An Investigation Into Carbon Flow Through the Metabolic Networks of Rhodobacter Sphaeroides

Home , Methylisocitrate lyase, Protein family, Reverse transcriptase, RNA polymerase, T7 RNA polymerase, Transposase

An Investigation into Carbon Flow through the Metabolic Networks of Rhodobacter sphaeroides

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

Michael Steven Carter

Graduate Program in Microbiology

The Ohio State University

2014

Dissertation Committee:

Dr. Birgit Alber, Advisor

Dr. F. Robert Tabita

Dr. Joseph Krzycki

Dr. Tina Henkin

Michael Steven Carter

2014

Abstract

Predicting carbon flow within an organism requires a complete knowledge of the pathways that compose the organism’s metabolic networks. Even in pathways that have been biochemically characterized, an incomplete knowledge of the regulation of the responsible genes and respective enzymes disallows prediction of carbon flow through the entire network. Therefore, in order to predict the flow of carbon molecules through metabolic networks, the corresponding enzymes must be characterized and their regulation must be elucidated.

Toward a better understanding of metabolic carbon flow, the following work investigated core metabolism in the purple nonsulfur bacterium Rhodobacter sphaeroides

2.4.1. As a member of the purple nonsulfur bacteria, R. sphaeroides is especially metabolically versatile, an indication of the diversity of pathways within its metabolic networks. By investigating the interplay of the pathways that compose the networks, this work intends to offer insights into the strategies employed by R. sphaeroides as it modulates carbon flow through its metabolic networks.

Chapter 2 begins the investigation by examining which enzymes participate in carbon flow through the C4/C3 node. The C4/C3 node is responsible for converting metabolic intermediates between the citric acid cycle (C4) and glycolysis/gluconeogenesis (C3). The growth of a series of strains with mutations in

genes of key enzymes of the node was examined across various carbon sources.

Specifically, strains with mutations in two different genes that encode malic enzymes – responsible for catalyzing the oxidative decarboxylation of malate to pyruvate – were compromised in their ability to grow with some substrates that require C4 to C3 conversion, such as succinate and (S)-malate, but their growth was unimpaired during growth with acetate which is assimilated via succinate and malate. As an additional apparent contradiction, a pyruvate phosphate dikinase mutant was unable to grow on acetate, suggesting that gluconeogenesis occurs exclusively from pyruvate during acetate growth. While the mode of C4 to C3 conversion in R. sphaeroides remains an open question, growth results with a pyruvate carboxylase mutant indicated that pyruvate carboxylase is primarily responsible for C3 to C4 conversion on substrates that are predicted to be assimilated through C3 intermediates.

Chapters 3 and 4 examine the role of carbon flow through intermediary pathways of short chain acyl-CoA assimilation in R. sphaeroides. R. sphaeroides employs the ethylmalonyl-CoA pathway for acetyl-CoA assimilation. The enzymes of the pathway are encoded by genes that are dispersed throughout the genome, and no previous information exists regarding strategies for their regulation. The pathway’s first committed reaction is catalyzed by crotonyl-CoA carboxylase/reductase (Ccr) and includes reactions that are shared with polyhydroxybutyrate biosynthesis. Transcript levels of ccr were observed to be 30-fold higher in extracts of acetate-grown cells than succinate-grown cells, and ccr promoter-reporter fusions were likewise regulated, demonstrating a transcriptional regulation of ccr expression. Mutating the gene that

iii

encodes PhaR, a transcriptional regulator of polyhydroxybutyrate synthesis, did not affect the observed regulation of Ccr activity.

Regulation of propionyl-CoA assimilation was also characterized here. PccR was identified as a transcriptional activator and repressor of pccB, the gene for the β-subunit of propionyl-CoA carboxylase of the methylmalonyl-CoA pathway for propionyl-CoA assimilation. Transcript levels of pccB in wild type R. sphaeroides were 11-fold higher

̅ during propionate/HCO3 growth compared to levels during succinate growth. When pccR was mutated, pccB transcript levels and propionyl-CoA carboxylase activities were equivalent in succinate- and acetate-grown cells but higher than levels in wild type cells grown with succinate. Mutation analyses with pccB promoter-reporter fusions revealed the PccR operator site within the pccB promoter as two proximal TTTGCAAA motifs.

Despite the presence of a single truncated palindrome (TTGCAA) upstream of ccr and the role of PccR in regulating the methylmalonyl-CoA pathway, a component of the ethylmalonyl-CoA pathway, ccr expression was unaffected by mutation of pccR.

Based on homology to PccR, a family of regulators of assimilation of short chain fatty acyl-CoA molecules was identified (the ScfR family). The relative sequence similarity among the proteins further classifies them into four different classes – RamB,

MccR, PccR, and IbcR. In nearly every case, genes for members of each class clustered with genes of the glyoxylate bypass (RamB), the methylcitrate cycle (MccR), the methylmalonyl-CoA pathway (PccR), or genes for the assimilation of isobutyryl-CoA

(IbcR). Three hundred twenty-seven proteins were identified, and a comparison of the

sequence immediately preceding their genes identified putative operator sequences for some of the classes.

The sum of the evidence presented herein continues the characterization of modes by which carbon flows through metabolic networks. The work in Chapter 2 highlights the obscurity that remains regarding the control of carbon flow through the C4/C3 node despite the biochemical characterization of its enzymes. Chapter 3 similarly seeks to initiate an intense study of the regulation of a single control point in the cell’s assimilation of acetyl-CoA. Chapter 4 turns the perspective outward as it identifies a source of regulation of propionyl-CoA and extrapolates the use of the regulatory strategy throughout many bacteria. Together, the sum of the contribution of the chapters serves to clarify possible schemes for metabolic network control while emphasizing the necessity for further clarification.

Dedication

Dedicated to my mom Ann Pendley, my wife Stacia Kock, and my best friend Einstein.

The unyielding determination modelled by my mom was a constant source of motivation for the completion of the work presented in and the construction of this document. All of which was performed through the sanity provided by the smiles, laughs, and love from

Stacia and Einstein.

Acknowledgments

My gratitude goes to Dr. Birgit Alber for the time that she committed to my training. The critical analysis that she provided through her unmatched attention to detail often allowed me to refine the precision of my reasoning and presentation of data.

For their advice, I also thank my dissertation committee – Dr. F. R. Tabita, Dr.

Joseph Krzycki, and Dr. Tina Henkin. Many of the experiments presented here were accomplished almost exclusively through the generosity and expertise of Dr. Tabita and the members of his laboratory. Specifically, I would like to thank Dr. Sriram Satagopan and Dr. Rick Laguna for advice on experimental and professional details. I am grateful for Dr. Krzycki’s ability to simultaneously explain scientific concepts while challenging me to fully incorporate their tenets. I also appreciate Dr. Henkin’s thoughtful professional advice, regular communication about career opportunities, and rigorous examination of my scientific assertions. As an unofficial member of my committee, Dr.

Charles Daniels often made himself available to analyze and critique my conclusions based on my data.

Within my laboratory, I am grateful to have worked with Dr. Marie Asao whose organizational techniques were a true model to the success that accompanies an organized mind. Also, I am glad to have worked with Dr. Kathrin Schneider who provided friendship and scientific conversation in my early graduate career. More currently, I

vii

appreciate the friendship offered by Steven Carlson. He often provided levity that brightened a sunless laboratory and an unfaltering but tolerant will that challenged my scientific understanding. I also appreciate the personal support of Dr. Lauren Branditz and Kelsey Baron who were undergraduate researchers in the laboratory at various times and were great friends to me. Much of my work builds upon the careful work that they completed during their time in the laboratory.

Finally, I thank Dr. Jessica Spears and members of the Alfonzo laboratory. Dr.

Spears provided an unparalleled combination of friendship and scientific discourse that was at the core of the growth that I experienced throughout graduate school. The other members of the Alfonzo laboratory, especially Dr. Juan Alfonzo and Dr. Mary Anne

Rubio, offered unprecedented inclusiveness and joviality to improve the scientific lifestyle.

viii

Vita

2007...... B.S. Microbiology, B.A. Biochemistry

Indiana University

Bloomington, IN

2007 to present ...... Graduate Teaching and Research Associate

Department of Microbiology

The Ohio State University

Columbus, OH

Publications

Schneider, K., M. Asao, M. S. Carter, B. E. Alber. 2012 Rhodobacter sphaeroides uses a reductive route via propionyl coenzyme A to assimilate 3-hydroxypropionate. J. Bact.

194(2): 225.

Fields of Study

Major Field: Microbiology

Table of Contents

Abstract ...... ii

Dedication ...... vi

Acknowledgments...... vii

Vita ...... ix

Table of Contents ...... x

List of Tables ...... xvii

List of Figures ...... xix

List of Abbreviations ...... xxii

Chapter 1: Introduction ...... 1

1.1 Introduction to Rhodobacter sphaeroides physiology ...... 1

1.2 Core Metabolism ...... 3

1.2.1 Precursor metabolites define core metabolism ...... 3

1.2.2 The C4/C3 node controls carbon flow through core metabolism ...... 4

1.3 Intermediary Metabolism ...... 7

1.3.1 Defining intermediary metabolism ...... 7

1.3.2 Acetyl-CoA assimilation requires intermediary metabolism ...... 7

1.3.3 Propionyl-CoA assimilation requires intermediary metabolism...... 13

Chapter 2: The C4/C3 Node of R. sphaeroides Core Metabolism ...... 18

2.1 Introduction ...... 18

2.2 Materials and Methods ...... 22

2.2.1 Bacterial strains and growth conditions...... 22

2.2.2 Plasmid transfer by conjugation...... 23

2.2.3 Isolation of RsΔmaeB1MC63...... 24

2.2.4 Bioinformatic search for oxaloacetate decarboxylase enzymes in R. sphaeroides 2.4.1 ...... 25

2.2.4 Random transposon mutagenesis ...... 28

2.3 Results ...... 30

2.3.1 The maeB1(rsp_1593) and maeB2 (rsp_1217) genes are required for optimal growth on (S)-malate and succinate but not acetate...... 31

2.3.2 The ppdK gene (rsp_1859) is required for optimal growth with succinate and acetate...... 33

2.3.3 The pycA gene (rsp_2090) is required for optimal growth with (R/S)-lactate. 34

2.4 Discussion ...... 35

2.4.1 Many reactions contribute to the generation of C3 intermediates during succinate and (S)-malate growth...... 35

2.4.2. Unimpaired acetate growth for strains in which either malic enzyme is mutated remains unexplained...... 37

2.4.3. Pyruvate carboxylase is responsible for C3 to C4 conversion in R. sphaeroides ...... 38

2.4.4. Suggested future directions ...... 39

Chapter 3: Control of Carbon Flow through the Ethylmalonyl-CoA Pathway ...... 42

3.1 Introduction ...... 42

3.2 Material and Methods...... 46

3.2.1 Bacterial strains and growth conditions...... 46

3.2.2 Preparation of cell extracts...... 47

3.2.3 Plasmid transfer by conjugation...... 48

3.2.4 Immunoblot...... 49

3.2.5 Crotonyl-CoA carboxylase/reductase assay...... 50

3.2.6 β-Galactosidase assay...... 51

3.2.7 RNA isolation...... 51

3.2.8 Quantitative reverse transcription PCR (qRT PCR)...... 52

3.2.9 Identification of the ccr transcriptional start site...... 53

3.2.10 DNA Motif Identification...... 53

3.2.11 Isolation of R. sphaeroides deletion mutants ...... 54

3.2.12 Isolation of R.sphaeroides reporter strains ...... 59

3.2.13 Identification of transposon insertion sites...... 61

3.2.14 Isolation of R. sphaeroides strains containing ccr-lac reporter plasmid fusions...... 62

3.3 Results ...... 66

3.3.1 Ccr activity, Ccr protein, and ccr transcript levels are upregulated during acetate growth...... 66

3.3.2 Multiple conserved motifs exist in the intergenic region between ecm and ccr...... 69

3.3.3 Expression from the ccr promoter is largely controlled via a sequence between 191 bp and 72 bp upstream of its transcriptional start...... 71

3.3.4 Interrupting carbon flow through the ethylmalonyl-CoA pathway affects Ccr activity...... 73

3.3.5 PhaR does not affect regulation of Ccr activity in photoheterotrophically succinate-, succinate/acetate-, or acetate-grown R. sphaeroides...... 74

xii

3.3.6 Loss of pccR does not affect regulation of Ccr activity in photoheterotrophically succinate-, succinate/acetate- or acetate-grown R. sphaeroides...... 75

3.3.7 Random transposon mutants have alternatively regulated ccr expression...... 75

3.4 Discussion ...... 79

3.4.1 Expression of ccr is primarily regulated by incremental control of transcription...... 79

3.4.2 Possible transcriptional regulators of ccr remain unknown...... 82

3.4.3 Other genes for acetyl-CoA assimilation might be regulated by the same repression...... 83

3.4.4 Ccr activity is affected by a product of the ethylmalonyl-CoA pathway...... 84

3.4.5 The significance of deregulation of β-galactosidase activity in transposon mutants of Rs ccr-lac1A is unknown...... 86

3.4.6 Suggested future directions ...... 91

Chapter 4: PccR Is a Member of the Newly Identified ScfR Family of Transcriptional Regulators ...... 93

4.1 Introduction ...... 93

4.2 Materials and Methods ...... 97

4.2.1 Materials...... 97

4.2.2 Bacterial strains and growth conditions...... 97

4.2.3 Plasmid construction...... 98

4.2.4 Isolation of mutant strains and complemented mutant strains...... 100

4.2.5 Preparation of cell extracts...... 101

4.2.6 Enzyme assays...... 101

4.2.7 RNA isolation...... 102

xiii

4.2.8 Quantitative reverse transcription PCR (qRT PCR)...... 103

4.2.9 Identification of the pccB transcriptional start site...... 103

4.2.10 In silico ScfR identification and DNA binding site recognition...... 104

4.3 Results ...... 106

4.3.1 The expression of pccB is regulated at the level of the transcript...... 106

4.3.2 The protein product of pccR is necessary for proper transcriptional activation of pccB...... 107

4.3.3 Activation of pccB requires both TTTGCAAA motifs ...... 111

4.3.4 PccR is a part of a proposed family of regulators for the assimilation of short chain fatty acyl-CoA (ScfR) molecules...... 112

4.4 Discussion ...... 118

4.4.1 PccR is the primary regulator responsible for controlling intracellular propionyl-CoA levels...... 118

4.4.2 PccR is also a transcriptional repressor...... 121

4.4.3 ScfR regulators uniquely respond to intracellular signals...... 122

4.4.4 Suggested future directions ...... 125

Chapter 5: Summary ...... 127

References ...... 133

Appendix A: Detailed Materials and Methods ...... 146

A.1. Crotonyl-CoA and propionyl-CoA synthesis ...... 147

A.2. Prepartation of small scale cell extracts by the cell mill ...... 150

A.3. Crotonyl-CoA carboxylase assay ...... 151

A.4. Radioactive carboxylation assay ...... 153

A.5. Propionyl-CoA assay ...... 156

A.6. β-Galactosidase assay ...... 158 xiv

A.7. Bradford protein concentration determination ...... 159

A.8. Protein production ...... 161

A.9. SDS Polyacrylamide gel electrophoresis for protein ...... 163

A.10. Tris-tricine polyacrylamide gel ...... 167

A.11. Silver staining of polyacrylamide gels ...... 169

A.12. Solubilizing inclusion bodies (Large Scale) ...... 171

A.13. Protein precipitation with Trichloroacetic Acid (TCA) ...... 173

A.14. Digest with recombinant Tobacco Etch Virus Protease (rTEVP): ...... 174

A.15. Immunoblot ...... 175

A.16. Polyacrylamide gel electrophoresis for DNA ...... 178

A.17. Site directed mutagenesis ...... 180

A.18. Electrophoretic mobility shifts (SYBR) ...... 182

A.19. Characterization of Δccr23KB ...... 184

A.20. Characterization of Δecm47KB ...... 186

A.21. Characterization of Δmcd11KB ...... 188

A.22. Characterization of Δmch49KB ...... 190

A.23. Characterization of Δmcl1_4KB ...... 192

A.24. Characterization of RsΔmaeB1MC63 ...... 194

A.25. Characterization of RsΔpccRMC12 ...... 195

A.26. Characterization of RsΔphaRMC43 ...... 197

A.27. Characterization of Rs ccr-lac1A ...... 199

A.28. Identification of location of transposon insertion site ...... 200

A.29. Reverse transcription polymerase chain reaction (RT PCR)...... 203

A.30. Large scale RNA isolation ...... 205

A.31. Quantitative polymerase chain reaction (qPCR) ...... 207

A.32. qPCR data analysis ...... 209

Appendix B: Additional Results ...... 210

B.1. Failed purification of soluble, intact PccR ...... 211

B.2. Analysis of spent media from R. sphaeroides, RsΔpccRMC12, and RsΔpccBSJC1A during succinate or acetate growth ...... 215

xvi

List of Tables

Table 2.1. Strains and plasmids used in Chapter 2 ...... 27 Table 2.2. Primers used in Chapter 2 ...... 27 Table 2.3. Doubling time of R. sphaeroides and malic enzyme mutant strains grown in various conditions ...... 32 Table 3.1. Previously observed regulation of the ethylmalonyl-CoA pathway ...... 44 Table 3.2. Primers used in Chapter 3 ...... 64 Table 3.3. Strains and plasmids used in Chapter 3 ...... 65 Table 3.4. Densitometric measurements of Ccr bands in the lanes of the immunoblot in Figure 3.2 ...... 67 Table 3.5. Crotonyl-CoA carboxylase/reductase (Ccr) activities in cell extracts of photoheterotrophically grown cells ...... 68 Table 3.6. Regulation of ccr transcripts during photoheterotrophic growth with various carbon sources ...... 68 Table 3.7. Conserved motif upstream of genes for acetyl-CoA assimilation in R. spheroies 2.4.1 ...... 70 Table 3.8. Regulation of Ccr (crotonyl-CoA carboxylase/reductase) and LacZ (β- galacatosidase) ...... 72 Table 3.9. Potential PhaR binding sites upstream of various genes in R. sphaeroides 2.4.1...... 74 Table 3.9. Transposon insertion sites in the genomes of selected mutant strains of Rs ccr- lac1A that displayed deregulation of lacZ expression from the ccr promoter during succinate growth ...... 76 Table 4.1. Primers used in Chapter 4 ...... 105

xvii

Table 4.2. Propionyl-CoA carboxylase activity in cell extracts of photoheterotrophically grown cells ...... 106 Table 4.3. β-Galactosidase and propionyl-CoA carboxylase activity in cell extracts of photoheterotrophically grown cells carrying derivatives of the pccB-lacZ translational fusion plasmid pMC85 ...... 110 Table A.1. Polyacrylamide gel components ...... 164 Table A.2. Polyacrylamide gel components ...... 178 Table B.1. Notes regarding failed PccR purification attempts ...... 213

xviii

List of Figures

Figure 1.1. The complete cycle of reactions of the glyoxylate bypass...... 8 Figure 1.2. The ethylmalonyl-CoA pathway and the methylmalonyl-CoA pathway...... 12 Figure 1.3. The complete set of reactions of the methylcitrate cycle...... 14 Figure 2.1. Known reactions within the C4/C3 node of central metabolism...... 19 Figure 2.2. Genotypic characterization of mutations in the two genes annotated to encode malic enzymes in R. sphaeroides...... 31 Figure 2.3. Phenotypic characterization of maeB1 and maeB2 R. sphaeroides mutants. 32 Figure 2.4. Genotypic and phenotypic characterization of Rs ccr-lacS9, a transposon mutant of Rs ccr-lac1A...... 34 Figure 2.5. Genotypic and phenotypic characterization of T17-143 (pycA::Tn), a transposon mutant of R. sphaeroides 2.4.1...... 35 Figure 3.1. The ethylmalonyl-CoA pathway...... 44 Figure 3.2. Immunoblot for detection of Ccr in cell extracts of R. sphaeroides...... 67 Figure 3.3. Standard curve of densitometric values from Table 3.5 for quantification of Ccr...... 67 Figure 3.4. Identification of conserved motifs in the intergenic region of ecm/ccr for various α-proteobacteria...... 69 Figure 3.5. Overlaid chromatograms that illustrate the sequencing reaction standards and the primer extension for identification of the ccr transcriptional start site...... 71 Figure 3.6. Organization of the ccr promoter and the truncations used to investigate the effects of various motifs on the expression from the ccr promoter...... 72 Figure 3.7. Illustrations of the locations of transposon insertions in ccr-lacZYA reporter strains isolated as blue colonies during growth on minimal media succinate supplemented with XGal...... 78 xix

Figure 3.8. Phenotypic characterization of transposon mutants derived from random mutagenesis of Rs ccr-lac1A...... 79 Figure 4.1. Comparison of propionyl-CoA carboxylase (PCC) activity as in Table 4.2 and pccB transcript abundance from R. sphaeroides 2.4.1 and RsΔpccRMC12 cells grown with succinate, acetate, and propionate/HCO3̅...... 107 Figure 4.2. Genomic context within several α-proteobacteria of pccB (propionyl-CoA carboxylase, β subunit; rsp_2189), pccR, and the consensus sequence identified in Figure 4.7 upstream of pccR (rsp_2186) genes...... 108 Figure 4.3. Photoheterotrophic growth of R. sphaeroides 2.4.1, RsΔpccRMC12, RsΔpccRMC12(pBBR), and RsΔpccRMC12(pMC66) with succinate, acetate, and ̅ propionate/HCO3 ...... 108 Figure 4.4. Photoheterotrophic growth of strains of R. sphaeroides 2.4.1 and RsΔpccRMC12 that carry no plasmid, pMC85, pMC85Δ1, pMC85Δ2, and

̅ pMC85Δ12 with succinate, acetate, or propionate/HCO3...... 111 Figure 4.5. Illustration of the mutated pccB upstream regions that are present on each plasmid in the pMC85 series...... 112 Figure 4.6. Alignment of select ScfR proteins from each class...... 114 Figure 4.7. Neighbor-joining tree of selected ScfR proteins...... 117 Figure 4.8. Topography of the scaled neighbor-joining tree with compressed clades that represent 327 proteins from the ScfR family...... 118 Figure A.1. Expected results from TRI extraction...... 206 Figure B.1. Relative retention times of standard molecules and of molecules in spent media from R. sphaeroides 2.4.1 at various timepoints during photoheterotrophic growth with succinate and acetate ...... 216 Figure B.2. Relative retention times of molecules in spent media from RsΔpccBSJC1A at various timepoints during photoheterotrophic growth with succinate and acetate ... 217 Figure B.3. Relative retention times of molecules in spent media from RsΔpccRMC12 at various timepoints during photoheterotrophic growth with succinate and acetate ... 218

Figure B.4. Quantified composition of spent media from photoheterotrophic cultures of R. sphaeroides 2.4.1, RsΔpccBSJC1A, and RsΔpccRMC12 grown with succinate or acetate ...... 219

xxi

List of Abbreviations

Aat Aspartate amino transferase AceA Isocitrate lyase; commonly abbreviated ICL AceB Malate synthase; commonly abbreviated MS AceK Isocitrate dehydrogenase kinase/phosphatase AcnB Aconitase AcnD Methylcitrate dehydratase Ack Acetate kinase Acs Acetyl-CoA synthetase ADP Adenosine diphosphate AMP Adenosine monophosphate ATP Adenosine triphosphate Aph Aminoglycoside aminotransferase for kanamycin resistance ATP Adenosine triphosphate CAP Catabolite activator protein (also CRP) Ccr Crotonyl-CoA carboxylase reductase CcrR Transcriptional activator of ccr in Methylobacterium extorquens cDNA DNA product of reverse transcription (complementary DNA) CitM Oxaloacetate decarboxylase with high amino acid sequence similarity to malic enzyme portion of MaeB Cra Cyclic-AMP independent catabolite repressor/activator (also FruR) CroR Crotonase CRP Cyclic-AMP receptor protein (also CAP) DNA Deoxyribonucleic acid Ecm Ethylmalonyl-CoA mutase Epi Epimerase

xxii

FadR Transcriptional repressor of genes for fatty acid degradation in Escherchia coli GDP Guanosine diphosphate GTP Guanosine triphosphate HibA Isobutyryl-CoA dehydrogenase HibB Methylacrylyl-CoA hydratase IbcR Class of ScfR regulators for assimilation of isobutyryl-CoA IclR Transcriptional repressor of the aceBAK operon in Escherichia coli IHF Integration host factor, a pleiotropic transcriptional regulator LacZ β-Galactosidase MaeB Fusion protein of malic enzyme and a phosphotransacetylase-like domain MccR Class of ScfR regulators of the methylcitrate cycle Mcd Methylsuccinyl-CoA dehydrogenase Mch Mesaconyl-CoA hydratase Mcl1 β-Methylmalyl-CoA/malyl-CoA lyase Mcl2 Malyl-CoA thioesterase Mcm Methylmalonyl-CoA mutase MmsA Methylmalonate semialdehyde dehydrogenase MmsB 3-Hydroxyisobutyryl-CoA dehydrogenase MmsR Transcriptional activator of the mmsAB operon in Pseudomonas NAD+ Nicotinamide adenine dinucleotide (oxidized) NADP+ Nicotinamide adenine dinucleotide phosphate (oxidized) NADH Nicotinamide adenine dinucleotide (reduced) NADPH Nicotinamide adenine dinucleotide phosphate (reduced) NCBI National center for biotechnology information Odx Oxaloacetate decarboxylase PCC Propionyl-CoA carboxylase activity PccA Propionyl-CoA carboxylase α-subunit (biotin carboxylase) PccB Propionyl-CoA carboxylase, β-subunit (carboxytransferase)

xxiii

PccR Transcriptional regulator of pccB in Rhodobacter sphaeroides; class of ScfR regulators of the methylmalonyl-CoA pathway PckA Phosphoenolpyruvate carboxykinase PEP Phosphoenolpyruvate PhaA β-Ketothiolase PhaB Acetoacetyl-CoA reductase PhaP Phasin protein for assembling polyhydroxyalkanoate granules PhaR Transcriptional regulator of polyhydroxyalkanoate synthesis PhaZ Polyhydroxyalkanoate depolymerase PpdK Pyruvate phosphate dikinase PrpB Methylcitrate lyase PrpC Methylcitrate synthase PrpD Methylcitrate dehydratase/hydratase PrpE Propionyl-CoA synthetase PrpF Methylaconitate cis-trans isomerase PrpQ* Former name of a class of regulators that includes PccR and MccR PrpR Transcriptional activator of the prpBCDE operon in Salmonella enterica Pta Phosphotransacetylase PycA Pyruvate carboxylase RamA Regulator of acetate metabolism protein A in Corynebacterium glutamicum RamB Regulator of acetate metabolism protein B first characterized in Corynebacterium glutamicum; class of ScfR regulators of the glyoxylate bypass RecA DNA repair recombinase RNA Ribonucleic acid RpoZ ω-Subunit of RNA polymerase ScfR Family designation of regulators of short chain fatty acyl-CoA molecules SfcA Malic enzyme SseA 3-Mercaptopyruvate sulfur transferase xxiv

Chapter 1: Introduction

1.1 Introduction to Rhodobacter sphaeroides physiology

Rhodobacter sphaeroides is a Gram-negative rod that is phylogenetically classified within the α-proteobacteria (130). However, it is best known for its membership in the nonphylogenetic group of purple nonsulfur bacteria, a nomenclature that is based on functional taxonomy rather than evolution. Originally, the group was defined by its members’ ability to perform anoxygenic phototrophy and their lack of sulfur granule accumulation during autotrophic growth with H2S (56). They are commonly isolated from the anaerobic portions of standing fresh water where detritus collects. As the organic material is decomposed by other organisms, an array of carbon molecules is released into the environment. As skilled scavengers, R. sphaeroides and other purple nonsulfur bacteria have evolved the capacity to assimilate the diverse carbon sources by growth modes that include photoautotrophy, photoheterotrophy, chemoautotrophy, and chemoheterotrophy.

The variety of possible growth modes and growth substrates of R. sphaeroides is immediately representative of the multitude of efficiently integrated pathways that form its optimized metabolic networks. The efficiency of the networks is expectedly dependent on a fine control of carbon flow that relies on overlapping layers of genetic and enzymatic regulation. Revealing the path of carbon through the networks therefore

requires a thorough understanding of the enzymes that form the pathways and the ways in which R. sphaeroides controls their operation.

The best studied metabolic networks in R. sphaeroides involve CO2 fixation (28,

32) and the construction and operation of the photosystems (11, 46, 82, 90, 133). While, of the two, only studies of CO2 fixation immediately offer insight into carbon flow through metabolic networks in R. sphaeroides, the broader interest has driven researchers to develop techniques that are essential for studying carbon flow. The result is that genetic tools are well-established, the genome sequence is published (70) and regularly updated (60), and growth media are optimized (86, 92, 116).

Another advantage of the history of broader study in R. sphaeroides is the thorough body of knowledge regarding the variety of regulatory strategies employed by

R. sphaeroides to control its physiology. Transcriptional regulation involving two component (33) and single component (28) systems by traditional regulator-DNA interaction is commonly observed. Transcription, transcript stability, translation, and product stability of the puf operon all appear to be points of regulation for R. sphaeroides

(31). There is also evidence for regulation of transcript stability by small noncoding

RNAs in response to photooxidative stress (7, 8). In total, evidence exists in R. sphaeroides for nearly all known strategies of controlling gene expression and protein functionality.

1.2 Core Metabolism

1.2.1 Precursor metabolites define core metabolism In order to study metabolic networks, the pathways involved must first be understood, beginning with the most central pathways of core metabolism. As with any series of reactions that define a metabolic pathway, core metabolism is composed of reactions that are involved in energy conservation and biosynthetic precursor synthesis.

In most organisms, core metabolism contains at least three components – glycolysis/gluconeogenesis, the citric acid cycle, and the C4/C3 node that reversibly converts intermediates between the other two. Studying the network of pathways within the three components of core metabolism requires quantitatively predicting the kinds of energy carriers and biosynthetic precursors that result from each pathway and relating them to the corresponding quantities required for cell biosynthesis. In the case of most organisms, the resulting analysis is a remarkably complex stoichiometric calculation that is best left to computer algorithms. Phototrophic growth in R. sphaerodies, however, offers a somewhat unique opportunity to essentially divorce energy metabolism from carbon metabolism. Because photophosphorylation allows for ATP production without necessitating additional oxidation of the carbon source for respiration, the cell is able to effectively devote its core metabolic pathways almost exclusively to production of biosynthetic precursors.

To construct an even more manageable analysis, studying the flow of carbon among the biosynthetic precursors within core metabolism can be simplified to six essential biosynthetic precursors – oxaloacetate, sugars, phosphoenolpyruvate, pyruvate,

acetyl-CoA, and α-ketoglutarate – defined here specifically as precursor metabolites.

Typically, if a cell is able to generate each of the precursor metabolites, standard biosynthetic pathways can convert them into each essential constituent of the cell.

Furthermore, because cellular constituents are nearly always required at specific concentrations within a cell, the ratio of the production of precursor metabolites is fixed.

The consequence is that carbon flow through the network of pathways in core metabolism must be properly adjusted to ensure that flow toward each precursor metabolite is sufficient to maintain the appropriate ratio of eventual production of cellular components.

1.2.2 The C4/C3 node controls carbon flow through core metabolism All precursor metabolites, except acetyl-CoA, can be divided into two classes – glycolytic/gluconeogenic and citric acid cycle intermediates. The C4/C3 node of core metabolism is the series of reactions responsible for directing carbon flow between the two classes. As the cell requires each precursor metabolite for the synthesis of different cellular constituents, reactions at the C4/C3 node must modulate the net direction of carbon flow. Therefore, a fine control of the C4/C3 node is essential to efficient cell viability through efficient precursor metabolite synthesis.

Before the control of the C4/C3 node can be studied, however, the enzymes that participate at the node must be identified. Multiple enzymes in the node catalyze seemingly redundant reactions, making it difficult to assign a role to each enzyme in vivo.

Genetic annotation introduces further ambiguity, as multiple genes are predicted to encode the same enzyme within a single organism. The relationship of PEP

carboxykinase (PckA) and malic enzyme is an excellent example. The activity of PckA is physiologically responsible for catalyzing the conversion of a C4 molecule

(oxaloacetate) into a C3 molecule (PEP) at the expense of the conversion of GTP/ATP to

GDP/ADP and the release of CO2 (125). Similarly, malic enzyme catalyzes a C4 to C3 conversion (malate to pyruvate), but its activity is coupled to the reduction of NAD(P)+ and the release of CO2 (10). The genomes of several organisms, including R. sphaeroides and E. coli, encode PckA and two malic enzymes.

An examination of the regulation of the three enzymes, as studied in E. coli, begins to reveal the fine control of carbon flow through the C4/C3 node. While evidence for allosteric control of the PckA protein is not available, expression of its gene is subject to catabolite repression (43, 44). Similarly, catabolite repression is assumed for the genes of the malic enzymes – sfcA and maeB – based on comparative transcriptome analysis

(104).

Additional control of the C4/C3 node occurs at the level of malic enzymes – SfcA and MaeB. SfcA is composed of two domains that are characteristic of malic enzymes and performs catalysis with either NAD+ or NADP+, but it operates optimally at physiological substrate concentrations with NAD+ (10). Conversely, MaeB exclusively employs NADP+ and contains an additional domain that is homologous to the active domain of a phosphotransacetylase but demonstrates no phosphotransacetylase activity

(10). The in vitro activity of both enzymes is reduced in the presence of oxaloacetate and increased in the presence of aspartate while SfcA is uniquely inhibited by long chain fatty acids and free CoA, and MaeB activity is uniquely and seemingly paradoxically elevated

in the presence of glucose-6-phosphate. Upon deletion of the phosphotransacetylase domain, however, MaeB activity becomes unaffected by any of the aforementioned effectors, except oxaloacetate (10). Overall, the activities of PckA, SfcA, and MaeB display their cumulative capacity for affecting C4 and C3 availability, the NADP(H) or

NAD(H) pools, and ATP/GTP pools. Furthermore, the details regarding their regulation demonstrate that the different paths through just one direction of the C4/C3 node are subject to distinct regulatory control in response to various signals via multiple strategies.

Their differential regulation emphasizes that understanding the control of carbon flux through the C4/C3 node is paramount to understanding the interactions of the metabolic networks of core metabolism.

The obvious physiological significance of the interplay of PckA, SfcA, and MaeB highlights the necessity for a thorough knowledge of the C4/C3 node, and it is at this node that the greatest gaps exist in the knowledge of R. sphaeroides core metabolism.

Certainly, regulation and activity of enzymes of the other two components of core metabolism remain uncharacterized in R. sphaeroides, yet it is exclusively at the C4/C3 node that predicting which enzymes are operating is currently impossible given the seemingly redundant genetic evidence. Because the operation of the C4/C3 node remains clouded, Chapter 2 will explore the reactions within the node in R. sphaeroides in an effort to offer clarity that can be extrapolated throughout the overall study of core metabolism.

1.3 Intermediary Metabolism

1.3.1 Defining intermediary metabolism Many carbon substrates are converted into metabolites of core metabolism through common intermediates. The common intermediates are ultimately converted to precursor metabolites through pathways that compose intermediary metabolism. An organism is able to grow with an external carbon source only if the complement of its intermediary and core metabolic pathways is sufficient to convert the given carbon source into each of the precursor metabolites

1.3.2 Acetyl-CoA assimilation requires intermediary metabolism The need for an intermediary pathway for acetyl-CoA assimilation may not be immediately apparent given that it can enter core metabolism directly via condensation with oxaloacetate to form citrate. However, in the absence of the capacity for reductive carboxylation of acetyl-CoA and without an intermediary pathway for assimilation, the carbon of acetyl-CoA will proceed through the citric acid cycle where the activities of isocitrate dehydrogenase and α-ketoglutarate dehydrogenase will catalyze its oxidation, each releasing one molecule of CO2. Given that only two carbons enter the cycle as acetyl-CoA and two carbons are released as CO2, the citric acid cycle is unable to perform any net assimilation of carbon. Pathways for acetyl-CoA assimilation, therefore, primarily serve to allow the biosynthesis of precursor metabolites while avoiding oxidation steps that would release carbon from acetyl-CoA as CO2.

The best known pathway for acetyl-CoA assimilation is the glyoxylate bypass

(also nominally referenced as the glyoxylate cycle or the glyoxylate shunt) (Figure 1.1)

(61). The overall reaction series converts oxaloacetate and two acetyl-CoA units into succinate and malate through enzymes of the citric acid cycle and two enzymes unique to the pathway, isocitrate lyase (AceA) and malate synthase (AceB). AceA is responsible for catalyzing the cleavage of isocitrate into succinate and glyoxylate (117). AceB subsequently catalyzes the condensation of the resulting glyoxylate with acetyl-CoA to form malate (63). Physiologically, isocitrate is derived from acetyl-CoA and oxaloacetate through standard reactions of the citric acid cycle. Therefore, isocitrate is a metabolic branching point between the citric acid cycle and the glyoxylate bypass.

Figure 1.1. The complete cycle of reactions of the glyoxylate bypass. Arrows indicate the physiologically relevant direction of the corresponding reactions. AceA (isocitrate lyase) and AceB (malate synthase) function to bypass two decarboxylation steps within the citric acid cycle. Although the entire cycle is illustrated, only enzymes unique to the pathway are named.

Studies regarding regulation of carbon flow through the glyoxylate bypass focus on acetate growth and span many different organisms, including Salmonella enterica,

Escherichia coli, and Corynebacterium glutamicum. The sum of the information indicates that control of carbon flow occurs in the form of transcriptional repression, transcriptional activation, and reversible enzyme modification. Regulation in acetate- grown S. enterica, for instance, begins with the finely controlled activation of acetate to acetyl-CoA by the reversible acetylation of acetyl-CoA synthetase (Acs) (118). The strategy is widespread, as it has additionally been identified in Rhodopseudomonas palustris (27) and mitochondria (109). In E. coli, acs is also transcriptionally activated by the cyclic AMP receptor protein (CRP; also known as catabolite activation protein,

CAP) that participates in the catabolite repression system (6).

Once the carbon is in the form of acetyl-CoA, it can be further assimilated by condensation with oxaloacetate to form citrate via catalysis by citrate synthase. As citrate, the carbon continues through standard citric acid cycle reactions until it reaches isocitrate. To appropriately direct carbon toward assimilation, E. coli reversibly inactivates isocitrate dehydrogenase by phosphorylation through the catalytic activity of isocitrate dehydrogenase kinase/phosphatase (AceK) (66). AceK is encoded by the distal coding region of the aceBAK operon. Its transcript is detected at lower levels than those of the preceding two genes of the operon, a disparity that has been attributed to variable transcriptional termination at a predicted stemloop between aceA and aceK (23).

To control the expression of the entire operon, E. coli has adapted a series of interconnected transcriptional regulators. The core of the transcriptional regulation is built around a transcriptional repressor that is the product of the autogenously repressed iclR (73). IclR binds the aceBAK promoter until intracellular pyruvate or glyoxylate

concentrations are sufficiently elevated (69). Because another source of acetyl-CoA for the cell is β-oxidation of fatty acids, E. coli has also adapted FadR as an activator of iclR expression (48). FadR operates as a transcriptional repressor of genes encoding enzymes for fatty acid degradation (115) while simultaneously preventing the expression of aceBAK by activating iclR expression. IHF further contributes to the overlapping control of IclR repression of aceBAK by disrupting IclR interaction with the aceBAK promoter during inducing conditions (101), thereby aiding in aceBAK derepression. Finally, independent of IclR, aceBAK receives additional transcriptional control through activation by Cra (formerly FruR) in the absence of fructose-1-phosphate (24).

Like E. coli, Corynebacterium glutamicum also assimilates acetyl-CoA by the glyoxylate bypass. However, unlike E. coli, no apparent IclR homolog exists in C. glutamicum. Instead, RamA and RamB (Regulators of acetate metabolism) operate as transcriptional regulators of genes responsible for acetyl-CoA assimilation. For example, acetate is first activated to acetyl-CoA following the catalytic action of acetate kinase

(Ack) and phosphotransacetylase (Pta) (100), whose genes are respectively activated and repressed by RamA and RamB (26, 42). Likewise, expression of the divergently transcribed aceA and aceB is also activated and repressed by RamA and RamB (26, 42).

Information about more extensive regulation of the genes and enzymes of the glyoxylate bypass in C. glutamicum is unknown, but transcriptional regulation by RamA and RamB has been observed across many metabolic pathways in C. glutamicum (3). The result is a regulatory scheme, unlike in E. coli, in which the transcriptional regulators directly control the interaction of core and intermediary metabolism.

1.3.2.1 The ethylmalonyl-CoA pathway is an intermediary pathway for acetyl-

CoA assimilation.

While E. coli and C. glutamicum have adapted alternative methods for the regulation of the same pathway for acetyl-CoA assimilation, other organisms such as

Methylobacterium extorquens and R. sphaeroides have developed an entirely novel pathway. In the ethylmalonyl-CoA pathway, three acetyl-CoA molecules and two CO2 equivalents are ultimately converted into one molecule of succinyl-CoA and one molecule of malate (see Figure 1.2 for an illustration of the pathway and descriptions of the associated abbreviations). In the first step, two acetyl-CoA molecules are condensed through catalysis by β-ketothiolase to form acetoacetyl-CoA (1). Then, acetoacetyl-CoA is reduced through catalysis by acetoacetyl-CoA reductase to generate 3- hydroxyburtyryl-CoA (96), which is dehydrated by the catalytic activity of an R-specific crotonase to make crotonyl-CoA. Crotonyl-CoA carboxylase/reductase then catalyzes the reductive carboxylation of crotonyl-CoA to (2S)-ethylmalonyl-CoA, which is epimerized by the catalytic activity of epimerase to (2R)-ethylmalonyl-CoA (34, 35). A carbon rearrangement of (2R)-ethylmalonyl-CoA catalyzed by ethylmalonyl-CoA mutase yields (2S)-methylsuccinyl-CoA (38). Methylsuccinyl-CoA is then oxidized through catalysis by methylsuccinyl-CoA dehydrogenase to form mesaconyl-CoA (37), which is hydrated through catalysis by mesaconyl-CoA hydratase to make β-methylmalyl-CoA

(132). The β-methylmalyl-CoA is cleaved into propionyl-CoA and glyoxylate through catalysis by β-methylmalyl-CoA/malyl-CoA lyase, which is also responsible for catalyzing the condensation of the glyoxylate with another acetyl-CoA to form malyl-

CoA (36). The thioesterase catalyzes the hydrolytic removal of the CoA group, and malate is left to enter the citric acid cycle (36). The propionyl-CoA generated by β- methylmalyl-CoA/malyl-CoA lyase continues into the methylmalonyl-CoA pathway as discussed below. Like the glyoxylate bypass, the ethylmalonyl-CoA pathway provides citric acid cycle intermediates directly from acetyl-CoA while avoiding decarboxylation steps.

Figure 1.2. The ethylmalonyl-CoA pathway and the methylmalonyl-CoA pathway. Abbreviations: PhaA, β-ketothiolase; PhaB, acetoacetyl-CoA reductase; CroR, crotonase; Ccr, crotonyl-CoA carboxylase/reductase; Epi, epimerase; Ecm, ethylmalonyl-CoA mutase; Mcd, methylsuccinyl-CoA dehydrogenase; Mch, mesaconyl-CoA hydratase; Mcl1, β-methylmalyl-CoA/L-malyl-CoA lyase; Mcl2, malyl-CoA thioesterase; PccAB, propionyl-CoA carboxylase; Mcm, methylmalonyl-CoA mutase.

Nearly each enzyme of the ethylmalonyl-CoA pathway has been characterized in detail, yet the investigation into the modes by which they are regulated is in its infancy.

In R. sphaeroides, many of the enzymes or their activities have been observed to be downregulated in conditions that do not require acetyl-CoA assimilation (1, 36, 37), yet 12

the mechanism that controls the observed regulation is not known. Conversely, a transcriptional activator has been identified in Methylobacterium extorquens, but it is reported to be responsible only for the regulation of ccr expression and is not present in

R. sphaeroides (54). Chapter 3 will explore the options for controlling intermediary metabolism via acetyl-CoA in R. sphaeroides. More specifically, the chapter will examine evidence regarding the regulation of expression of the gene that encodes Ccr, an indispensible enzyme of the ethylmalonyl-CoA pathway.

1.3.3 Propionyl-CoA assimilation requires intermediary metabolism. Propionyl-CoA, another short chain fatty acyl-CoA, is also known to be assimilated via multiple pathways, two of which are the methylcitrate cycle and the methylmalonyl-CoA pathway. The reactions of the methylcitrate cycle are similar to those of the glyoxylate bypass whereas the methylmalonyl-CoA pathway is a linear conversion of propionyl-CoA into citric acid cycle intermediates. The propionyl-CoA that enters either pathway can be derived directly from propionate or from cellular recycling processes such as odd chain fatty acid degradation, branched chain fatty acid degradation, and branched chain amino acid degradation.

1.3.3.1 The methylcitrate cycle is an intermediary pathway for propionyl-CoA assimilation.

Despite first being described in yeast (121), evidence for the operation of the methylcitrate cycle (Figure 1.3) can be found among diverse classes of bacteria and is best studied in S. enterica, E. coli, and Mycobacterium tuberculosis (see below).

Propionyl-CoA assimilation is often studied by adding propionate to the growth medium.

Consequently, the first step of assimilation is the activation of propionate to propionyl-

CoA by propionyl-CoA synthetase. In S. enterica and E. coli, reports have suggested that propionyl-CoA synthetase activity can be modulated by reversible covalent modification.

Propionyl-CoA synthetase from E. coli has been observed to be modulated in vitro via the reduction and oxidation of a disulfide bond between a pair of cysteine residues (49).

Similarly, in S. enterica, propionylation of the active site of propionyl-CoA synthetase has been observed to inhibit its activity in vivo and in vitro (40).

Figure 1.3. The complete set of reactions of the methylcitrate cycle. The cycle shares reactions with enzymes of the citric acid cycle. Only enzymes unique to the methylcitrate cycle are named. The combined activity of AcnD and PrpF replaces the activity of PrpD in some organisms. The reaction series is similar to that of the glyoxylate bypass, and the methyl group that chemically distinguishes the pathways is indicated in red. Abbreviations: PrpC, methylcitrate synthase; AcnD, methylcitrate dehydratase; PrpF, methylcitrate cis-trans isomerase; PrpD, methylcitrate hydratase/dehydratase; AcnB, aconitase; PrpB, methylcitrate lyase.

Upon activation to propionyl-CoA, the compound becomes a substrate for methylcitrate synthase (PrpC), an enzyme that catalyzes the condensation of propionyl-

CoA and oxaloacetate to form 2-methylcitrate (51, 124) (Figure 1.3). 2-Methylcitrate is subsequently dehydrated to form methylaconitate through catalysis by either methylcitrate dehydratase/hydratase (PrpD) (51, 122) or the combined activity of methylcitrate dehydratase (AcnD) (47) and methylaconitate cis-trans isomerase (PrpF)

(41). Following dehydration, methylaconitate is hydrated by the catalytic activity of aconitase (AcnB) to yield methylisocitrate (53), which is cleaved through catalysis by methylisocitrate lyase (PrpB in E. coli and S. enterica) (13, 51) or isocitrate lyase (AceA in M. tuberculosis) (45) to ultimately produce pyruvate and succinate.

Aside from regulation of propionyl-CoA synthetase activity, control of carbon flow through the methylcitrate cycle has almost exclusively been observed at the transcriptional level. The genes that encode the enzymes of the pathway are often observed in an operon (29, 52). The expression of the operon in S. enterica is σ54- dependent, requiring a corresponding transcriptional activator. The regulator, PrpR, activates expression of the prp operon in the presence of 2-methylcitrate and is encoded by a gene that is divergently transcribed from the operon (94). The expression of the operon in M. tuberculosis is transcriptionally activated by a homolog of RamB (29)

(discussed above). Additional evidence indicates that expression of the prp operon in M. tuberculosis, as in S. enterica, is dependent on an alternative σ factor although not σ54

(29). While a potential effector for the M. tuberculosis RamB homolog is not known, the

σ-factor suggested to be responsible for transcription of the prp operon, σE, responds to

extracellular stresses such as hypoxia, offering some slight insight into the signaling involved in regulation of carbon flow through the methylcitrate cycle in M. tuberculosis.

1.3.3.2 The methylmalonyl-CoA pathway is an intermediary pathway for propionyl-CoA assimilation.

The methylmalonyl-CoA pathway (Figure 1.2) is a comparatively simple pathway. The pathway has been biochemically well-characterized despite effectively no available information regarding its regulation. The pathway begins with the carboxylation of propionyl-CoA through catalysis by propionyl-CoA carboxylase

(PccAB), an enzyme composed of two subunits (55). The first subunit, PccA, contains a

̅ biotin cofactor, and the active site catalyzes the carboxylation of the biotin with HCO3 in a reaction that concomitantly hydrolyzes ATP into ADP and Pi (17). The second subunit,

PccB, catalyzes the carboxytransferase reaction, which removes the carboxyl group from biotin for use in carboxylating propionyl-CoA (103). The resulting (2S)-methylmalonyl-

CoA is epimerized to (2R)-methylmalonyl-CoA by the catalytic activity of a metal- dependent epimerase (68). (2R)-Methylmalonyl-CoA is then isomerized by the the catalytic activity of the cobalamin-dependent methylmalonyl-CoA mutase (Mcm or

McmAB) into succinyl-CoA (93).

Overall, the enzymes of the methylmalonyl-CoA pathway have been characterized in detail in humans and bacteria, but little evidence exists regarding the regulation of the expression of their respective genes. The work of Chapter 4 examines the regulation of carbon flow through the methylmalonyl-CoA pathway by following activity of propionyl-CoA carboxylase and expression of pccB. It also seeks to offer a

sense of how regulation of the pathway might be conserved across a diverse array of bacterial species.

Chapter 2: The C4/C3 Node of R. sphaeroides Core Metabolism

2.1 Introduction

Given its array of metabolic capabilities, R. sphaeroides offers researchers a tool for more deeply understanding carbon flow into and through central metabolic pathways.

Beginning their studies at the metabolic core, researchers initially measured and characterized the enzymes of glycolysis and the citric acid cycle (15, 20, 76, 80, 106). It quickly became evident, however, that for an organism with such metabolic diversity, the central metabolic pathways are also necessarily subject to variability. For example, by measuring enzyme activities in cell extracts and tracing incorporation of radiolabelled sugars in R. sphaeroides, researchers identified that photoheterotrophic growth with glucose almost exclusively employs the Entner-Doudoroff pathway whereas both the

Entner-Doudoroff pathway and the Embden-Meyerhof-Parnas pathway were used during photoheterotrophic growth with fructose (22).

While both pathways offer a version of glycolysis, they require alternative enzymatic steps and differing cofactors. The resulting variation in R. sphaeroides during growth with similar sugars highlights the possibility that carbon flow through other components of core metabolism may also occur via multiple enzymatic paths. Figure 2.1, for instance, illustrates the potential variability in the C4/C3 node based on reactions and enzymes characterized in many different organisms (see below). In R. sphaeroides,

specifically, the genetic evidence is especially ambiguous, as there are three immediate candidates that can participate in C4 to C3 conversion. Two genes are annotated to encode malic enzymes and a third is annotated to encode PEP carboxykinase (PckA).

Neither of the malic enzymes (herein named MaeB1 and MaeB2) has been characterized biochemically, so their unique contributions to C4 to C3 conversion in vivo remain purely speculative.

Figure 2.1. Known reactions within the C4/C3 node of central metabolism. Reactions represented in black are predicted to be present in R. sphaeroides 2.4.1. Predictions are based on genetic and reported biochemical evidence. Reactions in gray have been observed in other organisms. Arrows represent the proposed physiologically relevant direction of the respective reaction. Abbreviations (predicted locus tag): Mdh (RSP_0968), malate dehydrogenase; PckA (RSP_1680), PEP carboxykinase; Ppc, PEP carboxylase; PycA (RSP_2090), pyruvate carboxylase; Odx, oxaloacetate decarboxylase; MaeB (RSP_1593, RSP_1217), malic enzyme; PpdK (RSP_1859), pyruvate phosphate dikinase; PpsA, pyruvate, water dikinase.

The MaeB proteins are fusion proteins similar to MaeB that has been characterized in E. coli and Sinorhizobium melliloti (10, 127). Their sequences imply that they are malic enzymes fused to phosphotransacetylase, an enzyme typically involved in the reversible conversion of acetyl-CoA to acetyl-phosphate (14). In E. coli, however, the full length fusion protein demonstrates no phosphotransacetylase activity (10). Instead, in vitro

evidence suggests that modulation of malic enzyme activity in response to sensory molecules is greatly reduced if the phosphotransacetylase domain is removed (10). As a result, it seems that the role of the phosphotransacetylase domain is strictly regulatory rather than enzymatic.

The other likely candidate for participation in C4 to C3 conversion, PckA, was partially purified from phototrophically grown R. sphaeroides, and its activity was measured in both biochemical directions (125). The researchers observed similar Km values for PEP and oxaloacetate, suggesting that the enzyme might reversibly catalyze

C4 to C3 conversion in vivo. Another report, however, reveals that a pyruvate carboxylase mutant is unable to grow with glucose, lactate, or pyruvate – substrates that require C3 to C4 conversion – indicating that PEP carboxykinase is unable to sufficiently provide the cell with oxaloacetate from PEP (95).

Despite no current genetic evidence, additional contribution to C4 to C3 conversion may be provided by an as of yet unidentified oxaloacetate decarboxylase.

Three types of oxaloacetate decarboxylases have been described in the literature with the newest class being identified as late as 2010. The first and best studied are found in

Salmonella enterica (STM3352, STM3353, and STM3354) and Enterobacter aerogenes

(EAE_10935, EAE_10920, and EAE_10915) (30, 129). They are biotin-containing membrane complexes that generate a sodium gradient as they catalyze the formation of pyruvate from oxaloacetate decarboxylation. The second category of oxaloacetate decarboxylases contains two characterized members. The genes for both are clustered with genes for citrate metabolism and are designated citM in Lactococcus lactis (l3227)

and Enterococcus faecalis (ef3316) (39, 111). Although they lack the fused phosphotransacetylase domain, the amino acid sequences of the CitM proteins are homologous to the malic enzyme domains of MaeB, even in so far as they are annotated to contain an NAD(P)H binding domain. Neither characterized CitM, however, is capable of catalyzing the oxidative decarboxylation of malate. Instead, they exclusively catalyze the decarboxylation of oxaloacetate. The third type of described oxaloacetate decarboxylase contains a single member identified in C. glutamicum (Cgp_1458) (58) that lacks any sequence similarity to other known enzymes involved in C4/C3 nodes of metabolism.

For conversion of C3 to C4, R. sphaeroides only has an annotated pyruvate carboxylase. Its activity was measured in cell extracts of cells phototrophically and chemotrophically grown with acetate, glucose, pyruvate, (R/S)-malate, or lactate plus

(R/S)-malate (95). Between growth modes and among cultures grown with each carbon source in both growth modes, no more than a two-fold difference in pyruvate carboxylase activity was observed despite the prediction that it would be necessary only for glucose, pyruvate, and lactate growth. Although pyruvate carboxylase is clearly involved in the flow of carbon through the C4/C3 node, the slight variations in observed activity among growth conditions implies that the enzyme’s production is an unlikely control point of the

C4/C3 node in R sphaeroides.

Instead, the cell might control flow to C4 molecules from pyruvate by modulating pyruvate concentrations through the regulation of pyruvate kinase activity. After purifying pyruvate kinase from phototrophically grown R. sphaeroides cultures (106), the

enzyme’s activity was observed to be elevated in the presence of AMP, glucose-6- phosphate, and ribose-5-phosphate. ATP, citrate, C4 dicarboxylic acids, and inorganic phosphate were alternatively observed to reduce pyruvate kinase activity. No evidence was offered to suggest a regulation of production of the enzyme, but the reported regulation of its activity is consistent with its role in progressing carbon to pyruvate as citric acid cycle intermediates are depleted, energy reserves in the form of ATP are diminished, and sugar-phosphates accumulate.

Altogether, there are many clues as to the strategies that R. sphaeroides might use in controlling carbon flow through its C4/C3 node, yet they only provide sufficient evidence for a partial model of the enzymes involved. Even despite the characterization of some relevant enzymes, necessary information regarding their regulation is incomplete. The work presented here seeks to clarify the contribution of different enzymes to carbon flow through the C4/C3 node in various growth conditions by examining growth defects of strains that carry mutated genes of C4/C3 node enzymes.

2.2 Materials and Methods

2.2.1 Bacterial strains and growth conditions. Rhodobacter sphaeroides 2.4.1 (DSMZ 158) was grown aerobically in the dark or anaerobically in the light (3,000 lux) at pH 6.8 and 30°C in minimal media (MM) that contained 15 mM potassium phosphate buffer pH 6.7 and per liter: 0.2 g (0.8 mM)

MgSO4, 0.07 g (0.5 mM) CaCl2, 1.2 g (22 mM) NH4Cl, and 1.5 mL of vitamin solution

(per liter: 100 mg cyanocobalamin, 300 mg pyridoxamine-2∙HCl, 100 mg calcium-D(+)- pantothenate, 200 mg thiamine dichloride, 200 mg nicotinic acid, 80 mg 4-aminobenzoic

acid, 20 mg D(+)-biotin), 10 mL trace element solution (per liter: 500 mg disodium

EDTA, 300 mg FeSO4∙7H2O, 3 mg MnCl2∙4H2O, 5 mg CoCl2∙6H2O, 1 mg CuCl2∙2H2O,

2 mg NiCl2∙6H2O, 3 mg Na2MoO4∙2H2O, 5 mg ZnSO4∙7H2O, and 2 mg H3BO3, pH un3),

10 mM of each respective carbon source (in the case of two carbon sources, each was supplied at 10 mM). For R. sphaeroides mutant strains, 25 µg/mL and/or 20 µg/mL kanamycin was added. Media plates included 2.5% agar. Oxygen in liquid cultures was removed and replaced with N2 by repeated vacuuming and sparging. Growth in liquid cultures was monitored as an increase in optical density (OD) at 578 nm. Typically, 5 mL cultures in MM in stoppered, screw-capped (Hungate) tubes were used. Unless otherwise stated, 5 mL MM succinate precultures with appropriate antibiotics were grown to late exponential phase (OD578 nm ~ 1.5) before 0.1 mL was inoculated into the fresh medium to be used in growth studies. Escherichia coli strains DH5α, DH5α λpir,

S17-1 (114), and SM10 (114) were grown in Luria-Bertani (LB) broth at 37°C with 50

µg/mL spectinomycin or 50 µg/mL kanamycin if necessary.

2.2.2 Plasmid transfer by conjugation. Plasmids were transferred into R. sphaeroides strains by conjugation with either

E. coli S17-1 (for plasmids that encode kanamycin resistance) or E. coli SM10 (for plasmids that encode spectinomycin resistance). The strains to be mated were grown in

25 mL LB liquid to OD578 nm ~ 0.5. Cells were collected by centrifugation and resuspended in 1 mL LB. Cell densities in each suspension were estimated to be linearly related to the final OD578 nm of their cultures. Volumes of each cell suspension were mixed together to yield a final volume of 1 mL that contained approximately the same

number of cells of each strain. The cells were collected again by centrifugation before being resuspended in 100 µL LB. The entire volume was dropped into the middle of an

LB plate and allowed to incubate in nonselective conditions 24 hr at 30°C aerobically in the dark.

Approximately 15% of the spot was scraped from the surface of the LB plate and suspended in 100 µL MM succinate or lactate with appropriate antibiotics. The suspension was diluted 1:100 in MM succinate or lactate with appropriate antibiotics.

The diluted and the undiluted suspensions were spread on MM succinate or lactate plates with appropriate antibiotics to select for successful transconjugants.

2.2.3 Isolation of RsΔmaeB1MC63. Plasmid pKB99 was constructed for the inactivation of rsp_1593 by Kelsey Baron

(unpublished) to obtain strain RsΔmaeB1MC63. The pKB99 plasmid contains an in- frame deletion of 2,115 nt of rsp_1593 to yield a coding region that consists of 72 nt of the 5′ portion and 93 nt of the 3′ portion of rsp_0958 separated by a KpnI site. The hypothetical gene product is a 56 amino acid peptide. The 1,168 bp product of the primers deltaMeup_for2 and deltaMeup_rev1 was digested with KpnI and cloned into pUC19 to make pKB96. The 956 bp product of the primers deltaMedown_for1 and deltaMedown_rev2 was cloned into pUC19 via HindIII/KpnI digest and subsequent ligation to make pKB97. Following KpnI digest of both plasmids, a 1,153 bp fragment from pKB96 was ligated into pKB97 to make pKB98. The final construct, pKB99, is the result of ligating a 2,082 bp SphI/HindIII fragment into pK18mobsacB (provided as a gift by Dr. Izumi Orita) (105).

To obtain an in-frame maeB1 deletion mutant, pKB99 was transferred into R. sphaeroides by conjugation with E. coli S17-1(pKB99). Four single recombinant strains were isolated from kanamycin resistant colonies on MM lactate kanamycin plates.

Colonies of single recombinant strains were incubated in 100 µL MM lactate for 12 – 16 hr at 30°C aerobically in the dark. To select for cells that had lost the plasmid from the chromosome, the entire volume was plated on MM lactate with 10% sucrose and incubated anaerobically in the light at 30°C. Ninety-one isolated colonies that had grown in MM lactate were patched on MM lactate, MM lactate kanamycin, and MM lactate

10% sucrose plates. Double recombinant deletion strains were distinguished from wild type revertant strains by comparing colony PCR products of primers DmaeB1genUpR1 and DmaeBgenDnR1 (ΔmaeB = 236 bp; maeB = 2,345 bp). RsΔmaeB1MC63 was chosen and genetically characterized by sequencing genomic amplification products obtained using primers DmaeB1genUpF1 and DmaeB1genUpR1 (confirms chromosomal positions 186,833 – 186,926 and 189,041 – 190,049) and DmaeB1genDnF1 and

DmaeB1genDnR1 (confirms chromosomal positions 185,682 – 186,926 and 189,041 –

189,169).

2.2.4 Bioinformatic search for oxaloacetate decarboxylase enzymes in R. sphaeroides 2.4.1 To identify possible homologs of the multisubunit, sodium translocating oxaloacetate decarboxylase, each subunit of the Salmonella enterica Oad complex

(STM3351, STM3352, and STM3353) was queried against the R. sphaeroides 2.4.1 proteome in the NCBI database. Resulting proteins were only considered homologous if their alignment coverage included at least 95% of the query and the subject sequence.

To identify possible homologs of the single subunit oxaloacetate decarboxylase that resembles the malic enzyme domains of the MaeB protein, the CitM from

Lactococcus lactis (L3227) was used as a query for a BLAST search of the R. sphaeroides 2.4.1 proteome. Resulting proteins were only considered homologous if their alignment coverage included at least 95% of the query and the subject sequence.

To identify possible homologs of the single subunit oxaloacetate decarboxylase from Corynebacterium glutamicum, the Oxd protein (Cgp_1458) was used as a query of the R. sphaeroides 2.4.1 proteome. Resulting proteins were only considered homologous if their alignment coverage included at least 95% of the query and the subject sequence.

Table 2.1. Strains and plasmids used in Chapter 2 Name Relevant characteristics Reference E. coli

DH5α Cloning host DH5α λpir Replicates Pir-dependent plasmids SM10 RP4-2 Tc::Mu-Km::Tn7; donor strain for conjugation (114) S17-1 RP4-2 Tc::Mu Tpr Smr Scr; donor strain for conjugation (114) BW20767 Parental strain for random transposon mutagenesis (67)

R. sphaeroides 2.4.1 (DSMZ 158) Wild type Rs ccr-lac1A ccr promoter-lac operon chromosomal fusion, Smr Scr Chapter 3 (Rs::pMC26F) Rs ccr-lac1A transposon mutant; ppdK (rsp_1859)::Tn5-RL27(Kmr- Rs ccr-lacS9 Chapter 3 oriR6K), pos 456370 RsΔmaeB1MC63 in-frame deletion inactivation of maeB1 (rsp_1593) This work T17-143 pycA::Tn5-RL27 (Kmr-oriR6K), pos 686692 This work AU12-332-22-1 maeB2::Tn5-RL27 (Kmr-oriR6K), pos 2989396 This work Plasmids pMC26F Isolation of ccr promoter-lacZYA chromosomal fusion Chapter 3 pKB99 in-frame deletion inactivation of maeB1 This work

pJQSm2MCS Derived from pJQ200mp18 (removes sequence duplications, replaces This work Gmr) Scr Smr pK18mobsacB Replication incompetent in R. sphaeroides, Kmr, Sucroses (105)

Table 2.2. Primers used in Chapter 2 Primer Sequence (5′  3′)1 Use deltaMedown_for1 TCTAAGGTACCCTTCCCGGGTTTCGGAAACTCGTG maeB1 upstream fragment deltaMedown_rev2 GTGCAAAGCTTGATGCCGCCCGTCGAATGTTTGTC maeB1 upstream fragment deltaMeup_for2 GGTGGTACCCGCATGCAGCGGCCCGTAGCAGATATAG maeB1 downstream fragment deltaMeup_rev1 TACGGTACCAACCTCGCCCATATCGTAACCGCTTC maeB1 downstream fragment DmaeB1genUpF1 CCGACCCGAGATCATCGGCACATAG RsΔmaeB1MC63 genotyping DmaeB1genUpR1 GCCTCTGCTCAGCCGTAATGGGTAAC RsΔmaeB1MC63 genotyping DmaeB1genDnF1 AGAAGGTCTTCACCTTGTGGTTCTGCATC RsΔmaeB1MC63 genotyping DmaeB1genDnR1 CGGTTGCATGGACATTCAGATGGTTTAGTG RsΔmaeB1MC63 genotyping lacoperon_for1 AATGATAAGCTTGAATTCGATCCCGTCGTTTTACAACGTC ccr-lac operon fusion lacoperon_rev1 TGCACTGCAGTTAAACTGACGATTCAACTTTATAATCTTTGAAATAATAG ccr-lac operon fusion lacoperon_rev2 TGATATCGGTACCCTGCAGTTAAACTGACGATTC ccr-lac operon fusion JQm_for3 GTAACACCATGGCTGCTCCATAACATCAAACATC pJQSm2MCS plasmid construction JQm_rev3 AGAAGAGATCTACTTTGATATCGACCCAAGTACC pJQSm2MCS plasmid construction sm_for1 AGTCTCATCCATGGAGCGTAGCGACCGAGTGAG pJQSm2MCS plasmid construction sm_rev2 ATGTGGCGAGATCTCTTGAACGAATTGTTAGAC pJQSm2MCS plasmid construction JQMCS_for2 TCTAGAGTCGACCTGCAGGGTTCGCCCAGCTTCTGTATG pJQSm2MCS plasmid construction JQMCS_rev1 TCTAGAGGATCCCGGGTACCGAGCTCACGGCCGAGGTCTTCC pJQSm2MCS plasmid construction tpnRL 13-2 CAGCAACACCTTCTTCACGA plasposon sequencing tpmRL 17-1 AACAAGCCAGGGATGTAACG plasposon sequencing 1Restriction sites are underlined

2.2.4 Random transposon mutagenesis

2.2.4.1 Transposon mutagenesis of Rs ccr-lac1A.

Mutagenesis of Rs ccr-lac1A, a ccr-promoter β-galactosidase reporter strain, was performed to identify genes potentially involved in regulating ccr expression (Chapter 3).

One of the isolated mutants, Rs ccr-lacS9, also displayed growth defects that potentially provides information about carbon flow through the C4/C3 node of central metabolism.

The pRL27 plasmid was transferred into Rs ccr-lac1A by conjugation with

BW20767(pRL27) (67) as described above. The pRL27 plasmid is a suicide vector that contains the mini-Tn5 transposon, encodes a transposase outside of the transposon, and is replicated from a Pir-dependent origin of replication, oriR6K, which is not recognized by

R. sphaeroides. The cells scraped from the conjugation spot were suspended in 100 µL of MM succinate kanamycin/spectinomycin and diluted 1:10. The diluted and undiluted suspensions were spread on MM succinate kanamycin/spectinomycin plates that had been pre-spread with 50 µL of 2% 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (XGal).

Plates were incubated at 30°C anaerobically in the light until colonies formed. Rs ccr- lacS9 was isolated among the subset of colonies that were vibrant blue. The site of transposition in Rs ccr-lacS9 was identified in chromosome 1 at position 456,371, interrupting rsp_1859 annotated as pyruvate phosphate dikinase.

2.2.4.2 Random transposon mutagenesis of R. sphaeroides.

Random transposon mutagenesis of the R. sphaeroides genome was performed by students of the Microbiology 581 course at The Ohio State University under direction of

Dr. Kathleen Sandman.

Conjugation was performed via a variation of the protocol described above. Cells from 1 mL aliquots of freshly growth 25 mL LB overnight cultures of R. sphaeroides and

BW20767(pRL27) (OD578 nm = 0.5 – 1) were collected by centrifugation. Pellets were washed with 0.5 mL of LB without antibiotics before being collected again by centrifugation and suspended in 0.5 mL of LB. The suspensions of both strains were mixed and passed through a filter to concentrate the cells. The filter was incubated on nonselective LB plates for 48 hr at 30°C aerobically in the dark. Cells were washed from the filter with 2 mL sterile saline. An aliquot of 100 µL each was spread on four MM plates. Plates were incubated at 30°C aerobically in the dark. Resulting colonies were patched onto MM kanamycin plates that each contained a different carbon source.

Strain AU12-332-22-1 was isolated from a colony that grew with (R/S)-lactate but was unable to grow with (S)-malate. In the genome of AU12-332-22-1, a transposon insertion was identified at position 2,989,396 of chromosome 1, interrupting rsp_1217

(maeB2), which is annotated to encode a malic enzyme/phosphotransacetylase fusion protein. Strain T17-143 was isolated from a similar screen that included the following carbon sources: succinate, (R/S)-lactate, and acetate. T17-143 was able to grow with succinate and acetate but not with (R/S)-lactate. A transposon insertion was identified in its genome in chromosome 1 at position 686,692 within rsp_2090 (pycA), which is annotated to encode pyruvate carboxylase.

2.2.4.3 Identification of transposon insertion sites.

The genomic positions of transposon insertions were identified by fragmenting genomic DNA and performing intramolecular ligation to form circularized pieces of

genomic DNA. Fragments that contain the transposon also contain the Pir-dependent origin of replication, oriR6K, forming a transposon-containing plasmid (plasposon).

When inserting the transposon into the genome, the transposase duplicates 9 bp of the flanking genomic DNA. The reported transposon insertion sites represent the last position of genomic sequence of the forward strand (as numbered in the NCBI database) before transposon sequence was identified during sequencing. The stretch of 9 bp immediately upstream of the identified insertion site is duplicated following the last position of the transposon in the genome. The forward direction of the transposon represents the direction of the aph (for kanamycin resistance) gene within the transposon.

Genomic DNA (~ 1 µg) was digested with 10 U of NcoI in a 30 µL reaction for 1 hr at 37°C. The reaction was stopped by heat inactivation at 65°C for 20 min. A 15 µL aliquot of the fragmented genome was added into a 50 µL ligation reaction containing 1.5

µL of T4 Ligase (Fermentas). The ligation reaction was incubated at room temperature for 12 – 14 hr before a 25 µL aliquot was used to transform chemically competent DH5α

λpir cells. Plasposons were isolated from kanamycin resistant DH5α λpir cells. Primers tpnRL17-1 and tpnRL13-2, which anneal to opposite ends of the transposon and direct elongation out of the transposon, were used to sequence the regions of the plasposon that immediately flank the transposon.

2.3 Results

The results presented below include preliminary analysis of mutant phenotypes that have not been complemented.

2.3.1 The maeB1(rsp_1593) and maeB2 (rsp_1217) genes are required for optimal growth on (S)-malate and succinate but not acetate. Mutant strains were isolated in which either gene annotated to encode a malic enzyme was mutated (Figure 2.2). RsΔmaeB1MC63 is a mutant strain in which maeB1 is inactivated by an in-frame deletion. The resulting coding region encodes a 56 aa peptide, which is a truncated version of the 759 aa wild type protein. The maeB2 gene is interrupted by a transposon in the Au12-332-22-1 strain. Annotations of maeB1 and maeB2 predict their products to be fusion proteins that consist of an NAD(P)+-dependent malic enzyme at their amino termini fused to a phosphotransacetylase at their carboxy termini.

Figure 2.2. Genotypic characterization of mutations in the two genes annotated to encode malic enzymes in R. sphaeroides. The proposed reaction of the gene products of maeB1 (rsp_1593) and maeB2 (rsp_1217) (A). Genetic context of maeB1 and representation of the deletion of maeB1 in RsΔmaeB1MC63 (B). Genetic context of maeB2. The black arrow indicates the site and direction of transposon insertion in strain AU12-332-22-1 (maeB2::kan) (C). Gene annotations: cdd, cytidine deaminase; maeB1, malic enzyme; prpE, propionyl-CoA synthetase; cons., conserved gene; σ54 regulator, σ54-dependent transcriptional regulator; ilvEβ, β-subunit of branched chain amino acid aminotransferase; ilvEα, α-subunit of branched chain amino acid aminotransferase; rbsK, putative carbohydrate kinase; maeB2, malic enzyme; mutS, DNA mismatch repair protein.

While both malic enzyme mutants were able to grow in conditions that require conversion of C4 to C3 (Figure 2.3) – such as succinate, (S)-malate, and acetate – the doubling times of the mutants during photoheterotrophic and chemoheterotrophic growth with succinate or malate were significantly slower than identically grown R. sphaeroides

(Table 2.3) (Ashwin Lahiry, unpublished). The prediction would be that the requirement for C4 to C3 conversion during succinate and (S)-malate growth in R. sphaeroides would be similar.

Figure 2.3. Phenotypic characterization of maeB1 and maeB2 R. sphaeroides mutants. Chemoheterotrophic (aerobic in the dark) (A) and photoheterotrophic (anaerobic in the light) (B) growth of R. sphaeroides 2.4.1 (□), AU12-332-22-1 (maeB2::Tn) (▽), and RsΔmaeB1MC63(○) on indicated carbon sources. Strains were precultured on (R/S)-lactate. Values represent a single growth experiment.

Table 2.3. Doubling time of R. sphaeroides and malic enzyme mutant strains grown in various conditions1 Chemoheterotrophic Photoheterotrophic Strain (R/S)-Lactate Succinate (S)-Malate Acetate (R/S)-Lactate Succinate (S)-Malate Acetate R. sphaeroides 2.4.1 3.4 hr 2.7 hr 2.5 hr 5.3 hr 3.6 hr 3.0 hr 3.0 hr 3.9 hr AU12-332-22-1 3.4 hr 40 hr 84 hr 5.3 hr 3.6 hr 7.0 hr 14.5 hr 3.9 hr RsΔmaeB1MC63 3.4 hr 24 hr 13 hr 5.3 hr 3.6 hr 14 hr 17.5 hr 5.3 hr 1Values were determined based on the growth experiments presented in Figure 2.3 32

Thus, the comparable growth defects observed for either malic enzyme mutant with succinate and (S)-malate were expected. However, growth of either mutant with acetate, despite its assimilation via succinate and malate, was unaffected. The data therefore suggest that both enzymes contribute to the conversion of C4 to C3 during succinate and malate growth, but neither is exclusively required for acetate growth.

2.3.2 The ppdK gene (rsp_1859) is required for optimal growth with succinate and acetate. Pyruvate phosphate dikinase is encoded by ppdK and is responsible for the gluconeogenic conversion of pyruvate to phosphoenolpyruvate. Rs ccr-lacS9, a transposon mutant of Rs ccr-lac1A (see Chapter 3 for isolation), contains a transposon within ppdK (Figure 2.4). During succinate growth, the mutant strain reproducibly grew with a growth rate that was about 50% slower than that of Rs ccr-lac1A and R. sphaeroides. The reduced growth rate indicates that PpdK contributed to but was not exclusively responsible for production of C3 precursors during succinate growth. PpdK, however, was essential for acetate growth, as the Rs ccr-lacS9 strain was unable to grow with acetate. No growth in the absence of PpdK therefore implied that during growth with acetate, all gluconeogenic intermediates were likely derived from pyruvate.

Figure 2.4. Genotypic and phenotypic characterization of Rs ccr-lacS9, a transposon mutant of Rs ccr- lac1A. The proposed reaction catalyzed by the ppdK (rsp_1859) gene product (A). The local genetic context of ppdK in the R. sphaeroides chromosome. The black arrow indicates the site of transposon insertion and direction of the kanamycin resistance gene on the transposon (B). Photoheterotrophic growth of R. sphaeroides 2.4.1 (□), Rs ccr-lac1A (∆), and Rs ccr-lacS9 (▽) on succinate (left) and acetate (right) (C). Gene annotations: glyQ, α-subunit of glycyl-tRNA synthetase; hypo, hypothetical gene; glyS, β-subunit of glycyl-tRNA synthetase.

2.3.3 The pycA gene (rsp_2090) is required for optimal growth with (R/S)- lactate. The T17-143 strain was isolated following random transposon mutagenesis of R. sphaeroides. The genome of T17-143 contains a transposon insertion within the pycA gene (Figure 2.5). The predicted gene product of pycA, pyruvate carboxylase, is responsible for the conversion of C3 to C4 intermediates. The mutant strain and R. sphaeroides were observed to grow equivalently during growth with succinate and acetate, but T17-143 was compromised during growth with (R/S)-lactate (Figure 2.5).

Growth with succinate and acetate, which is assimilated via succinate, was not expected to require C3 to C4 conversion. However, lactate is expected to enter core metabolism as pyruvate. Thus, any C4 precursor metabolites must have been derived through C3 to C4 conversion reactions.

Figure 2.5. Genotypic and phenotypic characterization of T17-143 (pycA::Tn), a transposon mutant of R. sphaeroides 2.4.1. The proposed reaction of the pycA (rsp_2090) gene product (A). The local genetic context of pycA. The black arrow indicates the site and direction of transposon insertion (B). Photoheterotrophic growth of R. sphaeroides 2.4.1 (□) and T17-143 (○) on indicated carbon sources. Values are representative of a single growth experiment (C). Gene annotations: hypo, hypothetical gene; helicase; ATP-dependent DNA helicase; lepA, GTP-dependent proofreading elongation factor.

2.4 Discussion

2.4.1 Many reactions contribute to the generation of C3 intermediates during succinate and (S)-malate growth. Mutating either malic enzyme gene impairs but does not completely abolish growth with succinate or malate. The effects on growth were representative of contributions by both malic enzymes to the pool of available C3 precursors during 35

succinate and malate growth. However, interpreting the magnitude of their combined or individual contributions is complicated by other evidence. For example, if the combined effects of both malic enzymes were exclusively responsible for production of C3 precursors, all gluconeogenic carbon would pass through pyruvate. Therefore, continued gluconeogenic carbon flow would require the activity of pyruvate phosphate dikinase

(PpdK) for the generation of phosphoenolpyruvate, yet Rs ccr-lacS9 (containing a transposon interruption of ppdK) was able to grow with succinate, albeit at a reduced rate.

As a result, the overall production of C3 precursors must have been a cumulative task that involved the malic enzymes and other reactions that provide C3 precursors that do not require PpdK for their assimilation.

Reactions whose products could contribute to the C3 pool without further requiring PpdK include those of the reductive pentose phosphate cycle (Calvin-Benson-

Bassham cycle) and phosphoenolpyruvate carboxykinase (PckA). Contribution from the reductive pentose phosphate cycle is well precedented. Data by Laguna et al., for instance, indicate that R. sphaeroides photoheterotrophic growth with malate is dependent on the presence of a functional reductive pentose phosphate cycle (65). The requirement for the reductive pentose phosphate cycle during malate growth is due to the depletion of NAD+ as malate is converted through central metabolism to precursor metabolites in appropriate ratios. Operation of the reductive pentose phosphate cycle allows the cell to regenerate NAD+ while simultaneously producing 3-phosphoglycerate from 3 CO2 molecules. 3-Phosphoglycerate does not require the activity of PpdK for assimilation. As for potential contribution from PckA, its activity has been measured as

partially purified from cell extracts of cells grown photoheterotrophically with an unnamed carbon source (125), but no R. sphaeroides pckA mutants have yet been isolated.

2.4.2. Unimpaired acetate growth for strains in which either malic enzyme is mutated remains unexplained. Unlike with succinate and malate, the loss of either malic enzyme gene had no observable effect during acetate growth despite the assimilation of acetate via succinate and malate in R. sphaeroides. The immediate implication, therefore, was that neither malic enzyme was used for C4 to C3 conversion while growing with acetate. Instead, one explanation might be the operation of an oxaloacetate decarboxylase during acetate growth that is dormant during succinate or (S)-malate growth.

There are three known enzymes physiologically responsible for decarboxylation of oxaloacetate. The first is the multisubunit, biotin-containing, and membrane-bound

OadGBA from Enterobacter aerogenes and Salmonella enterica (30, 129). A BLAST search of the R. sphaeroides proteome only identifies a protein similar to the biotin- containing γ-subunit OadG. The similar protein in R. sphaeroides is a fragment of the biotin-dependent pyruvate carboxylase. A second prospective oxaloacetate decarboxylase is the soluble, single subunit Odx that was first characterized in

Corynebacterium glutamicum (58). Again, the R. sphaeroides chromosome encodes no homolog as determined using the criteria outlined in the materials and methods. Finally, the third type of oxaloacetate decarboxylase is indistinguishable from the malic enzyme domains of an MaeB-type malic enzyme (39). Given that no acetate growth defect is observed for either mutant of the two malic enzyme genes predicted in R. sphaeroides, it

seems unlikely that one of them is performing the proposed oxaloacetate decarboxylase during acetate growth.

Cursory analysis of the growth data of the ppdK mutant seems to further confuse possible explanations for which enzymes are responsible for catalyzing C4 to C3 conversions in R sphaeroides during acetate growth. Mutation of ppdK, whose product initiates gluconeogenesis from pyruvate, abolished acetate growth, suggesting that all C3 precursors in R. sphaeroides are derived from pyruvate. Thus, mutation of malic enzyme genes, responsible for catalyzing the production of pyruvate from malate, would be predicted to impair growth in conditions that require C4 to C3 conversion such as succinate, (S)-malate, or acetate. Acetate growth of the malic enzyme mutants, however, is equivalent to R. sphaeroides. The explanation for acetate growth becomes evident, though, as the necessary rates of pyruvate formation in the different growth conditions are considered. During R. sphaeroides succinate or (S)-malate growth, for example, a significant portion of the pyruvate generated by malic enzyme activity must be converted to acetyl-CoA for biosynthesis. Conversely, cells growing with acetate need only enough pyruvate for gluconeogenesis, as acetyl-CoA can be obtained directly from the carbon source. Thus, the combined activity of both malic enzymes might be unnecessary during acetate growth. As a result, a mutation in a single malic enzyme gene may be insufficient to affect acetate growth while sufficient to impair growth with succinate or (S)-malate.

2.4.3. Pyruvate carboxylase is responsible for C3 to C4 conversion in R. sphaeroides Growth results of T17-143 (Figure 2.5) confirm that R. sphaeroides (R/S)-lactate growth without pyruvate carboxylase was possible, albeit significantly impaired. Given

that (R/S)-lactate is predicted to be assimilated through pyruvate, the loss of pyruvate carboxylase would expectedly result in a complete inhibition of growth. Thus, other reactions must have been able to compensate for the necessary C4 to C3 conversion in the absence of pyruvate carboxylase. Indeed, during in vitro characterization, each of the enzymes assumed to operate in the C4 to C3 direction have also been observed to catalyze the reverse reaction. However, the reactions often require a high concentration of C3 substrate (10). Thus, it is likely that the malic enzymes and/or PckA were responsible for the growth of T17-143 with (R/S)-lactate, but their reverse activity was relevant only because of the physiological perturbation caused by the absent pyruvate carboxylase activity. It is improbable that the data represents a wild type physiological role for C3 to C4 conversion by the malic enzymes or PckA.

2.4.4. Suggested future directions In order to provide a more solid basis for continued investigation into the C4/C3 node in R. sphaeroides, the activities of potential reactions should be measured in cell extracts. Additional speculation regarding which enzymes are contributing to carbon flow through the node will be limited without a more thorough characterization of the reactions that occur in the cell. Malic enzyme, PEP carboxykinase, and oxaloacetate decarboxylase activities should be measured in extracts from succinate-, (S)-malate -,

(R/S)-lactate-, and acetate-grown R. sphaeroides cells. Measuring malic enzyme and

PEP carboxykinase activity might identify which activity is responsible for C4 to C3 conversion in which growth conditions. Attempting to measure oxaloacetate decarboxylase, despite an absence of genetic evidence for a known oxaloacetate

decarboxylase, will more definitively establish the potential for its role in the C4/C3 node.

To better clarify which reactions are responsible for C4 to C3 conversion in vivo, strains that contain in-frame, markerless deletions of ppdK, pckA, maeB2, and maeB1 and maeB2 should be isolated. Complementation of all growth defects, including the defects reported here for RsΔmaeB1MC63, should be completed to confirm that the observed phenotypes are a result of the corresponding mutations. The phenotypes of each mutant are expected to mirror the phenotypes reported here for the corresponding transposon mutants. An investigation of the phenotypes of the strain with mutations in both malic enzyme genes will address the possible redundancy of the two malic enzymes. Also, examining the growth of a PEP carboxykinase mutant may provide information about the enzyme’s contribution to C4 to C3 conversion.

Once the available reactions of the C4/C3 node are identified, the proteins responsible for the measured activities should be examined. The mutant studies described above provide candidate coding regions for malic enzymes, PEP carboxykinase, and pyruvate carboxylase. Each coding region should be heterologously expressed, and the resulting proteins should be purified and biochemically characterized.

Given that two predicted malic enzyme coding regions exist, biochemical characterization is expected to highlight distinctions that are representative of the role of either enzyme in the C4/C3 node. Also, their potential for operating as oxaloacetate decarboxylases can be addressed in vitro.

In spite of the open questions that remain regarding the flow of carbon through the C4/C3 node of R. sphaeroides, this work highlights the relevance of further characterization of the node and is consistent with previous studies regarding reductive pentose phosphate cycle operation in R. sphaeroides. The seeming discrepancies among succinate, (S)-malate, and acetate growth of the various mutants are only some of the indications that our knowledge regarding the node is inadequate. Without a more thorough model of the operation of the C4/C3 node, predictions regarding carbon flow through core metabolism will remain incomplete.

Chapter 3: Control of Carbon Flow through the Ethylmalonyl-CoA Pathway 3.1 Introduction

As with any intermediate, acetyl-CoA must be converted into other precursor metabolites for its biological assimilation. However, in the absence of pathways able to convert acetyl-CoA to other precursor metabolites, it is converted into citrate as it enters the citric acid cycle. Acetyl-CoA, a two-carbon molecule, is the only input of carbon into the citric acid cycle, which generates two CO2 molecules, thereby assimilating no net carbon. As citric acid cycle intermediates are withdrawn for cell biosynthesis, the cycle collapses without additional carbon input. Consequently, during growth on substrates

(such as fatty acids, waxes, and acetate) that are metabolized exclusively via acetyl-CoA, organisms require pathways for conversion of acetyl-CoA into citric acid cycle intermediates, a process known as anaplerosis (61).

A mechanism of acetyl-CoA assimilation was first characterized by Hans

Kornberg and Hans Krebs in Pseudomonas as they identified isocitrate lyase (AceA) and malate synthase (AceB) (61). Together with citric acid cycle enzymes, AceA and AceB catalyze the conversion of one molecule of oxaloacetate and two molecules of acetyl-

CoA to one molecule of succinate and one molecule of malate. Because glyoxylate is an intermediate in the reaction series that also bypasses the carboxylation steps of the citric acid cycle, the pathway is often referenced as the glyoxylate bypass. Shortly after

identifying the glyoxylate bypass, Kornberg published a report in which he revealed that

Rhodobacter sphaeroides was competent at acetyl-CoA assimilation yet demonstrated no

AceA activity (62). Another strategy of acetyl-CoA assimilation was later described in which pyruvate synthase (pyruvate:ferredoxin oxidoreductase) catalyzes the reductive carboxylation of acetyl-CoA to produce pyruvate (2), a source for all other precursor metabolites. Genome sequencing ultimately revealed that there is no genetic evidence for pyruvate synthase or the glyoxylate bypass in R. sphaeroides.

Recently, the ethylmalonyl-CoA pathway was recognized as the method by which

R. sphaeroides assimilates acetyl-CoA (Figure 3.1). The pathway is responsible for conversion of three acetyl-CoA units and two inorganic C1 units into two C4 molecules.

The pathway begins as acetyl-CoA molecules are condensed and converted to 3- hydroxybutyryl-CoA as in polyhydroxybutyrate synthesis (1, 96). 3-Hydroxybutyryl-

CoA is then dehydrated to crotonyl-CoA before the pathway’s characteristic enzyme crotonyl-CoA carboxylase/reductase catalyzes the reductive carboxylation of crotonyl-

CoA to form ethylmalonyl-CoA (34, 35). Through an oxidation step and several rearrangements, the carbon subsequently flows through a series of C5 molecules to β- methylmalyl-CoA before it is cleaved into glyoxylate and propionyl-CoA (36-38). The resulting glyoxylate is converted to malate after condensation with acetyl-CoA (36), and the resulting propionyl-CoA is assimilated via the methylmalonyl-CoA pathway (78).

Nearly all of the enzymes of the ethylmalonyl-CoA pathway and their corresponding genes have been identified and characterized. Furthermore, regulation of some enzymes in the pathway has been observed (Table 3.1), but the basis of their

regulation is not understood. Indeed, it is typical to find the genes encoding enzymes of the pathway dispersed throughout the genomes of different organisms, suggesting that there is no collective regulatory scheme for controlling the expression of the genes of the pathway.

Table 3.1. Previously observed regulation of the ethylmalonyl- CoA pathway Observed 1 Gene Regulation Enzyme (Locus Tag) Enzyme Protein Activity phaA β ketothiolase (rsp_0745) acetoacetyl-CoA phaB reductase (rsp_0747) crotonyl-CoA ccr 2 ≥60-fold carboxylase/reductase (rsp_0960) ecm ethylmalonyl-CoA mutase (rsp_0961) methylsuccinyl-CoA mcd 3 4 dehydrogenase (rsp_1679)  ≥100-fold

mch 3 mesaconyl-CoA hydratase  (rsp_0973) β methylmalyl-CoA/malyl-CoA mcl1 3 5 lyase (rsp_1771)  10-fold mcl2 malyl-CoA thioesterase 3 5 (rsp_0970)  4-fold pccA/pccB propionyl-CoA carboxylase (rsp_2189/ rsp_2191)

mcm 3 methylmalonyl-CoA mutase  (rsp_2192) ethylmalonyl- epi CoA/methylmalonyl-CoA (rsp_0812) Figure 3.1. The ethylmalonyl-CoA epimerase 1 pathway. See Figure 1.2 for Regulation indicates increased protein or enzymatic activity in extracts from acetate-grown cells compared to extracts from succinate- or glucose-grown cells. additional detail and Table 3.1 for a 2 description of the enzymes and the Reference (34) 3Reference (1) corresponding genes. 4 Reference (37) 5 Reference (36)

For example, CcrR, a transcription activator of ccr in Methylobacterium extorquens, has no homolog in R. sphaeroides, and there is no evidence that it affects expression of other ethylmalonyl-CoA pathway genes in M. extorquens (54).

In comparison, strategies for regulating acetyl-CoA assimilation through the glyoxylate bypass are well-studied in Escherichia coli and Corynebacterium glutamicum.

In E. coli, evidence indicates that there is a complex interplay of at least four different transcriptional regulators that combine with phosphorylation of isocitrate dehydrogenase to control how much carbon flows through the glyoxylate bypass during acetate growth

(24, 48, 73, 74, 101). For acetyl-CoA assimilation in C. glutamicum, a primary regulator is RamB (Regulator of acetate metabolism). By binding to the consensus DNA sequence

AAA(A/G)CTTTGCAAA, RamB represses expression of genes that encode proteins of the glyoxylate bypass and genes whose products are responsible for activating acetate to acetyl-CoA (42).

When considering potential regulatory strategies of acetyl-CoA assimilation in R. sphaeroides, it is important to recognize that the ethylmalonyl-CoA pathway shares two enzymatic steps (PhaA and PhaB) with polyhydroxybutyrate biosynthesis. Many organisms are known to produce polyhydroxybutyrate, and some aspects of the regulation of the process is understood. Reports indicate that the transcriptional regulator PhaR is responsible for repressing phaZ, encoding polyhydroxybutyrate depolymerase, in R. sphaeroides FJ1 (18), and phaP, encoding phasin, in R. sphaeroides FJ1 and Paracoccus denitrificans (18, 71). Moreover, in P. dentirificans and R. sphaeroides FJ1, in vitro work has demonstrated that PhaR binds sequences that contain a conserved inverted

repeat (CTGCN3/4GCAG) (18, 59, 72). Further in vitro work with R. sphaeroides FJ1

PhaR indicates that any sequence variation within the aforementioned repeat abolishes all binding by PhaR (19).

The following work seeks to identify regulatory schemes of the ethylmalonyl-

CoA pathway in R. sphaeroides. Because Ccr catalyzes the committed step of the ethylmalonyl-CoA, the work focuses on regulation of Ccr activity, ccr transcript levels, and ccr promoter expression in a variety of mutant and reporter strains. The results can ultimately be used to narrow the possible regulatory strategies of the overall pathway while beginning to offer insight into the precision required for optimal intermediary metabolism.

3.2 Material and Methods

3.2.1 Bacterial strains and growth conditions. Rhodobacter sphaeroides 2.4.1 (DSMZ 158) was grown aerobically in the dark or anaerobically in the light (3,000 lux) at pH 6.8 and 30°C in minimal media (MM) that contained 15 mM potassium phosphate buffer pH 6.7 and per liter: 0.2 g (0.8 mM)

MgSO4, 0.07 g (0.5 mM) CaCl2, 1.2 g (22 mM) NH4Cl, and 1.5 mL of vitamin solution

(per liter: 100 mg cyanocobalamin, 300 mg pyridoxamine-2∙HCl, 100 mg calcium-D(+)- pantothenate, 200 mg thiamine dichloride, 200 mg nicotinic acid, 80 mg 4-aminobenzoic acid, 20 mg D(+)-biotin), 10 mL trace element solution (per liter: 500 mg disodium

EDTA, 300 mg FeSO4∙7H2O, 3 mg MnCl2∙4H2O, 5 mg CoCl2∙6H2O, 1 mg CuCl2∙2H2O,

2 mg NiCl2∙6H2O, 3 mg Na2MoO4∙2H2O, 5 mg ZnSO4∙7H2O, and 2 mg H3BO3, pH3), 10 mM of each respective carbon source (in the case of two carbon sources, each was 46

supplied at 10 mM). For R. sphaeroides mutant strains, 25 µg/mL and/or 20 µg/mL kanamycin was added. Media plates included 2.5% agar. Oxygen in liquid cultures was removed and replaced with N2 by repeated vacuuming and sparging. Growth in liquid cultures was monitored as an increase in optical density (OD) at 578 nm. Media for R. sphaeroides contained 25 µg/mL spectinomycin and/or 20 µg/mL kanamycin as necessary. E. coli strains DH5α, S17-1 (114), and SM10 (114) were grown in Luria-

Bertani (LB) broth at 37°C with 50 µg/mL spectinomycin or 50 µg/mL kanamycin as necessary.

Cells to be lysed for generating cell extracts for enzyme assays and immunoblots were grown in 1 L bottles of minimal media as described above. Cultures were incubated with stirring at 30°C photoheterotrophically (anaerobically in the light) until an OD578 nm

= 0.35 – 0.55 was reached. Cells were collected by centrifugation at 8,000 x g for 10 min. Resuspended pellets were divided into five equal aliquots, and the excess media was removed. Pellets were stored at -80°C until use.

To obtain growth curves, the OD578 nm of 5 mL cultures in minimal media was observed in stoppered, screw-capped (Hungate) tubes. Unless otherwise stated, 5 mL sodium succinate precultures with appropriate antibiotics were grown to late exponential phase (OD578 nm ~ 1.5) before 0.1 mL was inoculated into the fresh medium to be used in growth studies.

3.2.2 Preparation of cell extracts. Frozen cell pellets (400 – 600 mg) were suspended in 600 µL of 50 mM Tris∙HCl pH 8.0, 5 mM MgCl2, and 0.1 mg/mL DNase I. After addition of ~ 1 g glass beads

(Retsch 0.1 – 0/25 mm diameter), the suspension was beaten at 30 Hz for 9 min with a bead beater (Retsch MM200). Insoluble cell material and beads were separated from cell extract by centrifugation at 15,800 x g for 10 min at 4°C. Protein concentrations were determined by the method of Bradford (12) (Appendix A.7). A series of dilutions of each sample was performed in 200 µL to which 900 µL of Bradford Reagent (per liter: 100 mg

Coomassie Blue G-250, 50 mL methanol, 100 mL 85% phosphoric acid, 850 mL water) was added. Reactions were incubated for 30 min at room temperature before the absorbance at 595 nm was recorded. Absorbance values were compared to a standard curve of 0 - 8 µg bovine serum albumin (BSA).

3.2.3 Plasmid transfer by conjugation. Plasmids were transferred into R. sphaeroides strains by conjugation with either

E. coli S17-1 (for plasmids that encode kanamycin resistance) or E. coli SM10 (for plasmids that encode spectinomycin resistance). The strains to be mated were grown in

LB liquid to OD ~ 0.5. Cells were collected by centrifugation and washed in 1 mL LB.

Cell densities in each suspension were estimated to be linearly related to the final OD of their cultures. Volumes of each cell suspension were mixed together to yield a final volume of 1 mL that contained approximately the same number of cells of each strain.

The cells were collected again by centrifugation before being resuspended in 100 µL LB.

The entire volume was dropped into the middle of an LB plate and allowed to incubate in nonselective conditions 24 hr at 30°C in the dark. Approximately 15% of the spot was scraped from the surface of the LB plate and suspended in 100 µL minimal media with appropriate antibiotics. Dilutions of the cell suspension were performed in 100 µL

minimal media that contained succinate and appropriate antibiotics. For strains that contained extrachromosomal plasmids, the suspension was diluted 1:10 and 1:1000, and both dilutions were spread on minimal media plates that contained succinate and appropriate antibiotic. For strains that contained a chromosomally recombined plasmid, the suspension was diluted 1:100, and the dilution and the undiluted suspension were spread on minimal media plates that contained succinate and appropriate antibiotics.

Isolated colonies were further streaked on minimal media plates that contained succinate and appropriate antibiotic. Plasmid-containing strains were isolated as single antibiotic resistant colonies after an additional passage on minimal media plates that contained succinate and appropriate antibiotic.

3.2.4 Immunoblot. Polyclonal anti-Ccr antibodies were generated by immunizing rabbits with purified recombinant Ccr from R. sphaeroides (Tobis Erb). Proteins in the cell extract were separated by 10% sodium dodecyl-sulfate polyacrylamide gel electrophoresis.

Proteins were transferred from the gel to a Millipore PVDF Immobilon-P membrane using a GeneMate Electroblotter. The membrane was incubated at room temperature for

1 hr in 0.01 g/mL instant milk powder (Carnation) in TTBS (0.2 M Tris pH 7.5, 0.15 M

NaCl, 0.1% Tween 20). The membrane was subsequently washed with TTBS before a

30 min incubation at room temperature with anti-Ccr antibodies diluted 1:10,000 in

TTBS. The membrane was again washed with TTBS before incubating it with the secondary antibody (Bio-Rad Goat Anti-Rabbit IgG conjugated to alkaline phosphatase) diluted 1:7,500 in TTBS.

Locations on the membrane that contained alkaline phosphatase activity were observed on a Storm phosphorimager by detection of light emission upon exposure to 1%

(v/v) AttoPhos Reagent (Promega). Signal quantification in each lane was accomplished using ImageQuant (GE) software to perform densitometric measurement of the Ccr band in each lane. A standard curve of densitometric values was assembled with relation to the amount of protein from extracts of acetate-grown cells (Figure 3.2 lanes 3, 4, 5, 6, 7).

Values from the linear portion (r2 ≥ 0.99) of the standard curve (values from lanes 5, 6, 7) were normalized to the amount of protein loaded into each respective lane. To estimate the relative quantities of detected Ccr between succinate and acetate growth conditions, the average of the normalized acetate values was compared to the normalized value of the densitometric measurement of the Ccr band in the lane loaded with extract from succinate-grown cells (Figure 3.2 lane 1). (Appendix A.15).

3.2.5 Crotonyl-CoA carboxylase/reductase assay.

̅ Activity was measured as the crotonyl-CoA-dependent, HCO3-dependent, and enzyme-dependent oxidation of NADPH at 30°C by observing the change in absorbance at 365 nm (ε = 3,400 M-1cm-1) in a cuvette with a 0.1 cm path length. The 200 µL reactions contained 3.3 mM NADPH, 100 mM Tris pH 7.8, 3.3 mM crotonyl-CoA, and

70 – 520 µg protein from cell extract and were initiated by the addition of 7 µL of 1 M

NaHCO3. Because detection of Ccr activity is limited by the sensitivity of the spectrophotometer to measure changes in absorbance, lower limits of Ccr activity vary depending on the protein content of each cell extract. (Appendix A.3).

3.2.6 β-Galactosidase assay. Activity was observed as the enzyme-dependent cleavage of ortho-nitrophenyl-β- galactosidase (ONPG), which was measured at 30°C by the change in absorbance at 412 nm (ε = 4500 M-1cm-1) in a cuvette with a 1 cm path length. A 500 µL reaction that contained 70 – 520 ng of protein from cell extract was initiated by the addition of 100 µL of 4 mg/mL ONPG in Z-buffer to 400 µL Z-buffer ( Z-buffer: 50 mM phosphate buffer

(pH 7.0), 40 mM KCl, 1 mM MgSO4, and 0.35% β-mercaptoethanol). Because detection of β-galactosidase activity is limited by the sensitivity of the spectrophotometer to measure changes in absorbance, lower limits of β-galactosidase activity vary depending on the protein content of each cell extract. (Appendix A.6).

3.2.7 RNA isolation.

RNA was isolated from 200 mL of culture at OD578 nm = 0.4 – 0.5. Cells were collected by centrifugation (8,000 x g, 10 min, 4°C) and immediately resuspended in 1.3 mL of TRI-Reagent (Sigma). After incubation at room temperature for 30 min, 650 µL of chloroform was added, and the vessel was briskly shaken to suspend the chloroform in the aqueous phase. The mixture was incubated at room temperature for 10 min before the aqueous and nonaqueous phases were separated by centrifugation (15,800 x g, 10 min,

4°C). The aqueous phase was added to 650 µL of fresh TRI-Reagent. Following incubation at room temperature for 5 min, 300 µL of chloroform was added, and the mixture was briskly shaken. The phases were separated again by centrifugation (15,800 x g, 10 min, 4°C). The aqueous phase was added to an equal volume (~1 mL) of isopropanol. After at least one hour incubation at -20°C, the precipitated nucleic acids were pelleted by centrifugation (15,800 x g, 10 min, 4°C) and dissolved in 400 µL 51

RNase-free water. DNA was degraded by adding 50 µL of 10X RNase-free DNase I buffer (Roche) and 500 U RNase-free recombinant DNase I (Roche). The reaction was incubated at 37°C for 1 hr. The RNA was precipitated again by addition of 500 µL of isopropanol and incubated at -20°C for at least 1 hr. Whole cell RNA was collected by centrifugation (15,800 x g, 10 min, 4°C) and dissolved in 200 µL RNase-free water.

(Appendix A.30).

3.2.8 Quantitative reverse transcription PCR (qRT PCR). Whole cell cDNA was produced in a 100 µL reaction by reverse transcribing 70

µL of whole cell RNA with 500 U Superscript III Reverse Transcriptase (Invitrogen) and

2 nmol of 10 nt oligomers of randomized sequence. Whole cell cDNA was diluted 44- fold to 352-fold in 2-fold steps in 22 µL PCR reactions that contained 14 µL IQ SYBR

SuperMix (BioRad) and 40 pmol of each respective primer. Amplification was performed for 40 cycles in a BioRad CFX96 themalcycer with the following amplification protocol: 97°C for 20 s, 64°C for 20 s, and 72°C for 10 s. To prevent detecting potential primer dimers, the reaction was heated to 81°C for 10 s before the fluorescence was recorded for each round of amplification. Each reaction was performed in triplicate for each dilution. Results of triplicates with threshold cycle (Ct) values that demonstrated 90% – 110% amplification efficiency were averaged and compared to standard curves in order to estimate the initial absolute value of cDNA template concentration. Averages from at least three sets of triplicates were used for each transcript. Standard curves were generated from Ct values of reactions containing known concentrations of ten-fold dilutions of purified PCR product that had been amplified from

R. sphaeroides chromosomal DNA with the same primers used in the experimental reactions. Primers used for detecting each cDNA are described in Table 3.2. Final absolute values were normalized to the average of the values of rpoZ (encoding the RNA polymerase omega subunit) and recA (encoding a recombinase) levels for comparison among biological replicates, strains, and growth conditions. (Appendix A.31).

3.2.9 Identification of the ccr transcriptional start site. A primer extension method was employed to establish the 5′ end of ccr as described (87). Briefly, MSACEccrR1, fluorescently labeled with 6-carboxyfluorescein

(FAM) on its 5′ terminus, was used to prime reverse transcription in whole cell RNA isolated from Rs(pMC19_2). The migration of the reverse transcription product in an

ABI 3770 capillary electrophoresis sequencer was compared to the migration of sequencing reactions performed on the corresponding DNA region with the same 5′ FAM labeled primer. Comigration with a given sequencing product was indicative of the 5′ position of the given transcript. (Appendix A.28).

3.2.10 DNA Motif Identification. DNA sequences suspected to contain similar features were compared using

Multiple Expectation-maximization for Motif Elicitation (MEME) (4). For motif identification upstream of ccr, the region between ccr and ecm was compared among

Rhodobacter sphaeroides 2.4.1, Ruegeria sp. TM1040, Jannaschia sp. CCS1,

Rhodobacter capsulatus SB1003, and Ruegeria pomeroyi DSS-3. Organisms were chosen from species with a close relationship to R. sphaeroides and with the full complement of genes of the ethylmalonyl-CoA pathway (35). The sequence comparison

yielded letter probability matrices of potential motifs that are displayed in Figure 3.4.

The letter probability matrix determined by MEME for the blue motif was used as a query in the Motif Alignment & Search Tool (MAST) (4) in order to scan the R. sphaeroides genome for other instances of the motif.

3.2.11 Isolation of R. sphaeroides deletion mutants 3.2.11.1 Plasmid construction for isolation of in-frame deletion mutants.

Each of the following plasmids was generated in order to delete a significant portion of the indicated gene, yielding an in-frame markerless inactivation of the coding region. In order to avoid disrupting any potential regulatory signals, small portions of each terminus of the respective coding region remain intact in the final mutant strain. In general, vectors were constructed by amplifying about 1500 bp of the upstream and downstream flanking regions of each respective gene. The amplified flanking regions were fused and inserted into pK18mobsacB (105), a suicide vector that does not contain an origin of replication that is recognized by R. sphaeroides. The pK18mobsacB vector also contains a kanamycin resistance marker and a counterselectable sucrose sensitivity marker. Deletion inactivation mutant strains of R. sphaeroides were isolated after double homologous recombination with the appropriate plasmid.

Plasmids denoted as pKB and the strains that were isolated after double recombination with pKB plasmids are a result of work performed by Kelsey Baron unless otherwise stated.

For inactivation of ccr (rsp_0960) in strain Δccr23KB, plasmid pKB15 was developed. The pKB15 plasmid contains an in-frame deletion of 1,132 nt of rsp_0960 to

yield a coding region that consists of 164 nt of the 5′ portion and 36 nt of the 3′ portion of rsp_0960 separated by a KpnI site. The hypothetical gene product is a 54 amino acid peptide. To construct pKB15, the 1636 bp product of deltaccr_KpnI_5′ and deltaccr_XbaI_3′ (nested PCR) and the 1533 bp product of deltaccrdown_for1 and deltaccrdown_rev1 (nested PCR) were digested with XbaI/KpnI and EcoRI/KpnI, respectively, prior to sequential ligation into pUC18 digested with XbaI/KpnI and

EcoRI/KpnI. The combined fragments were moved into pK18mobsacB via digest with

XbaI/EcoRI followed by ligation. (Appendix A.19).

Plasmid pKB22 was constructed for the inactivation of ecm (rsp_0961) in strain

Δecm47KB. The pKB22 plasmid contains an in-frame deletion of 1895 nt of rsp_0961 to yield a coding region that consists of 14 nt of the 5′ portion and 52 nt of the 3′ portion of rsp_0961 separated by a KpnI site. The hypothetical gene product is a 20 amino acid peptide. To construct pKB22, the 1,507 bp product of deltaecmup_for1 and deltaecmup_rev1 and the 1804 bp product of deltaecmdown_for1 and deltaecmdown_rev1 were digested with KpnI and HindIII/KpnI, respectively, before sequential ligation into pUC19 digested with KpnI then HindIII/KpnI. The combined fragments were moved into pK18mobsacB via digest with HindIII/PstI followed by ligation. (Appendix A.20).

The pKB4 plasmid was generated to inactivate mcd (rsp_1679) in strain

Δmcd11KB . The pKB4 plasmid contains an in-frame deletion of 1,528 nt of rsp_1679 to yield a coding region that consists of 8 nt of the 5′ portion and 111 nt of the 3′ portion of rsp_1679 separated by 4 nt of a BamHI site. The hypothetical gene product is a 40

amino acid peptide. To construct pKB4, the 1,510 bp product of deltamcd_EcoRIXbaI_5′ and deltamcd_KpnI_3′ and the 1567 bp product of deltamcd_XbaI_3′ and deltamcd_KpnI_5′were digested with EcoRI/KpnI and XbaI/KpnI, respectively, before sequential ligation into pUC19 digested with EcoRI/KpnI then

XbaI/KpnI. The combined fragments were moved into pK18mobsacB via digest with

XbaI followed by ligation. (Appendix A.21).

For inactivation of mch (rsp_0973) in strain Δmch49KB, the pKB23 plasmid was constructed. The pKB23 plasmid contains an in-frame deletion of 942 nt of rsp_0973 to yield a coding region that consists of 44 nt of the 5′ portion and 47 nt of the 3′ portion of rsp_0973 separated by 2 nt of a BamHI site. The hypothetical gene product is a 30 amino acid peptide. To construct pKB23, the 1,517 bp product of deltamchup_for1 and deltamchup_rev1 and the 1614 bp product of deltamchdown_for1 and deltamchdown_rev1 were digested with XbaI/BamHI and HindIII/BamHI, respectively, before sequential ligation into pUC19 digested with XbaI/BamHI then HindIII/BamHI.

The combined fragments were moved into pK18mobsacB via digest with HindIII/XbaI followed by ligation. (Appendix A.22).

Plasmid pKB11 was developed for the inactivation of mcl1 (rsp_1771) in strain

Δmcl1_4KB . The pKB11 plasmid contains an in-frame deletion of 1,225 nt of rsp_1771 to yield a coding region that consists of 19 nt of the 5′ portion and 116 nt of the 3′ portion of rsp_1771. The hypothetical gene product is a 44 amino acid peptide. To construct pKB11, the 1503 bp product of deltamcl1up_for1 and deltamcl1up_rev1 and the 1508 bp product of deltamcl1down_for1 and deltamcl1down_rev1 were digested with XbaI/SphI

and SphI/HindIII, respectively, before sequential ligation into pUC19 digested with

XbaI/SphI then SphI/HindIII. The combined fragments were moved into pK18mobsacB via digest with HindIII/XbaI followed by ligation. (Appendix A.23).

The pMC70 plasmid was generated to inactivate pccR (rsp_2186) in strain

RsΔpccRMC12. The pMC70 plasmid contains an in-frame deletion of 1,225 nt of rsp_2186 to yield a coding region that consists of 28 nt of the 5′ portion and 135 nt of the

3′ portion of rsp_2186 separated by a KpnI site. The hypothetical gene product is a 44 amino acid peptide. To construct pMC70, the 1513 bp product of D_ramB_upF2 and

D_ramB_upR2 and the 1838 bp product of D_ramB_downF2 and D_ramB_downR2 were digested with KpnI/BamHI and EcoRI/KpnI, respectively, before sequential ligation into pUC18 digested with KpnI/BamHI then EcoRI/KpnI. The combined fragments were moved into pK18mobsacB via digest with XbaI/BamHI followed by ligation. (Appendix

A.24).

The pMC69 plasmid was generated to inactivate phaR (rsp_0380) in strain

RsΔphaRMC43. The pMC69 plasmid contains an in-frame deletion of 456 nt of rsp_0380 to yield a coding region that consists of 75 nt of the 5′ portion and 24 nt of the

3′ portion of rsp_0380 separated by a KpnI site. The hypothetical gene product is a 34 amino acid peptide. To construct pMC69, the 1,553 bp product of DphaRUpF2 and

DphaRUpR2.2 and the 1511 bp product of DphaRDnF1.1 and DphaRDnR1 were digested with EcoRI/KpnI and HindIII/KpnI, respectively, before sequential ligation into pUC18 digested with EcoRI/KpnI then HindIII/KpnI. The combined fragments were

moved into pK18mobsacB via digest with XbaI/HindIII followed by ligation. (Appendix

A.26).

3.2.11.2 Isolation of mutant strains.

As described above, vectors carrying the inactivated gene fragments were introduced into R. sphaeroides by conjugation with E. coli S17-1 that had been transformed with the appropriate suicide vector. Single kanamycin resistant recombinant strains were genetically characterized by comparing PCR products of genomic regions that include the potential sites of recombination. Primer pairs for genotypic analysis were designed to anneal to the genome of the final mutant strain immediately outside of the upstream and downstream fragments that were included in the respective suicide vector.

When genomic DNA from single recombinant strains was used as template, product sizes indicated the presence of an upstream or downstream recombination event.

To isolate double recombinant strains, isolated colonies of single recombinant strains were incubated chemoheterotrophically overnight in 100 µL minimal media succinate without antibiotics. The entire volume was then spread onto a minimal media succinate plate with 10% sucrose. Isolated colonies were picked and patched to compare growth among minimal media that contained succinate, succinate and kanamycin, or succinate and sucrose plates. Strains in which a plasmid remains integrated into the chromosome were sensitive to the presence of sucrose because of the presence of sacB on the plasmid. Strains in which double recombination events had occurred did not grow on plates containing kanamycin. Among the kanamycin sensitive strains, colony PCR was used to amplify across the deleted region to identify which strains were wild type

revertants and which strains were deletion mutants. Genomic deletions were confirmed by PCR analysis and sequencing of the affected region.

3.2.12 Isolation of R. sphaeroides reporter strains

3.2.12.1 Isolation of the ccr-lac chromosomal fusion reporter strain, Rs ccr- lac1A.

The pMC26F plasmid was employed in the isolation of the Rs ccr-lac1A strain in which the lac operon is fused to the endogenous chromosomal ccr promoter. The pMC26F plasmid contains 638 nt of the ccr upstream region, including 24 nt of the ccr coding region, translationally fused to lacZ at an EcoRI site. The lac operon is followed by 494 nt of the ccr downstream region, including 231 nt of the ccr coding region separated from lacA by a PstI site.

Construction of pMC26F began with amplification of a 526 bp fragment of the ccr downstream with primers ccrmut_for7 and ccrmut_rev7. Due to difficulties in amplification from the R. sphaeroides genome, plasmid pccr03 was generously provided by Tobias Erb to be used as template for ccrmut amplification. Plasmid pccr03 contains the ccr coding region, 235 nt of its upstream, and 277 nt of its downstream region. After digesting with PstI/NdeI, the ccrmut product was ligated into pUC19 to make pMC22.

The lac operon was amplified from pMC1403 (112) with primers lacoperon_for1 and lacoperon_rev1. The lacoperon product (5062 bp) was digested with HindIII/PstI before ligation into pUC19 to make pMC27. A 5042 bp fragment was then removed from pMC27 by HindIII/PstI digest and ligated into pMC22 to make pMC23. The ccr upstream fragment, a 665 bp product of primers mutccr_for2 and mutccr_rev2 amplified

from the R. sphaeroides genome, was digested with EcoRI/KpnI before ligation into pUC18 to make pMC15_2. The ccr upstream fragment (684 bp) was removed from pMC15_2 via EcoRI/HindIII digest and ligated into pMC23 to make pMC24. The pMC26F (“F” indicates that the lac operon is encoded on the same strand as the spectinomycin resistance gene) plasmid is the result of moving the entire assemblage of the three fused fragments (6,193 bp) into pJQsm2MCS (described below) via XbaI digest and ligation. The final construct was mated into R. sphaeroides as described above, and single recombinant strains were isolated from single spectinomycin resistant colonies.

(Appendix A.27).

The pJQSm2MCS plasmid was constructed to replace pJQ200mp18 (99) that contained a 308 bp repeat on either side of its multiple cloning site and carried a gene for gentamicin resistance. The pJQSm2MCS5 plasmid replaces the region that contains the duplications and the multiple cloning site with a new multiple cloning site that includes

BamHI, XbaI, and PstI. It also substitutes the gentamicin resistance gene for a gene that imparts spectinomycin and streptomycin resistance. To construct pJQSm2MCS, a 5,276 bp PCR amplification product of pJQ200mp18 with primers JQm_for3 and JQm_rev3 and a 958 bp PCR amplification product of pBBRsm2MCS5 (107) with primers sm_for1 and sm_rev2 were digested with BglII and NcoI and subsequently ligated together. In order to insert a desired multiple cloning site, the resulting plasmid was PCR amplified with primers JQMCS_for2 and JQMCS_rev1, digested with XbaI, and intramolecularly ligated to yield pJQSm2MCS.

3.2.12.2 Transposon mutagenesis of strain Rs ccr-lac1A.

The pRL27 plasmid was transferred into Rs ccr-lac1A by conjugation with

BW20767(pRL27) (67) as described above. The pRL27 plasmid contains the mini-Tn5 transposon, encodes a transposase outside of the transposon, and is replicated from a Pir- dependent origin of replication, which is not recognized by R. sphaeroides. The cells scraped from the conjugation spot were suspended in 100 µL of minimal media succinate kanamycin/spectinomycin and diluted 1:10. The diluted and undiluted suspensions were spread on minimal media succinate kanamycin/spectinomycin plates that had been pre- spread with 50 µL of 2% 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (XGal).

Plates were incubated at 30°C anaerobically in the light. Nearly all colonies eventually became blue, so only colonies that rapidly presented the most vibrant blue were selected for additional screening. A transposon was identified in the genome of strain Rs ccr- lacS9 at position 456371. It interrupts a gene annotated as pyruvate phosphate dikinase.

3.2.13 Identification of transposon insertion sites. The genomic positions of transposon insertion were identified by cleaving genomic DNA and performing intramolecular ligation to form circularized genomic fragments. Fragments that contain the transposon also contain the Pir-dependent origin of replication, oriR6K, forming a transposon-containing plasmid (plasposon).

Genomic DNA (~ 1 µg) was digested with 10 U NcoI in a 30 µL reaction for 1 hr at 37°C. The reaction was stopped by heat inactivation at 65°C for 20 min. A 15 µL aliquot of the fragmented genome was added into a 50 µL ligation reaction containing 1.5

µL of T4 Ligase (Fermentas). The ligation reaction was incubated at room temperature

for 12 – 14 hr before a 25 µL aliquot was used to transform chemically competent DH5α

λpir. Plasposons were isolated from kanamycin resistant DH5α λpir cells. Primers tpnRL17-1 and tpnRL13-2, which anneal to opposite ends of the transposon, were used to sequence the regions of the plasposon that immediately flank the transposon. (Appendix

A.28).

3.2.14 Isolation of R. sphaeroides strains containing ccr-lac reporter plasmid fusions. The pMC83 plasmid series contains plasmid-borne fusions of varying truncations of the ccr upstream region fused to lacZ. Each plasmid in the series was constructed within the pMC76 scaffold that was built from pMC75. Plasmid pMC75 was based upon pBBRsm2MCS5 but removes the lacZ fragment within the multiple cloning site.

To develop pMC75, the lac operon was amplified from pMC27 with primers lacoperon_for1 and lacoperon_rev2 (5,071 bp). A 4193 bp fragment was also PCR amplified from pBBRsm2MCS5 (107) with primers MC75F3 and MC75R2. By way of

EcoRI/KpnI digest and subsequent ligation, both products were ligated together to form pMC75. To construct pMC76, the product of primers D_ramB_upF2 and

D_ramB_UpR2 (1,513 bp) was amplified from R. sphaeroides chromosomal DNA and cloned into pMC75 by XbaI/EcoRI digest and subsequent ligation. The presence of the

D_ramB_Up fragment serves to simplify future cloning, as its replacement by small promoter fragments is easily monitored throughout the cloning process. Fragments of the ccr upstream were amplified from the R. sphaeroides genome with the forward primer mutccr_for2 and reverse primers mutccrR72, mutccrR97, mutccrReg_rev2, or mutccrReg_rev1.2. Upstream fragments were ligated into pMC76 following cleavage

with XbaI or BamHI and EcoRI to generate pMC83-72, pMC83-97, pMC83-191, and pMC83-292, respectively. Each plasmid was mated into R. sphaeroides as described above. Plasmid-containing strains were isolated from single spectinomycin resistant colonies.

Table 3.2. Primers used in Chapter 3 Primer Sequence (5′  3′)1 Use mutccrR72 TGACGCTGCGTGGATCCAGACAGATTATTACAAAATGAGAC -72 bp ccr upstream fragment mutccrR97 GAAATCTAGACGCCCGCGGTGACGCTGC -97 bp ccr upstream fragment mutccrReg_rev2 TATCTATTCTAGAAACAGGATCCTGCGTCGCGCTCC -191 bp ccr upstream fragment mutccrReg_rev1.2 ACTCGTCTAGAGCAAGGATATTGCTGCATGTCGCATTTATG -292 bp ccr upstream fragment mutccr_for2 CGATTAAGAATTCATCGCTCTGCACGTCGAGGG ccr upstream fragment mutccr_rev2 TACCGAAGGTACCACGGAATCTGGTCGAACAGCATCCGCATGTC -561 bp ccr upstream fragment ccrmut_for7 CATCAGAAGCGTCTGCAGGGCAGCCACTTCG ccr downstream fragment ccrmut_rev7 ATCTCGAGCATATGTCTAGAGAGCCCGGCCGCCAGC ccr downstream fragment deltaccr_KpnI_5′ TAGGTACCCCGGATGGCCCAG ccr upstream for Δccr23KB deltaccr_XbaI_3′ GTTCTAGACGACGAGCCGCGC ccr upstream for Δccr23KB deltaccrdown_for1 GCTAGGTACCTTCGCCGACGTGCTC ccr downstream for Δccr23KB deltaccrdown_rev1 AGAGAATTCACCTGAGCGCCCTTGATG ccr downstream for Δccr23KB DphaRUpF2 GCCGAATTCTAGACCACGCTCACCGACTTTTCGGACCC phaR upstream for RsΔphaRMC43 DphaRUpR2.2 TCCTCGAGGGTGGTACCGTCGCTGGTCTCGGTGTTGTAGAG phaR upstream for RsΔphaRMC43 phaR downstream for DphaRDnF1.1 GCTGGCCGGTACCCAGAAGAAGCTCTCGAAGCTCTGAC RsΔphaRMC43 phaR downstream for DphaRDnR1 GCGGCCGTAAGCTTTCTCCGATCATCCTTCAGGGAGTGTTTGAC RsΔphaRMC43 D_ramB_upF2 CCGCCCGGATCCAGACCATCCAGGAGGTCATCAC pccR upstream for RsΔpccRMC12 D_ramB_upR2 GCTCGCGGTACCTCGCCCCGGCATAGAGTTTC pccR upstream for RsΔpccRMC12 pccR downstream for D_ramB_downF2 TGAAGGGGTACCTCGAGCCGATCGGCATCTCC RsΔpccRMC12 pccR downstream for D_ramB_downR2 GCTCGAATTCTAGAGTGGAGTATCCCGACATCCAGTATCACTTC RsΔpccRMC12 lacoperon_for1 AATGATAAGCTTGAATTCGATCCCGTCGTTTTACAACGTC ccr-lac operon fusion and pMC76 lacoperon_rev1 TGCACTGCAGTTAAACTGACGATTCAACTTTATAATCTTTGAAATAATAG ccr-lac operon fusion lacoperon_rev2 TGATATCGGTACCCTGCAGTTAAACTGACGATTC pMC76 plasmid construction JQm_for3 GTAACACCATGGCTGCTCCATAACATCAAACATC pJQSm2MCS plasmid construction JQm_rev3 AGAAGAGATCTACTTTGATATCGACCCAAGTACC pJQSm2MCS plasmid construction sm_for1 AGTCTCATCCATGGAGCGTAGCGACCGAGTGAG pJQSm2MCS plasmid construction sm_rev2 ATGTGGCGAGATCTCTTGAACGAATTGTTAGAC pJQSm2MCS plasmid construction JQMCS_for2 TCTAGAGTCGACCTGCAGGGTTCGCCCAGCTTCTGTATG pJQSm2MCS plasmid construction JQMCS_rev1 TCTAGAGGATCCCGGGTACCGAGCTCACGGCCGAGGTCTTCC pJQSm2MCS plasmid construction MC75F3 AGGAATTCTCTAGAAGCTTTAAACGCCTGGTGCTACGCCTGAATAAGTG pMC76 plasmid construction MC75R2 TGCGTATTGGGTACCTGCATAAAAACTGTTGTAATTCATTAAGCATTCTG pMC76 plasmid construction RTrpoZ_F2 ATCGACCGCGACAATGACAAGAAC detect rpoZ cDNA RTrpoZ_R2 CAGCGCCATCTGATCCTCTTCCG detect rpoZ cDNA RTrecA_F2 GGCAAGGGCTCGATCATGAAACTG detect recA cDNA RTrecA_R2 TTTCGGGGCCGTAGATCTCGATGATTC detect recA cDNA RTccr_for2 ATGTGCCGAAGGAGATGTATGCC detect ccr cDNA RTccr_rev3 GCGTCTCGACCACTTCGATCTG detect ccr cDNA ccr transcriptional start site MSACEccr_R1 GATGGCCCAGGCATACATCTCCTTCG identification deltaecmup_for1 TCAACTGGTTGGGACCCGACGCCTGCTTGAG ecm upstream fragment deltaecmup_rev1 GCCAGGCGGTACCCTTCTGGGTCATGGCAAG ecm upstream fragment deltaecmdown_for1 TTCGAGCTGAACGGTACCATGATGGATATCGTGGGTC ecm downstream fragment deltaecmdown_rev1 GGCTGGAAGCTTTGGCGCTCGGCCATTTG ecm downstream fragement deltamcd_EcoRIXbaI_5′ ATGAATTCTAGAAGCCGAACCCGAGATCTATGCCAC mcd upstream fragment deltamcd_KpnI_3′ ATAGGTACCGGTCATGGTCGATCCGTCCTTC mcd upstream fragment deltamcd_KpnI_5′ ATAGGTACCGCGCTGGAATATCAGATCAGCC mcd downstream fragment deltamcd_XbaI_3′ TACTCTAGAACTGCCCTACCAGCTCACCGTC mcd downstream fragment deltamchup_for1 GAAGAGGATCCTCTAGACCGCCGATTACGGCCATC mch upstream fragment deltamchup_rev1 GATGGTGGATCCCAGCCGGTAATCCTCGAAG mch upstream fragment deltamchdown_for1 AGTATGGATCCGGCGTCCTCCTGGATCTC mch downstream fragment deltamchdown_rev1 GTCTTCAAGCTTCCACGCCACCACGCCGGTG mch downstream fragment deltamcl1up_for1 GAAGAGGATCCTCTAGACCGCCGATTACGGCCATC mcl1 upstream fragment deltamcl1up_rev1 AGGCGGCGCATGCTGAAGGCGGAAGCTCATG mcl1 upstream fragment deltamcl1down_for1 GCGGCGATGCATGCGGCCAAGGCGAGGGGCGAG mcl1 downstream fragment deltamcl1down_rev1 CGCCCGAGCAAGCTTCGCCGATCACCACCGCCACC mcl1 downstream fragment tpnRL 13-2 CAGCAACACCTTCTTCACGA plasposon sequencing tpmRL 17-1 AACAAGCCAGGGATGTAACG plasposon sequencing

1Restriction sites are underlined.

Table 3.3. Strains and plasmids used in Chapter 3 Name Relevant characteristics Reference E. coli

SM10 RP4-2 Tc::Mu-Km::Tn7 (114) S17-1 RP4-2 Tc::Mu Tpr Smr Scr (114) BW20767 Parental strain for random transposon mutagenesis (67)

R. sphaeroides 2.4.1 (DSMZ 158) Wild type RsΔccrMC4 aph insertional deletion in ccr (107) Δccr23KB in-frame deletion inactivation of ccr (rsp_0960) This work Δecm47KB in-frame deletion inactivation of ecm (rsp_0961) This work Δmcd11KB in-frame deletion inactivation of mcd (rsp_1679) This work Δmch49KB in-frame deletion inactivation of mch (rsp_0973) This work Δmcl1_4KB in-frame deletion inactivation of mcl1 (rsp_1771) This work RsΔphaRMC43 in-frame deletion inactivation of phar (rsp_0380) This work RsΔpccRMC12 in-frame deletion inactivation of pccr (rsp_2186) This work Rs ccr-lac1A ccr promoter-lac operon chromosomal fusion, Smr Scr This work (Rs::pMC26F) Rs ccr-lacS1 Rs ccr-lac1A transposon mutant; sseA (rsp_0885)::Tn5-RL27(Kmr-oriR6K), pos. 2634504 This work Rs ccr-lacS2 Rs ccr-lac1A transposon mutant; sseA (rsp_0885)::Tn5-RL27(Kmr-oriR6K), pos. 2634865 This work Rs ccr-lacS3 Rs ccr-lac1A transposon mutant; sseA (rsp_0885)::Tn5-RL27(Kmr-oriR6K), pos. 2634954 This work Rs ccr-lacS9 Rs ccr-lac1A transposon mutant; ppdK (rsp_1859)::Tn5-RL27(Kmr-oriR6K), pos 456370 This work

Plasmids pJQ200mp18 Suicide vector (99) pJQSm2MCS Derived from pJQ200mp18 (removes sequence duplications, replaces Gmr) Scr Smr This work pBBRsm2MCS5 Broad host range vector, Scr Smr (107) pK18mobsacB Replication incompetent in R. sphaeroides, Kmr, Sucroses (105) pKB4 in-frame deletion inactivation of mcd This work pKB11 in-frame deletion inactivation of mcl1 This work pKB15 in-frame deletion inactivation of ccr This work pKB22 in-frame deletion inactivation of ecm This work pKB23 in-frame deletion inactivation of mch This work pMC19_2 ccr with endogenous promoter in pBBRsm2MCS5 (107) pMC26F Isolation of ccr promoter-lacZYA chromosomal fusion This work pMC69 in-frame deletion inactivation of phaR This work pMC70 in-frame deletion inactivation of pccR This work pMC76 cloning vector for plasmid-borne promoter-lacZYA fusions This work pMC83-72 ccr promoter (-72 – +77 bp)-lac operon fusion This work pMC83-97 ccr promoter (-97 – +77 bp)-lac operon fusion This work pMC83-191 ccr promoter (-191 – +77 bp)-lac operon fusion This work pMC83-292 ccr promoter (-292 – +77 bp)-lac operon fusion This work

3.3 Results

3.3.1 Ccr activity, Ccr protein, and ccr transcript levels are upregulated during acetate growth. Because acetyl-CoA assimilation via Ccr is required for acetate growth but is not involved in succinate growth, Ccr activity, Ccr protein levels, and ccr transcript levels were compared in acetate- and succinate-grown R. sphaeroides. In extracts from succinate grown cells, no Ccr activity was detected (Table 3.5). When cells were grown in the presence of equimolar succinate and acetate, observed Ccr activity increased to 110 nmol/min/mg. Observed Ccr activity further increased to 210 nmol/min/mg in extracts from cells grown with acetate as the sole carbon source. Based on quantification of Ccr immunodetection, >150-fold more Ccr protein was detected in extracts from acetate- grown cells than succinate-grown cells (Figure 3.2, Table 3.4, Figure 3.3). Similarly, ccr transcripts were 30-fold more abundant in extracts of acetate-grown cells than succinate- grown cells (Table 3.6). The observed upregulation at each level of ccr expression was consistent with regulation of transcription initiation.

Figure 3.2. Immunoblot for detection of Ccr in cell extracts of R. sphaeroides. Lane 1 contains 90 µg of protein from an extract of succinate-grown cells. Lanes 3, 4, 5, 6, and 7 contain 10, 5, 2.5, 1.3, and 0.6 µg, respectively, of protein from an extract of acetate-grown cells.

Table 3.4. Densitometric measurements of Ccr bands in the lanes of the immunoblot in Figure 3.2 Normalized Pixel Protein Loaded Lane Pixel Intensity Intensity (µg) (per µg protein) Succinate

1 90.0 1,500 16 Acetate

3 10.0 14,000 1,400 4 5.0 10,000 1,900 5 2.5 5,600 2,200 6 1.3 3,300 2,600 7 0.6 1,700 2,700

)

3 15

Pixel Intensity (x 10 (x Intensity Pixel 0 0.0 2.5 5.0 7.5 10.0 12.5 Protein (g)

Figure 3.3. Standard curve of densitometric values from Table 3.5 for quantification of Ccr. Values were calculated from Ccr bands detected in lanes containing various amounts of extract from acetate-grown R. sphaeroides as observed in the immunoblot in Figure 3.2. The line is the result of a linear regression calculation (r2 = 0.99) based on pixel intensities at 0.6, 1.3, and 2.5 µg protein.

Table 3.5. Crotonyl-CoA carboxylase/reductase (Ccr) activities in cell extracts of photoheterotrophically grown cells Ccr Specific Activity (nmol/min/mg)1,2 Strain Succinate Succinate/ Acetate Acetate R. sphaeroides 2.4.1 <6 (8) 110 ± 20 (8) 210 ± 30 (8) Δccr23KB <3 <4 NG3 RsΔccrMC4 <6 ND4 NG RsΔccrMC4(pMC19_2) 40 (3) 230 1800 (3) Rs(pMC19_2) 50 (4) 240 1900 (3) RsΔphaRMC43 <5 110 180 RsΔpccRMC12 <8 90 250 Δecm47KB <7 20 NG Δmcd11KB <4 60 NG Δmch49KB <6 20 NG Δmcl1_4KB <7 40 NG Rs ccr-lac1A <5 (3) 50 120 (3) Rs ccr-lacS1 60 40 100 Rs ccr-lacS2 30 30 90 Rs ccr-lacS3 40 (2) 60 100 (2) Rs ccr-lacS9 <10 50 NG Rs(pMC83-292) <4 (2) ND 220(2) Rs(pMC83-191) <6 (2) ND 250 (2) Rs(pMC83-97) <5 (2) ND 210 (2) Rs(pMC83-72) <5 (2) ND 190 (2) 1 Values are an average of the number of biological replicates indicated within the parentheses. Values that are not followed by parenthetical numbers are the result of a single sample. 2Errors represent standard deviations 3NG indicates no growth. 4ND indicates not determined.

Table 3.6. Regulation of ccr transcripts during photoheterotrophic growth with various carbon sources 1 Strain Relative ccr Transcript Levels ‾ Succinate Acetate Propionate/HCO3 R. sphaeroides 2.4.1 0.09 ± 0.03 2.66 ± 1.52 0.18 RsΔpccRMC12 0.12 ± 0.08 1.80 ± 0.90 NG2 1 Relative transcript levels are normalized to the average of the transcript levels of recA and rpoZ. Values that include a standard deviation represent the average relative transcript levels from at least three biological replicates. 2NG indicate no growth

3.3.2 Multiple conserved motifs exist in the intergenic region between ecm and ccr. The sequences between the coding regions of ecm and ccr were examined for motifs that were conserved among several chosen organisms that contain the entire complement of genes encoding the enzymes of the ethylmalonyl-CoA pathway. Three motifs were identified and are indicated by red, blue, and green boxes in Figure 3.4. The red motif was found upstream of ecm in three of the organisms. In the direction of ecm, it was the perfect consensus (TTGACA) for the minus 35 region of an E. coli σ70 promoter.

Figure 3.4. Identification of conserved motifs in the intergenic region of ecm/ccr for various α- proteobacteria. Conserved motifs and their relative locations are indicated as colored boxes. The consensus logo for each motif is illustrated within a correspondingly colored box. Gene annotations: ccr – crotonyl- CoA carboxylase/reductase, ecm – ethylmalonyl-CoA mutase, plsB – glycerol-3-phosphate acyltransferase, hyp – hypothetical.

The blue motif was characterized by the conserved inverted repeat

C(T/C)GCNNNGC(A/G)GC. The motif was found in three locations upstream of ccr in

R. sphaeroides. One was centered 228 bp upstream of the ccr transcriptional start site and immediately upstream of the red motif in the direction of ecm. Another was centered at 79 bp from the ccr transcription initiation site and contains a sequence

(CTGCNNNGCAG) that was a perfect match for inverted repeat within the PhaR binding site (CTGCN3/4GCAG) identified in R. sphaeroides FJ1 and Paracoccus denitrificans 69

(19, 59, 71). The final blue site was within the sequence that encodes the 5′ untranslated region of ccr.

In addition, the blue motif existed upstream of several other genes that are expected to be involved in acetyl-CoA assimilation (Table 3.7). The croR gene

(rsp_2305), for example, is the best candidate for the (R)-crotonase that is involved in the conversion of (3R)-hydroxybutyryl-CoA to crotonyl-CoA in the third step of the ethylmalonyl-CoA pathway. Also, the likely operon that would begin with mcl2 and continue with mch was preceded by an instance of the blue motif. Finally, an instance of the blue motif was upstream of the ppdK (pyruvate phosphate dikinase) and upstream of the likely operon that contains actP (acetate permease).

Table 3.7. Conserved motif upstream of genes for acetyl-CoA assimilation in R. spheroies 2.4.1 Distance from 1 Gene Locus Tag ATG (bp) Sites Consensus2 TTCTGCGNTGCGGCA

phaA rsp_0745 -45 TTCTGCACAGCGGCG croR rsp_2305 -36 TGCTGCATCGCGGCA ccr rsp_0960 -21 TTCTGCGACGCGGCG ecm rsp_0961 -71 TGCCGCGTCGCGGCG mcl2/mch3 rsp_0970/rsp_0972 -62 TTCTGCGCCGCGGCA ppdK rsp_1859 +9 TTCTGCACTGCGGCG hyp/actP4 rsp_3141/rsp_3142 -122 TGCTGCTCCGCGGCG 1 Positions that are highly conserved are underlined. 2Consensus refers to the blue motif identified in Figure 3.4. 3The mcl2 gene likely precedes mch in a single transcriptional element. 4An hypothetical gene likely precedes actP in a single transcriptional element.

To further contextualize the motifs upstream of ccr, the transcriptional start site of ccr was identified by primer extension (Figure 3.5). The primer extension product terminated at a guanosine residue located 54 nt upstream of the ccr translational start.

Centered 35 bp upstream of the position in the R. sphaeroides genome that corresponded 70

to primer extension termination was the green motif. The sequence of the green motif was similar to the consensus of the minus 35 region of the σ70 promoter in E. coli. It also contained the same palindromic sequence that is found within the binding site

(TTTGCAAA) of RamB in C. glutamicum (42).

Figure 3.5. Overlaid chromatograms that illustrate the sequencing reaction standards and the primer extension for identification of the ccr transcriptional start site. The open peaks represent relative retention times of fragments from the four sequencing reactions of the ccr upstream region with 5′FAM-labeled primer RTMSACEccr_R1. Each reaction contained a different dideoxynucleotide (Black, ddGTP; Red, ddTTP; Green, ddATP; Blue, ddCTP). The filled orange peak demonstrates the relative retention time of the 5′FAM-labeled cDNA fragment primed with the same primer.

3.3.3 Expression from the ccr promoter is largely controlled via a sequence between 191 bp and 72 bp upstream of its transcriptional start. In order to investigate which portions of the sequence upstream of the ccr coding region are involved in regulating expression from the ccr promoter, a reporter system was constructed in which truncated fragments of the ccr promoter were fused to the lac operon in the pMC83-series that is illustrated in Figure 3.6C.

Figure 3.6. Organization of the ccr promoter and the truncations used to investigate the effects of various motifs on the expression from the ccr promoter. The sequence of the upstream region of ccr in the chromosome of R. sphaeroides 2.4.1 (A). A diagram of the region of recombination of pMC26F in the chromosome of the reporter strain, Rs ccr-lac1A (B). An illustration of the truncated promoters that are fused to lacZYA in the pMC83 plasmid series (C). Colored regions represent colored motifs identified in Figure 3.4. The purple motif, within one of the blue motifs, is conserved upstream of phaR, phaZ, and phaP (see Table 3.9) and is similar to the PhaR binding sites in R. sphaeroides FJ1 (18) and P. denitrifcans (72). The orange sequence represents a palindromic motif of unknown function that was not identified during the in silico comparison that is illustrated in Figure 3.4.

Table 3.8. Regulation of Ccr (crotonyl-CoA carboxylase/reductase) and LacZ (β-galactosidase) activities in cell extracts of photoheterotrophically grown cells Ccr (nmol/min/mg)1 LacZ (nmol/min/mg)1

Strain Fold Fold Succinate Acetate Succinate Acetate Change Change R. sphaeroides 2.4.1 <6 (8) 210 (8) >35 <5 (3) <5 (3) -

Chromosomal Fusions Rs ccr-lac1A <5 (2) 120 (2) >24 10 (2) 1200 (2) 120

Rs ccr-lacS1 60 100 2 320 860 3 Rs ccr-lacS2 30 90 3 300 1000 3 Rs ccr-lacS3 40 100 3 360 1000 3

Plasmid Fusions Rs(pMC83-292) <4 (2) 220 (2) >35 100 (2) 1400 (2) 14

Rs(pMC83-191) <6 (2) 250 (2) >35 120 (2) 1600 (2) 16

Rs(pMC83-97) <5 (2) 210 (2) >35 60 (2) 660 (2) 11

Rs(pMC83-72) <5 (2) 190 (2) >35 80 (2) 300 (2) 3

1Values are an average of the number of biological replicates indicated within the parentheses. Values that do not include parentheses represent a single measurement.

Each resulting plasmid was transferred into R. sphaeroides. Each truncation sequentially removed motifs that were identified within the region.

When the lac operon was fused to the full length chromosomal ccr promoter (Rs ccr-lacS1A Table 3.8), β-galactosidase activity was upregulated 120-fold during acetate growth compared to succinate growth. However, when the Rs(pMC83-292) strain was grown with acetate, β-galactosidase was only 14-fold higher than during growth with succinate, indicating a loss of regulation, even with the longest plasmid-borne promoter fusion. Regulation of expression from the ccr promoter continued to decrease as the plasmid-borne promoter fragments decreased in length. For example, β-galactosidase activity was deregulated an additional 5-fold when the plasmid-borne promoter was truncated from 191 bp to 72 bp (Table 3.8). The intervening region includes the orange motif and a blue motif that contains a possible PhaR binding site (purple, Figure 3.6).

3.3.4 Interrupting carbon flow through the ethylmalonyl-CoA pathway affects Ccr activity. Relative to Ccr activity in extracts of R. sphaeroides grown with succinate/acetate, Ccr activity during succinate/acetate growth was decreased in any mutant strain in which a gene encoding a core enzyme downstream of Ccr in the ethylmalonyl-CoA pathway was inactivated (Table 3.5). The mutants display 40 – 80% less Ccr activity than R. sphaeroides during succinate/acetate growth. Despite the diminished Ccr activity levels, no detectable growth defect was observed (data not shown).

3.3.5 PhaR does not affect regulation of Ccr activity in photoheterotrophically succinate-, succinate/acetate-, or acetate-grown R. sphaeroides. Within the sequences between 191 bp and 72 bp upstream of the ccr promoter was a conserved blue motif that was centered at 79 bp from the ccr transcriptional start site and that resembles the PhaR binding site upstream of genes involved in polyhydroxybutyrate accumulation in R. sphaeroides FJ1 and P. denitrificans (19, 59,

72). In addition, comparable sites were found upstream of similar genes in R. sphaeroides 2.4.1 (Table 3.9). For the PhaR binding sites found in R. sphaeroides FJ1, it has been demonstrated in vitro that PhaR binding is dependent on the presence of the complete inverted repeat CTGCN3/4GCAG (19, 72).

Table 3.9. Potential PhaR binding sites upstream of various genes in R. sphaeroides 2.4.1 Distance from 1 Gene Locus Tag ATG (bp) Sites ccr rsp_0960 122 ACGCTGCGTTGCAGCAG phaZ rsp_0383 63 ATGCTGCCATGCAGCAT phaP rsp_0381 83 ATGCTGCGGTGCAGAAT phaR rsp_0380 17 CTTCTGCAGCCGCAGAA \1Underlined positions are necessary for PhaR binding to similar sites in R. sphaeroides FJ1(18) and are contained in PhaR binding sites in P. denitrificans (72).

In order to evaluate the effect of PhaR on the regulation of ccr expression, the

RsΔphaRMC43 mutant strain, containing an in-frame deletion inactivation of phaR, was isolated. Ccr activity in extracts of succinate-, succinate/acetate-, and acetate-grown cells did not significantly differ from Ccr activities in identically grown wild type R. sphaeroides cell extracts (Table 3.5), indicating that PhaR did not affect regulation of Ccr activity.

3.3.6 Loss of pccR does not affect regulation of Ccr activity in photoheterotrophically succinate-, succinate/acetate- or acetate-grown R. sphaeroides. The sequences between 191 bp and 72 bp upstream of the transcriptional start site of ccr contain a palindrome, illustrated in a green box in Figure 3.4, that is also found within the RamB binding site in C. glutamicum (42). In C. glutamicum, RamB is responsible for controlling, among other genes, the genes whose products are involved in the glyoxylate bypass and acetate activation to acetyl-CoA. The consensus for the RamB binding site is AAA(A/G)CTTTGCAAA (42). The gene that encodes the RamB homolog in R. sphaeroides, pccR, was inactivated by in-frame deletion in strain

RsΔpccRMC12. The loss of a functional pccR had no significantly detectable effect on

Ccr activities in extracts of succinate-, succinate/acetate-, and acetate-grown cells (Table

3.5). Consequently, PccR was not responsible for regulation of Ccr activity in the conditions observed here.

3.3.7 Random transposon mutants have alternatively regulated ccr expression. Rs ccr-lac1A is a reporter strain in which the endogenous chromosomal ccr promoter was fused to the lac operon, and a copy of the ccr promoter, containing 561 bp upstream of its transcriptional start, was fused to the endogenous ccr coding region

(Figure 3.6A). The strain was subjected to random transposon mutagenesis. Candidate mutants in which expression from the ccr promoter occurred in the absence of acetate were isolated from blue colonies on minimal media plates that contained succinate and

XGal. All characterized strains isolated from colonies that lacked a blue color on

succinate/acetate plates contained transposon insertions in lacZ, indicating that ccr

expression was likely unaffected.

After screening approximately 3,500 colonies, 51 strains were isolated from

colonies that initially appeared blue during growth on plates that contained succinate as

the sole carbon source. Upon additional screening, only 22 strains were noticeably bluer

than control strains when patched adjacent to control strains on plates containing

succinate as the sole carbon source. Of the 22 strains, the transposon insertion sites in the

genomes of the 11 bluest strains were characterized (Table 3.9).

Table 3.9. Transposon insertion sites in the genomes of selected mutant strains of Rs ccr-lac1A that displayed deregulation of lacZ expression from the ccr promoter during succinate growth Strain Tpn Insertion Genomic Locus Tag Annotated Gene Product Number Site Element S1 2634480 Chromosome 1 rsp_0885 SseA: 3-mercaptopyruvate sulfurtransferase S2 2634856 Chromosome 1 rsp_0885 SseA: 3-mercaptopyruvate sulfurtransferase S3 2634945 Chromosome 1 rsp_0885 SseA: 3-mercaptopyruvate sulfurtransferase S4 2330642 Chromosome 1 rsp_0587 Putative ABC transporter S8 248668 Chromosome 1 rsp_0742 Acyl-CoA dehydrogenase S9 456370 Chromosome 1 rsp_1859 PpdK: pyruvate phosphate dikinase S19 47465 Plasmid B rsp_7368 GntR family transcriptional regulator S22 3074004 Chromosome 1 rsp_1302 MotB: integral membrane flagellar subunit rsp_1667, Intergenic – Folk: 2-amino-4-hydroxy-6- S32 259508 Chromosome 1 hydroxymethyldihydropteridine rsp_1668 pyrophosphokinase;. conserved protein rsp_1667, Intergenic – Folk: 2-amino-4-hydroxy-6- S31 259467 Chromosome 1 hydroxymethyldihydropteridine rsp_1668 pyrophosphokinase; conserved protein S26 259216 Chromosome 1 rsp_1667 conserved protein

The regulation of Ccr and β-galactosidase activity was quantitatively

characterized in 4 of the 11 strains. For three strains – Rs ccr-lacS1, Rs ccr-lacS2, and Rs

ccr-lacS3 –transposon insertions were identified at three different locations within the

sseA (rsp_0885) coding region (Figure 3.7). For E. coli, SseA is well-characterized as a

rhodanese-like sulfur transferase. The CGSGVTA motif in the R. sphaeroides SseA distinctly indicates a preference for 3-mercaptopyruvate (instead of thiosulfate) as the thiol donor in its catalyzed reaction (21). In all reported cases, however, there is no evidence of the in vivo thiol acceptor. Immediately downstream of sseA in R. sphaeroides is aat, a gene that likely encodes aspartate aminotransferase.

The sseA and aat genes are separated only by the sequence CGAAGGA, which is a strong candidate for a ribosome binding site and is consistent with cotranscrption of sseA and aat. The transposon insertions in sseA occurred 207 bp, 583 bp, and 680 bp into the

849 bp sseA coding region (Figure 3.7A), and all transposon insertions were oriented such that their aph (aminoglycoside phosphotransferase for kanamycin resistance) is transcribed in the same orientation as the putative sseA/aat operon. In all three strains,

Ccr and β-galactosidase activity was elevated during succinate growth compared to the parent reporter strain, Rs ccr-lac1A (Table 3.8). There is no observed growth defect associated with the transposon insertions in these strains during succinate or acetate growth (Figure 3.8).

The fourth strain to be characterized, Rs ccr-lacS9, contains a transposon insertion in the pyruvate phosphate dikinase gene (ppdK; rsp_1859), whose protein product is responsible for the generation of phosphoenolpyruvate from pyruvate. At the translational start (GTG) of ppdK is an instance of a motif that is similar to the blue motif in Figure 3.4 (Table 3.7). The strain cannot grow with acetate yet can grow on succinate, albeit 60% more slowly than Rs ccr-lac1A (Figure 3.8). Ccr activity, β-galactosidase

activity, and potential growth defects during growth with succinate/acetate were not studied for Rs ccr-lacS9.

Figure 3.7. Illustrations of the locations of transposon insertions in ccr-lacZYA reporter strains isolated as blue colonies during growth on minimal media succinate supplemented with XGal. Locations of transposon insertions in the genomes of strains Rs ccr-lacS1, Rs ccr-lacS2, and Rs ccr-lacS3 (A). Location of transposon insertion the genome of strain Rs ccr-lacS9 (B). Below each illustrated gene locus is the likely reaction for which the gene products are responsible. Gene abbreviations and annotations: smpB (rsp_0883) – tmRNA binding protein; sseA (rsp_0885)– 3-mercaptopyruvate sulfurtransferase; aat (rsp_0886) – aspartate aminotransferase; amtB (rsp_088) – ammonium transporter; glnK (rsp_0889) nitrogen regulator; glyQ (rsp_1856) – glycine-tRNA ligase, α-subunint; hypo (rsp_1857) – hypothetical; glyS (rsp_1858) – glycine-tRNA ligase, β-subunit; ppdK (rsp_1859) – pyruvate phosphate dikinase.

Figure 3.8. Phenotypic characterization of transposon mutants derived from random mutagenesis of Rs ccr-lac1A. Photoheterotrophic succinate (left) and acetate (right) growth of R. sphaeroides 2.4.1 (□) and Rs ccr-lac1A (∆) compared to growth of Rs ccr-lacS1 (◊), Rs ccr-lacS2 (x), and Rs ccr-lacS3 (○) (A). R. sphaeroides 2.4.1 and Rs ccr-lac1A growth compared to growth of Rs ccr-lacS9 (▽) (B). Only Rs ccr- lacS9 demonstrated a growth defect. 3.4 Discussion

3.4.1 Expression of ccr is primarily regulated by incremental control of transcription. Transcriptional regulation was the most significant contribution to the observed control of ccr expression for R. sphaeroides. The chromosomal ccr-lac promoter fusion strain Rs ccr-lac1A, for instance, demonstrated a 120-fold upregulation of β-galactosidase activity during acetate growth compared to levels in succinate-grown cells. The upregulation was comparable to regulation of Ccr activity in extracts of wild type cells

(Table 3.5). In the Rs ccr-lac1A strain, the expression of the lacZ gene was driven by the chromosomal ccr promoter (Figure 3.6), yet its product (LacZ) should not have been 79

subject to any enzymatic regulation that the cell might use to control Ccr activity. Thus, any change in LacZ activity must have represented regulation of transcription, transcript stability, or translation. Truncating the ccr promoter without affecting the 5′ untranslated region of the ccr transcript (Figure 3.6), however, resulted in deregulation of expression

(Table 3.8). The results, therefore, suggest that regulation is due to control of transcription initiation rather than transcript stability or translation. Altogether, the data likely discounts significant contributions from any regulatory levels beyond transcription initiation. Consequently, the 30-fold increase in ccr transcript levels in acetate-grown wild type cells (Table 3.6) was likely a result of the transcriptional control of ccr expression.

β-Galactosidase activities in the strains carrying plasmids from the pMC83 ccr-lacZ fusion series offer additional information regarding the potential for transcriptional regulation of the ccr promoter. The β-galactosidase activities detailed in Table 3.8 indicate that there were multiple sites in the ccr upstream that were involved in controlling transcription. As the length of the upstream fragments were reduced among the plasmids, a correspondingly incremental reduction in β-galactosidase activities in acetate-grown cells were observed, implying that sites involved in activation were lost.

At no point, even in the strains carrying pMC83-72, did β-galactosidase activity in acetate-grown cells reduce to the levels of succinate-grown cells. If regulation of ccr is conserved among the organisms in Figure 3.4, the only remaining site able to contribute to regulation in the pMC83-72 plasmid is the blue site that is most proximal to the ccr translational start. Its presence within the transcribed region indicated that, as an operator

site, it was likely involved in repression, or it encoded a signal recognition element of the transcript. However, as discussed above, the evidence suggests that regulation occurs transcriptionally, implying that the site was probably used for transcriptional repression.

Furthermore, regulation of expression from truncated ccr upstream fragments of the pMC83 plasmid series also appeared to incompletely represent the regulation of ccr transcription. The quantitative regulation of β-galactosidase activity in Rs(pMC83-292), containing the longest promoter fragment, was only about 15-fold (Table 3.8) whereas the regulation of transcript abundance in wild type cells was 30-fold (Table 3.6). The discrepancy could be explained by unequal multicopy effects with respect to the multiple regulatory sites in the ccr upstream fragment. For example, a regulator responsible for repressing ccr expression might occur at higher intracellular concentrations than the activators of ccr expression. The result would be that the pool of repressors would be more available to repress expression from the additional plasmid-borne promoters than the proposed activators. As promoter concentrations increase within the cell, ample repressor would be available to regulate expression from the promoter, but the competing activation would be reduced. The overall effect would be a reduced capacity for upregulation in activating conditions. However, another explanation is more consistent with a deeper analysis of the data. Ccr activity in Rs ccr-lac1A cells grown on acetate was half of that of wild type cells. In the Rs ccr-lac1A strain, the chromosomal promoter that drives transcription of ccr only contains 561 bp upstream of the transcriptional start site (Figure 3.6B), indicating that a site involved in activation has been lost.

β-Galactosidase activities in acetate-grown Rs(pMC83-292) also appeared to be lacking full activation. Assuming a copy number effect of the plasmid of about 10, β- galactosidase activity in succinate-grown Rs(pMC83-292) was consistent with the β- galactosidase activity that represents expression of the endogenous ccr promoter that drives transcription of lacZ in Rs ccr-lac1A (Table 3.8). The expectation would then be that β-galactosidase activity in acetate-grown cells would be similarly elevated when the two strains are compared, yet the activities were actually nearly identical. Thus, β- galactosidase activities were about 10-fold lower than expected in Rs(pMC83-292) than if the expression of lacZ from the plasmid were subject to the complete transcriptional activation. Combined with the observations regarding incomplete regulation of Ccr activity in Rs ccr-lac1A, the β-galactosidase activity results of the pMC83 plasmid series imply that an additional site beyond 561 bp was involved in properly activating ccr expression.

3.4.2 Possible transcriptional regulators of ccr remain unknown. RamB was identified as a regulator of acetate metabolism in C. glutamicum where it is responsible for activating and repressing gene expression by binding to the consensus sequence AAA(A/G)CTTTGCAAA (3, 25, 42, 110). Upstream of the ccr coding region is a sequence AACTATTGCAAT that contains much of the RamB binding site, including most of the inherent palindrome. In addition, R. sphaeroides contains a RamB homolog encoded by rsp_2186, herein called PccR, which is discussed in Chapter 4 as a transcriptional regulator of propionyl-CoA assimilation. The pccR gene is also preceded by two strong matches (CTTTGCAAA and AACTTTGCAAAA) to the RamB binding

site. Although the above evidence is consistent with PccR autoregulation and regulation of ccr, transcript (Table 3.6) and activity levels (Table 3.5) in the pccR null mutant strain,

RsΔpccRMC12, are unchanged compared to wild type. As a result, PccR appears to have no role in regulating ccr in the conditions tested.

PhaR, a transcriptional regulator of genes involved in polyhydroxybutyrate accumulation (phaZ, phaP, and phaR) in other organisms (18, 72, 97, 131), is present in

R. sphaeroides 2.4.1 (encoded by rsp_0380) and is another candidate regulator of ccr. In

R. sphaeroides FJ1 (19) and Paracoccus denitrificans (72), the PhaR binding site includes the sequence CTGCN3/4GCAG. Similarly, Figure 3.4 illustrates a comparable conserved sequence 72 bp upstream of the transcriptional start of ccr, and Table 3.9 highlights that the same site is upstream of phaZ, phaP, and phaR in R. sphaeroides

2.4.1. Additionally, the ethylmalonyl-CoA pathway, for which Ccr is an essential enzyme, shares reactions with polyhydroxybutyrate synthesis. The sum of the evidence thereby suggests that PhaR could be involved in controlling the branching of carbon flow between the ethylmalonyl-CoA pathway or polyhydroxybutyrate accumulation.

However, R. sphaeroides did not use PhaR to control ccr expression in the conditions examined here. In the RsΔphaRMC43 strain, a phaR null mutant, Ccr activity was comparable to the wild type in each of the examined growth conditions (Table 3.5).

3.4.3 Other genes for acetyl-CoA assimilation might be regulated by the same repression. Sequences similar to the blue consensus were identified at three independent locations in the region between ccr and ecm. Of the three instances, the site most proximal to the ccr coding region contains the closest match to the overall blue consensus

and is likely used for repression of ccr expression. Table 3.7 displays additional selected sites that were identified upstream of genes involved in the ethylmalonyl-CoA pathway

(phaA, croR, ccr, ecm, mcl2/mch), conversion of C4 to C3 central metabolic intermediates (ppdK), and acetate import (actP). Collectively, repression of each listed gene would be expected in non-acetate growth conditions, as their products are involved in processes necessary for growth with acetate.

3.4.4 Ccr activity is affected by a product of the ethylmalonyl-CoA pathway. Because interrupting the flow of carbon through the ethylmalonyl-CoA pathway affected regulation of Ccr activity (Table 3.5; Δecm47KB, Δmcd11KB, Δmch49KB,

Δmcl1_4KB), it seems that some combination of products and/or intermediates of the pathway are involved in modulating the amount of active Ccr available in the cell.

Perhaps, during repressive conditions, constitutively low levels of ethylmalonyl-CoA pathway enzymes convert available acetyl-CoA to ethylmalonyl-CoA pathway intermediates. Upon accumulation of some number of intermediates and/or products of the pathway, the cell responds by increasing Ccr activity. As there is an abundance of evidence for transcriptional activation, one of the unidentified metabolites may participate as a coinducer for an activator that is involved in ccr expression.

Work in M. extorquens offers additional insight into which intermediates might be responsible for the observed effects on Ccr activity. When pccA is mutated in M. extorquens, mesaconyl-CoA and propionyl-CoA levels are observed at elevated levels when the strain is added to media in which the ethylmalonyl-CoA pathway is operational

(108). Levels of crotonyl-CoA were not reported, but levels of acetyl-CoA,

hydroxybutyryl-CoA, ethylmalonyl-CoA, and methylsuccinyl-CoA – intermediates upstream of mesaconyl-CoA in the pathway – were unchanged in the mutant strain compared to the wild type strain. The results indicate that the equilibrium of the reaction catalyzed methylsuccinyl-CoA dehydrogenase lies on the side of mesaconyl-CoA.

Therefore, upstream intermediates in the cell will quickly be converted to intermediates downstream of methylsuccinyl-CoA, leading to an accumulation of the downstream intermediates. The conclusion, then, is that it is unlikely that upstream intermediates would be useful signaling molecules for controlling the amount of active Ccr in the cell.

Extending the analysis to include the results regarding deregulation of Ccr activity during succinate/acetate growth in strains that contain mutations in genes for enzymes downstream of Ccr (Table 3.5), it appears that some number of products of the pathway are also likely involved in modulating the cell’s production of active Ccr. Ccr activity in extracts of each mutant strain (Δecm47KB, Δmcd11KB, Δmch49KB, Δmcl1_4KB) was reduced equivalently during succinate/acetate growth compared to levels in extracts from succinate/acetate-grown R. sphaeroides, suggesting that at least one eventual product of the pathway was involved in signaling to the cell how much active Ccr to maintain.

Taken with the metabolomics results reported in M. extorquens, the data is consistent with the assumption that the cell sensed a product of the ethylmalonyl-CoA pathway to properly adjust the amount of active Ccr in the cell.

The identity and role of the intermediates or products that are involved in signaling is unclear. While the sum of the evidence presented in this chapter indicates that the greatest influence on the observed regulation of Ccr activity occurred via

transcriptional regulation of ccr expression, the possibility remains for finer regulation at other levels. In order to drive more of its carbon to storage, for example, the cell might tune Ccr activity to allow 3-hydroxybutyryl-CoA, the precursor to crotonyl-CoA, to be funneled into polyhydroxybutyrate. As a result, even when the cell has sufficient biosynthetic precursors for growth, it is able to sequester available acetyl-CoA for future use.

3.4.5 The significance of deregulation of β-galactosidase activity in transposon mutants of Rs ccr-lac1A is unknown. Rs ccr-lacS1, Rs ccr-lacS2, Rs ccr-lacS3, and Rs ccr-lacS9 were isolated as strains that deregulated lacZ expression from the ccr promoter during succinate growth on plates. They are the result of random transposon mutagenesis of Rs ccr-lac1A, a strain in which a 561 bp fragment of the ccr upstream has been duplicated in the chromosome.

The expression of lacZYA is controlled by the endogenous chromosomal ccr promoter, and ccr expression is controlled by a duplicated promoter that includes 561 bp upstream of the transcriptional start (Figure 3.6B).

In Rs ccr-lacS9, the transposon insertion was identified within the gene encoding pyruvate phosphate dikinase (PpdK). PpdK works in concert with malic enzyme (MaeB) to begin gluconeogenesis from malate by converting pyruvate, the product of MaeB, to phosphoenolpyruvate. However, results from a previous study (128) offer evidence that

R. sphaeroides growth with succinate also relies on the refixation of metabolically produced CO2 through the reductive pentose phosphate cycle, which provides additional

C3 precursor metabolites.

Analysis of Figure 3.8 supports the conclusions of the study that demonstrated the role of the reductive pentose phosphate cycle in succinate growth. Rs ccr-lacS9 was still able to grow on succinate, albeit more slowly than R. sphaeroides, despite its inability to generate gluconeogenic intermediates from pyruvate. Instead, the cell produced gluconeogenic intermediates by the reduction of metabolically produced CO2 through the reductive pentose phosphate cycle. The slower growth rate of Rs ccr-lacS9 compared to

R. sphaeroides during succinate growth suggests that lower levels of phosphoenolpyruvate in Rs ccr-lacS9 might have limited the overall metabolic rate in the cell. Conversely, because the reductive pentose phosphate cycle does not function during acetate growth in R. sphaeroides (65), the only source of gluconeogenic intermediates in

Rs ccr-lacS9 and its parent strain, Rs ccr-lac1A, during acetate growth was through pyruvate phosphate dikinase. Rs ccr-lacS9, therefore, could not grow with acetate

(Figure 3.8) while Rs ccr-lac1A and R. sphaeroides – with functional pyruvate phosphate dikinase enzymes – were able to grow.

The relationship to ccr expression, however, is unclear. Although colonies of Rs ccr-lacS9 grown on succinate plates with XGal were reproducibly blue in direct comparison to Rs ccr-lac1A, the β-galactosidase and Ccr activity levels in extracts of cells grown in liquid media were not deregulated in extracts from succinate-grown cells.

Indeed, β-galactosidase and Ccr activities were indistinguishable from activities in extracts of succinate- or succinate/acetate-grown Rs ccr-lac1A (data not shown). In the absence of an observable quantitative deregulation, it is difficult to explain the apparent deregulation on plates. It might simply be due to the diffusion limitations of an unknown

metabolic intermediate that acted as a sensor molecule. Because diffusion occurs more slowly on plates than in liquid media, an intermediate more abundant in the mutant strain than the parent strain would have less opportunity to disperse. Regardless of growth condition, the concentration of the sensor molecule would be elevated compared to the parent strain, but the diminished diffusion might drive the immediate concentration above the necessary threshold for its concentration to have a regulatory effect.

Deregulation of expression from the ccr promoter was evident in Rs ccr-lacS1, Rs ccr-lacS2, and Rs ccr-lacS3 in liquid and on solid media (Table 3.8). Each strain carries a transposon insertion in sseA (rsp_0085), which is likely cotranscribed with a gene (aat; rsp_0886) encoding aspartate aminotransferase. Together, the activities of SseA and Aat might be responsible for the conversion of cysteine to pyruvate through a 3- mercaptopyruvate intermediate (Figure 3.7A) (85). According to work done on SseA from many organisms, the enzyme belongs to a class of rhodanese-like sulfur transferases. Rhodanese enzymes are well-studied sulfur transferases whose sulfur donor is thiosulfate. However, the SseA enzymes have a distinct motif in their active sites

(CGS(T)GVTA) that imparts a specificity for 3-mercaptopyruvate (88, 89). For E. coli,

SseA demonstrates a 50-fold higher Kd and a 130-fold higher kcat with 3- mercaptopyruvate compared to thiosulfate (21).

While the data confirm that the sulfur donor is 3-mercaptopyruvate, the in vivo sulfur acceptor for SseA enzymes is unknown, limiting the speculation about the effects of transposon interruption of sseA. An immediate hypothesis, in light of the discussion regarding Rs ccr-lacS9 in Chapter 2, involves the production of C3 molecules. However,

the biosynthesis of cysteine most often occurs from serine, which is synthesized from pyruvate, so there is likely no net yield of pyruvate following the activity of SseA.

With no obvious connection between the activity of SseA and the expected physiological shifts associated with acetate growth, considerations of possible polar effects on the downstream aat (rsp_0886) become more compelling. Predicting how polar effects on aat will manifest physiologically is difficult because prediction of the substrate specificity of the gene product is ambiguous, especially given that substrate promiscuity of each type of aminotransferase varies among different species. Primary structures of aminotransferases within a single evolutionary subgroup are often similar, but differences among the subgroups are well-defined (84). The product of the R. sphaeroides aat belongs to subgroup I, which includes aspartate, alanine, tyrosine, histidinol phosphate, and phenylalanine aminotransferases. Within subgroup I, Aat from

R. sphaeroides is most similar to aspartate and tyrosine aminotransferases. Aspartate aminotransferase from E. coli is known to efficiently catalyze the transamination of aspartate and to a lesser degree tyrosine and phenylalanine (98). Tyrosine aminotransferase is often assumed to be the primary enzyme responsible for catalyzing transamination of tyrosine and phenylalanine, and both enzymes use glutamate as an amino donor. Based on mutation experiments, specific residues have been identified that define the specificity of aspartate and tyrosine aminotransferases in E. coli (91), and Aat from R. sphaeroides contains only three (V35, K37, T43) out of four (V35, K37, T43,

N64) residues necessary for aspartate specificity. Moreover, there are no other annotated genes in R. sphaeroides whose predicted protein products share significant conservation

of amino acid positions that are characteristic of known aspartate and tyrosine aminotransferases. It would not be surprising, then, if Aat was actually responsible for catalyzing the transamination typically performed by the nominal aspartate aminotransferase and tyrosine aminotransferase. Such an enzyme has not yet been characterized from a natural source but is seemingly possible. For example, after only six amino acid substitutions, Onuffer and Kirsch were able to develop an E. coli Aat with equivalent efficiency for aspartate or phenylalanine while maintaining Kd values comparable to either wild type aminotransferase (91).

Indeed, if Aat is responsible for catalyzing the activity of aspartate and tyrosine aminotransferase, the loss of expression of aat might prevent aspartate, tyrosine, and phenylalanine production in the cell. It then becomes essential to note that in all three strains containing an interruption in sseA, the transposon was inserted such that its aph

(aminoglycoside phosphotransferase for kanamycin resistance) was transcribed in the same orientation as the sseA/aat putative operon. Depending on the expression from the aph promoter and the readthrough that occurs at its transcriptional terminator, the transcription of aat might have been dramatically reduced in the transposon mutants.

Diminished aat expression could manifest as a depletion of cellular aspartate as is likely experienced by R. sphaeroides during acetate growth. During the switch to acetate, oxaloacetate (the direct precursor to aspartate) might be depleted as it is consumed by the cell for biosynthesis before the acetyl-CoA assimilation enzymes can be produced. As a result, aspartate would be an ideal signaling molecule as the cell senses the necessity for the ethylmalonyl-CoA pathway.

3.4.6 Suggested future directions Continued characterization of the ccr promoter will be essential to recognizing the regions of the upstream sequence that participate in regulation of the expression of ccr.

Specifically, the motifs that are suggested here to participate in regulation from the ccr promoter should be mutated in promoter-reporter fusion plasmids. Comparing reporter activity in strains that carry fusions with the wild type sequence and the mutated sequence will more definitively illustrate the roles of the identified motifs.

Given that among the observed effects, the most significant deregulation was observed when a transposon was inserted into sseA, the physiological role of the putative sseA-aat operon should be explored. To establish if the observed deregulation is due to the inactivation of sseA or the potential polar effect on aat expression, an attempt should be made to complement the deregulation in an sseA mutant strain with a constitutively expressed sseA, a constitutively expressed aat, or both. Complementation in the presence of either coding region will reveal which gene is essential for appropriate regulation of ccr expression.

The strategy employed by R. sphaeroides for the regulation of ccr expression remains an open question. While it certainly involves transcriptional regulation, the evidence suggests that the overall transcriptional control is comprised of contributions from multiple regulators that might be repressors and/or activators. In a sense, the apparent complexity associated with the regulation of ccr is unsurprising, as the lack of genetic connection among the genes of the ethylmalonyl-CoA pathway implies their intricate regulation. Moreover, as a proxy for the ethylmalonyl-CoA pathway, Ccr is responsible for metabolism of acetyl-CoA, an intermediate that is the substrate for 91

pathways that span the metabolic spectrum, including acetyl-CoA assimilation, fatty acid biosynthesis, polyhydroxybutyrate biosynthesis, and oxidation through the citric acid cycle. Without a scheme capable of sensing signals associated with nearly all aspects of a cell’s physiology, the efficiency of the cell’s metabolic balance would collapse.

Chapter 4: PccR Is a Member of the Newly Identified ScfR Family of Transcriptional Regulators 4.1 Introduction

Rhodobacter sphaeroides belongs to the purple nonsulfur bacteria, a nonphylogenetic group of phototrophic bacteria known for their metabolic versatility

(126). Given their natural abundance in anaerobic portions of stagnant water, purple nonsulfur bacteria, including R. sphaeroides, are adept at growth with the carbon from fermentation products of other organisms, including propionate. The assimilation of propionate, however, must be closely monitored, as it is first activated to propionyl-CoA, a notable inhibitor of pyruvate dehydrogenase in R. sphaeroides (80). Additionally, propionyl-CoA is an intermediate of many metabolic pathways, including the degradation of branched chain amino acids (81), degradation of branched chain fatty acids, degradation of odd chain fatty acids, and acetyl-CoA assimilation via the ethylmalonyl-

CoA pathway (36). In R. sphaeroides, propionyl-CoA is assimilated via the methylmalonyl-CoA pathway, the enzymes of which have been thoroughly biochemically characterized in a variety of organisms (55, 64, 75, 83). Despite a strong understanding of the biochemistry of the pathway, its regulatory control remains largely unstudied.

In microorganisms, the assimilation of propionyl-CoA occurs via two known metabolic pathways. The first pathway, the methylcitrate cycle, is best characterized in

Salmonella enterica in which the enzymes of the pathway are encoded by the prp operon.

Expression of the operon is controlled by PrpR, a transcriptional activator that responds to intracellular 2-methylcitrate levels (94). Flow of carbon through the methylcitrate cycle in S. enterica is additionally controlled by reversible covalent propionylation of the propionyl-CoA synthetase (PrpE) active site (16, 118), modulating the amount of propionate that is activated to propionyl-CoA (50, 52). R. sphaeroides does not contain genes encoding homologs of the enzymes involved in the propionylation or depropionylation of PrpE, nor does the R. sphaeroides genome encode a homolog of

PrpR.

The second known pathway for propionyl-CoA assimilation, the methylmalonyl-

CoA pathway, operates in a range of organisms across all domains of life, including R. sphaeroides (78). The pathway is characterized by two enzymes that require distinct cofactors. The first enzyme is propionyl-CoA carboxylase, a biotin-dependent enzyme that catalyzes the carboxylation of propionyl-CoA to form (2S)-methylmalonyl-CoA at the expense of cleaving the γ-phosphate from ATP. The enzyme is composed of two subunits, PccA and PccB (55). PccA contains a biotin carboxylase domain and a biotin carboxyl carrier domain, whereas PccB includes the carboxytransferase domain responsible for catalyzing the transfer of the carboxyl group from the biotin cofactor in

PccA to propionyl-CoA, forming (2S)-methylmalonyl-CoA. Following carboxylation,

(2S)-methylmalonyl-CoA is epimerized to (2R)-methylmalonyl-CoA through the catalytic activity of an epimerase before the second unique enzyme of the pathway, methylmalonyl-CoA mutase, catalyzes the rearrangement of the carbon skeleton of (2R)- methylmalonyl-CoA to form succinyl-CoA in a cobalamin-dependent reaction.

One of the aforementioned sources of propionyl-CoA in R. sphaeroides is acetyl-

CoA assimilation (Figure 1.2). In the ethylmalonyl-CoA pathway, β-methylmalyl-

CoA/malyl-CoA lyase (Mcl1) cleaves β-methylmalyl-CoA into glyoxylate and propionyl-CoA (36). As a result, the methylmalonyl-CoA pathway is also a key component of acetyl-CoA assimilation. Despite the physiological link between the ethylmalonyl-CoA and the methylmalonyl-CoA pathways, little is published to date that might link their metabolic regulation in R. sphaeroides. A transcriptional regulator of ccr, a gene that encodes crotonyl-CoA carboxylase/reductase of the ethylmalonyl-CoA pathway, has been identified in Methylobacterium extorquens AM1 (54); howerever, the genome of R. sphaeroides encodes no obvious homolog. The regulator, CcrR, activates ccr expression in M. extorquens, but its binding site is not found upstream of any other genes for enzymes of the ethylmalonyl-CoA pathway or methylmalonyl-CoA pathway in

M. extorquens (54).

Other known strategies for regulating short chain acyl-CoA assimilation similarly offer limited insight into regulation of the methylmalonyl-CoA pathway in R. sphaeroides. For example, isobutyryl-CoA assimilation has been demonstrated in

Pseudomonas aeruginosa and Pseudomonas putida via the activity of, among other enzymes, methylmalonate semialdehyde dehydrogenase (MmsA; PA3570) (5) and 3- hydroxyisobutyrtate dehydrogenase (MmsB; PA3569) (77) to ultimately produce propionyl-CoA and CO2. In Pseudomonas, the mmsA and mmsB genes are encoded within an operon that is transcriptionally activated by MmsR (119). The genome of R. sphaeroides encodes an MmsA homolog (DddC; RSP_2962) and an MmsB homolog

(RSP_0154), but their genes are not clustered, and no MmsR homolog is encoded by the

R. sphaeroides genome.

A further strategy for regulating short chain acyl-CoA assimilation exists in the control of the glyoxylate bypass for acetyl-CoA assimilation in Escherichia coli. The pathway consists of two enzymes, isocitrate lyase (AceA) and malate synthase (AceB).

Together, they allow carbon to flow from acetyl-CoA to malate and succinate while bypassing the decarboxylations that occur at the isocitrate dehydrogenase and α- ketoglutarate dehydrogenase steps of the citric acid cycle. In E. coli, an operon encodes

AceB, AceA, and a kinase/phosphatase (AceK) responsible for modulating the activity of isocitrate dehydrogenase by reversible phosphorylation (66). Expression of the operon is largely controlled by IclR (73, 74) in response to pyruvate and glyoxylate (69). The R. sphaeroides genome, however, does not encode homologs of any of the Ace enzymes or

IclR.

As in E. coli, regulation of aceA and aceB expression in Corynebacterium glutamicum occurs transcriptionally although the genes are not within a single transcriptional element, and the genome of C. glutamicum does not encode an IclR homolog. Instead, aceA and aceB are divergently transcribed within a single genetic locus. Both are activated by the transcriptional activator RamA (26) and repressed by

RamB (42).

The R. sphaeroides genome encodes no RamA homolog, but it does encode a

RamB homolog. Indeed, the evidence presented here indicates that the RamB homolog, herein named PccR (RSP_2186), is an activator of pccB (rsp_2189), encoding a subunit

of propionyl-CoA carboxylase. This work further examines predicted proteomes of other organisms for homologs of RamB and PccR to reveal a family of regulators responsible for regulating pathways of short chain fatty acyl-CoA assimilation.

Of all of the above strategies for controlling short-chain acyl-CoA assimilation, only C. glutamicum RamB offers a potential clue to the strategy used by R. sphaeroides in controlling propionyl-CoA assimilation. The following study examines the role of the product of rsp_2186 (herein named pccR) as a transcriptional regulator of pccB.

Evidence is presented that activation by PccR is the primary mechanism for adjusting the flow of propionyl-CoA through the methylmalonyl-CoA pathway in R. sphaeroides. In addition, the ScfR class of transcriptional regulators, which contains PccR, is established to include four classes of regulators whose members are likely responsible for regulating pathways for short chain acyl-CoA assimilation in a variety of organisms.

4.2 Materials and Methods

4.2.1 Materials. Propionyl-CoA was synthesized from its anhydride as described (113).

4.2.2 Bacterial strains and growth conditions.

Rhodobacter sphaeroides 2.4.1 (DSMZ 158) was grown aerobically in the dark or anaerobically in the light (3,000 lux) at pH 6.8 and 30°C in minimal media (MM) that contained 15 mM potassium phosphate buffer pH 6.7 and per liter: 0.2 g (0.8 mM)

MgSO4, 0.07 g (0.5 mM) CaCl2, 1.2 g (22 mM) NH4Cl, and 1.5 mL of vitamin solution

(per liter: 100 mg cyanocobalamin, 300 mg pyridoxamine-2∙HCl, 100 mg calcium-D(+)- pantothenate, 200 mg thiamine dichloride, 200 mg nicotinic acid, 80 mg 4-aminobenzoic

acid, 20 mg D(+)-biotin), 10 mL trace element solution (per liter: 500 mg disodium

EDTA, 300 mg FeSO4∙7H2O, 3 mg MnCl2∙4H2O, 5 mg CoCl2∙6H2O, 1 mg CuCl2∙2H2O,

2 mg NiCl2∙6H2O, 3 mg Na2MoO4∙2H2O, 5 mg ZnSO4∙7H2O, and 2 mg H3BO3, pH 3), 10 mM of each respective carbon source (in the case of two carbon sources, each was supplied at 10 mM). For R. sphaeroides mutant strain, 25 µg/mL spectinomycin and/or

20 µg/mL kanamycin was added as necessary. Media plates included 2.5% agar.

Oxygen in liquid cultures was removed and replaced with N2 by repeated vacuuming and sparging. Growth in liquid cultures was monitored as an increase in optical density (OD) at 578 nm. Cells were harvested for enzyme assays and RNA isolation at OD578 nm = 0.4

– 0.5. Escherichia coli strains DH5α, S17-1 (114), and SM10 (114) were grown in Luria-

Bertani (LB) broth at 37°C with 50 µg/mL spectinomycin or 50 µg/mL kanamycin as necessary.

For growth studies, cell were pregrown to late exponential phase anaerobically in

5 mL minimal medium containing 10 mM sodium succinate (OD578 nm ~ 1.5), and 0.1 mL was transferred into stoppered, screw-capped (Hungate) tubes with 5 mL minimal medium and the appropriate carbon source.

4.2.3 Plasmid construction. All primers are listed in Table 4.1.

The suicide plasmid (pMC70) employed for the markerless inactivating deletion of pccR (rsp_2186) was constructed by amplifying ~1,500 bp of the upstream (primers:

D_ramB_upF2, D_ramB_upR2) and downstream (primers: D_ramB_downF2,

D_ramB_DownR2) regions of pccR by polymerase chain reaction (PCR) and cloning the

products in tandem into pK18mobsacB (105). The resulting plasmid (pMC70) contains an in-frame deletion of 1,224 bp. The remaining open reading frame includes 134 bp of the 3′ portion of the original coding region and 28 bp of the 5′ portion separated by a

KpnI site and encodes a 55 amino acid peptide.

For complementation of the RsΔpccRMC12 mutant, a plasmid was developed in which expression of the pccR coding region was driven by 162 nt of its own upstream region. A 1,584 bp fragment was amplified from R. sphaeroides 2.4.1 genomic DNA with primers 2186promF2 and 2186compR1. The fragment was digested with

XbaI/EcoRI and ligated into pBBRsm2MCS5 (107) to generate pMC66.

Plasmid pMC75 was constructed to remove the lacZ portion of the pBBRsm2MCS5 plasmid and was used as a scaffold for promoter-lacZ fusions. The lac operon was amplified from pMC1403 (112) with primers lacoperon_for1 and lacoperon_rev1 (5,062 bp) and inserted into pUC19 via HindIII/PstI digest to generate pMC27. A 4193 bp fragment was PCR amplified from pBBRsm2MCS5 (107) with primers MC75F3 and MC75R2. By way of EcoRI/KpnI digest and subsequent ligation, the above product was ligated to the 5,071 bp PCR product of primers lacoperon_for1 and lacoperon_rev2 whose template was pMC27.

Using primers pccBGSF0 and pccBGSR0, a fragment from the R. sphaeroides chromosome was amplified and cloned into pUC18 using BamHI/XbaI, resulting in plasmid pMC73. Mutated versions of pMC73 were constructed by amplification of pMC73 by PfuUltra II polymerase (Agilent) with the primers indicated in Table 4.1.

Methylated template pMC73 was removed by DpnI digest. A 230 bp product amplified

from pMC73, pMC73Δ1, pMC73Δ2, and pMC73Δ12 with primers pccBpromF1 and pccBpromR1 was cleaved with XbaI/EcoRI and ligated into pMC75 to generate pMC85, pMC85Δ1, pMC85Δ2, and pMC85Δ12, respectively. These plasmids contain 187 nt of either the original or the mutated pccB upstream region oriented immediately upstream of

27 bp of the pccB coding region fused to the lacZ gene. The variations in the upstream fragments available on each plasmid are detailed in Figure 4.5.

4.2.4 Isolation of mutant strains and complemented mutant strains. RsΔpccRMC12 was isolated by mating R. sphaeroides 2.4.1 with E. coli S17-1 transformed with pMC70. Single crossover strains were isolated as kanamycin resistant on minimal media succinate plates. To allow for the second crossover event, isolated colonies were grown chemoheterotrophically in 100 µL minimal media succinate overnight. Overnight cultures were spread on minimal media succinate plates supplemented with 10% sucrose and grown photoheterotrophically. Isolated colonies were patched on minimal media succinate plates with and without kanamycin.

Kanamycin sensitive deletion strains were separated from wild type revertants by comparing sizes of colony PCR products using primers DramB_seqUpR1 and

DramB_seqDnR1. RsΔpccRMC12 was genotyped by sequencing the PCR product of primers DramB_seqUpF1 and DramB_seqUpR1 and the product of primers

DramB_seqDnR1 and D_ramB_downR4. For complementation studies, pBBRsm2MCS5 (empty vector control) and pMC66 were independently conjugated into

RsΔpccRMC12 by mating with E. coli SM10 transformed with the respective plasmid.

100

4.2.5 Preparation of cell extracts. Frozen cell pellets (400 – 600 mg) were suspended in 600 µL of 50 mM Tris∙HCl pH 8.0, 5 mM MgCl2, and 0.1 mg/mL DNase I. After addition of ~ 1 g glass beads

(Retsch 0.1 – 0.25 mm diameter), the suspension was beaten at 30 Hz for 9 min with a bead beater (Retsch Model MM200). Insoluble cell material and beads were separated from cell extract by centrifugation at 15,800 x g for 5 min at 4°C. Protein concentration was measured by the method of Bradford (12), using bovine serum albumin (BSA) to generate a standard curve of known protein values.

4.2.6 Enzyme assays. Propionyl-CoA carboxylase was measured as the propionyl-CoA-dependent and

14 14 cell extract-dependent formation of soluble, acid stable C from NaH CO3. Assays were performed in 400 µL (100 mM Tris pH 8.0, 5 mM MgCl2, 10 mM KCl, 2.5 mM

14 DTT, 5 mM ATP, 0.38 mM propionyl-CoA, 12.5 mM NaH CO3[3.7 μCi], 70 – 520 µg cell extract). Reactions were initiated with the addition of propionyl-CoA. At time points of 0, 2.5, and 5 minutes, 100 µL of each reaction was stopped by addition to 50 µL of 100% propionic acid. All stopped reactions were centrifuged for 30 minutes to remove unincorporated CO2. Background values at T=0 were subtracted from other values. Incorporation of acid stable 14C was quantified by adding 75 µL of each stopped reaction to 3.0 mL of scintillation fluid and measured by a scintillation counter.

β-Galactosidase activity was observed as the enzyme-dependent cleavage of ortho-nitrophenyl-β-galactoside (ONPG) measured by the change in absorbance at 412 nm (ε = 4500 M-1cm-1) in a cuvette with a 1 cm path length. A 500 µL reaction was initiated by the addition of 100 µL of 4 mg/mL ONPG in Z-buffer to 400 µL Z-buffer 101

and 70 – 520 µg cell extract ( Z-buffer: 50 mM potassium phosphate buffer pH 7.0, 40 mM KCl, 1 mM MgSO4, 0.35% β-mercaptoethanol).

4.2.7 RNA isolation.

RNA was isolated from 200 mL of culture at OD578 nm = 0.4 – 0.5. Cells were collected by centrifugation (8,000 x g, 10 min, 4°C) and resuspended in 1.3 mL of TRI-

Reagent (Sigma). After incubation at room temperature for 30 min, 650 µL of chloroform was added, and the vessel was briskly shaken to suspend the chloroform in the aqueous phase. The mixture was incubated at room temperature for 10 min before the aqueous and nonaqueous phases were separated by centrifugation (15,800 x g, 10 min,

RNase-free water. DNA was degraded by adding 50 µL of 10X RNase-free DNase I buffer (Roche) and 500 U RNase-free recombinant DNase I (Roche). The reaction was incubated at 37°C for 1 hr. The RNA was precipated again by addition of 500 µL of isopropanol and incubated at -20°C for at least 1 hr. Whole cell RNA was collected by centrifugation (15,800 x g, 10 min, 4°C) and dissolved in 200 µL RNase-free water.

102

4.2.8 Quantitative reverse transcription PCR (qRT PCR). Whole cell cDNA was produced in a 100 µL reverse transcription reaction that contained 70 µL of whole cell RNA with 500 U Superscript III Reverse Transcriptase

(Invitrogen) and 2 nmol of 10 nt oligomers of randomized sequence. Whole cell cDNA was diluted 44-fold to 352-fold in 2-fold steps in 22 µL PCR reactions that contained 14

µL IQ SYBR SuperMix (BioRad) and 40 pmol of each respective primer. Amplification was performed for 40 cycles in a BioRad CFX96 themalcycer with the following amplification protocol: 97°C for 20 s, 64°C for 20 s, and 72°C for 10 s. To prevent detecting potential primer dimers, the reaction was heated to 81°C for 10 s before the fluorescence was recorded for each round of amplification. Each reaction was performed in triplicate for each dilution. Threshold cycle (Ct) values that demonstrated 90% – 110% amplification efficiency were averaged and compared to standard curves in order to estimate the initial absolute value of cDNA template concentration. Standard curves were generated from Ct values of reactions containing known concentrations of ten-fold dilutions of purified PCR product that had been amplified from R. sphaeroides chromosomal DNA with the same primers used in the experimental reactions. Primers used for detecting each cDNA are described in Table 4.1. Final absolute values were normalized to the average of the values of rpoZ and recA levels for comparison among biological replicates, strains, and growth conditions.

4.2.9 Identification of the pccB transcriptional start site. A primer extension method was employed to establish the 5′ end of pccB as described (87). Briefly, MSACEpccBRT1 that was fluorescently labeled with 6- carboxyfluorescein (FAM) on its 5′ terminus was used to prime reverse transcription in 103

whole cell RNA. The migration of the reverse transcription product in an ABI 3770 capillary electrophoresis sequencer was compared to the migration of sequencing reactions performed on the corresponding DNA region with the same 5′ FAM labeled primer. Comigration with a given sequencing product was indicative of the 5′ position of the given transcript.

4.2.10 In silico ScfR identification and DNA binding site recognition. Likely homologs of PccR were collected as the top 1,000 BLAST (Basic Local

Alignment Search Tool) results after querying the National Center for Biotechnology

(NCBI) non-redundant protein sequences database with the R. sphaeroides PccR sequence. Protein sequences that were not associated with a genome in the database were removed. The resulting 327 protein sequences were aligned using Muscle within the

MEGA5 software (123). Gaps were manually removed. Neighbor-joining trees were constructed using MEGA5. Any presented bootstrap values are predicted from 500 replicates and are based on the Jones, Taylor, Thornton statistical model (57).

To investigate possible DNA binding sites for each family of ScfR proteins, 200 bp fragments of sequence immediately upstream of coding regions for proteins from each family were compared using MEME (4). The parameters were adjusted to identify any number of 6 bp – 15 bp conserved motifs per query sequence. The resulting motifs were used to build the DNA logos presented in Figure 4.7.

104

Table 4.1. Primers used in Chapter 4 1,2 Primer DNA sequence (5′ -3′) Use D_ramB_upF2 CCGCCCGGATCCAGACCATCCAGGAGGTCATCAC pccR inactivation D_ramB_upR2 GCTCGCGGTACCTCGCCCCGGCATAGAGTTTC pccR inactivation D_ramB_downF2 TGAAGGGGTACCTCGAGCCGATCGGCATCTCC pccR inactivation D_ramB_downR2 GCTCGAATTCTAGAGTGGAGTATCCCGACATCCAGTATCACTTC pccR inactivation DramB_seqUpF1 GGGGCATCGCCTTCTTCGAATATCTG genotype RsΔpccRMC12 DramB_seqUpR1 CTGCTAATCGACAATCTCGTATGGCAG genotype RsΔpccRMC12 DramB_seqDnR1 CCGCTCATCCTCATCATGCTGTTCTC genotype RsΔpccRMC12 D_ramB_downR4 AGCCGGTCACGCTCTTCAAGCACTG genotype RsΔpccRMC12 pccBGSF0 GTCTGGATCCGGGCGGCCGAGAAATGGGTATGTC generate pMC73 pccBGSR0 CGAGCTCTAGAACACGGTGAAGTCCTGGCTGAAGACATAG generate pMC73 2186promF2 GCGTCTAGATGCCGCTCATCCTCATCATGCTGTTCTC ΔpccR complementation 2186compR1 ATGAGCGCGTGAATTCGAGAGGGCCTGCAG ΔpccR complementation site directed mutagenesis of pMC73-Δ1F1 GTTTGCGATTTGCAGGCGGGAGCGTAGCGGTCATTTGCAAATATC pccB upstream site directed mutagenesis of pMC73-Δ1R1 GATATTTGCAAATGACCGCTACGCTCCCGCCTGCAAATCGCAAAC pccB upstream site directed mutagenesis of pMC73-Δ2F1 CAGGCGGGATTTGCAAAGTCAGCGTAGCGTATCCCGAAATTTTC pccB upstream site directed mutagenesis of pMC73-Δ2R1 GAAAATTTCGGGATACGCTACGCTGACTTTGCAAATCCCGCCTG pccB upstream site directed mutagenesis of pMC73-Δ2F2 CAGGCGGGAGCGTAGCGGTCAGCGTAGCGTATCCCGAAATTTTC pccB upstream site directed mutagenesis of pMC73-Δ2R2 GAAAATTTCGGGATACGCTACGCTGACCGCTACGCTCCCGCCTG pccB upstream pccBpromF1 GGTCTAGAAGATGTTCGAGGCGAGAAGCAGACCGATGGTGACAAG promoter-lacZYA fusion pccBpromR1 GCGGAATTCCTCGAGTTCCTGGAGAATGTCTTTCATGGCCTTG promoter-lacZYA fusion lacoperon_for1 AATGATAAGCTTGAATTCGATCCCGTCGTTTTACAACGTC promoter-lacZYA fusion lacoperon_rev1 TGCACTGCAGTTAAACTGACGATTCAACTTTATAATCTTTGAAATAATAG promoter-lacZYA fusion lacoperon_rev2 TGATATCGGTACCCTGCAGTTAAACTGACGATTC promter-lacZYA fusion MC75F3 AGGAATTCTCTAGAAGCTTTAAACGCCTGGTGCTACGCCTGAATAAGTG promter-lacZYA fusion MC75R2 TGCGTATTGGGTACCTGCATAAAAACTGTTGTAATTCATTAAGCATTCTG promter-lacZYA fusion qRT PCR detection of recA RTrecAF2 GGCAAGGGCTCGATCATGAAACTG transcript qRT PCR detection of recA RTrecAR2 TTTCGGGGCCGTAGATCTCGATGATTC transcript qRT PCR detection of rpoZ RTrpoZF2 ATCGACCGCGACAATGACAAGAAC transcript qRT PCR detection of rpoZ RTrpoZR2 CAGCGCCATCTGATCCTCTTCCG transcript qRT PCR detection of pccB RTpccBF4 ACCCATGCGCAGAAGATCTGCAAG transcript qRT PCR detection of pccB RTpccBR5 GCCATGATGTTGCGCTGGAACACG transcript Identify pccB transcriptional MSACEpccBRT1 [FAM]-CGCACGAACATGTCGAATTCCTC start site 1 Restriction sites are underlined 2 Site directed mutations are italicized

105

4.3 Results

4.3.1 The expression of pccB is regulated at the level of the transcript. The product of pccB (rsp_2189) is the β-subunit of propionyl-CoA carboxylase

(PccAB). PccAB is essential for propionyl-CoA assimilation via the methylmalonyl-

CoA pathway, and the methylmalonyl-CoA pathway is a component of the ethylmalonyl-

CoA pathway for acetyl-CoA assimilation. Propionyl-CoA carboxylase activity was measured as the formation of soluble, acid-stable 14C upon incubation of cell extract with

14 ATP, propionyl-CoA, and NaH CO3. Table 4.2 indicates that propionyl-CoA carboxylase activity increased eight- and twenty-fold when extracts of cells grown with

̅ succinate are compared to extracts of cells grown with acetate or with propionate/HCO3, respectively. A similar trend is observed for pccB transcript levels (Figure 4.1), as they increased six- and eleven-fold when levels from extracts of succinate-grown cells are

̅ compared to acetate- or propionate/HCO3-grown cells. The correlation of trends of propionyl-CoA carboxylase activity and pccB transcript levels is consistent with transcriptional regulation of pccB.

Table 4.2. Propionyl-CoA carboxylase activity in cell extracts of photoheterotrophically grown cells Propionyl-CoA Carboxylase Activity Strain (nmol/min/mg) Succinate Acetate Propionate 1 R. sphaeroides 2.4.1 10 ± 1 76 ± 17 207 ± 8 RsΔpccRMC12 28 ± 6 35 ± 2 NG2 RsΔpccRMC12(pMC66) 18 60 191

1 Errors represent standard deviation of at least three biological replicates. 2NG indicates no growth.

106

Figure 4.1. Comparison of propionyl-CoA carboxylase (PCC) activity (right panel) as in Table 4.2 and pccB transcript abundance (left panel) from R. sphaeroides 2.4.1 (squares) and RsΔpccRMC12 (triangles) cells grown with succinate (filled), acetate (open), and propionate/HCO3̅ (hashed). All points represent the averages of values from at least three biological replicates, except for pccB transcript levels from R. ̅ sphaeroides cells grown with propionate/HCO3, which was only measured once. Error bars represent the standard deviation of at least three biological replicates for all other values. RsΔpccRMC12 did not grow ̅ with propionate/HCO3.

4.3.2 The protein product of pccR is necessary for proper transcriptional activation of pccB. The pccR gene (rsp_2186) is divergently transcribed and separated by two open reading frames from pccB in R. sphaeroides (Figure 4.2). When the pccR coding region was inactivated by an in-frame deletion of 1,224 bp of its original 1,386 bp, the resulting

̅ strain RsΔpccRMC12 was unable to grow with propionate/HCO3, but its growth was unaffected on succinate or acetate (Figure 4.3). The negative growth phenotype with

̅ propionate/HCO3 was complemented when the pccR coding region was expressed from its own promoter on plasmid pMC66 (Figure 4.3).

In the pccR mutant strain, regulation of expression from the pccB promoter was lost. Propionyl-CoA carboxylase activity and pccB transcript levels were detectable yet

107

unchanged between succinate- and acetate-grown RsΔpccRMC12 (Table 4.2 and Figure

4.1), albeit elevated in both conditions when compared to extracts of wild type R. sphaeroides grown with succinate. The level of propionyl-CoA carboxylase activity in the absence of PccR is therefore sufficient to optimally support acetate growth yet

̅ insufficient for propionate/HCO3 growth (Figure 4.3).

Figure 4.2. Genomic context within several α-proteobacteria of pccB (propionyl-CoA carboxylase, β subunit; rsp_2189), pccR, and the consensus sequence identified in Figure 4.7 (open boxes) upstream of pccR (rsp_2186) genes. Striped arrows represent genes annotated to encode homologous multidrug resistant transporters. Checkered arrows are conserved coding regions of unknown function. Open arrows indicate coding regions with no significant sequence identity to other genes shown.

Figure 4.3. Photoheterotrophic growth of R. sphaeroides 2.4.1 (□), RsΔpccRMC12 (∆), RsΔpccRMC12(pBBR) (x), and RsΔpccRMC12(pMC66) (○) with succinate (A), acetate (B), and ̅ propionate/HCO3 (C).

108

Similar to chromosomal regulation, observed β-galactosidase activity in strains carrying a translational fusion of pccB-lacZ on the pMC85 plasmid also indicates regulation of expression from the plasmid-borne pccB promoter (Table 4.3). β- galactosidase activity was elevated 13-fold in acetate-grown R. sphaeroides(pMC85) when compared to the same strain grown with succinate. Unexpectedly, however, R.

̅ sphaeroides(pMC85) failed to grow with propionate/HCO3, its growth was reduced in acetate media compared to all other strains (Figure 4.4), and propionyl-CoA carboxylase activity was undetectable in extracts from either growth condition (Table 4.3).

Conversely, in extracts from strains that carry a pMC85-derived plasmid, propionyl-CoA carboxylase activity levels were comparable to levels in extracts from the respective parent strain if the parent strain was a pccR mutant or if at least one TTTGCAAA palindrome on the strain’s plasmid was mutated (Figure 4.4). Therefore, the growth defect and the loss of observable propionyl-CoA carboxylase activity in R. sphaeroides(pMC85) was dependent on the presence of a functional pccR and the presence of both TTTGCAAA palindromes on the pMC85 plasmid. Altogether, the evidence indicates that PccR is a transcriptional activator of the pccB promoter, and the addition of extrachromosomal pccB promoters influences expression from the endogenous chromosomal pccB promoter.

109

Table 4.3. β-Galactosidase and propionyl-CoA carboxylase activity in cell extracts of photoheterotrophically grown cells carrying derivatives of the pccB-lacZ translational fusion plasmid pMC85

1 1 β-Galactosidase Activity Propionyl-CoA Carboxylase Activity (nmol/min/mg) (nmol/min/mg) Strain Propionate Propionate Succinate Acetate ̅ Succinate Acetate ̅ /HCO3 /HCO3 R. sphaeroides 2.4.1 <5 <5 <5 10 ± 0 80 ± 20 210 ± 10

2 pMC85 340 ± 30 4350 ± 1000 NG <5 <5 NG pMC85Δ1 10 ± 0 40 ± 0 90 ± 10 11 ± 1 80 ± 10 201 ± 1 pMC85Δ2 20 ± 0 30 ± 10 30 ± 20 14 ± 2 98 ± 26 185 ± 1 pMC85Δ12 30 ± 10 60 ± 20 60 ± 20 14 ± 0 92 ± 4 206 ± 14 RsΔpccRMC12 <5 <5 NG 30 ± 10 40 ± 0 NG pMC85 20 ± 10 50 ± 20 NG 27 ± 5 39 ± 5 NG pMC85Δ1 40 ± 20 50 ± 10 NG 31 ± 2 30 ± 6 NG pMC85Δ2 20 ± 10 40 ± 10 NG 32 ± 5 40 ± 1 NG pMC85Δ12 30 ± 10 60 ± 10 NG 38 ± 0 36 ± 4 NG

1 Errors represent the range of at least two biological replicates. 2 NG indicates no growth

110

Figure 4.4. Photoheterotrophic growth of strains of R. sphaeroides 2.4.1 (filled symbols; left panel) and RsΔpccRMC12 (open symbols; right panel) that carry no plasmid (■), pMC85 (▲), pMC85Δ1 (▼), ̅ pMC85Δ2 (♦), and pMC85Δ12 (●) with succinate (A), acetate (B), or propionate/HCO3 (C).

4.3.3 Activation of pccB requires both TTTGCAAA motifs In R. sphaeroides, the upstream regions of pccB and pccR each contain a proximal duplication of a TTTGCAAA motif, a palindrome that matches a consensus sequence

(TTTGCAAA) identified upstream of pccB and pccR in closely related organisms (Figure

4.2). In order to contextualize the palindromes with respect to the pccB promoter, primer extension was used to identify the transcriptional start site of pccB at a thymidine 29 bp upstream of the translational start site. For the functional study of the palindromes, a 215

111

bp fragment of the pccB upstream, including the first 27 bp of the pccB coding region, was fused to the lac operon in pMC85. Variations of pMC85 were constructed in which each palindrome was sequentially mutated to GCGTAGCG (Figure 4.5). The plasmids were transferred into wild type and RsΔpccRMC12 strains, and expression from the pccB promoter and its derivatives was measured as β-galactosidase activity (Table 4.3).

Figure 4.5. Illustration of the mutated pccB upstream regions that are present on each plasmid in the pMC85 series. Each plasmid contains a derivative of a 215 bp fragment of the pccB upstream fragment, including 27 bp of the pccB coding region, fused to lacZ. Regulation of expression from each putative promoter is presented in Table 4.3.

β-Galactosidase activity was nearly abolished in extracts from strains that contained pMC85-derived plasmids with one or both of the TTTGCAAA sites mutated and in strains derived from the pccR mutant strain RsΔpccRMC12. Regulation of β- galactosidase activity was therefore strictly dependent on the presence of an intact pccR and both TTTGCAAA palindromes. The results are consistent with the conclusion that regulation of expression from the pccB promoter involves an interaction between PccR and both palindromes.

4.3.4 PccR is a part of a proposed family of regulators for the assimilation of short chain fatty acyl-CoA (ScfR) molecules. An examination of other genomes for genes that encode homologs of PccR led to the recognition that PccR is a member of a large protein family. Genetic loci that contain genes for PccR homologs in other organisms also contain genes responsible for enzymes 112

in one of four pathways for short chain acyl-CoA metabolism. The collection of protein homologs is newly denoted here as the ScfR (Short chain fatty acyl-CoA assimilation

Regulator) family of regulators. ScfR proteins contain three highly conserved domains

(Figure 4.6). The N-terminal domain is an XRE-family helix-turn-helix domain for DNA binding. The distal portion of the C-terminus contains a domain of unknown function

(DUF2083) whose sequence conservation is remarkable among all of the ScfR proteins and maintains five 100% conserved cysteine residues (CX54CX26-28CYXCERXXC) where Y is usually R/K and the following residue is a branched chain amino acid. The final domain, which occurs immediately proximal to DUF2083, is another domain of unknown function (DUF955).

Based on the genomic context surrounding the genes encoding each ScfR protein, four classes were identified within the overall family. The classes and their names are defined by which metabolic gene cluster was found near the respective regulatory gene within the given genome. Some genomes encode multiple ScfR proteins.

113

1 PccR_R.s. 1 ------MAQKLYAGAKLRELRVKLGLTQKVFAERLGASLPYLNQMENNHRPVSATVVLALAQEFGVDVTKLTTSEAERIVTDMREAL 81 2 PccR_A.d. 1 ------MGAHLKTLREQRGLTQAALAQALKVSPSYLNQIENNQRPLTVNVLLRLQAAFGIDLQQFSEDSQARQLGQLRSAL 75 3 RamB-1_S.c. 1 ------MSKTYAGARLRRLREERRLSQAALARVLGISPSYLNQMEHDSRPLTVPVLLRLTETLGVDAAFFSERDTARLLADLREAL 80 4 RamB-1_H.o. 1 ------MSAEMRLGARVRALRRRENLRQAQLAKMLEISPSYLNLIEHDKRPLSAPLLLKLAKLFEIDLADFAAETDAQLEADLMEVF 81 5 RamB-2_M.u. 1 ------MSKTFVGSRVRQLRNERGFSQAALAQLLEI SPSYLNQIEHDVRPLTVAVLLRITEVFGVDATFFSSQDDTRLVAELREVT 80 6 RamB-2_R.c. 1 ------MAPRAEKLVIGQRLKVLRQSLGLTQAQMAAELGVSASYITLIEADQRPASVKLLMRLAEVYDLNISELSPAADAQLASDFAAAL 84 7 MccR_M.u. 1 MKGGARRVSHVPKTFAGARLRRLREDHGLTQVALARALGLSTSYVNQLENDQRPITVSVLLALAERFDLPTHYFAPDSDARLVSDLREVF 90 8 MccR_A.d. 1 ------MRKTFMGVRLRRLREERGLTQVALARALDISASYLNQIEQNQRPLTVPVLLKINAVFGVDVQRFSDDDEARLIAGLRDVL 80 9 IbcR_X.o 1 ----MPQSHAQLRHQLGLRLQRLRQRHGLTQAELARRLELSPSYLNQIERNQRPLTLAIQQRLKATLGDLDGLLDLDDPAALVEPLDQSL 86 10 XRE Family Helix-Turn-Helix 11 12 PccR_R.s. 82 ADPVFT---DSPPLADLRLVASNAPAFARAFLDLHRAYRQTHERLASL-----DEALG-RDEADLRPS--PWEEVRDFFHYCDNYLDAVD 160 13 PccR_A.d. 76 ADMPGG---DQVPLAETRILAEQLPAATRVLLGLHKRIRAAEERLSVIAE---NTDSGHAAHATSAPVQ-PYEEVREFFYAGHQHLALLD 158 14 RamB-1_S.c. 81 TGELAV---ARVSASDLAELASRMPAVAQVLIDLGRRNQLLSERLAGATD------GRDGDRSTAPS--PHEEIRDFFYRRQNYLHDTD 158 15 RamB-1_H.o. 82 GDPIFD--DFELSSPEVRELISTLPGVGKAVLQLYQRYREVHQTVASISD---ALSGGAAGIINIDPLRVPTEEVSDFIQKRRNHFPALE 166 16 RamB-2_M.u. 81 MDRDLD---ISVDPTEVAEMVSAHPSLARAMVNLHRRYRITTAQLAAATEERYSDGSGTGSITM------PHEEVRDYFYQRQNYLHELD 161 17 RamB-2_R.c. 85 KTPAAG--ADAVPRAEIEAVLQASPRIAAAFVRLQAAHRAAV--LAPMAEENPLTDRDKVEALGETSR--PVEAVRAWFYARRNHVDALD 168 18 MccR_M.u. 91 AEGPAT------PAQIEELVARMPAVGQTLVNLHRRLYDATAELEALH----SRATADVSAVSGQPM--PFEEVRDFFYDRKNYIGELD 167 19 MccR_A.d. 81 ADMPAGAAADAVSLAEIRELAASMPAVARALLALHRGQREAHERLEALSAHYGDERGGAAGAAHAPPM--AYEEVRDFFFARHNHIAELD 168 20 IbcR_X.o. 87 RSLGHT-----LLPAELRALTGNLPQVAQALLDLHRAHQHLLERNAAL-----ELQIGVEHVAVPSLS--PGEQVRDYFNRAHNYLPELD 164 21 22 23 PccR_R.s. 161 RAAEHYAAPG------GVRRDVFSAAMETLTRAGLDLQISDMPA------IRSREGNALRLSARAAAPTQRFQLLHQV 226 24 PccR_A.d. 159 EQAEQLYLQLHPENTDPHAPHAARLVAALEQRLRSLHGITVLRAAG------SPAVEQPVRMLERASHQLRLGAGLSAGQQAFQLGIQL 241 25 RamB-1_S.c. 159 LAAEHLAREI------GIRPGEVIGALTGRLADAHGVRLGAEPG------ERLHRYDRRGRTLHLSPRLRPGQRAFRMATQI 228 26 RamB-1_H.o. 167 DAAEAFSRQI------ELRPDDMYHAMSNYLREAHDIHIRYVRP------GDDRGAVRRFDPERRELVLSESLPRSSRKFQIAHQI 240 27 RamB-2_M.u. 162 AAAEDLTIQM------RLHHGDLRSELTRRLTEVHGVRITRRID------LGDTVLHRLDPATMTLEISNHLASGQQVFKMAAEL 234 28 RamB-2_R.c. 169 RAAEDLADEL------ALHRHEPHLALTGRLA-AHGVTVRIGQA------EAMRGQLRRYDPERRELLLSELLGQASRRFQIGVLL 241 29 MccR_M.u. 168 IAAEQMFGRH------GLHLGGLDAQLARLLGDNLGITVVLDDG------QSLNPNSKRMFQPESKTLYLARWLNRGQRAFQLATQI 242 30 MccR_A.d. 169 DAAERLAGQW------RLAPGASEAGLRQRLERAHGVRVSLPAEGLPGHGQDGAGEAMQRSFDAAARVLYLSPALRPAQRAFQMATQL 250 31 IbcR_X.o. 165 ERAEALYAEL------GLTPENLPLRLRQRLADRHGLLVQDAAD------LHRDKRSMDAQARVLWLAAHLRPGQQAFQMAAQL 236 32 DUF955 33 34 PccR_R.s. 227 ALLTQNDLLEATLDLARFQTAEAREIAKIGLANYFAGAALLPYRPFLQAAAETRHDLERLADLFGASIEQVAHRLSTLQRPGAKGVPFFF 316 35 PccR_A.d. 242 ALLESGPTIDGFTNAANFGSDEARALARIGFANHFAAALLLPNRLFQKTADELRYDIDLLGERFGVGFETVGHRLSTMRHASPHSIPFFF 331 36 RamB-1_S.c. 229 ALLEHDEALGRLAAQDFEPGSPAHALARIGIANYFAAALILPYTAFHAAAEEFRYDIERLTDHYGLGYETVGHRLSTLQRPRLRGVPFSF 318 37 RamB-1_H.o. 241 ALLTQSEVLDHIVEGAELKTEEAQRLCRVALANYYAGAVVMPYSRFSDSVRECRYDIELLGNRFGVSFEQVCHRMTTLQRPGHEGIPFHF 330 38 RamB-2_M.u. 235 AYLEFGDLIDAMVTDGKFTSDESRTLARLGLANYFAAA AVLPYRQFHDVAENFRYDVERLSAFYSVSYETIAHRLSTLQRPSMR GVPFSF 324 39 RamB-2_R.c. 242 ASLEQEPILTRLIAAAGLEDPAAAALMRVSLANYFAAALMMPYGRFLASCESHRYDVELLSHRFGTSFEQTAHRMSTLQRDGARGIPFFF 331 40 MccR_M.u. 243 ALLTQADLITGIIAGDDQLSDEARGVARIGLANYFAGALLLPYRPFLDAAASTRYDIDQLARRFEVGFETICHRLSTLQRPNARGVPFIF 332 41 MccR_A.d. 251 AFLEVPQELQRIVDAAQLSGDAARALARIGLANYFAGALLLPYAPFLQSAEALHYDIERLGQRWGVGFETVCHRLSTLQRPGARGVPFFF 340 42 IbcR_X.o. 237 ASLECAPLLDARIADAGFEDAERIALSRIGLSNYFAGALVMPYSAFLHSAQTSRYDIEWLADRFDVGFEAVCHRLSTLQRRGAAGLPIFF 326 43 DUF955 DUF2083 44 45 PccR_R.s. 317 VRVDQAGTITKRHSATRFQFARFGGACPLWNVHRAFETPGRFLRQLAQTPDGVRYLLLARDVSKPGGSFTAPVRRYAIGLGCEVQHADAL 406 46 PccR_A.d. 332 VRVDRAGNMSKRQSSVDFHFSRTGGTCPLWNVYDAFSQPGKILTQLARMPDERTYLWIARTVEHRRGGYGTPGRIFSVALGCDARHAHRL 421 47 RamB-1_S.c. 319 VRVDRAGNMSKRQSATGFHFSRAGGTCPLWNVYESFATPGRIHVQLSEMPDGQRYLWTARAVTRHRGGWGEPGKTYAIGLGCEIRHAHRL 408 48 RamB-1_H.o. 331 VRADVAGNIYKRFSASGIQISRFGGACPRWGLHAAFLTPGTIVTQLSEMHDGSGFFCMSRTVKRGARGYHAPRAVHAVTLGCQVKHARKL 420 49 RamB-2_M.u. 325 IRVDRAGNMSKRQSATGFHFSSSGGTCPLWNVYETFANPGKILVQ IAQMPDGRNYMWVARTVERRAARYGQPGKTFAIGLGCELRHAHRL 414 50 RamB-2_R.c. 332 VRVDRAGNLSKRFSAGRFPFSRFGGTCPLWNIHAAFETPEEVRTQVIRMPEGASYFSVARTVTRAGGTQGAPAQRLAIGLGCDVAYAPRL 421 51 MccR_M.u. 333 VRTDSAGNISKRQSATAFHFSRVGGNCPLWVIHQAFARPGQFLTQVAQMPDERTYFWIARTTTAEPSRYLGPGKSFAIGLGCDLAHADKL 422 52 MccR_A.d. 341 IRVDRAGNISKRQSATHFHFSKIGGTCPLWNVYEAFAQPGRIVRQLARMPDGRAYLWVARTVARSTGGWGAPGKTFSVALGCDVQHAPQL 433 53 IbcR_X.o. 327 MRVDRAGNVSKRHSATDFHFSHVGGACPLWIVYEAFNQPDRILTQIARMPDGRRYFWLARQVSSGPPGYGRPRKTFALAMGCDLRHADQL 416 54 DUF2083 55 56 PccR_R.s. 407 VYADGLDL----KGSFEPIGISCRICDRQECHQRSVPPLEKRLRVDPDRRGLLPYEIVD------461 57 PccR_A.d. 422 VYGDGLGL--DAPERAVPIGSGCKVCDRESCVQRAFPALGRPLSIDPNARRFAPYAPAS------478 58 RamB-1_S.c. 409 VYSDGLDL--DNASAATPIGMGCRVCERLDCPQRAAPPLGRALRIDPDSSTFVPYPVADPDSRPDRGAVRASADASKDRPPGL 489 59 RamB-1_H.o. 421 VYADGVDL--DNMTAAVPIGATCRLCERMDCEQRAFPRLHQPLNIDENVRGLSFYASASDGGDDF------483 60 RamB-2_M.u. 415 VYSEGLDLSGDPNIAATPIGAGCRVCER DNCPQRAFPALGRALDLDEHRSTVSPYLVKQP------474 61 RamB-2_R.c. 422 IYAEGIDL---SRTRPVEIGLNCYLCERADCTARAHAPVNRPLQVNERERSLALFRFEAG------478 62 MccR_M.u. 423 IYSVGVDL--TDTEAIAAIGAGCKICDRPSCPQRAFPYLGRPVRVDPHTSTDLPYPPAISTER------483 63 MccR_A.d. 431 VYARGLDL--ADPDAPVPIGMGCKVCERTACPQRAFPPMGRALEVNENRSSLVPYRVA------486 64 IbcR_X.o. 417 VYARGWDL--NAVDDAVPIGPGCLTCARSTCVQRAFPALPR------LPTSAR------461 65 DUF2083

Figure 4.6. Alignment of select ScfR proteins from each class. Except for the IbcR family, two proteins were chosen from each class. Entries were selected to represent the greatest sequence variation within the respective class. Residues highlighted in gray are greater than 70% identical among the sequences present in this figure. The cysteine residues highlighted in black are 100 % conserved among all proteins used to construct the full tree (Figure 4.8). Abbreviations (corresponding accession numbers): M.u., Mycobacterium ulcerans Agy99 (RamB YP_907961.1, MccR YP_906313.1); R.c., Rhodobacter capsulatus SB1003 (RamB YP_003579467.1); H.o., Haliangium ochraceum DSM 14365 (RamB YP_0032707981); S.c., Streptomyces coelicolor A3(2) (RamB NP_625277.1); R.s., Rhodobacter sphaeroides 2.4.1 (PccR YP_352239.1); A.d., Alicycliphylus denitrificans BC (PccR YP_004126909.1, MccR YP_004126537.1); X.o., Xanthomonas oryzae pv. oryzae PX099A (IbcR YP_001914226.1).

114

The PccR (Propionyl-CoA Carboxylase Regulator) class consists of proteins whose genes cluster with genes of the methylmalonyl-CoA pathway (pccA, pccB, and mcm) and tend to be found in α-proteobacteria where the homologs typically have greater than 70% sequence identity to R. sphaeroides PccR (RsPccR). The MccR (Methylcitrate

Cycle Regulator) class contains proteins encoded by genes that cluster with methylcitrate cycle genes (prpBCDE) and tend to have greater than 50% sequence identity to RsPccR and share no significant similarity with PrpR, the transcriptional regulator of the methylcitrate cycle genes in S. enterica (94). Proteins encoded by genes that cluster with genes of the glyoxylate bypass (aceA and aceB) were assigned to the RamB (Regulator of

Acetate Metabolism) (42) class and usually have greater than 30% sequence identity to

RsPccR. The proteins within the RamB class are further divided into two subclasses based on their organization in the tree in Figure 4.7. Finally, genes encoding members of the fourth class, IbcR (Isobutyryl-CoA assimilation Regulator), are found in the genera

Xanthomonas, Pseudoxanthomonas, and Stenotrophomonas. In these organisms, the respective regulatory gene is clustered with genes annotated to encode enzymes for the conversion of isobutyryl-CoA to propionyl-CoA and CO2 (mmsAB and hibAB). Among the genes in the cluster are mmsA and mmsB, yet IbcR proteins share no significant similarity with MmsR, a known transcriptional regulator of mmsAB in Pseudomonas

(119).

The clustering of selected ScfR proteins is illustrated in the neighbor-joining tree in Figure 4.7, an abbreviated version of Figure 4.8, which is constructed from 327 ScfR proteins. The genomic loci that encode each of the 327 proteins were inspected for the

115

gene clusters illustrated in Figure 4.7. Only 24 of the examined loci lacked one of the clusters corresponding to the type of assigned regulator. The Corynebacterium glutamicum ramB, for example, is not clustered with the aceA or aceB genes, but the role of RamB in regulation of their expression has been documented (42). Of the 327 analyzed loci, only two instances (YP_004126909.1 and YP_003186889.1) were observed in which the locus that encodes an ScfR protein contains a different metabolic gene cluster than would be consistent the protein’s organization in Figure 4.7. Otherwise, the organization of the proteins in Figure 4.7 correlates with the gene clusters present at the locus encoding each corresponding protein.

Of the ScfR classes, only the PccR proteins form a single branch within the tree.

Members of the MccR class and the RamB subclass 1 form distinct sub-branches within a single branch of the tree. Furthermore, the IbcR class forms an additional sub-branch within the branch that contains the MccR proteins. Members of the second RamB subclass are distributed among several sub-branches. A final sub-branch contains only unknown proteins. The genomic loci that encode the unknown proteins offer no information about their possible regulon.

116

Figure 4.7. Neighbor-joining tree of selected ScfR proteins. Nodes with bootstrap values greater than 50 % are labeled. Each entry includes the family name assigned to the given protein based on the genomic locus of its respective gene. A question mark indicates that the corresponding genetic locus does not contain any genes of the indicated metabolic gene clusters. An asterisk indicates a protein that clusters in the tree with proteins of a different class. Following the protein name is the NCBI accession number for the given protein and the species in which the protein is found. To the right of the tree is an illustration of the general genomic locus that encodes proteins from the corresponding clade. A DNA logo (4) is included if a conserved motif was identified upstream of the genes responsible for the proteins in the corresponding clade. See Figure 4.8 for the extended tree. Gene annotations: pccB, propionyl-CoA carboxylase β- subunit; pccA, propionyl-CoA carboxylase α-subunit; mcm, methylmalonyl-CoA mutase; prpB, 2- methylcitrate lyase; prpC, 2-methylcitrate synthase; prpD, 2-methylcitrate dehydratase; prpE, propionyl- CoA synthetase; mmsA, methylmalonate semialdehyde dehydrogenase; hibA, isobutyryl-CoA dehydrogenase; hibB, methylacrylate hydratase; mmsB, 3-hydroxyisobutyrate dehydrogenase; aceA, isocitrate dehydrogenase; aceB, malate synthase.

117

In an effort to predict potential operator sites of the different regulator classes, it was assumed that the regulators are likely autoregulatory. A 200 bp portion of genomic sequence was collected from the upstream regions of 37 pccR genes, 24 mccR genes, 27 ramB-1 genes, and 14 ramB-2 genes. Motifs upstream of the genes of each class were identified with MEME (4). Logos in Figure 4.7 illustrate the motifs that were identified upstream of genes encoding a given class of ScfR regulator. No consensus motif was identified upstream of ramB-2 genes.

Figure 4.8. Topography of the scaled neighbor-joining tree with compressed clades that represent 327 proteins from the ScfR family. Distances are estimated by the Poisson correction method.

4.4 Discussion

4.4.1 PccR is the primary regulator responsible for controlling intracellular propionyl-CoA levels. For R. sphaeroides, pccB transcript levels were upregulated 11-fold during acetate growth compared to levels during succinate growth. In the pccR deletion strain, however, 118

pccB transcript levels were deregulated in extracts of succinate- and acetate-grown cells.

Also, expression from and regulation of the pccB promoter on pMC85 was dependent on the presence of a full length pccR and both TTTGCAAA motifs within the promoter.

The resulting conclusion is that PccR is a transcriptional activator of pccB and interacts with both TTTGCAAA sites to perform its regulatory function.

An examination of the R. sphaeroides genome (both chromosomes and each of the five plasmids) identified only two positions that contain a proximal duplication of the proposed PccR binding site, TTTGCAAA, and only five additional positions with a single TTTGCAAA. The two regions that contain duplications of the palindrome are located upstream of pccR and pccB. Of the five additional positions, only two are within intergenic regions. Given that the data presented here indicates that two proximal sites are required for appropriate regulation, none of the additional sites are likely involved in regulation by PccR. Without evidence for additional sites of regulation, it seems that evolution of the genome avoided the introduction of additional binding sites, implying a very specific regulation by PccR.

In addition to being specific, regulation by PccR is predicted to be finely tunable.

Because PccR controls propionyl-CoA carboxylase activity, which is responsible for the consumption of propionyl-CoA, regulation by PccR likely finely tunes expression of pccB to avoid the accumulation and possible toxic effects of propionyl-CoA in R. sphaeroides (79, 80). In other bacteria, control of propionyl-CoA accumulation is achieved by the reversible modification of propionyl-CoA synthetase (16, 27, 49, 118).

Known enzymes necessary for modifying the synthetase, however, are not encoded

119

within the R. sphaeroides genome. Instead, R. sphaeroides likely modulates cellular propionyl-CoA levels primarily by precisely tuning propionyl-CoA assimilation rates.

For instance, during succinate growth, insignificant propionyl-CoA levels are

̅ expected, whereas acetate growth and propionate/HCO3 growth produce propionyl-CoA as a direct metabolic intermediate. During acetate growth in R. sphaeroides, the reversible β-methylmalyl-CoA/L-malyl-CoA lyase is responsible for generating propionyl-CoA, whereas it is likely to be a physiologically irreversible propionyl-CoA

̅ synthetase that generates propionyl-CoA during propionate/HCO3 growth. The probable effect of propionyl-CoA production in the two growth conditions is that propionyl-CoA levels are elevated in acetate-grown cells and further elevated in propionate-grown cells.

The cell’s response is a tuned increase of PccR-dependent pccB promoter activation

(Figure 4.1) that likely mirrors suggested propionyl-CoA levels during growth with

̅ acetate and propionate/HCO3.

A gradient of propionyl-CoA accumulation during the observed growth conditions is also consistent with the growth data of the pccR deletion strain

RsΔpccRMC12. Given that propionyl-CoA inhibits pyruvate dehydrogenase activity

̅ (80) and that growth with propionate/HCO3 requires pyruvate dehydrogenase activity for

̅ production of acetyl-CoA, RsΔpccRMC12 cannot grow with propionate/HCO3 because propionyl-CoA production outpaces the strain’s reduced capacity for propionyl-CoA assimilation. Growth with acetate, however, does not require pyruvate dehydrogenase, as the cell’s supply of acetyl-CoA can be provided immediately from its supplied carbon source.

120

4.4.2 PccR is also a transcriptional repressor. Together, the correlation of pccB expression to suggested propionyl-CoA levels in

R. sphaeroides and the comparison of pccB expression between R. sphaeroides and

RsΔpccRMC12 imply that PccR is responsible for activating pccB by recognizing an inducer that is tied to propionyl-CoA levels. However, a more exhaustive examination of the data also suggests that in the absence of the appropriate inducer, PccR further functions as a transcriptional repressor. For example, pccB transcript levels and propionyl-CoA carboxylase activity levels in extracts from RsΔpccRMC12 cells grown with succinate were greater than levels in extracts of succinate-grown R. sphaeroides.

Additionally, conditions that upregulated β-galactosidase activity in R. sphaeroides(pMC85) (expressed from an extrachromosomal pccB promoter) completely eliminated any observable propionyl-CoA carboxylase activity (expressed from the chromosomal pccB promoter). It seems that an excess of regulation-competent pccB promoters titrates an inducing element responsible for PccR-dependent activation of propionyl-CoA assimilation. The titratable factor is unlikely to be PccR given that the effects were not observed with RsΔpccRMC12. Nevertheless, the capacity for upregulation in response to the suggested factor was dependent on the TTTGCAAA palindromes on pMC85. If either palindrome was mutated on pMC85, the observed propionyl-CoA carboxylase activity was comparable to the parent strain that lacks the respective pMC85 derivative. As a result, in the absence of the titratable inducing element, PccR may bind to the chromosomal pccB promoter and repress its expression entirely.

121

In addition to unexpected inhibition of expression from the chromosomal pccB promoter, the R. sphaeroides(pMC85) strain was impaired when growing with acetate

̅ and unable to grow with propionate/HCO3 – growth defects that are is consistent with the absence of propionyl-CoA carboxylase activity. Without propionyl-CoA carboxylase, R. sphaeroides(pMC85) was predictably unable to assimilate any propionyl-CoA and

̅ consequently unable to grow with propionate/HCO3. During acetate growth, glyoxylate and propionyl-CoA are simultaneously generated from a single intermediate in the ethylmalonyl-CoA pathway (36). Optimal acetate growth, therefore, requires the assimilation of both intermediates. However, in the absence of propionyl-CoA carboxylase, only glyoxylate can be assimilated. As observed with a pccB deletion strain, the metabolically generated propionyl-CoA must be secreted as propionate (see Appendix

B.4.), reducing the efficiency of the cell’s consumption of the acetate. Therefore, because acetate was incompletely assimilated, growth occurred at a reduced rate and to a lower yield (Figure 4.4).

4.4.3 ScfR regulators uniquely respond to intracellular signals. An array of PccR-like proteins was identified by querying the NCBI genome database for genomes that encode PccR homologs. The resulting collection of regulators, designated here as the ScfR family of regulators, includes proteins whose apparent regulons encode assimilatory pathways for at least three different short-chain acyl-CoA molecules through four different pathways. Proteins within the ScfR family fall into four classes illustrated in Figure 4.7. The boundaries of each class are derived from the sequence relationships of its members as demonstrated by their organization in the tree.

122

The boundaries also tend to correspond with presumed functional similarities. Functional relationship is inferred from the genomic context of genes encoding the regulators and the genes of their respective proposed regulons.

The link between the PccR and MccR classes was first identified in work published by Suvorova et al. (120). The authors performed in silico analysis of regulators of propionyl-CoA assimilation to identify a group of regulators that they referenced as PrpQ*. The name is described by the authors to encompass regulators of the methylcitrate cycle and the methylmalonyl-CoA pathway (called the citramalate pathway by Suvorova el al.). The work identifies a potential binding site for PrpQ* proteins (TTTGCRAA) that is nearly identical to the motif identified upstream of pccR genes and is consistent with one of the inverted repeats found upstream of the mccR genes. Here, the PrpQ* nomenclature is replaced by PccR and MccR.

Despite the general relationship that leads to the functional organization of the regulators within the tree, each class contains exceptions. The genome of C. glutamicum, for example, encodes a RamB and an MccR. The MccR is yet unstudied, but its gene is clustered with the apparent prp operon as predicted. However, its ramB is not clustered with the genes of the glyoxylate bypass, and the RamB regulon extends far beyond only the glyoxylate bypass genes (9, 25, 110). Also, its binding site (AA/GAACTTTGCAAA)

(42) is markedly more similar to that of RsPccR (TTTGCAAA) than to the motif identified upstream of genes encoding other members of the same RamB cluster.

While some members of different classes may share mixed properties, the continued presence of multiple ScfR proteins in a single organism highlights the

123

necessity for a tightly conserved correlation between ScfR regulons and the clades formed by the ScfR proteins in Figure 4.7. The structural distinctions that lead to the clustering in Figure 4.7 surely allude to distinctions regarding the diverse responses of the various ScfR proteins to cellular signaling. The distinctions likely prevent potentially detrimental redundancy among different ScfR regulators within the same cell. As examples, the genomes of Paracoccus denitrificans and Rhodobacter capsulatus encode a PccR, MccR, and RamB. The gene of each ScfR protein, except pccR in P. denitrificans, is appropriately clustered with its respective gene cluster and is presumably responsible for control of the corresponding two- or three-carbon short chain acyl-CoA assimilatory pathway. The structural distinctions that are apparent by the clustering in

Figure 4.7 are likely responsible for the ability of each ScfR protein in both organisms to uniquely control expression of the appropriate pathway in response to the appropriate signal.

The similarity of the pathway substrates and the similarity of the probable C- terminal effector recognition domain of each ScfR protein indicate that the effectors are expected to have similar chemical structures (possibly acetyl-CoA, propionyl-CoA, and/or isobutyryl-CoA). However, it seems unlikely that both propionyl-CoA assimilation pathways in P. denitrificans and R. capsulatus would respond to the same signaling strategy in the same organism. Such blatant metabolic redundancy is rare in biology. Furthermore, if the pathways do respond to the same signal, it would be additionally superfluous to have two regulators performing identical tasks. A significant

124

difference in signal detection and/or DNA binding likely exists among the classes, even if it is only the affinity for an identical effector of the regulator.

4.4.4 Suggested future directions The data are consistent with the conclusion that the two TTTGCAAA palindromes upstream of pccB compose the operator site through which PccR acts to activate and repress pccB expression. However, because no attempts to purify PccR were successful, the direct interaction between the proposed operator and the PccR protein was not observed. In pursuit of evidence of physical interaction, in vivo crosslinking experiments should be performed. DNA fragments from in vivo crosslinking coupled to

PccR-specific immunoprecipiation should be examined for an enrichment of the pccB promoter. Comparing coprecipitation rates of wild type pccB promoters with pccB promoters that are mutated in the putative operator site will indicate the relevance of the palindromes on the potential for PccR to associate with the DNA. Furthermore, in vivo crosslinking combined with immunoprecipitation will provide the opportunity to investigate if other proteins associate with PccR during its operation in the cell.

To demonstrate the specificity of the ScfR regulators, coding regions for RamB,

MccR, and PccR from R. capsulatus should be expressed from plasmid-borne pccR promoters in the R. sphaeroides pccR deletion strain. The expected differential ability of

̅ each protein to complement the propionate/HCO3-negative growth phenotype of

RsΔpccRMC12 will highlight the distinct nature of each regulator.

The sum of the evidence indicates that regulators identified here as homologs of

R. sphaeroides PccR are responsible for transcriptionally controlling the assimilation of

125

short chain fatty acyl-CoA molecules in a remarkable diversity of bacteria. The work presented regarding PccR, specifically, indicates its role as a transcriptional activator and repressor that responds to a signal associated with propionyl-CoA accumulation.

Extrapolated to the larger ScfR family, the data represents another step into a larger understanding of strategies for controlling carbon flow through intermediary metabolism in bacteria.

126

Chapter 5: Summary

The data and discussion contained within this document seeks to clarify strategies for carbon assimilation in biological systems by using Rhodobacter sphaeroides 2.4.1 as a model system. The goal is to incorporate the results of this work into the continued construction of a complete model of the metabolic pathways that form the metabolic networks within organisms. To achieve this goal, this document examines the roles of a variety of proteins and enzymes in controlling carbon flow through the metabolic networks of R. sphaeroides.

Chapter 2 focuses on a preliminary discussion of enzymes involved in the C4/C3 node of central metabolism by examining phenotypes of mutant strains in which genes for enzymes of the node have been mutated. The chapter identifies genes that are proposed to encode pyruvate carboxylase, pyruvate phosphate dikinase, and two malic enzymes. Results indicate that gluconeogenesis during growth with acetate occurs from the pyruvate that is the substrate for pyruvate phosphate dikinase, yet gluconeogenesis during growth with succinate operates with additional C3 precursors derived from reduction by the reductive pentose phosphate cycle of metabolically generated CO2. The production of the pyruvate used for gluconeogenesis in R. sphaeroides during succinate and acetate growth is suggested here to be the result of the activity of two malic enzymes.

Mutating the gene for either malic enzyme impairs growth with succinate or (S)-malate, but does not affect growth with acetate. Growth with acetate is predicted to require less flow to pyruvate from malate given that it does not require oxidation of pyruvate to generate acetyl-CoA for biosynthesis; therefore, the activity of a single malic enzyme is

127

sufficient to support optimal growth. Finally, a pyruvate carboxylase mutant is unable to grow with lactate, a substrate that requires C3 to C4 conversion, but is able to grow with succinate, which does not require C3 to C4 conversion. Thus, the conclusion is that C3 to C4 conversion occurs exclusively through pyruvate carboxylase in R. sphaeorides.

While the mutant phenotypes were not complemented, the preliminary results initiate an investigation of the potential genes and the roles of their protein products in the conversion of precursors through the C4/C3 node.

Chapter 3 examines potential factors that influence the control of acetyl-CoA assimilation in R. sphaeroides by observing changes in expression of the gene that encodes crotonyl-CoA carboxylase/reductase (Ccr), a key enzyme of the ethylmalonyl-

CoA pathway for acetyl-CoA assimilation. The chapter first establishes that ccr expression is regulated by identifying that when acetate growth was compared to succinate growth, Ccr activity was >30-fold upregulated, Ccr protein was ≥150-fold upregulated, and ccr transcript was 30-fold upregulated.

Random transposon mutagenesis was performed in a reporter strain, Rs ccr-lac1A, that contains a genomic ccr promoter-lacZ fusion. Mutants were screened for a deregulation of expression from the ccr promoter during succinate growth. Of the mutants that were isolated during the screen, only four strains were characterized. Three strains had independent transposon insertions in the sseA gene, whose protein product is involved in cysteine conversion to pyruvate. Each of the three strains exhibited 30-fold higher β-galactosidase activity than the parent reporter strain during succinate growth. A fourth mutant contained a transposon insertion in the gene encoding pyruvate phosphate

128

dikinase, but despite its apparent deregulation of β-galactosidase activity during succinate growth on plates, no deregulation was observed during liquid growth. Overall, the genes identified in the screen provided no obvious connection to acetyl-CoA assimilation.

An investigation of the ccr upstream region identified three motifs that were conserved in closely related organisms. Two motifs were similar to known binding sites of RamB (regulator of acetate metabolism) from Corynebacterium glutamicum and PhaR

(regulator of polyhydroxybutyrate accumulation) from R. sphaeroides FJ1 and

Paracoccus denitrificans. When the genes encoding the RamB homolog or PhaR homolog in R. sphaeroides 2.4.1 was mutated, no detectable change was observed in the regulation of Ccr activity.

The contribution of the various motifs upstream of ccr were investigated by inserting truncated promoter fragments into lacZ reporter plasmids and observing the regulation of β-galactosidase activity in R. sphaeroides between succinate and acetate growth. The strain carrying the plasmid that contained a 272 nt promoter fragment exhibited a 14-fold upregulation of β-galactosidase activity during acetate growth, which is 9-fold lower than was observed in the Rs ccr-lac1A strain (mentioned above). The strain carrying the plasmid that contained a 72 nt promoter fragment exhibited only a 3- fold upregulation of β-galactosidase activity during acetate growth. The incremental reduction of regulation as sites were lost from the promoter fragments indicates that multiple sites are responsible for regulating ccr expression.

Overall, the results from Chapter 3 indicate that full regulation of ccr expression is accomplished via multiple upstream sites. Given the diversity of the resulting hits

129

during the random mutagenesis and their unclear connection to acetyl-CoA assimilation, there are likely also multiple signals involved in controlling acetyl-CoA assimilation.

Therefore, there are probably multiple regulators that integrate the influence of multiple signals into the regulation of expression of ccr.

Chapter 4 presents data that identifies PccR as a transcriptional activator and repressor that ultimately serves to control propionyl-CoA assimilation in R. sphaeroides.

In R. sphaeroides, propionyl-CoA assimilation occurs via the methylmalonyl-CoA pathway, which is a component of acetyl-CoA assimilation and includes the activity of propionyl-CoA carboxylase. Propionyl-CoA carboxylase is composed of two subunits –

PccA and PccB. The levels of pccB transcripts are upregulated 6- or 11-fold when extracts of succinate-grown cells are compared to acetate- or propionate-grown cells, respectively. When pccR is mutated, no regulation of pccR transcripts is observed

̅ between succinate and acetate growth, and the cells fail to grow with propionate/HCO3.

Upstream of pccB and pccR are two TTTGCAAA motifs. Promoter fragments that contained mutations of either or both motifs were fused to lacZ in a reporter plasmid, and the different resulting plasmids were transferred into R. sphaeroides and a pccR deletion strain. When both motifs and pccR were present, a 13-fold upregulation of β- galactosidase was observed between succinate and acetate growth. If either TTTGCAAA motif was mutated or if pccR was mutated, effectively no β-galactosidase activity was observed during succinate or acetate growth. Taken cumulatively, that data indicates that

PccR is responsible for regulating pccB expression through an interaction with both

TTTGCAAA motifs upstream of pccB.

130

The sequence of PccR was compared to other proteins in the NCBI database to identify a protein family that includes proteins that appear to regulate different modes of assimilation of different short chain fatty acyl-CoA molecules. The family was therefore named the ScfR family. ScfR regulators were subdivided into four class – IbcR, PccR,

MccR, and RamB – based on the genomic context of their respective genes. Genes for

ScfR regulators tend to be found near genes for the assimilation of isobutyryl-CoA

(IbcR), the methylmalonyl-CoA pathway for propionyl-CoA assimilation (PccR), the methylcitrate cycle for propionyl-CoA assimilation (MccR), or the glyoxylate bypass for acetyl-CoA assimilation (RamB).

The sum of the work presented in this document expands the understanding of the pathways within the metabolic networks of R. sphaeroides and the strategies for controlling carbon flow through the pathways. The accompanying discussion begins to more precisely define carbon flow through the C4/C3 node of central metabolism while providing more concrete hypotheses about the enzymes involved. The work also thoroughly explores techniques for identifying how the cell accomplishes the control of acetyl-CoA assimilation. Broad possibilities for the mechanism of controlling acetyl-

CoA assimilation are provided, but the experimental results offer solid direction for further investigation. Finally, the cell’s strategy for controlling carbon flow through the methylmalonyl-CoA pathway is outlined by its use of PccR to transcriptionally regulate the assimilation of propionyl-CoA. Furthermore, investigation into the biological conservation of PccR revealed a large family of transcriptional regulators responsible for regulation of various short chain fatty acyl-CoA molecules. Altogether, the work refines

131

the current understanding of the metabolic networks of R. sphaeroides while offering a new classification of proteins that likely participate in the control of carbon flow through the metabolic networks of hundreds of other organisms.

132

References

1. Alber, B. E., R. Spanheimer, C. Ebenau-Jehle, and G. Fuchs. 2006. Study of an alternate glyoxylate cycle for acetate assimilation by Rhodobacter sphaeroides. Mol. Microbiol. 61:297-309.

2. Andrew, I. G., and J. G. Morris. 1965. The biosynthesis of alanine by Clostridium kluyveri. Biochem. Biophys. Acta. 97:176-179.

3. Auchter, M., A. Cramer, A. Hüser, C. Rückert, D. Emer, P. Schwarz, A. Arndt, C. Lange, J. Kalinowski, and V. F. Wendisch. 2011. RamA and RamB are global transcriptional regulators in Corynebacterium glutamicum and control genes for enzymes of the central metabolism. J. Biotechnol. 154:126-139.

4. Bailey, T. L., M. Boden, F. A. Buske, M. Frith, C. E. Grant, L. Clementi, J. Ren, W. W. Li, and W. S. Noble. 2009. MEME Suite: tools for motif discovery and searching. Nucleic Acids Res. 37:202-208.

5. Bannerjee, D., L. E. Sanders, and J. R. Sokatch. 1970. Properties of purified methylmalonate semialdehyde dehydrogenase of Pseudomonas aeruginosa. J. Biol. Chem 245:1828-1835.

6. Beatty, C. M., D. F. Browning, S. J. W. Busby, and A. J. Wolfe. 2003. Cyclic AMP receptor protein-dependent activation of the Escherichia coli acsP2 promoter by a synergistic class III mechanism. J. Bacteriol. 185:5148-5157.

7. Berghoff, B. A., J. Glaeser, C. M. Sharma, J. Vogel, and G. Klug. 2009. Photooxidative stress-induced and abundant small RNAs in Rhodobacter sphaeroides. Mol. Microbiol. 74:1497-1512.

8. Berghoff, B. A., J. Glaeser, C. M. Sharma, M. Zobawa, F. Lottspeich, J. Vogel, and G. Klug. 2011. Contribution of Hfq to photooxidative stress resistance and global regulation in Rhodobacter sphaeroides. Mol. Microbiol. 80:1479-1495.

9. Blombach, B., A. Cramer, B. J. Eikmanns, and M. Schreiner. 2009. RamB is an activator of the pyruvate dehydrogenase complex subunit E1p gene in Corynebacterium glutamicum. J. Mol. Microbiol. Biotechnol. 16:236-239.

133

10. Bologna, F. P., C. S. Andreo, and M. F. Drincovich. 2007. Escherichia coli malic enzymes: two isoforms with substantial differences in kinetic properties, metabolic regulation, and structure. J. Bacteriol. 189:5937-5946.

11. Braatsch, S., O. V. Moskvin, G. Klug, and M. Gomelsky. 2004. Responses of the Rhodobacter sphaeroides transcriptome to blue light under semiaerobic conditions. J. Bacteriol. 186:7726-7735.

12. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254.

13. Brock, M., C. Maerker, A. Schütz, U. Völker, and W. Buckel. 2002. Oxidation of propionate to pyruvate in Escherichia coli. Eur. J. Biochem. 269:6184- 6194.

14. Brown, T. D. K., M. C. Jones-Mortimer, and H. L. Kornberg. 1977. The enzymic interconversion of acetate and acetyl-coenzyme A in Escherichia coli. J. Gen. Microbiol. 102:327-336.

15. Buzdygon, B. E., J. E. Braginski, and A. E. Chung. 1973. Isocitrate dehydrogenase from Rhodopseudomonas spheroides: kinetic mechanism and further characterization. Arch. Biochem. Biophys. 159:400-408.

16. Chan, C. H., J. Garrity, H. A. Crosby, and J. C. Escalante-Semerena. 2011. In Salmonella enterica, the sirtuin-dependent protein acylation/deacylation system (SDPADS) maintains energy homeostasis during growth on low concentrations of acetate. Mol. Microbiol. 80:168-183.

17. Chou, C., L. P. C. Yu, and L. Tong. 2009. Crystal structure of biotin carboxylase in complex with substrates and implications for its catalytic mechanism. J. Biol. Chem 284:11690-11697.

18. Chou, M. E., W. T. Chang, Y. C. Chang, and M. K. Yang. 2009. Expression of four pha genes involved in poly-beta-hydroxybutyrate production and accumulation in Rhodobacter sphaeroides FJ1. Mol. Genet. Genomics.

19. Chou, M. E., and M. K. Yang. 2010. Analyses of binding sequences of the PhaR protein of Rhodobacter sphaeroides FJ1. FEMS Microbiol. Lett. 302:138-143.

20. Chung, A. E., and J. E. Braginski. 1972. Isocitrate dehydrogenase from Rhodopseudomonas spheroides: purification and characterization. Arch. Biochem. Biophys. 153:357-367.

134

21. Colnaghi, R., G. Cassinelli, M. Drummond, F. Forlani, and S. Pagani. 2001. Properties of the Escherichia coli rhodanese-like protein SseA: contribution of the active-site residue Ser240 to sulfur donor recognition. FEBS Lett. 500:153-156.

22. Conrad, R., and H. G. Schlegel. 1977. Influence of aerobic and phototrophic growth conditions on the distribution of glucose and fructose carbon into the Entner- Doudoroff and Embden-Meyerhof pathways in Rhodopseudomonas sphaeroides. J. Gen. Microbiol. 101:277-290.

23. Cortay, J. C., F. Bleicher, B. Duclos, Y. Cenatiempo, C. Gautier, J. L. Prato, and A. J. Cozzone. 1989. Utilization of acetate in Escherichia coli: structural organization and differential expression of the ace operon. Biochimie. 71:1043-1049.

24. Cortay, J. C., D. Negre, M. Scarabel, T. M. Ramseier, N. B. Vartak, J. Reizer, M. H. Saier Jr, and A. J. Cozzone. 1994. In vitro asymmetric binding of the pleiotropic regulatory protein, FruR, to the ace operator controlling glyoxylate shunt enzyme synthesis. J. Biol. Chem 269:14885-14891.

25. Cramer, A., M. Auchter, J. Frunzke, M. Bott, and B. J. Eikmanns. 2007. RamB, the transcriptional regulator of acetate metabolism in Corynebacterium glutamicum, is subject to regulation by RamA and RamB. J. Bacteriol. 189:1145-1149.

26. Cramer, A., R. Gerstmeir, S. Schaffer, M. Bott, and B. J. Eikmanns. 2006. Identification of RamA, a novel LuxR-type transcriptional regulator of genes involved in acetate metabolism of Corynebacterium glutamicum. J. Bacteriol. 188:2554- 2567.

27. Crosby, H. A., K. C. Rank, I. Rayment, and J. C. Escalante-Semerena. 2012. Structural insights into the substrate specificity of the Rhodopseudomonas palustris protein acetyltransferase RpPat: identification of a loop critical for recognition by RpPat. J. Biol. Chem 287:41392-41404.

28. Dangel, A. W., J. L. Gibson, A. P. Janssen, and F. R. Tabita. 2005. Residues that influence in vivo and in vitro CbbR function in Rhodobacter sphaeroides and identification of a specific region critical for co-inducer recognition. Mol. Microbiol. 57:1397-1414.

29. Datta, P., L. Shi, N. Bibi, G. Balázsi, and M. L. Gennaro. 2011. Regulation of central metabolism genes of Mycobacterium tuberculosis by parallel feed-forward loops controlled by sigma factor E (sigmaE). J. Bacteriol. 193:1154-1160.

30. Dimroth, P. 1981. Characterization of a membrane-bound biotin-containing enzyme: oxaloacetate decarboxylase from Klebsiella aerogenes. Eur. J. Biochem.

135

31. Donohue, T., P. Kiley, and S. Kaplan. 1988. The puf operon region of Rhodobacter sphaeroides. Photosynthesis Res. 19:39-61.

32. Dubbs, J. M., and F. R. Tabita. 2003. Interactions of the cbbII promoter- operator region with CbbR and RegA (PrrA) regulators indicate distinct mechanisms to control expression of the two cbb operons of Rhodobacter sphaeroides. J. Biol. Chem 278:16443-16450.

33. Eraso, J. M., J. H. Roh, X. Zeng, S. J. Callister, M. S. Lipton, and S. Kaplan. 2008. Role of the global transcriptional regulator PrrA in Rhodobacter sphaeroides 2.4.1: combined transcriptome and proteome analysis. J. Bacteriol. 190:4831-4848.

34. Erb, T. J., I. A. Berg, V. Brecht, M. Muller, G. Fuchs, and B. E. Alber. 2007. Synthesis of C5-dicarboxylic acids from C2-units involving crotonyl-CoA carboxylase/reductase: the ethylmalonyl-CoA pathway. PNAS 104:10631-10636.

35. Erb, T. J., V. Brecht, G. Fuchs, M. Muller, and B. E. Alber. 2009. Carboxylation mechanism and stereochemistry of crotonyl-CoA carboxylase/reductase, a carboxylating enoyl-thioester reductase. PNAS 106:8871-8876.

36. Erb, T. J., L. Frerichs-Revermann, G. Fuchs, and B. E. Alber. 2010. The apparent malate synthase activity of Rhodobacter sphaeroides is due to two paralogous enzymes, (3S)-malyl-CoA/beta-methylmalyl-CoA lyase and (3S)-malyl-CoA thioesterase. J. Bacteriol. 192:1249-1258.

37. Erb, T. J., G. Fuchs, and B. E. Alber. 2009. (2S)-Methylsuccinyl-CoA dehydrogenase closes the ethylmalonyl-CoA pathway for acetyl-CoA assimilation. Mol. Microbiol. 73:992-1008.

38. Erb, T. J., J. Retey, G. Fuchs, and B. E. Alber. 2008. Ethylmalonyl-CoA mutase from Rhodobacter sphaeroides defines a new subclade of coenzyme B12- dependent acyl-CoA mutases. J. Biol. Chem 283:32283-32293.

39. Espariz, M., G. Repizo, V. Blancato, P. Mortera, S. Alarcón, and C. Magni. 2011. Identification of malic and soluble oxaloacetate decarboxylase enzymes in Enterococcus faecalis. FEBS Journal. 278:2140-2151.

40. Garrity, J., J. G. Gardner, W. Hawse, C. Wolberger, and J. C. Escalante- Semerena. 2007. N-Lysine propionylation controls the activity of propionyl-CoA synthetase. J. Biol. Chem. 282:30239-30245.

41. Garvey, G. S., C. J. Rocco, J. C. Escalante-Semerena, and I. Rayment. 2007. The three-dimensional crystal structure of the PrpF protein of Shewanella

136

oneidensis complexed with trans-aconitate: Insights into its biological function. Protein Sci. 16:1274-1284.

42. Gerstmeir, R., A. Cramer, P. Dangel, S. Schaffer, and B. J. Eikmanns. 2004. RamB, a novel transcriptional regulator of genes involved in acetate metabolism of Corynebacterium glutamicum . J. Bacteriol. 186:2798-2809.

43. Goldie, A. H., and B. D. Sanwal. 1980. Genetic and physiological characterization of Escherichia coli mutants deficient in phosphoenolpyruvate carboxykinase activity. J. Bacteriol. 141:1115-1121.

44. Goldie, H. 1984. Regulation of transcription of the Escherichia coli phosphoenolpyruvate carboxykinase locus: studies with pck-lacZ operon fusions. J. Bacteriol. 159:832-836.

45. Gould, T. A., H. Van De Langemheen, E. J. Muñoz-Elías, J. D. McKinney, and J. C. Sacchettini. 2006. Dual role of isocitrate lyase 1 in the glyoxylate and methylcitrate cycles in Mycobacterium tuberculosis. Mol. Microbiol. 61:940-947.

46. Gregor, J., and G. Klug. 2002. Oxygen-regulated expression of genes for pigment binding proteins in Rhodobacter capsulatus. J. Mol. Microbiol. Biotechnol. 4:249-253.

47. Grimek, T. L., and J. C. Escalante-Semerena. 2004. The acnD genes of Shewenella oneidensis and Vibrio cholerae encode a new Fe/S-dependent 2-methylcitrate dehydratase enzyme that requires prpF function in vivo. J. Bacteriol. 186:454-462.

48. Gui, L., A. Sunnarborg, and D. C. LaPorte. 1996. Regulated expression of a repressor protein: FadR activates iclR. J. Bacteriol. 178:4704-4709.

49. Guo, Y., and D. J. Oliver. 2012. E. coli propionyl-CoA synthetase is regulated in vitro by an intramolecular disulfide bond. Appl. Biochem. Microbiol. 48:257-261.

50. Horswill, A. R., and J. C. Escalante-Semerena. 1999. The prpE gene of Salmonella typhimurium LT2 encodes propionyl-CoA synthetase. Microbiology. 145:1381-1388.

51. Horswill, A., and J. C. Escalante-Semerena. 1999. Salmonella typhimurium LT2 catabolizes propionate via the 2-methylcitric acid cycle. J. Bacteriol. 181:5615-5623.

137

52. Horswill, A., and J. C. Escalante-Semerena. 1997. Propionate catabolism in Salmonella typhimurium LT2: two divergently transcribed units comprise the prp locus at 8.5 centisomes, prpR encodes a member of the sigma-54 family of activators, and the prpBCDE genes constitute an operon. J. Bacteriol. 179:928-940.

53. Horswill, A. R., and J. Escalante-Semerena. 2001. In vitro conversion of propionate to pyruvate by Salmonella enterica enzymes:2-Methylcitrate dehydratase (PrpD) and aconitase enzymes catalyze the conversion of 2-methylcitrate to 2- methylisocitrate. Biochemistry (N. Y.). 40:4703-4713.

54. Hu, B., and M. Lidstrom. 2012. CcrR, a TetR family transcriptional regulator, activates the transcription of a gene of the ethylmalonyl coenzyme A pathway in Methylobacterium extorquens AM1. J. Bacteriol. 194:2802-2808.

55. Huang, C. S., K. Sadre-Bazzaz, Y. Shen, B. Deng, Z. H. Zhou, and L. Tong. 2010. Crystal structure of the alpha6beta6 holoenzyme of propionyl-coenzyme A carboxylase. Nature. 466:1001-1005.

56. Imhoff, J. F., H. G. Trüper, and N. Pfennig. 1984. Rearrangement of the species and genera of the phototrophic “Purple Nonsulfur Bacteria”. Int. J. Syst. Bacteriol. 34:340-343.

57. Jones, D. T., W. R. Taylor, and J. M. Thornton. 1992. The rapid generation of mutation data matrices from protein sequences. Comput. Appl. Biosci. 8:275-282.

58. Klaffl, S., and B. J. Eikmanns. 2010. Genetic and functional analysis of the soluble oxaloacetate decarboxylase from Corynebacterium glutamicum. J. Bacteriol. 192:2604-2612.

59. Kojima, T., T. Yamane, and H. Nakano. 2006. In vitro selection of DNA binding sites for transcription factor, PhaR, from Paracoccus denitrificans using genetic library on microbeads and flow cytometry. J. Biosci. Bioeng. 101:440-444.

60. Kontur, W. S., W. S. Schackwitz, N. Ivanova, J. Martin, K. LaButti, S. Deshpande, H. N. Tice, C. Pennacchio, E. Sodergren, G. M. Weinstock, D. R. Noguera, and T. J. Donohue. 2012. Revised sequence and annotation of the Rhodobacter sphaeroides 2.4.1 genome. J. Bacteriol. 194:7016-7017.

61. Kornberg, H. L., and H. A. Krebs. 1957. Synthesis of cell constituents from C2-units by a modified tricarboxylic acid cycle. Nature. 179:988-991.

62. Kornberg, H. L., and J. Lascelles. 1960. The formation of isocitratase by the Athiorhodaceae. J. Gen. Microbiol. 23:511-517.

138

63. Kornberg, H. L., and N. B. Madsen. 1958. The metabolism of C2 compounds in microorganisms: Synthesis of malate from acetate via the glyoxylate cycle. Biochem. J. 68:549-557.

64. Kühnl, J., T. Bobik, J. B. Procter, C. Burmeister, J. Höppner, I. Wilde, K. Lüersen, A. E. Torda, R. D. Walter, and E. Liebau. 2005. Functional analysis of the methylmalonyl-CoA epimerase from Caenorhabditis elegans. FEBS Journal. 272:1465- 1477.

65. Laguna, R., F. R. Tabita, and B. E. Alber. 2011. Acetate-dependent photoheterotrophic growth and the differential requirement for the Calvin-Benson- Bassham reductive pentose phosphate cycle in Rhodobacter sphaeroides and Rhodopseudomonas palustris. Arch. Microbiol. 193:151-154.

66. LaPorte, D. C., and T. Chung. 1985. A single gene codes for the kinase and phosphatase which regulate isocitrate dehydrogenase. J. Biol. Chem. 260:15291-15297.

67. Larsen, R. A., M. M. Wilson, A. M. Guss, and W. W. Metcalf. 2002. Genetic analysis of pigment biosynthesis in Xanthobacter autotrophicus Py2 using a new, highly efficient transposon mutagenesis system that is functional in a wide variety of bacteria. Arch. Microbiol. 178:193-201.

68. Leadlay, P. F. 1981. Purification and characterization of methylmalonyl-CoA epimerase from Propionibacterium shermanii. Biochem. J. 197:413-419.

69. Lorca, G. L., A. Ezersky, V. V. Lunin, J. R. Walker, S. Altamentova, E. Evdokimova, M. Vedadi, A. Bochkarev, and A. Savchenko. 2007. Glyoxylate and pyruvate are antagonistic effectors of the Escherichia coli IclR transcriptional regulator. J. Biol. Chem 282:16476-16491.

70. Mackenzie, C., M. Choudhary, F. W. Larimer, P. F. Predki, S. Stilwagen, J. P. Armitage, R. D. Barber, T. J. Donohue, J. P. Hosler, J. E. Newman, J. P. Shapleigh, R. E. Sockett, J. Zeilstra-Ryalls, and S. Kaplan. 2001. The home stretch, a first analysis of the nearly completed genome of Rhodobacter sphaeroides 2.4.1. Photosynth Res. 70:19-41.

71. Maehara, A., Y. Doi, T. Nishiyama, Y. Takagi, S. Ueda, H. Nakano, and T. Yamane. 2001. PhaR, a protein of unknown function conserved among short-chain- length polyhydroxyalkanoic acids producing bacteria, is a DNA-binding protein and represses Paracoccus denitrificans phaP expression in vitro. FEMS Microbiol. Lett. 200:9-15.

139

72. Maehara, A., S. Taguchi, T. Nishiyama, T. Yamane, and Y. Doi. 2002. A repressor protein, PhaR, regulates polyhydroxyalkanoate (PHA) synthesis via its direct interaction with PHA. J. Bacteriol. 184:3992-4002.

73. Maloy, S. R., and W. D. Nunn. 1982. Genetic regulation of the glyoxylate shunt in Escherichia coli K-12. J. Bacteriol. 149:173-180.

74. Maloy, S. R., and W. D. Nunn. 1981. Role of gene fadR in Escherichia coli acetate metabolism. J. Bacteriol. 148:83-90.

75. Mancia, F., G. A. Smith, and P. R. Evans. 1999. Crystal structure of substrate complexes of methylmalonyl-CoA mutase. Biochemistry (N. Y.). 38:7999- 8005.

76. Markwell, J. P., and J. Lascelles. 1978. Membrane-bound, pyridine nucleotide-independent L-lactate dehydrogenase of Rhodopseudomonas sphaeroides. J. Bacteriol. 133:593-600.

77. Marshall, V. D., and J. R. Sokatch. 1972. Regulation of valine catabolism in Pseudomonas putida. J. Bacteriol. 110:1073-1081.

78. Maruyama, K. 1979. The metabolic pathway of propionate in Rhodopseudomonas spheroides S. Agric. Biol. Chem. 43:2385-2386.

79. Maruyama, K., and H. Kitamura. 1975. Some effects of propionate on the growth of Rhodopseudomonas spheroides S. Agric. Biol. Chem. 39:1521-1526.

80. Maruyama, K., and H. Kitamura. 1985. Mechanisms of growth inhibition by propionate and restoration of the growth by sodium bicarbonate or acetate in Rhodopseudomonas sphaeroides S. Journal of Biochemistry. 98:819-824.

81. Massey, L. K., J. R. Sokatch, and R. S. Conrad. 1976. Branched-chain amino acid catabolism in bacteria. Bacteriological Reviews. 40:42-54.

82. Masuda, S., and C. E. Bauer. 2002. AppA Is a blue light photoreceptor that antirepresses photosynthesis gene expression in Rhodobacter sphaeroides. Cell. 110:613- 623.

83. McCarthy, A. A., H. M. Baker, S. C. Shewry, M. L. Patchett, and E. N. Baker. 2001. Crystal structure of methylmalonyl-coenzyme A epimerase from P. shermanii: a novel enzymatic function on an ancient metal binding scaffold. Structure. 9:637-646.

140

84. Mehta, P. K., T. I. Hale, and P. Christen. 1993. Aminotransferases: demonstration of homology and division into evolutionary subgroups. European Journal of Biochemistry. 214:549-561.

85. Meister, A., P. E. Fraser, and S. V. Tice. 1954. Enzymatic Desulfuration of ß-mercaptopyruvate to pyruvate. J. Biol. Chem. 206:561-575.

86. Meister, M., S. Saum, B. E. Alber, and G. Fuchs. 2005. L-Malyl-Coenzyme A/ß-methylmalyl-Coenzyme A lyase is involved in acetate assimilation of the asocitrate lyase-negative bacterium Rhodobacter capsulatus. J. Bacteriol. 187:1415-1425.

87. Merighi, M., D. R. Majerczak, M. Zianni, K. Tessanne, and D. L. Coplin. 2006. Molecular characterization of Pantoea stewartii subsp. stewartii HrpY, a conserved response regulator of the Hrp type III secretion system, and its interaction with the hrpS promoter. J. Bacteriol. 188:5089-5100.

88. Nagahara, N., and T. Nishino. 1996. Role of amino acid residues in the active site of rat liver mercaptopyruvate sulfurtransferase. cDNA cloning, overexpression, and site-directed mutagenesis. J. Biol. Chem 271:27395-27401.

89. Nagahara, N., T. Okazaki, and T. Nishino. 1995. Cytosolic mercaptopyruvate sulfurtransferase is evolutionarily related to mitochondrial rhodanese: striking similarity in active site amino acid sequence and the increase in the mercaptopyruvate sulfurtransferase activity of rhodanese by site directed mutagenesis. J. Biol. Chem. 270:16230-16235.

90. Oh, J. I., and S. Kaplan. 2001. Generalized approach to the regulation and integration of gene expression. Mol. Microbiol. 39:1116-1123.

91. Onuffer, J. J., and J. F. Kirsch. 1995. Redesign of the substrate specificity of Escherichia coli aspartate aminotransferase to that of Escherichia coli tyrosine aminotransferase by homology modeling and site-directed mutagenesis. Protein Sci. 4:1750-1757.

92. Ormerod, J. G., K. S. Ormerod, and H. Gest. 1961. Light-dependent utilization of organic compounds and photoproduction of molecular hydrogen by photosynthetic bacteria; relationships with nitrogen metabolism. Arch. Biochem. Biophys. 94:449-463.

93. Padmakumar, R., R. Padmakumar, and R. Banerjee. 1997. Evidence that cobalt-carbon bond homolysis is coupled to hydrogen atom abstraction from substrate in methylmalonyl-CoA mutase. Biochemistry. 36:3713-3718.

141

94. Palacios, S., and J. C. Escalante-Semerena. 2004. 2-Methylcitrate- dependent activation of the propionate catabolic operon (prpBCDE) of Salmonella enterica by the PrpR protein. Microbiology. 150:3877-3887.

95. Payne, J., and J. G. Morris. 1969. Pyruvate carboxylase in Rhodopseudomonas spheroides. J. Gen. Microbiol. 59:97-101.

96. Peoples, O. P., and A. J. Sinskey. 1989. Poly-beta-hydroxybutyrate biosynthesis in Alcaligenes eutrophus H16. Characterization of the genes encoding beta- ketothiolase and acetoacetyl-CoA reductase. J. Biol. Chem. 264:15293-15297.

97. Potter, M., M. H. Madkour, F. Mayer, and A. Steinbuchel. 2002. Regulation of phasin expression and polyhydroxyalkanoate (PHA) granule formation in Ralstonia eutropha H16. Microbiology. 148:2413-2426.

98. Powell, J. T., and J. F. Morrison. 1978. The purification and properties of the aspartate aminotransferase and aromatic-amino-acid aminotransferase from Escherichia coli. European Journal of Biochemistry. 87:391-400.

99. Quandt, J., and M. F. Hynes. 1993. Versatile suicide vectors which allow direct selection for gene replacement in Gram-negative bacteria. Gene. 127:15-21.

100. Reinscheid, D. J., S. Schnicke, D. Rittmann, U. Zahnow, H. Sahm, and B. J. Eikmanns. 1999. Cloning, sequence analysis, expression and inactivation of the Corynebacterium glutamicum pta-ack operon encoding phosphotransacetylase and acetate kinase. Microbiology. 145:503-513.

101. Resnik, E., B. Pan, N. Ramani, M. Freundlich, and D. C. LaPorte. 1996. Integration host factor amplifies the induction of the aceBAK operon of Escherichia coli by relieving IclR repression. J. Bacteriol. 178:2715-2717.

102. Rocco, C. J., K. L. Dennison, V. A. Klenchin, I. Rayment, and J. C. Escalante-Semerena. 2008. Construction and use of new cloning vectors for the rapid isolation of recombinant proteins from Escherichia coli. Plasmid. 59:231-237.

103. Rodriguez, E., and H. Gramajo. 1999. Genetic and biochemical characterization of the a and ß components of a propionyl-CoA carboxylase complex of Streptomyces coelicolor A3(2). Microbiology. 145:3109-3119.

104. Sauer, U., and B. J. Eikmanns. 2005. The PEP-pyruvate-oxaloacetate node as the switch point for carbon flux distribution in bacteria. FEMS Microbiol. Rev. 29:765-794.

142

105. Schafer, A., A. Tauch, W. Jager, J. Kalinowski, G. Thierbach, and A. Puhler. 1994. Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19; selection of defined deletions in the chromosome of Corynebacterium glutamicum. Gene. 145:69-73.

106. Schedel, M., J. H. Klemme, and H. G. Schlegel. 1975. Regulation of C3- enzymes in facultative phototrophic bacteria: the cold-labile pyruvate kinase of Rhodopseudomonas sphaeroides. Arch. Microbiol. 103:237-245.

107. Schneider, K., M. Asao, M. S. Carter, and B. E. Alber. 2012. Rhodobacter sphaeroides uses a reductive route via propionyl coenzyme A to assimilate 3-aydroxypropionate. J. Bacteriol. 194:225-232.

108. Schneider, K., E. Skovran, and J. A. Vorholt. 2012. Oxalyl-Coenzyme A reduction to glyoxylate is the preferred route of oxalate assimilation in Methylobacterium extorquens AM1. J. Bacteriol. 194:3144-3155.

109. Schwer, B., J. Bunkenborg, R. O. Verdin, J. S. Andersen, and E. Verdin. 2006. Reversible lysine acetylation controls the activity of the mitochondrial enzyme acetyl-CoA synthetase 2. PNAS. 103:10224-10229.

110. Seibold, G. M., C. T. Hagmann, M. Schietzel, D. Emer, M. Auchter, J. Schreiner, and B. J. Eikmanns. 2010. The transcriptional regulators RamA and RamB are involved in the regulation of glycogen synthesis in Corynebacterium glutamicum. Microbiology. 156:1256-1263.

111. Sender, P. D., M. G. Martı́n, S. Peirú, and C. Magni. 2004. Characterization of an oxaloacetate decarboxylase that belongs to the malic enzyme family. FEBS Lett. 570:217-222.

112. Shapira, S. K., J. Chou, F. V. Richaud, and M. J. Casadaban. 1983. New versatile plasmid vectors for expression of hybrid proteins coded by a cloned gene fused to lacA gene sequences encoding an enzymatically active carboxy-terminal portion of β- galactosidase. Gene. 25:71-82.

113. Simon, E. J., and D. Shemin. 1953. The preparation of S-succinyl Coenzyme A. J. Am. Chem. Soc. 75:2520-2520.

114. Simon, R., U. Priefer, and A. Puhler. 1983. A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram negative bacteria. Nat Biotech. 1:784-791.

115. Simons, R. W., P. A. Egan, H. T. Chute, and W. D. Nunn. 1980. Regulation of fatty acid degradation in Escherichia coli: isolation and characterization of

143

strains bearing insertion and temperature-sensitive mutations in gene fadR. J. Bacteriol. 142:621-632.

116. Sistrom, W. R. 1960. A requirement for sodium in the growth of Rhodopseudomonas spheroides. Journal of General Microbiology. 22:778-785.

117. Smith, R. A., and I. Gunsalus. 1954. Isocitritase: a new tricarboxylic acid cleavage system. J. Am. Chem. Soc. 76:5002-5003.

118. Starai, V. J., and J. C. Escalante-Semerena. 2004. Identification of the protein acetyltransferase (Pat) enzyme that acetylates acetyl-CoA synthetase in Salmonella enterica. J. Mol. Biol. 340:1005-1012.

119. Steele, M. I., D. Lorenz, K. Hatter, A. Park, and J. R. Sokatch. 1992. Characterization of the mmsAB operon of Pseudomonas aeruginosa PAO encoding methylmalonate-semialdehyde dehydrogenase and 3-hydroxyisobutyrate dehydrogenase. J. Biol. Chem. 267:13585-13592.

120. Suvorova, I. A., D. A. Ravcheev, and M. S. Gelfand. 2012. Regulation and evolution of malonate and propionate catabolism in proteobacteria. J. Bacteriol. 194:3234-3240.

121. Tabuchi, T., and H. Uchiyama. 1975. Methylcitrate condensing and methylisocitrate cleaving enzymes; evidence for the pathway of oxidation of porpionyl- CoA to pyruvate via C7-tricarboxylic acids. Agric. and Biol. Chem. 39:2035-2042.

122. Tabuchi, T., H. Aoki, H. Uchiyama, and T. Nakahara. 1981. 2- Methylcitrate dehydratase, a new enzyme functioning at the methylcitric acid cycle of propionate metabolism. Agric. Biol. Chem. 45:2823-2829.

123. Tamura, K., D. Peterson, N. Peterson, G. Stecher, M. Nei, and S. Kumar. 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28:2731-2739.

124. Textor, S., V. F. Wendisch, A. A. De Graaf, U. Muller, M. I. Linder, M. I. Linder, D. Linder, and W. Buckel. 1997. Propionate oxidation in Escherichia coli: evidence for operation of a methylcitrate cycle in bacteria. Arch. of Microbiol. 168:428- 436.

125. Uchida, K., and G. Kikuchi. 1966. Phosphoenolpyruvate carboxykinase from Rhodopseudomonas spheroides and its possible role in light-stimulation of glucogenesis. J. Biochem. 60:729-732.

144

126. van Niel, C. B. 1944. The culture, general physiology, morphology, and classification of the non-sulfur purple and brown bacteria. Microbiol. Mol. Biol. Rev. 8:1-118.

127. Voegele, R. T., M. J. Mitsch, and T. M. Finan. 1999. Characterization of two members of a novel malic enzyme class. Biochim. Biophys. Acta. 1432:275-285.

128. Wang, X., D. L. Falcone, and F. R. Tabita. 1993. Reductive pentose phosphate-independent CO2 fixation in Rhodobacter sphaeroides and evidence that ribulose bisphosphate carboxylase/oxygenase activity serves to maintain the redox balance of the cell. J. Bacteriol. 175:3372-3379.

129. Wifling, K., and P. Dimroth. 1989. Isolation and characterization of oxaloacetate decarboxylase of Salmonella typhimurium, a sodium ion pump. Arch. Microbiol. 152:584-588.

130. Woese, C. R., E. Stackebrandt, W. G. Weisburg, B. J. Paster, M. T. Madigan, V. J. Fowler, C. M. Hahn, P. Blanz, R. Gupta, K. H. Nealson, and G. E. Fox. 1984. The phylogeny of purple bacteria: the alpha subdivision. Syst. Appl. Microbiol. 5:315-326.

131. York, G. M., J. Stubbe, and A. J. Sinskey. 2002. The Ralstonia eutropha PhaR protein couples synthesis of the PhaP phasin to the presence of polyhydroxybutyrate in cells and promotes polyhydroxybutyrate production. J. Bacteriol. 184:59-66.

132. Zarzycki, J., A. Schlichting, N. Strychalsky, M. Muller, B. E. Alber, and G. Fuchs. 2008. Mesaconyl-coenzyme A hydratase, a new enzyme of two central carbon metabolic pathways in bacteria. J. Bacteriol. 190:1366-1374.

133. Zeilstra-Ryalls, J. H., M. Gomelsky, A. A. Yeliseev, J. M. Eraso, and S. Kaplan. 1998. Transcriptional regulation of photosynthesis operons in Rhodobacter sphaeroides 2.4.1. Methods Enzymol. 297:151-166.

145

Appendix A: Detailed Materials and Methods

146

A.1. Crotonyl-CoA and propionyl-CoA synthesis

Reagents:

 40 mL anaerobic H2O in 100 mL CLEAN stoppered bottle with stir bar  80 mg CoA o In glass vial covered with Kimwipe o Bring to room temperature before opening

 0.336 g NaHCO3 (final concentration to be 0.1 M) o In glass vial covered with Kimwipe  22 µL anhydride in ~500 µL acetonitrile o AVOID WATER! o Prepare immediately before use

Procedure:

Adapted from Simon, F.J. and D. Shemin (1953) J. Am. Chem. Soc. 75, 2520

1. Move anaerobic water, CoA, NaHCO3 to anaerobic bag 2. Open bottle of water 3. Dissolve NaHCO3 4. Add CoA 5. Reseal bottle 6. Remove from anaerobic bag 7. Add anyhydride/acetonitrile to sealed bottle with a syringe 8. Stir on stir plate at room temperature for 30 min 9. Check free CoA concentration of 40 µL of reaction 10. Open bottle and adjust pH to 3 with 1 M HCl a. Allow all of CO2 to evolve before moving to next step b. Be careful to minimize the amount of additional salt added while adjusting pH 11. Extract twice with diethylether a. Add ~1 volume of diethylether per 1 volume of aqueous b. Add to glass separatory funnel c. Shake briskly a few times holding stopper tightly 147

d. Release pressure e. Repeat shaking and releasing 3 – 5 times f. Allow phases to separate g. Collect aqueous (bottom) phase 12. Secure Kimwipe over top of bottle 13. Incrementally (-20  -80 °C) move bottle to -80 °C 14. Lyophilize overnight 15. Scrape dried powder from walls of bottle and store in a sealed glass vial 16. Add 500 µL H2O to bottle after everything is scraped out a. Swirl water around to dissolve remaining powder 17. Determine % free CoA a. 40 µL for Ellman’s Test b. Test for adenosine concentration i. Make 1:1500 dilution in cuvette -1 -1 ii. ε260 = 15,400 M cm Lyophilization:

1. Turn on refrigeration >30 min before use 2. Place bottle inside glass lyophilization container/tube 3. Seal lid onto lyophilization container/tube with rubber connector a. Be sure that hole in neck is sealed 4. Turn on vacuum pump 5. Attach neck of lyophilization container/tube to one lyophilizer valve 6. Open valve 7. After lyophilization, release vacuum on system before turning off vacuum pump c. BE CAREFUL! When vacuum is released, lyophylization container/tube can fall apart Ellman’s Test for Free Thiols:

A colorimetric method for determining low concentrations of mercaptans” Ellman, G.L. (1958) Arch. Biochem. Biophys. 74, 443-445.

250 µL 200 mM MOPS pH 7.2 225 - x µL H2O 25 µL Ellman’s Reagent x µL Crotonyl-CoA synthesis

-1 -1 ε412 nm= 13,600 M cm

148

Ellman’s Reagent (Light sensitive, store at 4 °C) 10 mM DTNB (5,5’-dithiobis(2nitrobenzoate)) 1 mM EDTA 0.5 M phosphate buffer pH7.3 Author: Michael Carter

Adapted from: Henrik 2000

149

A.2. Prepartation of small scale cell extracts by the cell mill

Note

This protocol will yield 200-500 µL of cell extracts at about 8 - 10 mg/mL. If greater volume or concentrations are required, use a French Pressure Cell.

Reagents  Cracking Buffer o 50 mM Tris pH 8 o 5 mM MgCl2  0.1-0.25 mm Retsch glass beads  Retsch MM200 Bead Beater Procedure

1. With one tube of harvested cells (~200 mL of culture, pelleted), resuspend cells in 600 µL Cracking Buffer 2. Add glass beads to about 0.5 in from the top of the tube (~1 µg) 3. Shake at 30 Hz for 9 min in Bead Beater 4. Spin 3 min at 15.8 K x g at 4 °C 5. Supernatant = Cell Extract

Author: Michael Carter

Adapted from: Birgit Alber

150

A.3. Crotonyl-CoA carboxylase assay

Required Reagents:

 100 mM Tris pH 7.8  100 mM NADPH  Crotonyl-CoA o 25 mg/100 µL ~ 100 mM o Determine concentration -1 -1 . A(1:1500)260nm ε = 16,400 M cm . Subtract free CoA concentration  Ellman’s o 250 200 mM MOPS pH 7.2 o 220 H2O o 15 Ellman’s Reagent o 5 Crotonyl-CoA -1 -1 o A412nm ε = 13,600 M cm  Note: High (>5%) free CoA interferes with reaction

 1 M NaHCO3 (Freshly Made)

Procedure Note: Reactions are performed in 0.1 cm quartz cuvette to account for the high extinction coefficient of NADPH 1. Reaction:  x µL Cell Extract  6 µL 100 mM NADPH (3.3 mM)  187 – x – y µL Buffer (100 mM Tris pH 7.8) Record background activity

 y µL Crotonyl-CoA (3.3 mM)

 7 µL 1 M NaHCO3 (35 mM) 2. Add cell extract to one side of cuvette opening 3. Add NADPH to other side of cuvette opening 4. Add buffer to same side as cell extract to aid in mixing 5. Mix by inversion, cover top of cuvette with parafilm

151

6. Record background activity Perfom quickly:

7. Add crotonyl-CoA 8. Invert once to get crotonyl-CoA into reaction 9. Add NaHCO3 with Hamilton syringe. Mix well with syringe. 10. Begin following reaction at 365 nm

Data Analysis

1. Estimate ΔA/min based on the settings of the chart reader -1 -1 a. εNADPH @ 365 nm = 3400 M cm

Author: Michael Carter

Adapted from: Birgit Alber

152

A.4. Radioactive carboxylation assay

Principle Radioactive bicarbonate is mixed with cold bicarbonate to yield a very low concentration of radioactive bicarbonate to be used as substrate in the reaction. Several reactions are started in succession at given time intervals. Then, the reactions are stopped after a given time by adding acid. The acid denatures the enzyme and also causes the evolution of the bicarbonate as CO2 gas. The only radioactive carbon that remains is what has been incorporated into the molecules in solution. The remaining solution is measured for radioactivity in a scintillation counter.

This assay is more sensitive than corresponding spectrophotometric assays. Therefore, less enzyme and less substrate can be used. The assay is discontinuous over a few minutes, so the concentrations must be such that the reaction will not run out of substrate and the concentration of products will not inhibit the reaction. Several time points must be used to establish that the reaction is linear within the time frame used. Because all of the reactions are done simultaneously, it is relatively easy to conduct replicates.

Scintillation Counter Scintillation fluid fluoresces in the presence of radioactivity. The amount of fluorescence is directly proportional to the amount of radioactivity present. The resulting data is given in counts per minute (cpm). The fluorophores in the scintillation fluid are known to have certain efficiencies for recognizing different radioisotopes. Using a conversion factor to account for the efficiency of detection, the disintegrations per minute (dpm) can be calculated. Most often, the disintegrations per minute are not useful because all of the work is done relatively, so the counts per minute are sufficient.

The machine is loaded by placing glass scintillation vials inside of plastic bottles. The plastic bottles are loaded into a tray that is labeled with a card containing a pattern of black and white spots that indicate to the machine which program to use. When using the machine in the Tabita lab, the program card “User 1” indicates to the computer to use “RuBisCO S.A.” which is just the name of the program used to follow radioactivity of 14C.

NaHCO3 as Substrate 1. Do no prepare NaHCO3 until everything else is ready. a. To avoid losing too much substrate to CO2 b. Only work with heavy NaHCO3 in the radioactive hood. It should be the last thing removed from its stock location.

153

2. Dilute heavy HCO3 1:50 with cold HCO3 (20 µL heavy in 980 µL 50 mM cold) = “Working HCO3” a. Contribution of heavy HCO3 to the total concentration of HCO3 is negligible 3. Determine the amount of radioactivity per concentration of HCO3 a. Dilute Working NaHCO3 1:100 in 1 mL b. Mix 100 µL with 3 mL scintillation fluid c. Determine radioactivity with the scintillation counter as described above i. Typical = 30,000 cpm ii. Back-calculate to determine cpm/nmol 1. Desire ~ 600 cpm/nmol total HCO3 Reaction 125 µL Enzyme 100 µL Working NaHCO3 25 µL Substrate

Stop with 100 µL Propionic Acid

Note: Be sure to include a negative control that lacks the enzyme. It is common for there to be slight, albeit detectable, amounts of contaminating radioactive compounds in the NaHCO3.

1. Start reactions at given time intervals. a. Example: For 1 min reactions i. Start Rxn 1 at T=0 ii. Start Rxn 2 at T=15 s iii. Start Rxn 3 at T=30 s iv. Start Rxn 4 at T=45 s v. Stop Rxn 1 at T=60 s vi. Stop Rxn 2 at T=1:15 vii. Stop Rxn 3 at T=1:30 viii. Stop Rxn 4 at T=1:45 ix. Repeat for next series of four reactions 2. Spin reactions for >30 min to allow time for HCO3 to evolve as CO2 3. Add 100 µL of stopped reaction to 3 mL scintillation fluid a. Keep in mind that the results are only going to indicate counts for 100 µL of the 350 µL reaction, so to determine activity from the whole reaction, the results must be multiplied by 3.5 4. Subtract out the background radioactivity determined by negative controls. 5. Use the aforementioned amount of radioactivity per nmol of HCO3 to calculate the nanomoles of HCO3 that were assimilated 6. Divide by assay time and amount of protein loaded

154

Author: Michael Carter

Adapted from: Sriram Satagopan

155

A.5. Propionyl-CoA assay

Required Reagents:

 PCC Buffer (10 mL) o 1 M Tris HCl pH 8.0 o 50 mM MgCl2 o 100 mM KCl o 25 mM DTT (77 mg Freshly Added)  50 mM ATP (1 mL) o 28 mg

 80 mM NaHCO3 (10 mL) o 42 mg  1.5 mM Propionyl-CoA (1 mL) o 6.5 mg

Procedure

1. Prepare Hot NaHCO3 (Goal is 40,000 DPM/µL) a. Add 15 µL Hot NaHCO3 to 985 µL NaHCO3 = Working Stock b. Add 10 µL Working Stock to 990 µL NaHCO3 = 1:100 Working Stock c. Add 100 µL to 3 mL Scintillation Fluid x 2 2. Reaction (Final Concentration): a. Prepare each reaction in small, open culture tubes on ice

 220 – x – y µL H2O  40 µL PCC Buffer  40 µL ATP (5 mM)  x µL Cell Extract

 100 µL Working Stock NaHCO3 (20 mM) Add to start reaction

 y µL Propionyl-CoA (0.38 mM) or H2O (Be very precise about Propionyl-CoA concentration. Too much is inhibitory!)

156

b. Prepare small, open culture tubes with 50 µL 100% propionic acid (13.5 M)  # tubes = 3 x # reactions c. Start reactions at 15 s intervals d. @ 0 min, 2 min, 4 min remove 100 µL of reaction and add to propionic acid to stop reaction  Avoid splashing. Add volume directly to propionic acid, not down the wall of the tube. Need to make sure reaction is stopped efficiently.

 Acid will convert excess HCO3 to CO2, releasing into the atmosphere. e. Spin all stopped reactions for >30 min f. Resuspend pellets g. For each reaction: Remove 75 µL and add to 3 mL of Scintillation Fluid in Scintillation vial. h. Count 14C

Data Analysis

1. Determine counts or disintegrations per minute based on counts from 1:100 Working Stock NaHCO3. 2. Use that value to estimate how much 14C was fixed in negative controls (background) and experimental. 3. For each time point, subtract background from experimental to calculate the actual amount of propionyl-CoA-dependent 14C that was fixed by cell extract.

Author: Michael Carter

Adapted from: Kathrin Schneider

157

A.6. β-Galactosidase assay

Required Reagents:

 Z-Buffer o 50 mM Potassium Phosphate pH 7.0 o 40 mM Potassium Chloride o 1 mM MgSO4  4 mg/mL ONPG in Z-Buffer Procedure

1. Add β-mercaptoethanol to an aliquot of Z-Buffer to a concentration of 0.35%

Reaction at 412 nm in a 1 cm cuvette at 30°C: 400 – X µL Z-Buffer X µL Cell Extract Record background activity 100 µL ONPG

-1 -1 ε412 nm = 4500 M cm

Author: Michael Carter

158

A.7. Bradford protein concentration determination

Reagents

 10 mg/mL Bovine Serum Albumin  Bradford Reagent o 100 mg Coomassie Blue G-250 o 50 mL Methanol o 100 mL 85% Phosphoric Acid o 850 mL H2O Procedure Notes:

1. Protein staining with Coomassie Blue varies slightly each time. Therefore, it is important to perform a standard curve alongside any given set of protein concentration determination reactions. 2. The reaction between protein and the Coomassie dye is time dependent. Accordingly, all protein concentration determination reactions should be incubated for the same amount of time within a given experiment. Because this is impractical, two identical series of reactions for the standard curve are assembled. The first series of standard reactions is recorded prior to recording the experimental reactions. The second series of standard reactions is recorded after recording the experimental reactions. The values from the two standard series are averaged together to assemble the standard curve. 3. At least two concentrations for each experimental sample should be recorded. The reported protein concentration should be the average concentration determined by each concentration. Typical Protein Determination Reactions Standard (Perform in duplicate)

 Dilute 10 mg/mL BSA to 0.1 mg/mL  To water, add the following volumes of 0.1 mg/mL: 0, 25, 50, 75, 100 µL  Add 900 µL Bradford Reagent

159

Experimental

 x = 0  200 µL of 1:200 cell extract  200 – x µL of water  Add 900 µL Bradford Reagent Protocol

1. Add the water and protein necessary for all reactions. 2. Add Bradford Reagent to each reaction in the same sequence as they are to be recorded. 3. Add Bradford Reagent to empty cuvette. 4. Record the A595 for each sample using prestained cuvette.

Author: Michael Carter

Adapted from: Birgit Alber

160

A.8. Protein production

Reagents

 4 x LB + Chloramphenicol (34 µg/mL) Ampicillin (100 µg/mL) plates  Competent Rosetta(DE3)  Bradford Reagent o 100 mg Coomassie Blue G-250 o 50 mL Methanol o 100 mL 85% Phosphoric Acid o 850 mL H2O  2 x 125 mL LB + Chloramphenicol (34 µg/mL) Ampicillin (100 µg/mL)

Procedure

Notes:

 Rosetta strains are derivatives of BL21 strains. Like the BL21 strain, the Rosetta strains have an IPTG iducible gene for the T7 RNA polymerase encoded in the chromosome. Unlike the BL21 strain, Rosetta cells also contain a plasmid that carries the genes for rare tRNAs and chloramphenicol resistance.  Expression vectors that are pET-based are organized such that the inserted coding region will be expressed from a T7 RNA polymerase dependent promoter. Also, the coding region is proximal to and in-frame with a series of six histidine codons. This allows the resulting protein to be produced such that it includes a hexa- histidine tag. The tag is used in nickel affinity chromatography to purify the protein of interest from the respective cell extract.

Procedure Preliminary Note: This step is used to establish the efficiency of the production of soluble protein (protein not in inclusion bodies). It will also establish how long the overproduction should be allowed to continue before the cells are lysed and the protein is extracted.  The procedure is designed to minimize the amount of time that the cells spend in stationary phase in an effort to avoid adding any additional stress to the cells.  A population of cells is maintained (instead of working with a single colony) to avoid the effects that might occur from the expression of a potentially toxic 161

protein. The likelihood of successful expression is greater if a wider array of genetic backgrounds is used.

Perform two independent transformations of Rosetta(DE3) with 5 – 10 µL of vector

a. Parent vector b. Expression vector Note: Rosetta cells transform inefficiently. When performing a standard transformation, the entire 200 µL of the transformed cells must be plated. Divide the transformed cells evenly across two plates. 2. Incubate plates at 37 °C overnight. Avoid allowing colonies to grow too large. (typically less than 14 hr 3. Add 5 mL LB +Cm/Amp to the surface of each plate. 4. Liberate the colonies on the plates into the media. 5. Add suspended colonies from each strain (2 plates per strain) into the respective 125 mL LB+Cm/Amp 6. Incubate at 37°C until an OD578 = 0.4 – 0.6 7. Incubate on ice for 15 min a. Remove 1500 µL from both cultures and spin. Store pellets. = Lysate #1 8. Add 125 µL 0.4 M IPTG to each culture. 9. Incubate at 30°C. a. At 1.5 hour intervals, remove 1500 µL and store pellet. Continue for remainder of day. = Lysate #2, Lysate #3, Lysate #4 . . . 10. After overnight, remove 1500 µL from each culture. Spin and store pellet. = Lysate F 11. Spin remaining cultures and aliquot pellets into 2 microcentrifuge tubes per culture. 12. Extract protein with bead beater. = Extract F 13. Resuspend Lysate #1 in 200 µL of SDS Loading Dye 14. Resuspend Lysate #2, Lysate #3, Lysate #4 . . . and Lysate F in 250 µL SDS Loading Dye 15. Aliquot out 5 µL Extract F for a gel 16. Load 10 µL of the Lysates and all of the aliquotted Extract F on a 10% polyacrylamide gel

Author: Michael Carter

Adapted from: Birgit Alber

162

A.9. SDS Polyacrylamide gel electrophoresis for protein

Reagents

 30% acrylamide/0.8% bisacrylamide  1.5 M Tris-Cl (pH8.8)  10% Ammonium Persulfate (APS; freshly prepared)  Tetramethylethylenediamine (TEMED)  1 X SDS Buffer (Running Buffer) o 25 mM Tris o 0.1 M Glycine o 0.1% SDS  Coomassie Stain o 0.25% Coomassie blue R-250 o 30% Methanol o 20% Acetic Acid  Destain o 30% Methanol o 20% Acetic Acid  Drying Solution o 30% Ethanol o 3% Glycerol  5X SDS Loading Buffer (50 mL) o To 15 mL H2O add: . 1.9 g Tris . 25 mL Glycerol . 2.5 g SDS . pH to 6.8 . 3 mg Bromophenol Blue o Aliquot as necessary, adding 50 µL β-mercaptoethanol to 500 µL dye Protocol

Note: Two layers of gel are prepared. The sample first runs through the Stacking Gel. This is a very low percentage gel with a lower pH. The lower pH in the Stacking Gel serves to keep the amino group of the glycine in the buffer protonated. Thus, the glycine 163

maintains a net neutral charge. This allows the proteins to move more quickly through the gel until they reach the second layer. Then, in the second layer – the Separating Gel – the pH is higher, deprotonating the amino group of the glycine in the buffer. Now, with a net negative charge, the glycine acts to shield the proteins from the effects of the voltage across the gel, decreasing the speed of the proteins through the gel. The decrease in speed causes the proteins to accumulate as they enter the Separating Gel, creating a concentration effect and improving the resolution of the bands.

1. Prepare the Separating Gel according to:

Table A.1. Polyacrylamide gel components

Acrylamide 5 6 7 8 9 10 11 12 13 15 Concentration (%)

30% acrylamide/ 2.50 3.00 3.50 3.75 4.0 4.50 5.00 6.00 6.50 7.50 0.8% bisacrylamide

10% SDS 0.150 0.150 0.150 0.150 0.150 0.150 0.150 0.150 0.150 0.150

1.5 M Tris-HCl 3.75 3.75 3.75 3.75 3.75 3.75 3.75 3.75 3.75 3.75 pH 8.8

H2O 8.75 8.25 7.75 7.50 7.25 6.75 6.25 5.25 4.75 3.75

10% Ammonium Persulfate 0.14 0.14 0.14 0.14 0.14 0.14 0.14 0.14 0.14 0.14

TEMED 0.014 0.014 0.014 0.014 0.014 0.014 0.014 0.014 0.014 0.014

All volumes in mL

a. Add APS and TEMED just before pouring the gel b. Concentration of separating gel should be chosen dependent on the size of expected proteins i. For proteins <500 Da, a Tris-Tricine Gel should be used 2. Assemble glass gel plates in BioRad Casting Stand a. Wet interfacing regions of glass plates with water to help develop a good seal 3. Using a syringe, add the liquid gel between the plates such that it runs down the sides of the space between the plates. This helps to prevent air bubbles. a. Add until 2/3 – 3/4 full 4. Add about 1 in. of water to facilitate the formation of a flat, even top interface 5. Allow >30 min to polymerize 6. Prepare the Stacking Gel a. 0.65 mL 30% acrylamide/0.8% bisacrylamide 164

b. 1.25 mL 0.5 M Tris-HCl pH 6.8 c. 50 µL 10% SDS d. 2.85 mL ddH2O e. 35 µL 10% ammonium persulfate f. 7 µL TEMED 7. Fill the remaining volume between the glass plates with Stacking Gel 8. Slide the well comb into place at the top of the space between the plates 9. Allow >30 min for gel to polymerize. 10. Mount the assembled gel into the electrode assembly a. The smaller glass plate should be facing inward b. Pay attention to the orientation of the buffer dam if only running a single gel 11. Place the electrode assembly into the gel box a. Pay attention to the color coding on the gel box 12. Fill the inside of the electrode assembly with 1 X Running Buffer a. The entirety of the small glass plate of the assembled gel should be submerged in buffer 13. Add <15 µL of sample to each well a. Samples should be boiled in Loading Buffer before loading on gel 14. Once it is confirmed that there are no leaks from the electrode assembly, fill the gel box halfway with 1 X Running Buffer. 15. Run at 10 mA until the dye front has moved into the Separating Gel. a. This is obvious as the dye front is diffuse in the Stacking Gel. As it enters the Separating Gel, it converges into a finer band. 16. Once the dye front has condensed in the separating gel, increase the current to 20 mA. 17. Alternatively, run the sample at 50 V until the dye front condenses in the separating gel. 18. Increase the voltage to 150 V until dye front runs off the gel 19. Continue with Coomassie Staining unless the gel is to be used for Western Blot 20. Remove Stacking Gel 21. Stain in Coomassie Stain overnight 22. Destain in 10 min rounds with Destain a. The Destain can be reused for the initial few rounds b. Used Destain should be passed over activated charcoal to filter it before it is reused c. Continue rounds of Destain until the gel reaches desired visual clarity 23. Soak gel in Drying Solution for 5 min

165

24. Sandwich gel between two sheets of clear cellophane in gel drying frame, avoiding air bubbles a. The cellophane must be saturated in Drying Solution 25. Allow drying overnight

Author: Michael Carter

Adapted from: Current Protocols 1987

166

A.10. Tris-tricine polyacrylamide gel

Stock Solutions Polyacrylamide Gel: . 40% Acrylamide solution (37.5 :1) . 3 M Tris HCl pH 8.45 . 50% Glycerol . 20% SDS . 10% APS . TEMED 10X Upper (Cathode) Buffer: 1 M Tris, 1 M Tricine, 1% SDS (do not adjust pH as it should be about 8.25) 10X Lower (Anode) Buffer: 1 M Tris pH 8.9 (adjust pH with concentrated HCl)

Procedure

1. Pour separating gel: 1 M Tris 8.45, 12-15% Acrylamide, 10% Glycerol

12% gel 15% gel for 10 mls: H2O 1.3 mls 0.6 mls 3 M Tris 8.45 3.3 mls 3.3 mls 40% 3.0 mls 3.7 mls Acrylamide 50% glycerol 2.4 mls 2.4 mls 10% APS 50 ul 50 ul TEMED 7 ul 7 ul . Assemble plates and spacers and seal bottom with 1% agarose . Before adding APS and TEMED, remove 1 ml of solution to microfuge tube . Add 5 ul of APS and 5 ul TEMED to 1 ml solution and pour into bottom of plate assembly to quickly seal . When acrylamide plug has polymerized, add APS and TEMED to the remaining solution and pour separating gel . Overlay top with 0.05% SDS and allow to polymerize

167

2. Pour stacking gel: 0.75 M Tris 8.45, 4% Acrylamide, 0.1% SDS

4% gel for 3 mls: H2O 1.95 mls 3 M Tris 8.45 0.75 mls 40% Acrylamide 0.30 mls 20% SDS 12 ul 10% APS 30 ul TEMED 6 ul

. Pour off 0.05% overlay from polymerized separating gel and rinse with ddH2O . Add APS and TEMED to stacking gel solution and pour gel to with 0.5 cm of top . Insert comb and fill in remaining space with stacking gel solution . Allow to polymerize

3. Electrophoresis

. Assemble electrophoresis unit . Add Upper (Cathode) Buffer to upper reservoir . Add Lower (Anode) Buffer to lower reservoir . Remove comb and rinse wells with Upper Buffer using a 5 ml syringe and 21 gauge needle . Add samples to wells using 22 gauge Hamilton syringe . Run samples through stacking gel at 40-50 volts . Increase voltage to 50-100 volts once samples have completely entered the separating gel . Continue electrophoresis until tracking dye has reached the bottom of the gel . Dissemble unit and stain gel accordingly

Author: Chad Rappleye

168

A.11. Silver staining of polyacrylamide gels

Reagents

 Solution A (50 mL) o 0.02% NaS2O3-5H2O  Solution B (50 mL) o 0.1% AgNO3  Solution C (50 mL) o 6% NaCO3 o 0.02% of 37% Formaldehyde o 0.0004% NaS2O3-5H2O (Solution A)  Fixing Solution (Coomassie Destain) o 30% Methanol o 20% Acetic Acid

Protocol

1. Incubate gel in Fixing Solution 30 min – overnight 2. Rinse in ddH2O 4 x 5 min a. This was is vital! The acid from the fixing solution must be removed for subsequent steps to be successful. 3. Incubate in Solution A 5 min 4. Rinse in ddH2O 2 x 5 min 5. Incubate in Solution B 40 min – 1 hr a. The gel will develop a pale translucent yellow/brown color 6. Rinse BRIEFLY in ddH2O at least 3 times to remove excess silver a. Add water to container and swirl and discard the water. Excessive washing can remove the silver b. Remaining silver will precipitate in the next step if not properly rinse off 7. Add half the prepared volume of Solution C a. The basicity of NaCO3 will cause the Ag to precipitate, developing the bands b. Remaining silver outside the gel might precipitate. If this is observed, merely discard the spent Solution C and replace with the remaining prepared volume of Solution C c. It will generally take about 15 min for any bands to become visible. Usually, all developing will have occurred before 40 min. Excess

169

developing will not result in more intense bands, but will cause dark specks to form on the gel, making it harder to see the bands. 8. Quench the reaction by removing the Solution C and adding Fixing Solution. a. The acid will cause the evolution of CO2 9. If no bands develop, the procedure can be repeated. However, be sure to thoroughly wash the NaCO3 out of the gel. Otherwise, the Ag will precipitate immediately upon addition to the gel, and no staining will be possible without the addition of more Ag after washing the NaCO3 out of the gel.

Author: Michael Carter

Adapted from: Chad Rappleye

170

A.12. Solubilizing inclusion bodies (Large Scale)

Required Reagents

 Cracking Buffer o 50 mM Tris pH 8.0 o 5 mM MgCl2  Wash Buffer 1 o 50 mM Tris pH 8.0 o 1 mM EDTA o 1% Triton X-100  Wash Buffer 2 o 50 mM Tris pH 8.0 o 1 mM EDTA o 0.5 M Urea  Unfolding Buffer o 50 mM Tris pH 8.0 o 8 M Urea o 50 mM β-mercaptoethanol

Procedure

1. Resuspend 4 g of induced cells harvested after induction in 10 mL of Cracking Buffer + 0.1 mg/mL DNase I 2. Crack cells with French Press 3. Incubate at room temperature 15 min to allow DNase to act Note: The following steps serve to dissolve lipids and associated proteins that are within the insoluble pellet. 4. Centrifuge @ 15,700 x g for 10 min 5. Resuspend pellet in 10 mL of Wash Buffer 1 6. Run suspension through French Press 7. Centrifuge @ 15,700 x g for 10 min 8. Resuspend pellet in 10 mL of Wash Buffer 1 9. Incubate at room temperature 5 min

171

10. Repeat steps 7 – 9 Note: The following steps serve to dissolve proteins associated with the inclusion body. 11. Resuspend pellet in 10 mL of Wash Buffer 2 12. Incubate at room temperature 5 min 13. Centrifuge @ 15,7000 x g for 10 min 14. Repeat steps 11 – 13 twice 15. Resuspend pellet in 10 mL of Unfolding Buffer 16. Incubate at room temperature 1 hr 17. Centrifuge @ 15,700 x g for 10 min (Supernatant = unfolded, dissolved inclusion body)

Author: Michael Carter

172

A.13. Protein precipitation with Trichloroacetic Acid (TCA)

Reagents

 100% Trichloroacetic Acid (6.1 M)

Protocol

1. Add Trichloroacetic acid to final concentration 12% 2. Incubate at 4 °C >1 hr 3. Spin at 15.8 K x g for 10 min 4. Remove supernatant

Author: Michael Carter

173

A.14. Digest with recombinant Tobacco Etch Virus Protease (rTEVP):

 Great review of rTEVP: Waugh. Protein Expression and Purification. 80 (2011) 283-293.  rTEVP is produced from plasmid pMHT238Δ o Residues 238 – 242 (C-terminal residues) are not included to improve solubility o Produces the S219V variant that is 100-fold more resistant to autocleavage o MHT = MalE-His-TEVP . The MalE portion is autocatalytically cleaved in vivo during protein production in the host cell to yield an N-terminal His- tagged TEVP  TEVP cleavage site: ENLYFQˇG o See Waugh 2011 for possible subsitutions  Cleavage of each respective fusion protein should be optimized independently o Optimal reaction time, temperature, and molar ratio of fusion protein to protease varies o Typical effective molar ratios range from 10:1 – 100:1

Author: Michael Carter

Adapted from: Chris Rocco

174

A.15. Immunoblot

Required Reagents:

 Whatman 3MM blotting paper  Transfer Membrane slightly larger than gel o Millipore Catalog # IPVH00010 o PVDF Immobilon-P o 0.45 µm pore size  Transfer Buffer (100 mL) o 25 mM Tris (0.5 g) don’t adjust pH o 0.2 M Glycine (2.5 g)  TTBS(Tween 20, Tris Buffered Saline) (500 mL) o 0.2 M Tris pH 7.5 (12.1 g) o 0.15 M NaCl (4.38 g) o 0.1% Tween 20 (500 µL)  Blocking Solution o 0.5 g Skim Milk Powder o 50 mL TTBS  0.1 M Tris-aceate pH 9.5 (0.6 g Tris in 50 mL)  DEA Buffer pH 10 (10mL) o 25% (v/v) Diethanolamine (2.5 mL)

o 0.2 µM MgCl2∙6H2O (2 µL 1M MgCl2) o pH to 10.0 with NaOH  Attophos Reagent o 0.06% (m/v) Attophos in DEA Buffer . AttoPhos: Promega Catalog # S1011  Attophos Solution (50 mL) o 10 mM MgCl2 (500 µL 1 M MgCl2) o 1% (v/v) Attophos Reagent (500 µL Attophos Reagent) o 0.1 M Tris-acetate pH 9.5 (0.6 g Tris)  Primary Antibody Solution o 105 dilution (depends) of Primary Antibody in TTBS  Secondary Antibody Solution

175

o 1:7,500 dilution of Secondary Antibody in TTBS . Secondary Antibody: Bio-Rad Goat Anti-Rabbit IgG (H+L)-AP Conjugate Catalog # 170-6518

Procedure

Note:

All incubations are carried out agitating at room temperature unless indicated otherwise. Wear gloves when handling the membrane.

1. Run Protein on polyacrylamide gel 2. Rinse Transfer Membrane with EtOH 3. Equilibrate Blotting Paper, Transfer Membrane, and gel in Transfer Buffer a. Keep gel separate from Blotting Paper and Transfer Membrane 4. Assemble transfer apparatus a. Add TTBS buffer between each layer to be help prevent air bubbles and to keep the layers saturated b. Place 1 piece of Blotting Paper on the bottom plate of the GeneMate Electroblotter c. Place the Transfer Membrane on the Blotting Paper d. Place the gel on the Transfer Membrane e. Place another piece of Blotting Paper on top of the gel f. AVOID AIR BUBBLES BETWEEN LAYERS! 5. Blot at 5.5 mA/cm2 of gel for 1.5 hr 6. Remove membrane from transfer apparatus a. Keep membrane moist! 7. Wash the Transfer Membrane 3 times in TTBS for 5 min 8. Incubate the Transfer Membrane in Blocking Solution 1 hr to overnight 9. Wash the Transfer Membrane 3 times in TTBS for 5 min 10. Incubate the Transfer Membrane in primary antibody 1 hr a. Do not exceed 1 hour incubation 11. Wash the Transfer Membrane 3 times in TTBS for 5 min 12. Incubate the Transfer Membrane in Secondary Antibody Solution 1 hr 13. Wash the Transfer Membrane in 0.1 M Tris-acetate pH 9.5 for 5 min 14. Incubate in Attophos Solution for 2 min a. Avoid over incubation 15. Expose on Storm a. Use setting for fluorescence emission at 520 nm 176

b. Adjust PMT voltage to adjust the amount of background detected

Author: Michael Carter

177

A.16. Polyacrylamide gel electrophoresis for DNA

Reagents

 30% acrylamide/0.8% bisacrylamide  1.5 M Tris-Cl (pH 8.8)  10% Ammonium Persulfate  TEMED  1 X TBE Buffer (Running Buffer) o 0.9 M Tris o 0.9 M Boric Acid o 20 mM EDTA pH 8.0 Protocol

Note: Polyacrylamide gels are more sensitive at detecting lower amounts of DNA than agarose gels. 12% Polyacylamide gels should be used to separate DNA that is between 700 and 90 bp. Anything larger should be separated on an agarose gel.

Note: While assembly of the gel is similar for protein gels, there are slight differences detailed here that distinguish the process from preparing and running a protein gel.

1.Prepare the separating gel according to:

Table A.2. Polyacrylamide gel components

Acrylamide 5 6 7 8 9 10 11 12 13 15 Concentration (%)

30% acrylamide/ 2.50 3.00 3.50 3.75 4.0 4.50 5.00 6.00 6.50 7.50 0.8% bisacrylamide

1.5 M Tris-HCl 3.75 3.75 3.75 3.75 3.75 3.75 3.75 3.75 3.75 3.75 pH 8.8

H2O 8.75 8.25 7.75 7.50 7.25 6.75 6.25 5.25 4.75 3.75

10% Ammonium Persulfate 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07

TEMED 0.014 0.014 0.014 0.014 0.014 0.014 0.014 0.014 0.014 0.014

All volumes in mL 178

a. Add APS and TEMED just before pouring the gel

b. Adjust acylamide concentration depending on the fragment sizes to be separated 2. Assemble glass gel plates in BioRad Casting Stand a. Wet interfacing regions of glass plates with water to help develop a good seal 3. Using a syringe, add the liquid gel between the plates such that it runs down the sides of the space between the plates. This helps to prevent air bubbles. a. Fill until about ¼ in. from the top of the front plate 4. Slide the well comb into place at the top of the space between the plates 5. Allow >30 min for gel to polymerize. 6. Mount the assembled gel into the electrode assembly a. The smaller glass plate should be facing inward b. Pay attention to the orientation of the buffer dam if only running a single gel 7. Place the electrode assembly into the gel box a. Pay attention to the color coding on the gel box 8. Fill the inside of the electrode assembly with 1 X TBE a. The entirety of the small glass plate of the assembled gel should be submerged in buffer 9. Add <15 µL of sample to each well 10. Once it is confirmed that there are no leaks from the electrode assembly, fill the gel box halfway with 1 X TBE. 11. Run at 150 V for 1.25 hour or until dye front approaches the end of the gel 12. Disassemble the gel and submerge in 0.1% ethidium bromide solution for >1 min 13. Visualize with UV light

179

A.17. Site directed mutagenesis

Principle

PCR amplification, primed by oligonucleotides that contain desired mutations, of a circular plasmid isolated from E. coli is catalyzed by a nonstrand-displacing polymerase. Following PCR, complementary product strands contain the mutations present in the primers and may anneal to each other to form a double-stranded, circular molecule that is nicked once on each strand and whose sequence varies from the template plasmid only at the sites mutated in the primers. Given that E. coli methylates its DNA, addition of DpnI selectively degrades the original template, and the final product is used directly for transformation.

Primer Design

1. Forward and Reverse primers should be equal in length and perfectly complementary to each other 2. Length: 25 – 45 bases 3. Melting Temperature: ≥ 78 °C 4. The desired mutation should be centered in each primer Protocol

Reaction (50 µL)

100 ng Template Plasmid 1 µL 10 µM Forward Primer 1 µL 10 µM Reverse Primer 1 µL 10 mM dNTPs 5 µL 10X Buffer 1 µL Pfu Ultra II Fusion x µL Water

180

Thermalcycling Program

1. 95 °C 5 min 2. 95 °C 30 s 3. 55 °C 30 s 4. 68 °C x s (Repeat steps 2-4 for 18 cycles)

5. Add 1 µL DpnI 6. Incubate at 37 °C 1 hr 7. Transform cells with 1 µL a. Concentrate transformed cells and plate entire volume Notes

1. The frequency of picking colonies that contain the appropriately mutated plasmid is about 50%. 2. If DpnI is not added, the transformation plate will be nearly a lawn. 3. In screening for the appropriate mutation, try to use primers that will provide results of ≥150 bp of the regions that flank the mutation. ~1/10 will include a duplication or insertion that might be missed if the sequencing focuses too closely on only the mutated portion. 4. Remember that, most often, the only way to distinguish the parent plasmid from the mutated plasmid will be by sequence analysis. Most mutations will not affect the digest pattern. Be especially careful to work and label carefully!

Author: Michael Carter

Adapted from: Kathleen Sandman. Microbiology 581 Lab Manual.

181

A.18. Electrophoretic mobility shifts (SYBR)

Reagents

 Cold TBE o Per Liter . 10.9 g Tris (do not adjust pH) . 5.5 g Boric Acid . 4.0 mL 0.5 M EDTA pH 8.0 o Note: Do not store TBE in cold conditions. A precipitate forms that is impossible to return to solution and is nearly impossible to clean out of the bottle. Place the bottle at 4 °C approximately 30 minutes before running the gel. Store at room temperature  1.5 M Buffer  30% Acrylamide/0.8% Bisacrylamide  10% Ammonium Persulfate (Freshly prepared)  TEMED  Loading dye: 50% Glycerol, 1:2000 SYBR o Story in dark conditions o Make fresh loading dye frequently o SYBR excites at 497 and emits at 520 when bound to double stranded DNA Procedure Notes:

4. Gels must be loaded quickly! As samples sit, noncovalent protein-DNA complexes will dissociate, and the resulting bands with become increasingly diffuse. 5. Adjust the pH of the gel to the appropriate pH for promoting DNA-protein interaction. a. Unless required for the DNA-protein complex, no additional salts are necessary. 6. A balance must be struck. While running the gel, the DNA-protein complex will dissociate. More time spent running the gel will result in increasingly diffuse bands; therefore, the gel must be run quickly. However, a faster gel requires a 182

higher voltage. The result is more heat being provided to the gel which will also cause the DNA-protein complex to dissociate. 7. Use small DNA fragments (100 – 200 bp). a. Smaller fragments run faster on the gel, allowing for shorter run times. b. Shifts will be easier to resolve from unbound probe. 8. Keeping the gels cold as they run slows the dissociation of the DNA-protein complex. 9. Appropriate DNA:protein molar ratios must be determined experimentally. a. A value should be chosen that clearly demonstrates a lack of a shift with a negative control DNA fragment yet is high enough that in more dilute protein conditions, a shift can be detected. It is crucial to be able to establish that the amount of shifted DNA varies according to the amount of protein added. 10. Reaction volumes should be kept small. Larger volumes will yield more diffuse bands. Example Protocol (His-MalE-PccR:pccB, pccR, rpoZ upstream fragments)

5. Assemble a 5% polyacrylamide gel according to “Polyacrylamide Gel Electrophoresis for DNA” a. Use the appropriate buffer in place of “1.5 M Tris-HCl pH 8.8” 6. Reaction a. 5 µL 20 mM MOPS pH 7.0 b. 4 µL 0.1 pmol/µL DNA fragment (0.4 pmol) c. 2 µL Diluted His-MalE-PccR (1.7 – 7 pmol) i. Mix thoroughly! 7. Incubate at room temperature for 20 min 8. Add 5 µL Loading Dye 9. Load gel quickly! a. Keep close watch on the wells. It is difficult to visualize which wells have been loaded. 10. Run gel at 100 V for 45 min. 11. Without removing the gel from the glass plates, immediately visualize the gel on the Storm

Author: Michael Carter

183

A.19. Characterization of Δccr23KB

Isolation: Kelsey Baron

Conjugation experiment: 6/6/11, pg 1-20, 40-49, plasmid pKB15 # of original transconjugates: 10 isolated reddish-brown colonies on 1:100 dilution plate Picked 6 colonies and inoculated minimal medium succinate (~100 μl aerobically) and three phase streak each colony on LB·kan (keep as potential single crossover).

3/50 (6%) KanS, sucroseR, acetateR potential double crossovers. 1/50 (2%) KanR single crossovers.

Clone: Δccr23KB Primer 504 (genotccrup_2) ATCGCGACATAGGCGAGGCGTTTCC Primer 221 (deltaecmup_rev1) GCCAGGCGGTACCCTTCTGGGTCATGGCAAG Template: Genome of Δccr23KB Product: Δccrupmutant

Primer # 505(genotccrdown_2) CGATCGAGGCCGGCATCACATTC Primer # 203 (ccrgeno_for3) CGCTCAGCACGTCGAGGATCCAGTC Template: Genome of Δccr23KB Product: Δccrdownmutant

Sequenced Region: Target Region: pos 2716054 to 2720329 Deletion: pos 2717572 to 2718704 Sequenced: 2715759 – 2720543 completely sequenced (across deletion and beyond recombined fragment: +295 nucleotides and +214 nucleotides)

Sequencing Results Δccrupmutant: primer #270 (RTheli_R1) CCGCCTCCATCATCAAGATCGAGAAGC pos 2715759 – 2716068 primer #254 CTCGCCGGGCTTCTCGATCTTG: pos 2715995 – 2717067 (10/27/11) primer #88 CTGACCTTCCATATGTTCTAGACCACCTCGAGCCCGGCCTCGATCAG: pos 2716961 – 2717572 and 2718704 – 2719035 (10/27/11)

184

Δccrdownmutant: primer #204 (ccrgeno_rev3) GGTTGGTGCGATAGAGCGCATTCGAG: pos 2717340 – 2717572 and 2718704 – 2719174 (11/17/11) primer #124 (ecmgeno_rev1) AAGCGGCGGTATTTCTCTTC: pos 2718873 – 2719841 (11/17/11) primer #505 (genotccrdown_2) CGATCGAGGCCGGCATCACATTC: pos (11/17/11)

185

A.20. Characterization of Δecm47KB

Isolation: Kelsey Baron

Plasmid used: pKB22 Conjugation experiment: 3/23/11, pg. 41-45 (notebook “Kelsey Baron 2011”) # of original transconjugates: >100 isolated reddish-brown colonies. There was a lawn in the background of these isolated colonies because the proper dilution was not performed before plating the conjugated R.s./E.coli cells. Continued with 6 potential single cross over colonies.

2/50 (4%) KanR single cross overs 10/50 (20%) KanS, Succinate+, Acetate- potential double cross overs Carried on with 2 clones: Δecm47KB, Δecm49KB

Clone: Δecm47KB Amplified region for sequencing Primer #411 (ecmmutantup_for1) GGGCTTGTGCTGGTTTCTGTACATCTTC Primer #412 (ecmmutantup_rev1) GCCGGGACGCGAGGGATCCTATTC Template: Δecm47KB Product: Δecmupmutant

Primer #413 (ecmmutantdown_for1) CGCATAAATGCGACATGCAGCAATATCC Primer #414 (ecmmutantdown_rev1) GGACCAAGATGACCGCCGAACTCAC Template: Δecm47KB Product: Δecmdownmutant

Sequenced Region: Target Region: bp 2717764-2722929 Deletion: bp 2719210-2721105 Sequenced: (+87) 2717677 – 2719210 and 2721105 – 2723218 (+289)

Δecmupmutant: Primer # 411 (ecmmutantup_for1) GGGCTTGTGCTGGTTTCTGTACATCTTC: pos 2717677-2718744 (6/2/11) Primer # 412 (ecmmutantup_rev1) GCCGGGACGCGAGGGATCCTATTC: pos 2718191-2719210c and 2721106-2721123c (6/2/11) Primer # 424 (ecmmutantdownF_seq1) AGCCGGGACGCGAGGGATCCTATTC: pos 2719140-2719210c and 271105-2721121c 186

Δecmdownmutant: Primer # 413 (ecmmutantdown_for1) CGCATAAATGCGACATGCAGCAATATCC: pos 2721105-2721781 (6/2/11) Primer # 414 (ecmmutantdown_rev1) GGACCAAGATGACCGCCGAACTCAC: pos 2722153-2723218c (6/2/11) Primer #425 (ecmmutantdownR_seq1) ATATTCGACGACCGTCACCTTGG: pos 2722189-2723012 (6/14/11)

187

A.21. Characterization of Δmcd11KB

Isolation: Kelsey Baron

Plasmid: pKB4 Conjugation experiment: 5/31/12 pg 115

Clone: Δmcd11KB Amplified region for sequencing Primer #757 (deltamcdPCRdown_rev1) GCTGCTACGCCAAGACGATCAATCTC Primer #755 (deltamcdgenot_for1) ATCTCCGCTGGGAGATGCGTCTCTTG Template: Δmcd11KB purified chromosomal DNA Product: DmcdUp

Primer #261(mcddown_for2) CCCTGGTTCTATCCCGAGGAC Primer #756 (deltamcdgenot_rev1) ATGACCGCCACATCGAATTGAGAGC Template: Δmcd11KB purified chromosomal DNA Product: DmcdDn

Sequenced Region: Target Region: pos 266754 – 271326 Deletion: pos 268307 to 269833 Sequenced: 266698 – 271356 (across deletion and beyond recombined fragment: +56 bp and +32 bp)

DmcdUp: Primer #755 (deltamcdgenot_for1) ATCTCCGCTGGGAGATGCGTCTCTTG : 266834 – 270290, 267877 – 268306 (8/21/13) Primer #907 (DmcdUpSeqR2) CGCAGCTCCGTCTGACGGATG: 270041 – 270864, (08/28/13) Primer #757 (deltamcdPCRdown_rev1) GCTGCTACGCCAAGACGATCAATCTC: 270552 – 271314, (08/21/13) Primer #906 (DmcdUpSeqR1) CCGCTCTCGGACGCGTTCGAGATATAG: 271205 – 271356 (08/28/13)

DmcdDn: Primer #757 (deltamcdPCRdown_rev1) GCTGCTACGCCAAGACGATCAATCTC: 266698 – 267537 (8/21/13) Primer #908 (DmcdDnSeqR1) CCGGGCCTCTCCGAGTGACTG: 267444 – 268010 (08/28/13) 188

Primer #756 (deltamcdgenot_rev1) ATGACCGCCACATCGAATTGAGAGC: 2677652 – 268306, 269834 – 270156 (08/21/13)

189

A.22. Characterization of Δmch49KB

Isolation: Kelsey Baron

Plasmid: pKB23 Conjugation experiment: pg 115

Clone: Δmch49KB Amplified region for sequencing Primer #94 (RTmcl2_for1) TATTCCCGGCTCGAAGG Primer #759 (deltamchgenot_rev1) GCGCCCTTTCAGATGATGATGACGG Template: Δmch49KB MM Suc Cell Suspension Product: DmchUp

Downstream: PCR primer #758 (deltamchgenot_for1) ACTCTACCGCTACCTGACCGAAGAC PCR primer #230 (mchdown_rev2) CGTGAGCTTGGCGTTGAAGA Template: ΔmchKB MM Suc Cell Suspension Product: DmchDn

Sequenced Region: Target Region: pos 2732335 – 2736366 Deletion: pos 2733828– 2734768 Sequenced: pos 2732251 – 2736405 (across deletion and beyond recombined fragment: +84 bp and +39 bp)

SeqTemplate: Primer #94 (RTmcl2_for1) TATTCCCGGCTCGAAGG: 2732251 – 2733093 (8/21/13) Primer #226 (deltamchup_rev1) GATGGTGGATCCCAGCCGGTAATCCTCGAAG: 2732685 – 2733780 (08/29/13) Primer #759 (deltamchgenot_rev1) GCGCCCTTTCAGATGATGATGACGG: 2733665 – 2733827, 2734769 – 2735367 (08/21/13)

SeqTemplate: Primer #758 (deltamchgenot_for1) ACTCTACCGCTACCTGACCGAAGAC: 2733632 – 2733827, 2734769 – 2735451 (8/21/13) Primer #230 (mchdown_rev2) CGTGAGCTTGGCGTTGAAGA: 2735360 – 2736360 (08/21/13) 190

Primer #909 (DmchDnSeqF1) TCTACGGCCAGTCGGTCAAGAACAAC: 2736306 – 2736405 (08/28/13)

191

A.23. Characterization of Δmcl1_4KB

Isolation: Kelsey Baron

Plasmid: pKB11 Conjugation experiment: 8/16/11, pg. 21-34 # of original transconjugates: 100 (1:100); 20 (1:1000) Continued with 5 (2 from 1:100, 5 from 1:1,000) potential single cross over colonies.

9/50 (18%) KanS, KanS, AcetateR Potential double cross overs Carried on with 3 clones

Clone: Δmcl1_4KB Amplified region for sequencing Primer #577 (mcl1up_for2) CGGCAGCAGGGTCTGGAGATCG Primer #565 (mcl1PCRup_rev1) GCATGATCGGCGTCTATTGGGACAACC Template: Δmcl1_4KB genomic DNA Product: ΔMcl1upmutant

Primer #566 (mcl1PCRdown_for1) GCGGCTTCCGCAATTATCTTCGCAG Primer #567 (mcl1PCRdown_rev1) ATCTTCTCGGCATCCTTCCACAGGATCG Template: Δmcl1_4KB, 63°C, PCR Conditions 11/22/11, pg. 29 Product: Δmcl1downmutant

Sequenced Region: Target Region: pos 353280–357067 Deletion: pos 354761-355584 Sequenced: (+ 98) 353182 -354761 355584-357109 (+42)

Δmcl1upmutant: Primer #577 (mcl1up_for2) CGGCAGCAGGGTCTGGAGATCG: 353182-353761 (12/12/11) Primer #566 (mcl1PCRdown_for1) GCGGCTTCCGCAATTATCTTCGCAG: 354616- 354761 and 355584-356101, across deletion includes SphI site (12/12/11) Primer #222 (deltamcl1down_for1) GCGGCGATGCATGCGGCCAAGGCGAGGGGCGAG: 355628-356102 (12/14/11) Primer #224 (deltamcl1up_rev1) AGGCGGCGCATGCTGAAGGCGGAAGCTCATG: 353677-354721c (12/16/11)

192

Δmcl1downmutant: Primer #565 (mcl1PCRup_rev1) GCATGATCGGCGTCTATTGGGACAACC: 354554- 354761c and 355584-356065c (11/18/11) Primer #228 (mcl1down_rev2) GGCGAGATCACCGAATAGAC: 356075-357109c (11/28/11) Primer #601(mcl1down_seq1) TATCGGGTCATTGTCCGATGAGTTGAGGAG: 354566-354761c and 355584-356231c (12/14/11)

193

A.24. Characterization of RsΔmaeB1MC63

Isolation: Michael Carter

Plasmid: pKB99 Conjugation experiment: Michael Carter DNA Work pKB99 11/5/12 # of original transconjugates: 30 on undiluted plate Continued with 4 potential single cross over colonies.

67/91 (74%) KanS, Lactate+ Potential double cross overs Carried on with 4 clones

Clone: RsΔmaeB1MC63 Amplified region for sequencing Primer (DmaeB1genUpF1) CCGACCCGAGATCATCGGCACATAG Primer (DmaeB1genUpR1) GCCTCTGCTCAGCCGTAATGGGTAAC Template: chromosome of strain RsΔmaeB1MC63 Product: DmaeB1genUp

Primer (DmaeB1genDnF1) AGAAGGTCTTCACCTTGTGGTTCTGCATC Primer (DmaeB1genDnR1) CGGTTGCATGGACATTCAGATGGTTTAGTG Template: chromosome of strain RsΔmaeB1MC63 Product: DmaeB1genDn

Sequenced Region: Target Region: pos 185785 – 189974 Deletion: pos 186927 – 189040 Sequenced: pos (+54) 185731 – 186926, 189041-190002 (+28)

DmaeB1genUp: Primer (DmaeB1genUpF1) CCGACCCGAGATCATCGGCACATAG: pos 186891 – 186926 (12/13/12 Req: 558279) Primer (DmaeB1genUpR1) GCCTCTGCTCAGCCGTAATGGGTAAC: pos 189041 – 189794; 186880 – 186926 (12/13/12 Req: 558280)

DmaeB1genDn: Primer (DmaeB1genDnF1) AGAAGGTCTTCACCTTGTGGTTCTGCATC: (12/13/12 Req: 558281) pos 185731 – 186714 Primer (DmaeB1genDnR1) CGGTTGCATGGACATTCAGATGGTTTAGTG: pos 186066 – 186926; 189041 – 189123 (12/13/12 Req: 558282) 194

A.25. Characterization of RsΔpccRMC12

Isolation: Michael Carter

Plasmid: pMC70 Conjugation experiment: Michael Carter DNA Work pMC70 02/24/12 # of original transconjugates: 10 big colonies (undiluted), ~500 small colonies (1:100) Continued with 2 big and 2 small potential single cross over colonies.

22/25 (88%) KanS, MM Suc+ Potential double cross overs (All originate from big single crossover colonies) 100/112 (89%) KanS, LB+ Potential double cross overs (48 from big single crossover colonies; 40 from small single crossover colonies) Carried on with 6 clones: RsΔpccRMC12, 15, 19: From MM Suc, originally big single crossover colony RsΔpccRMC34, 52: From LB, originally big single crossover colony RsΔpccRMC106: From LB, original small single crossover colony

Clone: RsΔpccRMC12 Amplified region for sequencing Primer (DramB_seqUpF1) GGGGCATCGCCTTCTTCGAATATCTG Primer (DramB_seqUpR1) CTGCTAATCGACAATCTCGTATGGCAG Template: chromosome of RsΔpccRMC12 Product: D_ramB_up2

Primer (DramB_seqDnR1) CCGCTCATCCTCATCATGCTGTTCTC Primer (D_ramB_downR4) AGCCGGTCACGCTCTTCAAGCACTG Template: chromosome of RsΔpccRMC12 Product: D_ramB_down2

Sequenced Region: Target Region: pos 791155 – 795679 Deletion: pos 792967 – 794190 Sequenced: pos (+88) 791067 – 792966, 794191 – 795705 (+26)

D_ramB_up2: Primer (DramB_seqUpF1) GGGGCATCGCCTTCTTCGAATATCTG: pos 794857 – 795705 (04/04/12 Req: 511182)

195

Primer (DramB_seqUpR1) CTGCTAATCGACAATCTCGTATGGCAG: pos 792880 – 792966; 794191 – 794936 (04/04/12 Req: 511183) Primer (DramBseqUp1) CTGCGAGGAAGACAGGTGGAAGATCTC: pos 794955 – 795483 (04/30/12 Req: 514632)

D_ramB_down2: Primer (D_ramB_downR4) AGCCGGTCACGCTCTTCAAGCACTG: pos 791067 – 791771 (04/04/12 Req: 511185) Primer (DramB_seqDnR1) CCGCTCATCCTCATCATGCTGTTCTC: pos 792741 – 792966; 794191 – 794326 (4/04/12 Req: 511184) Primer (D_ramB_downF) TATTAGGTACCCTGCCATACGAGATTGTCGATTAG: pos 792301 – 792826 (04/06/12 Req: 511351) Primer (D_ramB_downR) ATTATAGAATTCTAGAGATCCGCTTCAACTACATGTCG: pos 791346 – 791999 (04/06/12 Req: 511352) Primer (DramBseqDn4) AGACATTGCCCTTGATCGCGCACTC: pos 791725 – 792345 (04/19/12 Req: 5113151)

196

A.26. Characterization of RsΔphaRMC43

Isolation: Michael Carter

Plasmid: pMC69 Conjugation experiment: Michael Carter DNA Work pMC69 04/24/12 Continued with 4 potential single crossover colonies.

77/79 (97%) KanS, MM Suc+ Potential double cross overs 46/52 (89%) KanS, LB+ Potential double cross overs Carried on with 7 clones:: RsΔphaRMC43, 53, 74 from MM Suc+ RsΔphaRMC144, 153, 205, 207 from MM LB+

Clone: RsΔphaRMC43 Amplified region for sequencing

Primer (DphaRgenoUpF5) CGACTACATCCGCGAAGGCTACATGC Primer (DphaRgenoDnR5) GGTCTTCGCCGAAGTCGCCTGAC Template: chromosome of strain RsΔphaRMC43 Product: DphaRgenoUp

Primer (DphaRgenoUpF2) AGCCGGTCACGCTCTTCAAGCACTG Primer (DphaRgenoDnR3) AGGATCTTCGAGGCAGGACAGTCTCTTTC Template: chromosome of strain RsΔphaRMC43 Product: DphaRgenoDn

Primer (DphaRgenoUpF1.1) CCTTTCTAGATGATCGACCGTGCCCTAGTCTCTTCTG Primer (DphaRgenoDnR1.1) ATGACGGATCCGCAGATGGACGAGATGTTCGAGAAG Template: chromosome of strain RsΔphaRMC43 Product: pMC77 (Inserted into pUC19, secondary structure in PCR products prevented sequencing)

Sequenced Region: Target Region: pos 2110172 – 2113636 Deletion: pos 2111657 – 2112112 Sequenced: (+24) 2110148 – 211656, 2112113 – 2113743 (+107)

197

DphaRgenoUp: Primer (DphaRgenoUpF5) CGACTACATCCGCGAAGGCTACATGC: pos 2113170 – 2113743 (06/04/12 Req: 522036) Primer (DphaRgenoUpF6) AGCGGATAACAATTTCACACAGG: pos 2112763 – 2113723 (06/21/12 Req: 525491) Primer (DphaRUpR2.2) TCCTCGAGGGTGGTACCGTCGCTGGTCTCGGTGTTGTAGAG: pos 2112149 – 2113061 (06/29/12 Req: 525492) Primer # (DphaRgenoUpF2) GTGGGTATGGCCGAGACTGACAAG: pos 2111551 – 2111656 (06/29/12 Req: 525490)

DphaRgenoDn: Primer (DphaRgenoUpF2) AGCCGGTCACGCTCTTCAAGCACTG: pos 2110961 – 211656 (06/05/12 Req: 522038) Primer (DphaRgenoDnR3) AGGATCTTCGAGGCAGGACAGTCTCTTTC: pos 2110148 – 2111048 (06/04/12 Req: 522039) pMC77: Primer (pUC-Reverse) AGCGGATAACAATTTCACACAGG: pos 2111503 – 2111656; 2112113 – 2112202 (10/17/12 Req: 548030)

198

A.27. Characterization of Rs ccr-lac1A

Isolation: Michael Carter

Plasmid: pMC26F (lac operon is in the same direction as specR gene) Conjugation experiment: Michael Carter DNA Work 12/14/10

Clone: Rs ccr-lac1A Amplified region for sequencing Primer (ccr-fusUp_seqF1) GGCTGAGATATTCCTTCATCAGATCGTTCTG Primer (ccr-lacUp_seqR1) CAGTCACGACGTTGTAAAACGAC Template: chromosome of strain Rs ccr-lac1A Product: Upstream

Note: The pMC26F plasmid and Rs ccr-lac1A strain were originally designed to be a part of developing the RsΔccr::lac44A strain, requiring the inclusion of a downstream fragment of ccr. The downstream fragment of ccr was not involved in the recombination the resulted in Rs ccr-lac1A strain and is not addressed here.

Sequenced Region: Target Region: pos 2718803 – 2719440 Sequenced: pos (26 bp of lacZ) 2718803 – 2719570 (+130)

Upstream: Primer (ccr-fusUp_seqF1) GGCTGAGATATTCCTTCATCAGATCGTTCTG: pos 2718803 – 2719543 (3/19/11 Req 445965) Primer (ccr-lacUp_seqR1) CAGTCACGACGTTGTAAAACGAC: pos 2718812 – 2719570 (3/19/11 Req 445966)

199

A.28. Identification of location of transposon insertion site

Protocol: Transposon insertion site identification and freezer stock culture preparation of Rhodobacter sphaeroides 2.4.1 mutants obtained using the “plasposon” pRL27 (Larsen et al., 2002)

Liquid LB + kanamycin (20 μg/ml) culture 1. Inoculate a colony of R. sphaeroides transposon mutant grown on an LB + kanamycin (20 μg/ml) plate into a 5-ml LB + kanamycin (20 μg/ml) tube. 2. Incubate the tube aerobically on a shaker at 30°C until the culture turns pink- orange turbid (usually takes 2–3 days).

Genomic DNA isolation 1. Transfer 1.5 ml fully-grown culture (above) into an eppendorf tube. 2. Centrifuge the culture at 9,000 rpm at 4°C for 5 min and discard the supernatant. The cell pellet can be stored at –20°C until genomic DNA isolation is performed. 3. Genomic DNA is isolated from this cell pellet using the GenEluteTM Bacterial Genomic DNA Kit (Sigma-Aldrich, St. Louis, MO) following the manufacturer’s instruction. Genomic DNA can be stored at –20°C or proceed directly to NcoI digestion (see below).

Freezer stock culture preparation 1. About 3.5 ml LB + kanamycin culture remains after genomic DNA isolation. This culture is transferred aseptically into a sterile eppendorf tube and centrifuged at 9,000 rpm at 4°C for 2–5 min each until all cells pelleted. 2. Discard the supernatant completely and resuspend the cells in 0.9 ml sterile LB medium. Transfer this cell suspension into a sterile cryogenic tube. 3. Add 100 μl sterile dimethylsulfoxide (DMSO) or 200 μl sterile 50% (w/w) glycerol to the cell suspension. Gently invert mix the cell suspension several times and place the tube on ice. 4. Store the culture at –74°C.

NcoI endonuclease digestion of genomic DNA 1. Following reaction mixture (Vtotal = 30 μl) is prepared in an eppendorf tube.

10× NEB3 Buffer + BSA (3+) 3 μl Genomic DNA 26 μl NcoI 1 μl

2. Incubate the reaction mixture at 37°C for 1 h. 200

3. Incubate the reaction mixture at 65°C for 20 min (for enzyme inactivation) and store the digest at 4°C until agarose gel electrophoresis is run.

Agarose gel electrophoresis Run 5 μl each of the genomic DNA and its digest on a 0.8% agarose gel at 80 V for 1 h to check for isolation and digestion, respectively.

Intramolecular ligation of the NcoI digest 1. Once the agarose gel shows successful digestion, proceed to perform intramolecular ligation. 2. Prepare a following reaction mixture (Vtotal ≈ 50 μl).

ddH2O 29 μl 10X ligase buffer 5 μl NcoI digest 15 μl T4 DNA ligase 1.5 μl

3. Incubate the reaction mixture at room temperature (about 17°C) overnight (14– 18h). 4. Store the product at 4°C or –20°C until transformation is performed.

Transformation 1. Obtain a tube of 200 μl competent E. coli DH5α-λpir from –80°C and thaw on ice. 2. Add 25 μl of the ligation product (above) to the above E. coli culture and gently mix by tapping. 3. Incubate the cells on ice for 20 min. 4. Incubate the cells at 42°C for 2 min. 5. Incubate the cells on ice for 10 min. 6. Add 1 ml LB medium to the tube. 7. Incubate the culture on a shaker at 37°C for 1 h. 8. Centrifuge the culture at 9,000 rpm at 4°C for 10 min and discard the supernatant. 9. Resuspend the cells in the residual medium and spread it all on an LB + kanamycin (20 μg/ml) plate. 10. Incubate the plate at 37°C overnight and then store it at 4°C. 11. Inoculate a colony into a 5-ml LB + kanamycin (20 μg/ml) tube and incubate on a shaker at 37°C overnight. 12. Store the culture at 4°C until plasmid isolation is performed.

Plasmid DNA isolation 1. Harvest all the culture (i.e. 5 ml) of E. coli DH5α-λpir clone (above) by centrifuging at 9,000 rpm at 4°C for 1–2 min each until all cells pelleted.

201

2. Plasmid is isolated from the above cell pellet using the GeneJETTM Plasmid Miniprep Kit (Fermentas, Burlington ,Ontario, Canada).

Plasmid DNA sequencing 1. Following primers are used in sequencing the plasmid isolated above.

tpnRL17-1 (5’– AAC AAG CCA GGG ATG TAA CG –3’) tpnRL13-2 (5’– CAG CAA CAC CTT CTT CAC GA –3’)

2. Note that tpnRL17-1 anneals to the oriR6K end of the transposon, whereas tpnRL13-2 anneals to the KanR gene end of the transposon. 3. Conduct Blastn search (NCBI website) of the plasmid sequences against the genome of Rhodobacter sphaeroides 2.4.1. You will find the overlap region (usually 8–10 bp) from your tpnRL17-1 and tpnRL13-2 sequences. This is the location of transposon insertion.

Reference

Larsen RA, Wilson MM, Guss AM, and Metcalf WM (2002) Genetic analysis of pigment biosynthesis of Xanthobacter autotrophicus Py2 using a new, highly efficient transposon mutagenesis system that is functional in a wide variety of bacteria. Arch Microbiol 178: 193–201

Author: Marie Asao

Adapted from: Dr. Kathleen Sandman, Microbiology 581 Lab Manual

202

A.29. Reverse transcription polymerase chain reaction (RT PCR)

Reagents

 5X First Strand Buffer  0.1 M Dithiothreotol (DTT)  10 mM dNTPs  Superscript III Reverse Transcripase  10 µM RT10-mer (random oligomer) Reverse Transcription (cDNA prep)

 4 µL H2O  20 µL 5X First Strand Buffer (Invitrogen)  1 µL 10 µM RT10-mer  70 µL total RNA  2 µL 0.1 M DTT o Incubate at 65 ºC for 10 min o Allow reaction mix to cool to 50 ºC with heat block  1 µL 10 mM dNTPs

Remove 5 µL for a –RT control

 2 µL Superscript III (Invitrogen) Run reaction at 50 ºC for 1 hr “cDNA”

Notes: o Use 1 µL of cDNA as template for PCR to detect background undigested DNA o PCR conditions are specific to given primers and product size.

203

PCR Template 1 µL Buffer 2.5 µL Reaction Conditions F-Primer (10 µM) 1 µL 97°C 20 s R-Primer (10 µM) 1 µL 64°C 20 s dNTP (10 mM) 0.25 µL 72°C 20 s Taq 0.75 µL 40 cylces Water 18.5 µL

Author: Michael Carter

204

A.30. Large scale RNA isolation

Reagents:

 TRI Reagent  Chloroform  Isopropanol Protocol:

1. Grow culture to desired OD (usually mid-exponential phase) 2. Spin cells in 200-250 mL aliquots at 8,600 x g (7,500 RPM) for 5 min at 4 °C 3. Resuspend in left over media after decanting. Transfer to 2mL microcentrifuge tube. 4. Spin and remove remaining media For each aliquot:

5. Remove ALL supernatant and resuspend in 1.3 mL of TRI Reagent per aliquot of cells a. Will become very viscous. Higher OD cultures (>0.5) might require more TRI b. Lower OD (<1.0), two aliquots should be combined and carried through remainder of protocol c. Use 2 mL microcentrifuge tubes 6. Incubate at room temp for 20 min 7. Add 0.65 mL chloroform 8. Shake briskly by flipping wrist 9. Incubate at room temp for 10 min 10. Centrifuge at 15,800 x g (13,000 RPM) for 10 min at 4 °C 11. Remove top phase to a fresh, RNase free tube a. Be very careful to avoid the interphase and the bottom phase, even if it means leaving some of the top phase 12. Add 0.6 mL TRI, mix by inversion 13. Incubate at room temp 5 min 14. Add 0.6 mL chloroform 15. Shake briskly by flipping wrist 16. Incubate at room temp for 5 min 205

17. Centrifuge at 15,800 x g (13,000 RPM) for 10 min at 4 °C 18. Move top phase to a fresh, RNase free tube a. Again, avoid the interphase. 19. Add equal volume of isopropanol (1 mL) 20. Incubate at -20 °C >20 min 21. Centrifuge at 8630 x g (7500RPM) for 10 min 22. Allow pellet to dry inverted for 10 min 23. Dissolve pellet in 400 µL RNase-free H2O. 24. Add 50 µL DNase I (RNase-free) and 50 µL DNase I buffer a. Incubate for 1 hour 25. Isopropanol precipitate 26. Invert and allow dry for 10 min 27. Dissolve in 200 µL H2O Notes

1. TRI extraction or isopropanol precipitation seems to be selective for small RNAs 2. Uniquely, in many Rhodobacteriaceae, the 23S rRNA is processed into 16S-like, 14S, and 5.8S fragments. Therefore, the whole cell RNA on a gel will appear different than E. coli. Typically, quality of RNA is assessed by comparing the 23S band to the 16S band. However, here, only the 16S band is visible, and it contains the processed product of the 23S rRNA, so it is deceptively intense.

Figure A.1. Expected results from TRI extraction.

Author: Michael Carter 206

A.31. Quantitative polymerase chain reaction (qPCR)

Reagents:

 SYBR Supermix (BioRad)  20 µM! F and R primer  Highly purified water Principle

This protocol encompasses the techniques involved in setting up a 96-well plate for qPCR using cDNA that has been acquired through the Reverse Transcription protocol.

Notes:

 qPCR is remarkably sensitive! It will detect any DNA contamination that is present. Some DNA contamination is inevitable if the Large Scale RNA Preparation technique is used. For this reason, everything should be clean, and only the highest purity of water should be used.  Be extremely careful to add volumes exactly, and repeat technique of addition of volumes exactly! Any variation will be apparent in the final Ct values. This technique has been adjusted to optimize reproducibility among technical replicates. Protocol:

Make 2-fold serial dilution of each cDNA template. Use 0.2 mL PCR tubes in a rack that imitates the 96-well plate. Repeat the following protocol for each cDNA to be quantified. It is absolutely essential that the utmost care be used for these volumes! Any variation will be obvious in the end. 28. Add 70 µL of water to the first tube. (tube 1) 29. Add 40 µL of water to the next four tubes. (tube 2 – 5) 30. Add 10 µL of cDNA to the first tube. Mix well! 31. Add 40 µL from tube 1 to tube 2. Mix well! 32. Add 40 µL from tube 2 to tube 3. Mix well! 33. Repeat until tube 5. After mixing in tube 5, remove 40 µL from tube 5. 34. Add 36 µL water to tube 6. 35. Add 4 µL –RT control to tube 6.

207

Prepare SYBR Supermix/primers mastermix.

Prepare master mix of 2 µL of each primer (20 µM) and 10 µL IQ SYBR Supermix for each reaction. Use 0.2 mL PCR tubes in a separate rack that imitates a 96-well plate. Repeat this procedure for each cDNA to be quantified. 1. Add 227.5 µL IQ SYBR Supermix 2. Add 45.5 µL of F-Primer 3. Add 45.5 µL of R-Primer 4. Add 49 µL of the master mix to tubes 1 – 6 a. Remember: This is to be a separate rack than the one containing the template. The SYBR supermix is viscous and causes problems with reproducible pipetting. By keeping these separate, the template can be added reproducibly to the 96-well plate that will be used in the thermal cycler for quantification. The reproducible addition of SYBR and primers is far less pertinent than template.

Prepare the 96-well plate for the thermal cycler. Here, a multichannel micropipettor will be used. Every reaction will be performed in triplicate to average out any pipetting errors. Each round of pipetting should use fresh, prepacked tips to maximize reproducibility.

1. With a multichannel micropipettor, add 8 µL of template to columns 1 – 3, replacing the tips between each column. 2. With a multichannel micropipettor, add 14 µL of SYBR Supermix/Primer master mix. a. Do not mix by pipetting up and down! Because of the viscosity, some volume will inevitably get caught in the pipette tip. The sequestered volume will contain template, changing the amount available for amplification in the well. 3. The plate is ready for the thermal cycler.

Author: Michael Carter

208

A.32. qPCR data analysis

1. Plot the Ct value vs the log of the amount of template loaded in a given reaction. Make separate graphs for each condition, and include all values within a single condition. The amount of template can be measured as a volume of template or number of molecules.

Figure A.2. Example plot for determining efficiency of amplification.

Determine which values are most reasonable by including those that will fit a curve with a slope as close to -3.3219 as possible.

a. The efficiency of amplification = 10(-1/slope) – 1. b. Only move forward if >3 points are available with an acceptable slope. 2. To establish actual values for the amount of cDNA in each reaction, compare Ct values to a standard curve that was constructed with purified PCR product as template. 3. To establish relative values between conditions, compare Ct values of samples with respect to the amount loaded. Notes

 Ct (Threshold Cycle) values are the result of proprietary algorithms that move all curves to the same baseline. Then, the algorithm will find the cycle in all curves that represents the first time at which amplification is reliably observed.  Twice as much template should result in an increase of 1 in the Ct value.  Detectable amplification is not necessarily specific product. Use the melt curves to assign specificity to the product.  Products should be no larger than 150 bp. Author: Michael Carter 209

Appendix B: Additional Results

210

B.1. Failed purification of soluble, intact PccR

Initially, efforts to purify PccR surrounded purification of the N-terminally His-tagged His-PccR. The fused His tag originates from the pTEV5 (102) scaffold vector that was used to generate pMC74. Initial efforts of small-scale production of His-PccR in Rosetta(DE3) resulted in extremely low yields of a soluble protein of expected size (3/4/12, 2/29/12). The results were never reproduced in large-scale.

Produce Soluble PccR Instead, alternative strategies were explored for increasing the production of soluble His- PccR. First, the protein was produced simultaneously with various combinations of the chaperones GroES, GroEL, Tig, DnaK, DnaJ, and GrpE from the plasmids pGro7, pG- Tf2, pTf16, and pG-KJE8 (Takara) (7/2/12). In all cases, there was no observable effect on His-PccR solubility. Another strategy was to adjust the rate of transcription of the gene encoding the His-PccR protein. The strategy relies on reduced production rates to permit each His-PccR molecule to properly fold before encountering another partially folded His-PccR. In the Lemo21(DE3) strain (New England Biolabs), transcription by T7 RNA polymerase is modulated by the rhamnose-inducible production of T7 lysozyme which is a natural inhibitor of the polymerase. At small-scale, after standard induction of His-Pccr production in the presence of 0 – 2.0 mM rhamonse, there was some evidence for soluble His-PccR at 0.75 and 1.5 mM rhamnose (5/18/13). Repeating His-PccR production in larger scale in the presence of 1.5 mM rhamnose yielded very low amounts of detectable His-PccR after Ni2+-NTA column chromatography (6/3/13). However, the purified protein did not remain soluble overnight. Purify and Refold Insoluble PccR Efforts to purify soluble His-PccR from cell extract were abandoned in favor of potentially refolding the protein from inclusion bodies of Rosetta(DE3) cells. Table B.1.1 illustrates the buffer systems that were employed during the various refolding strategies. Denatured immobilized metal affinity chromatography (IMAC), in principle, keep individual unfolded His-PccR molecules separated as they refold, potentially avoiding their aggregation throughout the intermediate stages of folding. The His-PccR was added to Ni2+ NTA agarose resin equilibrated with 50 mM TAPS (pH 8.5) and 8 M urea. The resin was washed sequentially with 50 mM TAPS (pH 8.5) buffers that contained decreasing urea concentrations (4, 2, 1, 0.5, and 0.1 M urea). The column was eluted with 50 mM TAPS (pH 8.5) buffers that contained 50, 100, and 250 mM imidazole. No

211

protein was detected in any of the imidazole wash buffers. The original flowthrough was not analyzed. Stepwise dialysis gradually decreases the denaturant (urea in this case) in the solution containing the protein to be refolded. The goal is to allow ample opportunity for the protein to sample different potential intermediate folding stages as it proceeds toward the properly folded state. In addition, the continued presence of denaturant prevents the nonspecific interactions that lead to aggregation. Finally, once the denaturant is removed, the protein should be properly folded and should not aggregate with other versions of itself. For stepwise dilution of His-PccR, two concentrations of protein were placed within two different dialysis membranes and were sequentially incubated for 1 hr at 4°C in 4, 2, 1, and 0.5 M urea buffers that also contained 10 mM TAPS (pH 8.5) and 0.1 mM CoCl2. The sample of higher protein concentration (140 µg/mL) precipitated after only 5 min of dialysis with 1 M urea. Precipitate was apparent in the sample of lower protein concentration (14 µg/mL) gradually after it was dialyzed against the final buffer containing no urea. After centrifugation of the samples, no protein was observable in the supernatant. Rapid dilution is another strategy that attempts to spatially separate protein molecules as they fold in order to avoid molecules in intermediate folding stages from interacting with each other. A 5 µL drop of His-PccR (7 mg/mL) was added into 1 mL of a series of 20 mM MOPS pH 7 buffers. Each buffer contained a different additive to potentially aid the protein in properly folding into a state that was soluble. The additives were 2.5 mM GSH/GSSG, 500 mM potassium glutamate, 200 mM urea, 400 mM arginine, 100 mM guanidine-HCl, and 100 mg/mL PEG. Only arginine was able to maintain soluble protein, and it only maintained about 10 % of the protein as soluble. Additional rapid dilution strategies included the use of heparin. Overnight solubility in the presence of 23 µg/mL heparin was 30 – 40 % efficient. However, the soluble protein was unable to shift the pccR upstream fragment in an electrophoretic mobility shift assay.

212

Table B.1. Notes regarding failed PccR purification attempts Denatured IMAC Refolding Date Buffer Experimental Notes Results 3/5/13 50 mM TAPS pH 8.5 The column was washed with gradually No evidence that His-PccR was ever bound to 4, 2, 1, 0.5, 0.1 M Urea decreasing urea concentrations the column Stepwise dialysis 3/6/13 10 mM TAPS pH 8.5 1 hr incubation in each buffer at 4°C. 140 µg/mL: Precipitated after 5 min in 1 M urea 0.1 mM CoCl2 14 µg/mL: Precipitated in 0 M urea 4, 2, 1, 0.5 M Urea Rapid Dilution 4/29/13 20 mM MOPS pH 7 Drop 5 µL His-PccR (7 mg/mL) into 1 mL All immediately insoluble 2.5 mM GSH, GSSG 20 mM MOPS pH 7 Drop 5 µL His-PccR (7 mg/mL) into 1 mL All immediately insoluble 500 mM K-glutamate 20 mM MOPS pH 7 Drop 5 µL His-PccR (7 mg/mL) into 1 mL All immediately insoluble 200 mM urea 20 mM MOPS pH 7 Drop 5 µL His-PccR (7 mg/mL) into 1 mL ~10 % was still soluble after 1 hr 400 mM Arginine 20 mM MOPS pH 7 Drop 5 µL His-PccR (7 mg/mL) into 1 mL All immediately insoluble 100 mM Guanidine HCl 20 mM MOPS pH 7 Drop 5 µL His-PccR (7 mg/mL) into 1 mL All immediately insoluble 100 mg/mL PEG 4/29/13 20 mM MOPS pH 7 Drop 5 µL His-PccR (7 - .09 mg/mL) into 1 mL ~40 % soluble after overnight (4°C) 594 µg/mL Heparin 4/30/13 20 mM MOPS pH 7 Drop 1 µL His-PccR (3.5 mg/mL) into 100 µL ~40 % soluble after overnight (4°C) 23.1 µg/mL Heparin 5/16/13 20 mM MOPS pH 7 Drop 1 µL His-PccR (3.5 mg/mL) into 100 µL ~ 30 % soluble immediately 30 µg/mL Heparin No observable gel shift with pccR upstream

Purify PccR from Soluble, Cleavable Fusion to MalE In order to produce PccR in a soluble form, its gene was fused to malE (maltose binding protein) in pKLD116 to encode a His-tagged MalE fused to PccR. The fusion product (His-MalE-PccR [HMP]) contains a TEV protease cleavage site at the junction between MalE and PccR. The fusion protein can be cleaved with a His-tagged TEV protease (rTEVP). Then, the His-containing proteins can be removed to yield purified PccR. The cleavage event leaves a GLSSRCMWTSRASHG extension on the amino-terminus of PccR. Cleavage was observed to occur optimally after 1.5 hours with a mass ratio of 1 µg rTEVP:8 µg HMP. Nearly every cleavage attempt was 50 – 80 % efficient, yet the protein was either insoluble or unable to shift the pccR or pccB upstream fragments in electrophoretic mobility shift assays. Soluble PccR was optimally observed after cleavage reactions at pH 8.5 and pH 9. The isoelectric point of PccR is predicted to be 8.0. No change in protease cleavage efficiency was observed from pH 6 – pH 9 (1/25/13), and cleavage appeared to be unimpeded by any of the additives used in efforts to maintain PccR solubility. Additives that were tested include DTT, aginine, NaCl, guanidine-HCl, and GSH:GSSG (3:1, 1:1, 1:2). Only cleavage reactions in the presence of arginine yielded soluble PccR. However, 65 mM arginine (the lowest solubilizing concentration) interferes with removal of the remaining His-tagged proteins (2/1/13). Results of cleavage in the presence of

213

metals (Cu2+, Ni2+, Co2+, Zn2+; 2/18/13) similarly did not indicate that a contribution to maintaining soluble PccR.

214

B.2. Analysis of spent media from R. sphaeroides, RsΔpccRMC12, and RsΔpccBSJC1A during succinate or acetate growth

Background: No propionyl-CoA carboxylase activity is detectable in extracts from succinate- or acetate-grown Rs(pMC85) or RsΔpccBSJC1A (data not shown). Both strains also grow with a doubling time of about 10 hr in acetate media compared to a 3.5 hr doubling time as observed for R. sphaeroides (Figure B.4). Given that the methylmalonyl-CoA pathway for propionyl-CoA assimilation is a component of the ethylmalonyl-CoA pathway (required for acetate assimilation), it is surprising that either strain would be able to grow with acetate. A possible explanation is that some eventual product of propionyl-CoA is secreted, possibly propionate. Purpose: Identify potential organic acids that might be secreted by RsΔpccBSJC1A during acetate growth. Principle: Grow R. sphaeroides 2.41, RsΔpccBSJC1A, and RsΔpccRMC12 (reduced propionyl-CoA carboxylase activity during acetate growth compared to wild type) with succinate and acetate. Collect aliquots of spent media at different timepoints during growth. Analyze the spent media by HPLC to attempt to identify potential components of the spent media Protocol: 1. Growth a. Grow cultures in 50 mL serum bottles. b. Collect ~2mL of cultures at timepoints plotted in Figure B.4. i. 1 mL: spin, and freeze supernatant and pellet separately ii. 1 mL: record OD578 nm 2. HPLC a. Column: Phenomenex Luna 5 µ C18(2) 100A b. Sample: 95 µL of timepoint supernatants + 2% 30 mM phosphate buffer pH 2.8 c. Elution: i. Buffer: Isocratic with 30 mM phosphate buffer pH 2.8 ii. Flow rate: 0.7 mL/min iii. Time: 35 min for each sample

215

Results:

Figure B.1. Relative retention times of standard molecules (A) and of molecules in spent media from R. sphaeroides 2.4.1 at various timepoints during photoheterotrophic growth with succinate (B) and acetate (C)

216

Figure B.2. Relative retention times of molecules in spent media from RsΔpccBSJC1A at various timepoints during photoheterotrophic growth with succinate (A) and acetate (B)

217

Figure B.3. Relative retention times of molecules in spent media from RsΔpccRMC12 at various timepoints during photoheterotrophic growth with succinate (A) and acetate (B)

218

Figure B.4. Quantified composition of spent media from photoheterotrophic cultures of R. sphaeroides 2.4.1 (A), RsΔpccBSJC1A (B), and RsΔpccRMC12 grown with succinate (left) or acetate (right). OD is illustrated with a red line, concentration of acetate is illustrated in blue, and the orange line represents propionate concentrations.

Conclusions: The growth rate and final yield of RsΔpccBSJC1 is reduced compared to the other two strains. This is consistent with propionate accumulation in the spent media of RsΔpccBSJC1A during acetate growth. Relative to the other strains, RsΔpccBSJC1A is suspected to lose access to ~ 20% - 30% of the total available carbon as the strain secretes the carbon as propionate.

It is also important to note that RsΔpccRMC12 does not demonstrate signs of secreting propionate. This further supports the model that the reduced rate of PCC (compared to wild type) is sufficient to prevent accumulation of propionyl-CoA in the cell during acetate growth. 219