Transposon Mutagenesis in Purple Sulfur Photosynthetic Bacteria: Identification of hypF , Encoding a Protein Capable of Processing [NiFe] Hydrogenases in α, β, and γ Subdivisions of the Proteobacteria

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Abstract
A random transposon-based mutagenesis system was optimized for the purple sulfur phototrophic bacterium Thiocapsa roseopersicina BBS. Screening for hydrogenase-deficient phenotypes resulted in the isolation of six independent mutants in a mini-Tn 5 library. One of the mutations was in a gene showing high amino acid sequence similarity to HypF proteins in other organisms. Inactivation of hydrogen uptake activity in the hypF -deficient mutant resulted in a dramatic increase in the hydrogen evolution capacity of T. roseopersicina under nitrogen-fixing conditions. This mutant is therefore a promising candidate for use in practical biohydrogen-producing systems. The reconstructed hypF gene was able to complement the hypF -deficient mutant of T. roseopersicina BBS. Heterologous complementation experiments, using hypF mutant strains of T. roseopersicina , Escherichia coli , and Ralstonia eutropha and various hypF genes, were performed. They were successful in all of the cases tested, although for E. coli , the regulatory region of the foreign gene had to be replaced in order to achieve partial complementation. RT-PCR data suggested that HypF has no effect on the transcriptional regulation of the structural genes of hydrogenases in this organism.

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