Mutagenesis of hupT Gene and H(2)-uptake Hydrogenase Expression in Photosynthetic Bacterium SDH2

HupT(-) insertion mutants, SDHT1 and SDHT2, were constructed by the homologous double exchange between the host, Rodobacter sp. SDH20, and the hupT gene fragment with a kan(R) gene inserted on pSE8, a suicide plasmid, which had been introduced into the host by using triparental conjugation. The HupT(-) mutants were verified by Southern hybridization. H(2)-uptake hydrogenase activity analysis revealed that the H(2)-uptake hydrogenase activities of HupT(-)mutants were two-fold as that of wild type strain SDH20 when grown anaerobically in MN medium and aerobically in MG or MN medium, and had no distinct improvement relative to wild type strain when grown anaerobically in MG medium. The expression analysis of hupS confirmed that the HupT- mutants exhibited two- to three-fold increase of the expression of hupS relative to wild type strain when grown anaerobically in MN medium and aerobically in MG or MN medium, but no obvious increase when grown anaerobically in MG medium. These results demonstrate that hupT regulates negatively the synthesis of H(2)-up-take hydrogenase in the photosynthetic bacterium strain SDH2.