Transposon mutagenesis in purple sulfur photosynthetic bacteria: Identification of hypF, encoding a protein capable of processing [NiFe] hydrogenasesin alpha, beta, and gamma subdivisions of the proteobacteria

A random transposon-based mutagenesis system was optimized for the purple s ulfur phototrophic bacterium Thiocapsa roseopersicina BBS, Screening for hy drogenase-deficient phenotypes resulted in the isolation of six independent mutants in a mini-Tn5 library. One of the mutations was in a gene showing high amino acid sequence similarity to HypF proteins in other organisms, In activation of hydrogen uptake activity in the trypF-deficient mutant result ed in a dramatic increase in the hydrogen evolution capacity of T, roseoper sicina under nitrogen-fixing conditions. This mutant is therefore a promisi ng candidate for use in practical biohydrogen-producing systems. The recons tructed hypF gene was able to complement the hypF-deficient mutant of T, ro seopersicina BBS, Heterologous complementation experiments, using hypF muta nt strains of T, roseopersicina, Escherichia coli, and Ralstonia eutropha a nd various hypF genes, were performed. They were successful in all of the c ases tested, although for E. coli, the regulatory region of the foreign gen e had to be replaced in order to achieve partial complementation. RT-PCR da ta suggested that HypF has no effect on the transcriptional regulation of t he structural genes of hydrogenases in this organism.