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Three classes of Hup‒ mutants of Azotobacter chroococcum were obtained by N-methyl-N′-nitro-N-nitrosoguanidine mutagenesis and screening by H3H uptake: (1) those with no discernible H2-uptake or H2-evolving activity, (2) those showing no uptake but some H2 production and (3) those leaky for both activities. One mutant strain, MCD-124, expressed hydrogenase activity similar to the solubilized wild-type enzyme in O2 sensitivity, sedimenting behaviour and pH optimum. All the other mutants were probably mutated in the hydrogenase structural or processing (methylene blue) genes rather than in genes for hydrogenase-linked respiratory proteins. Four mutants chosen from the first category were complemented for hydrogenase activity by conjugation with Escherichia coli carrying plasmid pHU1 containing Rhizobium japonicum hydrogenase genes. A pHU1 transconjugant of strain MCD-124, on the other hand, did not express any additional hydrogenase activity.
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