Summary: DNA fragments from and were isolated which restored hydrogenase activities in both and mutant strains of The and genes, which map near minute 59 of the genome map, 17 kb distant from each other, are not structural hydrogenase genes, but mutation in either of these genes leads to failure to synthesize any of the hydrogenase isoenzymes. The smallest DNA fragments which restored hydrogenase activity to both mutant strains were 4.7 kb from and 2.3 kb from These fragments were cleaved into smaller fragments which did not complement either of the mutations. The cloned heterologous genes also restored formate hydrogenlyase activity but they did not restore activity in or mutant strains of The cloned genes, on plasmids, did not lead to the synthesis of proteins of sufficient size to be the hydrogenase catalytic subunit. The hydrogenase proteins synthesized by and mutant strains of transformed by cloned genes from and were shown by isoelectric and immunological methods to be hydrogenase. Thus, these genes are not hydrogenase structural genes.


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