The genetic diversity of bacterial populations nodulating Lupinus luteus (yellow lupine) in Northern Tunisia was examined. Phylogenetic analyses of 43 isolates based on recA and gyrB partial sequences grouped them in three clusters, two of which belong to genus Bradyrhizobium (41 isolates) and one, remarkably, to Microvirga (2 isolates), a genus never previously described as microsymbiont of this lupine species. Representatives of the three clusters were analysed in-depth by multilocus sequence analysis of five housekeeping genes (rrs, recA, glnII, gyrB and dnaK). Surprisingly, the Bradyrhizobium cluster with the two isolates LluI4 and LluTb2 may constitute a new species defined by a separate position between Bradyrhizobium manausense and B. denitrificans. A nodC-based phylogeny identified only two groups: one formed by Bradyrhizobium strains included in the symbiovar genistearum and the other by the Microvirga strains. Symbiotic behaviour of representative isolates was tested, and among the seven legumes inoculated only a difference was observed i.e. the Bradyrhizobium strains nodulated Ornithopus compressus unlike the two strains of Microvirga. On the basis of these data, we conclude that L. luteus root nodule symbionts in Northern Tunisia are mostly strains within the B. canariense/B. lupini lineages, and the remaining strains belong to two groups not previously identified as L. luteus endosymbionts: one corresponding to a new clade of Bradyrhizobium and the other to the genus Microvirga.

Rhizobium leguminosarum bv. viciae expresses an uptake hydrogenase in symbiosis with peas (Pisum sativum) but, unlike all other characterized hydrogen-oxidizing bacteria, cannot express it in free-living conditions. The hydrogenase-specific transcriptional activator gene hoxA described in other species was shown to have been inactivated in R. leguminosarum by accumulation of frameshift and deletion mutations. Symbiotic transcription of hydrogenase structural genes hupSL originates from a −24/−12 type promoter (hupS_p). A regulatory region located in the −173 to −88 region was essential for promoter activity in R. leguminosarum. Activation of hupS_p was observed in Klebsiella pneumoniae and Escherichia coli cells expressing the K. pneumoniae nitrogen fixation regulator NifA, and in E. coli cells expressing R. meliloti NifA. This activation required direct interaction of NifA with the essential −173 to −88 regulatory region. However, no sequences resembling known NifA-binding sites were found in or around this region. NifA-dependent activation was also observed in R. etli bean bacteroids. NifA-dependent hupS_p activity in heterologous hosts was also absolutely dependent on the RpoN σ-factor and on integration host factor. Proteins immunologically related to integration host factor were identified in R. leguminosarum. The data suggest that hupS_p is structurally and functionally similar to nitrogen fixation promoters. The requirement to coordinate nitrogenase-dependent H₂ production and H₂ oxidation in nodules might be the reason for the loss of HoxA in R. leguminosarum and the concomitant NifA control of hup gene expression. This evolutionary acquired control would ensure regulated synthesis of uptake hydrogenase in the most common H₂-rich environment for rhizobia, the legume nodule.

The Type VI secretion systems (T6SSs) allow bacteria to translocate effector proteins to other bacteria or to eukaryotic cells. However, little is known about the role of T6SS in endosymbiotic bacteria. In this work we describe the T6SS of Rhizobium etli Mim1, a bacteria able to effectively nodulate common beans. Structural genes and those encoding possible effectors have been identified in a 28-gene DNA region of R. etli Mim1 pRetMIM1f plasmid. Immunodetection of Hcp protein, a conserved key structural component of T6SS systems, indicates that this secretion system is active at high cell densities, in the presence of root exudates, and in bean nodules. Rhizobium etli mutants affected in T6SS structural genes produced plants with lower dry weight and smaller nodules than the wild-type strain, indicating for the first time that the T6SS plays a positive role in Rhizobium–legume symbiosis.

Summary: The Tat (twin‐arginine translocation) system mediates export of periplasmic proteins in folded conformation. Proteins transported via Tat contain a characteristic twin‐arginine motif in their signal peptide. Genetic determinants (tatABC genes) of the Tat system from Rhizobium leguminosarum bv. viciae were cloned and characterized, and a tatBC deletion mutant was constructed. The mutant lacked the ability for membrane targeting of hydrogenase, a known Tat substrate, and was impaired in hydrogenase activity. Interestingly, in the absence of a functional Tat system, only small, white nodules unable to fix nitrogen were induced in symbiosis with pea plants. Analysis of nodule structure and location of green fluorescent protein (GFP)‐tagged bacteria within nodules indicated that the symbiotic process was blocked in the tat mutant at a stage previous to bacteria release into cortical cells. The R. leguminosarum Tat‐deficient mutant lacked a functional cytochrome bc₁ complex. This was consistent with the fact that R. leguminosarum Rieske protein, a key component of the symbiosis‐essential cytochrome bc₁ complex, contained a typical twin‐arginine signal peptide. However, comparative analyses of nodule structure indicated that nodule development in the tat mutant was arrested at an earlier step than in a cytochrome bc₁ mutant. These data indicate that the Tat pathway is also critical for proteins relevant to the initial stages of the symbiotic process.

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.