Diversity and evolution of hydrogenase systems in rhizobia

Abstract

Uptake hydrogenases allow rhizobia to recycle the hydrogen generated in the nitrogen fixation process within the legume nodule. Hydrogenase (hup) systems in Bradyrhizobium japonicum and Rhizobium leguminosarum bv. viciae show highly conserved sequence and gene organization, but important differences exist in regulation and in the presence of specific genes. We have undertaken the characterization of hup gene clusters from Bradyrhizobium sp. (Lupinus), Bradyrhizobium sp. (Vigna), and Rhizobium tropici and Azorhizobium caulinodans strains with the aim of defining the extent of diversity in hup gene composition and regulation in endosymbiotic bacteria. Genomic DNA hybridizations using hupS, hupE, hupUV, hypB, and hoxA probes showed a diversity of intraspecific hup profiles within Bradyrhizobium sp. (Lupinus) and Bradyrhizobium sp. (Vigna) strains and homogeneous intraspecific patterns within R. tropici and A. caulinodans strains. The analysis also revealed differences regarding the possession of hydrogenase regulatory genes. Phylogenetic analyses using partial sequences of hupS and hupL clustered R. leguminosarum and R. tropici hup sequences together with those from B. japonicum and Bradyrhizobium sp. (Lupinus) strains, suggesting a common origin. In contrast, Bradyrhizobium sp. (Vigna) hup sequences diverged from the rest of rhizobial sequences, which might indicate that those organisms have evolved independently and possibly have acquired the sequences by horizontal transfer from an unidentified source.

FIG. 1.

Genetic organization of hydrogenase clusters in Bradyrhizobium japonicum 122DES and Rhizobium leguminosarum bv. viciae UPM791. Grey arrows indicate hup and hyp genes common to both species. Black arrows show genes present and functional in only one microorganism. Thick lines above genes show the positions of DNA probes used in Southern hybridizations.

FIG. 2.

Genomic DNA hybridizations of Bradyrhizobium sp. (Lupinus) strains with hup, hyp, and hox DNA probes. Panels A and B and panels C and D show Southern hybridizations using hupS and hypB probes from R. leguminosarum and hupUV and hoxA probes from B. japonicum, respectively. Genomic DNA was restricted with EcoRI enzyme. Strains: R. leguminosarum UPM791 (lane 1), B. japonicum 122DES (lane 2), R. leguminosarum PRE (lane 3), Bradyrhizobium sp. (Lupinus) 624 (lane 4), IM43B (lane 5), Z89 (lane 6), 466 (lane 7), and UPM860 (lane 8). Numbers on the left indicate molecular sizes of markers (in kilobases).

FIG. 3.

Hybridization profiles of hup genes in Bradyrhizobium sp. (Vigna) strains. EcoRI-digested genomic DNAs were hybridized with Rhizobium leguminosarum hupS (A) and hypB (B) probes and with a hoxA probe from B. japonicum (C). Strains: R. leguminosarum UPM791 (lane 1), B. japonicum 122DES (lane 2), R. leguminosarum PRE (lane 3), Bradyrhizobium sp. (Vigna) M2 (lane 4), M5 (lane 5), M18 (lane 6), M21 (lane 7), M43 (lane 8), B78 (lane 9), B96 (lane 10), B97 (lane 11), and 32H1 (lane 12). Numbers on the left indicate molecular sizes of markers (in kilobases).

FIG. 4.

Southern hybridizations of genomic DNA from Azorhizobium sp. and A. caulinodans strains with hup and hyp DNA probes. Panels A and C show Southern hybridizations using R. leguminosarum hupS and hypB probes, respectively. Panel B shows the hybridization signals obtained with a hupUV probe from B. japonicum. In all cases, genomic DNAs were restricted with EcoRI enzyme. Strains: R. leguminosarum UPM791 (lane 1), B. japonicum 122DES (lane 2), R. leguminosarum PRE (lane 3), Azorhizobium caulinodans ORS571 (lane 4), Azorhizobium sp. ORS552 (lane 5), Azorhizobium caulinodans ORS591 (lane 6), Azorhizobium sp. SD02 (lane 7), and Azorhizobium sp. SG05 (lane 8). Numbers on the left indicate molecular size of markers (in kilobases).

FIG. 5.

DNA hybridization with hup and hyp probes and plasmid profiles of R. tropici Hup⁺ strains. (A and B) EcoRI-digested genomic DNAs were hybridized with the R. leguminosarum hupS (A) and hypB (B) probes. Strains for panels A and B: R. leguminosarum UPM791 (lane 1) and PRE (lane 2), Rhizobium tropici USDA 2734 (lane 3), USDA 2786 (lane 4), USDA 2738 (lane 5), USDA 2787 (lane 6), USDA 2793 (lane 7), USDA 9030 (lane 8), USDA 2801 (lane 9), USDA 2840 (lane 10), USDA 2838 (lane 11), USDA 2813 (lane 12), and USDA 2822 (lane 13). Numbers on the right indicate molecular sizes, in kilobases. (C) Plasmids were resolved by the Eckhardt procedure (see Materials and Methods) (lanes 1), transferred to a membrane, and hybridized to R. leguminosarum hupS (lanes 2) or nifH (lanes 3) gene probes. Subpanels: (a) R. leguminosarum UPM791 (control strain); (b to f) R. tropici strains USDA 9030 (b), USDA 2840 (c), USDA 2838 (d), USDA 2813 (e), and USDA 2822 (f). Numbers on the left indicate molecular sizes (in megadaltons) of R. leguminosarum UPM791 plasmids.

FIG. 6.

Phylogenetic trees derived from hup and 16S rDNA sequences of rhizobia. Partial hupS and hupL sequences from Bradyrhizobium sp. (Lupinus), Bradyrhizobium sp. (Vigna), Rhizobium tropici, Azorhizobium sp., and Azorhizobium caulinodans strains were obtained and aligned with the corresponding sequences from Rhizobium leguminosarum bv. viciae UPM791, Bradyrhizobium japonicum 122DES, two other α-proteobacteria (Rhodobacter capsulatus and Rhodobacter sphaeroides), and hyaA and hyaB (hydrogenase 1 structural genes) from E. coli (used as the outgroup). Minimum-distance trees were generated for hupS (A) and hupL (B) by using CLUSTALX and TREEVIEW software. A similar tree was constructed from 16S rDNA sequences of the rhizobial strains mentioned above or database 16S rDNA sequences from strains belonging to the same taxa (C). Tree scales are indicated as per site substitutions. Figures at nodes indicate bootstrap values (per 1,000). The accession numbers of the sequences obtained from databases are as follows: R. leguminosarum bv. viciae UPM791 (hupS and hupL, gi:1167855; 16S rDNA, AY072787), B. japonicum USDA110 (16S rDNA, gi:534881), 122DES (hupS and hupL, gi:152100), E. coli (hyaA and hyaB, gi:146419; 16S rDNA, gi:174375), R. capsulatus (hupS and hupL, gi:46032; 16S rDNA, gi:1944502), R. sphaeroides (hupS and hupL, gi:4539150; 16S rDNA, gi:303817), R. tropici USDA9030 (16S rDNA, gi:1895079), A. caulinodans ORS571 (16S rDNA, gi:870816). Abbreviations: Azoca, Azorhizobium caulinodans; Braja, Bradyrhizobium japonicum; Bralu, Bradyrhizobium sp. (Lupinus); Bravi, Bradyrhizobium sp. (Vigna); Ecoli, Escherichia coli; Rhilv, Rhizobium leguminosarum bv. viciae; Rhtro, Rhizobium tropici; Rhoca, Rhodobacter capsulatus; Rhosh, Rhodobacter sphaeroides.