Construction of a Rhizobium japonicum gene bank and use in isolation of a hydrogen uptake gene

Abstract

A gene bank of Rhizobium japonicum DNA was constructed by using the broad host range conjugative cosmid pLAFR1. Eighty-three percent of the clones in the bank contained cosmids with insert DNA averaging 22.6 kilobase pairs in length. A series of cosmids containing a hydrogen uptake (hup) gene was identified by transferring the gene bank into a H(2) uptake-negative (Hup(-)) R. japonicum point mutant (PJ17nal) and screening tetracycline-resistant colonies for the ability to grow chemolithotrophically and to reduce methylene blue in a recently devised colony assay. Hup(+) transconjugants arose at a frequency of approximately 6 x 10(-3). Plasmid DNAs from II of the Hup(+) transconjugants were isolated and used to transform Escherichia coli. EcoRI digests of all plasmids isolated from Hup(+) transconjugants had three DNA fragments in common. Eight of the E. coli transformants containing hup gene cosmids were conjugated with PJ17nal and another Hup(-) point mutant, PJ18nal. All PJ17nal transconjugants were Hup(+). The frequency of Hup(+) transconjugants with PJ18nal was approximately 10(-3). The results indicate that the hup gene cosmids may contain one gene and a portion of another.