Role of the Global Transcriptional Regulator PrrA in Rhodobacter sphaeroides 2.4.1: Combined Transcriptome and Proteome Analysis

ABSTRACT The PrrBA two-component regulatory system is a major global regulator in Rhodobacter sphaeroides 2.4.1. Here we have compared the transcriptome and proteome profiles of the wild-type (WT) and mutant PrrA2 cells grown anaerobically in the dark with dimethyl sulfoxide as an electron acceptor. Approximately 25% of the genes present in the PrrA2 genome are regulated by PrrA at the transcriptional level, either directly or indirectly, by twofold or more relative to the WT. The genes affected are widespread throughout all COG (cluster of orthologous group) functional categories, with previously unsuspected “metabolic” genes affected in PrrA2 cells. PrrA was found to act as both an activator and a repressor of transcription, with more genes being repressed in the presence of PrrA (9:5 ratio). An analysis of the genes encoding the 1,536 peptides detected through our chromatographic study, which corresponds to 36% coverage of the genome, revealed that approximately 20% of the genes encoding these proteins were positively regulated, whereas approximately 32% were negatively regulated by PrrA, which is in excellent agreement with the percentages obtained for the whole-genome transcriptome profile. In addition, comparison of the transcriptome and proteome mean parameter values for WT and PrrA2 cells showed good qualitative agreement, indicating that transcript regulation paralleled the corresponding protein abundance, although not one for one. The microarray analysis was validated by direct mRNA measurement of randomly selected genes that were both positively and negatively regulated. lacZ transcriptional and kan translational fusions enabled us to map putative PrrA binding sites and revealed potential gene targets for indirect regulation by PrrA.

[1]  T. Donohue,et al.  Activation of the global gene regulator PrrA (RegA) from Rhodobacter sphaeroides. , 2006, Biochemistry.

[2]  J. Vieira,et al.  The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. , 1982, Gene.

[3]  Mahbuba Rahman,et al.  Effect of rpoS gene knockout on the metabolism of Escherichia coli during exponential growth phase and early stationary phase based on gene expressions, enzyme activities and intracellular metabolite concentrations , 2006, Biotechnology and bioengineering.

[4]  J. Mekalanos,et al.  Repressor binding to a regulatory site in the DNA coding sequence is sufficient to confer transcriptional regulation of the vir-repressed genes (vrg genes) in Bordetella pertussis , 1993, Journal of bacteriology.

[5]  Samuel Kaplan,et al.  Proteomic Characterization of the Rhodobacter sphaeroides 2.4.1 Photosynthetic Membrane: Identification of New Proteins , 2007, Journal of bacteriology.

[6]  S. Kaplan,et al.  Localization and structural analysis of the ribosomal RNA operons of Rhodobacter sphaeroides. , 1990, Nucleic acids research.

[7]  Ljiljana Paša-Tolić,et al.  An accurate mass tag strategy for quantitative and high‐throughput proteome measurements , 2002, Proteomics.

[8]  D. K. Hawley,et al.  DNA determinants of promoter selectivity in Escherichia coli. , 1983, Cold Spring Harbor symposia on quantitative biology.

[9]  S. Kaplan,et al.  Transcriptional Regulation of puc Operon Expression in Rhodobacter sphaeroides , 1995, The Journal of Biological Chemistry.

[10]  M. Choudhary,et al.  DNA sequence analysis of the photosynthesis region of Rhodobacter sphaeroides 2.4.1. , 2000, Nucleic acids research.

[11]  Samuel Kaplan,et al.  Transcriptome Dynamics during the Transition from Anaerobic Photosynthesis to Aerobic Respiration in Rhodobacter sphaeroides 2.4.1 , 2007, Journal of bacteriology.

[12]  Samuel Kaplan,et al.  Application of the accurate mass and time tag approach to the proteome analysis of sub-cellular fractions obtained from Rhodobacter sphaeroides 2.4.1. Aerobic and photosynthetic cell cultures. , 2006, Journal of proteome research.

[13]  Frederick R. Blattner,et al.  Genome-Wide Expression Analysis Indicates that FNR of Escherichia coli K-12 Regulates a Large Number of Genes of Unknown Function , 2005, Journal of bacteriology.

[14]  Stephen J. Callister,et al.  Comparison of aerobic and photosynthetic Rhodobacter sphaeroides 2.4.1 proteomes. , 2006, Journal of microbiological methods.

[15]  Jonathan W Willett,et al.  Regulation of hem gene expression in Rhodobacter capsulatus by redox and photosystem regulators RegA, CrtJ, FnrL, and AerR. , 2004, Journal of molecular biology.

[16]  F. Tabita,et al.  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 * , 2003, The Journal of Biological Chemistry.

[17]  C. Dorman,et al.  A global role for Fis in the transcriptional control of metabolism and type III secretion in Salmonella enterica serovar Typhimurium. , 2004, Microbiology.

[18]  Lucy Shapiro,et al.  Genes directly controlled by CtrA, a master regulator of the Caulobacter cell cycle , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[19]  G. Klug,et al.  Thioredoxin can influence gene expression by affecting gyrase activity. , 2004, Nucleic acids research.

[20]  S. Kaplan,et al.  Interacting regulatory networks in the facultative photosynthetic bacterium, Rhodobacter sphaeroides 2.4.1. , 2005, Biochemical Society transactions.

[21]  C. B. V. Niel,et al.  THE CULTURE, GENERAL PHYSIOLOGY, MORPHOLOGY, AND CLASSIFICATION OF THE NON-SULFUR PURPLE AND BROWN BACTERIA , 1944 .

[22]  P. Kiley,et al.  Posttranscriptional control of puc operon expression of B800-850 light-harvesting complex formation in Rhodobacter sphaeroides , 1989, Journal of bacteriology.

[23]  A. Pühler,et al.  A new family of RSF1010-derived expression and lac-fusion broad-host-range vectors for gram-negative bacteria. , 1990, Gene.

[24]  Xueqiao Liu,et al.  Probing the ArcA-P Modulon of Escherichia coli by Whole Genome Transcriptional Analysis and Sequence Recognition Profiling* , 2004, Journal of Biological Chemistry.

[25]  A. D. de Koning,et al.  Effects of Fis on Escherichia coli gene expression during different growth stages. , 2007, Microbiology.

[26]  Samuel Kaplan,et al.  Postgenomic adventures with Rhodobacter sphaeroides. , 2007, Annual review of microbiology.

[27]  T. Conway,et al.  Microarray expression profiling: capturing a genome‐wide portrait of the transcriptome , 2003, Molecular microbiology.

[28]  Haluk Resat,et al.  Combining microarray and genomic data to predict DNA binding motifs. , 2005, Microbiology.

[29]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[30]  D. Kobayashi,et al.  Improved broad-host-range plasmids for DNA cloning in gram-negative bacteria. , 1988, Gene.

[31]  C. Bauer,et al.  Regulatory factors controlling photosynthetic reaction center and light-harvesting gene expression in Rhodobacter capsulatus , 1992, Cell.

[32]  J. M. Pemberton,et al.  Sequencing, chromosomal inactivation, and functional expression in Escherichia coli of ppsR, a gene which represses carotenoid and bacteriochlorophyll synthesis in Rhodobacter sphaeroides , 1994, Journal of bacteriology.

[33]  C. Li,et al.  Model-based analysis of oligonucleotide arrays: expression index computation and outlier detection. , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[34]  T. Donohue,et al.  Activation of the cycA P2 promoter for the Rhodobacter sphaeroides cytochrome c2 gene by the photosynthesis response regulator , 1999, Molecular microbiology.

[35]  V. Wendisch,et al.  Genome-Wide Analysis of the General Stress Response Network in Escherichia coli: σS-Dependent Genes, Promoters, and Sigma Factor Selectivity , 2005, Journal of bacteriology.

[36]  P. Kiley,et al.  Molecular genetics of photosynthetic membrane biosynthesis in Rhodobacter sphaeroides. , 1988, Microbiological reviews.

[37]  S. Kaplan,et al.  Molecular genetic analysis suggesting interactions between AppA and PpsR in regulation of photosynthesis gene expression in Rhodobacter sphaeroides 2.4.1 , 1997, Journal of bacteriology.

[38]  M. Simon,et al.  Signal transduction pathways involving protein phosphorylation in prokaryotes. , 1991, Annual review of biochemistry.

[39]  U. Simonis,et al.  Pyrroloquinoline quinone preserves mitochondrial function and prevents oxidative injury in adult rat cardiac myocytes. , 2007, Biochemical and biophysical research communications.

[40]  S. Kaplan,et al.  Generalized approach to the regulation and integration of gene expression , 2001, Molecular microbiology.

[41]  S. Kaplan,et al.  prrA, a putative response regulator involved in oxygen regulation of photosynthesis gene expression in Rhodobacter sphaeroides , 1994, Journal of bacteriology.

[42]  Matthew E Monroe,et al.  Probability-based evaluation of peptide and protein identifications from tandem mass spectrometry and SEQUEST analysis: the human proteome. , 2005, Journal of proteome research.

[43]  Mark Gomelsky,et al.  Transcriptome Analysis of the Rhodobacter sphaeroides PpsR Regulon: PpsR as a Master Regulator of Photosystem Development , 2005, Journal of bacteriology.

[44]  A. Travers,et al.  A DNA architectural protein couples cellular physiology and DNA topology in Escherichia coli , 1999, Molecular microbiology.

[45]  T. Donohue,et al.  Transcriptional Activation of the Rhodobacter sphaeroides Cytochrome c2 Gene P2 Promoter by the Response Regulator PrrA , 2002, Journal of bacteriology.

[46]  Miriam L. Land,et al.  Construction and Validation of the Rhodobacter sphaeroides 2.4.1 DNA Microarray: Transcriptome Flexibility at Diverse Growth Modes , 2004, Journal of bacteriology.

[47]  S Kaplan,et al.  Aerobic and anaerobic regulation in Rhodobacter sphaeroides 2.4.1: the role of the fnrL gene , 1995, Journal of bacteriology.

[48]  C. Condon.  Maturation and degradation of RNA in bacteria. , 2007, Current opinion in microbiology.

[49]  T. Donohue,et al.  Construction, characterization, and complementation of a Puf- mutant of Rhodobacter sphaeroides , 1988, Journal of bacteriology.

[50]  W. A. Havelka,et al.  A broad-host-range vector system for cloning and translational lacZ fusion analysis. , 1988, Plasmid.

[51]  Matthew E Monroe,et al.  A proteomic study of the HUPO Plasma Proteome Project's pilot samples using an accurate mass and time tag strategy , 2005, Proteomics.

[52]  G. Klug,et al.  In vivo and in vitro analysis of RegA response regulator mutants of Rhodobacter capsulatus. , 2000, Journal of molecular microbiology and biotechnology.

[53]  Thomas D. Schneider,et al.  Computation-Directed Identification of OxyR DNA Binding Sites in Escherichia coli , 2001, Journal of bacteriology.

[54]  R. Rappuoli,et al.  In vitro selection of high affinity HspR-binding sites within the genome of Helicobacter pylori. , 2002, Gene.

[55]  Stephen J. Callister,et al.  Normalization approaches for removing systematic biases associated with mass spectrometry and label-free proteomics. , 2006, Journal of proteome research.

[56]  S. Kaplan,et al.  Role of the fnrL Gene in Photosystem Gene Expression and Photosynthetic Growth of Rhodobacter sphaeroides 2.4.1 , 1998, Journal of bacteriology.

[57]  C. Patten,et al.  Microarray analysis of RpoS-mediated gene expression in Escherichia coli K-12 , 2004, Molecular Genetics and Genomics.

[58]  F. Tabita,et al.  Regulators of nonsulfur purple phototrophic bacteria and the interactive control of CO2 assimilation, nitrogen fixation, hydrogen metabolism and energy generation. , 2004, FEMS microbiology reviews.

[59]  Samuel Kaplan,et al.  Effects of Oxygen and Light Intensity on Transcriptome Expression in Rhodobacter sphaeroides 2.4.1 , 2004, Journal of Biological Chemistry.

[60]  S. Kaplan,et al.  Oxygen-insensitive synthesis of the photosynthetic membranes of Rhodobacter sphaeroides: a mutant histidine kinase , 1995, Journal of bacteriology.

[61]  S. Kaplan,et al.  Redox flow as an instrument of gene regulation. , 2002, Methods in enzymology.

[62]  S. Kaplan,et al.  Effects of light, oxygen, and substrates on steady-state levels of mRNA coding for ribulose-1,5-bisphosphate carboxylase and light-harvesting and reaction center polypeptides in Rhodopseudomonas sphaeroides , 1985, Journal of bacteriology.

[63]  G. Klug,et al.  Expression of the trxA gene for thioredoxin 1 in Rhodobacter sphaeroides during oxidative stress , 2003, Archives of Microbiology.

[64]  Javier Arsuaga,et al.  Genomic transcriptional response to loss of chromosomal supercoiling in Escherichia coli , 2004, Genome Biology.

[65]  M. Choudhary,et al.  DNA sequence analysis of the photosynthesis region of Rhodobacter sphaeroides 2.4.1T , 2000 .

[66]  J. K. Lee,et al.  Isolation and characterization of trans-acting mutations involved in oxygen regulation of puc operon transcription in Rhodobacter sphaeroides , 1992, Journal of bacteriology.

[67]  Darren A. Natale,et al.  The COG database: an updated version includes eukaryotes , 2003, BMC Bioinformatics.

[68]  S. Ouchane,et al.  Global Regulation of Photosynthesis and Respiration by FnrL , 2007, Journal of Biological Chemistry.

[69]  S. Kaplan,et al.  Interacting Regulatory Circuits Involved in Orderly Control of Photosynthesis Gene Expression in Rhodobacter sphaeroides 2.4.1 , 2000, Journal of bacteriology.

[70]  C. Bauer,et al.  Expression of Uptake Hydrogenase and Molybdenum Nitrogenase in Rhodobacter capsulatus Is Coregulated by the RegB-RegA Two-Component Regulatory System , 2000, Journal of bacteriology.

[71]  G. Cohen-bazire,et al.  Kinetic studies of pigment synthesis by non-sulfur purple bacteria. , 1957, Journal of cellular and comparative physiology.

[72]  A. Pühler,et al.  A Broad Host Range Mobilization System for In Vivo Genetic Engineering: Transposon Mutagenesis in Gram Negative Bacteria , 1983, Bio/Technology.

[73]  C. Bauer,et al.  RegB/RegA, a Highly Conserved Redox-Responding Global Two-Component Regulatory System , 2004, Microbiology and Molecular Biology Reviews.

[74]  S. Kaplan,et al.  appA, a novel gene encoding a trans-acting factor involved in the regulation of photosynthesis gene expression in Rhodobacter sphaeroides 2.4.1 , 1995, Journal of bacteriology.

[75]  A. Khodursky,et al.  RecA can stimulate the relaxation activity of topoisomerase I: Molecular basis of topoisomerase-mediated genome-wide transcriptional responses in Escherichia coli , 2006, Nucleic acids research.

[76]  G. Ditta,et al.  Broad host range DNA cloning system for gram-negative bacteria: construction of a gene bank of Rhizobium meliloti. , 1980, Proceedings of the National Academy of Sciences of the United States of America.

[77]  Richard D. Smith,et al.  The Utility of Accurate Mass and LC Elution Time Information in the Analysis of Complex Proteomes , 2005, Journal of the American Society for Mass Spectrometry.

[78]  A. Gerber,et al.  Post-transcriptional gene regulation: From genome-wide studies to principles , 2007, Cellular and Molecular Life Sciences.

[79]  Navdeep Jaitly,et al.  VIPER: an advanced software package to support high-throughput LC-MS peptide identification , 2007, Bioinform..

[80]  H. B. Stewart,et al.  Hormonal Modulation of Key Hepatic Regulatory Enzymes in the Gluconeogenic/Glycolytic Pathway a , 1986, Annals of the New York Academy of Sciences.