Pigments accumulation via light and oxygen in Rhodobacter capsulatus strain XJ-1 isolated from saline soil.

A Rhodobacter capsulatus strain, designated XJ-1, isolated from saline soil, accumulated almost only one kind of bacteriochlorophyll a anaerobically in the light, aerobically in the light and dark, and the relative contents of the bacteriochlorophyll a were 44.61, 74.89, and 77.53% of the total pigments, respectively. A new purple pigment appeared only in aerobic-light grown cells, exhibited absorption maxima at 355, 389, 520, 621, and 755 nm, especially distinctly unusual peak at 621 nm, whereas vanished in anaerobic-light and in aerobic-dark culture. Spheroidene and OH-spheroidene predominated in anaerobic phototrophic cultures. Spheroidenone was the sole carotenoid when exposed to both light and oxygen. The second keto-carotenoids, OH-spheroidenone, presented only in aerobic-dark culture in addition to spheroidenone. Strain XJ-1 would be a good model organism for the further illustration of the regulation of bacteriochlorophyll biosynthesis gene expression in response to unique habitat.

Coordinated, long-range, solid substrate movement of the purple photosynthetic bacterium Rhodobacter capsulatus.

The long-range movement of Rhodobacter capsulatus cells in the glass-agar interstitial region of borosilicate Petri plates was found to be due to a subset of the cells inoculated into plates. The macroscopic appearance of plates indicated that a small group of cells moved in a coordinated manner to form a visible satellite cluster of cells. Satellite clusters were initially separated from the point of inoculation by the absence of visible cell density, but after 20 to 24 hours this space was colonized by cells apparently shed from a group of cells moving away from the point of inoculation. Cell movements consisted of flagellum-independent and flagellum-dependent motility contributions. Flagellum-independent movement occurred at an early stage, such that satellite clusters formed after 12 to 24 hours. Subsequently, after 24 to 32 hours, a flagellum-dependent dispersal of cells became visible, extending laterally outward from a line of flagellum-independent motility. These modes of taxis were found in several environmental isolates and in a variety of mutants, including a strain deficient in the production of the R. capsulatus acyl-homoserine lactone quorum-sensing signal. Although there was great variability in the direction of movement in illuminated plates, cells were predisposed to move toward broad spectrum white light. This predisposition was increased by the use of square plates, and a statistical analysis indicated that R. capsulatus is capable of genuine phototaxis. Therefore, the variability in the direction of cell movement was attributed to optical effects on light waves passing through the plate material and agar medium.

The critical role of tryptophan-116 in the catalytic cycle of dimethylsulfoxide reductase from Rhodobacter capsulatus.

In dimethylsulfoxide reductase of Rhodobacter capsulatus tryptophan-116 forms a hydrogen bond with a single oxo ligand bound to the molybdenum ion. Mutation of this residue to phenylalanine affected the UV/visible spectrum of the purified Mo(VI) form of dimethylsulfoxide reductase resulting in the loss of the characteristic transition at 720 nm. Results of steady-state kinetic analysis and electrochemical studies suggest that tryptophan 116 plays a critical role in stabilizing the hexacoordinate monooxo Mo(VI) form of the enzyme and prevents the formation of a dioxo pentacoordinate Mo(VI) species, generated as a consequence of the dissociation of one of the dithiolene ligands of the molybdopterin cofactor from the Mo ion.

A [2Fe-2S] ferredoxin (FdVI) is essential for growth of the photosynthetic bacterium Rhodobacter capsulatus.

The physiological function of Rhodobacter capsulatus FdVI, a [2Fe-2S] ferredoxin, was investigated by the cloning, sequence analysis, and mutagenesis of its structural gene, called fdxE. The DNA region surrounding fdxE was mapped, and the nucleotide sequence of a 4.2-kb fragment was determined. fdxE is preceded by a sequence that is very similar to a sigma54 recognition site and is followed by a putative transcription stop signal, suggesting that fdxE forms a separate cistron. Two open reading frames were identified upstream and downstream of fdxE and were named ORFE0 and ORFE1, respectively. The former may encode a polypeptide having 34% similarity with HtrA, a serine protease found in enteric bacteria. ORFE1 is homologous to purU, a gene involved in purine biosynthesis. Interposon mutagenesis of fdxE was unsuccessful when attempted on the wild-type strain B10. Disruption of fdxE could be achieved only in strains harboring an additional copy of fdxE on a plasmid. Mutants obtained in this way and carrying a plasmid-borne copy of fdxE under the control of the nifH promoter grew only in N-free medium, thus demonstrating that fdxE expression is required for growth. Nevertheless, such mutants were found to spontaneously revert at a frequency of 5 x 10(-6) to an apparent wild-type phenotype, although they contained no detectable amount of FdVI. Taken together, the results indicate that FdVI is required for an essential metabolic function in R. capsulatus and that this FdVI dependence could be relieved by a single-mutation event. In accordance, FdVI biosynthesis was found to be constitutive in R. capsulatus.

Phenotypic characterisation and genetic complementation of dimethylsulfoxide respiratory mutants of Rhodobacter sphaeroides and Rhodobacter capsulatus.

Two chlorate resistant mutants of Rhodobacter sphaeroides were isolated which were deficient in dimethylsulfoxide reductase activity. Immunoblotting experiments showed that the phenotype of these mutants and that of Rhodobacter capsulatus strain DK9, a mutant unable to reduce dimethylsulfoxide, was correlated with low or undetectable levels of the dimethylsulfoxide reductase apoprotein. All three mutants were complemented by a cosmid from a library of Rhodobacter sphaeroides genomic DNA. Further genetic complementation analysis revealed that functions required for restoration of dimethylsulfoxide reductase activity in the Rhodobacter sphaeroides mutants were encoded on an 9 kb EcoR1 DNA fragment derived from this cosmid. Expression of this 9 kb DNA fragment in Escherichia coli showed that it encoded the dimethylsulfoxide reductase structural gene of Rhodobacter sphaeroides.

Site-directed mutagenesis of the target arginine for ADP-ribosylation of nitrogenase component II in Rhodobacter capsulatus.

The role of the conserved residue arginine-102 in the functioning and the regulation of the nitrogenase component II protein in Rhodobacter capsulatus has been studied by site-directed mutagenesis. The arginine at position 102 was replaced by thirteen different amino acids, and the effect of these substitutions on diazotrophic growth, in vivo and in vitro nitrogenase activity, and ADP-ribosylation of the component II protein was tested. The results show that although arginine is the optimal amino acid at this position, it is not essential for activity. However, the mutant proteins were not modified by ADP-ribosylation, either in the dark or after addition of NH4+, consistent with the specificity of the post-translational regulatory mechanism for the Arg-102. Indirect evidence suggest that this residue may be involved in interaction with the in vivo low-potential electron donor.