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 mu tagenesis of its structural gene, called fdxt. The DNA region surround ing 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 sigma(54) recognition site and is followed by a putative transcript ion stop signal, suggesting that fdxE forms a separate cistron, Two op en reading frames were identified upstream and downstream of fdxE and were named ORFE0 and ORFE1, respectively. The former may encode a poly peptide having 34% similarity with HtrA, a serine protease found in en teric bacteria, ORFE1 is homologous to purU, a gene involved in purine biosynthesis. Interposon mutagenesis of fdxE was unsuccessful when at tempted on the wild-type strain BIG. Disruption of fdxE could be achie ved only in strains harboring an additional copy of fdxE on a plasmid. Mutants obtained in this way and carrying a plasmid-borne copy of fdx E under the control of the nifH promoter grew only in N-free medium, t hus demonstrating that fdxE expression is required for growth. Neverth eless, such mutants were found to spontaneously revert at a frequency of 5 x 10(-6) to an apparent wild-type phenotype, although they contai ned no detectable amount of FdVI, Taken together, the results indicate . that FdVI is required for an essential metabolic function in R. caps ulatus and that this FdVI dependence could be relieved by a single-mut ation event, In accordance, FdVI biosynthesis was found to be constitu tive in X. capsulatas.