In vitro activation and repression of photosynthesis gene transcription in Rhodobacter capsulatus

Abstract

SummaryIt has been known for over half a century that anoxygenic photosynthetic bacteria maximally synthesize their photosystems in the absence of oxygen. During the last decade, it has become clear that this regulation is largely at the transcriptional level, with photosynthesis genes expressed only under anaerobic conditions. We describe here in vitro reconstitution of activation and repression of three photosynthesis promoters, bch (bacteriochlorophyll biosynthesis), puc (light-harvesting II apoproteins) and puf (reaction centre and light-harvesting I apoproteins) using puri®ed transcription factors and RNA polymerase from Rhodobacter capsulatus. Previous genetic results have indicated that each of these three promoters is differentially regulated by three key regulators: CrtJ acting as a repressor of bch and puc and the two-component regulators RegA/RegB, which are activators of puc and puf. These regulators are distinct from those that mediate oxygen control in enteric bacteria. Our in vitro studies show that these puri®ed regulators directly control the expression of the housekeeping RNA polymerase at these promoters. High-level basal expression of the bch promoter is shown to be repressed by CrtJ. The puc promoter is activated by the RegB-phosphorylated RegA protein and additionally repressed by CrtJ. At the puc promoter, CrtJ effectively competes for promoter binding with RegA, while at the bch promoter, repression appears to be by competition for the RNA polymerase binding site. In contrast to what has been suggested previously, the RegA-activated puf promoter is demonstrated as being recognized by the housekeeping RNA polymerase. We also discuss evidence that RegA,P activation of the puc and puf promoters involves recruitment of RNA polymerase by different modes of protein±protein interaction.

Transcriptional Activation of the Rhodobacter sphaeroides Cytochrome c 2 Gene P2 Promoter by the Response Regulator PrrA

“…This is the first demonstration of activation by a wild-type regulator in this family since in a previous transcription analysis with R. capsulatus RegA the workers used a gain-of-function mutant with altered in vitro properties (RegA*) (5). In this regard, it is significant that more than 10-fold more R. capsulatus RegA* protein than wild-type PrrA was needed to activate expression from cycA P2 (27).…”

“…In studies of response regulators that are closely related to PrrA, it has been shown that Bradyrhizobium japonicum RegR binds to the promoter region of the fixR-nifA operon (2,12) and Rhodobacter capsulatus RegA directly interacts with the promoters of more than 20 target genes (3,5,9,10,48,50). The apparent binding sites for these proteins at target promoters are located at different distances from their transcriptional start sites, making the mechanism of transcription activation an interesting question (3,5,11,48).…”

Redox property and regulation of PpsR, a transcriptional repressor of photosystem gene expression in Rhodobacter sphaeroides

“…PrrA, phosphorylation by PrrB; FnrL, reassembly of cofactor (Eraso & Kaplan, 1995;Zeilstra-Ryalls & Kaplan, 1998). Also, there was a report that RegA, a PrrA homologue in R. capsulatus, competed with CrtJ to bind to the bchC promoter region (Bowman et al, 1999). Therefore, it is proposed that an anaerobic activator regulates photosystem gene expression under these conditions.…”

Rhodobacter capsulatus nifA1 Promoter: High-GC −10 Regions in High-GC Bacteria and the Basis for Their Transcription

“…Plasmids pRGK301, pHTT7f1-NH␣, pMKSe2, and pT7␤Ј were described previously (3,4,25,27,30). pRGK325, which allows overexpression of the R. capsulatus ␣ subunit of RNAP, was made by PCR of the rpoA gene from the chromosome of R. capsulatus SB1003 by use of the upstream oligonucleotide 5Ј-GAGGCAGAGCATATGATCCACAAGA ATTGG-3Ј and the downstream oligonucleotide 5Ј-CGGGATCCTCAGAACT GGTCTTCGAAGCGCTTGGCC-3Ј.…”

“…Kranz et al previously noted that R. capsulatus and other ␣-proteobacteria (see reference 15 for a review) may have Ϫ10 features different from those of organisms with a lower GϩC content (3,4). For another ␣-proteobacterium, Sinorhizobium meliloti, most of the 70 promoters that have been characterized are not transcribed by the E. coli RNAP in vivo or in vitro, but the S. meliloti RNAP can initiate transcription at typical E. coli 70 promoters (23).…”