Generation of New Hydrogen-Recycling Rhizobiaceae Strains by Introduction of a Novel hup Minitransposon

Hydrogen evolution by nitrogenase is a source of inefficiency for the nitrogen fixation process by the Rhizobium-legume symbiosis. To develop a strategy to generate rhizobial strains with H(2)-recycling ability, we have constructed a Tn5 derivative minitransposon (TnHB100) that contains the ca. 18-kb H(2) uptake (hup) gene cluster from Rhizobium leguminosarum bv. viciae UPM791. Bacteroids from TnHB100-containing strains of R. leguminosarum bv. viciae PRE, Bradyrhizobium japonicum, R. etli, and Mesorhizobium loti expressed high levels of hydrogenase activity that resulted in full recycling of the hydrogen evolved by nitrogenase in nodules. Efficient processing of the hydrogenase large subunit (HupL) in these strains was shown by immunoblot analysis of bacteroid extracts. In contrast, Sinorhizobium meliloti, M. ciceri, and R. leguminosarum bv. viciae UML2 strains showed poor expression of the hup system that resulted in H(2)-evolving nodules. For the latter group of strains, no immunoreactive material was detected in bacteroid extracts using anti-HupL antiserum, suggesting a low level of transcription of hup genes or HupL instability. A general procedure for the characterization of the minitransposon insertion site and removal of antibiotic resistance gene included in TnHB100 has been developed and used to generate engineered strains suitable for field release.