Transcriptome Analysis of the Rhodobacter sphaeroides PpsR Regulon: PpsR as a Master Regulator of Photosystem Development

Abstract

PpsR from the anoxygenic phototrophic bacterium Rhodobacter sphaeroides has been known as an oxygenand light-dependent repressor of bacteriochlorophyll and carotenoid biosynthesis genes and puc operons involved in photosystem development. However, the putative PpsR-binding sites, TGTN 12 ACA, are also located upstream of numerous nonphotosystem genes, thus raising the possibility that the role of PpsR is broader. To characterize the PpsR regulon, transcriptome profiling was performed on the wild-type strain grown at high and low oxygen tensions, on the strain overproducing PpsR, and on the ppsR mutant. Transcriptome analysis showed that PpsR primarily regulates photosystem genes; the consensus PpsR binding sequence is TGTcN 10 gACA (lowercase letters indicate lesser conservation); the presence of two binding sites is required for repression in vivo. These findings explain why numerous single TGTN 12 ACA sequences are nonfunctional. In addition to photosystem genes, the hemC and hemE genes involved in the early steps of tetrapyrrole biosynthesis were identified as new direct targets of PpsR repression. Unexpectedly, PpsR was found to indirectly repress the puf and puhA operons encoding photosystem core proteins. The upstream regions of these operons contain no PpsR binding sites. Involvement in regulation of these operons suggests that PpsR functions as a master regulator of photosystem development. Upregulation of the puf and puhA operons that resulted from ppsR inactivation was sufficient to restore the ability to grow phototrophically to the prrA mutant. PrrA, the global redox-dependent activator, was previously considered indispensable for phototrophic growth. It is revealed that the PrrBA and AppA-PpsR systems, believed to work independently, in fact interact and coordinately regulate photosystem development.Rhodobacter sphaeroides is a facultatively phototrophic anoxygenic alphaproteobacterium. Under high oxygen tension, whether in the light or in the dark, this bacterium uses aerobic respiration for energy generation. When oxygen tension decreases, R. sphaeroides induces synthesis of photosynthetic apparatus, an alternate energy generation system functional under anoxic-light conditions (28,30,32,36). The photosynthetic apparatus is comprised of one type II photosystem (PS). Development of PS involves synthesis of photosynthetic pigments, bacteriochlorophyll (Bchl) and carotenoids (Crt), membrane proteins of the reaction center (RC), and two light-harvesting (LH) complexes as well as assembly factors. The RC and LH complexes are housed in the specialized intracytoplasmic membrane system. A decrease in oxygen tension triggers a significant increase in transcription of PS genes, which is required for PS development. Major regulatory components involved in oxygen control of PS development have been identified previously (1,44). However, precise roles of individual regulators and interactions between the regulatory pathways are not yet fully understood. This work is aimed at characterizing the role of one majo...

Combining microarray and genomic data to predict DNA binding motifs

“…These 11 predicted PpsR-targeted genes, together with their fold changes between expression levels at 10 W m 22 light intensity (without DMSO) versus expression under aerobic conditions (30 % O 2 ), are listed in Table 2. Ten of the 11 predicted genes are known to be regulated by PpsR (Choudhary & Kaplan, 2000;Moskvin et al, 2005;Zeng et al, 2003), an observation that supports our approach. Strikingly, with the exceptions of argD and bchC, nine of Fig.…”

“…Very recently, two divergently transcribed genes involved in the early steps of tetrapyrrole biosynthesis, haem (RSP0680) and hemC (RSP0679), were experimentally identified to be targeted by PpsR (Moskvin et al, 2005). Their observed PpsR binding sites are all located within the coding regions for these two genes.…”

“…The detected consensus sequences for PpsR, FnrL and PrrA were then used to predict their potential regulons on a genome-wide scale. Of 11 genes detected by MAST to be regulated by PpsR (Table 2), 10 have been reported earlier to be potentially regulated by PpsR (Choudhary & Kaplan, 2000;Moskvin et al, 2005;Zeng et al, 2003). These 10 genes are indicated in Table 2 and their encoded products are all related to the photosynthetic metabolic function; examples are bacteriochlorophyll biosynthesis (bch genes), carotenoid biosynthesis (crt genes), light-harvesting complex (puc genes), and the regulation of photosynthesis genes (ppaA).…”

“…Therefore, in the highly unlikely event that our predictions were perfect, we would always underestimate the number of genes regulated by PrrA. This problem is compounded by the fact that binding sites can be buried within the coding regions of 'stand-alone' genes (Moskvin et al, 2005), and operons will also be missed in our method, resulting in an underestimation of genes controlled by a regulator.…”

“…Thus, we suggest that argD may be a false-positive detection, or that PpsR may under certain conditions function as an activator (Jaubert et al, 2004). Based on our motif-searching results, we conclude that PpsR in R. sphaeroides is to a large extent specific for the regulation of photosynthesis-related genes (Moskvin et al, 2005).…”