Organization and Expression of Hydrogen-Uptake (hup) Genes of Rhizobium Leguminosarum

Abstract

Genes involved in H2-uptake (hup genes) in Rhizobium leguminosarum strain UPM791 are clustered in a DNA region of the symbiotic plasmid. A frag ment of 20.7 kilobases from this region has previously been cloned in cosmid pAL618. Analysis of 25 Tn5 insertions generated by marker exchange into the UPM791 genome indicates that at least 18 kilobases of insert DNA in pAL618 are essential for H2-uptake in R. leguminosarum, Transposon Tn3-HoHol, which contains lacZ gene (Stachel et al. 1985. EMBO J. 4, 891–898) was used to create lac fusions within the hup region of pAL618. High β-galactosidase activity was induced in symbiosis with peas in six Tn3-HoHol-generated lac fusions. The corresponding Tn3-HoHol insertions were located in hup-specific DNA and all of them contained the lacZ gene in the left to right orientation. Two of these insertions mapped in a region showing homology to the structural genes of the B. japonicum hydrogenase, and the other four mapped in a region located outside and to the right of the putative structural region. These last four fusions also induce β-galactosidase activity in free-living cells incubated under low-oxygen conditions. These results demonstrate that we have created lac fusions within hup genes cloned in pAL618, and that these genes are expressed in a leftward to rightward orientation. It is also evident from this work that some genes located in pAL618 insert DNA are inducible in free-living cells and may be involved in regulation of hup genes expression.