Construction of an R‐prime plasmid carrying the hup genes of Azorhizobium caulinodans ORS571 and its transfer to and stability in Rhizobium meliloti

Abstract

An R-prime plasmid (pJB3JI-18) carrying a 10 kb region of ,4iorhiiohiun1 c~uu/inodurrs ORSS71 DNA which codes for uptake hydrogenase activity was isolated from a Km' derivative of R68.45. R-prime plasmids were obtained by transferring R68.45 to ORSS71. retransferring it to E. coli. and selecting transconjugants for hydrogenase activity by means of a colour test based on methylene bluc. The R-prime plasmid DNA was hybridized with a DNA region which contained the Imp genes of Brarlrrliizohium juponicuiii, indicating homology between the heterologous lzirp genes. Transfer of the R-prime plasmid to different Hup-mutants restored the Hup' phenotype. When it was introduced t o R. meli/ori strains, it maintained its molecular weight and did not affect indigenous plasmids in two of three recipients. In the third recipient cointegrate formation and incompatibility with indigenous plasmids were found. Molecular hydrogen is a by-product in the biological reduction of dinitrogen to ammonia. The formation of hydrogen by nitrogenase implies loss of energy and reduction equivalents. In bacteroids, hydrogen formation may consume 40 to 60"; of the electron flux to nitrogenase (SCHUBERT and EVANS 1976). A number of nitrogen-fixing Rltizobiutn strains reoxidize hydrogen via an hydrogen uptake system. In most cases the hydrogenase activity is insufficient to recycle all the hydrogen formed. Previously, the fast-growing strain ORS571 of Azorhizobiunz caulinodans was isolated from stem nodules of the tropical legume Sesbania rostrata (DREYFUS and DOMMERGUES 1981). This strain forms nodules on both the stem and the roots of the host plant. Nitrogen-fixing cultures of ORS571 show considerable hydrogenase activity (STAM et al. 1984). In this special case, hydrogen oxidation is coupled with ATP formation, which is not found in other Rhizobiunz species or strains (NELSON 1983). These features prompted us to look for suitable cloning procedures, because the transfer of the /?up genes of ORS571 to other rhizobia, e.g. R. meliloti would be beneficial for improving nitrogen fixation. Transmissible plasmids carrying DNA segments have been isolated in several bacterial species by using in vivo procedures. These prime plasmids have been of great value in the study of chromosomal genes (Low 1972, HOLLOWAY 1979 HOLLOWAY 1976) to induce recombination with the chromosome was expoited in these studies. ORS571 is a plasmidless strain, and therefore the /zup genes must be located on the chromosome.Here we report the isolation and characterization of an R-prime plasmid containing a 10 kb fragment of A . caufinodans ORS571 DNA. This fragment includes the hup genes.Moreover, we show the stability of the R-prime plasmid after transfer to several R. tneliloti strains and the expression of the hup genes in this species.