Mutagenesis of a Rhizobium Plasmid Carrying Hydrogenase Determinants

Transposon Tn5-mob, a Tn5 derivative containing the mobilization site of plasmid RP4, was introduced into Rhizobium leguminosarum strain 128C53 on the suicide vector pSup501 I. Transposon insertions into the plasmids of strain 128C53 were selected after mating the mutagenized population with a non-nodulating R. leguminosarum recipient. In this way, a tenfold enrichment was effected for plasmid-linked mutations. Transconjugants that had received mutagenized symbiotic plasmids were applied individually to the roots of pea seedlings and, after three weeks, the resulting nodules were screened for the absence of hydrogenase activity. Eight hydrogenase-deficient (Hup-) mutants of R. leguminosarum strain 128C53 were isolated. Physical and genetic analyses suggest that each Hup- mutant was due to a single insertion of Tn.5-mob into the symbiotic plasmid, pRL6JI. All mutations were suppressible by pHU 1, a cosmid clone carrying some of the hup genes of Rhizobium japonicum. I NTRODUCTI ON Uptake hydrogenase systems have been found in many nitrogen-fixing bacteria, both free-living and symbiotic. Many possible beneficial roles have been suggested for these enzymes (for a review see Eisbrenner & Evans, 1983) yet they are not essential for bacterial growth unde