Conserved Plasmid Hydrogen-Uptake ( hup )-Specific Sequences within Hup ⁺ Rhizobium leguminosarum Strains

Abstract

Thirteen Rhizobium leguminosarum strains previously reported as H 2 -uptake hydrogenase positive (Hup + ) or negative (Hup − ) were analyzed for the presence and conservation of DNA sequences homologous to cloned Bradyrhizobium japonicum hup -specific DNA from cosmid pHU1 (M. A. Cantrell, R. A. Haugland, and H. J. Evans, Proc. Natl. Acad. Sci. USA 80:181-185, 1983). The Hup phenotype of these strains was reexamined by determining hydrogenase activity induced in bacteroids from pea nodules. Five strains, including H 2 oxidation-ATP synthesis-coupled and -uncoupled strains, induced significant rates of H 2 -uptake hydrogenase activity and contained DNA sequences homologous to three probe DNA fragments (5.9-kilobase [kb] Hin dIII, 2.9-kb Eco RI, and 5.0-kb Eco RI) from pHU1. The pattern of genomic DNA Hin dIII and Eco RI fragments with significant homology to each of the three probes was identical in all five strains regardless of the H 2 -dependent ATP generation trait. The restriction fragments containing the homology totalled about 22 kb of DNA common to the five strains. In all instances the putative hup sequences were located on a plasmid that also contained nif genes. The molecular sizes of the identified hup-sym plasmids ranged between 184 and 212 megadaltons. No common DNA sequences homologous to B. japonicum hup DNA were found in genomic DNA from any of the eight remaining strains showing no significant hydrogenase activity in pea bacteroids. These results suggest that the identified DNA region contains genes essential for hydrogenase activity in R. leguminosarum and that its organization is highly conserved within Hup + strains in this symbiotic species.

Molecular and functional characterization of the ORS571 hydrogenase gene cluster

“…rostrata seeds were surface sterilized and germinated on 1% water agar plates [13]. Seedlings were grown in Leonard jar-type assemblies under bacteriologically controlled conditions with a nitrogen-free plant nutrient solution [14] supplemented with 20 lM NiCl 2 from day 10 after seedling inoculation. Acetylene reduction and hydrogen evolution in intact nodules were determined by gas chromatography.…”

“…Acetylene reduction and hydrogen evolution in intact nodules were determined by gas chromatography. Hydrogenase activity in bacteroid suspensions was measured using an amperometric method with oxygen as electron acceptor [14]. Determinations of protein content of bacteroid suspensions and cell cultures were done by the bicinchoninic acid method [15].…”

“…Restriction enzyme digestions, PCR amplifications, agarose gel electrophoresis and Southern blot transfers were carried out by standard protocols [16]. Genomic DNA of A. caulinodans ORS571 was extracted as previously described [14]. The R. leguminosarum UPM791 hupS and hypB, and the B. japonicum hupUV gene probes were generated by PCR and labelled with digoxigenin using DIG-11-dUTP (Roche Molecular Biochemicals, Mannheim, Germany) at 40 lM final concentration [10].…”

Hydrogenase genes are uncommon and highly conserved in Rhizobium leguminosarum bv. viciae

“…Previous surveys from the literature suggest that the Hup trait is rare within R. leguminosarum bv. viciae [4], and few of the reported Hup + strains have been substantiated by genetic analysis [13].…”

“…Furthermore, our group had previously obtained evidence that major hup cluster restriction enzyme cut sites are conserved within the standard Hup + collection strains of R. leguminosarum bv. viciae [13], and that hup genes from strains R. leguminosarum bv. viciae UPM791 and B10 are very strongly conserved [10].…”