Alteromonas macleodii Deep ecotype is a marine, heterotrophic, gammaproteobacterium isolated in the Mediterranean Sea between depths of 1000 and 3500 m. The sequenced strain was previously reported to contain a [NiFe] hydrogenase. We verified the presence of this hydrogenase in other strains of A. macleodii Deep ecotype that were previously isolated from several bathypelagic microenvironments. We developed a system for the genetic manipulation of A. macleodii Deep ecotype using conjugation and used this system to create mutant strains that lack the [NiFe] hydrogenase structural genes (hynSL). The mutants did not possess hydrogenase activity, and complementation of the mutant strain with the hynSL genes successfully restored hydrogenase activity. Both the mutant and the wild-type strains grew at the same rate in a variety of media and under different environmental conditions, indicating little effect of the hydrogenase mutation under the conditions tested.