Isolation of regulatory mutants in photosynthesis gene expression in Rhodobacter sphaeroides 2.4.1 and partial complementation of a PrrB mutant by the HupT histidine-kinase

Abstract

The photosynthetic bacterium Rhodobacter sphaemides responds to the transition from aerobiosis to anaerobic photosynthesis by increasing the expression of the photosynthesis genes. Mutants have been isolated based on their inability, following such a transition, to increase transcription of the puc operon encoding the apoproteins of the light-harvesting complex II. Mutant D5, a representative of one mutant class, described here, although remaining photosynthetically competent, produced only low levels of the photosynthetic spectral complexes. Complementation analysis revealed that either the gene for the photosynthesis response regulator prrA or the gene encoding its cognate sensor kinase, prrB, was capable of rescuing this mutant. However, partial complementation of this mutant was achieved by placing in trans additional copies of other defined genes from the cosmid library of R. sphaeroides. We describe this effect in detail, attributable to the hupT gene, which has been proposed to encode a histidine-kinase for the hydrogen uptake system in Rhodobacter capsulatus. The effect of HupT on the expression of the photosynthesis genes was mediated through PrrA and independent of a functioning hydrogen uptake system. Thus, we raise the possibility that HupT can participate in phosphorylation of the heterologous response regulator PrrA by so-called cross-talk and therefore partially compensate for the defect in the mutant described. The observation of cross-talk, together with the complementation analysis, allowed us to assign the original mutation to the prrB gene; this was confirmed by DNA sequencing. Analysis of cross-talk in the wild-type, prrB and prrA genetic backgrounds suggested that besides kinase activity, PrrB may possess phosphatase activity toward PrrA. We also report the cloning, organization and structure of some of the hup genes from R. sphaeroides and construction of a Hup-strain.

Oxygen-insensitive synthesis of the photosynthetic membranes of Rhodobacter sphaeroides: a mutant histidine kinase

“…The alteration in PrrB is then manifest by either an increase in histidine kinase activity or a decrease in phosphatase activity or any combination of the two, leading to the increase steady-state phosphorylated state of PrrA. Additionally, at least one (19) and possibly other (52) histidine kinases are capable of phosphorylating PrrA, which can normally be rendered noneffective in the presence of the PrrB-mediated phosphatase activity when oxygen is present.…”

“…Therefore, the insertion mutation in PRRB2 was not polar to prrA and thus defined another coding sequence required for photosynthesis gene expression. Additionally, when multiple copies of prrA were placed in trans in PRRB2, the photosynthetic defect was bypassed, as might be anticipated since we have shown that prrA can be made active by at least one noncognate kinase, notably HupT (19). Therefore, the locus upstream of prrA was designated prrB, for photosynthetic response, regulator B.…”

Identification and in vivo characterization of PpaA, a regulator of photosystem formation in Rhodobacter sphaeroides

“…Cosmid pUI8714 was identified in the genetic screen for activators of PS gene expression in R. sphaeroides described previously (Gomelsky & Kaplan, 1995b). When provided in trans in the PrrB or PrrA null strains, it increased puc expression and pigmentation (Fig.…”

“…Plasmid mobilization into R. sphaeroides and P. denitrificans was carried out from E. coli S17-1 as described previously (Gomelsky & Kaplan, 1995a) Construction of the PPA1 mutant. A deletion/substitution mutation in ppaA from R. sphaeroides 2.4.1 was constructed by replacing the internal NcoI fragment with an VKm r cartridge from pUI1637 (Gomelsky & Kaplan, 1995b) to generate plasmid p714BgHDNc : : Km (see Fig. 1b).…”

Activation of the Global Gene Regulator PrrA (RegA) from Rhodobacter sphaeroides

“…The presence of consensus-like PrrA DNA binding sites (15) in promoter regions of genes regulated by R. capsulatus RegA, for genes regulated under anaerobic conditions like ccoNOPQ and cycA (1,16), suggests that RegA and PrrA bind as dimers in all cases. RegA and PrrA might be able to form a dimer when unphosphorylated in the presence of a co-regulator, or might be activated by other kinases than their cognate histidine kinase (44).…”