Mutagenesis of hupT Gene and H2-uptake
Hydrogenase
Expression in Photosynthetic Bacterium SDH2
LI Qiang and WU Yong-Qiang*
( Shanghai Institute of Plant physiology, the Chinese Academy of Sciences,
Shanghai 200032, China )
Abstract 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 kanR
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. H2-uptake hydrogenase activity
analysis revealed that the H2-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 H2-uptake hydrogenase in the
photosynthetic bacterium strain SDH2.
Key Words Photosynthetic bacterium； H2-uptake
hydrogenase；
negative regulative gene； insertion mutage-nesis
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