Role of the Global Transcriptional Regulator PrrA inRhodobacter sphaeroides2.4.1: 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 wholegenome 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.Rhodobacter sphaeroides 2.4.1 is a purple nonsulfur photosynthetic bacterium which is well studied for its remarkable metabolic versatility. It can grow aerobically, anaerobically in the presence of external electron acceptors, such as dimethyl sulfoxide (DMSO), photosynthetically in the light, and fermentatively and lithotrophically in the presence or absence of oxygen (72). As a reflection of its metabolic versatility, R. sphaeroides has a branched electron transport chain (ETC) that utilizes several terminal respiratory pathways. The expression of genes encoding components of these pathways is coordinately regulated, depending on the prevailing redox conditions and the terminal electron acceptor present.The concentration of oxygen, when used as a terminal electron acceptor, regulates membrane biogenesis in R. sphaeroides; below approximately 3% oxygen, the intracytoplasmic membrane is synthesized (34). This organelle houses components necessary for the photosynthetic lifestyle, such as the various photosystems and electron carriers. In addition to O 2 , incident light intensity also regulates intracytoplasmic membrane abundance and composition in the absence of oxygen.Gene expression in R. sphaeroides is controlled by several well-defined regulatory elements. The expression of th...

A Two-Component Regulatory System Integrates Redox State and Population Density Sensing in Pseudomonas putida

“…The sequence CGCTGCCAGCC was identified as a potential RoxR box, with the underlined hexamer highly conserved. This sequence differs from sequences proposed in Bradyrhizobium japonicum and Rhodobacter sphaeroides, where a degenerate hexamer followed by a pentamer located at a variable distance has been described as a binding site for the RoxR orthologs RegR and PrrA (10,19). Sequences resembling the consensus can be found both in the cioA and the ddcA promoters (CCC TGCGATCG and CGTGCCATGCC, respectively; nucleotides identical to the consensus sequence are underlined).…”

Regulation of bacterial photosynthesis genes by the small noncoding RNA PcrZ

“…More recently CryB, a member of a newly described cryptochrome family (15), was shown to affect expression of photosynthesis genes in R. sphaeroides and to interact with AppA (16,17). Remarkably, the different signaling pathways for control of photosynthesis genes are also interconnected, e.g., the appA gene is controlled by PrrA (18,19) and a PpsR binding site is located in the ppaA promoter region (20). Thus, a complex network comprising several regulatory proteins controls the formation of the photosynthetic apparatus.…”

Regulation of the Rhodobacter sphaeroides 2.4.1 hemA Gene by PrrA and FnrL

“…Recently, Eraso et al provided an elegant description of the transcriptome and proteome of cells lacking PrrA (10). We hope that the present study will provide the impetus for further transcriptome and proteome investigations directed toward resolving the gene sets subject to each of the two modi operandi of PrrA.…”

“…A combination of microarray and other investigations have shown that the PrrA regulon includes tetrapyrrole biosynthesis genes (10,11,27,30,41), as well as genes with roles in many other biological processes, including photosynthesis, carbon and nitrogen fixation, denitrification, formaldehyde dehydrogenase activity, and aerotaxis (2,8,13,18,31). For hemA, two PrrA binding sites that are centered at bp Ϫ163 and Ϫ67 relative to the P1 transcription start site have been identified.…”