ methyltransferase and γ-tocopherol methyltransferase, respectively, are probably genotypically heterogeneous mixtures of secondary suppressor mutants. These mutants with defects in tocopherol biosynthesis were originally selected and maintained in the presence of glucose, and although the targeted genes were completely disrupted, a variety of colony growth morphologies have now been observed, which is consistent with genetic heterogeneity. When these mutant lines were reconstructed by a second round of transformation, followed by selection and maintenance in the absence of glucose, the resulting mutant lines (here denoted as
“authentic” mutants to distinguish them from the original mutants previously selected in the presence of glucose) were also found to be completely disrupted at each respective locus, but the observed colony morphology and growth characteristics were now uniform.
It was found that the “authentic” mutants are highly sensitive to the presence of glucose at pH values less than 7.2, and that the growth of each mutant strain ceased within 24 h following transfer to medium containing glucose under those conditions. These results clearly show that α-tocopherol is conditionally essential for the survival of Synechocystis sp. PCC 6803 in the presence of glucose. It is likely that the selection and culturing of the original transformants in the presence of glucose caused the selection of secondary suppressor mutations that permitted survival under these otherwise suboptimal or lethal conditions. Although the exact nature of the suppressor mutations has yet to be 154 determined, one can propose a few possibilities that enable the glucose-sensitive cells to
become glucose tolerant: inactivation of the glucose transporter, alteration of flux through the glycolytic and/or oxidative pentose phosphate pathways, or the alteration of the
regulatory pathway(s) that controls glucose metabolism (Beuf et al., 1994, Hihara et al.,
1997, also see below).
The glucose toxic phenotype was found to be dependent on the pH of medium and
occurs at pH ~7.2 or lower. PS II inactivation and the reduction of PBP contents were
also found to be pH-dependent when glucose is present in the medium and α-tocopherol
synthesis is interrupted. Similar pH-dependent growth defects and reduction of
photosynthetic activities were observed for the cotA mutant (Katoh et al., 1996). CotA is involved in light-induced proton extrusion, and its absence has been shown to lead to severe growth defects at pH 7.0 or lower. Therefore, it is possible that the glucose toxicity observed for the “authentic” tocopherol mutants are related to defects in proton extrusion. However, a simple comparison between the cotA mutant and the tocopherol mutants is not appropriate, because of the significant phenotypic differences between them. The cotA mutant shows pH-dependent growth defects in the absence of glucose, whereas the tocopherol mutant does not under the same conditions. Growth rate of the cotA mutant is slower than wild type (60-70% of the wild-type level) at pH 8.0 in the absence of glucose, whereas the tocopherol mutants grow similarly to wild type under the same conditions (visual inspection). Therefore, the pH-dependent glucose toxicity of the tocopherol mutants seems to be very complex and involves one or more factors that are associated with the pH homeostasis of the cells. Further studies are required to resolve 155 the molecular mechanism of the pH-dependent glucose toxicity in the tocopherol mutants.
Trebst and co-workers reported that the herbicide-mediated loss of α-tocopherol led to the complete loss of PS II activity with concomitant degradation of the D1 protein following high light treatment in the green alga Chlamydomonas reinhardtii (Trebst et al., 2002). This was certainly not the case in the tocopherol mutants of Synechocystis sp.
PCC 6803, as the level of the D1 protein remained similar to the wild type even when the
PS II activity was completely lost. The growth light intensity (50 µE m-2 s-1) used in this study was less than one half of the saturating intensity, and this value seems unlikely to promote a photoinhibitory oxidative stress. In fact, the glucose toxicity observed for these mutants is light-independent, since it occurs over a wide range of light intensity, from < 5 to 300 µE m-2 s-1, as described in the Results. Given that the transcript level of the sodB gene is largely unaffected in the mutants under these lethal conditions, it is highly unlikely that the inactivation of PS II and the death of the cells are directly associated with an oxidative stress. Furthermore, the deleterious effects of glucose on the slr1736, sll0418, and slr0089 mutants were virtually indistinguishable despite the varying compositions and contents of tocopherols in each mutant. Should α-tocopherol function solely as an antioxidant, an increasing susceptibility to glucose with decreasing tocopherol content would reasonably be expected among the various tocopherol mutants, but this is not observed. Therefore, it appears most likely that α-tocopherol plays a role other than, or at least in addition to, that as a bulk antioxidant in the survival of
Synechocystis sp. PCC 6803 in the presence of glucose. Taking all of these considerations 156 together, it was concluded that the observed metabolic perturbation, PS II inactivation, and cell death of the tocopherol mutants grown in the presence of glucose at pH values below 7.2 are unlikely to be directly associated with the antioxidant activity of α- tocopherol in Synechocystis sp. PCC 6803. Moreover, it appears most likely that α- tocopherol plays a role other than as a bulk antioxidant in the survival of Synechocystis sp. PCC 6803 in the presence of glucose.
PBPs are the major constituent of the light harvesting antennae in cyanobacteria, and they can constitute nearly half of the total soluble protein (Grossman et al., 1994).
When cyanobacteria are subjected to macronutrient deprivation conditions for carbon, nitrogen, phosphorus, sulfur, and iron, the synthesis of PBPs ceases and their active degradation begins (Allen and Smith, 1969; Ihlenfeldt and Gibson, 1975; Foulds and
Carr, 1977; Yamanaka and Glazer, 1980; Wood and Haselkorn 1980; Stevens et al.,
1981; Schmidt et al., 1982; Sherman and Sherman, 1983; Jensen and Rachlin, 1984;
Elmorjani and Herdman, 1987). It is generally believed that the degradation of these proteins provides amino acids for energy production and maintenance protein synthesis, while reducing the potential for photooxidative stress due to light stress at PS II.
Therefore, it is highly likely that the observed, dramatic decrease in the PBP contents in the “authentic” tocopherol mutants during growth in the presence of glucose represents a response to a perceived state of nutritional deprivation. Supporting this interpretation is the observation that transcription of the nblA gene, known to accumulate in response to nitrogen deprivation (Richaud et al., 2001), also accumulated in the tocopherol-deficient mutant in response to glucose. The combined results hence suggest that the complete loss 157 of, or a substantial decrease of, α-tocopherol perturbs metabolic processes related to
nitrogen, carbon, and sulfur, and that this disruption ultimately leads to phycobilisome
degradation and cell death.
Recent studies in mammalian systems have demonstrated the involvement of α- tocopherol in modulating the production of signaling molecules and pathways (Chan et al., 2001; Azzi et al., 2002; Ricciarelli et al., 2002). For example, α-tocopherol has been shown to bind to phospholipase A2 specifically at the substrate-binding pocket and act as
a competitive inhibitor, thereby decreasing the release of arachidonic acid for eicosanoid
synthesis (Chandra et al., 2002). α-Tocopherol has also been suggested to modulate the
phosphorylation state of protein kinase Cα in rat smooth-muscle cells, possibly via
phosphorylation of protein phosphatase 2A (Ricciarelli et al., 1998). α-Tocopherol is also
directly involved in transcriptional regulation in animals, including the expression of the
genes encoding liver collagen αI, α-tocopherol transfer protein, and α-tropomyosin
collagenase (Yamaguchi et al., 2001; Azzi et al., 2002). Therefore, it seems possible that,
in addition to acting as a bulk lipid-soluble antioxidant, α-tocopherol could also play a
regulatory role in Synechocystis sp. PCC 6803, possibly in the regulation of metabolic
processes such as those involved in carbon and nitrogen metabolism.
Two glucose-tolerant mutants reported previously appear to be relevant to the
observations reported here. The pmgA (sll1986) and icfG (slr1680) loci have been shown
to be determinants for glucose sensitivity (Hihara and Ikeuchi, 1997; Beuf et al., 1994). 158 Inactivation of the pmgA locus has been shown to result in a glucose-sensitive phenotype
and enhanced photoautotrophic growth capability (Hihara and Ikeuchi, 1997).
Inactivation of the icfG locus, on the other hand, results in a glucose-sensitive phenotype
under low CO2 conditions (Beuf et al., 1994). The “authentic” tocopherol mutants, whose
phenotypes are characterized in this chapter, are glucose-sensitive both under high and
low CO2 environments but only at pH values less than 7.2. The amino acid sequences deduced from the pmgA and icfG coding regions show weak similarity to RsbT/W (a
serine/threonine protein kinase) and RsbU/X (a protein phosphatase and a negative
regulator of sigma factor B) in Bacillus subtilis. These proteins form a signal transduction
network with other Rsb proteins and regulate the activity of sigma factor B through
phosphorylation/dephosphorylation interactions upon perception of environmental stress
(Price 2000; Woodbury et al., 2004). Homologous protein sequences of other Rsb
proteins are also found in the Synechocystis sp. PCC 6803 genome: Slr2031 (RsbU/X),
Ssr1600 (RsbS/V), Slr1856 (RsbS/V), and Slr1861 (RsbT/W). It is therefore plausible
that an Rsb-like pathway is present in this organism and may participate in the regulation
of glucose metabolism. In fact, studies have shown that recombinant Slr1861
phosphorylates Slr1856 and IcfG dephosphorylates the phosphorylated Slr1856 (Shi et
al., 1999). Whether the role of α-tocopherol is related to this hypothetical pathway is an
open question and is a subject of continuing investigation. 159 SUMMARY
The tocopherol mutants (slr1736, sll0418, and slr0089), which had been previously isolated in the presence of glucose, were shown to be phenotypically and genotypically heterogeneous populations. Newly isolated “authentic” tocopherol mutants were glucose-sensitive and were not able to grow in the presence of glucose after 24 h.
This glucose toxicity was shown to occur when the mutants were grown at pH values below 7.2. Under these conditions, the PS II activities were significantly reduced or not detected at all in the tocopherol mutants. 160 MATERIALS AND METHODS
Growth Conditions and Strains
The slr1736, sll0418, and slr0089 mutants were generated by targeted insertion
with the aphII gene, which confers kanamycin resistance, as described previously
(Shintani and DellaPenna, 1998; Collakova and DellaPenna, 2001; Shintani et al., 2002).
The wild-type strain used for transformation and further analysis was a glucose-tolerant
strain of Synechocystis sp. PCC 6803 (Williams, 1988). Medium BHEPES, pH 8.0, was
used for selection, maintenance, and growth measurements of wild type and mutants.
This medium is prepared by supplementing BG-11 medium (Stainer et al., 1971) with 4.6 mM HEPES [4-(2-hydroxyethyl)piperazine-N’-(2-ethnesulfonic acid)]-KOH and 18 mg
L-1 ferric ammonium citrate. Medium BEHEPS40, modified medium BEHEPS after supplementing with 40 mM HEPES for higher buffer strength, was used for pH-
dependent or pH-controlled growth analyses. Wild-type cells were maintained on solid B-
HEPES medium containing 1.5 % (w/v) agar and 5 mM glucose, and the “authentic”
tocopherol mutants were maintained on solid medium BHEPES containing 1.5 % (w/v)
agar, 50 µg kanamycin ml-1, and importantly, no glucose. Prior to all growth
measurements and preparations for subsequent analyses, cells were transferred from solid
to liquid medium BHEPES and allowed to grow exponentially for several generations in the absence of glucose. For determination of growth characteristics, late-exponential
phase cultures were diluted in fresh liquid medium BHEPES to an OD730 nm of
approximately 0.05 cm-1. The diluted cultures were grown at 32 °C with continuous 161
bubbling with air containing 1% or 3% (v/v) CO2. Growth was monitored by measuring the optical density at 730 nm. For growth in the presence of glucose, the medium was supplemented with 5 mM glucose. The growth light intensity was 50 µE m-2 s-1, unless
otherwise noted. For growth measurements in the presence of inhibitors of the electron
transport chain, the following concentrations were used: 0.03 µM DCMU (3-(3,4-
dichlorophenyl)-1,1-dimethyl-urea); 2, 20, or 200 µM potassium cyanide. Pre-
conditioned medium was prepared from a culture of an authentic tocopherol mutant that
had been grown in the presence of glucose for 72 h. The cells were removed by
centrifugation at 8000 × g, and the supernatant was filtered through a sterile 0.22 µm
pore-size membrane. The filtrate was then mixed with five-fold strength medium
BHEPES in a four-to-one ratio (v/v) to achieve a normal, one-fold-strength medium.
Isolation of Tocopherol Mutants
Synechocystis sp. PCC 6803 wild type was transformed with genomic DNA
extracted from the slr1736, sll0418, and slr0089 mutants selected in the presence of
glucose as previously described (Williams, 1988). Segregation of mutant alleles from the
wild type allele was carried out in the absence of glucose and in the presence of 50 µg
kanamycin ml-1. Oligonucleotide primers used for PCR analysis were as follows; slr1736
forward primer (5’-GGCTTCTCCTACCCGGAATTCTACTTCCTG-3’), slr1736 reverse
primer (5’-GCTTTCTAAGTGTACATCTAGACTCCGCCA-3’), sll0418 forward primer
(5’-ATGCCCGAGTATTTGCTTCTGCC-3’), sll0418 reverse primer (5’-
GCACTGCTTTGAACATACCGAAG-3’), slr0089 forward primer (5’- 162 TCTACCGGAAATTGCCAACTACCA-3’), and slr0089 reverse primer (5’-
CCTAGGAGATTGTGGACTTCAA-3’).
Oxygen Evolution and Consumption Measurements
Cells grown in the absence and in the presence of glucose for 24 h were harvested
by centrifugation at 8,000 × g at room temperature and resuspended in a 25-mM HEPES
buffer, pH 7.0, to obtain an optical density of 1.0 cm-1 at 730 nm. For whole-chain
oxygen evolution and consumption measurements, the cell suspensions were incubated in
the dark in the presence of 10 mM NaHCO3 for at least 15 min prior to measurements.
For the Photosystem II (PS II)-dependent oxygen evolution measurements, 1 mM 1,4-
benzoquinone and 0.8 mM K3Fe(CN)6 were added instead of NaHCO3 and measurements
were performed immediately. The excitation light intensity was approximately 3 mE m-2
s-1. The oxygen concentration was measured polarographically with a Clark-type
electrode as previously described (Sakamoto and Bryant, 1998).
Quantification of Pigments and Quinones by High Performance Liquid Chromatography
Chl a in whole cells was extracted with methanol, and its molar content per unit
optical density at 730 nm was determined using the molar absorption coefficient 82 L g-1
cm-1 (MacKinney, 1941). For quinone analyses, cells were harvested by centrifugation at
8,000 × g, at 4 °C for 6 min, washed in 1 ml of 50 mM Tris-HCl buffer, pH 8.0, and
centrifuged at 18,000 × g for 1 min to obtain tight cell pellets. Pigments and quinones
were extracted by ultrasonication in 400 µl of cold acetone-methanol (7:2, v/v) in the 163 dark. High performance liquid chromatography (HPLC) analysis was performed with a
Agilent 1100 system equipped with a diode array detector (Agilent Technologies,
Wilmington, DE, USA). One hundred microliters of extract was mixed with 10 µl of 1 M
ammonium acetate and injected onto a NovaPak C18 column (3.9 × 300 mm, 4 µm
packing, Waters, Milford, MA). Analytes were eluted by a linear gradient program consisting of solvent A (100% methanol) and solvent B (100% isopropanol) using the
following protocol: [100% A] for 10 min, [100% A] to [20% A : 80% B] in 20 min, [20%
A: 80% B] for 5 min, and [20% A : 80% B] to [100% A] for 5 min. The flow rate was
0.75 ml min-1. The Chl a, phylloquinone, and plastoquinone contents of each sample
were determined based on integrated peak areas and their molar absorption coefficients,
which are 17.4 mM-1 cm-1 at 618 nm, 18.9 mM-1 cm-1 at 270 nm (Dunphy and Brodie,
1971), and 15.2 mM-1 cm-1 at 254 nm (Crane and Dilley, 1963), respectively. The absorption coefficient of Chl a at 618 nm was calculated from the ratio of the absorption peaks at 618 nm and 666 nm in methanol-isopropanol (6:4, v/v) and from its absorption coefficient at 666 nm in methanol (MacKinney, 1941). The quinone content per unit optical density at 730 nm was calculated by multiplying the molar ratio of quinone to Chl a by the molar Chl a content per optical density at 730 nm.
Estimation of Relative Content of Phycobiliproteins
Cells were harvested by centrifugation at 8000 × g for 6 min, and pellets were
resuspended in the 25 mM HEPES buffer, pH 7.0, to obtain 2 ml of the cell suspensions
with an optical density of 0.5 cm-1 at 730 nm. One milliliter of this suspension was heated 164 at 100 °C for 1 min. A modification was made in the method previously described (Zhao and Brand, 1989) for estimation of the relative PBP content. The absorbance at 635 nm and 730 nm were recorded for unheated and heated samples, and the data were then inserted into the following equation: the relative PBP content = (∆OD635nm –
∆OD730nm)/OD730nm·unheated, where ∆ indicates ODunheated sample minus ODheated sample
SDS-Polyacrylamide Gel Electrophoresis and Immunoblotting of the D1 protein
Cells were grown in the absence of glucose to mid-exponential phase or in the presence of glucose for 24 h, and harvested by centrifugation at 8,000 × g at 4 °C for 6 min. The resulting cell pellets were immediately frozen in liquid N2 and stored at –80 °C until being used. Frozen cells were thawed on ice and resuspended in the 25 mM HEPES buffer, pH 7.0, at an optical density of 100 cm-1 at 730 nm. An equal volume of glass beads was added to the cell suspension, which was allowed to stand on ice for 5 min.
Using a home-built bead-beater, the samples were vigorously shaken 4 times for 30 s at 4
°C with 30 s intervals on ice. An aliquot (10 µl) of each sample was mixed with an equal volume of loading buffer, and the mixture was incubated at 65 °C for 20 min, and applied onto a discontinuous SDS-polyacrylamide gel with 10% (w/v) acrylamide in the separating gel as described (Schägger and van Jagow, 1987). Electrophoresis was carried out at 50 V for 12 h. The antibodies raised against the amino acids 234-242 of the D1 protein of Synechocystis sp. PCC 6803 were a kind gift from Prof. Eva-Mari Aro of the
University of Turku, Finland. The antibodies raised against the PsbO protein of
Synechocystis sp. PCC 6803 were a kind gift from Prof. Robert Burnap of the Oklahoma 165 State University, USA. After electrophoresis, proteins were transferred to a nitrocellulose
membrane. Proteins were detected by immunoblotting by using an enhanced
chemiluminescence reagents and analysis system (Amersham Biosciences, Piscataway,
NJ) according to manufacturer’s specifications.
Isolation of total RNA and RT-PCR analyses
Total RNA was isolated from cells using the Mini-to-Midi RNA isolation kit
(Invitrogen Corp., Carlsbad, CA) followed by an RNase-free DNase treatment, and
repurification by using the same kit. The RNAs obtained were adjusted to a final
concentration of 50 ng mL-1 in RNase free double distilled water and store at -80°C until
being used. RT-PCR reactions were carried out by using the One-Step RT-PCR kit
(Qiagen Inc., Valencia, CA) for detecting the rnpB transcript. The reaction volume was
25 µL, and 4 ng µL-1 of the total RNAs was used. For detecting the nblA and sodB
transcripts, 400 ng of total RNA was first converted to cDNA by the RT reaction using
M-MLV reverse transcriptase (Promega, Madison, WI). The reaction volume was 20 µL
for each sample. The resulting cDNA was diluted with 80 µL of TE buffer, and stored at
–20 °C until being used. Two microliters of cDNA stock were used for PCR (final volume, 25 µL) using Hot-Start Taq polymerase (Qiagen Inc., Valencia, CA). RNasin
(Promega, Madison, WI), an RNase inhibitor, was present in one-step RT-PCR reactions and during the cDNA synthesis.
166 ACKNOWLEDGEMENTS
The authors thank Prof. Eva-Mari Aro (University of Turku) for kindly providing the D1
antibodies, Prof. Robert Burnap (Oklahoma State University) for kindly providing the
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Fig 4.1: Biosynthetic pathway of α-tocopherol in Synechocystis sp. PCC 6803. HPPD, 4-hydroxyphenylpyruvate dioxygenase; HPT, homogentisate phytyltransferase; MPBQ MT, 2-methyl-6-phytyl-1,4-benzoquinone methyltransferase; TC, tocopherol cyclase; γ-TMT, γ-tocopherol methyltransferase. 177
Fig 4.2: Colony morphologies of three tocopherol mutants previously isolated in the presence of glucose. Cells were grown in the absence of glucose (A-D) and in the presence of glucose (E-H). Wild type (A, E), the slr1736 mutant (A, E), the sll0418 mutant (C, G), and the slr0089 mutant (D, H), on solid BHEPES medium. 178
Fig 4.3: Growth curves of wild type and cell lines derived from small (A, C) and large colonies (B, D) of tocopherol mutants selected in the presence of glucose (see -2 -1 Fig 4.2) at 32 °C, 50 µE m s with 3% (v/v) CO2. The data shown for each strain are averages of at least three independent cultures, and standard error bars are shown. Growth in the absence of glucose (A, B) and in the presence of glucose (C, D). (A, C) Wild type (filled circles) and a cell line derived from a small colony slr0089 mutant selected in the presence of glucose (open circles) (see Fig 4.2). (B, D) Wild type (filled circles) and cell lines derived from large colonies the slr0089 (open circles), sll0418 (open triangles), and slr1736 mutants (open squares) selected in the presence of glucose (see Fig 4.2). 179
Fig 4.4: PCR analysis of the genomic DNAs extracted from the newly isolated tocopherol mutants selected in the presence of glucose. (A) The slr1736 mutant, (B) the sll0418 mutant, and (C) the slr0089 mutant. Lane 1 and lane 2 in each panel show PCR products amplified from the wild-type and mutant genomic DNA templates, respectively. The DNA fragments amplified from the mutant templates are 1.3-kb longer than those from the wild-type template because of the insertion of the aphII cassette encoding resistance to kanamycin. 180
Fig 4.5: Growth curves of wild type and the “authentic” tocopherol mutants (A) in the absence and (B, C) in the presence of glucose at 32 °C, 50 µE m-2 s-1, 3% (v/v) CO2. Closed circles indicate the wild-type strain; open squares, triangles, and circles indicate the authentic slr1736, sll0418, and slr0089 mutant strains, respectively. The data shown for each strain are averages of three independent cultures, and standard error bars are shown. 181
Fig 4.6: Growth curves of wild type and the “authentic” slr1736 mutant in the presence of glucose at 32 °C, 3% (v/v) CO2. Growth (A) at 300 µE m-2 s-1, (B) at < 5 µM m-2 s-1, (C) in the presence of 0.03 µM DCMU at 50 µE m-2 s-1, and (D) in the presence of potassium cyanide (2 µM and 20 µM) at 50 µE m-2 s-1. Closed circles and open squares indicate the wild-type and the “authentic” slr1736 mutant strains, respectively. Data shown for each strain are averages of three independent cultures, and standard error bars are shown. Dotted lines in (D) and (E) indicate the presence of DCMU and potassium cyanide, respectively. 182
F
Fig 4.7: Growth analyses of wild type and the “authentic” slr1736 mutant of -2 -1 Synechocystis sp. PCC 6803 at 32 °C, 50 µE m s , 3% CO2 (v/v), in the presence various carbon sources. (A) 5 mM glucose, (B) 5 mM succinate, (C) 5 mM pyruvate, (D) 5 mM fumarate, (E) 15 mM glutamate, and (F) 15 mM ornithine. Filled circles and open squares indicate the wild-type and the slr1736 mutant strains, respectively. Data shown here average of three independent measurements, and standard error bars are shown. In panel F, the tube on the left is the wild-type strain grown in the presence of 15 mM ornithine, while the tube on the right is the slr1736 mutant grown under the same conditions. 183
Figure 4.8: Chlorophyll a (Chl a) contents, and quinone contents in wild type and the “authentic” tocopherol mutants grown in the absence and in the presence of glucose. (A) Chl a content, (B) phylloquinone (PhyQ) content, and (c) plastoquinone (PQ) content. The data shown are averages of three determinations, and standard error bars are shown. Cultures grown in the absence of glucose (shows at 0 h) were transferred to media containing glucose for 24, 48, and 72 h. Wild type, the slr0089, the sll0418, and the slr1736 mutants are shown in black, diagonally slashed, checked, and open columns, respectively.
184
1200
-1
h L) 900
730nm
600
(OD
2
mol O
300 µ
N.D. N.D. N.D. N.D. N.D. N.D. 0 WT slr1736 sl0418 slr0089
Fig 4.9: Oxygen-evolution activities of the whole-chain and the Photosystem II- dependent electron transport chains in wild type and the “authentic” tocopherol mutants of Synechocystis sp. PCC 6803. - O2-evolution activities of whole-chain electron transport (H2OÆHCO3 ) were measured for cells grown in the absence of glucose (black columns) and in the absence of glucose (diagonally slashed columns). O2-evolution activities of PSII-dependent electron transport (H2OÆ1,4-BQ/K3Fe(CN)6) were measured for cells grown in the absence of glucose (checked columns) and in the presence of glucose (open columns). Data shown here average of five independent measurements, and standard error bars are shown. The cells were grown for 24 h under the designated conditions before measurements. N.D., not detectable. 185
Fig 4.10: Immunoblotting analysis for the D1 and PsbO proteins in whole cells of wild type and three “authentic” tocopherol mutants grown in the absence of glucose (Photoautotrophic) and in the presence of glucose (Photomixotrophic) at 32 °C, 50 -2 -1 µE m s , with 3% (v/v) CO2. Proteins from equal amount of cells (10 µL of cell suspension with OD730 nm =100) was loaded in each lane. After electrophoresis, proteins were transferred to a nitrocellulose membrane. Proteins were detected by immunoblotting as described in the Materials and Methods. 186
Fig 4.11: Time-dependent RT-PCR analysis of the sodB and nblA transcripts in wild type and the “authentic” slr1736 mutant of Synechocystis sp. PCC 6803 The cells were initially grown in the absence of glucose (shown at 0 h) and transferred to media containing 5mM glucose and grown for 4, 8, 24 h at 3% CO2 (v/v), 32°C, 50 µE m-2 s-1. rnpB, encoding the catalytic RNA subunit of RNase P, was used as control. 187
1 A
0.8 rotein content
p 0.6
cobili 0.4
y
h
p
0.2
0
Relative WT slr1736 sll0418 slr0089
1.2 B
0.8
0.4
0
Relative phycobiliprotein content 0 4 8 24 Time (h)
Fig 4.12: Relative phycobiliprotein contents in wild type and the “authentic” tocopherol mutants of Synechocystis sp. PCC 6803 grown at 3% (v/v) CO2, 32°C, 50 µM E m-2 s-1. (A) Cells grown in the absence of glucose (black columns) were transferred to glucose- containing BHEPES media and grown for 24 h (diagonally slashed columns). (B) Time- course analyses in wild type (closed circles) and the slr1736 mutant (open squares) grown in the absence of glucose (shown at 0 h), and transferred to medium containing glucose and grown for 4, 8, and 24 h. The values represent the ratio relative to wild type grown in the absence of glucose. Data shown here are the average of six independent measurements, and standard error bars are shown. 188
Fig 4.13: Growth curves of wild type and the “authentic” slr1736 mutant in the presence of glucose at 3% (v/v) CO2 and air. Closed circles and open squares indicate the wild-type and the slr1736 mutant strains, respectively. Solid and dotted lines indicated the presence of 3% (v/v) CO2 and air in medium, respectively. Cells were grown at 32°C, 50 µE m-2 s-1. 189
Fig 4.14: Cultures of wild type and the “authentic” tocopherol mutants of Synechocystis sp. PCC 6803 grown at various pH for 2 days at 1% (v/v) CO2, 32°C, 50 µE m-2 s-1. Cells were grown in the absence (above) or in the presence (below) of glucose in medium BHEPES40 at pH 6.8, 7.0, 7.2, 7.6, and 8.0.
190
Fig 4.15: Growth curves of wild type and the “authentic” slr1736 mutant of Synechocystis sp. PCC 6803 at various pH values in the presence of glucose in air and 1% (v/v) CO2. Cells were grown in BHEPES40 medium at (A) pH 8, (B) pH 7.6, (C) pH 7.2, (D) pH 7.0, (E) pH 6.8, in 1% (v/v) CO2 (circles) and air (squares). Wild type and the “authentic” slr1736 mutant strains are indicated with filled symbols and open symbols, respectively. Growth analyses were carried out at 50 µE m-2 s-1, 32°C. 191
Fig 4.16: Cultures of wild type and the “authentic” slr1736 mutant of Synechocystis sp. PCC 6803 grown at various conditions for 48 h at pH 7. (Top) Growth in the presence of 5 mM pyruvate, fumarate, succinate, and 3-O-methyl- glucose at pH 7. (Bottom) Growth in the presence of 5 mM glucose at pH 7 with 5 mM NaCl (left), at room temperature (middle), and cultures pre-conditioned at pH 8. Growth -2 -1 analyses were carried out at 50 µE m s , 32°C, 1% (v/v) CO2 using medium BHEPES40. 192
Fig 4.17: PS II-dependent O2-evolution activities of wild type and the “authentic” slr1736 mutant grown in the absence (closed symbols) and in the presence (open symbols) of glucose. Wild type and the slr1736 mutant are indicated by circles and squares, respectively. Cells were grown in medium BHEPES40 at the designated pH. O2 evolution was measured for 2 mL of cell suspension (OD 730nm = 1.0) in 50 mM HEPES buffer, pH 7.0, using 1mM 2,6-dimethyl-1,4-benzoquinone as the electron acceptor. Excitation light intensity was ca. 3 mE m-2 s-1. Temperature of the measurement chamber was maintained at 30 °C. A Clark-type electrode equipped with a magnetic stirrer was used for the assay. 193
Fig 4.18: Phycobiliprotein contents in wild type and the tocopherol mutants of -2 -1 Synechocystis sp. PCC 6803 grown at 1% (v/v) CO2, 32°C, 50 µE m s . (A) Wild type, and the slr1736, sll0418, and slr0089 mutants were grown for 24 h at pH 7 in the absence (black columns) and in the presence (open columns) of glucose. (B) Wild type and the slr1736 mutant were grown for 24 h at various pH values in the presence of glucose. 194
Chapter 5
Transcriptional Regulation of Metabolic and Sigma Factor Genes in the Vitamin E-
Deficient Mutant of Synechocystis sp. PCC 6803
Publication:
Yumiko Sakuragi, Dean DellaPenna, Donald A. Bryant, manuscript in preparation
195 ABSTRACT
Transcriptional regulation of metabolic genes and sigma factor genes was studied in the
vitamin E (α-tocopherol)-deficient mutant (slr1736) of Synechocystis sp. PCC 6803.
When glucose was present in growth medium at pH 8.0, the transcripts of nblA (a factor
essential for the controlled degradation of PBP), sbpA (the periplasmic sulfate binding
protein), and sigB, sigC, sigD, sigE, sigH, sigI (alternative sigma factors) were shown to
accumulate in the tocopherol-less mutant cells. These results indicate that the α-
tocopherol mutant responds to the presence of glucose as though nitrogen, carbon, and
sulfur were limiting in the environment. When glucose is present in the growth medium
at pH 7.0, the tocopherol mutant ceased growth after 20 h. Under these conditions, the
amounts of the sigA (the principal sigma factor), rbcL (the RuBisCO large subunit), and ccmK1 and ccmL (carboxysome shell proteins) transcripts diminished after 4 h. Because the amounts of these transcripts remained unaltered when cells were grown in the absence of glucose or in the presence of glucose pH 8.0, it was concluded that the pH- dependent, glucose-induced misregulation of the sigA and rbcL are largely responsible for the glucose-induced lethality of the tocopherol mutants under these conditions. Thin- section electron micrographs revealed the absence of carboxysomes in the tocopherol mutant grown in the presence of glucose at pH 7.0. The transcription of slr2031 (a putative protein phosphatase that shows sequence similarity with RsbU and that is essential under sulfur- and nitrogen-limiting conditions) and ndhF3, ndhR, sbtA
(inorganic carbon uptake mechanisms) were shown to be constitutively down-regulated under all conditions tested in the tocopherol mutant. This result demonstrates that α- 196 tocopherol is involved in the transcriptional regulation of these metabolic genes in
Synechocystis sp. PCC 6803. Thin-section electron micrographs of the tocopherol-less
mutant showed that a large number of glycogen granules accumulated in the cells when
grown even in the absence of glucose. The targeted interruption of the pmgA gene (a putative anti-sigma factor homologous to RsbW) resulted in a similar pH-dependent, glucose-induced lethal phenotype. Based on these results, it is concluded that α- tocopherol acts as a global regulatory molecule in carbon, sulfur, and nitrogen metabolism in Synechocystis sp. PCC 6803. 197 ABBREVIATIONS
CCM inorganic carbon concentrating mechanisms
DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl-urea
HEPES 4-(2-hydroxyethyl)-piperazine-N’-(2-ethanesulfonic acid)
PBP phycobiliprotein
PS II Photosystem II
RT-PCR reverse transcriptase-polymerase chain reaction
RuBisCO ribulose-1,5-bisphosphate carboxylase/oxygenase
198 INTRODUCTION
α-Tocopherol is synthesized only in oxygenic phototrophs including
cyanobacteria, green algae and higher plants (Threlfall and Whistance 1971; Collins and
Jones, 1981). Given that α-tocopherol constitutes an essential component of our diet,
most of our knowledge concerning the roles and functions of this molecule has been
obtained from studies in animal systems. Based on results gathered in animals, animal
cell cultures, and artificial membranes, it has been shown that tocopherols function as
antioxidants and scavenge or quench various reactive oxygen species and lipid oxidation
byproducts that would otherwise propagate lipid peroxidation chain reactions in
membranes (Kamal-Eldin and Appelqvist, 1996). This antioxidant role of α-tocopherol
has now been demonstrated in eukaryotic phototrophs owing to studies in the past several
years. The herbicide-mediated interruption of the α-tocopherol biosynthesis pathway has
been shown to render PS II more susceptible to oxidative stress under extremely high
light illumination in the green alga Chlamydomonas reinhardtii (Trebst et al., 2002). The presence of α-tocopherol has been shown to suppress the propagation of lipid peroxidation during seedling germination in Arabidopsis thaliana (Sattler et al., 2004).
Thus, there is little doubt that α-tocopherol functions as a bulk antioxidant in the eukaryotic phototrophs as well as in animals. The antioxidant role of α-tocopherol is also being investigated in the cyanobacterium Synechocystis sp. PCC 6803, and the results obtained so far support an antioxidant role of α-tocopherol in this organism (H. Maeda,
Y. Sakuragi, D. A. Bryant, and D. DellaPenna, personal communication) 199 Perhaps the most novel and exciting functions of α-tocopherol have been uncovered by various research groups during the last few years. In addition to the antioxidant functions, other “non-antioxidant” functions related to modulation of signaling and transcriptional regulation in mammals have also been reported (Chan et al.,
2001; Azzi et al., 2002; Ricciarelli et al., 2002). For example, α-tocopherol has been shown to bind to phospholipase A2 specifically at the substrate-binding pocket and to act as a competitive inhibitor, thereby decreasing the release of arachidonic acid for eicosanoid synthesis (Chandra et al., 2002). α-Tocopherol is also directly involved in transcriptional regulation in animals, including the expression of the genes encoding liver collagen αI, α-tocopherol transfer protein, and α-tropomyosin collagenase (Yamaguchi et al., 2001; Azzi et al., 2002). The presence of α-tocopherol resulted in a modulation of the phosphorylation state of protein kinase Cα in rat smooth-muscle cells, possibly via phosphorylation of protein phosphatase 2A (Ricciarelli et al., 1998). None of these non- antioxidant functions of α-tocopherol has yet been experimentally demonstrated in oxygenic phototrophs.
In the previous studies described in Chapter 4, the role(s) of α-tocopherol in cyanobacteria was investigated by using three mutants, slr1736, sll0418, and slr0089, deficient in enzymes involved in the α-tocopherol biosynthetic pathway (see Fig 4.1).
The tocopherol mutants are able to grow similarly to wild type at pH values between 6.8 and 8.0 in the absence of glucose (control conditions), under which conditions the PS II activities and the phycobiliprotein (PBP) contents were comparable to those of wild type.
After transfer to a medium containing glucose at pH 8.0, the tocopherol mutants also 200 grew similarly to wild type and the PS II activity remained unaltered, although the PBP
content decreased by ~30 % (permissive conditions). After transfer to medium containing
glucose at pH 7.0, however, the tocopherol mutants ceased growth by 24 h, and the PS II
activity was completely lost. Under these conditions the PBP content was reduced by ≥
60 % (lethal conditions). The involvement of oxidative stress in the PS II inactivation was largely ruled out, because the levels of the D1 protein and the sodB transcript were comparable between wild type and the tocopherol mutants under these conditions. Based on the observed reduction in the PBP content and the accumulation of the nblA transcript, which is known to accumulate in response to nutrient limitation (Collier and Grossman,
1994; Richaud et al., 2001), it was suggested that the tocopherol mutants are experiencing macronutrient starvation. This led to the hypothesis that α-tocopherol plays a role as a regulatory molecule in macronutrient metabolism in Synechocystis sp. PCC 6803.
In this chapter, a study of the transcriptional regulation of metabolic genes and sigma factor genes in a tocopherol-deficient mutant (slr1736) is described. Of 39 genes
initially analyzed, the amounts of the transcripts of 10 genes were shown to respond
either to the presence of glucose alone or to the presence of glucose and pH in the
medium, whereas the amounts of the transcripts of 4 genes were shown to be
constitutively down regulated upon loss of α-tocopherol. Based on these observations, it
is concluded that α-tocopherol is involved in transcriptional regulation of some key
metabolic genes and plays a role in the global coordination of macronutrient metabolism
in Synechocystis sp. PCC 6803.
201 RESULTS
Transcription of nblA and sbpA
Given the significant reduction of the PBPs in response to glucose and lowered pH (see Chapter 4), the nblA transcript was analyzed in wild type and slr1736 mutant cells grown at pH 7.0 and pH 8.0 (Fig 5.1). In the absence of glucose, the wild-type strain barely accumulated detectable nblA transcripts both at pH 7.0 and pH 8.0, and only a small increase followed in response to glucose in the growth medium. This appeared somewhat contradictory to the observed behavior of the PBP content in the wild type, in which the PBP content increased by 20% after transfer to medium containing glucose when cells were grown at pH 7.0 (see Chapter 4, Fig 4.18). Furthermore, the medium used for culturing these cells contains a growth-rate saturating concentration of nitrate
(ca. 18 mM) (Stainer 1971; see Materials and Methods). Thus, it is inconceivable that these cells were actually limited by the availability of nitrogen. The presence of glucose in the medium might lead to an increase in the intracellular carbon pool and, as a result, facilitate the faster growth of this organism. These “carbon-saturated”, fast-dividing cells perhaps are not capable of metabolizing nitrogen at an equivalent rate, and thus respond as though they are limited by nitrogen in their environment.
The slr1736 mutant accumulated a higher amount of nblA transcript in the absence of glucose as compared to the wild-type control (Fig 5.1, shown at 0 h), which suggests that the slr1736 mutant is “perceiving” nitrogen limitation even in the absence of glucose. In response to glucose, the amount of the nblA transcripts increased dramatically in the slr1736 mutant after 4 h when cells were grown in medium at pH 7.0 202 (Fig 5.1). Similar although more moderate increases were observed when cells were
grown in the presence of glucose at pH 8.0. These results indicate that the slr1736 mutant
is “perceiving” a severe nitrogen limitation at pH 7.0 and moderate nitrogen limitation at
pH 8.0. This is consistent with the dramatic reduction of PBP contents at these pHs (ca.
40% of the wild-type level at pH7.0 and ca. 70% of the wild-type level at pH 8.0).
The sbpA transcript, encoding the periplasmic sulfate binding protein, is known to accumulate in response to sulfate limitation (Laudenbach et al., 1991). No detectable sbpA transcripts were present in wild-type cells grown at pH 7.0 and pH 8.0 in the absence or presence of glucose (Fig 5.1). In the slr1736 mutant cells grown at pH 7.0, a
small yet discernible amount of the sbpA transcripts was detected in the absence of
glucose, and these transcripts dramatically increased by 8 h after transfer to medium
containing glucose. When the slr1736 mutant cells were grown at pH 8.0, no sbpA
transcripts were detectable in the absence of glucose, and a gradual increase was
observed in 24 h after transfer to a medium containing glucose. These results indicate that
the slr1736 mutant is “perceiving” sulfur limitation even in the absence of glucose at pH
7.0, and the response is exacerbated after transfer to a medium containing glucose. It is
noteworthy that the amounts of the sbpA and nblA transcripts accumulated at pH 7.0 and
8.0 coincided well with the degree of reduction of the PBP contents at these pHs in the presence of glucose (See Chapter 4, Fig 4.18, and Fig 5.1). The combined results hence demonstrate that the reduction of the PBP content is associated with “perceived” nitrogen and sulfur limitation in the slr1736 mutant. 203 The cpcA transcripts, encoding the phycocyanin α-subunit, which is a subunit of
the most abundant PBPs, were found to decrease gradually in the tocopherol mutant in
response to glucose when the slr1736 mutant cells were grown at pH 7.0. At pH 8.0 its level remained largely unaltered both in wild type and the slr1736 mutant before and
after addition of glucose. Therefore, it was concluded that the mechanism of the
reduction of the PBP content at pH 7.0 and 8.0 in the slr1736 mutant grown in the
presence of glucose are different. At pH 8.0, the reduction of the PBP content primarily
involves active degradation of the PBP proteins, while at pH 7.0 it involves both the
active degradation, as characterized by the accumulation of the nblA transcript, and the
transcriptional down regulation of the genes encoding PBPs, which further supports the
conclusion that the cells are perceiving nutrient limitation and responding to it (Grossman
et al., 1994; also see references therein)
Transcription of Alternative Sigma Factor Genes
The Synechocystis sp. PCC 6803 genome encodes eight σ70-type alternative sigma
factors (SigB through I) in addition to the principal sigma factor (SigA), and they play
roles as transcription factors that are activated in response to various kinds of stresses
(Caslake et al., 1997; Gruber and Bryant, 1998; Huchauf et al., 2000; Muro-Pastor et al.,
2001; Imamura et al., 2003a, b). Fig 5.2 shows the results of the RT-PCR analysis of
sigB, sigC, sigD, sigE, sigH, and sigI in wild type and the slr1736 mutant in the absence
of glucose and in the presence of glucose. SigB has been suggested to be involved in a
variety of stress responses, including dark response (Imamura et al., 2003a) and heat-
shock response (Huchauf et al., 2000) in Synechocystis sp. PCC 6803, and carbon and 204 nitrogen starvation responses in Synechococcus sp. PCC 7002 (Caslake et al., 1997).
When cells were grown at pH 7.0, the amount of the sigB transcripts was largely unaltered in wild type before and after addition of glucose to the medium (Fig 5.2). In the slr1736 mutant, the amount of the sigB transcript was somewhat higher in the absence of glucose as compared to the wild-type control, followed by a large increase by 4 h after transfer to medium containing glucose. A similar response was observed for the slr1736 mutant grown at pH 8.0. Therefore the amount of the sigB transcripts responded to glucose alone and was not influenced by the pH of the medium. Similar accumulation patterns of transcripts were also observed for sigC, sigH, and sigI. SigH is involved in the heat-shock response (Hackauf et al., 2000), while SigC, which is related to SigE in
Synechococcus sp. PCC 7002, is likely to be involved in the post-exponential phase of growth (Gruber and Bryant, 1998). The role of SigI has not yet been elucidated. The results suggest that the slr1736 mutant is experiencing a mixture to stresses in response of glucose.
Transcription of sigE is under the control of NtcA, a global nitrogen regulator, which binds to its characteristic binding site located in the upstream flanking sequence of the sigE transcription initiation site and induces transcription of sigE in response to nitrogen deprivation (Muro-Pastor et al., 2001). Interestingly, the amount of the sigE transcripts increased in response to glucose in wild type both at pH 7.0 and 8.0 (Fig 5.2).
This is consistent with the pattern of the accumulation observed for the nblA transcripts
(Fig 5.1, see above). Although the reason for this glucose-induced nitrogen limitation response has not yet been defined, it is possible that this is due to an altered intracellular 205 carbon-nitrogen balance caused by increased carbon flux induced by the glucose metabolism (see also above). In the slr1736 mutant, the amount of the sigE transcripts increased more dramatically than for the wild type in response to glucose. This glucose- induced accumulation of the sigE transcripts occurred both at pH 7.0 and pH 8.0, which is consistent with the results obtained for the nblA transcripts (Fig 5.1, see above) and which indicates that the slr1736 mutant is “perceiving” a nitrogen limitation in response to glucose.
In contrast to sigE, the amount of the sigD transcripts was largely unaffected by the presence of glucose in wild type (Fig 5.2). In the tocopherol mutant, the amount of the sigD transcript was lower than that of wild type in the absence of glucose and increased by several-fold in response to glucose (by visual inspection). SigD is highly expressed during exposure to high light (Imamura et al., 2003a,b). It has been suggested that this light-dependent behavior of sigD expression is regulated by the redox state of PS
II (Imamura et al., 2003a). Because the growth conditions used in this study employ continuous illumination of the cultures with a moderate light intensity (50 µE m-2 s-1), and because the PS II activity is nearly at the wild-type level for the slr1736 mutant in the absence of glucose at pH 7.0 and 8.0 or in the presence of glucose at pH 8.0, the observed alteration of the sigD transcription can not be explained simply by the previously suggested light-responsive role of SigD.
Taking these results together, it was firmly concluded that the transcription of these alternative sigma factor genes is modulated in the slr1736 mutant in response to glucose and that pH plays little role in the glucose-induced response of the transcription 206 of the alternative sigma factors. This indicates that α-tocopherol plays a role in regulating the stress response related at least to carbon, nitrogen and sulfur metabolism in
Synechocystis sp. PCC 6803.
Transcription of sigA and Ci Genes
The sigA gene encodes the principal sigma factor, which is responsible for the transcription of genes encoding proteins essential for growth and reproduction in eubacteria including cyanobacteria (Gruber and Bryant, 1997; Caslake and Bryant, 1996).
The amount of the sigA transcripts was analyzed for wild type and the slr1736 mutant grown at pH 7.0 and 8.0 (Fig 5.3). At pH 7.0, the sigA transcripts in wild type remained constant after addition of glucose to medium, which is consistent with the fact that the wild-type cells grow normally under these conditions. The sigA transcript level in the slr1736 mutant at pH 7.0 was virtually identical to that for the wild type in the absence of glucose, which is also consistent with the fact that the slr1736 mutant grows similarly to wild type in the absence of glucose at pH 7.0. The level of the sigA transcripts remained unaltered for 4 h when the slr1736 mutant was transferred to medium containing glucose at pH 7.0. However, after 8 h the sigA transcript level significantly diminished. Such a dramatic reduction in the amount of the sigA transcript is likely to cause a reduction of the content of SigA available for transcription of house-keeping genes, which would in turn severely impair the growth capacity of the cells. Indeed, the tocopherol mutants are unable to grow in the presence of glucose at pH 7.0 (see Chapter 4, Fig 4.14 and Fig
4.15). In contrast, when the slr1736 mutant was grown in the presence of glucose at pH
8.0, the amount of the sigA transcript was virtually unaltered and was very similar to that 207 in the wild type control. This is consistent with the fact that the slr1736 mutant grew
similarly to wild type under these conditions (see Chapter 4, Fig 4.14 and Fig 4.15). The
direct correlation between the amounts of the sigA transcripts and the growth behavior of
the slr1736 mutant in the absence and in the presence of glucose at pH 7.0 and 8.0
strongly indicate that the pH-dependent glucose toxicity is caused by the pH-dependent,
glucose-induced alteration of the sigA transcript level in the slr1736 mutant.
Accompanying the reduction of the amounts of the sigA transcript, the amounts of
the transcripts of carboxysome-related genes (rbcL, ccmK1, ccmL), a carbon-
concentrating mechanism (CCM) gene (ndhF4), and sigG were also decreased in a pH-
and glucose-dependent manner in the slr1736 mutant (Fig 5.3). rbcL encodes the large
subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), which is
responsible for the fixation of CO2 and is essential for the survival of the cells, because
this gene cannot be completely inactivated in cyanobacteria (Pierce et al., 1989). ccmK1 and ccmL encode carboxysome shell proteins, which form the outer shell of carboxysomes and encapsulate RuBisCO within the carboxysomal structure (Price et al.,
1993). ndhF4 encodes a subunit of a multi-subunit complex that is a low-affinity constitutive CO2 uptake transporter (Shibata et al., 2001). The reduction in the levels of
the carboxysome transcripts most likely leads to the reduction in the amount of
carboxysomes in the cells; as a result, the ability to fix CO2 would be severely impaired
(Price et al., 1993).
208 Intracellular Structures
Thin-section electron micrographs of wild-type and slr1736 mutant cells grown at
pH 7.0 in the absence and the presence of glucose were recorded. Fig 5.4 shows the
electron micrographs of wild type grown in the absence (Fig 5.4A) and in the presence of glucose (Fig 5.4B). In the wild-type cells grown in the absence of glucose, the space
between the thylakoid membranes was filled with electron-opaque material, which most
likely represents the protein-rich phycobilisomes (Fig 5.9A). In the cytoplasm, electron-
opaque polyhedral bodies are visible, which are characteristic of carboxysomes. Slightly
larger electron-transparent bodies with round or oval shapes are most likely due to poly-
β-hydroxybutyrate (Stainer, 1988). In the wild-type cells grown in the presence of
glucose (Fig 5.4B), on the other hand, the intermembrane space is filled with small
electron-transparent objects with irregular shapes, which are characteristic of glycogen
granules. Glycogen granules serve as general carbohydrate reserves (Stainer, 1998). The
cellular content of glycogen accounts for 10-20 % of the dry cell weight when cells are
growing photoautotrophically and exponentially, whereas it accounts for up to 60 % of
dry cell weight when cells are subjected to nitrogen-limiting conditions (Stainer, 1988;
Smith 1982). That few glycogen granules are observed in the wild-type cells grown in the
absence of glucose is consistent with the fact that these cells grow in a balanced manner
and optimally under these conditions. The observed accumulation of glycogen in the
wild-type cells grown in the presence of glucose most likely reflects a carbon-saturated,
or nitrogen-limited metabolic state of the cells. 209 Fig 5.5A shows an electron micrograph of an slr1736-mutant cell grown in the absence of glucose at pH 7.0. The intracellular structures, such as the presence of thylakoid membranes, carboxysomes, and poly-β-hydroxybutyrate, are very similar to those of the wild-type control grown under the same conditions (Fig 5.4 A). However, in the slr1736 mutant, the intermembrane space was filled with glycogen granules. This clearly contrasts with the situation in the wild-type cells, in which only a small number of glycogen granules was detectable (Fig 5.4A). Rather, this feature of the slr1736 mutant cell resembles that of the wild-type cell grown in the presence of glucose (Fig 5.4B). This suggests that the slr1736 mutant accumulates an unusually large amount of carbohydrate even in the absence of glucose. Therefore, the slr1736 mutant cells seem either to be experiencing nitrogen limitation, or to be performing CO2 fixation or glycogen synthesis much more efficiently than the wild-type cells. The former is not likely to be the case as the mutant cells grow similarly to wild type under these conditions. Therefore, it is most likely that the mutant is overproducing photosynthate. In either case, it is obvious that the
“metabolic state” of the slr1736 mutant is different from that of the wild type even in the absence of glucose.
When the slr1736 mutant was grown in the presence of glucose, the appearance of the intracellular structures changed dramatically (Fig 5.5B). The intermembrane space is no longer electron-opaque, which is consistent with the significant reduction in the PBP content. No carboxysomes were observed in the majority of the cells, which supports the results that the transcripts of the carboxysome genes are substantially reduced in the slr1736 mutant under these conditions. The number of layers formed by the thylakoid 210 membranes, however, does not seem to be affected significantly. Interestingly, new oval structures with an electron-opaque appearance are visible between the thylakoid membranes; these structures were absent in wild type grown in the absence and in the presence of glucose and in the slr1736 mutant grown in the absence of glucose.
Constitutive Alteration of the CCM Genes and slr2031 Expression
Given the alteration of the “metabolic state” in the slr1736 in the absence of glucose, further transcription analyses were carried out in order to understand the involvement of α-tocopherol in macronutrient metabolism. Of 39 genes analyzed for their transcript level, 4 genes (ndhF3, ndhR, sbtA, slr2031) showed constitutive down- regulation both in the presence and in the absence of glucose (Fig 5.6). ndhF3, ndhR, and sbtA encode components of high-affinity CO2 uptake transporters (Klughammer et al.,
1999; Shibata et al., 2001, 2002), a repressor of the ndhF3 operon, and the sodium- dependent bicarbonate transporter, respectively, and all participate in inorganic carbon concentrating mechanisms (CCM) under limited inorganic carbon environments. The transcripts of these genes were detected in wild type both in the absence and the presence of glucose at pH 7.0 and 8.0. On the contrary, the transcript levels for these genes were significantly reduced in the slr1736 mutant both in the presence and in the absence of glucose both at pH 7.0 and 8.0. Thus, the constitutive alteration of these CCM genes is directly linked to the loss of α-tocopherol.
These CCM genes are known to be upregulated in response to low inorganic carbon environments, and are hardly detectable under high CO2 conditions as analyzed by Northern-blot hybridization (Figge et al., 2001). This raises a question as to whether 211
the detection of the CCM genes in this study [at 1% (v/v) CO2] is due to an artifact such
as contamination with genomic DNA during the RNA isolations. The RNA preparation is carried out through three steps, including isolation, RNase-free DNase treatment, and finally purification; thus, it is unlikely that genomic DNA is present in the preparation.
Indeed, PCR analyses on the same RNA templates used for RT-PCR analyses using primers targeted to the rnpB gene resulted in no product formation, which confirms that the RNA preparations are free of genomic DNA contamination. Currently, we do not have an explanation other than attributing the discrepancy to a difference in the sensitivity of the detection methods. Typically one-step RT-PCR analyses using the
Qiagen One-Step RT-PCR kit (Qiagen inc., Valencia, CA) requires 50 pg µL-1 to 5 ng
µL-1 of total RNA in order to detect the target transcripts, whereas typical Northern-blot
hybridization reactions require several micrograms of RNA. Indeed, when more sensitive
methods were employed, other groups have also detected the presence of the ndhF3 and sbtA transcripts in RNA preparations obtained from cells grown at high CO2 level [3-5%
(v/v) CO2] (Shibata et al., 2002; McGinn et al., 2003).
slr2031 encodes a putative protein phosphatase (similar to RsbU) involved in the
regulation of SigB in B. subtilis. In wild-type cells, the amount of slr2031 transcripts
were low yet detectable in the absence of glucose, and transcript levels substantially
increased by 8 h in response to glucose both at pH 7.0 and pH 8.0 (Fig 5.6). In the
slr1736 mutant, the slr2031 transcript is hardly detectable in the absence of glucose, and
only a slight induction was observed in response to glucose at pH 7.0. At pH 8.0, the
slr2031 transcript behaved similarly at pH 7.0, although a moderate increase was 212 observed in the presence glucose by 24 h. In Synechocystis sp. PCC 6803, Slr2031 has
been shown to be somehow involved in sulfur and nitrogen metabolism, and its absence
is deleterious under sulfur- and nitrogen-limiting conditions (Huchauf et al., 2000). An
independent study has shown that the presence of Slr0231 is also essential when the cotA
gene is inactivated (Katoh et al., 1995). CotA has been suggested to be involved in light-
induced proton extrusion and CO2 transport in Synechocystis sp. PCC 6803 (Katoh et al.,
1996a,b). Thus, it is possible that Slr2031 is involved in the transcriptional regulation of
genes that are involved in regulating processes including carbon, nitrogen, and sulfur
metabolism. The fact that the expression of slr2031, as well as ndhF3, ndhR, and sbtA, is
constitutively down-regulated in the absence of α-tocopherol demonstrates that α- tocopherol plays a role in the transcriptional regulation of metabolic genes in
Synechocystis sp. PCC 6803.
Is the significant reduction of slr2031 transcription directly related to the cause of
the pH-dependent glucose toxicity of the tocopherol mutant? In order to answer this
question, an slr2031 disruption mutant was generated by insertion of the aadA gene cassette, conferring spectinomycin resistance, into the unique MfeI site of the slr2031
coding region. Complete segregation of the mutant allele from the wild-type allele was
demonstrated by PCR analysis (Fig 5.7). Using the primers designed to target and
amplify a 0.9-kb N-terminal portion of the slr2031 loci resulted in the amplification of a
0.9-kb DNA fragment when the genomic DNA isolated from wild type was used as the
template. No 0.9-kb product was detectable when the genomic DNA isolated from the
slr2031::addA transformant was used as the template. Instead, a product of 2 kb was 213 detected. The size difference between the two products corresponds to the size of the
addA gene cassette, which demonstrates that the slr2031::addA transformant is homozygous and free from the slr2031 wild-type allele.
A preliminary growth analysis was carried out at pH 7.0 and pH 8.0. The slr2031
mutant (slr2031::aadA) was able to grow both at pH 7.0 and pH 8.0 in the absence of
glucose. When the cells were transferred to medium containing glucose, the slr2031
mutant was able to grow similarly to wild type both at pH 7.0 and pH 8.0 (Fig 5.8 A and
B). An slr1736 slr2031 double mutant behaved similarly to the slr1736 mutant at both
pHs in the presence of glucose (Fig 5.8 A and B). Therefore, it was concluded that the
constitutive down-regulation of slr2031 transcription is not the direct cause of the pH-
dependent glucose toxicity observed for the tocopherol mutant.
As mentioned in Chapter 4, the pmgA mutant of Synechocystis sp. PCC 6803 is
unable to grow in the presence of glucose, which indicates that PmgA plays a role in the
regulation of photomixotrophic growth (growth on CO2 and glucose as the carbon source)
(Hihara and Ikeuchi, 1997). To test whether the role of PmgA is related to the role of α-
tocopherol, a pmgA inactivation mutant was constructed by inserting the accC1 gene
cassette, conferring gentamicin resistance, into the unique SpeI site within the pmgA
coding region. The complete segregation of the mutant allele from that of wild type was
demonstrated by PCR analysis (Fig 5.7). PCR using the primers designed to target and
amplify a 0.8-kbp of the pmgA locus resulted in the amplification of a 0.8-kbp fragment
when genomic DNA isolated from wild-type cells was used as the template. No 0.8-kb
product was detectable for the PCR product when genomic DNA isolated from the 214 pmgA::aacC1 transformant was used as the template. Instead, a product of 1.9 kb was detected. The size difference between the two products corresponds the size of the aacC1
gene cassette, which demonstrates that the pmgA::aacC1 transformant is homozygous
and free from the pmgA wild-type allele.
A preliminary growth analysis was carried out in the absence of glucose and in
the presence of glucose at pH 7.0 and pH 8.0. The results showed that the pmgA mutant
grew similarly to wild type in the absence of glucose at pH 7.0 and at pH 8.0 (by visual
inspection). In the presence of glucose, the pmgA mutant grew similarly to wild type in
medium at pH 8.0; however, its growth was found to be severely impaired in the presence
of glucose at pH 7.0. This apparent similarity in the growth behavior between the
tocopherol and pmgA mutants in response to glucose and pH suggests that α-tocopherol
and PmgA play roles in the same regulatory pathway. PmgA shows similarity to
RsbW/RsbT, protein serine/threonine kinase, in B. subtilis (Price CW, 2000; Woodbury
et al., 2004). RsbW functions as an anti-SigB factor, while RsbT functions as an activator
of RsbU, which in turns activates SigB in the signal transduction cascade that regulates
the activity SigB. They act together with other protein phosphatases and kinases
including RbsU, an Slr2031 homologue (Price CW, 2000; Woodbury et al., 2004). More
than 6 Rsb-like proteins were found to be conserved in the genome of Synechocystis sp.
PCC 6803, and PmgA, Slr2031, and Ssr1600 are also found to be present in the genomes
of Thermosynechococcus elongatus BP-1, Nostoc sp. PCC 7120, Nostoc punctiforme,
Trichodesmium erythraeum, Synechococcus sp. PCC 7002, Prochlorococcus marinus sp.
MED4, P. marinus sp. MIT9313, P. marinus sp. SS120, and Synechococcus sp. 215 WH8102. Thus, it is highly plausible that a pathway similar to the Rsb pathway in B. subtilis is present in cyanobacteria and regulates the balance between carbon, nitrogen and sulfur metabolism.
216 DISCUSSION
The potential involvement of α-tocopherol in macronutrient metabolism was
investigated. RT-PCR analyses performed on the cells grown at pH 7.0 and 8.0 in the
absence and in the presence of glucose demonstrated that the transcripts of the alternative
sigma factors accumulate in the slr1736 mutant in response to glucose, which indicated
that the slr1736 mutant is experiencing a variety of stresses when grown in the presence of glucose (Caslake et al., 1997; Gruber and Bryant, 1998; Huchauf et al., 2000; Muro-
Pastor et al., 2001; Imamura et al., 2003a,b). The nblA and sigE transcripts, known to
accumulate in response to nitrogen limitation (Mulo-Pastor, 2001; Richaud 2001), also
accumulated in response to glucose in the slr1736 mutant. The sbpA transcript, known to accumulate in response to sulfur limitation (Laudenbach and Grossman, 1992), also accumulated in response to glucose in the slr1736 mutant. In combination with the results obtained for the PBP content as described in Chapter 4, these results demonstrate that slr1736 mutant is experiencing macronutrient starvation when grown in the presence of glucose, and that α-tocopherol is somehow influencing carbon, nitrogen, and sulfur metabolism in Synechocystis sp. PCC 6803.
When cells were grown at pH 7.0 in the presence of glucose, the level of transcripts of sigA, ndhF4, cpcA, and carboxysome genes (rbcL, ccmK1, ccmL) decreased substantially. These genes were constitutively expressed, and their products play housekeeping roles for the survival of cyanobacteria. Thus, it is possible that the reduction of the ndhF4, and cpcA, and carboxysome transcripts is caused by the reduction of the cellular content of SigA (Pierce et al., 1989; Caslake and Bryant, 1996; Gruber and 217 Bryant, 1997). Because the functions of SigA and RuBisCO are absolutely indispensable
for the survival of cyanobacteria, the observed pH-dependent, glucose-induced
transcriptional misregulation of sigA and rbcL is a likely cause of the cell death of the
tocopherol mutant in the presence of glucose in medium at pH 7.0.
The mechanism of the pH-dependent, glucose-induced misregulation of sigA
transcription has yet to be determined. A study has suggested that sigA transcription is
regulated by the activity of the photosynthetic electron transport chain, and in the
presence of DCMU or in the dark the amount of sigA transcripts is rapidly lowered
(Tuominen et al., 2003). Interestingly, the activity of PS II, and thus the activity of the
photosynthetic electron transport chain, is reduced in a pH-dependent manner in the
tocopherol mutant in response to glucose, and PS II activity was completely lost at pH
7.0 (see Chapter 4, Fig 4.17). The PS II content in the tocopherol mutant under these
conditions appeared to be largely unaltered based on the contents of the D1 and PsbO
proteins (see Chapter 4, Fig 4.10). Thus, the inactivation of PS II is thought to be related
either to the loss of electron transfer cofactors or to the generation of metabolites that
inhibit the activity of PS II. Decades of studies and recent X-ray crystallographic
structures of the PS II complexes from the cyanobacterium Synechococcus elongatus have confirmed the presence of one bicarbonate molecule in the vicinity of the non-heme iron located between the two bound plastoquinone molecules (QA and QB) (Blubaugh and
Govindjee, 1986; Petrouleas et al., 1994; Ferreira et al., 2004, see also reviews by
Blubaugh and Govindjee, 1988; Govindjee and van Rensen, 1993; van Rensen et al.,
1999, and references therein ). It has been demonstrated that this bicarbonate molecule is 218 displaced in vitro by other molecules such as formate, NO, glycolate, glyoxylate, and oxalate (Petrouleas et al., 1994). Glycolate is a metabolite generated by the oxygenase activity of RuBisCO, which occurs when the intracellular CO2 concentration is substantially reduced. Although, there is no evidence suggesting a significant reduction in the intracellular Ci pool in the tocopherol mutant, this possibility needs to be examined nevertheless.
Perhaps the most exciting outcome of the RT-PCR analyses is the discovery that the expression of slr2031 and CCM genes (ndhF3, ndhR, sbtA) is constitutively down- regulated in the tocopherol mutant in the presence and absence of glucose both at pH 7.0 and 8.0. This direct effect of the loss of α-tocopherol on gene expression is a strong indication that α-tocopherol is involved in transcriptional regulation in cyanobacteria.
Because the function of these products are directly related to macronutrient metabolism, this further leads to the conclusion that α-tocopherol is involved in the regulation of the macronutrient metabolism as a transcriptional regulator in cyanobacteria. It is noteworthy that, in mammalian systems, it has been demonstrated that α-tocopherol is directly involved in transcriptional regulation of the genes encoding liver collagen αI, α- tocopherol transfer protein, and α-tropomyosin collagenase (Yamaguchi et al., 2001;
Azzi et al., 2002). The mechanisms of these transcriptional regulation processes are not completely understood; however, it is a reasonable assumption that it involves a modulation of a signal transduction cascade that regulates the expression of these genes.
Indeed, α-tocopherol has been suggested to modulate the cell signaling pathway by modulation of the phosphorylation state of protein kinase Cα in rat smooth-muscle cells, 219 possibly via phosphorylation of protein phosphatase 2A (Ricciarelli et al., 1998). In light of the observed involvement of α-tocopherol in cell signaling in the animal system, it is
intriguing to recognize the phenotypic similarity between the tocopherol mutant and the
pmgA mutant in Synechocystis sp. PCC 6803. As mentioned earlier, the pmgA gene has been demonstrated to be a determinant of glucose tolerance in this organism (Hiahara and
Ikeuchi, 1997), and its deduced amino acid sequence shows similarity to a serine/threonine protein kinase, which has been characterized as the anti-sigma factor
(RsbW) or the activator for the anti-RsbW (RsbT) in the regulatory pathway of SigB in B. subtilis (Price, 2000; Woodbury et al., 2004). Thus, it is highly plausible that α- tocopherol plays a regulatory role through this Rsb-like pathway in Synechocystis sp.
PCC 6803 as summarized in Fig 5.9. This is the first time, to the best of my knowledge,
that the involvement of α-tocopherol in transcriptional regulation has been
experimentally demonstrated in the oxygenic phototrophs. This newly discovered role of
α-tocopherol in Synechocystis sp. PCC 6803 thus strongly indicates that α-tocopherol
plays a regulatory role in addition to a role as a bulk antioxidant in some cyanobacteria.
The next question is whether such a regulatory role is evolutionarily
conserved among green algae and plants. Recent studies have shown that a mutant
in maize, originally isolated because of its deficiency in sucrose transport through
plasmodesmata, is also deficient in tocopherol cyclase (Porfirova et al., 2002;
Sattler et al., 2003). This suggests that α-tocopherol plays a role in sugar
metabolism in maize. It is therefore highly plausible that α-tocopherol has similar
regulatory functions in macronutrient metabolism in plants. 220 SUMMARY
It was shown that a α-tocopherol mutant responds to the presence of glucose as though nitrogen and sulfur were limiting in the environment. This conclusion is based on the accumulation of stress-responsive gene transcripts. When glucose is present in growth medium at pH 7.0, the amounts of the sigA and rbcL transcripts diminished after 4 h.
Because the amount of these transcripts remained unaltered when cells were grown in the absence of glucose or in the presence of glucose pH 8.0, it was concluded that the pH- dependent glucose-induced misregulations of the sigA and rbcL are largely responsible for the glucose-induced lethality of the tocopherol mutants under these conditions. The transcription of metabolic genes (slr2031, ndhF3, ndhR, and sbtA) is shown to be constitutively down regulated under all conditions tested in the tocopherol mutant. This result demonstrates that α-tocopherol is involved in transcriptional regulation of these metabolic genes in Synechocystis sp. PCC 6803. Thin-section electron micrographs of the tocopherol mutant showed that large amounts of glycogen granules accumulated in the cells. The targeted interruption of the pmgA gene (a putative anti sigma factor homologous to RsbW) resulted in the similar pH-dependent glucose-induced lethal phenotype. Based on theses results, it was concluded that α-tocopherol is involved as a regulatory molecule in the global regulation of carbon, sulfur, and nitrogen metabolism in
Synechocystis sp. PCC 6803.
221 MATERIALS AND METHODS
Strains and Growth Conditions
The wild-type strain used for transformation and further analyses was a glucose- tolerant stain of Synechocystis sp. PCC 6803 (Williams 1988). Medium BHEPES, pH
8.0, was used for maintenance of the wild type and mutant strains. This medium is
prepared by supplementing BG-11 medium (Stainer et al., 1971) with 4.6 mM of HEPES
[4-(2-hydroxyethyl)piperazine-N’-(2-ethnesulfonic acid)]-KOH and 18 mg L-1 ferric ammonium citrate. Medium BHEPES40 is prepared by supplementing the medium
BHEPES with 40 mM HEPES. Prior to all growth measurements and the preparation of cells for subsequent analyses, cells were transferred from solid to liquid medium
BHEPES and allowed to grow exponentially for several generations in the absence of
glucose. For the determination of growth characteristics, late-exponential phase cultures were diluted in fresh liquid medium BHEPES40, pH 7.0 or 8.0, to an OD730 nm of
approximately 0.05 cm-1. The diluted cultures were grown at 32 °C with continuous bubbling with air containing 1% (v/v) CO2. For growth in the presence of glucose, the
medium was supplemented with 5 mM glucose. The light intensity was 50 µE m-2 s-1,
unless otherwise noted. For isolation of RNA, late-exponential phase cultures were
-1 diluted in fresh liquid medium BHEPES40 to an OD730 nm of approximately 0.3 cm in a total volume of 80 mL. The diluted cultures were grown under the same conditions as described above.
222 Generation of the slr2031 and the pmgA Mutants
The 5’-terminal portion of the slr2031 locus was amplified by PCR using primers designed to target this genomic region [slr2031-F, 5’-
AGGAGTTGGTGGCTAAGCTTTACCGGGAACAAA-3’, including an engineered
HindIII cleavage site (underlined); slr2031-R, 5’-
TGACAAAGCGATGGGAATTCTCCAGGTCGGCA-3’, including an engineered
EcoRI cleavage site (underlined)]. The pmgA locus was amplified by PCR using primers
designed to target this genomic region [pmgA-F, 5’-
TTCTCTGTGCCGAAAGCTTCTATG-3’, including an engineered HindIII cleavage site
(underlined); pmgA-R, 5’-CACCATGGTGGCGAATTCAGCC-3’, including an
engineered EcoRI cleavage (underlined)]. The amplified DNA fragments were digested
with HindIII and EcoRI and ligated with pUC19 obtained after digestion of pUC19 with
HindIII and EcoRI. An EcoRI fragment of pSRA2, containing the aadA gene conferring
spectinomycin resistance, was inserted into the unique MfeI site within the 5’ slr2031
coding region. An XbaI fragment of pMS266, containing the accC1 gene conferring
gentamicin resistance, was inserted into the unique SpeI site within the pmgA coding
region. The resulting constructs were linearized after digestion with EcoRI and used to
transform wild-type Synechocystis sp. PCC 6803 cells. Selection of transformants was
carried out on solid medium BHEPES, pH 8.0, in the presence of 50 µg mL-1
spectinomycin or in the presence of 20 µg mL-1 gentamicin at 27 °C, under a moderate
light intensity (~50 µE m-2 s-1). PCR analyses were carried out using the same sets of
primers used for the cloning. 223 Isolation of Total RNA and RT-PCR Analyses
Total RNA was isolated from cells using the Mini-to-Midi RNA isolation kit
(Invitrogen corp., Carlsbad, CA) followed by an RNase-free DNase treatment and
repurification by using the same kit. The final concentration of the purified RNA
preparations was 50 ng mL-1 in RNase free double-distilled water; the RNA samples were
stored at -80°C until being used. RT-PCR reactions were carried out by using the One-
Step RT-PCR kit (Qiagen Inc., Valencia, CA) for detecting the rnpB, spbA, sigC, sigE,
sigI, ndhF4, ndhF3, ndhR, sbtA and slr2031 transcripts. The reaction volume was 25 µL, and 4 ng µL-1 of the RNA preparation was used for each reaction. For detecting the other
transcripts, 400 ng of total RNA was first converted to cDNA by a reverse transcription
reaction using M-MLV reverse transcriptase (Promega, Madison, WI). The reaction
volume was 20 µL for each sample. The resulting cDNA was diluted with 80 µL of TE
buffer, and stored at –20 °C until being used. Two microliters of cDNA stock were used
for each PCR reaction (final volume, 25 µL) using Hot-Start Taq polymerase (Qiagen
Inc., Valencia, CA). RNasin (Promega, Madison, WI), an RNase inhibitor, was present
in the one-step RT-PCR reactions and during the cDNA synthesis.
Transmission Electron Microscopy
Cells grown in the absence or in the presence of glucose for 24 h were harvested
and immediately fixed in a 2.5 % glutaraldehyde solution prepared in a 0.1 M cacodylate
buffer, pH 7.4, over night at 4°C. The fixed cells were washed in the 0.1 M cacodylate
buffer for three times at room temperature, and subjected to the second fixation in a 1%
osmium tetroxide solution prepared in the 0.1 M cacodylate buffer for 1. 5 h at room 224 temperature. The cells were washed twice in the 0.1 M cacodylate buffer and then once in double distilled water. After incubation in a 2 % uranyl acetate solution for 2 h at room temperature, the cells were washed in the following concentration of ethanol: 50% (v/v),
70% (v/v), 90% (v/v), 95% (v/v) ethanol in water followed by two washes in 100% (v/v) ethanol. Final washes were performed three times in 100% ethanol (EM grade) and then three times in acetone at room temperature before infiltration. The washed cells were incubated in the presence of the following concentration of the resin Spurr (Spurr 1969) over night at room temperature: 50% (v/v) and 72 % (v/v) in acetone and twice in 100%
(v/v) Spurr. The samples were then polymerized at 60°C over night. Thin sections (ca.
50-60 nm thickness) were stained with 2 % (v/v) uranyl acetate before examination under a JEM 1200 EXII transmission electron microscope (Joel USA Inc., Peabody, MA,
USA).
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Fig 5.1: Time-course RT-PCR analysis of the cpcA, nblA, and sbpA transcripts in wild type and slr1736 mutant grown at pH 7.0 and pH 8.0. Cell were grown in the absence of glucose (shown at 0 h) and in the presence of glucose -2 -1 for 4, 8, and 24 h, at 1% (v/v) CO2, 32 °C at 50 µE m s . 234
Fig 5.2: Time-course RT-PCR analysis of alternative sigma factor transcripts in wild type and slr1736 mutant grown at pH 7.0 and pH 8.0. Cell were grown in the absence of glucose (shown at 0 h) and in the presence of glucose -2 -1 for 4, 8, and 24 h, at 1% (v/v) CO2, 32 °C at 50 µE m s . 235
Fig 5.3: Time-course RT-PCR analysis of transcripts for sigA, sigG and genes involved in Ci uptake and fixation in wild type and slr1736 mutant grown at pH 7.0 nd pH 8.0. Cell were grown in the absence of glucose (shown at 0 h) and in the presence of glucose -2 -1 for 4, 8, and 24 h, at 1% CO2 (v/v), 32 °C at 50 µE m s .
236
Fig 5.4. Thin-section electron micrographs of the wild-type cells grown (A) in the absence and (B) in the presence of glucose for 24 h -2 -1 Cells were grown in medium BHEPES40, pH 7.0, 1% (v/v) CO2, 32 °C, 50 µE m s , at 32 °C. Cells in the mid-exponential growth phase (A) were transferred to medium containing glucose and allowed to grow for 24 h (B). 237
Fig 5.5: Thin-section micrographs of the slr1736 mutants grown (A) in the absence and (B) in the presence of glucose for 24 h Arrows in (B) show the novel, proteinaceous structure (see text). Cells were grown in -2 -1 medium BHEPES40, pH 7.0, 1% (v/v) CO2, 32 °C, 50 µE m s , at 32 °C. Cells in the mid-exponential growth phase (A) were transferred to medium containing glucose and allowed to grow for 24 h (B). 238
Fig 5.6: Time-course RT-PCR analyses of the slr2031 and various CCM gene transcripts in wild type and slr1736 mutant grown at pH 7.0 and pH 8.0, 1% (v/v) -2 -1 CO2, 32 °C at 50 µE m s . Cell were grown in the absence of glucose (shown at 0 h) and in the presence of glucose for 4, 8, and 24 h.
239
Fig 5.7: PCR analysis of the genomic DNAs extracted from the slr2031::aadA and pmgA::accC1 mutants. Reaction s were carried out using (A) the primer set slr2031-F and slr2031-R, and (B) the primer set pmgA-F and pmgA-R (see Materials and Methods). Left and right lanes in each panel shows PCR products amplified from the wild type (WT) and mutant genomic DNAs. 240
Fig 5.8: Cultures of wild type, slr1736, slr2031, and pmgA mutants of Synechocystis sp. PCC 6803 grown in the presence of glucose (A) at pH 8.0 and (B) at pH 7.0 -2 -1 Cell were grown at 1% (v/v) CO2, 32 °C at 50 µE m s . 241
Fig 5.9: Summary and a model of the non-antioxidant role of α-tocopherol in Synechocystis sp. PCC 6803 A Rsb-like regulatory pathway, which involves PmgA, appears to exist in Synechocystis sp. PCC 6803 and possibly in other cyanobacteria (see text). In this model, α-tocopherol either directly or indirectly interacts with the Rsb-like pathway, possibly via the function of PmgA. Through such interaction, it is hypothesized that α-tocopherol affects the transcriptional regulation of some metabolic genes, which may be required for photomixotrophic growth of this organism. 242
Chapter 6
Comparative Genome Analyses and Genetic Studies of the Plastoquinone
Biosynthesis Pathway in Cyanobacteria
Publication:
Yumiko Sakuragi and Donald A. Bryant, manuscript in preparation 243 ABSTRACT
The biosynthetic pathway of plastoquinone-9 (PQ-9) was studied by means of
comparative genome analyses in 14 cyanobacteria. The presence of genes homologous to
ubiA, ubiC, ubiD, ubiE, ubiH, and ubiX involved in ubiquinone-8 (UQ) biosynthesis in
Escherichia coli was detected in cyanobacterial genomes, which suggests that PQ-9
biosynthesis in cyanobacteria occurs from 4-hydroxybenzoate through a pathway similar
to that for the UQ biosynthesis in proteobacteria. Attempted insertional inactivation of
the ubiA, ubiX, and ubiH homologs in Synechocystis sp. PCC 6803 resulted in an
incomplete segregation of the alleles, indicating that the products of these genes are
essential for the survival of this cyanobacterium. Of the several homologs of ubiE genes
found in the cyanobacterial genomes, only two are conserved among 14 species.
However, inactivation mutagenesis of these two conserved ubiE homologs, sll1653 and
sll0418, as well as others, including sll0829, slr1039, sll0487, slr0407, and slr1618 had
little effect on the cellular PQ-9 content. These results therefore demonstrate that these
UbiE homologs are probably not involved in PQ-9 biosynthesis in cyanobacteria. After exhaustive database searching, the presence of ubiC homologs, which are closely related
to the plastid ycf21 gene, was detected in many cyanobacterial genomes. Taking these
observations together, a hypothetical PQ-9 biosynthetic pathway in cyanobacteria is
proposed.
244 ABBREVIATIONS
PQ-9 plastoquinone-9
UQ-8 ubiquinone-8
MSBQ 2-methyl-6-solanyl-1,4-benzoquinone
MPBQ 2-methyl-6-phytyl-1,4-benzoquinone
ORF open reading frame
4-HBA 4-hydroxybenzoate
PCR polymerase chain reaction
HPLC high performance liquid chromatography
SAM S-adenosyl-L-methionine
245 INTRODUCTION
Plastoquinone (PQ-9, 2-3-dimethyl-5-solanyl-1,4-benzoquinone) is an isoprenoid
quinone that is only synthesized in oxygenic phototrophs including cyanobacteria, algae, and higher plants (Threlfall and Whistance, 1971; Collins and Jones, 1981). It serves as a one-electron carrier (QA) as well as a two-electron carrier (QB) within Photosystem II and
+ - stabilizes the charge separation between P680 and QB upon photoexcitation (Barry et al., 1994; Diner and Babcock, 1994; Bricker and Ghanotakis, 1996; Britt, 1996). PQ-9 also shuttles electrons from NADH dehydrogenase to cytochrome b6f complex, therefore serving as an essential membrane-associated, lipid-soluble electron carrier both in photosynthetic and respiratory electron transport chains.
Studies of PQ-9 biosynthesis have been carried out since the 1960’s in higher plants by using isotopic tracers and direct enzyme activity assays (see a review by
Threlfall and Whistance, 1970) as well as by genetic methods (Collakova and
DellaPenna, 2001; Shintani et al., 2002; Cheng et al., 2003). The combined results have shown that PQ is synthesized from homogentisate after two enzymatic reactions: condensation of solanyl diphosphate and homogentisate by a solanyl transferase resulting in 2-methyl-6-solanyl-1,4-benzoquinone (MSBQ), and a methylation reaction at the C3
position of the benzoquinoid ring system by MSBQ methyltransferase. This MSBQ
methyltransferase is also responsible for methylation of 2-methyl-6-phytyl-1,4-
benzoquinone (MPBQ) in α-tocopherol biosynthesis, and therefore it is a bifunctional
enzyme in higher plants (Shintani et al., 2002; Cheng et al., 2003). Given the frequent
similarity of biochemical pathways in plants and cyanobacteria, it might be assumed that 246 the same pathway functions in cyanobacteria. However, recent studies (Dänhardt et al.,
2002; Cheng et al., 2003) showed that neither the synthesis of homogentisate nor the presence of MPBQ methyltransferase activity is required for PQ-9 biosynthesis in the cyanobacterium Synechocystis sp. PCC 6803. These results suggested that PQ-9 biosynthesis in cyanobacteria is different from that in higher plants and that the PQ-9 biosynthetic pathways convergently evolved in cyanobacteria and higher plants (Cheng et al., 2003).
How is PQ synthesized in cyanobacteria? A preliminary inspection of the whole genome sequence of Synechocystis sp. PCC 6803 has shown the presence of ORFs that show sequence similarity to enzymes responsible for ubiquinone-8 (UQ-8, 2,3- dimethoxy-5-methyl-6-octaprenyl-1,4-benzoquinone) biosynthesis. UQ-8 and PQ-9 are both members of the alkyl-substituted benzoquinone family and contain a 1,4- benzoquinoid ring with a polyprenyl side chain (45 carbon units for PQ-9 and 40 carbon units for UQ-8) at the C6 position of the benzoquinoid ring system. UQ-9 biosynthesis occurs as follows (Fig 6.1, see a review by Meganathan, 2001, and references therein).
The first committed step of the pathway is the aromatization of chorismate to 4- hydroxybenzoate (4-HBA), which is catalyzed by chorismate-pyruvate lyase (UbiC).
Prenylation by 4-HBA octaprenyltransferase (UbiA) and decarboxylation by 3- octaprenyl-4-HBA decarboxylase (UbiD and UbiX) results in the synthesis of 2- octaprenyl phenol, which is converted to UQ after three hydroxylation reactions that alternate with three methylation reactions. A hydroxyl group is first introduced by 2- octaprenyl-phenol monooxygenase (UbiB) (at the position ortho to the C1 OH), which is 247 subsequently methylated by an O-methyltransferase (UbiG) resulting in 2-methoxy-6-
octaprenyl phenol. Secondly a hydroxyl group is introduced by a monooxygenase (UbiH)
at the position para to the C1 hydroxyl group, resulting in the synthesis of 2-methoxy-6-
octaprenyl-1,4-benzoquinol. UbiE, a C-methyltransferase, delivers a methyl group to a
position ortho to the C6 prenyl side chain, resulting in the synthesis of 2-methoxy-5-
methyl-6-octaprenyl-1,4-benzoquinol. Further hydroxylation by a monooxygenase (UbiF)
and methylation by UbiG at the C3 position of the ring system results in UQ-8. In the
structure of PQ-9, the two methoxy groups are replaced by two methyl groups (see Fig
6.1), and no additional methyl group is present at the ring position ortho to the prenyl
side chain. Therefore, it is possible that the PQ-9 biosynthesis occurs by a pathway that is
similar to the UQ-8 biosynthesis pathway in proteobacteria, at least in that part of the
pathway that leads to the formation of the prenyl benzoquinone.
To investigate the PQ-9 biosynthesis pathway in cyanobacteria, comparative
genome analyses using 14 cyanobacterial genome sequences were performed in
combination with a reverse genetic approach. The results suggest that the PQ-9
biosynthesis occurs by a pathway similar to that for the UQ-8 pathway in proteobacteria. 248 RESULTS
Comparative Genome Analyses
As of April 26, 2004, largely completed genomic sequences of 14 cyanobacteria are available: eight complete sequences exist for Synechocystis sp. PCC 6803 (Kaneko et al., 1996), Nostoc sp. PCC 7120 (Kaneko et al., 2001), Thermosynechococcus elongatus
BP-1 (Nakamura et al., 2002a,b), Gloeobacter violaceus (Nakamura et al., 2003),
Prochlorococcus marinus MED4 and Prochlorococcus marinus MIT9313 (Rocap et al.,
2003), Prochlorococcus marinus SS120 (Dufresne et al., 2003), Synechococcus sp.
WH8102 (Palenik et al., 2003); and incomplete sequences exist for Synechococcus sp.
PCC 7002 (J. Marquardt, T. Li, J. Zhao, and D. A. Bryant, unpublished), Nostoc punctiforme (Gene Bank accession number: NZ_AAAY00000000), Trichodesmium erythraeum (Gene Bank accession number: NZ_AABK00000000), Synechococcus elongatus PCC 7942 (Gene Bank accession number: NZ_AADZ01000001), Anabaena variabilis ATCC 29413 (Gene Bank accession number: NZ_AAEA01000001), and
Crocosphaera watsonii WH8502 (Gene Bank accession number: NZ_AADV01000004).
Database searches using the E. coli UbiA, UbiB, UbiC, UbiD, UbiE, UbiF, UbiG, UbiH, and UbiX protein sequences against these genomes revealed the presence of homologs for UbiA, UbiD, UbiX, UbiE, and UbiH. Similarities between the sequences of E. coli
Ubi proteins and the cyanobacterial homologs were about 50% for UbiA (Table 6.1), 30-
50% for UbiH (Table 6.2), 60% for UbiD (Table 6.3), and 50% for UbiX (Table 6.4).
Multiple UbiE/UbiG homologues were detected, and the number varied depending on the species (Table 6.5). The observed conservation of these Ubi proteins among 249 cyanobacteria and between cyanobacteria and E. coli suggests that the part of the UQ-8 biosynthesis pathway involving prenylation, decarboxylation and the introduction of a hydroxyl group is probably functioning in cyanobacteria. The only exception to this is P. marinus MED4, in which no UbiD and UbiX homologs were detected. Reverse-phase
HPLC analyses of the whole cell extracts confirmed that P. marinus MED4 synthesizes about two molecules of PQ-9 per 100 chlorophyll a molecules. Similar value was obtained for the whole cell extracts from Synechocystis sp. PCC 6803. Therefore, it is safely concluded that P. marinus MED4 synthesizes PQ-9 and it is suggested that the carboxylation reaction is probably catalyzed by yet unknown enzymes.
UQ-8 biosynthesis in E. coli involves eight enzymes and two of these were absent in cyanobacteria: UbiB, and UbiF. The absence of UbiB and UbiF, the monooxygenases that introduce hydroxyl groups into ring system (which are subsequently methylated by
UbiG), is consistent with the absence of the methoxy substituents in PQ-9. In combination with the results described above, it was hypothesized that biosynthesis of
PQ-9 in cyanobacteria occurs as shown in Fig. 6.2. In this pathway, 4-hydroxybenzoate is converted by UbiA to 2-solanyl-4-hydroxybenzoate, which is decarboxylated by
UbiD/UbiX giving rise to 2-solanyl phenol. It is proposed that 2-solanyl phenol is first hydroxylated by UbiH, followed by two methylation reactions performed by one or more of the UbiE homologs, although it is entirely possible that one or both of the methylation reactions occurs before hydroxylation, as is the case for O-methylation by UbiG, which precedes hydroxylation by UbiH during UQ-8 synthesis (see Meganathan, 2001, and Fig
6.1). 250 UbiC, chorismate lyase, is responsible for the first committed step in the pathway
(Fig 6.1). The ubiC gene in some β-proteobacteria and γ-proteobacteria, including
Neisseria meningitis Z2491, Pseudomonas aeruginosa PA01, Salmonella enterica subsp. enterica serovar Typhi, Shigella flexneri str. 301 setotype 2a, Yersinia pestis C092 and E. coli, is located immediately upstream of the ubiA gene based on genome analyses performed on these organisms. In cyanobacteria, no ubiC homolog was found in the vicinity of the ubiA gene, and no obvious ubiC homologs were found in their genomes.
These observations appeared to indicate that homologs of this gene were absent in the cyanobacteria, and that synthesis of 4-hydroxybenzoate occurs through an alternative pathway (see discussion). After exhaustive database searching, however, ORFs that showed very weak similarity to the UbiC sequences of E. coli (< 30 %) were detected
(Table 6.6). These ORFs were highly conserved among cyanobacteria (50 –80% similarity) and showed amino acid sequence similarity to Ycf21 proteins, which are also found in the plastid genomes of eukaryotic phototrophs including Porphyra purpurea and
Cyanophora paradoxa. Fig 6.3 shows an alignment of sequences of the UbiC protein in
E. coli and the Yfc21 homologs in the cyanobacteria and eukaryotic phototrophs. In the alignment, a glutamate residue at the position 155 (UbiC) was found to be conserved among the Ycf21 homologs in both the cyanobacteria and eukaryotic phototrophs. This residue constitutes a portion of the catalytic site and is hydrogen-bonded to the hydroxyl oxygen of 4-hydroxybenzoate as previously shown in the 1.4-Å resolution X-ray crystal structure of the UbiC protein (Gallagher et al., 2001). An arginine residue at the position
79, which has also been shown to constitute a portion of the active site, was conserved among many cyanobacteria, although not all. Therefore, it is possible that the Ycf21 251 homologs catalyze the conversion of chorismate to 4-hydroxybenzoate in the PQ-9
biosynthesis in cyanobacteria.
Mutagenesis of ubiA, ubiX, and ubiH
An attempt was made to inactivate the ORFs slr0926 (ubiA), slr1099 (ubiX), and slr1300 (ubiH) in Synechocystis sp. PCC 6803 by insertion of the aphII gene conferring
kanamycin resistance. These attempts, however, failed to lead to homozygous mutants
and resulted in incomplete segregation of the alleles under photoautotrophic conditions
even under selective conditions in the presence of 50 µg mL-1 kanamycin (data not
shown). Addition of glucose to growth medium, which provides electrons to the electron
transport chains through NADH (thereby bypassing Photosystem II), likewise did not
lead to complete segregation. Addition of various quinoid compounds to the growth
medium either to inhibit the Photosystem II activity (atrazine) or to compensate the loss
of PQ-9 (2,6-dimethyl-1,4-benzoquinone, PhyQ, UQ-0, UQ-1, and UQ-2) did not lead to complete segregation (data not shown). Merodiploid mutants were unstable both under photoautotrophic and photomixotrophic conditions under illumination at a normal light intensity (20 - 50 µE m-2 s-1), as no further growth was observed after several transfers on
solid media. These results indicates that the ubiA, ubiX, and ubiH genes are indispensable
for the survival of Synechocystis sp. PCC 6803, which is consistent with the anticipated
essential role of plastoquinone in cyanobacteria.
252 Phylogenetic Reconstruction with Ubi proteins of Cyanobacteria and Proteobacteria
Evolutionary relationships between the Ubi homologues of cyanobacteria and proteobacteria (E. coli, Rhodopseudomonas palustris, Rhodospirillum rubrum and
Rhodobacter sphaeroides) were analyzed by constructing phylogenetic trees based on deduced amino acid sequences and by comparing the results to the phylogenetic tree based on 16S ribosomal RNA (rRNA) (Fig 6.4). 16S rRNA is universally present and highly conserved among prokaryotes and thus is often used as a molecular marker to reflect the evolutionary clock (Woese, 1987). Phylogenetic trees based on the nucleotide sequence of 16S rRNA showed that the cyanobacteria and proteobacteria formed independent groups, which is consistent with previous results (Woese, 1987). Within the cyanobacteria, two major clades were observed: clade A consisted of Synechocystis sp.
PCC 6803, Synechococcus sp. PCC 7002, Nostoc sp. PCC 7120, N. punctiforme, and T. erythraeum, and clade B consisted of Synechococcus sp. WH8102 and the three
Prochlorococcus species (Fig 6.4). Phylogenetic trees based on the amino acid sequences of UbiA, UbiD, and UbiX showed similar branching orders and topologies to the tree for
16S rRNA. The trees were supported by high bootstrap values (Fig 6.4), suggesting that the ubiA, ubiD, and ubiX genes have evolved through vertical heritage from the last common ancestor of both cyanobacteria and the proteobacteria. The topology of the tree based on the UbiH sequences appeared different, however. Within the group formed by all cyanobacteria, the proteobacteria formed a cluster with clade A, which may indicate the lateral transfer of the ubiH gene between these two groups of organisms. However, the bootstrap value assigned to the branch connecting a node where cyanobacteria in clade A and clade B bifurcate and a node where the proteobacteria deviate from the 253 cyanobacteria in clade A is only 52, which only poorly supports the likeliness of this branching order. Alternatively, it is possible that the out-group used to construct the tree
(Mlr2092, a putative UbiH homologue in Mesorhizobium loti) may be distorting the topology and thereby gives rise to an artifact. This happens if the sequence of the out- group is more closely related to the cyanobacterial sequences than to the proteobacterial sequences. As shown in Table 6.2, the amino acid sequence of Mlr2092 that was used as an out-group is equally distantly related to any of the UbiH sequences analyzed. The replacement of Mlr2092 by another protein sequence, NahG (salicylate hydroxylase in
Pseudomonas putida ) or Bsu1050 (a putative monooxygenase in Bacillus subtilis), did not alter the topology of the tree. Therefore, it is possible that the proteobacteria form a group within the cyanobacteria and a lateral exchange of ubiH took place between proteobacteria and the cyanobacteria in clade A. This evolutionarily close relationship of
UbiH homologs between the two groups of organisms provides further support for the involvement of these proteins in the PQ-9 biosynthesis in cyanobacteria.
UbiE
Database search of the genomes of cyanobacteria showed the presence of multiple
UbiE homologs (Table 6.5, Fig 6.5). There are two ORFs that are conserved in all 14 cyanobacteria: these are homologs of Sll1653 and Sll0418 of Synechocystis sp. PCC
6803. Note that the incomplete genome sequence of N. punctiforme only contained a homolog of Slr0089, which encodes γ-tocopherol methyltransferase. Whether a homolog of Sll0418 is present in this organism is yet to be determined. Homologs of Sll0829 and
Sll0487 are conserved in all cyanobacteria except P. marinus MED4 and P. marinus 254 SS120. Slr1618 and Slr0407 seem to be specific to Synechocystis sp. PCC 6803 and C. watsonii, or Synechocystis sp. PCC 6803, A. variabilis, and G. violaceus, respectively
(Table 6.5). Amino acid sequences of all these homologs contain the conserved S- adenosyl-L-methionine binding motif (data not shown). UbiE in E. coli is a bifunctional enzyme and is responsible for the C-methylation reactions in both UQ-8 and menaquinone biosyntheses (Lee et al., 1997). Phylogenetic analyses conducted on the
UbiE homologues of cyanobacteria showed that Sll1653 forms a cluster with the E. coli
UbiE (Fig 6.5). Furthermore, as described in Chapter 2 and Chapter 3 (also see Sakuragi et al., 2002), this ORF is responsible for transferring a methyl group to 2-prenyl-1,4- naphthoquinone in phylloquinone and menaquinone-4 biosyntheses in cyanobacteria.
Therefore, it was expected that Sll1653 would be responsible for at least one of the methylation reactions in PQ-9 synthesis. Whole-cell extracts from the sll1653 mutant were analyzed by reverse-phase HPLC. PQ-9 eluted at 36 min using the current analytical system (see “Materials and Methods”, see also Chapter 2). Whereas the chemically synthesized 2-methyl-6-solanyl-1,4-benzoquinone (a kind gift from Prof. DellaPenna in the Michigan State University, see Shintani et a., 2000), a predicted intermediate in the
PQ-9 synthesis, eluted at 33 min as shown in Fig 6.6A. The HPLC profile of the whole- cell solvent extracts from the sll1653 mutant showed a peak at 35.5 min. The absorption spectrum of the component eluting at this retention time showed a maximum at 256 nm
(Fig 6.6B, solid line), which is characteristic of PQ-9 (Dunphy and Brodie, 1971) and is a few nanometers red shifted as compared to the absorption maximum observed for the
MPBQ standard (Fig 6.6B, dotted line). The results therefore demonstrate that PQ-9 is present in the sll1653 mutant cells. The contents of PQ-9 were analyzed based on the 255 integrated peak area in the HPLC chromatograms and were expressed as relative to the
cellular contents of chlorophyll a (Fig 6.7). The results showed that the PQ-9 content in
the sll1653 mutant is comparable to that in wild type. Therefore, it was concluded that
Sll1653 is not involved in the PQ-9 biosynthesis or that a redundant activity conferred by another methyltransferase(s) is present.
An in vitro study has previously demonstrated that recombinant Sll0418, MPBQ methyltransferase, can catalyze methylation of MSBQ and lead to the synthesis of PQ-9
(Shintani et al., 2002). Because this ORF is also highly conserved among the cyanobacteria (Table 6.5, Fig 6.5), it was predicted that Sll0418 is either responsible for transferring of one or two of the methyl groups in the PQ-9 biosynthesis or that it provides a redundant activity with Sll1653. Targeted inactivation and complete segregation of the mutated allele was performed and confirmed by PCR (see Chapter 3).
The sll0418 mutant was, however, capable of synthesizing the wild-type level of PQ-9 as shown by HPLC analysis on the whole-cell lipid extracts (Fig 6.7).
Double and triple mutants of the UbiE homologs were constructed in
Synechocystis sp. PCC 6803 in further attempt to identify methyltransferase(s) involved in the PQ-9 biosynthesis. Combinations of mutated loci are as follows: double mutations were constructed for sll1653 and sll0418, sll1653 and slr0407, sll1653 and sll0829, sll0418 and sll0829, sll0418 and slr0407, and sll0418 and slr0089; and triple mutations were constructed for of sll1653, sll0418 and sll0829; and sll1653, slr0407, and sll0829.
Antibiotic resistance cartridges were used to insertionally inactivate these genes (Fig 6.8).
The full segregation of the mutated alleles from the respective wild-type alleles was 256 analyzed by PCR as shown in Fig 6.9. PCR using the designed primers (Table 6.7) that
are targeted to the sll0829, sll01653, sll0418, slr0407, slr0089, slr1618 resulted in
products of 0.84 kbp, 0.3 kbp, 0.92 kbp, 0.94 kbp, 1.2 kbp, and 1.8 kbp, respectively (Fig
6.9, lane 1), which are expected based on the restriction maps as shown in Fig 6.8. No
product with corresponding wild-type size was detected for the mutants (Fig 6.9, lanes 2); instead products with larger sizes were detected in all cases. The difference between the mutants and the wild-type corresponds either to the size of antibiotic resistance cartridges or to the size of antibiotic resistance cartridges minus the deleted regions (Fig 6.8).
Therefore, it was concluded that all the mutants are free of respective wild-type alleles and were homozygous in the allele for which the drug cartridges had been introduced.
Reverse-phase HPLC analyses on whole-cell lipid extracts showed that none of the double and triple mutants showed a significant difference from the wild type in the amount of PQ-9 accumulated in the cells (Fig 6.7). The fact that the sll1653 sll0418 double mutant did not affect the PQ-9 content strongly indicates that Sll1653 and
Sll0418, despite their conservation among all the cyanobacteria and their predicted functioning in PhyQ/MQ synthesis (or the demonstration of function in vitro PQ-9 synthesis), do not play major roles in the PQ-9 biosynthesis. This is further supported by the construction of the sll1635 sll0418 double mutant, which also synthesized a wild-type level of PQ-9 (Fig 6.7). The possibility that another UbiE homolog is providing an additional activity to Sll01653 and/or Sll0418 was tested by analyzing the PQ-9 contents in the sll0418 slr1618, sll0418 slr0407, sll0418 sll0829, sll1653 sll0829, and sll1653 slr0407 double mutants by reverse-phase HPLC. The results showed that all of the double 257 mutants contained essentially the wild-type level of PQ-9, which indicates that Sll0829,
Slr0407, and Slr1618 are not involved in the PQ-9 biosynthesis. This is further supported by the result that the two triple mutants, sll1653 sll0418 sll0829 and sll1653 slr0407 sll0829, contained a wild-type level of the PQ-9 in the whole-cell solvent extracts.
Participation of Slr0089 in the PQ synthesis was also ruled out as the sll0418 slr0089
double mutant synthesized the wild-type level of PQ-9. Slr1039 and Slr0487 are two
hypothetical proteins with an S-adenosyl-L-methionine binding motif, and they are well
conserved among most of the cyanobacteria (Fig 6.5, Table 6.5). Targeted inactivation of
these ORFs, however, did not affect the PQ-9 biosynthesis. In combination, these results
strongly suggest that the UbiE homologs do not play a major role in the PQ-9
biosynthesis in Synechocystis sp. PCC 6803.
258 DISCUSSION
Database searches of the whole genome sequences of 14 cyanobacteria (8
complete and 6 incomplete) revealed the presence of the UbiA, UbiC, UbiD, UbiE, UbiH, and UbiX homologs, which are proteins involved in the UQ-8 biosynthetic pathway in
proteobacteria. Attempted insertional inactivation of the ubiA, ubiH, and ubiX genes in
Synechocystis sp. PCC 6803 resulted in the generation of unstable merodiploids, which
indicates that these genes are essential for the survival of this organism and which is
consistent with the presumed essential role of PQ-9 in cyanobacteria. Based on these
findings, it is proposed that the PQ biosynthesis occurs in cyanobacteria as shown in Fig
6.2. In this proposal, UbiA catalyzes the condensation of 4-hydroxybenzoate and
solanyldiphosphate resulting in 3-solanyl-4-hydroxybenzoate, which is decarboxylated by
UbiD/UbiX resulting in 2-solanyl phenol. Hydroxylation by UbiH is expected to take
place at the C4 position of the ring system, resulting in 2-solanyl-1,4-benzoquinol.
Whether the two methylation reactions at the C2 and C3 position take place before or after
the hydroxylation is an open question. These methylation reactions, however, are not
catalyzed by the UbiE homologs (Sll1653, Sll0418, Sll0829, Slr0407, Sll0487, Slr1618
and Slr1039) in Synechocystis sp. PCC 6803 as demonstrated by insertional mutagenesis
and subsequent HPLC analyses. Phylogenetic analyses have indicated that the presence
of the UbiA, UbiD, and UbiX homologs in cyanobacteria are the result of vertical
heritage from the common ancestor of cyanobacteria and proteobacteria, suggesting a
possible common evolutionary origin of the UQ-8 and PQ-9 biosyntheses. UbiE
(Sll1653) is probably inherited from the common ancestor and evolved in cyanobacteria 259 to be specific to the methylation reaction in the PhyQ/MQ biosynthesis, while another methyltransferase(s) has been recruited to function for the PQ-9 biosynthesis.
The methyltransferase(s) that function in the PQ-9 biosynthesis is/are still a mystery. The fact that mutants of 7 UbiE homologs contained wild-type levels of PQ-9 suggests i) that a SAM (S-adenosyl-L-methonine)-dependent methyltransferase(s) that is only very distantly related to UbiE is responsible for these reactions, or ii) that a SAM- independent methyltransfrase(s) is responsible for the reactions. Of the few SAM- independent methyltransferases detected in the Synechocystis sp. PCC 6803 genome, none is conserved among cyanobacteria except for slr0212, which is annotated as encoding the methionine synthase and uses 5-methyltetrahydrofolate as the methyl donor
([EC 2.1.1.13], see http://www.chem.qmul.ac.uk/iubmb/enzyme/EC2/1/1/13.html). This
ORF is, however, highly conserved among other bacteria and eukaryotes and is present in the genome as a single copy. Therefore, this gene product most likely functions as methionine synthase and is unlikely to be involved in the PQ biosynthesis. When the genome was searched for SAM-binding proteins, ORFs sll1407, slr1115, slr0303, slr1071, sll1693, slr1815 were detected. Of these, slr0303 is the only ORF that is conserved among all the cyanobacteria. The possible involvement of this hypothetical protein in PQ-9 biosynthesis needs to be tested.
The presence of conserved Ycf21 homologs in cyanobacteria, which are weakly similar to UbiC in E. coli, suggested that 4-hydroxybenzoate derives from chorismate by the action of chorismate-lyase activity possibly conferred by these Ycf21 homologs. This was thought to be a highly plausible hypothesis because catalytic residues of the UbiC 260 protein are largely conserved among the Ycf21 homologs in cyanobacteria. However, the
poor amino acid sequence similarity between the Ycf21 homologs and UbiC proteins
leaves a possibility that an alternative pathway exists that leads to the synthesis of 4- hydroxybenzoate from a compound other than chorismate. Genome analyses have revealed that many organisms that synthesize UQ lack UbiC, including higher plants, animals, and even some proteobacteria such as Agrobacterium tumefaciens, Brucella melitensis, Rhodopseudomonas palustris, Chromobacterium violaceum, and
Xanthomonas axonopodis. There are three pathways that are known to lead to the synthesis of the intermediate 4-hydroxybenzoate in these organisms: i) the UbiC- dependent chorismate pathway in E. coli and some β- and γ-proteobacteria; ii) the 4- hydroxyphenyllactate pathway in rat (see review by Meganathan, 2001, and references therein); and iii) the aromatic amino acid pathway in higher plants (see a review by
Threlfall and Whistance, 1971, and references therein). In UQ biosynthesis, 4- hydroxybenzoate is synthesized from the aromatic amino acid tyrosine or phenylalanine via p-coumarate (Loscher and Heide, 1994). It has also been reported that the cyanobacterium Anacystis nidulans converts the aromatic amino acid tyrosine to 4- hydroxybenzoate, and that this conversion may be carried out by thylakoid-bound enzyme complexes consisting of tyrosine ammonia-lyase and 4-hydroxybenzoate synthase (Löffelhardt, 1976). Therefore, it is plausible that a similar aromatic amino acid pathway may exist in other cyanobacteria and could provide 4-hydroxybenzoate for PQ-9 biosynthesis as proposed in Fig 6.10. 261 Lastly, the fact that the sll0418 mutation did not affect the cellular PQ content in
Synechocystis sp. PCC 6803 indicates that Sll0418 is only involved in α-tocopherol biosynthesis. However, this ORF is conserved in nearly all cyanobacteria whose genome sequence is so far available, including those that are predicted not to be capable of α- tocopherol synthesis (Synechococcus sp. WH 8102 and three Prochlorococcus species, see Chapter 5). What is the function of the Sll0418 homologs in these cyanobacteria? Is it possible that the Sll0418 homologs are involved in PQ biosynthesis in these cyanobacteria, given that the recombinant Sll0418 can catalyze the methylation of MSBQ and generate PQ-9. Genetic analyses of the role of Sll0418 needs to be performed in these other organisms. 262 SUMMARY
The PQ-9 biosynthesis pathway was studied by means of comparative genome
analyses in 14 cyanobacteria. The presence of UbiA, UbiD, UbiE, UbiH, and UbiX
homologs, which are required for UQ-8 biosynthesis in E. coli, was detected in the cyanobacteria. The presence of Ycf21 homologs, which show weak similarity to UbiC, was detected after exhaustive database searching in cyanobacteria. The results suggest that PQ-9 synthesis occurs similarly to UQ-8 biosynthesis pathway. Targeted inactivation of six UbiE homologs (methyltransferases), however, did not affect PQ-9 biosynthesis in
Synechocystis sp. PCC 6803. This suggested that enzymes that are not evolutionarily related to UbiE in E. coli catalyze the methylation reactions in PQ-9 biosynthesis in cyanobacteria. 263 MATERIALS AND METHODS
Growth Conditions
The wild-type strain used for transformation and further analysis was a glucose-
tolerant stain of Synechocystis sp. PCC 6803 (Williams, 1988). Medium BHEPES, pH
8.0, was used for selection, maintenance, and growth measurements of wild type and
mutants. This medium is prepared by supplementing BG-11 medium (Stainer et al., 1971)
with 4.6 mM of HEPES [4-(2-hydroxyethyl)piperazine-N’-(2-ethanesulfonic acid)]-KOH
and 18 mg L-1 ferric ammonium citrate. The wild-type and ubiA, ubiH, and ubiX mutant
strains were grown on solid medium BHEPES containing the following additions: 5 mM
glucose alone; 5 mM glucose and various concentrations (5 µM, 50 µM, 500 µM, 1 mM, and 10 mM) of ubiquinone-0 (2,3-dimethoxy-5-methyl-1,4-benzoquinone), ubiquinone-1
(2,3-dimethoxy-5-methyl-6-(3-methyl-2-butenyl)-1,4-benzoquinone), ubiquinone-2 (2,3- dimethoxy-5-methyl-6-geranyl-1,4-benzoquinone), or phylloquinone; 5 mM glucose and
5 µM atrazine (2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine); or 5 mM
glucose, 5 µM ubiquinone-2, and 20 µM 2,6-di-O-methyl-β-cyclodextrin. The cells on
these solid media were grown at room temperature at moderate light intensity (ca. 50 µE
m-2 s-1) or at low light intensity (ca. 5 µE m-2 s-1).
Constructions of Single and Double Methyltransferase Mutants
DNA fragments containing the sll0829, sll0418, slr0407, slr1618, slr1039, and
slr0089 genes were amplified from isolated genomic DNA by PCR using a set of forward and reverse primers (see Table 6.7 for sequences and Fig 6.8 for positions). The 264 constructions of the insertional inactivation of these alleles are described in Table 6.8.
The aacC1 gene, conferring gentamicin resistance, derived from the pMS266 plasmid
(Becker et al., 1995) and the aadA gene, conferring spectinomycin resistance, derived
from the pSRA2 plasmid (Frigaard et al., 2004) were inserted into the selected site within
these gene coding regions (see Table 6.8 and Fig 6.8). The resulting p829Gm and
p1039Sp plasmids were linearized and used to transform the wild-type Synechocystis sp.
PCC 6803 to obtain the homozygous sll0829::accC1 and slr1039::aadA mutants.
Genomic DNA isolated from the sll0418::aphII mutant (Chapter 4) and the
sll1653::aphII mutant (Chapter 3) were used to transform the homozygous
sll0829::accC1 mutant to generate the sll0418::aphII sll0829::accC1 and sll1653::aphII
sll0829::accC1 double mutants. The linearized p418Sp and p407Sp were used to
transform the homozygous sll1653::aphII sll0829::accC1 double mutant to generate the
sll1653::aphII sll0418::aadA sll0829::accC1 and sll1653::aphII slr0407::aadA
sll0829::accC1 triple mutants. The linearized p418Sp was used to transform the
homozygous sll1653::aphII mutant to generate the sll0418::aadA sll1653::aphII double
mutant. The linearized p1618Sp, p407Sp, and p89Gm were used to transform the
homozygous sll0418::aphII mutant to generate the sll0418::aphII 1618::aadA,
sll0418::aphII slr0407::aadA, and sll0418::aphII slr0089::accC1 double mutants. A
homozygous sll0487::aphII mutant was constructed in a previous study (W. M.
Schluchter, G. Shen, and D. A. Bryant, unpublished results). Complete segregation of the
each mutated alleles from the corresponding wild-type allele was confirmed by PCR.
Primer used for the PCR analyses are summarized in Table 6.7.
265 Quinone and Chlorophyll Analysis
Cells were harvested from photoautotrophically grown cultures and recovered in a
tight pellet. Pigments and quinones were extracted with 400 µL of cold acetone:methanol
(7:2 v/v) after brief ultrasonication. After centrifugation and filtration through a PTFE
filter membrane with a 0.2-µm pore size (Whatman International Ltd., Maldstone, UK),
the extract in the organic phase were directly injected into an Agilent Technology 1100
series HPLC system (Agilent Technology, Palo Alto, CA, USA) equipped with
® SUPELCO (Sigma-Aldrich Corp., St. Louis, MO, USA) Discovery C18 column (25 cm x 4.6 mm, 5 µm). Analyses were carried out using the following protocol: 80%A/20%B from 0 to10 min, a linear change to 20%A/80%B in 40 min, and 20%A/80%B for 5 min, where solvent A and solvent B are 100% methanol and 100% isopropanol, respectively.
The flow rate was 0.75 ml min-1. Detection of eluates was performed with a diode array
detector (Agilent 1100 series). The chlorophyll a, phylloquinone, and plastoquinone
contents of each sample were determined based on integrated peak areas and their molar
absorption coefficients, which are 17.4 mM-1 cm-1 at 618 nm, 18.9 mM-1 cm-1 at 270 nm
(Dunphy and Brodie, 1971), and 15.2 mM-1 cm-1 at 254 nm (Crane and Dilley, 1963),
respectively. The absorption coefficient of chlorophyll a at 618 nm was calculated from
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able 6.1: Amino acid sequence similarities between UbiA homologs in 14 cyanobacteria and E. coli (expressed as %)
Organism Locus tag 6803 7002 Nost Npun Tery Telon 8102 Selo Avar Cwat MED MIT S120 Gloe Eco Synechocystis 6803 Slr0926 100 Synechococcus 7002* ORF 76 100 Nostoc 7120 Alr3553 79 77 100 N. punctiforme* Npun4339 80 76 89 100 T. erythraeum* Tery4006 76 74 77 76 100 T. elongatus BP-1 Tlr0402 67 64 66 66 65 100 Synechococcus 8102 Synw1847 54 53 55 55 56 53 100 S. elongatus 7942* Selo021251 71 71 73 73 69 68 56 100 A. variabilis 29413* Avar020964 80 78 80 80 77 69 57 74 100 C. watsonii 8501* Cwat098001 83 76 98 89 79 68 54 72 80 100 P. marinus MED4 Pmm0456 53 52 54 51 55 55 64 57 54 55 100 P. marinus MIT Pmt9313 54 54 54 55 56 57 75 57 57 57 67 100 P. marinus SS120 Pro0455 56 54 56 56 57 53 70 55 58 59 68 75 100 G. violaceus 7421 Glr3428 71 70 70 70 69 67 52 71 73 72 53 53 56 100 E. coli K-12 UbiA 51 52 55 53 53 51 48 51 56 52 48 46 50 51 100
6803, Synechocystis sp. PCC 6803; 7002, Synechococcus sp. PCC 7002; Nost, Nostoc sp. PCC 7120; Npun, Nostoc punctiforme; Tery, Trichodesmium erythraeum; Telon, Thermosynechococcus elongatus BP-1; 8102, Synechococcus sp. WH8102; Selo, Synechococcus elongatus PCC 7942; Avar, Anabaena variabilis ATCC 29413; Cwat, Crocosphaera watsonii WH8501; Prochlorococcus marinus MED4; MIT, Prochlorococcus marinus MIT9313; S120, Prochlorococcus marinus SS120; Gloe, Gloeobacter violaceus PCC 7421; Eco, E. coli. Pairwise alignments were generated with BLOSUM30 using ClustalW. * Incomplete genome sequences. 272
Table 6.2: Amino acid sequence similarities between UbiH homologs in 14 cyanobacteria and E. coli (expressed as %) UbiH Locus tag 6803 7002 Nost Npun Tery Telon 8102 Selo Avar Cwat MED MIT S120 Gloe Eco Synechocystis 6803 Slr1300 100 Synechococcus 7002* ORF 64 100 Nostoc 7120 All4274 63 65 100 N. punctiforme* Npun3352 62 64 91 100 T. erythraeum* ------T. elongatus BP-1 Tlr2289 56 57 57 58 - 100 Synechococcus 8102 Synw0816 42 42 40 41 - 39 100 S. elongatus 7942* Selo021343 63 64 65 64 - 43 45 100 A. variabilis 29413* Avar025218 66 65 98 90 - 58 42 65 100 C. watsonii 8501* ------P. marinus MED4 Pmm0834 37 38 35 36 - 36 48 41 36 - 100 P. marinus MIT Pmt0812 35 43 42 44 - 42 63 49 45 - 47 100 P. marinus SS120 Pro0906 42 38 36 39 - 36 50 42 37 - 50 41 100 G. violaceus 7421 Gll2827 31 32 33 33 - 29 30 38 37 - 27 33 31 100 E. coli UbiH 47 52 47 49 - 45 40 49 49 - 38 42 40 29 100 Mesorhisobium loti* Mlr2092 33 31 33 34 - 32 26 33 32 - 28 31 27 29 29
6803, Synechocystis sp. PCC 6803; 7002, Synechococcus sp. PCC 7002; Nost, Nostoc sp. PCC 7120; Npun, Nostoc punctiforme; Tery, Trichodesmium erythraeum; Telon, Thermosynechococcus elongatus BP-1; 8102, Synechococcus sp. WH8102; Avar, Anabaena variabilis ATCC 29413; Selo, Synechococcus elongatus PCC 7942; Cwat, Crocosphaera watsonii WH8501; Prochlorococcus marinus MED4; MIT, Prochlorococcus marinus MIT9313; S120, Prochlorococcus marinus SS120; Gloe, Gloeobacter violaceus PCC 7421; Eco, E. coli. Pairwise alignments were generated with BLOSUM30 using ClustalW. * Incomplete genome sequences.* Used as an out-group for phylogenetic reconstruction (Fig 6.3). 273
Table 6.3: Amino acid sequence similarities between UbiD homologs in 14 cyanobacteria and E. coli (expressed as %)
Locus tag 6803 7002 Nost Npun Tery Telon 8102 Selo Avar Cwat MED4 MIT S122 Gloe Eco Synechocystis 6803 Sl0936 100 Synechococcus 7002* ORF 92 100 Nostoc 7120 Alr1108 93 92 100 N. punctiforme* Npun1887 93 92 98 100 T. erythraeum* ------T. elongatus BP-1 Tlr2225 88 87 88 87 - 100 Synechococcus 8102 Synw1006 81 79 81 80 - 81 100 S. elongatu 7942* Selo022640 89 89 90 90 - 88 82 100 A. variabilis 29413* Avar403501 93 92 98 97 - 87 81 90 100 C. watsonii 8501* ------P. marinus MED4 ------P. marinus MIT Pmt0397 78 78 80 79 - 79 90 80 80 - - 100 P. marinus SS120 Pro1047 79 77 79 79 - 79 85 80 80 - - 88 100 G. violaceus 7421 Gll3010 85 83 84 84 - 84 77 85 84 - - 76 78 100 E. coli K-12 UbiD 60 60 60 60 - 59 58 61 61 - - 59 59 59 100
6803, Synechocystis sp. PCC 6803; 7002, Synechococcus sp. PCC 7002; Nost, Nostoc sp. PCC 7120; Npun, Nostoc punctiforme; Tery, Trichodesmium erythraeum; Telon, Thermosynechococcus elongatus BP-1; 8102, Synechococcus sp. WH8102; Selo, Synechococcus elongatus PCC 7942; Avar, Anabaena variabilis ATCC 29413; Cwat, Crocosphaera watsonii WH8501; Prochlorococcus marinus MED4; MIT, Prochlorococcus marinus MIT9313; S120, Prochlorococcus marinus SS120; Gloe, Gloeobacter violaceus PCC 7421; Eco, E. coli. Pairwise alignments were generated with BLOSUM30 using ClustalW. * Incomplete genome sequences. 274
Table 6.4: Amino acid sequence similarities between UbiX homologs in 14 cyanobacteria and E. coli (expressed as %)
Organism Locus tag 6802 7002 Nost Npun Tery Telon 8102 Selo Avar Cwat MED4 MIT S120 Gloe Eco Synechocystis 6803 Slr1099 100 Synechococcus 7002* ORF 81 100 Anabaena 7120 Alr1241 85 83 100 N. punctoforme* Npun3020 87 84 92 100 T. erythraeum* (Tery2980) (64) - - - - T. elongatus BP-1 Tlr2225 80 78 80 79 - 100 Synechococcus 8102 Synw1201 69 67 66 68 - 67 100 S. elongatus 7942* Selo021196 79 76 78 77 - 78 64 100 A. variabilis 29413* (Avar334101) (91) ------C. watsonii 8501* ------P. marinus MED4 ------P. marinus MIT9313 Pmt0767 70 65 64 66 - 68 84 64 - - - 100 P. marinus SS120 Pro0989 71 66 66 65 - 68 78 66 - - - 78 100 G. violaceus 7421 Gll0529 78 77 80 80 - 78 77 74 - - - 68 68 100 E. coli UbiX 52 52 50 52 - 54 48 52 - - - 50 55 52 100
6803, Synechocystis sp. PCC 6803; 7002, Synechococcus sp. PCC 7002; Nost, Nostoc sp. PCC 7120; Npun, Nostoc punctiforme; Tery, Trichodesmium erythraeum; Telon, Thermosynechococcus elongatus BP-1; 8102, Synechococcus sp. WH8102; Selo, Synechococcus elongatus PCC 7942; Avar, Anabaena variabilis ATCC 29413; Cwat, Crocosphaera watsonii WH8501; Prochlorococcus marinus MED4; MIT, Prochlorococcus marinus MIT9313; S120, Prochlorococcus marinus SS120; Gloe, Gloeobacter violaceus PCC 7421; Eco, E. coli. Pairwise alignments were generated with BLOSUM30 using ClustalW. * Incomplete genome sequences. The N-terminal 107 and 62 residues were truncated in the tery2980 and Avar334101 sequences were 107 and 62 residues as compared to the Slr1099 sequence. 275
Table 6.5: List of the UbiE homologs in 14 cyanobacteria
Synechocystis 6803 Sll1653 Sll0418 Sll0829 Slr0407 Slr1618 Slr1039 Sll0487 Synechococcus 7002 * 89-c70244 80-162268 89-46731 - - aAAD26590 aAAC14722 Nostoc PCC 7120 Alr5252 All2121 All3750 - - All3016 All0012
N. punctiforme * Npun3758 - NPun0871 - - - Npun5885 T. erythraeum * Tery3321 Tery4209 Tery1223 - - - Tery3855 T. elongatus BP-1 Tll2373 Ttll11726 Ttll1604 - - - Tll1948 Synechococcus 8102 Synw1674 Synw2141 Synw1749 - - Synw1148 Symw0487 S. elongatus 7942* Selo20383 - Selo232101 - - - Selo213301 A. variabilis 29413* Avar287101 Avar184601 Avar395201 Avar025741 - Avar020095 - C. watsonii 8501* Cwat179201 Cwat194101 Cwat026375 - Cwat020748 - Cwat023036 P. marinus MED4 Pmm0431 Pmm1505 - - - Pmm0662 Pmm1243 P. marinus MIT Pmt0276 Pmt1785 Pmt1242 - - Pmt0792 Pmt1474 P. marinus SS120 Pro0427 Pro1661 - - - Pro0816 Pro0816 G. violaceus 7421 Gll0127 Glr3039 Gll1483 Glr2042 - Glr3970 Gll1180
*Incomplete genome sequences. Locus tags were used to identify the proteins. For proteins in Synechococcus sp. PCC 7002, proteins are designated with a contig number and translation initiation site separated with a hyphen, or a Gene Bank accession numbers are provided if available. 276
277 Table 6.6: Ycf21/UbiC homologs in cyanobacteria and eukaryotic phototrophs
Organism Locus tag
Synechocystis sp. PCC 6803 Sll1797
Crocosphaera watsonii WH 8501 Cwat020398
Nostoc sp. PCC 7120 All0938
Nostoc punctiforme Npun5760
Anabaena variabilis ATCC 29413 Avar303301
Trichodesmium erythraeum Tery3876
Thermosynechococcus elongatus Tll1562
Synechococcus elongatus sp. PCC 7942 Selo236201
Synechococcus sp. WH8102 Synw2384
Prochlorococcus marinus sp. MIT9313 Pmt2179
Prochlorococcus marinus sp. MED4 Pmm1676
Prochlorococcus marinus sp. SS120 Pro1837
Porphyra purpurea PopuCp195 (red algae) Cyanophora paradoxa CypaCp147 (a photoautotrophic protist) 278 Table 6.7: Sequences of primers used for PCR analyses Lower case letters indicate heterologous nucleotides that are introduced in order to engineer restriction enzyme cleavage sites, which are indicated with under lines. Primer Sequence
sll0829-F 5’-GGTCCGCCAAGATTTAGCAGTAAG-3’
sll0829-R 5’-AGATTGGGACAGTAATCGAAGGC-3’
sll0418-F1 5’-CGACTGAGGAAACGGTTGAaTTCCCGCACC-3’ (EcoRI cleavage site).
Used for cloning the sll0418 coding region
sll0418-R1 5’-CTTACGTGGCAATTTAAGCTTGAGTGGCGT-3’
Used for cloning the sll0418 coding region
sll0418-F2 5’-ATGCCCGAGTATTTGCTTCTGCC-3’
Used for verification of segregation
sll0418-R2 5’-GCACTGCTTTGAACATACCGAAG-3’
Used for verification of segregation
slr0089-F 5’-TCTACCGGAAATTGCCAACTACCA-3’
slr0089-R 5’-CCTAGGAGATTGTGGACTTCAA
slr0407-F 5’-CTCTGGAAACATTTGAAAgCTTTATCCG-3’ (HindIII cleavage site)
slr0407-R 5’-TACGTTACTATTCCGCCGAaTTCCTCT-3’ (EcoRI cleavage site)
slr1618-F 5’-TCTATTCTGGATATGCTGGCACA-3’
slr1618-R 5’-TTGTTTGCCTTCGGCTTTAGC-3’
slr1039-F 5’-GGGTGTTTACAATAACAGGGC-3’
slr1039-R 5’-ACTGGTGGGAGGTTGGTGCC-3’
279 Table 6.8: Insertional inactivation constructs of the methyltransferase mutants in Synechocystis sp. PCC 6803. HindIII (blunt) indicates that the 3’-overhand generated after digestion with HindIII is filled by Klenow to yield a blunt end. Target Restriction Site of insertion in allele Insertion of drug cartridge digestions pUC19 (Construct) sll0829 aacC1 between the BxtXI HindIII, HincII HindIII, HincII (p829Gm) sites sll0418 aadA at the unique SmaI EcoRI, HindIII EcoRI, HindIII (p418Sp) site slr0407 aadA between tow HincII EcoRI, HindIII EcoRI, HindIII (p407Sp) sites slr1618 aadA at the unique NheI HindIII, HincII HindIII, HincII (p1618Sp) site slr1039 aadA at the unique MfeI EcoRI, HindIII EcoRI, HindIII (p1039Sp) site slr0089 HindII (blunt), HindII (blunt), aacC1 between the two (p89Gm) KpnI KpnI PstI sites 280
Fig 6.1 UQ biosynthesis pathway in E. coli
281
Fig 6.2. Hypothetical PQ-9 biosynthesis pathway in cyanobacteria The question mark indicates the possibility that an alternative route exists for 4- hydroxybenzoate synthesis. 282
Fig 6.3: An alignment of the Ycf21/UbiC homologs in cyanobacteria and eukaryotic phototrophs. Two catalytic residues (arginine at the position 76 and glutamate at position 155) of the UbiC protein are shown by black arrows.
283
284 Fig 6.4: Phylogenetic analyses based on nucleotide sequences of 16S rRNA and amino acid sequences of Ubi proteins in cyanobacteria and proteobacteria. Multiple alignments were generated with BLOSUM30 using ClustalW. Phylogenetic reconstruction was carried out using the Neighbor Joining method using PAUP* 4.0 beta. Bootstrap values were obtained after 1000 times repetitions. Ctep, Chlorobium tepidum; Cpar, Cyanophora paradoxa; Cwat, Crochosphaera watsonii WH 8501; Ecol, E. coli; Gvio, G. violaceus PCC 7421; Nostoc, Nostoc sp. PCC 7120; Npun, N. punctiforme; PMM, P. marinus MED4; PMT, P. marinus MIT 9313; S122, P. marinus SS120; Ppur, Porphyra purpurea; Rpal, Rps. palustris; Rrub, Rs. rubrum; Rsphe, Rb. sphaeroides; S6803, Synechocystis sp. PCC 6803; S7002, Synechococcus sp. PCC 7002; Synw, Synechococcus sp. WH 8102; Selon, Synechococcus elongatus PCC 7942; Tery, T.. erythraeum; Telon, T. elongatus. Aeropyrum pernix K1 (locus tag: Ape1674), C. tepidum (locus tag: CT1511), Bacillus halodurance (locus tag: Bh3930), and Mesorhizobium loti (locus tag: Mlr2092) were used as out-groups. 285
Fig 6.5: Phylogenetic trees based on amino acid sequences of UbiE homologs in cyanobacteria. Multiple alignments were generated with BLOSUM30 using ClustalW. This phylogenetic tree was constructed by the Neighbor Joining method using PAUP* 4.0 beta. Locus tags (Prefix followed by a 4-digit number) are used to indicate organisms and sequence identity (summarized in Table 6.6). Sll or Slr, Synechocystis sp. PCC 6803; All or Alr, Nostoc sp. PCC 7120; Npun, N. punctiforme; Tery, Td. erythraeum; Tll or Tlr, Ts. elongatus; Gll or Glr, G. violaceus; Pmm, P. marinus MED4; Pmt, P. marinus MIT9313; Pro, P. marinus SS120; Synw, Synechococcus sp. WH8102.
286
Fig 6.6: HPLC analysis of the sll1653 mutant of Synechocystis sp. PCC 6803 and the 2-phytyl-1,4-benezoquinone (MPBQ) standard. (A) Chromatograms of the whole-cell extract of the sll1653 mutant of Synechocystis sp. PCC 6803 (solid line) and the MPBQ standard (dotted line). (B) Absorption spectra of PQ-9 eluting at 35.5 min in the sll1653 mutant sample (dotted line) and of MPBQ eluting at 33 min. 287
3.5 )
-2 3.0 10 x (
a 2.5
2.0
1.5
1.0
0.5 mol PQ-9 / mol chlorophyll PQ-9 / mol mol
0.0 7 7 WT sll0418 sll1653 sll0829 slr1039 sll048 sll0418 sll1653 sll0418 sll0829 sll1653 sll0829 sll0418 slr0089 sll0418 slr040 sll0418 slr1618 sll1653 sll0418 sll0829 sll1653 slr0407 sll0829
Fig 6.7: PQ-9 content in whole cells of the wild-type and methyltransferase mutant strains of Synechocystis sp. PCC 6803 An error bar for the wild-type strain represents the standard error derived from 6 determinations. At least two independent samples were analyzed for each mutant and, although somewhat varied, similar results were obtained. 288
Fig 6.8: Restriction maps of coding regions and their flanking sequences for the UbiE homologs in Synechocystis sp. PCC 6803. Primers used for PCR for both cloning and PCR verifications are shown by black arrows. F1 and R1 primers were used for cloning the sll0418 coding region, while F2 and R2 primers were used for verification of segregation. 289
Fig 6.9: PCR analyses of genomic DNA isolated from the single, double, and triple methyltransferase (UbiE homolog) mutants of Synechocystis sp. PCC 6803. Primers used for the analyses are shown by black arrows in Fig 6.5. The thin arrows indicate the relationship between parental strains and mutant strains. Identities of mutants are shown above the black lines and the primers used for the analyses are shown below. PCR reactions carried out using genomic DNA isolated from mutants are shown in lane 2 and reactions carried out using genomic DNA isolated from wild type are shown in lane 1 for comparison. 290
Fig 6.10: Aromatic amino acid biosynthesis and an alternative proposal for 4- hydroxybenzoate synthesis in cyanobacteria. CM, chorismate mutase; PD, prephenate dehydratase; PPAT, prephenate amino transferase; ADH, arogenenate dehydrogenase; TAL, tyrosine amino-lyase; PAT, phenylalanine aminotransferase; PAL, phenylalanine amino-lyase; CH, chorismate hydratase. The pathways leading to tyrosine and phenylalanine biosynthesis in cyanobacteria are as previously described (Bonner et al., 1995). 291 Appendix
A.1. Amino acid sequence alignments of phylloquinone (Men) and plastoquinone (Ubi) biosynthetic enzymes.
A.1.1. MenA homologs in cyanobacteria and other organisms
A.1.2. MenB homologs in cyanobacteria and other organisms
A.1.3. MenD homologs in cyanobacteria and other organisms
A.1.4. UbiA homologs in cyanobacteria and other organisms
A.1.5. UbiD homologs in cyanobacteria and other organisms
A.1.6. UbiH homologs in cyanobacteria and other organisms
A.1.7. UbiX homologs in cyanobacteria and other organisms
A.1.8. UbiE homologs in Synechocystis sp. PCC 6803
A.1.9. Sll1653 homologs in cyanobacteria
A.1.10. Sll0418 homologs in cyanobacteria
A.1.11. Sll0829 homologs in cyanobacteria
A.1.12. Sll0487 homologs in cyanobacteria
A.1.13 Slr1039 homologs in cyanobacteria
A.2. Photosystem I (PS I) complexes
A.2.1. Polypeptide composition of PS I complexes isolated from the
menG, menF, and rubA menB mutants of Synechococcus sp. PCC
7002.
A.2.2. Illustration of genetically engineered PS I in cyanobacteria
292 A.1.1. An amino acid sequence alignment of MenA homologs in cyanobacteria and other organisms
Slr1518 1 MTESSPLAPS TAPATRKL W LAA I K P P M Y T V A V 32 MenA7002 1 MLTENPPTS SPKATRRL W LAA I K P P I Y T V A V 31 Alr0033 1 M LNCLPQLMTTKQISHPKIKL W MAA I K P P M Y S V A I 35 Tlr2196 1 MTSSS ESIDRSRL W WAA I K L P L Y S V A V 27 Gll1578 1 MSGSPLRPW LNSFNP LIY TASV 22 Synw2307 1 MVEPQAVAS RYADRRLL W KAA I K W P M Y S V A V 31 Pmm0176 1 MNERNKSL W KQA I K W P L Y S V A I 22 At1g60600 1 M Y S V A L 6 OJ1217B0918 1 M P L A G I A L A P L F V S H L A P P H H R R S S V A S A A A A A A R R R P R A V Q C S A TATAASGEGAGDDVELSRGTL LWRA A K L P I Y S V A L 80 CymeCp095 1 MNKIVTLFLA SRP KTLLV SF 20 B3930 1 MTEQQISRTQAW LESLRP KTLPLA F 25 Bsu38490 1 M NQTNKGEGQTAPQKESMGQILWQLTRP HTLTASF 35 CT1511 1 MSVSSS SQLSAFQAW MLA I R P KTLPAGA 28 AF2036 1 MEKLKALIEVTK P KQTFLLM 20 MPLAGIALAPLFVSHLAPPHHRRSSVASAAAAAARRRPRAVQCSAM S LW AIKPP Y.VA.
Slr1518 33 V P ITV G S A V A YGLTG QWHGDVFTIFL LSA I AIIAW I N L S N D V F D SDT G I D VR------K AHS V V N LTGNRNLVFLISN 104 MenA7002 32 T P VIV TTA A A YGEFG V F NPVRFGLFL GAA I CLIAW M N L S N D V F D ADT G I D VN------K ATS V V N LTGDRPLVFGIAL 103 Alr0033 36 M P IWV G G A V A FAGNKVF NLAVFFTFIVAA I L ILAW E N ISN D V F D SET G I D VN------K HHS L V N LTKKKTLVFWLGN 107 Tlr2196 28 M P IWLG T A V A IAKTG RGHWWPFGLFL TAA V L ILAW L N L S N D V F D AET G I D RH------K YHS V V N LTGKKQLIFWLSN 99 Gll1578 23 I P ILV G T A F A WWQGG V F DLQRFALTL AGLVFVHAW I N L T N D V F D WDT G V D RN------K LNS L V RTTGSRSQVLWVGN 94 Synw2307 32 M P VLLAAGWRIGAVG SLRWTQLFGFL LAA I L LLLW E N L S N D L F D ADT G V D TTG------K PHS V V N LTGRRDRVALASL 104 Pmm0176 23 L P VFISGA YTLNSFKNVKIYNLIAFTIAA I L ILIW E N L T N D L F D SET G I D EF------K FHS I V N LVRSKTIVSITAY 94 At1g60600 7 V P LTV G ASAA YLETG L F LARRYVTQL LSSI L IITW L N L S N D VYD FDT G A D KN------K MES V V N LVGSRTGTLAAAI 78 OJ1217B0918 81 V P LTV G S A C A YHHVG S F FGKRYFVLL VASVL VITW L N L S N D VYD SDT G A D KN------K KES V V N IVGSRTMTQYAAN 152 CymeCp095 21 CCWLSVSSLQHAF----SIPALLLAIG----LQIW A N WVN D YLD FMNG I D HA------LRQGPTRWTSRLTPSFMKII 84 B3930 26 AAIIV G T A L A WWQ-G H F DPLVALLAL ITA G L LQILSN L A N D YGD AVKG S D KPDR I G P L R GMQKGV ITQQEMKRALIITVV 104 Bsu38490 36 V P VLLG TVLA MFY-VKVDLLLFLAML FSCLWIQIATN L F N EYYD FKRG L D TAES------VGIGGAIVRHGMKPKTILQL 108 CT1511 29 M P VVIG A A L A AAS-G V F KPLPALVAL ICA LGIQIATN FIN EIYD FRKG A D TAER L G P T R TVAAGIITEQTMIRVSIVLGV 107 AF2036 21 ITFLV SYIVA RGG-ADF NFVIAAISMFLA I SGTTAIN MWLD R-D IDAIMPRT------RKRPV PAGILKPSECAAFGA 90 .P..VG.A.A . G F . ... L..AIL...W NL.ND.FD .TG.D R GP R K SVVN......
Slr1518 105 FFL LAG VLGLMSMSWRAQD -WTVLEL IGVAIFLG Y T Y Q G P P F R L G Y L G L G E LIC LITF G P L A IAAAYYSQ SQS------176 MenA7002 104 GFL GLG ITGIALISYLQQD -WMVFNL LLLATAIAY T Y Q G P P F R L G Y L G I G E FVC FLAYWITGQGVFYS-Q AQT------174 Alr0033 108 LCL ISG LLGILAIATWQQD -PTVIGIILLCCGLG Y M Y Q G P P F R L G Y Q G L G E ILC FFAF G P LGVSAGYYSQ TQT------179 Tlr2196 100 LCL LLG LLGIAAISWLQQD -GTVLGL VLLCCALG Y S Y Q G P P F R L G Y L G L G E PIC FICF G P L A IAAAYYSQ VQT------171 Gll1578 95 IFL SIG YV----CFWALQD -WWLLAFGSLGIFLG Y A Y Q G P P F R L A Y K G L G E WISSSCF G P I A VLTATRAQ TGS------162 Synw2307 105 ASL GSG LALMAWLAWCSS--VLVLAL VLLCCGLG Y I Y Q G P P F R L G Y R G L G E PLC WLAF G P C A TAAALMVLEPQ-----GA 177 Pmm0176 95 TSL LIG LVVIAIISISTS--INVMLL VGACCFLG Y L Y Q G P P F R L G Y Q G L G E PLC WLAF G P F A YAAALIALNPSD---IYM 169 At1g60600 79 TSL ALG VSGLVWTSLNASN-IRAILL LASAILCG Y V Y Q C P P F R L S Y Q G L G E PLC FAAF G P F A TTAFYLLLGS---S S EMR 154 OJ1217B0918 153 ISL LFG FMGLFWAFAQAGD -ARFILSVTCAIICG Y V Y Q C P P F R L S Y R G L G E PLC FAAF G P L A TTAFYFSNSSRNI S S GTA 231 CymeCp095 85 LCL WCCLLFVCASY------FKMKIL VGILILIAWFY S-----AWLGFAG E WLSFFACG P L A TYLFSSIYQVS------146 B3930 105 LICLSG LALVAVACHTLAD FVGFLIL GGLSIIAAITY TVGNRPYGY I G L G DISVLVFF G WLSVMGSWYLQ AHT------177 Bsu38490 109 ALASYG IAILLGVYICASSSWWLALIGLVGMAIG Y L Y T G G P LPIAY TPFG E LFSGICMG SVFVLISFFIQ TDK------181 CT1511 108 SVFVLG LYLVAIGG------WPILLIGVLSLLFAWAY T G G P F PIAY S G L G DVFVFIFF G LVA VGGTYYVQ ALS------174 AF2036 91 AIFAIG QVLAFLVSLEFGLVVFFGLFFDIVVYTILLKRKSP YSIVLGG FAGAMPALG-G WVA VQGFTL------157 . L..G...... D ...L...... GY.YQGPPFRL.Y GLGE .C...FGP.A... . .Q . ISS
Slr1518 177 ---FSWNLLTPSVFVG IST AII L F C S H F H Q VEDD LAAG K K S P I V R L G TKLGSQVLTLSVVSLY LITAIGVLCHQAP WQ-T 252 MenA7002 175 ---LTLNAVWASAWVALTT SII L F C S H F H Q AEDD LAAG K K S P I V R L G TYQGAQVLTYSLGAVLVLTCLLVAVGTWSPW-M 250 Alr0033 180 ---WSITSLAASVIVG IVT SLVL F C S H F H Q VDDD IKAG K R S P I V R L G TANGAKVLTWFTASIY PFSLLFVLLGFFP VW-T 255 Tlr2196 172 ---FSASIWPVAILNG LTT TLI L F C S H F H Q VADD LAAG K R S P V V R L G TARSAQLVYGACGLFY GVLVASVLWGLLP WP-T 247 Gll1578 163 ---WELGALLAGIAVG SWT ATI L YAHH F Y Q YEDD REFDK RTP V V R W G PELAAKRFWWVISPSY VVLLLAVIGGFLP WT-V 238 Synw2307 178 NVIPWATAWMLGAGPAVAT SLVL F C S H F H Q VSED SAHG K R S P V V R L G TARAAALIPWLVALTLALEWLPMCRGDWP LT-V 256 Pmm0176 170 ISIPWKESLLLGSGSSLAT TLVL F C S H F H Q IKED KEHG K N S P L V L L G AKKGAKIIPWIVFIIY VFQLFLIINGFIP IL-C 248 At1g60600 155 HLPLSGRVLSSSVLVG FTT SLI L F C S H F H Q VDGD LAVG K Y S P L V R L G TEKGAFVVRWTIRLLY SMLLVLGLTRILP LPC T 234 OJ1217B0918 232 LLPLSKTVIASSILVG LTT TLI L F C S H F H Q IDGD RAVG K M S P L V R I G TKTGSRLVTLGVVTLY VLLAAFGMSKSLP SAC T 311 CymeCp095 147 --FWQWDLLLLSIING SWSAALL MANNMRDRISD THSPK K S TCV WFG QDWASMQYQMFILMCIWCLFAWLIFRPSWFW-L 223 B3930 178 ---LIPALILPATACG LLATAVL NINNLRDINSD RENG K NTLVV R L G EVNARRYHACLLMGSLVCLALFNLFSLHSLW-G 253 Bsu38490 182 ---INMQSILISIPIAILVGAI NLSNNIRDIEED KKGG RKTLAILMG HKGAVTLLAASFAVAY IWVVGLVITGAASPW-L 257 CT1511 175 ---LPMEVLVAAAAPG AFSVCI L LVNNIRDIDTD RKVG K MTLPAR I G APAARALYVALVVLAY LVP-FYMISTGYSLW-C 249 AF2036 158 -----PGFIIAAIVLLWIPSHI WYIS MHYEEDYRMANIPMYP L V -VG MERASWAIVFATAAMLVLAASLYVLLPLGIFYL 231 ...... G. T..ILFCSHFHQ...D .GK.SP.VRLG ...... Y...... P C
Slr1518 253 L L IIASLP WAVQL IRHVGQYHDQP EQVSNCKFIA VNLHFFSG M L MAAG YGWAGLG 307 MenA7002 251 L L TFASAP FALSL INHVRLNHDQP DKVSNCKFFA VKFHFASG L L L AIAYVLSYYNLEF LSV T G L G Y N 317 Alr0033 256 L L SWLSLP YAFKL CRHVQHNHNQP EKVSNCKFIA VAVHFWSCLL L G L G FVIAGV 309 Tlr2196 248 L L ALSSLP WAIYL CQRMAQFHSVP EQIKNSKFIA VQLHFWSSLL L G L G YLL 298 Gll1578 239 L AALATIP LAVSL VSFVRENATNP AVVVGALPLA ARFHLVSG A L L T L G LLAGTLLPR 295 Synw2307 257 L L SGLGLP AGLAL IRLMQRCHDQP DRISGSKFLA LRFQAWNG LGL A L G LALGRL 310 Pmm0176 249 V L FLISFP QSLKL INLLKYSYNKP EAIKNCKFIA IKFQTLNG IGL IAG FIINYLIYK 305 At1g60600 235 L MCFLTLP VGNLVSSYVEKHHKDNGKIFMAKYYCVRLHALLG AAL S L G LVIAR 287 OJ1217B0918 312 V L CALTLP VGKWVVDYVLKNHEVTSDLTYNLDANL 346 CymeCp095 224 L MMCVFLP YALRL CMMSLTTPT--ALLYPTLSFIFRYCFLFSFCASYS 269 B3930 254 W L FLLAAP LLVKQARYVMREMDP-VAMRPMLERTVKGALLTNLL FVL G IFLSQWAA 308 Bsu38490 258 FVVFLSVP KPVQAVKGFVQNEMP-MNMIVAMKSTAQTNTFFG F L L SIG LLISYFR 311 CT1511 250 L L SLLSIP LAIGMVRTLYASEG--QALNAVLAGTGKVLTVHG L L FSL G LVIPNIISIF RP 307 AF2036 232 VISTSAVAFFLYKAVKFALSPDR-VKARKMYKLA SMTLGLVYFSL L L G VFL 281 LL ....P .. L. . P . .A.. . G.LL.LG .. F VTGLGYN The multiple alignment was generated by BLOSUM30 using Clustal W. The locus tags used to indicate organisms are summarized in Table 2.1, except for the following: Cymecp095 in Cyanidiochyzon merolae and OJ1217B09.18 in Oryza sativa. AF2036, a hypothetical protein in the archaeon Archeoglobus fulgidis DSM4304, was used as an out-group in phylogenetic construction as shown in Fig 2.3.
293
A.1.2. An amino acid sequence alignment of MenB homologs in cyanobacteria and other organisms
Sll1127 1 MDW HI 5 MenB7002 1 MESLAW QA 8 All2347 1 MQIDW QS 7 Tll2458 1 MDSVHW RQ 8 Synw0998 1 MAELRQVLPGDPAAAW TA 18 Pmm0608 1 MKILPGETNSNW TE 14 At1g60550 1 M A D S N E LGSA S R R L SVV T N H L I P IGFSPARA DSVEL CS---A S SMDDRFHKVHGEVPTHEVVWKKTDFFG 67 P0671D0118 1 MDAA G R R L ARV T A H L L P SSLPLPLA SAPTL APS P A A S PASDSYRRVHGDVPSEPPEWRAATDES 64 CymeCp092 1 0 B2262 1 MIYPDEAMLYAPVEWHDCSEG---- 21 CT1846 1 MPVEPGQRRFSSTTIEISDMSTVNW IT 27 Bsu3075 1 MAEW KT 6 AF0435 1 0 MADSNE A RRL VT HL P A L SPAAS . W
Sll1127 6 ---AK H Y D D I L Y Y K AG--G I A K I V I N R P H K R N A F R P Q T V F E LYDA F CNA R E D NRI G V VLL T G A G P H S D G K 70 MenB7002 9 ---VK A Y E D I L Y Q K LD--G I A K I T I N R P H K R N A F R P Q T IFE M YEA F C D A R E D QDI G V ILL T G A G P H T D G K 73 All2347 8 ---TK T Y E D I L Y D K AD--G M A K I T I N R P H K R N A F R P K T V F E LYDA F C D A R E D TSI G V VLFT G A G P H T D G K 72 Tll2458 9 ---VHNY T D I L Y H K TT--G I A K I T I N R P H K R N A F R P Q T IVE M SQA F E D A R N D PTI G V VLL T G A G P H T D G K 73 Synw0998 19 ---WGTY T D I L VDRCSE-G I A RVAI N R P A K R N A F R P Q T V V E LCDA F SRIR D D RSI G V VLFT G V G P AAD G G 84 Pmm0608 15 ---WK S Y E D I L FHNSGD-G I A R I A I N R P E K R N A F R P K T V C E M LDA F NLVR N D EKI G V VLL T G A G P DKKG V 80 At1g60550 68 E G D N K EFVD I I Y E K ALDE G I A K I T I N R P ERR N A F R P Q T V K E LMRA F N D A R D D SSVG V IIL T G K G -----T 132 P0671D0118 65 ---GK GFVD I L Y D K AVGE G I A K I T I N R P DRR N A F R P L T V K E LMRA F E D A R D D SSI G V IIL T G K G -----T 126 CymeCp092 1 M D I K Y E K -KE-G I A T I T I N R P HVLN A F R P Q T V KQM LEA MID A R W D DDI G V VIL T G E G -----T 56 B2262 22 ------FED I R Y E K STD-G I A K I T I N R P QVR N A F R P L T V K E M IQA LAD A R Y D DNI G V IIL T G A G -----D 79 CT1846 28 ---AGEY S D I L Y H K TEE-G I A K I T I N R P ERR N A F R P Q T V DQM IEA LQD A R N D SQI G V IIL T G A G -----D 88 Bsu3075 7 ---KRTY DEI L Y ETYN--G I A K I T I N R P EVHN A F T P K T V A E M IDA F A D A R D D QNVG V IVL A G A G -----D 66 AF0435 1 MGERVKLELDG--EI A VAT L N R P E K L N A LDTKT RME LAEVIEGI--EEVARV LIIT G S G ------55 EGD K Y.DILY.K EGIAKITINRP KRNAFRPQTV EM .AF DAR.D IGV..LTGAGPH DG
Sll1127 71 Y A F C S G G D Q S V R G EGG - Y I D DQG TPR L N V L D L Q R L I R SMP K V V I A L V A G Y A I G G G H V L H LVC D L T I A A D N 139 MenB7002 74 Y A F C S G G D Q S V R G EGG - Y V D QAG VPR L N V L D L Q KLI R SMP K V V I A L V A G Y A I G G G H V L H LLC D L T I A A D N 142 All2347 73 Y A F C S G G D Q S V R G QAG - Y V D DAG IPR L N V L D L Q R L I R SMP K V V I A L V A G Y A I G G G H V L H LIC D L T I A A D N 141 Tll2458 74 Y A F C A G G D Q SIR G EAG - Y L D EQG VAR L N V L D L Q R Q I R SLP K V V I A L V A G Y A I G G G H V L H LVC D L T I A A E N 142 Synw0998 85 F A F C S G G D Q S V R G DGG - Y VGDDG LPR L N V L D L Q R I I R SLP K V V I A L V A G Y A M G G G Q V L H LLC D L SLA A D N 153 Pmm0608 81 FSF C S G G D Q SIR G QNG - Y E D DEG TQR L N V L E L Q R L I R TLP K V V I A L V P G F A I G G G Q V L QLVC D L S I A SEN 149 At1g60550 133 K A F C S G G D Q ALR TQDG - Y A D PNDVGR L N V L D L Q VQI R RLP K P V I A M V A G Y A V G G G H I L H MVC D L T I A A D N 201 P0671D0118 127 QSF C S G G D Q ALR DADG - Y V D FDSFGR L N V L D L Q VQI R RLP K P V I A M V A G Y A V G G G H V L H MVC D L T I A A D N 195 CymeCp092 57 R A F C T G G D Q K V R G TKG - Y K D ENG KQSL N V L Q L Q R E I R TIP K P V I A K V A G Y A I G G G QIL QMLC D L T I A A D N 125 B2262 80 K A F C S G G D Q K V R G DYG G Y K D DSG VHHL N V L D F Q R Q I R TCP K P V V A M V A G Y S I G G G H V L H MMC D L T I A A D N 149 CT1846 89 L A F C S G G D Q KIR G NAG - Y A D EKG VNKL N V L D F Q R D I R TCP K P I I A M V A G Y A I G G G H V L H MLC D L T I A A E N 157 Bsu3075 67 K A F C S G G D Q K V R G HGG - Y VGDDQIPR L N V L D L Q R L I R VIP K P V V A M V S G Y A I G G G H V L H IVC D L T I A A D N 135 AF0435 56 K A F AAG A D INELLQRDAIKAFEATKLG--TD L FSRI EELEIP V I A A V N G Y TLG G G CEL AMAC D IRI A SEK 123 AFCSGGDQ VRG .G Y.D. G. RLNVLDLQR IR..PKPVIA.VAGYAIGGGHVLH..CDLTIAADN
Sll1127 140 A I F G Q T G P K V G S F D G G F G S S Y L A R I V G Q K K A R E I W Y L C R Q Y S A Q E A ERM G M V N T V V P VDRL E E E GIQW A K 209 MenB7002 143 A I F G Q T G P K V G S F D G G F G A S Y M A R I V G Q K K A R E I W F L C R Q Y D A Q Q A L Q M G L V N Q V V P IAQL E A E GVQW A R 212 All2347 142 A I F G Q T G P K V G S F D G G F G A S Y L A R I V G Q K K A R E I W F L C R Q Y D A Q Q A L E M G L V N C V V P I E Q L E A E GIKW A G 211 Tll2458 143 A I F G Q T G P K V G S F D A G F G A S Y L A R I V G Q K K A R E I W F L C R Q Y T A Q E A L A M G L V N A V V P V E A L E A E GIRW A Q 212 Synw0998 154 A V F G Q T G P K V G S F D G G F G A G Y L A R V V G Q R K A R E I W F L C R R Y G A D E A L R M G L V N A V V P L DQL E A E GVRW A R 223 Pmm0608 150 A I F G Q T G P R V G S F D A G F G S S Y L A R L V G Q R K A R E I W F L C R K Y NSKE A L E M G L V N AITKIE E L E A E GVIW A R 219 At1g60550 202 A I F G Q T G P K V G S F D A G Y G S S IMSR L V G P K K A R E M W F MTR F Y T A S E A EKM G L I N T V V P L E D L E K E TVKW C R 271 P0671D0118 196 A I F G Q T G P K V G S F D A G Y G T S IMSR L V G P K K A R E M W F L S R F Y T A D E A DRM G L V N V V V P L ADL E R E TVKW C R 265 CymeCp092 126 A I F G Q A G P R V G S F D G G Y G A S Y M A R I V G Q K R A R E I W F L C R Q Y S A Q E A L Q M G WIN A V V P L E E L DQHTKAW A K 195 B2262 150 A I F G Q T G P K V G S F D G G W G A S Y M A R I V G Q K K A R E I W F L C R Q Y D A KQA L D M G L V N T V V P L ADL E K E TVRW C R 219 CT1846 158 A R F G Q T G P R V G S F D G G W G A S Y M A R L V G Q K K A R E I W Y L C R Q Y N A Q E A L D M G L V N T V V P L E K L E E E TIQW C R 227 Bsu3075 136 A I F G Q T G P K V G S F D A G Y G SGY L A R I V G H K K A R E I W Y L C R Q Y N A Q E A L D M G L V N T V V P L E Q L E E E TIKW CE 205 AF0435 124 A K F G Q PEINLAIIPG AGG TQRL P R L V G LGMA KKLVLTGEIIDA Q T A L RIG L V EEV V EHE R L MERAKEVA A 193 AIFGQTGPKVGSFDGG.GASYLARIVGQKKAREIWFLCRQY AQEAL MGLVN VVPLE LE E...WAR
Sll1127 210 E I L SKS P L A I R C L K A A F N A D C D G --Q A G L Q E L A G N A T L L Y Y M T E E GSE G KQA F L E K R P P D F S QYP WLP 275 MenB7002 213 E V L SKS P I A I R C L K A A L N A D C D G --Q A G L Q E L A G N A T M L Y Y M T E E GRE G KQA F L E K R S P N F RQYP WLP N 279 All2347 212 E I L EKS P I A I R C L K A A F N A D C D G --Q A G L Q E L A G N A T L L Y Y M T E E GAE G KQA F L E K R P P N F RDF P WLP 277 Tll2458 213 E I L SKS P L A I R C L K A A F N A D C D G --Q A G L Q E L A G N A T L L Y Y L T Q E SAE G KTA F L E K R P P N F QQF P WRP 278 Synw0998 224 E V L QHS P T A I R C L K A A F N A ETD G --LA G I Q E L A G N A T H L F Y R T D E A L E G RNA F L E K R P P D F S ETGWLP 289 Pmm0608 220 E I L RNS P T A I R I L K A SFN A E C D G --IA G I Q E L S G YTT Q L F Y S T D E A K E G RDA F L E K R P P D F S DYGWTP 285 At1g60550 272 E I L RNS P T A I R V L K A A L N A VDD G --HA G L Q G L G G D A T L L F Y G T E E A T E G RTA YMHRR P P D F S K F HRRP 337 P0671D0118 266 K I L RNS P T A I R V L K S A L N A ADD G --HA G L Q E L G G N A T L I F Y G T E E A K E G KNA YME R R R P D F S K F P RKP 331 CymeCp092 196 Q I L EHS P T A LSML K A A L N A D C D G --Q A G L Q E L A G L A T H L F Y G T E E A Q E G LNA F L E K R N P K F RRRKALDEN 263 B2262 220 E M L QNS P M A L R C L K A A L N A D C D G --Q A G L Q E L A G N A T M L F Y M T E E GQE G RNA F NQK R Q P D F S K F KRNP 285 CT1846 228 E I L ANS P L A I R C L K A A L N A D C D G --Q A G L Q E L A G N A T L L Y Y MSE E GQE G RNA F V E K R K P D F S K F P KRP 293 Bsu3075 206 E M L EKS P T A L R F L K A A F N A D T D G --LA G I Q QFA G D A T L L Y Y T T D E A K E G RDSF K E K R K P D F GQF P RFP 271 AF0435 194 K I IEKS P L A VKVAK K A L N A SINMP L KEG L RYEA SLFAL L F SS-E D A K E G MRA F L E K R K P E F RGR 256 EIL .SP AIRCLKAALNADCDGPLQAGLQELAGNATLLFY TEEA EG..AFLEKR PDFS FP P N
The multiple alignment was generated by BLOSUM30 using Clustal W. The locus tags used to indicate organisms are summarized in Table 2.1, except for the following: Cymecp092 in Cyanidiochyzon merolae and P0671D01.18 in Oryza sativa. AF0435, a hypothetical protein in the archaeon Archeoglobus fulgidis DSM4304, was used as an out-group in phylogenetic construction as shown in Fig 2.3.
294 A.1.3. An amino acid sequence alignment of MenD homologs in cyanobacteria and other organisms
Sll0603 1 0 Alr0312 1 0 Tll0130 1 0 Synw0997 1 0 Pmm0607 1 0 At1g68890 1 M R S S F L V S N P P F L P S L I P R Y S S R K S I R R S R E R F S F P E S L R V S L L H G I R R N I E V A Q G V Q F D G P I M D R D V N L D D D L V V Q V C V 80 CymeCp094 1 0 B2264 1 0 ML2270 1 0 MRSSFLVSNPPFLPSLIPRYSSRKSIRRSRERFSFPESLRVSLLHGIRRNIEVAQGVQFDGPIMDRDVNLDDDLVVQVCV
Sll0603 1 0 Alr0312 1 0 Tll0130 1 0 Synw0997 1 0 Pmm0607 1 0 At1g68890 81 T R T L P P A L T L E L G L E S L K E A I D E L K T N P P K S S S G V L R F Q V A V P P R A K A L F W F C S Q P T T S D V F P V F F L S K D T V E P S Y K S L Y 160 CymeCp094 1 0 B2264 1 0 ML2270 1 0 TRTLPPALTLELGLESLKEAIDELKTNPPKSSSGVLRFQVAVPPRAKALFWFCSQPTTSDVFPVFFLSKDTVEPSYKSLY
Sll0603 1 0 Alr0312 1 0 Tll0130 1 0 Synw0997 1 0 Pmm0607 1 0 At1g68890 161 V K E P H G V F G I G N A F A F V H S S S V D S N G H S M I K T F L S D E S A M V T A Y G F P D I E F N K Y S T V N S K D G S S Y F F V P Q I E L D E H E E V S 240 CymeCp094 1 0 B2264 1 0 ML2270 1 0 VKEPHGVFGIGNAFAFVHSSSVDSNGHSMIKTFLSDESAMVTAYGFPDIEFNKYSTVNSKDGSSYFFVPQIELDEHEEVS
Sll0603 1 0 Alr0312 1 0 Tll0130 1 0 Synw0997 1 0 Pmm0607 1 0 At1g68890 241 I L A V T L A W N E S L S Y T V E Q T I S S Y E K S I F Q V S S H F C P N V E D H W F K H L K S S L A K L S V E E I H P L E M E H M G F F T F S G R D Q A D V K 320 CymeCp094 1 0 B2264 1 0 ML2270 1 0 ILAVTLAWNESLSYTVEQTISSYEKSIFQVSSHFCPNVEDHWFKHLKSSLAKLSVEEIHPLEMEHMGFFTFSGRDQADVK
Sll0603 1 MVDFTN P N TLAA S V L V E T L F R L G L QQA V I C P G S R S S P L T V A L A RHG---GI DCV VSL D E R S A S F F A L G 65 Alr0312 1 MSNLM PIAYKN I N QLW A Y I FTE T L K R L G L AYA V I C P G S R S T P L A V A F A QQAP--NI E A I SIL D E R S A A F F A L G 71 Tll0130 1 MILTEN L N VLW A S V L F E T L Y R L G L RTV V L S P G S R S G P L A I A A A AHP---HL E A L PIL D E R S A A F F A L G 65 Synw0997 1 MTRL V L C P G S R S G P L A A A A G LLAAR G D L A L TTAI D E R S A A F L A L G 45 Pmm0607 1 M TSHIECQN FFRSLQL L D LFTK I G V KNL I L C P G S R S G P L A I A A G ELNKR RVL N V FNSI D E R S A G F HSL G 69 At1g68890 321 E L L N R E T EVSNFLRDEAN I N AVW A S A I I E ECTR L G L TYFCV A P G S R S S H L A I A A A NHP---LTTCL ACFD E R S L A F H A I G 397 CymeCp094 1 M LLFCSFSLIDW SEL I I KLL A H Y G FQYKFI A P G A R S S L L A R A A L LHG-----NCV VHFD E R S L A F A A L G 64 B2264 1 MSVSAFN RRW A A V I L E A L T R H G V RHI C I A P G S R S T P L T L A A A ENS---AFIHHTHFD E R G L G HLA L G 64 ML2270 1 M N PSTTQA R V V V D E L I R G G V RDV V L C P G S R NAP L A F A L QDADHAG R I R L HVRI D E R T A G Y L A I G 64 ELLNRET M N N WA ...E L R.G. .V.CPGSRS.PLA.AAA RG . .. .DERSAAF ALG
Sll0603 66 H G K R------T G Q P V A L V C T S G T A A A N F L P A I I E A HYS Q V P L L V L T G D R P P R L R H C R A G Q T I D Q TKLY G HYPQW QT 135 Alr0312 72 I A K A ------T NRP V A I V C T S G T A G A N FYP A V I E A QES R V P L L L L T A D R P P E L R D C HSGQ T I D Q MKLY G S YPNW Q A 141 Tll0130 66 L V QQ------QG R P V A L L C T S G T A A A N FYP A I I E A SLS H L P L I V L S A D R P P E L R F C Q A G Q A I D Q VHLY G HAVR HYR 135 Synw0997 46 M A T A ------GG RAV A V V T T S G T A V A N L L P A A V E A DRS TQP L L L L T A D R P ARL K N C G A N Q T V N Q EQFLQPVCR W L G 115 Pmm0607 70 I STA ------S G DVSL V V T T S G T A V G N L L P A A I E A DKS CKSI V F I T A D R P LRL K N C G S N Q T V N Q EEFLNS VCR SNL 139 At1g68890 398 Y A K G ------S L K P A V I I T S S G T A V S N L L P A V V E A SEDFL P L L L L T A D R P P E L QGVG A N Q A I N Q INHF G S FVR F FF 467 CymeCp094 65 V G K A S Q I Q T S K T N A S KTKA I L I V T S G T A V G N L W P A V M E A SLS QTSL I L L T A D R P Y E L R DTSA N Q T L D Q VKLF NGYVR W QF 144 B2264 65 L A K V ------S KQP V A V I V T S G T A V A N L Y P A L I E A GLT GEKL I L L T A D R P P E L IDC G A N Q A I R Q PGMF A S HPTHSI 134 ML2270 65 L A V A ------AG A P V C V A M T S G T A V A N L G P A V V E A NYARV P L I V L S A N R P Y E L LGTG A N Q T M E Q LGYF G T QVR AAI 134 .AKASQIQTSKTNA.G PVA.. TSGTAVANL.PA.IEA S .PL.LLTADRPPEL. CGANQTI.Q .G. R. .
Sll0603 136 E L A L P EPNMDYCHY L RQT A LHSWQK CFWPGL------G V V H L N C P F D E P L VPLEHAR--ESL QHL AEEF SKEHFY RGL T 206 Alr0312 142 E L A L P VSDMGML A Y L RQT L VHSWYR MQA PTP ------G P V H L N I P F R D P L APIP DG---TDL SYL LAKF HPEEFF AGI T 211 Tll0130 136 E L S L P --ELPLL P Y L RQT L CHSWQTA LWPDP ------G P V H L N I P L R D P L D L RSQAN--FHG ELPKGFF DQVQP F VPPR 204 Synw0997 116 H G APEGLASQPPQAL FDLA HEA WRH CHG RPP ------G P V Q L N L P F E E P L H G A P EDQ--LSL QVSGLQRSAPAP SSDSP 186 Pmm0607 140 STNL NGIHENNDDDI LKIV ETV KKQI LQ-SP ------G P I H L N I A F EKP L D I SLKNK--KKNFEV FERF YLNKTY KFL K 209 At1g68890 468 N L PPP -TDLIPV RMV LTT V DSA LHWA T G SAC------G P V H L N C P F R D P L D G S P TNWSSNCL NGL DMWMSNAEP F TKYF 539 CymeCp094 145 D V PPP -HHELDA D W L AST L AYA AFK A SD------G P V H L N L M F R E P FASYP IP------MPKKLVEYVP ASTA T 205 B2264 135 S L PRP -TQDIPA R W L VST I DHA LGTL H A ------G G V H I N C P F A E P L Y G EMDDTGLSWQQRL GDWW QDDKP W LREA 203 ML2270 135 S L G L AEDAPERL DSL NAT WRSA TCR V L V AATG S R T A N A G P V QFDI P L R E P L IPDP EP----HG AITPQGRPGGKP W TYI P 210 L LP . .L T. A .. . PGSRTANAGPVHLN.PFREPL . P . . . P. .
Sll0603 207 TFTTMNRGEAISLPVNFS I F S FPSPQLDFS PLG L I L V G VMPG G E TPS--LLTDI L A I A RGL G Y P V L C D A L CSL R N ----- 279 Alr0312 212 DTTPLPHHSPLSIPP------EWLQS QRG I I I A G VAQPQQPQE--YCRA I A R L SQTL Q W P V L A E G L S P V R N ----- 274 Tll0130 205 VVTSLPWQT------WQQMQRG L I I A G PSHG V D PLA--EAAA I DRL SRFL Q W P V L A D A L S S A R ------259 Synw0997 187 -LGAAPQLD------PNQ--AG V V V A G PWRG LAPALPAYQQA L L QWL ARSGW P L L A D P L AAI P------240 Pmm0607 210 NDNQNKNIQFSEKFFKR------INLS NPG I I I V G PYQG STKDLFSFNSA L KKL QEITGW P V F V D P V S G V S------274 At1g68890 540 QVQSHKSDGVTTGQIT-----EILQVIKEAKKG L L L I G AIHTED EIW-----A S L L L A KEL M W P V V A D V L S G V R L R K L F K 609 CymeCp094 206 GFEWSLDWQ------QK---G L F I V A QAMDDE SFH-----L L L Q L A QQWSW P I L V E V FCHA R ------253 B2264 204 PRLESEKQRD------WFFWRQKRG V V V A G RMSA E E GK------KV A LWA QTL G W P L I G D V L S QTG------257 ML2270 211 QVTFDQPLE------IDLS ADTV V I A G HGAG VHPN------LT-ELP I V A E PTAPFG------254 SIFS S G..IAG G . A.. L. L WPVLAD.LS .RNRKLFK
295
Sll0603 280 --Y DDGQTALI TNY D F L I RCPR WAEQL V-PE Q I I Q I G ELPT S K A L RHW L G-TIDCPRYI L NFHGENL D P L Q G QTIYL S A S 355 Alr0312 275 --Y ADFNPYLI STY D L I L RNQQL ATRL A-PD M V I Q I G DMPT S K E L RNW I D-THQPRRWV I D PSDQNL D P L H G RTTHL R I R 350 Tll0130 260 -----GLPHGI SHY D L L L RDAH L REYL R-PE A V I Q L G PLPT S K A L REW L S-ACDPLIWCL D PTGDNNNP L H G RCQML A I A 332 Synw0997 241 ----ADCPGQL DGW E L Q L DRLQL AP----GSPV L R L G PLPAS R R L EAW L QRQTGPQLLI S E GEPRGL D P L G L ADQWSGG L 312 Pmm0607 275 ----AELKGLV ENW E L I L K--K NKNII Q-CD Q I L RFG PLSS S NYL EDF L LNFEGLQILV K E NNIRKL D P I K KSLEYDFG I 347 At1g68890 610 P F VEKLTHVFV DHLD H A L FSDSV RN-L IEFD V V I Q V G SRIT S K R V SQML EKCFPFAYIL V D KHPCRHD P S H L VTHRV QSN 688 CymeCp094 254 -----YQYKHSCVIAYHETICH KTMPFPDANII I Q F G ERLIS NHL ITIMK--KVNQYVV V SSSTVRV D P L K A VTTRI C V T 326 B2264 258 ------QPL PCAD L W L GNAK A TSEL QQAQIV V Q L G SSLT G K R L LQW QASCEPEEYWI V D DIEGRL D P A H HRGRRL I A N 329 ML2270 255 ------PYRPY P L HPLALPL LH----PKQV I M L G RPTLHR P V SALL A---DPQVPV YALTTGPCWP DVSGNSQA A G T 318 PFY . .DL.L .. L . VIQ.G TSK L WL ... LDPL.. . .
Sll0603 356 V A QVA EYIHN--QGFIPDAEQKNYAH---SWLEK Q R QSQTIII S A LADA HTPLMV AQL AHCL P P Q TNL F V A N S L P V R W L E 430 Alr0312 351 V EELG YQGVE--EDKSSVSEYL QLWC---NAETK V R VNV DETL DKMEDL V E CKA A WLL SQML P P ETPL F I A N S M P V R D V E 425 Tll0130 333 PQAVDCP-----PDPLPPNPYL KDWQ---DQDQR V R EQL KRTFEA IDWFS E VKL I YHL PQWL P S Q TAI F V A S S M P V R D V E 404 Synw0997 313 A A WWA EQNQD--FPGASTPEQL MPQS---GIASLL R ----QRL P L QGAV N E PAL A HWL PLLL P P Q LPV M L A A S S P V R D WL 383 Pmm0607 348 SNFVNQLLGAFLNHKKKSKPLI NLGQV L I KEGAK I K EIL KEQL FSNTEI T E YKL A NFV PKIWP E N YPI M L SAS S P I R D WL 427 At1g68890 689 I V QFA NCVLKSRFPWRRSKLHG HLQA----LDGAI AREMSFQI S A ESSL T E PYV A HML SKAL TSKSAL F I G N S M P I R D V D 764 CymeCp094 327 N V QSL LKFWFKTLRPTTKPQWL SWWQ---HLNFCV GYKL RQYFAQEIKL S E PGV L RMCFQYA K P --LI F V G N S M P I R D A N 401 B2264 330 I A DWL ELHPAE----KRQPWCV EIPR------LA EQAMQAVI ARRDAFGE AQL A HRI CDYL P E Q GQL F V G N S LVV R L I D 398 ML2270 319 R A VTTGT------PHPAWL QRCA---EMNQH A ISTV RSQL A A HPLI T GLHV A AAV ADAL R P GDQL V L G A S N P V R D MA 386 .. . L VLI ...... E .A L LPP. LF..NS PVRD..
Sll0603 431 F F WPANGDH------HR I F V N R G A N G I D G T L S T A M G I A H RSRG------ET V L L T G D L S L L H D 481 Alr0312 426 F F WKPNNL R------VR SHFN R G A N G I D G T L S T A L G I S H RQQ------SS V L I T G D L A L L H D 475 Tll0130 405 SVWQGSDRR------HR F Y F N R G A N G I D G T L S T A L G V A H RGQ------P T L L I T G D L A C L H D 454 Synw0997 384 I W GGLQAQN------RR C F SFR G A S G I D G T L S L A M G L A MEQG------P M V L V T G D L A L L H D 433 Pmm0607 428 T F SENATL T------RECF SFR G A S G I D G T L S L A L G I A R I TK------P L L L V T G D L A L I H D 477 At1g68890 765 M Y GCSSENSSHV V D M M L S A E L P C Q W -IQV TGN R G A S G I D G L L S S A T G F A V G CKK------RVV C V V G D I SFL H D 831 CymeCp094 402 H F VECHHL HDP------VH V F A N R G V S G I D G N L A T SMG V SYA LQE------P LMCI L G D L A F L H D 454 B2264 399 ALSQLPAG Y------PV Y S N R G A S G I D G L L S T A A G V Q R A SGK------P T L A I V G D L S A L Y D 448 ML2270 387 LVGLSTDG I------QV RSN R G V A G I D G T V S T A I G A A L A YERS P G Y A G S T D R P A R T V A L I G D L A F V H D 448 . . VVDMMLSAELPCQW ... NRGASGIDGTLSTA.G.A.. SPGYAGSTDRPAP.VL.TGDLA.LHD
Sll0603 482 S N G F L NQSQ --MRG N L T I I L L N N N G G G I F QTL P I A QCE DV--FE T Y F A T P Q G V D F GQL CRTY G V EHK I I TNLWD L KEQWP 557 Alr0312 476 T N G F L I RNK--FVG H L T I I L I N N N G G G I F EML P I A KFE PP--FE E F F G T P Q D I D F AQL CTTY N V QHELI HSWVHL QQRLN 551 Tll0130 455 T N G W L I TPQ --FQG C L T V L L I N N R G G G I F EHL P I RQFD PP--FE A F F A T P Q Q V D F SYL A A A Y G I PYH C L KDWAD V EAQLR 530 Synw0997 434 S N G W L H G A Q --G NPPL L V L L I D N Q G G G I F QQL P I QSKQ----FD RLF A M P Q R V NPLAL A A A HGI DGR Q V ACLED L PEALE 507 Pmm0607 478 I N G F L I ENA--I E L N L T I L L I N N N G G N I F NNL YKNNLD EE-ELKKLF I M P KSI N W ENL A K G Y Q V PIK N V SDLNKL REAFE 554 At1g68890 832 T N G L A I L K Q RIA RKPMT I L V I N N R G G G I F RLL P I A KKTEPSV LNQY F Y T AHDI SIENL CLA HG 894 CymeCp094 455 LTSLHL L R Q --L TKHI W V I V I N N Q G G G I F ELI P N V FNH------Y FHHHDH-F EHV A KQF DFHYYAV NNWYE L ALHLQ 523 B2264 449 L N A L A L L R Q --V S A P L V L I V V N N N G G Q I F SLL P TPQSE ----RE R F Y L M P Q N V H F EHA A AMF E L KYH RPQNWQE L ETAFA 522 ML2270 449 S S G L L I G PTEPTPQQL K I V V S N D N G G G I F ELL EQG DPRLSAV SSRIF G T P HDV DVGAL CRA Y H V EGR Q I EVDKLREALDE 528 .NG.LI. Q . . LTI.LINNNGGGIF LPI. . V . .F.TPQ V F LA .Y . . . .L
Sll0603 558 SNNS-SPIRV L E I IGDR HQEA Q WLKSL QAQFCCADSLIQ 595 Alr0312 552 PLPN-TGIRV L E L RTNR KIDA Q W R RDNLSNFAADNII 587 Tll0130 531 QTPW-PKIRL L E FRSDR HRNA Q W R QQV LAHL GG 562 Synw0997 508 WGLAQGRPAL L R L ATDR EADA RLR TQL RSAA QNAEPLL 545 Pmm0607 555 WSLSMQKSVI I K V DINVENEMKGR NLI LKKI LTS 588 At1g68890 895 894 CymeCp094 524 NTKG---ANL I E I QTNQLENA TLH KKL TQYTNLFSVSFHS D 561 B2264 523 DAWRTPTTTV I E MVVNDTDGA Q TLQQL LAQV SHL 556 ML2270 529 PGVG---MRV L E V KADR SSLRQ L H AAI TATL 556 .LE.RAQ... SD
The multiple alignment was generated by BLOSUM30 using Clustal W. The locus tags used to indicate organisms are summarized in Table 2.1, except for the following: Cymecp094 in Cyanidiochyzon merolae. ML2270, a MenD homolog in Mycobacterium leprae, was used as an out-group in phylogenetic construction as shown in Fig 2.3.
296 A.1.4. An amino acid sequence alignment of UbiA homologs in cyanobacteria and other organisms
10 20 30 40 50 60 70 S6803 MVAQTPSSPPLWLTIIYL L R W H K - P A G R L I L M I P A L W AVCL A AQGLP P L ----PL LGTI ALG T L ATS GL S7002 MVSEPTWKKIIYL L R W D K - P A G R L I L I I P A L W AVFL A AEAKP DW----KL VLVI ILG AITTS A A A7120 MLETHQ-EPIWLTIFRL L R W H K - P E G R L I L M I P A L W AVFL A AAGKP P L ----PL VGVI VLG T L ATS A A Npunc L RMPERPQ-PPVWLVIIRL L R W H K - P E G R L I L M I P A L W AVFL A ASGKP P L ----PL VGVI ILG T L ATS A A Tery MTQAASPESTLMKVIRL L R W D K - P A G R F I L M I P A L W AVFL A THAMP PI----PL VGVI VVG T L ATS A A Telon NSQVSRTSVSTWVAIAQL L R W H K - P A G R L I L L I P A L W SLTL A SEGLP S L ----KL LLII TM G AIATS A A S8102 MDSIRERLGPWLEL L R W T K - P T G R L I L L I P A G W SLW L SPSAPP G L ----DL LLQI VVG G L AVS G A PmarMED4 M IKKDQNSKINILFKL L R W N K - P T G R L I L L I P A G W SLYL TPESNP SI----YMLLKI LIG G L LVS GL PmarMIT9313 MTSRSFSNGLRQWLEL L R W H K - P S G R L I L L V P A G W SLW L TPDAPP LA----GL VSLI VVG G L MVS G A PmarSS122 MKSFLLTKKLLNIFHL L R W N K - P T G R L I L L I P A G W SLW L TPNAPP SK----EL VTLI IAG AISVS G A Gvio MSTAPTDWRAKADAVFRL L R W D K - P A G R L I L M I P A L W GVFAA AGGSP SP----LL VLVMVVG TIATS A A Rpal NWVDNHAPLWTRPYLRL A R FDR-P I G SW L L L M P C L W SAAL A AGMAHDL S R L P LFCALFFIG SFVMRGA Rrub DGWVDRMAPGVIRPYLKL M R LDR-P I G TW L L L F P CW W SQSMA AQGW P D L ----W L AALFAAG A L VM RGA Ecol MEWSLTQNKLLAFHRL M R TDK - P I G A L L L L W P T L W ALWVA TPGVP Q L ----W ILAVFVAG VW LM R A A CT1511 MSVSSSSQLSAFQAWMLAIR PKTL P A G AMPVVI G A ALAAASG-VFKP LP------ALVALICAL GIQIA L . ...LLRW KLP .GRLILLIPALW ...LA . P LSRLP L ...I..G.L. S .A
80 90 100 110 120 130 140 S6803 G C V V N D L W D - R D I D PQV E R T K-QR P L A ARAL SVQVGIGVALVAL L C AAGL AFYL T----PL S F W L C V A A V S7002 G C V V N D L W D - R D I D PQV E R T K-VR P L A SKAL TVKVGIITAI AFFL C AAGL AFYFN PW T N P L S F W L C V A A V A7120 G C V V N D L W D - R D I D PEV E R T R-DR P L A ARTL SIKVGIAVGI IAL F C AAIL AYYL N ----SL S F W L S V A A V Npunc G C V V N D L W D - R D I D PEV E R T R-DR P L A SRAL SVKVGIVVAI VSL A C AAIL AFYL N ----PL S F W L C V A A V Tery G C V V N D L W D - R D I D SEV E R T R-DR P L TSRAL TIQTGIIIVI IAFAC AGVIAL Y L N ----PL S F W L S V A A V Telon G C IVN D C W D - R N I D PHV Q R T Q-NR P L A SRRL SLGVA LGLMVIAL L C AW G L TFYL T----PL GYW L A V A A V S8102 G C IAN D L W D - R RFD GRV E R T K-QR P L A RGAIRPTSA LVLLI VLL SLSLAVVL S L DEASRQL CLLL SIGA L PmarMED4 G C V A N D I W D -KRI D QKV L R T K-NR P L A ANKISTKTA YLILI FLIIC SFFL T L S L PENGRLL S LSL AFFA L PmarMIT9313 G C IAN D L W D - R R I D RQV E R T R-ER P L A KGSIRICTA VGLLI VLL L LSLLVVASL PTPSQRL CLAL AILA L PmarSS122 G C IAN D L W D - R K I D SKV K R T K-NR P L A NGSL SIVEA FSLLI FLL IISLKVIL L L PTPSQNL S LKL AAMA L Gvio G C V I N D L W D - R D I D PLV E R T R-AR P L A SREL AVPVA VVTFAVSL L C AW G L TFFL N ----PFTFW L C V A A V Rpal G C TW N D ITD - R D L D DKV E R T R-SR P IPAGQVTAKQA AVFMVLLCL IGLVVLL QFN ----SFAIATGISSL Rrub G C TFN D IVD - R D F D AQV A R T A-AR P IPSGAVSRKKA VVFLGAQL L VGLAVLL S L N ----GFS I W L G V A S L Ecol G C V V N D YAD - R KFD GHV K R T A-NR P L PSGAVTEKEA RALFVVLVL ISFLL V L T L N ----TMTILL SIA A L CT1511 TNFIN EIYD F R KGADTAE R LGP T R TVA AGIITEQTM IRVSI VLGVSVFVL G L Y L VA IGGW P ILL IGV LSL GCV .NDLWDFRDID VERT .P RPLA .L.. A ....I..LLC ...L.L.LN LS .WL VAAL
150 160 170 180 190 200 210 S6803 P V I VAY P G A K R V F PVP Q L V L SIA W G F A V L I S W S A V TGDL T--DATWVL W G A T VFW T L G F D T V Y A --M A D R S7002 P V I IAY P L A K R F F PVP Q L V L AIA W G F A V L I S W S A V TQNL T--AATWLL W G A T VFW T L G F D T I Y A --LSD R A7120 P V I LLY P G A K R V F PVP Q L V L SIA W G F A V L I S W S A V TQNIS--QATWLL W G A T LLW T L G F D T V Y A --M S D R Npunc P V I VLY P G A K R V F PVP Q L V L SIA W G F G V L I S W S A V TQTIT--QPTWLL W G A T VLW T L G F D T V Y A --M S D K Tery P V I ICY P L A K R F F PVP Q L V L SIA W G F A V L I S W S A AVAKL D--SATW IL W G A T IVW T L G F D T V Y A --M S D R Telon P V I LLY P L A K R VLPIP Q L V L AVA W G F A V L I P W A A V QGRL T--LTTAGL W A A VVMW T L A F D T V Y A --M P D R S8102 P A I LLY P S A K R W F AYP Q A V L AFCW G F A V L I P W A A AERSL TLQPALIGCW L A T LLW T F G F D T V Y A --M A D R PmarMED4 P L I LIY P S A K R W F KYP Q FIL SICW G F A V V I P W A A NEGNIN-SIVLLFCW L A T IFW T F G F D T V Y A --LAD K PmarMIT9313 P P I LLY P S A K R W F AYP Q A V L ALCW G F A V L I P W A A SQANL KGGWPLLGCW L A T LMW T F G F D T V Y A --M A D R PmarSS122 VPI LIY P SSK R W F RYP Q VLL AFCW G F A V L I P W A A SEHSL NGGFPLLTCW L A T MVW T F G F D T V Y A --M A D K Gvio P V I ALY P G A K R V F PVP Q L V L SLA W G F A A L I S W S A V TGGL E--TPAWW L W G A T VLW T L G F D T V Y A --LSD R Rpal IIVAAY P FM K R ITYW P Q F V L GLA FSWGAL MGFA GTFERL D--LVAFAL YAGSIAW VIG Y D T V Y A --HQD A Rrub ILVFGY P FM K R ITYW P Q AW L GLTFTYGAL MGW A A V RGSL D--PAPLLL YAA CFFW T L HYD T I Y A --HQD K Ecol ALAWVY P FM K R YTHLP Q V V L GAA F G W S IPM AFA A V SESVP--LSCWLMFLA NILW AVAYD T Q Y A --M V D R CT1511 LFAWAY TGGPFPIAYSGL GDVFVFIF FGL VAVGGTYYVQALSLPMEVL VAA AAPGAFSVCILLVN N IRD I P .I..YP .AKR F .PQLVL ..AWGFAVLI W AAV L . ..LW .AT ..WTLGFDTVYANNM DR
220 230 240 250 260 270 280 S6803 E D D RRI G V N S S A L F F G QYVGEA VGI FFAL T IGCL FYLG M ILM L NPLYW -LSL AIAIVGWVIQ YIQLSAPT S7002 D D D LKI G I N S S A IFF G KFA PEA VGI FFVL T ATCL A ILG KTLNINLLIYGIAWAIAVGLWFKQ YLKIRKPN A7120 E D D QRI G V N S S A L F F G DYA PLA IG I FFLGT IICL TW L G VAIHL HLAF W -ITL GIASVGW IW Q VLRLRQPQ Npunc E D D RRI G V N S S A L F F G NYA PVA IG I FFAGT ILLL A WLG LLINL HFAF W -ISL AVATIGWVW Q SLRLRQRD Tery E D D QRLG I N S S A L F F G KYA NNA VGI CFFL T TIFL GLLG IKM EL HTGF W -ITL GIATVFWFIQ YKKLGQED Telon P D D RRLG V N S S A L F F G AYA PLA IALFYAL T VVFL MMVG VRAGL TW R F W -LAL AVTTYFWLRQ SLQIYTHE S8102 R D D ALI G L N S S A L SLG NRA VVTVRACYVL T VVAL GVAAASAGVHPLF W IFW L GASLLMQISCQSLN-HRN PmarMED4 KYD IQ I G V N S S A VNLAYNTKKTIQI CYFL T SSFL A ICAFINQL NFVF W PIWL ITGFLM QKD ILK IFPESK PmarMIT9313 R D D ANLALKS S A L SLG SHA LKTVAFSYAL ACTFL A SSAVSAGVGWAF W PFW L IASIGMQKETWTLR-GSN PmarSS122 D D D KKLG LQS S A L SLG QKA LKSVFI CYAL T STLIA FSALTKDIGIIF W PFW L IASIGMQKEVFSLK-ELE Gvio E D D LKI G I N S S A IFF G RFTPEA IAAFFAGAALCL A VVG YQLEL GSIF Y-VCL IVATLFWWREYRSLVTGD Rpal E D D LM VG IKS T A L L F G ENTKLA LVLLFGVAVSLIGVALKLADAGWFAW -IGL AAFAAHLVQQ IVRVDIHD Rrub E D D LLVG VKS S A L ALG SNTRPA LVVFSTL MMALVA AAG WQAGL SW P F W -ALL GLPAAHLAW Q IW R V D I H D Ecol D D D VKI G IKS T A ILF G QYDKLIIGI LQIGVLALMA IIG ELNGL GWGYYWSIL VAGALFVYQQ KLIANRER CT1511 DTD RKVG KMTLPARIG --A PAA RALYVAL VVLAYLVPFYM ISTGYSLW CLLSLLSIPLAIGMVRTLYASE .DD ..IG .NSSAL FG .A A ..I...LT .. LA ..G . L ..FW . L ...... Q . .
290 300 310 320 330 340 350 S6803 P------EPKLYG QIF GQN V IIG FVL L A G MLLG WL S7002 L------PRKAYNRLF EEN V TIG FIL L S G M I SSFIF A7120 L------PNPAYG EM F RQN V WIG FIVL A G M I F G SL Npunc L------PNSVYG KM F RQN V WIG FIL L A G M I A G SF Tery ------ILKPYAQIF RQN V WIG FIL L A G M I T G LIF Telon RRD T Q T T T LPIQTYG RYF NEN V WLG FLL WVG MW CPRP S8102 A------TMASFG LHF RRQV QLG SLL L L G L I VSRGLSG PmarMED4 Q------SIKKIG DHF KNQSIYG GLIL L G F I IAS PmarMIT9313 Q------PITTYG QHF QHQV LLG AM L L L G L I L G RIS PmarSS122 A------NTNKFG KHF RNQV FLG GLIL F G L I L G NF Gvio ------DPARFNRIF NEN V TVG FVIL T G L I G G TLI Rpal S------PLCLKLF KSN RDAG LLL FGG LVADAVSRSMA Rrub S------PVCLM IF KSN RFVG WLL L AAIVAG RAPGGV Ecol E------ACFKAF MNN NYVG LVL FLG LAMSYWHF CT1511 GQ------ALNAVLAGTGKVLTVHG LLFSLG LV IPN IIS IFRP DTQTTT ..G F . NV .G..LL .G.I.G . P
The multiple alignment was generated by BLOSUM30 using Clustal W. S6803, Synechocystis sp. PCC 6803; S7002, Synechococcus sp. PCC 7002; A7120, Nostoc sp. PCC 7120; Npun, Nostoc punctiforme; Tery, Trichodesmium erythraeum; Telon, Thermosynechococcus elongatus BP-1; S8120, Synechococcus sp. WH8102; PmarMED4, Prochlorococcus marinus MED4; PmarMIT9313, P. marinus MIT9313; PmarSS122, P. marinus SS120; Gvio, Gloeobacter violaceus PCC7421; Rpal, Rhodopseudomonas palustris; Rrub, Rhodospirillum rubrum; Ecol, E. coli; CT1511, Chlorobium tepidum.
297 A.1.5. An amino acid sequence alignment of UbiD homologs in cyanobacteria and other organisms
10 20 30 40 50 60 70 80 S6803 M ARD L R G F IQL L E T R G Q L R R I T A E V D P D L E V A E I SNR M L QAG G P G L L F E N V K-----G S PFP V A V N L M G T V E S7002 M ARD L R A F IKL V E E R G Q L R R I K A L V D S D L E I A E I ANR M L QVG G P A L L F E N V R-----G T S V P V V V N L L G T V E A7120 M ARD L R G F IK IL E E R G Q L Q R I S A L V D P D L E I A E I SNR M L QKG G P G L L F E N V K-----G A S F P V A V N L M G T V E Npunc M ARD L R G F IK IL E EKG Q L R R I S A L V D P E L E I A E I SNR M L QKG G P G L L F E N V K-----G A S F P V A V N L M G T V E Telon M TKD L R HYL Q L L E Q R Q Q L R R I TVP V D P D L E M A E I CNR L L AAG G P A L L F E N V I-----G S PYP V A I N L L G T L E S8102 M A L F R SGP ATRD L R G F L Q L L DQR G Q L K R I T A A V D P D L E L A A I ADR V L SQG G P A L L F E N V I-----G S S M P V A V N TLG T V E PmarMIT9313 M P L L R PGP ASQD M R D F L A L L E Q R G Q L R R I T A P V E P D L E L A A I TDR V L GLG G P A L L F E N V I-----G S S M P V A V N L M G T L E PmarSS122 M NVFQNGVGSQD M R D F L S L L E KKG Q L R R I SKP V D P D L E L A A I SDR V L SM G G P A L L F E N V I-----G S S M P V A I N L L G T I E Gvio M GRD L R T F L Q L L E S R G Q L R R I S A P V ERD L E V A E I ANR L L AAG G P A L L F E N V K-----G S PFP V A V N L L G T V E Rpal M L S R VKP PFPD L R A F SGYL E S R G Q L H R I RKP V SVVHDLTE I HRR V L HAG G P A L L I E N PIK ADGT P S EM P ILV N L F G T V E Rsphe M R SLP IFPD L R A F L DWCARKG D L S R I AEP V SLRHE TTAVASKIL RGG G P V L R F E S---LRDT SATMP LVAN L F G T R E Ecol MDAMKYND L R D F L T L L E QQG E L K R I TLP V D P H L E ITE I ADR T L RAG G P A L L F E N ----P-KG Y S M P V LCN L F G T PK BH3930 M YQNL QECINDL E KHG H L I R I REEV D P Y L E M A A I HLKVYEAG G P A L L F E N V K-----G S NYQAVSN L F G T M E M L R PM .DLR.FL LLE RGQLRRI.APVDPDLE .AEI R .L .GGPALLFENV K TGSS PVAVNL GTVE
90 100 110 120 130 140 150 160 S6803 R ICW A M NMDHPLEL E D L G K K L A L L Q Q P K P P K KISQA IDF GKVL F D V L K A K P G-RNFFP P C Q E V V IDG ENLD L NQIP L I R P S7002 R ICW A M KMEKPEEL E D L G R K L A M L Q Q P K P P K KISQA IDF GKVL F D V L K A K P G-GSFFP P C Q Q V V VEQEELD L GLIP MLR P A7120 R ICW A M NMQHPQEL E T L G K K L SM L Q Q P K P P K KISQA IDF GKVL F D V L K A K P G-RDFFP A C Q Q V V VQG DDV D L NTL P L I R P Npunc R ICW A M NMQHPEEL E T L G K K L SM L Q Q P K P P K KISQA IDF GKVL F D V V K A K P G-RDFFP A C Q Q V V LQG NDLD L NKL P L I R P Telon R V C W A M NMDDPLEL E T L G E K L GKL Q Q P K P P K TLSQA LDF GKIL F D V VRA K P S-RDLLP P C Q Q V V ITAPDLD L RQL P L I R P S8102 R V V W S M G LERAEQL E D L G SRL A L L Q Q P R P P K GLSETKQF ARVFW D LVK A K P D-RDLTP P C R Q QIFKG DAV N L YNIP L I R P PmarMIT9313 R V V W S M G LDNAQQL E D L G TRL A L L Q Q P R P P K GLQETRQF ASVFW D LIK A R P D-LDLTP P C H Q Q V LRG DALNL DNL P L I R P PmarSS122 R V V W S M G LSSQDEL E G L G K K L SYL Q Q P E P P K GIKKTIEF GGIL W D L L Q A R P D-LDLTP P C H Q RLLKG EDV N L DKL P L I R P Gvio R V V W S M G LEEQQQL E A L G E K L GKL Q K P R P P RSLQDA LEF APLL F D V I K A R P GRVLFAP E C Q Q V V LEG KAV D L DRIP M I R P Rpal R V A W GLG -ILPENL SRL G EAL A EM REP A P P QSLTDA LSKLPMAKAAL AM RP K-LAKSAP V Q E V V LTG DAV D L GRL P VQIP Rsphe R V AAGLG -LGLEQIPEL G AFL A A L RAP A P VAGMRDA LSRWPQL QAAL NTRAK-IVRSAEAQ E V V HEG AAV D L GM IP VPTC Ecol R V AM GM G QEDVSAL REVG KLL A F L KEP E P P K GFRDLFDKLPQFKQV L NMPTK-RLRGAP C Q Q KIVSG DDV D L NRIP IM T C BH3930 R SKFIFR-----QTWQSAENVVAL RN-DP MSALKHPFAHARTALAASK A L P LKKSRLP AGFEEITISD------L P L I QH RV .W.MG . LE LG KLA LQQP .PPK .. A ..F...L.DVLKA.P . PPCQQVV . G . VDL LPLIRP
170 180 190 200 210 220 230 240 S6803 Y P G D A G KII T L G L V I T K D C-----E T G T P N V G V Y R L Q L Q S -----KTT M T V H W L S V R G G A R H L R K A A EQG --K K L E V A I A S7002 Y P K D A G KNI T L G L V I T K D C-----E T G V P N V G V Y R L Q L Q S -----KNT M T V H W L S V R G G A R H L R K A A ENG --K K L E V A I A A7120 Y P S D A G KII T L G L V I T K D C-----E T G T P N V G V Y R L Q L Q S -----KNT M T V H W L S V R G G A R H L R K A A ERG --K K L E I A I A Npunc YVG D A G KII T L G L V I T K D C-----E T G T P N V G V Y R L Q L Q S -----KNT M T V H W L S V R G G A R H L R K A A ERG --K K L E V A I A Telon Y P K D A G KIMT L G L V I T K D C-----E T G I P N V G I Y R L Q L Q S -----PTT M T V H W L S V R G G A R H L R K A A AQG --K K L E V A V A S8102 W P G D A G GVI T L G L V I T K D P -----E T G V P N V G V Y R L Q R Q S -----VNT M T V H W L S V R G G A R H L R K A A AM G --K K L E V A V A PmarMIT9313 W P G D A SGVI T L G L V I T K D P -----E T G V P N V G V Y R L Q R Q S -----PKT M T V H W L S V R G G A R H L R K A A AM G --QK L E V A I A PmarSS122 W P G D A G KII T F G L V I T K D P -----E T NTP N V G V Y R L Q Q Q S -----INT M T V H W L S V R G G A R H L R K A SAMG --K K L E V A I A Gvio YAG D A G RILT F G L V I T H D P -----E D G T P N V G I Y R L Q Q Q S -----RDT M T V H W L S V R G G A R H L R K A A ERG --QK L P V A I A Rpal W P G EPAPLI T W G L V F T K P P ---PGAHG TDN V G V Y R M Q VLG-----KDRLIMRW L AHR G G A K H HHQWKADK--REMPV A I V Rsphe W P G D A G PLVT WPVV L T RPHGTS AEE T LHYN A G V Y R A Q VIG-----RDRLIMRW L AHR G G A G H C R SW M R A G --EPMPV A L A Ecol W P E D A APLI T W G L TVT RGP H K ---E --RQN L G I Y R Q Q L IG-----KNKLIMRW L S H R G G A LDYQEWCAAHP G ERFPV SVA BH3930 W P D D G G AFI T L PQV YSED P D K --PGIMNAN L G M Y R V Q L TGN E Y E L DQEVGLH YQIHR G IGVH QTK A NQKG --EPL K V S I F W PGDAG ..ITLGLVITKDP KS ETG PNVGVYRLQLQSNEYEL ..TMTVHWLSVRGGARHLRKAA GPGKKLEVAIA
250 260 270 280 290 300 310 320 S6803 L G V D P L I I M A A A T P I P V D L S E W L F A G L Y G G S G V A L AKC K T V D L E V P A D S E F V L E G T I T P G E M L P D G P F G D H M G Y Y G G V E D S7002 V G I D P IL I M A A A T P I P V D L S E W L F A G L Y G G S G V A L TKC K T V D L E V P A DAE F I L E G T I T P G E M L P D G P F G D H M G Y Y G G V E D A7120 L G V D P L I I M A A A T P I P V D L S E W L F A G L Y G G S G V Q L AKC K T V D L E V P A D S E F V L E G T I T P G E I L P D G P F G D H M G Y Y G G V E D Npunc L G V D P L I I M A A A T P I P V D L S E W L F A G L Y G G S G V Q L AKC K T V D L E V P A D S E F V L E G T I T P G E V L P D G P F G D H M G Y Y G G V E D Telon V G V H P L I I M A A A T P I P V D L S E W L F A G L Y G G G G IHL AKC K T L D L E V P A Q S E F V L E G T I T P G E V L P D G P C G D H M G Y Y G G V E D S8102 I G V H P L LVM A A A T P I P V Q L S E W L F A G I Y A G E G V R L TPC K T L D L Q V P SHS E V V L E G T I T P G E V L P D G P F G D H M G F Y G G V E D PmarMIT9313 I G V H P L L I M A A A T P I P V Q L S E W L F A G L Y A G E G V R L TGC K T L D L K V P SHS E V V L E G T I T P G E E L E D G P F G D H M G F Y G G V E S PmarSS122 I G V H P L ILM A A A T P I P V T L S E W L F A G L Y A G K G V R L SNC K T VNL Q V P SHS E I V L E G T I A P G E EM ND G P F G D H M G F Y G G I E K Gvio V G V D P L LVM A A A T P I P V E L S E W V F A G L Y A G E G V Q L AQC R T S D L L V P A H S E F V L E G T I T P G E V L P D G P F G D H M G Y Y G G V E D Rpal I G A D P SM I LSA VLP L P ETVS E IKF A G L LGG ERPSL TPC Q T IP ISV P A DAE I V L E G FVSP T E TAP E G P Y G D H T G Y Y NAV E E Rsphe L G C D P ALLLA A A L P L P EQVS E LTF S G VLRG ARTPL VAGRT VPL M V P A TAE I V V E G WVHP G DM AP E G P F G D H T G Y Y NSV E D Ecol L G A D P ATI LGA V T P V P DTL S E YAF A G L LRG TKTEVVKC ISND L E V P A SAE I V L E G Y I EQG E TAP E G P Y G D H T G Y Y NEV DS BH3930 V G GPP AHSLSA VM P L P EGL S E MTF A G L LSG R--RFRYSYVDGYCISHDADF V ITG E I P P G DTKP E G P F G D H L G Y Y SLIHD .GVDPL.IMAAATPIPV LSEWLFAGLY .G GV L . CKT .DL VPA SEFVLEGTITPGE LPDGPFGDHMGYYGGVED
330 340 350 360 370 380 390 400 S6803 S P L V R F Q C L T H R KNP V Y L T T F S G R P P K E E A M M A I A L N R I Y T P I L R Q Q V S E I T D F F L P M E A L S Y K A A I I S I D K A Y P G Q A K R S7002 S P L V R F H C L T H R K D P I Y L A T F S G R P P K E E A M M A I A L N R I Y T P I L R Q Q V S E I V D F F L P M E A L S Y K A A I I S I E K A Y P G Q A R R A7120 S P L I R F Q C M T H R K D P I Y L T T F S G R P P K E E A M M A I A L N R I Y T P I L R Q Q V S E I V D F F L P M E A L S Y K A A I I S I D K A Y P G Q A R R Npunc S P L I R F E C M T H R KNP I Y L T T F S G R P P K E E A M M A I A L N R I Y T P I L R Q Q V S E I V D F F L P M E A L S Y K A A I I S I D K A Y P G Q A R R Telon S P VIHF H C L T H R RNP I Y L T T F S G R P P K E E A M I A L A L N R I Y T P I L R Q Q V P E I V D F F L P M E A L S Y K A A I I S I D K A Y P G Q A R R S8102 S P L V R F H C M T Q R R D P VFL T T F S G R P P K E E A M L A I A L N R I Y T P I L R Q Q I P E I T D F F L P M E A L S Y K L A V I S I D K A Y P G Q A K R PmarMIT9313 S P L V R F H C V T Q R R D P I F L T T F S G R P P K E E A M L A I A L N R I Y T P I L R Q Q V S E I V D F F L P M E A L S Y K L A V I A I D K S Y P G Q A K R PmarSS122 S P L I R F H C I T S R K D P I Y L T T F S G R P P K E E A M L A I A L N R I Y T P I L R R Q INE I T D F F L P M E A L S Y K L A I I S I D K S Y P G Q A R R Gvio S P L V R I H TVT H R H D P I Y H T T F S G R P P K E E A M I A I A L N R I Y T P L L R A Q V P E VRD F F L P M D A L S Y K L A VLS I K K A Y P G Q A R R Rpal F P VM R ITA IT M R RHP I Y L S T YTG R P P D E PSRLGEA F N DVFLP VAR R Q F P E I V D LW L P P E A C S Y RIA VAS I K K R Y P G Q A R R Rsphe F P VLR VSAIT H R K D P L Y L T T HTG R P P D E PSVIGEVFN DLAMP VFR Q Q I P E VKD LYL P PAA C S Y RIA I V S I D K R Y P G Q A R R Ecol F P VFTVTHIT Q R E D A I Y HST YTG R P P D E P A VLGVA L N EVFVP I L QKQ F P E I V D F Y L P P E GCS Y RLA VVTI K K Q Y A G H A K R BH3930 F P VM KVH KVYAKQGAI WPFT VVG R P P Q E DTSFGALIHELTGDAVKLEIP GVKEVHAVDAA GVHPLLFAIGSERY TPYQKV SPL .RFHC .THR .DPIYLTTFSGRPPKEEAM .AIALNRIYTPILRQQVPEIVDFFLPMEALSYK .AIISIDKAYPGQARR
410 420 430 440 450 460 470 480 S6803 A A L A F W S A L P ------Q F T Y T K F V I V V D KSI N I R D P R ---Q V V W A I S S K V D P V R D V F I L PET P F D S L D F A S E K I G L G G R S7002 A A L A F W S A L P ------Q F T Y T K F V I V V D K D V N I R D P R ---Q V V W A I S S K V D P V R D V F I L PDT P F D T L D F A S E K I G M G G R A7120 A A L A F W S A L P ------Q F T Y T K F V I V V D K D I N I R D P R ---Q V V W A I S S K V D P T R D V F I L PNT P F D T L D F A S E K I G L G G R Npunc A A L A F W S A L P ------Q F T Y T K F V I V V D K D I N I R D P R ---Q V V W A I S S K V D P V R D V F I L PNT P F D T L D F A S E K I G L G G R Telon A A L A F W S A L P ------Q F T Y T K F V I V V D KEI N I R D P R ---Q V V W A I S S K V D P S R D V F I L GET P F D S L D F A S E K I G L G G R S8102 A A M A F W S A L P ------Q F T Y T K F V V V V D KHI N V R D P R ---Q V V W A I AAQV D P Q R D L F T L ADT P F D S L D F A S E QLG L G G R PmarMIT9313 A A M A F W S A L P ------Q F T Y T K F V V V V D ANI N V R D P R ---Q V V W A I AAQV D P Q R D L F V L ENT P F D S L D F A S E HLG L G G R PmarSS122 A A M A F W S A L P ------Q F T Y T K F V V V V D STI N V R D P R ---Q V V W A I S S L V D P Q R D LLVMENT P F D S L D F A S E N I G L G G R Gvio A A M A F W T A L A------Q F T Y T K F V I V V D E D I DIR D P S---Q V V W A I A S K V D P E R D L F VIPRT P F D T L D F A C E R I G L G A R Rpal LMM GLW S M L P ------Q F S Y T K LLI I V D D D VDVR D W A---DV M W A V S TRCD TSR D MVSISDT P I D Y L D F A S PKSG L G G K Rsphe VMM A L W GM L A------Q F S Y T K M V I V V D E D I N P R D W D---DV A W A MATRMD P S R D VVLL EKT P M D Y L D F A S PEPG L A G K Ecol VMM GVW S F L R------Q F M Y T K F V I V C D D D V N A R D W N---DV I W A I TTRMD P A R D TVLVENT P I D Y L D F A S PVSG L G SK BH3930 KQPA ELLTIAN R I L G T G Q LSLAK YLFITAEQDKPLD THK E E EFLTYLLERID LHR D IHFQTNT TID T L D YSGTGLNTG SK AAMAFWSALPNRILGTGQFTYTKFVIVVD DIN .RDPRKEEQVVWAISS .VDP RD .F.L TPFD .LDFASE .IGLGGR
490 500 510 520 530 540 550 560 S6803 M G I D A T T K I P P E T D H E W G E V L E-----SD P AMAEQV SQR W A E Y G L G D INLTEV N P N L F G Y DV S7002 M G I D A T T K I P P E T D H E W G E V L E-----SD P KIAERCDRR W A E Y G L A D IDLTAV D P NKF G Y EM N A7120 M G I D A T T K I P P E T D H E W G A P L E-----SD ADVAAMV ERR W A E Y G L A D LQLGEV D P N L F G Y DM K Npunc M G I D A T T K I P P E T D H E W G S P L E-----SD P DVAAMV ERR W A E Y G L AELQLGEV D P N L F G Y DM K Telon M G I D A T T K I P P E T D H P W G D P L T-----SD P EVARRV TER W Q E Y G L G D IDLTAV D ATRF G Y ELDPAFRWR S8102 LAI D A T T K VGP E KNH D W G E P L S-----RPADLEERV SAR W S E L G L DGLGQDEPD P S L F G Y ALDRLIQGLKTSP PmarMIT9313 M A I D A T T K I G P E KRH E W G E P L S-----RD ADLESRV DAR W Q E L G L E D LGSEEPD P S L F G Y VMESLCRQAMVKKT PmarSS122 LAI D A T T K VGP E KNH E W G N P L I-----HSSNLSKKIDER W N E L G L S D LKPNHAD P S L F G Y VIDEIIKFNQITKS Gvio L G I D A T T K I P P E T D H A W G E P L R-----AD P ATAEQV SKR W A E Y G L G D IPLGGV D P RAW G Y SGK Rpal L G I D A T N K I GTE T ERE W G KVL E-----MD KDVIARV DAMW TSLG L SPEHQPAAGQRRLIR Rsphe I G I D A T N K I G P E T HRE W G E VMT-----QSP EAEAFADRLIAKLRIGA Ecol M G L D A T N K W P G E T QRE W G R P IK-----KD P DVVAHIDAIW D E LAIFNNGKSA BH3930 VVI A A YGDKKRE LCQE VPE LFKG L Q T F QNP RLVMPGVVALEGPSFTSYERAQEEFRAF SETITQNGELPSCPM I MGIDATTKIPPET .HEWGEPL GLQTF DP ... .V. RW E GL .D. VDP LFGY
298 The multiple was generated by BLOSUM30 using Clustal W. S6803, Synechocystis sp. PCC 6803; S7002, Synechococcus sp. PCC 7002; A7120, Nostoc sp. PCC 7120; Npun, N. punctiforme; Telon, T. elongatus BP-1; S8120, Synechococcus sp. WH8102; PmarMIT9313, P. marinus MIT9313; PmarSS122, P. marinus SS120; Gvio, G. violaceus PCC7421; Rpal, R. palustris; Rsphe, Rhodobacter sphaeroides; Ecol, E. coli; BH3930, Bacillus halodulans.
299 A.1.6. An amino acid sequence alignment of UbiH homologs in cyanobacteria and other organisms
10 20 30 40 50 60 70 80 S6803 MTFVAASITDNQFD VAI A G G G VVG LVL A AGL RH---TG LKI S7002 MEKSHLGFD VII V G G G IVG VTL A ACL KR---TNLRI A7120 MALTQLTQTISPQPTPPELRKYDYD LVI V G G G IVG LTL A S A L KD---SG LNI Npunc MALTQLEQPLSPQPTPPDLRGYEYD LVI V G G G IIG LTL A S A L KD---SG LSI Telon MMTSAFAPSEQSLVASPQDLPAVD VAI V G A G IVG LSL A C A L RN---SG LAI S8102 MATSSTTFHI L G A G PTG SLAA I A L AS---TG CSV PmarMED4 MNNYLNFKI V G S G PSG LLL A ISL AK---LNFNI PmarMI9313 MVDHLNRFHSAAKGSLPVDIRVKI L G A G PTG TLL A L A L AR---LG NSV PmarSS122 MSNLHIKVIG A G PSG SLL A LSL VG---NSNAV Gvio MAFNNRNTLAAERGKAALQAD CII I G G G PAG AVL SLLL AR---QG VSV Rpal M P R G H A G D F P P P A L S V R N R PGNSGLVLETCFAQLRSGRKDHGARVRNMTAPRSIVI G G G AFAG LAL A L A L RQG--LG PEV Rrub MEYGVSEPLLRGLAAGDPPSATGPVTGSADKVAD VLI V G G G LVG GTL A C A L AE---KG VSV Ecol MSVII V G G G MAG ATL A L A ISRLS H GALPV Mlr2092 MQAQDDQQKGKDMTTAAAGPTEVD VAI I G A G MAG TTL A TLL GN---AG RKV MPRGHAGDFPPPALSVRNR D ..I.GGG .G. LA .AL SH G . V
90 100 110 120 130 140 150 160 S6803 AII E A LPKEQALTK---PQA Y A ISLLS GKILAGLG V W ENIKDSIGHFERIQISD NDYRGTV P F AKED VDE-----LALG H S7002 AIVE A QPLDVVTQR---QR A Y A MSLLS QRL FQELG V W EVIAARSGKYRNIQLSD EDFSGVV R F SRQD L KT-----DFLG F A7120 L L I E A KVASAAVAK---GQA Y A VHLLS ALIYQGIG I W DKILPQIAKYRQVRLSD ADYPGVV E F ESTD L GT-----PELG Y Npunc L L I E A KVASAAVAK---GQA Y A VHQLS ALIYQGIG V W DKILPQIAKYRRVRLSD ADYPNVV E F TTDD IHT-----AELG Y Telon A L I E A TPYRRENTK---GQA Y A LHQVS RYFFEEIG V W DKL MPHVQPFEVVQLSD ERFPLTV H F SPAD L GT-----EALG Y S8102 V L TDPLTRKELLSR---SR A Y A ITHSS RRL LTDLNLW TSL QGSLTAFSFLDLRD SACGGRV V F GLDD L PNSN GRHEAIG W PmarMED4 YITDLLTREKLINK---DKTY A ITHSTRKILSKFNLW SKL KSYLYKFDSLSISD SVTSDFTILTTSD L DKDICSLDTIG W PmarMI9313 T L CDPLTVEELPAR---SR A Y A LTHSS RRL LQSLDLW ASL IPHLAPFTRLRLDD QELNRHTYF EVDD L SSQN QLHGAVG W PmarSS122 SIYE A KSKEQILLR---DR T Y A INHSS RRL LQRIG I W DDL NEFM IPFDSLSLED YSVNQRLVLYNKD L VNLN SSYKEVG W Gvio I L F E A QKDFDRDFR-----GDSLHAVVMEL MDDLRLTERL LEHKHSKLKTLTMVSAVGAARVADFSRL PT----HHNYMT Rpal PVI V A DPALALRPS-RD P R A T A IVAACRRL FEALG V W DEVAPTAQPIIDMVVTD SHLEDATRPVFLTFAGEVQPGEPFAH Rrub VVI DGEDPEALLAAG Y D G R CSA IALACQRL LDTIG L W DLL GGESQPILDIRVVD GGSPLFLHYAQAEAQG------PMG Y Ecol H L I E A TAPESHAHPG F D G R A I A LAAGTCQQLARIG V W QSL ADCATAITTVHVSD RGHAGFV TLAAED YQL-----AALG Q Mlr2092 A L I DPHRVHHDEFR-----A EKIGADQMQL FEKLG LDKMVMPLVTPFTDIDVFRLG-----QF FARE--K------KWEY .LIEA .. .G D RAYA . ..S .L .G.W. L ...... D.VFDLN .G.
170 180 190 200 210 220 230 240 S6803 VAEH PVILQAL ENCVEQCPRIAWFRP AELISFTAGENHKQVTL QQEGR----EITLQTKL L V A A D G ARS HTR SLAG I QTK S7002 SGQH QVVLETL QASLRGYGNIRWFCP AQV TAIHYESEQAIVEL SSETQG ---QIKIQGAL V V G A D G PRS LVR QGASI KTR A7120 VAEH QAL LEPL QEFVQNCPNVTYLCP AEV VSTNYQPNEVVIAVKIADQ----LNTIRSKL L V A A D G SRS PIR QAAG I KTH Npunc VAEH QAL LQPL QEFVQDCPNVTYLCP AEV VNTQYQKDIVAVDIKVADQ----LYTVRSKL LIA A D G ARS PIR QAAG I KTS Telon VGEH SAIAETL QSVLQSAGNVTFYCP WRV VTNEVHGDRAQLTL ISG DPG E P K VAHLAARL V V A A D G GKS PLR QQMG I EPK S8102 ILDH QPL MKLL MDRLHQHHRVELALG-CTAPTPGIDD-----L ------IV A A D G PRS TTR SQWDI GCW PmarMED4 VVKH SDL MNVFFDEVDNVDNIFFKSSLDLSFNDIKFD------FEFVSTG ANPNHKKNRNFIDI PmarMI9313 ILDH RPL MELL IDRLQTSPKVLLNLGRFDDHHLDGHD-----L ------VV A A D G PRS LTR QAW G I NTW PmarSS122 TLDH KLL MEYL FYKISKSSNINIKFSSKDNDANALYD------YTFA A D G TNS RYR DLWKFKSY Gvio MVAQKDFLNFMVAEAKRYANFRVFMATSV QRLIEEEGEIRGVVFNG PDG ---PQEARCAL T V G A D G RSS RTR QM AG I DLV Rpal MVENRYL VEAL AKRAEAEGVELRATP --V TSYDARTEAIGVTL GDG S------VIDASL L V A A D G AKS KLR QRAG I ATY Rrub MVENRLL RQAILTRLGRLPAATLLAP ARMTALRRDLDGVSATL SDG Q------TVRARL V V G A D G RRS QVR ESAG I GIR Ecol VVELHNVGQRL FALLRKAPGVTLHCP DRV ANVARTQSHVEVTL ESG E------TLTGRVLV A A D G THS ALATACG VDWQ Mlr2092 AFSYGAL INGL RDALPPQVPITIGKVAEV STGPNRQR---LVL ADG ------AVIDARL L V V A T G YSELVR RAIG VERI ...H .L. L . . P V . . . L G GEPK . L .VAADG .S .R ..GI
250 260 270 280 290 300 310 320 S6803 GW K Y W Q S C VAFTIQHQAP DNTTA F E R F CDTG P ------MGIL P L PGDR AQIV W TM---P HHKAHTLVNL PEADF ITE L S7002 GW K Y W Q S C VTCVV EHDAP RNDVA F E R F WGTG P ------MGVL P L TENR CQIV W TN---P HAEAQRLKEL PPEVF L EYL A7120 GW K Y W Q S C IVAFV KPEKP HNDTA Y E R F WPSG P ------FA I L P L PENR CRIV W TA---P HDEAKALCAL NDEEF L R E L Npunc GW K Y W Q S C IVAFV KPEKP HNNTA Y E R F WSSG P ------FA I L P L PENR CRIV W TA---P HEEAKALCAL DDEQF L A E L Telon GW Q Y P Q S C VVATLDVAHP QPVIA Y E R F WPTG P ------MGVL P L PRNR YRVV W TL---P HPEAEAVAAL DDRSF L ATL S8102 KFRY R Q G C LTSKIALRGVKPGMA Y E L F RPEG P ------FA I L P L GDGIFQVV W SA---P W TQCQRRADL PTPEF L D E L PmarMED4 RKSY N Q S C LTFEV LVRGNVPRRA Y E I F RKEG P ------LA L L P L DKNKYQIIW TA---TTSKSMDRLNSSKSFLL DNL PmarMI9313 NHPY QNGC LTAKV LLRGADPLMA Y E L F RAEG P ------LA V L P MGGEVFQMV W SG---P LRRCQERAGL SPSCF L DHL PmarSS122 RFKY N Q E C ITFKALLRTP FTHRA Y E I F RKDG P ------MA I L P MANDLYQIV LSM---P PLKSDYLLNL TTSHF L DTV Gvio KFSQ-AVDVLWFRLPRYP HEPEGVLYRVGQG A------VMGQADRSDVWQISYIIR KGTYQKLRAQGIEGLRRSVTTL Rpal GW D Y D Q S GIVVTV EHERDHNGCA E E H F LPAG P ------FA I L P L TGRR SSLV W SE---SRRDAERIVAL PEKEF QRE L Rrub TLGY G Q TAIVLTV EHERSHRGCA V E H F LPAG P ------FA I L P MPGNR SSLV W TE---RSDLVPGLLAL PAEHF QAE L Ecol QEPY E Q LAVIANV ATSVAHEGRA F E R F TQHG P ------LA M L P MSDGR CSLV W CH---P LERREEVLSWSDEKF CRE L Mlr2092 ELSKAHS LSMGFDLAIAP R-DCA Y RAVTCYG K R A S D R V A Y LTVFP IGDKMRANMFVYR TVAEQWTRDFRADPQK-ML C E L . Y QSC ... V P . AYE .F GPRASDRVAY A .LPL R .VW . R P . ...L FL EL
330 340 350 360 370 380 390 400 S6803 RQRIGDRLG EFHLINARRLF PVQL MQSDCYV-QPR LAL V G D A A H CCH P V G G Q G L N L G I R D GAAL A QVIATAH-SQG E D W G S7002 EQHTGPQLG RLKLLGDRQVF PVQL MQGDR YV-DHR LAL V G D A A H CCH P V G G Q G L N L G I R D AAAL A QTLIEAQ-QRG Q D L G A7120 SQRFGQQMG KLELLGDRFVF QVQL MQSDR YV-LPR LAL V G D A A H NCH P V G G Q G L N L G I R D V AAL A QVIQKAH-QAG E D I G Npunc TRRYGDQMG KLELLGDRFVF QVQL MQSDR YV-LPR LAL I G D A A H NCH P V G G Q G L N L G I R D AAAL A QVIQTAH-QAG K D I G Telon QPYLDPQMAEITAVGDRFVF PAQFMQVNSYA-ADR FVVIG D A A H RCH P V G G Q G L N L G L R D V WNL A QQILAS---PLEEIG S8102 AAVLPAGIEPDLLLDTPRAF SQQWSMARR LS-RGR GVL L G E A G H RCH P V G G Q G L N L CW R D V ASL RNLAEHGG------T PmarMED4 STILPDNFKLDQINSDINIF PVSL SFRLPIFN FKKVVFVG D SFH TFH P V G G Q G L N TCW R D V NVIYDIFNRN--FPVSNRA PmarMI9313 AAVLPDGLQPDVLLDRPAAF PLQL AFAFR LQ-RGR GVL V G ESGH RCH P V G G Q G L N L CW R D V STL MNLVKNVDE---GKLP PmarSS122 ATYLPIGMEIDTLINKPQKF SLSL NVPIKLQ-DYNRFL I G ESLH SLH P V G G Q G L N L SIR D IDDIMNLLTSSN------Gvio VPELADRVG LLKDWSQASLLSVESGYVRR W Y-RPGLLL I G D A A H VM SP F G G V G ISYAIQD AVAAA NVLAGPLR ---SGRV Rpal EKRFGLRLG DIKPLDKPKSF PLGYFVAQSFI-APR LAL I G D A A H VIH P IAG Q G L N M G L R D V AAL A EVVVDAAR -LG I D P G Rrub ERRFGDHLG W VRPVGPRFSYRLTL QAANR YV-DHR LAL V G D A A H GM H P V A G Q G M N Y G L R D V AVL A ERLVAAQR -LG L D P G Ecol QSAFGWRLG KITHAGKRSAYPLAL THAAR SI-THR TVL V G N A A QTLH P IAG Q G F N L G M R D V MSL A ETLTQAQ-ERG E D M G Mlr2092 MPEIATQCG NFEVASPVEVRQVNL TTTQGHR-RDGVVFIG D A FVTTCP TPG V G IGRVMVD V DQL HSVHIPR------WL .. .G . .. .F ..L . R ..N R ..L.GDAAH .HPVGGQGLNLG.RDV..LA... . R G D G
410 420 430 440 450 460 470 480 S6803 SLAVL KRY EHW R KPEN-WLILGFT D L L D R F F S SHWLPAIALR RFG L EVL RLVPPAKKLAL RLM T G LLGRKP ---QL ATGQ S7002 SLAVL KRY ERW R KREN-LVILGFT D F L N R L F S N RIWPVVWVR RLG L IVMAKFNPL RLFAL KLM T G QKGRQP ---RILA A7120 NIRVL KRY ESW R QKEN-LAILGFT D M L D R M F S N QFPPL VFLR RLG L WFL RSVPVL KTFML KLM I G LKGKTP ---EL AKR Npunc DIQIL KGY ERW R KLEN-LTILGFT D L L D R M F S N NFLPVVVVR RLG L WFL QRVPLL KVFML KLM I G LKGRTP ---EL AKR Telon DRRHL MHFHRQR WW QN-VLTLAFT D F L N R L F S N TGWPL VAVR RCG L GLL CLLPPL KRLLL RFM A G LLLPLP GDREFPRGR S8102 SQRLARRY GRSR WAD L-LMVGLAT D L L V R L F S N QQPW L LPFR RIG L KAMARFSW L RRISL LAM TDGPTQLL--KPL PD PmarMED4 LNIFKYKY YFIR CLD I- IST IVVT D S L IQFF A N NKFFL LPFR KFSFLLL NKFIFMRRIIL NHM TKSLIYSSIK PmarMI9313 VEKLPQVY ARKR IFD L-VLVGLFT D LIVR F F S SRNILL LM VR LPLMFLL ARLSLL RQLLL KAM TDGPITVI--RLL PE PmarSS122 ----L LQY KICR YID I-YTTSFLT HM L IV IF S SNNIIL KVFKLSLFTIL RKSILFRKIVL SLM TDGIPHIFK Gvio GVGHL AAVQRRR ELPTRLSQYYQAM IQDQLLATAANEGIPPKPTLL RRL LRLPGL RELPTRLLAFGLWPARLRIKNSIPP Rpal QVDVL ERY QRW R RFD T-VAMGVAT NGL NFLF S N NSPLL RGIR DIG L GLVDRLPPL KGAFIRQAAG LTGEIP ---RL LKGE Rrub APALL AEY EALR RPD N-LLMLAIT D A L V R L F S N DIAPVALAR RLG IGAVERMGPL KRLFMRHAMG TLKLGP EPPRL MRGV Ecol DYGVL CRY QQRR QSD R-EATIGVT D S L VHLF A N RW A P L VVGR NIG L MTMELFTPARDVLAQRTLG W VAR Mlr2092 ETPGMAADKIGAFYD DPVKVAADKAGMRVSF YAKQITTETGLEWRVRRL RNNAARQLM IIGRKMRHLGPRR---EPGMA .L .Y . R D .. .. TD.L R.FSN L. .R. GL .L . L. ..L..M G... P L
490 500 510 520 530 540 550 560 S6803 S L VSQ S7002 A7120 Npunc Telon S8102 PmarMED4 PmarMI9313 PmarSS122 Gvio NIHR Rpal A L Rrub P L Ecol Mlr2092 LQ
300 The multiple alignment was generated by BLOSUM30 using Clustal W. S6803, Synechocystis sp. PCC 6803; S7002, Synechococcus sp. PCC 7002; A7120, Nostoc sp. PCC 7120; Npun, N. punctiforme; Telon, T. elongatus BP-1; S8120, Synechococcus sp. WH8102; PmarMED4, P. marinus MIT9313; PmarMIT9313, P. marinus MIT9313; PmarSS122, P. marinus SS120; Gvio, G. violaceus PCC7421; Rpal, R. palustris; Rrub, R. rubrum; Ecol, E. coli; Mlr2092, Mesorhizobium loti.
301 A.1.7. An amino acid sequence alignment of UbiX homologs in cyanobacteria and other organisms
10 20 30 40 50 60 S6803 MAQP LIL G V S G A S G L I Y A V R AIKHL L A A D-YTIEL V S7002 MDRP LTVG I S G A S G L I Y A V R TLKYL L E A N-LTVDVV A7120 MSQHNKP LIIG V S G A S G L I Y A V R ALKYL L T A D-YEIEL V Npun MSQKSKP LIL G V S G A S G L I Y A V R AIKFL L E A D-YSIEL V Telon M IAVVTSSSLP I V L G V T G A S G L I Y A V R AIKFL L Q A N-YPIQL V S8102 MHP Y V L A V T G A S AQPLA E R TLQLL L Q A G-RSVHL V PmarMIT9313 MDP Y V L A V S G A S AQPLA E R SLQLL L ENN-RDVHL I PmarSS122 MSSIV L AITG A S AQI L A E R SIDLL L RCN-QKLDVI Gvio MQTQAPSSRP I V L G V A G A S G L I Y A V R TVRFL L A A G-HAVDL V Ecol MKRLIVG I S G A S G A I Y G V R LLQVL RDVTD IETHL V Rpal MSTQHRIV V G I S G A S G AAVGLR VVELL ASVD-CEVHL V Rsphe M G R G H D A V A R G R S L R R P ADREAEDRSMRVV L G V S G A S G AALA AACARHL SALG-AEIDL V Ape1647 MSCRPSSVSIAV T G S S G VRVA L R LLEAL KGEV-EIRGII MGRGHDAVARGRSLRRP .P.VLGVSGASG .IYAVR ... LL A D ..LV
70 80 90 100 110 120 S6803 A S R A SYQV W QAE QNIQMP GEP SAQ AEF W R S Q A G V EKGG K L I C H R W G D V G A T I A S G S Y R CA S7002 A S K A SFMV W QAE N G ITM P ADP DHQ AKF W R D Q A G V PEKG I L R C H R W G D V G A G I A S G S Y R T K A7120 A S K STYMV W QAE QETRMP SEP KQQ EQF W R E Q A G V ALSG K L R C H P W S D V G A N I A S G S F R T L Npun A S K STYMV W QSE QEIRMP PEP TQQ EQF W R E Q A G V ALSG K L R C H P W G D V G A G I A S G S F A T L Telon A S K A A HQV W RAE Y G LTLP TQP DKQ ELF W R E Q A Q V W-EG C L T C H RPTD V A A A I A S G S F R T L S8102 L S RGA YAV FQAE Q G VQVP VNP ERQ ASF W R E RLNCS-EG E L F C H R W N D QSVGI A S G S F R T S PmarMIT9313 F S HGA LQV W KAE R G IEVP VDP SDQ EKF W R DHLQV K-NG N L T C H R W N D QSA C I A S G S F R T R PmarSS122 I S K G A YEV W KSE MNVNIP AEGAKQ ATF W R NRLKNN-QG EIIC H K W N D NSA T I A S G S F R T K Gvio A S R A SFMV W REE M G TAMP VEP AEQ ERF W R E Q S G ES-GG K L R C H AFANLAA S I A S G S Y R T R Ecol M S Q A A RQTLSLE T-DFSLREVQALA------DVTH DARD IAA S I S S G S F Q T L Rpal V S K A A RRTIAYE V G PNALDHAADLV------HRFH KIED V G A C I A S G S F R T S Rsphe I S K A GERTLVEE I G LEAADALRAQ A------TRVH AIMNV G A A I A S G S APVA Ape1647 LTRGA EEV ARYE E G IEPEDLRRLLR------SYAPLYMEND MSSPLA S S S NQPD .SKAA. VW .E G . P .P Q . FWREQAGV G L CH .W DV .A IASGSFRT
130 140 150 160 170 180 S6803 G M VVLP C S M S T V AKLA V G M S S D L L E R A A D V QIK E G K P L V VVP R E T P L S L I H L R N L T S L A E S7002 G M LVIP C S M S T V AKI A N G L S S D L L E R A A D V SIK E G R KVV VVP R E T P F S L I H L R N L T Q L A E A7120 G M IV IP C S M S T V AKLA G G L S S D L L E R A A D V H L K E G R K L V IVP R E T P F S L I H L R N L T T L A E Npun G M I I I P C S M S T V AKLA V G L S S D L L E R A A D V Q L K E G R K L V IVP R E T P F S L I H L R N L T S L A E Telon G M V I L P C S M S T V AKLA A G L S S D L L E R V A D V H L K E H R P L V LVP R E T P F S L I H L Q N L T Q L A M S8102 G M L I V P C S M G T AGRI QAG VAMD L I E R C A D V H L K E G R P L LIAP R E M P F N L I H L R N L T A L A E PmarMIT9313 G M A I V P C S M G T IGR I NAG V S I D L I E R C A D V H L K E G R P L V IAP R DM P W S I I H L R N L T S L A E PmarSS122 G M V I V P C S M G T IGR I A S G S S INL I E R C A D V H L K E G R P L IISP R E S P F N L I H L R N M T T L C E Gvio G M LVIP C S M S T V GKLA A G L S S D L L E R A A D V Q L K E G R P L V LVP R E T P F S L I H L R N L T A L A E Ecol G M V I L P C S IKT LSGI VHSYTDGL L T R A A D V V L K E R R P L V LCVR E T P LHL G H L R LM T QAA E Rpal G M IVAP C S IRT LSAI A H G DLDNL L V R A A D V Q L K E R R R L V LM LR E T P LHL G H I R SMAQATE Rsphe G M IVAP C S M RSLAAI A H G LDDNL L T R A A S V Q L K E R R R L V LLAR E S P LTL A H L R N M T AVTE Ape1647 A M A I V P A S M K T V GLI A R G IPS S L PAR A A LAVL RLG R R L V VAP R E T P LGVVEL E N MLAIA R GM .I.PCSM TV ..IA .G.SSDLLERAADV LKEGRPLV ..PRETPFSLIHLRNLT LAE
190 200 210 220 230 240 S6803 A G VRI V P A I P A W Y HQP QSVE D L V D F V V A R A L D Q L AID CVP LQR W Q G GMEGE S7002 V G VKI V P A I P A W Y HQP Q T I H D L V D F V V A R A L D Q FEID CVP LKR W KEEAASQKSEN A7120 T G VRI V P A I P A W Y HNP Q T I E D L V D F V V A R T L D Q L DID CVP LKR W E G GKHNS Npun V G VRI V P A I P A W Y HNP Q T I E D L V D F V V A R A L D Q L DID CIP IQR W E G RR Telon A G A R I V P A I P A W Y HRP Q T I E D L V D F VIAR A L D Q L GLD CVP LQR W Q G PLS S8102 A G A R I AAPI P A W Y TQP R T L E EM V D F I V I R L L D GFEDD LAP LQR W T G PIK PmarMIT9313 A G A K I A P P I P A W Y TKP N T I E EM V D F L V V R LFD LFDERLAP INR W N G PSQ PmarSS122 A G A K I I P C I P A W Y SKP KDLE EVID F M V V R LYD LFDLNMKDINR W K G N Gvio A G A R I V P A I P A W Y QQP K T I E D L V D F VIGR A L D Q L DID HSLFER W HPDT Ecol I G A V I M P PVP A F Y HRP QSLDD VINQTV N R V L D Q FAITLPEDLFARWQGA Rpal I G A IVAP LSP A F Y QRP RSI SEMV D HM ALR A IGLL GLNDLELSAPEWSDDTPPTCPK P Rsphe M G GIVAP PVP A F Y LRP ESLAAAV EQIAAR A V D L L GLGRPQAQAW E Ape1647 M G GIVV P LTLSFY IKP SSVE D L V D F AAGKVL D A L GVKVDVYRR W R G PEEGD .GA .IVP .IPAWY .P.TIEDLVDF .V.RALDQL ..D .P..RW G KP
The multiple alignment was generated by BLOSUM30 using Clustal W. S6803, Synechocystis sp. PCC 6803; S7002, Synechococcus sp. PCC 7002; A7120, Nostoc sp. PCC 7120; Npun, N. punctiforme; Telon, T. elongatus BP-1; S8120, Synechococcus sp. WH8102; PmarMIT9313, P. marinus MIT9313; PmarSS122, P. marinus SS120; Gvio, G. violaceus PCC7421; Rpal, R. palustris; Rsphe, R. sphaeroides; Ecol, E. coli; Ape1647, Aeropyrum pernix K1.
302 A.1.8. An amino acid sequence alignment of UbiE homologs in Synechocystis sp. PCC
6803
10 20 30 40 50 60 Sll1653 M SNSLLTQPTQESVQQIFARIAP------QYDDLN TFL Sll0418 M P E Y L L L P A G L ISLSLAIAAGLYL LTARGYQSSDSVANAYDQWTED G I L E Y YWGDHIHL G Sll0829 MATIFRTWSYQYPWVYALVS------RLATLN --- Sll0487 M L SNSDQHRAVQALYNTYPFPPE------PLLQEPP-- Slr1039 M L ERILEPEVMDTEAEAMDYDAM------DFQAVN --- UbiE MVDKSQETTHFGFQTVAKEQKADMVAHVFHSVAS------KYDVMN D L M MPEYLLLPAGL ML ...... DGILEY . N L
70 80 90 100 110 120 Sll1653 S F G QH---HIWKAMAVK------WSG VS---P G DRLL D VCC G S G DLAFQGAKVVGTRG K Sll0418 HYG DPP V A KDFIQSKIDN V H A M A QW G G LDTLPP G TTV L D VGC G I G G---SSRILAKDYG N Sll0829 -VG GE---ERFHQLPLE------NLAIS---P G QKV L D LCC G G G ----QATVYLAQSG A Sll0487 -PG YN---WRWQWTAAHN -----FCLG RRPANQKVRIL D AGC G T G VGT-EYLVHLNPEAE Slr1039 -LA------FAQRACQ------LG PS----KATV L D AGTG TARIP-ILLAQLRPAWQ UbiE S F G IH---RLWKRFTID------CSG VR---RG QTV L D LAGG T G DLTAKFSRLVGETG K S G PVA . . NVHAMA .G. PG VLD ..CG.G . . . G
130 140 150 160 170 180 Sll1653 V V GLD FCAELLA IAAGKHKSK-YAHLPMQWLQGDA LAL P FSDNEF D GATMGYGL RNVG-- Sll0418 N V TG-ITIS PQQVKRATELTP-P-DVTAKNAVDDA MAL SNPDG S F D VVWSVEAGPHMP-- Sll0829 T V VG-LDAS PKA LGRAKIN-----VPQATYVQGLA EDL P FGEG E F D LVHTSVAL HEMTP A Sll0487 VHAVD ISEGALA VAQTRLQKSG VVCDRVHFHHLSLENL AHLPG Q F D YINSVGVL HHLP-- Slr1039 ITA ID FARS MLA IAKENVIAA-GCETQICLEFVDA KQL P YANG S F D GVIANSLCHHLP-- UbiE V V LAD INES MPKMGREKLRNI-GVIGNVEYVQANA EAL P FPDNTF D CITISFGL RNVT-- .V..D.S A..... G ....A LP..GFD.. .L..PA
190 200 210 220 230 240 Sll1653 NIPQALTELQR V L K P G KKVAILD FHQ------P GN--ALAANFQRWYLA---- Sll0418 DKAVNAKELLR V V K P G G ILVVAD W NQRDDRQVPL NFWEKP VM--RQL LDQWSHPAN---- Sll0829 QLQSIISGVHR V L K P G G IFALVD LHR------P ------SNWLFWPPL---- Sll0487 DPVAGIQAVAEKL A P G G LFHIFVYAEIGRWEIQL MQKAIAILQG KKRGDYQDGVAVG R E I Slr1039 RPLDFLREVKR V L K P H G FLLIRD LIR------P ESP-EKL ADFVTAIGS---- UbiE DKDKALRSMYR V L K P G G RLLVLEFSK------P II--EPL SKAYDAYSF---- .. . RVLKPGG ....D. L P . G L . . . GREI
250 260 270 280 290 300 Sll1653 ------NVVVPMAKQWR------LTEEYAYLQPSLDRFPTGPKQVQFALEVGF--A Sll0418 ------ASIEGNAENLEA------TGLVEGQVTTADWTVPTLPAWLDTIWQGIIRPQ Sll0829 ------AIFMGLFETET------AWQLINTDLGSLLDQAG-FTVVRKHLY--A Sll0487 F A S L P E Y N RLVKREKERWSLENH R D E S FADMYVHPQETDYNIDTLFELIDSAGLEFLGFS Slr1039 ------DYNDQQRKLFADSLH ----ASLTIAEIQDYLQHLGWVGDDGPNLELHLYQ-S UbiE ------HVLPRIGSLVAN------DADSYRYLAESIRMHPDQDTLKAMMQDAGF--E FASLPEYN .. HRDES ......
310 320 330 340 350 360 Sll1653 KAVHYPIA------AGLMGVLVAEK Sll0418 GWLQYGIR------GNIKSVREVPTILL MRL ANG--VGLCRF GMFKAVRKNATQAN Sll0829 GGSLQVIQ------ARANKTVN Sll0487 NPDYWQLDR L L G K A P D L M ERAKDLSEKERYRL IEL LDPE I THYEFF LAKPPIQISDWTDD Slr1039 SDRHWTLE------KKFQSSTIVQR UbiE SVDYYNLT------AGVVALHRGYKF . . RLLGKAPDLM . . . . . L L EI F
370 380 390 400 410 420 Sll1653 Sll0418 Sll0829 Sll0487 Q T L L K A L P N L H P C L T G W P S R S L F D Y N Y Q P L E L T E E E F K F L Q A V E A Q T E I Q A N V E K V L T D L Slr1039 UbiE QTLLKALPNLHPCLTGWPSRSLFDYNYQPLELTEEEFKFLQAVEAQTEIQANVEKVLTDL
430 440 450 460 470 480 Sll1653 Sll0418 Sll0829 Sll0487 G D R S I D L A L V R S L Q T R Q L L T L G N S Slr1039 UbiE GDRSIDLALVRSLQTRQLLTLGNS
The multiple alignment was generated by BLOSUM30 using Clustal W. Sequences with “Sll” and “Slr” prefixes indicate the UbiE homologs of Synechocystis sp. PCC 6803. UbiE indicate the E. coli sequence.
303 A.1.9. An amino acid sequence alignment of Sll1653 homologs in cyanobacteria
10 20 30 40 50 60 Sll1653 MSNSLLTQP-TQESV QQI F ARI A P Q Y D D L N TFL S F G Q H HIW K A M A V K W SG 89-c70244 M SKPNAATEIQGI F NRI A P Q Y D A L N EW IS L G Q H R I W K K M A V K W SG Alr5252 M TN-----KIRAI F DRI A P V Y D Q L N D W L S L G Q H R I W K E M A IKW TG Npun3758 M TN-----EV QSI F NRI A P V Y D Q L N D W L S L G Q H R I W K E M A V K W SA Tery3321 M TDD--SIEV KAI F NKI A P V Y D Q L N D L L S L G I H KVW K Q M A V R W SE Tll2373 M TN------IQALF ERI A P L Y D R L N D Q L S F G L H HVW K Q M A V D W LE Synw1674 M KPG-DPAAV EQLF DSVA SRY D Q L N D L L S L G L H R Q W K RQLQCLLR Pmm0431 M RYK-KTNEV KSI F NNI SCSY D F L N SLL S L G L H KYW K KTLV DLLK Pmt0276 Pro0427 M KPQ-DPIAIKELF DSVSKNY D F L N D LFS L G F H R I W K RQLLTW LK Gll0127 M QGS---QEIQGI F NAI A P V Y D QIN D RM S F G L H R V W K K M A IRW TD UbiE M V D K S Q E T T H FGFQTVAKEQKADMV AHVF HSVA SKY D VM N D LM S F G I H R L W K RFTIDCSG MVDKSQETTH M V IF IAP YD LND LSLG HR .WK MAV W .
70 80 90 100 110 120 Sll1653 VSPG DRLL D V C C G S G D L A FQGA KVV G TRG K V V G L D F CAELL AIA AGKHKSKYAHLP-MQW 89-c70244 VTGG DLAL D V C C G S G D L TLLL A KTV G VYG KTVG I D F ASQQL AIA AQKQNRLCP HLD-I Q W Alr5252 AKPG D T C L D L C C G S G D L A LRL A R R V G STG Q V S G V D F SANLL ETA KQR AQSQYP QPN-I S W Npun3758 AKSG N T A L D L C C G S G D L A LRL A R R V G ATG Y V Y G V D F SCNLL ETA KER SQKQYP QPA-I A W Tery3321 PDYG S T C L D L C C G S G D L A YM L A KHV G NTG H V F G V D F SREQL AIA RNR EPPLLNKISPI H W Tll2373 LPQG A T A L D L C C G T G D L TRLL A R R V G RQG R V V G L D F AAAPL AIA RQR SD----HYPQI E W Synw1674 PAAG E T W L D L C C G T G D L A LEL A R R V RPGG S V L G L D AAAAPL ELA RHR QSRQ--PWLNVVF Pmm0431 PING ENWAD L C C G T G D L SFLIFKKV RPNG S V T G I D SAKEIL NIA KEKSKLI--ENKFI S W Pmt0276 M A RRR AAVE--PWLPLSW Pro0427 PASG EKW ID L C C G T G D L SLPL A R L V RPFG S V I G V D F SRAQINCA HKR SLKE--PWLPI S W Gll0127 CKPG D T V L D L C C G T G D L A FLL A R R V G VAG T V W G V D F AAAQL AQA RRR D-----REGRVRW UbiE VRRG Q T V L D L AGG T G D L TAKFSR L V G ETG K V VLAD INESMPKMGREKLRNIG-VIGNVEY G .T LDLCCGTGDLA LAR .VG G V G .DF L A . R P I W
130 140 150 160 170 180 Sll1653 LQGD A L A L P F S D NEF D G A T M G Y G L R N V GNI P QAL T E L Q R V L K P G KKVAI L D F HQP G-NAL 89-c70244 QEGD A L A L P YPD NHF D G A T M G Y G L R N V T D I P QAL R E L Q R V L K P G KKVAI L D F HRP D-NPG Alr5252 VEANVL D L P F K D NQF D AAT M G Y G L R N V T D I P RSL Q E L H R V L K P NAKA A I L D F HRP N-NQQ Npun3758 VEAD V L N L P F D D NQF D AAT M G Y G L R N V K D I P RSL Q E L H R V L K P G AKA A I L D F HRP S-NPQ Tery3321 VEANA L S L P F PHNYF D CAT M G Y G L R N V SNI P LCL Q E L Y R V L K P NAKA A I L D MHCP S-SST Tll2373 LQGD A L AVP F APQTF Q G I T I G Y G L R N V V D I P QAL R E MFR L L V P G GRA A I L D F SHP Q-TSA Synw1674 QQGD A L STGLPD ASAD G IVM A Y G L R N LAD PVVGL K E MAR L L K P G RLA GVL D F NRLTTAGP Pmm0431 EM QD I L EIDEDLKNYD G ICM S Y G L R N LVNVEEGL KKVFNLL SDTGRA GFL D F NHSKFNSI Pmt0276 LQGD A L DTGLPSHRF D G AVM A Y G L R N LAD PGAGL I E L R R L L R P G ARA GVL D F NRMGEGSL Pro0427 LNED V L KAGLPSSTF D G VVM A Y G L R N LSSPEAGL K E IHR L L K P G ARA GVL D F NHTIEGSK Gll0127 LEAD A L D L P F A D DSF D AVT Q G F G L R N V V D I P ACFAQL R R V L K P G GRA V I L D LHAP A-DAG UbiE VQANA EAL P F P D NTF D CIT ISFG L R N V T D KDKAL RSMYR V L K P G GRLLVL E F SKP IIEPL . .DAL LPF D . FDG .TMGYGLRNV DIP .L EL RVLKPG ..A.ILDF .P .
190 200 210 220 230 240 Sll1653 AANF Q RW Y L ANVV V P M A KQWRL TEE Y A Y LQP S L DRF P T G PKQ VQFA LEVG F AKA V H Y P IA 89-c70244 KQMF Q QW Y L DRFV V P L A ERFQL TPE Y A Y IQP S IERF P T G KTQ EKL A KEAG F RNA I H Y GIV Alr5252 FRTF Q QW Y L DSIV V P L A DRLG VKEE Y A Y ISP S L DRF P I G KEQ VEIA LKVG F TSA T H Y P IA Npun3758 LRAF Q QLY L NSFV V P V A NYLG L KEE Y A Y ISP S L DRF P I G KEQ IEL A RQVG F AVA T H Y P IA Tery3321 IRW F Q KW Y L DTVV V P V A SNLG L TKE Y A Y ISP S L TKF LTG KDQ VAL A YKLG F VNA K H Y P II Tll2373 LEQF Q QW Y L QQW V V P T A RHYG L AAE Y D Y LW P S IQAF P TPPTLCAL IQQAG F ERVKH Y P LL Synw1674 AADF Q RFY L RRLV V P V A SAAG L KEE Y A Y LEES L KRF P D G TAQ ERL A LEAG F AEA R H HTLV Pmm0431 ADLF Q KIY L RFVV V P ISK IFRL SKE Y S Y IEKS IKNF P K G NELMLIA KGVG F KKVEYRTIF Pmt0276 AARF Q RFY L RRLV V TVA AQVG L REHY A Y LEAS L QQF P K G EVQ ERL A RDAG F AAA S H R P LA Pro0427 NSFF Q KFY L RNFV V P I A SKMG L KEE Y A Y LEES L KKF P V G LIQ EQIA INVG F QEA NYKTLA Gll0127 WRAF Q RW Y L EGW V TALGRAQG L EAE Y A Y IAP S L ERF P T G DEQ VRL A QQAG F SQA S H Y P LI UbiE SKAYDAYSFHVLPRIGSLVANDADSY R Y LAES IRM HP DQDTLKAMMQDAG F ESVDYY NLT . FQ ..YL .VVP .A GL .EYAY . PSL .FP G Q LA .GF A HYP ..
250 260 270 280 290 300 Sll1653 A G L M G V L VAEK 89-c70244 GDMM G V L VATK N Alr5252 N G M M G V L IISK Npun3758 N G M M G V L VVSK F Tery3321 G G M M G I L VITK Tll2373 G G L M AITVAQK Synw1674 A G Q M G M L LLKR Pmm0431 FNQM G I L ILEK Pmt0276 A G Q M G A L LLTA Pro0427 G G Q M G A L LLRA Gll0127 G G TVG V L VAQ UbiE A G VVALHRGYK F .G M G .L . . K F
The multiple alignment was generated by BLOSUM30 using Clustal W. Locus tags are used to indicate each organism (see also Table 6.5): Sll1653, Synechocystis sp. PCC 6803; Alr5252, Nostoc sp. PCC 7120; Npun3758, N. punctiforme; Tery3321, T. erythraeum; Tll2373, T. elongatus BP-1; Synw1674, Synechococcus sp. WH8102; Pmm0431, P. marinus MED4; Pmt0276, P. marinus MIT9313; Pro0427, P. marinus SS120; Gll0127, G. violaceus PCC7421. 89-c70244 indicates a tentative locus in Synechococcus sp. PCC 7002.
304 A.1.10. An amino acid sequence alignment of Sll0418 homologs in cyanobacteria
10 20 30 40 50 60 Sll0418 M PEYL LLPAGLISLSLAIAAGLYL L T A R G Y Q S S DSV A NAY D Q W T E D G I L E YYW G D H I H Slr0089 M V YHVRPKHAL FLAFYCYFSLLTMASAT IASADLYEKIKNFY D --DSSG LW E DVW G E H M H 80a16268 MNIL LLAL GISATILAIALISYL L T A R R Y Q S ADTV A DSY D E W T E D G I L E YYW G E H I H All2121 MSWL FSTL VFFLTLLTAGIALYL I T A R R Y Q S S NSV A NSY D Q W T E D G I L E FYW G E H I H Npun3758 M T NEVQSIFNRIA PVY D ------QLNDW LSLGQ Tery4209 MYL YYAL GF ILLLVALG IV IYL I T P R S Y E S S NTV A NSY D D W T Q D G I L E FYW G E H I H Tll1726 M SHLGLILVITVVALVLLGLALYL LFPR K Y E S ARSV A ESY D N W T K D G I L E FYW G E H I H Synw2141 M L AGLL LLTGAAGATALLIW L QRDR R Y H S S DSV A AAY D A W T D D QLL E RLW G D H V H Pmm1505 M SIFL ISSL VIFLTLLFSSLILWRINTR K Y I S S RTV A TAY D S W T Q D KLL E RLW G E H I H Pmt1785 M ILAGTTLTSAVVIWTQRDR R Y K S S ASV A SAY D A W T N D QLL E RLW G E H V H Pro1661 MIAFLL PIVSLLVLSLIFLW L FNDR K Y K S S ESV SSAY D S W T N D RLL E KLW G E H I H Glr3039 MGERTVLNENIRRFY D --ASSG LW E EVW G E H M H UbiE MVDKSQETTHFGFQT VAKEQKADMV A HVFHSVASKYDVMNDLMSFGI MVM L L ...... L.T R .Y SS ..VA .YD W T DG .LE W GEHIH
70 80 90 100 110 120 Sll0418 L G H Y G D P P VAK D - F I Q S K I D N V H AM AQW G G L D T L P P G TTV L D V G C G I G G S S R I L A K D Y G N Slr0089 H G Y Y G PHGTYRID R R Q A QID LIKE LLAW AVPQNSAKPRKIL D L G C G I G G S S LYL A QQHQA 80a16268 L G H Y G N P P RAK N-F LKA K A D F V H E M V R W G G L D Q L P P G TKV L D V G C G I G G S S R I L A R D Y G F All2121 L G H Y G S P P QRK D - F LVA K S D F V H E M V R W G G L D K L P P G TTLL D V G C G I G G S S R I L A R D Y G F Npun3758 HRIWK------EMAV K W SAAK---SG NTAL D LCC G S G DLALRL A R RVG A Tery4209 L G H Y G S P P RRK D - F L Q A K A D F V H E M V K W G G L D K L P R G TTV L D V G C G I G G S S R I L A KEY E F Tll1726 L G H Y G L P P RPK D - F R Q A K V D F V H E M V R W A G L D R L P P G TTV L D V G C G I G G S S R I L A R D Y G F Synw2141 L G H Y G N P P GSVD - F R Q A K EAF V H E L V R W S G L D Q L P R G SRV L D V G C G I G G S A R I L A R D Y G L Pmm1505 L G F Y P-LNKNID - F R E A K VQF V H E L V S W S G L D K L P R G SRIL D V G C G I G G S S R I L A NYY G F Pmt1785 L G Y Y G K P P STRD - F R A A K Q D F V H E L V Q W S G L AQL P R G SRV L D V G C G I G G S A R I L A R D Y N F Pro1661 L G Y Y ENSYKTK D - F R Q A K I D F V H KLAHW S G L STL P K G SRIID I G C G I G G S S R I L A K D Y G F Glr3039 H G H W EVGEADK D -RR V A QVD L V VRLLDW A G ---IDRAESIVD V G C G I G G S S LFL A ERFG A UbiE HRLWKR------FTIDCSG VR---RG QTV L D LAGG T G DLTAKFSR LVG E LGHYG PP KDDFRQAK .DFVHE .V.W.GLD LP .G..VLDVGCGIGGSSRILARDYGF
130 140 150 160 170 180 Sll0418 N--V T G I T I S P Q Q V K R A T E L T P P---DVTAKNAV D D A MAL SNPD G S F D V V W S V E A G P H M P Slr0089 E--V M G ASLS P V Q V E R A G E RARALGLGSTCQF Q V ANA L D L P F ASDS F D W V W S L E S G E H M P 80a16268 D--V T G I T I S P K Q V E R A TQL T P P---GLTAKF A V D D A MNL S F A D G S F D V V W S V E A G P H M P All2121 A--V T G I T I S P Q Q V Q R A Q E L T P Q---ELNAQF L V D D A MAL S F P D N S F D V V W S I E A G P H M P Npun3758 T G Y V Y G VDFS CNLLETA K E RSQKQYPQPAIAWVEAD V L N L P F D D NQF D AATMGYGLRNVK Tery4209 E--V T G V T I S P K Q V Q R A T E L T P Q---GVTAKF Q V D D A L A L S F P D N S F D V V W S I E A G P H M P Tll1726 H--V T G I T I S P E Q V R R A R E L T P A---ELNVRF QLDD A L A L S F P D A S F D V V W S I E A G P H M P Synw2141 D--V L G VSI S P A Q IRR A T E L T P A---GLSCRF E V M D A L N L QLPD RQF D A V W TVE A G P H M P Pmm1505 N--V T G I T I S P A Q V K R A K E L T P Y---ECKCNF K V M D A L D L K F EEGIF D G V W S V E A G A H M N Pmt1785 D--V L G I T I S P A Q V K R A SQL T P E---GMTCQF Q V M D A L D L KLANGS F D A V W S V E A G P H M P Pro1661 D--V V G I T I S SEQ V K R A NQL T P K---ELKCHF EIMNA L N L K F E D G S F D G V W S V E A G P H IL Glr3039 R--V E G I T L S P V Q CKR A A E RAREHHLDGRAHF Q V A D A HRMPF A D GRF D L V W S L E S G E H M A UbiE T G K V VLADI NESMPKMGRE KLRNIGVIGNVEYVQANA EAL P F P D NTF D CITISFGLRNVT GVGITISPQV.RAELTP ...FVDALLFD.SFD.VWS.EAGPHMP
190 200 210 220 230 240 Sll0418 D K AVNA K E L L R V V K P G G I L V V A D W N Q R --D DRQVP L N FW E KPV M R Q L L D Q W S H P ANAS I E Slr0089 N K AQF LQE AW R V L K P G G R L ILA T W CHR P I D PGNGP L TADE RRHLQAIYD VYCLP YVVS LP 80a16268 D K AIF A Q E L L R V L K P G G K L V V A D W N Q R --D DRQIP L N WW E KPV M T Q L L D Q W A H P K F S S I E All2121 D K AIF A K E L M R V L K P G G IM V L A D W N Q R --D DRQKP L N FW E KPV M Q Q L L D Q W S H P A F S S I E Npun3758 D IPRSLQE L H R V L K P G AKAAILD FHRP------SN PQLRAFQQLYL NSFVVP -VANYL Tery4209 D K VKYGSE MMR V L K P G G I L V V A D W N Q R --D DRQKP L N YW E KPV M R Q L L D Q W S H P A F S S I E Tll1726 D K QQF A K E L L R V L K P G G I L V V A D W N Q R --D DRQQP L N FW E RLIM R Q L L D Q W A H P A F A S I E Synw2141 D K QRF A D E L L R V L R P G G C L AAA D W N R R --APKDGAMN STE RW V M R Q L L N Q W A H P E F A S I S Pmm1505 N K TKF A DQML R T L R P G G Y L ALA D W N S R --D LQKQP PSM IE KIILKQ L L E Q W V H P K F I S I N Pmt1785 D K QRYA D E L L R V L R P K G V L A V A D W N R R --D YEDGEMTSLE RW V M R Q L L D Q W A H P E F A S I K Pro1661 N K QLF A D E M L R V L R P G G V L A V A D W N R R --D YAKKEIGFLNSLV LKQ L L N Q W S H P D F ATI Y Glr3039 D K AQF LRE CHR V L R P G G RFVFVTW CCR -----HGAL DARDQKWLGAIYRIYHLP YILS I E UbiE D K DKALRSMYR V L K P G G R L L V LEFSKP------IIEPLSKAYD AYSFHVLPRI G DK FA ELLRVLKPGG L .VADWN RPID PLN E . VM .QLLDQW HP F .SI
250 260 270 280 290 300 Sll0418 G NAENL EATG L V E G Q V T T A D W T VPT L P A W L D T I WQG IIR P QGWLQYG IRG NIK SVR E V P T Slr0089 DYEAIARECG --FG EIKT A D W SVAVAP F W DRVI ESAF-DP RVLWALG QAG -PK IINAALC 80a16268 G F SELL EETG L V QDQV INAD W SQET L P S W LHTI WVG AIR P WGWLQYG LPG FIK SVR E I P T All2121 G F SELL AATG L V E G E V I T A D W T KQT L P S W L D S I WQG IVR P EGLVRFG LSG FIK SLR E V P T Npun3758 G LKEEYAYIS------PSLD R------FP -----IG KEQ-IELAR QVGF Tery4209 G F SEQIAETG L V E G E V A T A D W T QET L P S W FES I WQG IVR P KGLIKFG FSG FIK SLR E V P T Tll1726 G F AEAL AATG L V A G E V M T A D W T QET L P S W L D S I WQG IVR P EGLIRFG LPG LVK SLR E V P T Synw2141 G F RANL EASPHQRG LIST G D W T LAT L P S W F D S I AEG LRR P W AVLGLG PKAVLQGLR E T P T Pmm1505 E F SSIL INNKNSSG Q V ISSNW NSFT N P S W F D S I FEG MRR P NSILSLG PGAIIK SIR E I P T Pmt1785 G F RRNL LHSPFACG T V ESDD W T RSIL P S W N D S I LEG FRR P GAVLGLG PAALVK GFR E I P T Pro1661 G F RNNL SDSIYSAG R V E T D D W T KYT I P S W N D S I IEG IRR P NVFFDLG LGSFFK AIR E V P T Glr3039 SYTQLL GETG --FSGIRT T D W SDRVARFW SLVI DSAL-EP AVLWKVIAQG -PTVIKGALA UbiE SLVANDADSY------RYLAESIRMHP ------DQ------DTLKAMMQDAGF GF L . .GLV G V T .DWT TLPSW DSI G . RP ... .G G .K .RE.PT
310 320 330 340 350 360 Sll0418 I L L M R L A NGVG L C R F G M F KAVRKNATQAN Slr0089 LRL M KWGYERG L V R F G LLTGIKPLV 80a16268 I L L M R L A F GTG L C R F G M F QAVRGEGNPV TKNRTTQQAISRN All2121 L L L M R L A F GTG L C R F G M F RALRADTVRSS A E QTSAIKVAQK Npun3758 AVATHYPIANG MMGVLVVSKF Tery4209 M L L M R LGF GAG L C R F G M F RAVKSNSVPV S T E TATTEVANA Tll1726 F L L M R I A F GM G L C R F G M F RAVRAEIPAV S L E PAPQVNC Synw2141 L L L M HW A F ATG L MQF G V F RLSR Pmm1505 I L L M DW A F KKG L MEF G VYKCRG Pmt1785 I L L M R W A F AHG L MQF G V F RSRD Pro1661 IVL M R W A F HTG L MQF G V F RSRG Glr3039 MQL M R RSYARG L V R F G V F AAQKAEG UbiE ESVDYYNLTAG VVALHRGYKF .LLMR .AF. GL .RFG.F.. . VS E
305 The multiple alignment was generated by BLOSUM30 using Clustal W. Locus tags are used to indicate each organism (see also Table 6.5): Sll0418 and Slr0089, Synechocystis sp. PCC 6803; All2121, Nostoc sp. PCC 7120; Npun3758, N. punctiforme; Tery4209, T. erythraeum; Tll1726, T. elongatus BP-1; Synw2141, Synechococcus sp. WH8102; Pmm1505, P. marinus MED4; Pmt1785, P. marinus MIT9313; Pro1661, P. marinus SS120; Gll3039, G. violaceus PCC7421. 80a16268 indicates a tentative locus in Synechococcus sp. PCC 7002.
306
A.1.11. An amino acid sequence alignment of Sll0829 homologs in cyanobacteria
10 20 30 40 50 60 Sll0829 M A T I F R -TW S Y QYPW V Y ALVS ----RLA T L N V G G E E R F HQL P L ENL AI 89a46731 M A T F L R -TLS Y RYQW L Y D T I S ----RLA A L T V G G E T R F R N L A L QGL TG All3750 M A T I F R -DLS Y RYQW L Y D S I S ----RVA A L T V G G E A R F R K L A L QGL TI Npun0871 M A T I L R -DW S Y RYQW L Y D S I S ----RLA A L S V G G E A R F R Q L A L QAL TI Tery1223 M A T I L R -DW S Y RYQW L Y D G I S ----RLA S L S V G G E T R F R Q L A L EGL II Tll1604 M A T I L R -TW S Y QSPW L Y D T I S ----ALA A IAV G G SDR LHRL A WQDL NL Synw1749 M SSFL R -PLAY RHRW I Y D LVT----AVSSL S V G G VAR L R G L G L EAL GP Pmt1242 M TSPL R -TFAY EHRW F Y D LVT----TISA L S V G G VKR L R S L G L TAL QD Gll1483 MEAPAWNNALA T I L R -DW S FACAPL Y EAI T----SGA A VLAG G E RHF R QAA W QNVSL UbiE M V D KSQETTHFGFQT VAKE QKADMVAHVFHSVAS K Y D VM NDL MSFG IHR LWKRFTIDCSG MVD MATILRE .SY W LYD ISSKYD .AAL.VGGE RFR LAL .L .
70 80 90 100 110 120 Sll0829 SP--GQKV L D L C C G G G QAT VYL AQSGAT---V V G L D A S P --KAL G R A KIN----V P Q A T Y 89a46731 DK--SLKIL D L C C G A G QTT QFL TQYSDD---V T G L D I S P --LAIER A KKN----V P Q A N Y All3750 QA--NTSV L D V C C G S G QAT QLL VKSSQN---V T G L D A S P --LSL Q R A RQN----V P E A N Y Npun0871 HS--DTQV L D L C C G S G QTT QFL VKISQN---V T G L D A S P --KSL Q R A RLN----V P E A S Y Tery1223 EE--NTQIL D L C C G S G QGT NFL AKYSQS---V T G L D A S P --LSINR A KKN----V P S A K Y Tll1604 PR--TAVV L D L C C AHG IVT QAL TQAFDQ---V T G L D AAP --KAIAR A RER----V P Q A T Y Synw1749 HLNPDAAV L D L C C G S G EAAAPWLEAGYR---V T G L D I S P --RAL ALA AQR----HP AM TR Pmt1242 KISKGAPV L D L C C G S G ETAAPW IEAGFA---V T G L D L S P --KAL ALA AER----TP QLQC Gll1483 SA--ASSV L D L C C G P G GAT RYL AATGAR---V T G L D R S E--GSL RHA RAR----V P G A HF UbiE VR-RGQTV L D L AGG T G DLT AKFSRLVGET G K V VLAD INES M PKMGR EKLRN I G V IGNVEY VLDLCCG .G .T L . TGKVTGLD .SPSM L RA . NIGVVP A Y
130 140 150 160 170 180 Sll0829 V QGLA E DLP F GEGEF D L V H T S V A L H E M TPAQ L QSI ISGV H R V L K P G G I F A L V D L H R P S N W 89a46731 V VAPA E K M P LPD QQF D L V H T S A A L H E M TPTQ L SQI FQE V Y R V L K P G G I F TFID L H Q P T N P All3750 V EAFA E K M P F P D NQF D I V H T S A A L H E M EPQQ L REI IQE V Y R V L K P G G V F T L V D F H T P T N P Npun0871 V EAFA E E M P F T D NLF D V V H I S V A L H E M QPQQ L RKI IDE V Y R V L K P G G I F T L V D F H A P T N P Tery1223 V EGFA E D M P F SSNQF D L V H T S A A L H E M NYEQ L RQI IQE V Y R V L K P S G I F TFVD F H S P T N P Tll1604 V QAFA E K M P F A D ATF D L V H T S M A L H E M TAAQ RDAI LAE V W R I L K P G G W F A L I D F H R P QVP Synw1749 V EGLA E DPP LAD GSF AAIQLS V A L H E FPRSDREAVLRSCLR L L Q P G G W LVVVD L H - P AGP Pmt1242 IEGM A E KPP LNTSQF AAIQIS L A L H E FSSEERQQVLKACMR L L Q P G G WLVL I D L H - P AGP Gll1483 V QGRA E A M P F AGASF D L V H T S V A L H E M EPTQ RRTI IRE V L R V L K P G G T F A L I D Y H R P T N P UbiE V QANA E ALP F P D NTF D CITIS FGL RNVTDK--DKALRSMYR V L K P G G RLLVLEFSKP IIE V . AE MPF D FD .VHTS .ALHEM QL I. EV .RVLKPGG F .L.D.H P .NP
190 200 210 220 230 240 Sll0829 LFW P PLAIF MGL F E T E T A W Q L INT D L GSLL DQAG F TVVR-----KHL Y A G G S L Q V I Q A RA 89a46731 FFVP SLYTF MFL F E T E T A W Q L IKT N L AEKL TTTG F EIHK----Q-EQYA G G S L Q M I Q SRK All3750 IFW P GLTVF LLL F E T E T A W Q L L K T D L AGLL TEIG F EAG-----QSIL Y A G G S L Q V I Q A K K Npun0871 ILW P GISLF LLL F E T E T A W Q L L K T D L AGLL TETG F DVS-----KPIL Y A G G S L Q V I Q A K K Tery1223 LFW P GFAMF LW L F E T E T S W KFINT NILELL TTIG F NLVDTKLPLPIL Y A G G S L Q V I Q VQK Tll1604 LLW P GIALF FW L F E T E T A W Q L L Q T D L AEQL RLQG F TVER-----QTYHLG R S L Q VLHGRK Synw1749 W LQLPQQLF CAL F E T D T A TAML EDD L PAQL KQLG F SAVN-----QEL L A G QAL Q R I T A TR Pmt1242 CLKLPQQLF CAL F E T E T A LTML QAD L PKQL REMG YSTIE-----QEL L A G RAL Q R I T A RL Gll1483 LYW P GLALF LW V F E T H T A W E L L A T D L PGLL AECG F STLE-----QQL L A G G S L Q TVQ A R K UbiE PLSKAYDAYSFHVLPRIGSLVANDADSYRYLAESIRMHPDQDTLKAMMQDAGFESVDYYN . W P .. .F LFETETAW LL TDL . L GF .. L AGGSLQ IQA .K
250 260 270 280 290 300 Sll0829 NKTVN 89a46731 P GSEPLKN All3750 Npun0871 Tery1223 Tll1604 P C A ND Synw1749 SAA STS Pmt1242 P Gll1483 P UbiE LTA GVVALH R G Y K F P A HRGYKF
The multiple alignment was generated by BLOSUM30 using Clustal W. Locus tags are used to indicate each organism (see also Table 6.5): Sll0829, Synechocystis sp. PCC 6803; All3750, Nostoc sp. PCC 7120; Npun0871, N. punctiforme; Tery1223, T. erythraeum; Tll1604, T. elongatus BP-1; Synw1749, Synechococcus sp. WH8102; Pmt1242, P. marinus MIT9313; Gll1483, G. violaceus PCC7421. 89a46731 indicates a tentative locus in Synechococcus sp. PCC 7002.
307 A.1.12. An amino acid sequence alignment of Sll0487 homologs in cyanobacteria
10 20 30 40 50 60 Sll0487 M LSN---SDQHRA V QAL Y N T Y P F P P E P L LQE P P P G Y N W R W Q W TAA HNF C All0012 M SDP---QAVSSA V AKL Y D T Y P F P P E P ILD E P P P G Y N W R W N W LAA HSF C Npun5885 M SDS---QTVSAA V AKL Y N T Y P F P P EAL L D E P P P G Y N W R W N W LAA HNF C Tery3855 M KDR---INISSA V EGL Y D T F P F P P D P L T D E A P P G Y N W R W S W PVA YSF C Tlr1948 M TSA---AEITAA V RQL Y N T Y P F P P E P L L D E P P P G Y N W R W S W PAA YSF C Synw0487 M AEPGNRDAATPVV SAFY DRFP F P G D P L Q D G P P P G Y N W R W CHQSVLAAV Pmt1474 MSSVAQP--CDEATPVV SAFY DRFP Y P G D P L Q D G P P P G Y N W R W SVDMA YAVC Glr1180 MDITRA V QSL Y ERY P F P P D P L S D K P P P G W N W R W SYSFA HSF C UbiE M V D K S Q E T THFGFQTVAKEQKADMV AHVFHSVASKYD VM ND LM SFG IHRLW KRFTID--C MVDKSQET M .. AV LY .TYPFPPDPL DEPPPGYNWRW W .A FC
70 80 90 100 110 120 Sll0487 L G RRP ----ANQKVR I L D A G C G T G V G T E Y L V H L N P E-A E V HAVD I S E G A L A V A QTR L Q K S All0012 T G RKP ----AKQDIR I L D A G C G S G V G T E Y L V H L N P Q-A Q V VGI D L S A G T L A V A KVR C Q R S Npun5885 T G QKP ----QKQDIR I L D A G C G T G V S T E Y L V H L N P Q-A S V VGI D L S T G A L D V A K E R C Q R S Tery3855 T G QKP ----KKQDIR I L D A G C G T G S S T D Y L V H L N P Q-A S V VGI D L S S G A L R V A K E R C S R S Tlr1948 T G RYP ----SQLDVAI L D A G C G T G V G T E Y L A H L N P Q-A KITALD L S E A A L A I A C E R C R R S Synw0487 H G AVP ---A GTVRPR I L D A G C G T G V S T D Y L C H L N P G-A E V LGI D I S E G A L A V A R E R L Q R S Pmt1474 T G SVVR K C A GSEPLKI L D A G C G T G V S T D Y L A H L N P G-A EILAVD I S L G A L E V A R E R L Q R S Glr1180 R G WRP ----EPRPIR I L D A G C G T G V S T E Y L AVQN P E-A Q V TAI D I S E ASL A L A KRR C AGH UbiE S G VRR------GQTVL D LAGG T G DLT AKFSRL VGET GKV VLAD I N E SM PKM GRE KLRNI TG .PRKCA .RILDAGCGTGVSTEYL .HLNP TA V ..IDISEGALAVA .ERCQRS
130 140 150 160 170 180 Sll0487 G VVCD--RV H F H H L S L ENLAHL P G Q F D Y I N S V G V L H H L P D P VAG I Q A V A E K L A P G G L F H I All0012 G A N----RV E F H H L S L Y D VEQL P G E F D L I N C V G V L H H L P D P IRG I Q A L A K K L A P G G L M H I Npun5885 G A N----RV E F H H L S L F D VEQL P G E F D L I N C V G V L H H TSD P IRG I Q A L A Q K L A P G G L M H I Tery3855 G A S----RV D F H H L S IYD VGRL E G E F KFI N C V G V L H H L P D P IRG I Q N L A L K L A P G G IM H I Tlr1948 G A T----NV Q F H H L S L EEVAQL GQTF QM I N C V G V L H H L PEP QRG I Q A L A DVL A P G G IVH I Synw0487 G A AAQ V SQLRQEQRS L L D LESE-G P F D Y I N S V G V L H H L DQP ESG LRSL A GRL A P D G L L H L Pmt1474 G GNDQ -AHV RIENQS L LELEDE-G Q F D Y I N S V G A L H H L RQP EAG LKA L A SRL K P G A L L H L Glr1180 ANV------QF AQMS L T D AGDL E G Q F D L I N C V G V L H H L A D P KAG LAA L A A K L A P G G L L H I UbiE G VIG---NV EYVQANAEALPFPDNTF D C I TISFGL RNVTD KDKALRSMYRVL K P G G RLLV GA QV .V FHHLSL D . L G FD INCVGVLHHL DP .GIQALA KLAPGGL .HI
190 200 210 220 230 240 Sll0487 F V Y A E I G R W E I Q L M Q K A I A I L Q G KKRG D Y Q D G V AVG R EIF A S L P E Y N R L V K R E K E R W SLE All0012 F V Y G E L G R R E I Q L M Q K A I A L L Q NEKQG D Y R D G V QVG R KIF A S L P E N N R L V K R E K E R W AM E Npun5885 F V Y G E L G R W E I Q L M Q K A I A L L Q G DKKG D Y R D G V QVG R QIF A S L P E N N R I V K Y E KQR W SLE Tery3855 F V Y A E L G R W E I R L M Q Q A I A I L Q G NERG N Y K D G V KIG R Q L F A S L P E N N R I V K Y E K E R W SW E Tlr1948 F V Y G E Y G R W E I K L M Q Q A I A L L Q G SDRHNY REG V AIG R Q L F A A L P E N N R L K K R E Q E R W SLE Synw0487 F L Y ADAG R W E I HRTQ Q A LTL L D---VG TGREG LRLG R E L L A S L P E G N R L ARHHCE R W AVD Pmt1474 F L Y A E A G R W E I HRIQ RFLSSL G---VG TDEQG LRLAR Q L F EIL P E H N R L RLNHEQR W AID Glr1180 F V Y G E L G R W E I R L M Q E A I R L L R-DGRDPLAD G LKVG R A L F EAL P E D N R L KSADR-R W VLE UbiE LEFSKP---II EPLSKA YDAYS------F HVL P RIGSL V ANDADSYRYL FVY .E.GRWEI.LMQ .AIALLQG .G Y .DGV .GR LFASLPE NRLVK E .ERW .E
250 260 270 280 290 300 Sll0487 N HRD E S F A D M Y V H P Q E TDY N I D T L F E L I DSAG L E F L G F S N P DYW Q L D R L L G K A P D L M E R A All0012 N QRD E C F A D M Y V H P Q E VDY N I D T L F E L I D A S G L E F I G F S N P SFW N L D R L L G K A P E L I E R S Npun5885 N HKD E H F A D M Y V H P Q E IDY N I E T L F E L I D A S G L E F I G F S N P SFW D L E R L L S K A P E L V E R A Tery3855 N KKD E C F A D M Y V H P Q E IHY N I D T L F E L I E A S G L E F L G F S N WEYW D L G R L L G K S P E L I E R A Tlr1948 N QRD E C F A D M Y V H P Q E IDY T I ASL F E L I R A S G L E F I G F S N P QVW Q L E R L L G SQP E L LQR A Synw0487 CAAD ANF A D M Y L H P Q E TSY DLQRL F AFI E A ADL H F A G F S N AEVW DPAR L L NG--E L L E R A Pmt1474 CVAD VNF A D M Y L H P Q E TSY N LERL F AFVASADL E F V G F S N P KVW DPVR L L QG--E L L E M A Glr1180 N HHD TCF V D M Y V QVQ E IRY T I P T L F D L I ASS G L G F L G F S N P GFW R L E R L V G K A P W L L E R A UbiE AES------IRMH P DQ-----DT L KAMMQDAG F E SVDYYN ------L TAG------V N .DE FADMYVHPQE . YNI.TLFELI.ASGLEF .GFSNP .W L .RLLGKAPEL .ERA
310 320 330 340 350 360 Sll0487 KDL SEKERY R L I E L L D P E-ITH Y E F F L AKP P IQ ISD W T D D QTL L K A L P N L H P C LTG W P S R All0012 EKL SDRQ R Y R L I E L L D P E-VTH Y E F F L GRP P IKKAD W SND NAL L Q A I P E L N P C IDG F P S Q Npun5885 KEL SDRQ L Y R L I E L L D P E-VTH Y E F F L GRP P LVKTD W S D D KAL L A A I P E L N P C IEG F P S Q Tery3855 IDL NDQERY R L I E L L D P Q-ISH Y E F F L GRP P LQKVD W SHD ELL K A A I P E L S P C IQG W P S E Tlr1948 QQL DPIQ Q Y Q L I E C L D P E A MTH F E F F L AKP P LPRHD W AAD REL L A A I P WRHP C IDG W P GA Synw0487 QAL PQRQ QW L L V E Q L D P N-ISH F E F F L SSQP VQPASW S-D AAL Q A A KGLRQP C LW G E P -D Pmt1474 LAL PQRQ QW Q L I E Q L D P D-ISH F E F F L SKGP QASLGW T D D QEL L A A TGKL SIC LW G W P GQ Glr1180 ATL SDKERFR L I E L L D P V-VAH Y E F F L GAP P LERLELDD D -RL D A A L P VRSP YLYPW P ER UbiE VAL HRG------YKF L ..Q YRLIELLDP .A..HYEFFL ..PP . ..DW .DD LLAA .P L PC . GWPS
370 380 390 400 410 420 Sll0487 SLF D YNY QPLEL TEEE FKF L QAVEAQTE--IQANV EKVL TDLGD RSIDLALV R S L QTR Q L All0012 CIF NYDY QIVNL S ALE F E F L QKCDG------NST V AEIL VSVQ---LSLDEV R S L IQQQ L Npun5885 CFF NYNY QIVNL S EPE F E F MQKCDA------NST V AEIL ADVQ---LGLDEV R K L LQQQ L Tery3855 NIF D GDY RLVNL S RPE Y E F L EACTQGVSN K SQST V GEIL TDVP---VGVETV R S L QSR L L Tlr1948 CVF NCHY EVIHL NQAE Q E F L NAASG------KAT V AELL AQQPD --FNLAEV R S L LAR H L Synw0487 PILD RNMQPLQL S DAE RQLL RRVDEQ-----PNT PLGAMAE------PAVIR D L AAR Q L Pmt1474 SLLD FEMAPLEL S VEQVE L L KAIEAT-----PDT ALGCL PLGW D SKRISSLAR D L QRR Q V Glr1180 QVF D HEY ELVEL S EAE VAF L QRCNG------RQT LGQLRGDIAQ---ARIRELLEHFLLQ UbiE .FD Y .. LS E EFL . .. NK TV . .L... D . VR L RQL
430 440 450 460 470 480 Sll0487 L T L GNS All0012 VM L I P GAKGYAS Npun5885 I L L A P A Tery3855 I L L S P NSGYH Tlr1948 L L L GVKE Synw0487 L L L KA Pmt1474 L L L S P GNVAST Glr1180 L APPP GLLSSKMR V UbiE LLL P RV
The multiple alignment was generated by BLOSUM30 using Clustal W. Locus tags are used to indicate each organism (see also Table 6.5): Sll0487, Synechocystis sp. PCC 6803; All0012, Nostoc sp. PCC 7120; Npun5885, N. punctiforme; Tery3855, T. erythraeum; Tlr1948, T. elongatus BP-1; Synw0487, Synechococcus sp. WH8102; P. marinus MIT1474; Glr1180, G. violaceus PCC7421.
308 A.1.13. An amino acid sequence alignment of Slr1039 homologs in cyanobacteria
10 20 30 40 50 60 Slr1039 M LER ILE P E ----VM DTEAEA MDY D A M D F QAV NL--AFAQRACQLGPSK------91c179267 M ALTR ILE P E ----VM DDPAEA I A Y D A M D F RAV NQ--DFADLVVTTYSQET------All3016 MHR QLE P E ----VM DSWEEA SEY D A M D F TEV NN--AFAEEAVACGLSEH------Tlr1953 M PLPR ILE P E ----VM ETPETA A A Y D A M D F TAV NT--AFCDDLAAVLPRVDQP------Synw1148 MQR LPE P E ----LM VDPAQVLA Y A A A D F SGGDQ--RTLDRIEALLSSASGRAA----- Pmm0662 MER TVE P E ----LM EREDQVDSY AKAD F SEGEN--NLISQINHYLIKNNIYLS----- Pmt0792 MPE P E ----LM DDPLQA R A Y AEAD F SCGDE--ALIQRLQEYLISLCKIPS----- Pro0816 MQR VTE P E ----LM LAPDQVQA Y AEAD F SSSEA--SMLSEVDLLIRDRGMHID----- Glr3970 M LER VLE P E ----VM DSQAEA E A Y D A M D HGEV NA--RFVADVLALGPADG------UbiE M VDKSQE TTH F G F QTVAKEQKADMVA HVF HSV ASK Y DVMNDLMSFGIHRLWKRFT I D C S MM R ..EPEHFGF .M. A AY A DF . V KY . . . TIDCS
70 80 90 100 110 120 Slr1039 -----ATVL D A G T G TARI PILL AQLRP A-WQITAID FARSM L AIA KENVI---AAG CETQ 91c179267 -----AFVL D L G T G TAQI PILL GQQRP Q-WQIKG T D LAQSM L ALGQKNVV---AAG LTAQ All3016 -----GLVL D A G T G TARI PVLICQKRP Q-WQLVAID MAENM L QIA TQHVQ---QSG LQEH Tlr1953 -----LKVL D V G T G N G R I LQLL HQRYP H-WQLTG I D LSPAM L AIA RHHS------PQ Synw1148 --SDPAVIL D L G C G P G N I SLPL AKRFP E-SQVIG V D GSRAM L QVA RDR-----ANQQGLS Pmm0662 --EK-ELIVD L G C G P G N I SEKL STKW P N-ANVIG I D GSKEM IR IA ELNKKNS L NRSRLKN Pmt0792 --PG-SM IVD L G C G P G N I CERL QRLW P E-VMVLG I D GAQAM L DHA LQRQKA--MAAELKR Pro0816 --RK-KLIVD L G C G P G N I TQRL ADQW P E-AKVLG L D DSPEM L MYA RKKQKE--KISFLER Glr3970 ------PW L D L G T G PAHLPVLL ASERP D-IRITAVD LAAPM L AIA RRRVE---AAG LAGR UbiE G V RRGQTVL D L AGG T G DLTAKFSRLVGET GKVVLAD INESM PKMGREKLR---NIG VIGN GV .LDLG G G I . L P T ...G.D. ML .A. . SL .G .
130 140 150 160 170 180 Slr1039 I CLEFVD A K Q L PYANGSF DGVIA----N S L C H H L PRP LDFLREVKR VLKP H G FLLIR D L I 91c179267 I ELVYAD A K N L PWPDQSF DVIIS ----N S L I H H L P D P LPCFQEMRR LLKP Q G NIIVR D L F All3016 I RLELVD A K R L PYEDGIF DLVVS ----N S L V H H L P D P LPFFAEIKR VCKP Q G GIF I R D L L Tlr1953 LNFIEGD A K T L PFAAASF DVVIS ----N S L V H H L A D P QPALGEMLR VLRP Q G TLF I R D L C Synw1148 I DLRCSTLQDL ALEPVDLIVS------N S L L H H L HEP GLLWGLTRELAAP GCRVLHR D L R Pmm0662 LRYICAD I K S L KSSDISF EKNIS L L V S N S L I H H ITYLDDFFNCIKR LSSDLTINF HKD L K Pmt0792 LTYRCSNLSSL VNQCVDLKCSAAL V V S N S L L H H L H D P NLLWQVTKYLAAP GAFVF H R D L R Pro0816 I TYKKINISQIANGDFPF RECADL V V S N S L I H H IHD P LIFIKALKNISKHGAIHF H R D L R Glr3970 I TLVHGD A K APALGAARF ACVFS ----N S L V H H L PQP APFWQACAR LLAP G G VLF V R D L A UbiE VEYVQANA EAL PFPDNTF DCITI----SFGLRNVTD KDKALRSMYR VLKP G G RLLVLEFS I .DAK L . F. ..SLVVSNSL.HHL DP. . .R...P.G .F.RDL
190 200 210 220 230 240 Slr1039 R P ESPEKLADFVTAIGSDYNDQ-QRKLF ADS L H A SLT IAE IQDYLQHLGWVGDDGP N L EL 91c179267 R P DSTAAIDQIVAAAEGPGFDARQRQLF WDS L H A A F T VPE IEAIATEAGL TK------A All3016 R P EDEATMNALVASIGNEYDDY-QKKLF RDS L H A A L T LDE VNQLIITAGL TG------V Tlr1953 R P QTLAELRALVEQYAGQDTPQ-QQKLF ADS L H A A L T LRE MRHLLRTLPQSPQ-----RW Synw1148 R P AA-LAEVHRLQQLHCFDAPAVLIQDF CAS L V A A F T PEE VQQQLALAEL DG------L Pmm0662 R P ID-EQSALYLKEKCGEKYNEILTNDYYAS L K A SYT SKE LKDFIFENKL SS------L Pmt0792 R P LS-TKHAIALQQKHMQEAPSILIRDYLAS L H A A F T LEE VQSQLVHAGL DH------L Pro0816 R P SS-LEEALAIKQKHLPTAPSVMEKDF IAS L K A A Y T SRE ISQYLKDSEL NS------F Glr3970 R P DSLSRRDALVEQYAAGCDDD-QRRLF AES L Q A A LIPAE VASQARAAGL TG------A UbiE K P II-EPLSKAYDAYSFHVLPRIGSLVANDADSYRYLAESIRMHPDQDTL KAMMQ----D RP ...... F .SL.AA .T. E . . . L PNL .
250 260 270 280 290 300 Slr1039 HLYQSSD R HWTLEKKFQSSTIVQR 91c179267 Q V YQSSER HWTLIFPQPN All3016 EIYQSSD R HWTAKRSWTN Tlr1953 QAILTSD R HWTVVAQR Synw1148 T V EM EDD R YLVVSGLVD Pmm0662 E V FEEGD QYLVIYGKV Pmt0792 H V IEVED R YLDVFGTI Pro0816 K V VEVDD R YLDVFGVII Glr3970 Q V AM SSD R HWTLIRRGAV UbiE AGFESVD YYNLTAGVVALHRGYKF V.DR...
The multiple alignment was generated by BLOSUM30 using Clustal W. Locus tags are used to indicate each organism (see also Table 6.5): Slr1039, Syechocystis sp. PCC 6803; All3016, Nostoc sp. PCC 7120; Tlr1953, T. elongatus BP-1; Synw1148, Synechococcus sp. WH8102; Pmm0662, P. marinus MED4; Pmt0792, P. marinus MIT9313; Pro0816, P. marinus SS120; Glr3970, G. violaceus PCC7421. 91a179267 indicates a tentative locus in Synechococcus sp. PCC 7002.
309 A.2.1. Polypeptide composition of isolated PS I complexes isolated from the wild-type, menG, menF, rubA, rubA menG, and rubA menB mutants of Synechococcus sp. PCC
7002
M and T indicate monomeric and trimeric PS I complexes isolated from the respective wild type (WT) and mutant strains. SDS-PAGE gel containing 16% acrylamide and 6 M urea were used to resolve the polypeptides (electrophoresis at 80 mV for 12 h at room temperature). Small blue, yellow, and red arrows indicate polypeptide bands that are not detectable in the wild-type PS I trimeric forms. The polypeptide bands were visualized after silver staining.
310 Presence of all the polypeptides was confirmed in the PS I complexes isolated
from menF and menG mutants. Immunoblotting analysis showed that PsaA and PsaB
polypeptides were present in the PS I complexes isolated from the rubA menB and rubA menB double mutants, while PsaC, PsaD, and PsaE were absent. The absence of the latter three polypeptides is expected as a result of the inactivation of the rubA gene, as previously reported [Shen G, Antonkine ML, van der Est A, Vassiliev IR, Brettel K, Bittl
R, Zech SG, Zhao J, Stehlik D, Bryant DA, Golbeck JH (2002) J Biol Chem 277: 20355-
20366]. In the rubA menB mutant, no PsaL was detectable when the isolated PSI complexes were analyzed, while it was detectable when thylakoid membranes was analyzed. This suggests that PsaL was lost during the preparation of PS I complexes. The absence of PsaL is also visible in the SDS-PAGE gel (a band immediately below PsaF is missing). It has been reported that PsaL is required for the formation of trimeric forms of
PS I complexes [Chitnis VP and Chitnis PR (1993) FEBS Lett 336: 330-334]. Indeed, after the first sucrose-density gradient centrifugation, most of the chlorophyll (an thus, PS
I complexes) was collected in the PS I monomeric fraction from the rubA menB mutant.
311 A.2.2. Illustration of genetically engineered PS I in cyanobacteria.
Strain names are shown below the respective PS I complexes. WT, wild-type strains of Synechocystis sp. PCC 6803 (containing PhyQ) and Synechococcus sp. PCC 7002 (containing MQ-4); DMQ, demethylmenaquinone-4; DphQ, demethylphylloquinone; DMPBQ, 2,3-dimethyl-6-phytyl-1,4- benzoquinone; PQ, plastoquinone-9; AQ, 9,10-anthraquinone. The PS I core is shown in green, while the PsaC subunit is shown in blue. P700 and A0 are indicated by purple circles, A1 is indicated by red diamonds, and [4Fe-4S] (FX, FA, FB) clusters are indicated by yellow squares. All the PS I complexes shown here have been generated, except for the DMPBQ-containing complexes, which are expected to be produced in a menF cyclase (in α-tocopherol biosynthetic pathway) double mutant that is yet to be constructed. For constructions of the mutants, see Chapter 2 and 3. The rubA menG mutant in Synechococcus sp. PCC 7002 was generated by transforming the rubA mutant with the menG::accC1 construct (see the construction of the rubA menB double mutant described in Chapter 2). The PS I complex devoid of quinone and [4Fe-4S] clusters has been recently generated by treating the PS I complexes isolated from the rubA menB mutant with AQ followed by extensive washing (Y. Sakuragi, B. Zybailov, G.
Shen, R. Balasubramanian, B.A. Diner, I. Karygina, Y. Pushkar, D. Stehlik, D.A. Bryant, and J.H. Golbeck, unpublished).
CURRICULUM VITAE
YUMIKO SAKURAGI
Date of Birth: April 2, 1973 Nationality: Japanese Place of Birth: Tokyo, Japan
EDUCATION
1996 B. S., Biology, Department of Biology, Tokyo Metropolitan University, Japan 1999 M. S., Biology, Department of Biology, Tokyo Metropolitan University, Japan 2004 Ph.D. in Biochemistry, Microbiology, and Molecular Biology, Department of Biochemistry and Molecular Biology, The Pennsylvania State University, USA
HONORS
1996 Danish Government Scholarship, Denmark 1999 Braddock Graduate Fellowship, The Pennsylvania State University 2000 First place in the competition for poster presentation, American Association for Microbiology, Allegheny Branch Meeting, Pennsylvania, USA 2001 Invited speaker at the 7th Cyanobacterial Workshop, California, USA 2002 Center for Biomolecular Structure & Function Travel Award, The Pennsylvania State University 2003 Second place in the competition for oral presentation in Environmental Engineering, Ecology and Soil Science, 6th Annual Environmental Chemistry Symposium, The Pennsylvania State University 2003 Invited speaker at the 11th International Symposium on Phototrophic Prokaryotes, Tokyo, Japan 2003 Alumni Association Dissertation Award, The Pennsylvania State University
PROFESSIONAL MEMBERSHIPS
2000 American Society of Microbiology 2002- American Association for the Advancement of Science 2004