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

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Abstract
A gene bank of R. japonicum DNA was constructed by using the broad host range conjugative cosmid pLAFR1. Of the clones in the bank, 83% 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 H2 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 .apprx. 6 .times. 10-3. Plasmid DNA from 11 of the Hup+ transconjugants were isolated and used to transform Escherichia coli. EcoRI digests of all plasmids isolated from Hup+ transconjugants had 3 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 .apprx. 10-3. The hup gene cosmids may contain one gene and a portion of another.