In vivo and in vitro analysis of RegA response regulator mutants of Rhodobacter capsulatus.

In the facultative photosynthetic bacterium Rhodobacter capsulatus, the transcription of genes encoding pigment binding proteins is tightly regulated in response to the oxygen partial pressure by the RegB/ RegA two component system. After a shift from high to low oxygen tension, the response regulator RegA enhances transcription of the puf and puc operon coding for the reaction center, light-harvesting complex I (LHI), and LHII proteins. Various regA mutant strains were analyzed in this study. In a RegA deficient strain, activation of puf and puc transcription is severely impaired which consequently leads to the synthesis of only a few photosynthetic complexes. Strains carrying a mutation in the helix-turn-helix domain of RegA or a mutation of the phosphorylation site, Asp63, show a phenotype like the RegA deficient mutant, although the RegA(D63K) mutant protein showed the same DNA binding behavior as the wild type protein. In contrast, the puf and puc mRNAs still reach about 50-70 % of the wild type level after reduction of oxygen tension in strains which synthesize the C-terminal RegA activator domain only or a hybrid protein composed of the RegA activator and the FixJ receiver domain, while both mutant proteins are impaired in DNA binding. Our data suggest that phosphorylation is not required for DNA binding but rather plays a role for efficient initiation of transcription.

DNA binding of wild type RegA protein and its differential effect on the expression of pigment binding proteins in Rhodobacter capsulatus.

The transcription of genes encoding pigment binding proteins in the facultative photosynthetic bacterium Rhodobacter capsulatus is regulated in response to oxygen partial pressure. Previous results identified RegA and RegB as members of a two component system involved in oxygen dependent synthesis of the photosynthetic apparatus. Here we demonstrate that RegA differentially controls the transcription of the puf and pucoperons which encode proteins of the LHI and LHII antenna complexes, respectively. In a regA mutant strain the level of puf specific mRNA reaches about 30% of the wild type levels and transcription is still responsive to oxygen tension. In contrast, the level of puc specific mRNA is very low and is no longer oxygen regulated. RegA binds to DNA sequences upstream of both the puf and puc operons, although with different affinities. We provide experimental evidence that a putative helix-turn-helix motif in the C-terminal region of RegA is responsible for its specific binding to the puf and puc promoter regions. In contrast to many other response regulators, the affinity of RegA for the target DNA is only slightly modified by phosphorylation.

Molecular analysis of the molybdate uptake operon, modABCD, of Escherichia coli and modR, a regulatory gene.

The nucleotide sequence of a 6.8-kb chromosomal subfragment of plasmid pHW100 complementing an Escherichia coli modC (chlD) mutant has been determined. This DNA region encodes the genes of a high-affinity uptake system for molybdate arranged in an operon with the genes modABCD. Since the modA product has a signal peptide at the N-terminus it probably is the periplasmic binding-protein for molybdate. The products of modB (chlJ) and modC (chlD) have been described earlier as the inner membrane protein and the ATP-binding protein of the molybdate transport system, respectively. At present, there is no information on possible functions of the fourth gene of the operon, modD. Upstream of the mod operon, two other gene loci, termed modR and an open reading frame ORF6 could be identified. ModR shares homology with a molybdenum-pterin binding protein of Clostridium pasteurianum. ORF6 has extensive homology to ModC and other nucleotide-binding proteins of E. coli. Insertional inactivation of modR and ORF6 using a gentamicin resistance gene cartridge has no effect on molybdoenzyme activities, indicating that none of the two gene products is essential for molybdate uptake or molybdenum cofactor synthesis. However, by using a plasmid carrying a modA-lacZ gene fusion we observed that inactivation of modR releases repression of the mod operon independent of the molybdate concentration in the medium. This indicates that modR is a component of the mod operon regulation or the repressor itself.

Isolation of Escherichia coli mutants defective in uptake of molybdate.

For the study of molybdenum uptake by Escherichia coli, we generated Tn5lac transposition mutants, which were screened for the pleiotropic loss of molybdoenzyme activities. Three mutants A1, A4, and M22 were finally selected for further analysis. Even in the presence of 100 microM molybdate in the growth medium, no active nitrate reductase, formate dehydrogenase, and trimethylamine-N-oxide reductase were detected in these mutants, indicating that the intracellular supply of molybdenum was not sufficient. This was also supported by the observation that introduction of plasmid pWK225 carrying the complete nif regulon of Klebsiella pneumoniae did not lead to a functional expression of nitrogenase. Finally, molybdenum determination by induced coupled plasma mass spectroscopy confirmed a significant reduction of cell-bound molybdenum in the mutants compared with that in wild-type E. coli, even at high molybdate concentrations in the medium. A genomic library established with the plasmid mini-F-derived cop(ts) vector pJE258 allowed the isolation of cosmid pBK229 complementing the molybdate uptake deficiency of the chlD mutant and the Tn5lac-induced mutants. Certain subfragments of pBK229 which do not contain the chlD gene are still able to complement the Tn5lac mutants. Mapping experiments showed that the Tn5lac insertions did not occur within the chromosomal region present in pBK229 but did occur very close to that region. We assume that the Tn5lac insertions have a polar effect, thus preventing the expression of transport genes, or that a positively acting regulatory element was inactivated.