Proteome Analysis Reveals a Significant Host-Specific Response in Rhizobium leguminosarum bv. viciae Endosymbiotic Cells.

The Rhizobium-legume symbiosis is a beneficial interaction in which the bacterium converts atmospheric nitrogen into ammonia and delivers it to the plant in exchange for carbon compounds. This symbiosis implies the adaptation of bacteria to live inside host plant cells. In this work, we apply RP-LC-MS/MS and isobaric tags as relative and absolute quantitation techniques to study the proteomic profile of endosymbiotic cells (bacteroids) induced by Rhizobium leguminosarum bv viciae strain UPM791 in legume nodules. Nitrogenase subunits, tricarboxylic acid cycle enzymes, and stress-response proteins are among the most abundant from over 1000 rhizobial proteins identified in pea (Pisum sativum) bacteroids. Comparative analysis of bacteroids induced in pea and in lentil (Lens culinaris) nodules revealed the existence of a significant host-specific differential response affecting dozens of bacterial proteins, including stress-related proteins, transcriptional regulators, and proteins involved in the carbon and nitrogen metabolisms. A mutant affected in one of these proteins, homologous to a GntR-like transcriptional regulator, showed a symbiotic performance significantly impaired in symbiosis with pea but not with lentil plants. Analysis of the proteomes of bacteroids isolated from both hosts also revealed the presence of different sets of plant-derived nodule-specific cysteine-rich peptides, indicating that the endosymbiotic bacteria find a host-specific cocktail of chemical stressors inside the nodule. By studying variations of the bacterial response to different plant cell environments, we will be able to identify specific limitations imposed by the host that might give us clues for the improvement of rhizobial performance.

Hydrogen-uptake genes improve symbiotic efficiency in common beans (Phaseolus vulgaris L.).

Hydrogen-uptake (Hup) activity is implicated in the mitigation of energy losses associated with the biological nitrogen fixation process, and has been related to productivity increases in some legume hosts. However, in common bean (Phaseolus vulgaris L.) the expression of hydrogenase is rare. In this study an 18-kb hup gene cluster from Rhizobium leguminosarum bv. viciae encoding a NiFe hydrogenase was successfully transferred to three common bean rhizobial strains lacking hydrogenase activity (Hup-) but symbiotically very effective and used in commercial inoculants in Brazil: one strain originally from Colombia (Rhizobium tropici CIAT 899), and two strains from Brazil (R. tropici H 12 and Rhizobium freirei PRF 81). The inclusion of NiCl2 in the nutrient solution did not increase hydrogenase activity, indicating that common bean plants allow efficient nickel provision for hydrogenase synthesis in the bacteroids. The symbiotic performance-evaluated by nodulation, plant growth, N accumulation and seed production-of wild-type and Hup+ derivative strains was compared in experiments performed with cultivar Carioca under greenhouse conditions, in sterile substrate and in non-sterile soil. Statistically significant increases in one or more parameters were observed for all three Hup+ derivatives when compared to the respective wild-type strain. Differences were found mainly with the Brazilian strains, reaching impressive increases in nodule efficiency and seed total N content. The results highlight the potential of using Rhizobium Hup+ strains for the design of more energy-efficient inoculants for the common bean crop.

Diverse Bacteria Affiliated with the Genera Microvirga, Phyllobacterium, and Bradyrhizobium Nodulate Lupinus micranthus Growing in Soils of Northern Tunisia.

The genetic diversity of bacterial populations nodulating Lupinus micranthus in five geographical sites from northern Tunisia was examined. Phylogenetic analyses of 50 isolates based on partial sequences of recA and gyrB grouped strains into seven clusters, five of which belong to the genus Bradyrhizobium (28 isolates), one to Phyllobacterium (2 isolates), and one, remarkably, to Microvirga (20 isolates). The largest Bradyrhizobium cluster (17 isolates) grouped with the B. lupini species, and the other five clusters were close to different recently defined Bradyrhizobium species. Isolates close to Microvirga were obtained from nodules of plants from four of the five sites sampled. We carried out an in-depth phylogenetic study with representatives of the seven clusters using sequences from housekeeping genes (rrs, recA, glnII, gyrB, and dnaK) and obtained consistent results. A phylogeny based on the sequence of the symbiotic gene nodC identified four groups, three formed by Bradyrhizobium isolates and one by the Microvirga and Phyllobacterium isolates. Symbiotic behaviors of the representative strains were tested, and some congruence between symbiovars and symbiotic performance was observed. These data indicate a remarkable diversity of L. micranthus root nodule symbionts in northern Tunisia, including strains from the Bradyrhizobiaceae, Methylobacteriaceae, and Phyllobacteriaceae families, in contrast with those of the rhizobial populations nodulating lupines in the Old World, including L. micranthus from other Mediterranean areas, which are nodulated mostly by Bradyrhizobium strains.IMPORTANCELupinus micranthus is a legume broadly distributed in the Mediterranean region and plays an important role in soil fertility and vegetation coverage by fixing nitrogen and solubilizing phosphate in semiarid areas. Direct sowing to extend the distribution of this indigenous legume can contribute to the prevention of soil erosion in pre-Saharan lands of Tunisia. However, rhizobial populations associated with L. micranthus are poorly understood. In this context, the diversity of endosymbionts of this legume was investigated. Most Lupinus species are nodulated by Bradyrhizobium strains. This work showed that about half of the isolates from northern Tunisian soils were in fact Bradyrhizobium symbionts, but the other half were found unexpectedly to be bacteria within the genera Microvirga and Phyllobacterium These unusual endosymbionts may have a great ecological relevance. Inoculation with the appropriate selected symbiotic bacterial partners will increase L. micranthus survival with consequent advantages for the environment in semiarid areas of Tunisia.

Maturation of Rhizobium leguminosarum hydrogenase in the presence of oxygen requires the interaction of the chaperone HypC and the scaffolding protein HupK.

[NiFe] hydrogenases are key enzymes for the energy and redox metabolisms of different microorganisms. Synthesis of these metalloenzymes involves a complex series of biochemical reactions catalyzed by a plethora of accessory proteins, many of them required to synthesize and insert the unique NiFe(CN)2CO cofactor. HypC is an accessory protein conserved in all [NiFe] hydrogenase systems and involved in the synthesis and transfer of the Fe(CN)2CO cofactor precursor. Hydrogenase accessory proteins from bacteria-synthesizing hydrogenase in the presence of oxygen include HupK, a scaffolding protein with a moderate sequence similarity to the hydrogenase large subunit and proposed to participate as an intermediate chaperone in the synthesis of the NiFe cofactor. The endosymbiotic bacterium Rhizobium leguminosarum contains a single hydrogenase system that can be expressed under two different physiological conditions: free-living microaerobic cells (∼ 12 µm O2) and bacteroids from legume nodules (∼ 10-100 nm O2). We have used bioinformatic tools to model HupK structure and interaction of this protein with HypC. Site-directed mutagenesis at positions predicted as critical by the structural analysis have allowed the identification of HupK and HypC residues relevant for the maturation of hydrogenase. Mutant proteins altered in some of these residues show a different phenotype depending on the physiological condition tested. Modeling of HypC also predicts the existence of a stable HypC dimer whose presence was also demonstrated by immunoblot analysis. This study widens our understanding on the mechanisms for metalloenzyme biosynthesis in the presence of oxygen.

Bradyrhizobium paxllaeri sp. nov. and Bradyrhizobium icense sp. nov., nitrogen-fixing rhizobial symbionts of Lima bean (Phaseolus lunatus L.) in Peru.

A group of strains isolated from root nodules of Phaseolus lunatus (Lima bean) in Peru were characterized by genotypic, genomic and phenotypic methods. All strains possessed identical 16S rRNA gene sequences that were 99.9% identical to that of Bradyrhizobium lablabi CCBAU 23086(T). Despite having identical 16S rRNA gene sequences, the Phaseolus lunatus strains could be divided into two clades by sequence analysis of recA, atpD, glnII, dnaK and gyrB genes. The genome sequence of a representative of each clade was obtained and compared to the genomes of closely related species of the genus Bradyrhizobium. Average nucleotide identity values below the species circumscription threshold were obtained when comparing the two clades to each other (88.6%) and with all type strains of the genus Bradyrhizobium (≤92.9%). Phenotypes distinguishing both clades from all described and closely related species of the genus Bradyrhizobium were found. On the basis of the results obtained, two novel species, Bradyrhizobium paxllaeri sp. nov. (type strain LMTR 21(T) = DSM 18454(T) = HAMBI 2911(T)) and Bradyrhizobium icense sp. nov. (type strain LMTR 13(T) = HAMBI 3584(T) = CECT 8509(T) = CNPSo 2583(T)), are proposed to accommodate the uncovered clades of Phaseolus lunatus bradyrhizobia. These species share highly related but distinct nifH and nodC symbiosis genes.

Cytisus villosus from Northeastern Algeria is nodulated by genetically diverse Bradyrhizobium strains.

Fifty-one rhizobial strains isolated from root nodules of Cytisus villosus growing in Northeastern Algeria were characterized by genomic and phenotypic analyses. Isolates were grouped into sixteen different patterns by PCR-RAPD. The phylogenetic status of one representative isolate from each pattern was examined by multilocus sequence analyses of four housekeeping genes (16S rRNA, glnII, recA, and atpD) and one symbiotic gene (nodC). Analysis of 16S rRNA gene sequences showed that all the isolates belonged to the genus Bradyrhizobium. Phylogenetic analyses based on individual or concatenated genes glnII, recA, and atpD indicated that strains cluster in three distinct groups. Ten out of the sixteen strains grouped together with Bradyrhizobium japonicum, while a second group of four clustered with Bradyrhizobium canariense. The third group, represented by isolates CTS8 and CTS57, differed significantly from all other bradyrhizobia known to nodulate members of the Genisteae tribe. In contrast with core genes, sequences of the nodC symbiotic gene from all the examined strains form a homogeneous group within the genistearum symbiovar of Bradyrhizobium. All strains tested nodulated Lupinus angustifolius, Lupinus luteus, and Spartium junceum but not Glycine max. From these results, it is concluded that C. villosus CTS8 and CTS57 strains represent a new lineage within the Bradyrhizobium genus.

Phylogenetic evidence of the transfer of nodZ and nolL genes from Bradyrhizobium to other rhizobia.

Nod factor modifications mediated by nodZ and nolL gene products (fucosylation and acetylation of fucose residues, respectively) were probably later acquisitions in the nodulation process. Novel phylogenetic analyses suggest that nodZ and nolL genes were transferred from Bradyrhizobium to other nodule bacteria. These bradyrhizobial genes are highly diverse while rhizobial, sinorhizobial and mesorhizobial nodZ and nolL genes are represented by few branches among those from bradyrhizobia. These genes in novel rhizobial backgrounds may have favored efficient nodulation in legume hosts commonly associated with Bradyrhizobium strains.

Dual role of HupF in the biosynthesis of [NiFe] hydrogenase in Rhizobium leguminosarum.

BACKGROUND: [NiFe] hydrogenases are enzymes that catalyze the oxidation of hydrogen into protons and electrons, to use H2 as energy source, or the production of hydrogen through proton reduction, as an escape valve for the excess of reduction equivalents in anaerobic metabolism. Biosynthesis of [NiFe] hydrogenases is a complex process that occurs in the cytoplasm, where a number of auxiliary proteins are required to synthesize and insert the metal cofactors into the enzyme structural units. The endosymbiotic bacterium Rhizobium leguminosarum requires the products of eighteen genes (hupSLCDEFGHIJKhypABFCDEX) to synthesize an active hydrogenase. hupF and hupK genes are found only in hydrogenase clusters from bacteria expressing hydrogenase in the presence of oxygen. RESULTS: HupF is a HypC paralogue with a similar predicted structure, except for the C-terminal domain present only in HupF. Deletion of hupF results in the inability to process the hydrogenase large subunit HupL, and also in reduced stability of this subunit when cells are exposed to high oxygen tensions. A ΔhupF mutant was fully complemented for hydrogenase activity by a C-terminal deletion derivative under symbiotic, ultra low-oxygen tensions, but only partial complementation was observed in free living cells under higher oxygen tensions (1% or 3%). Co-purification experiments using StrepTag-labelled HupF derivatives and mass spectrometry analysis indicate the existence of a major complex involving HupL and HupF, and a less abundant HupF-HupK complex. CONCLUSIONS: The results indicate that HupF has a dual role during hydrogenase biosynthesis: it is required for hydrogenase large subunit processing and it also acts as a chaperone to stabilize HupL when hydrogenase is synthesized in the presence of oxygen.

Rhizobium leguminosarum hupE encodes a nickel transporter required for hydrogenase activity.

Synthesis of the hydrogen uptake (Hup) system in Rhizobium leguminosarum bv. viciae requires the function of an 18-gene cluster (hupSLCDEFGHIJK-hypABFCDEX). Among them, the hupE gene encodes a protein showing six transmembrane domains for which a potential role as a nickel permease has been proposed. In this paper, we further characterize the nickel transport capacity of HupE and that of the translated product of hupE2, a hydrogenase-unlinked gene identified in the R. leguminosarum genome. HupE2 is a potential membrane protein that shows 48% amino acid sequence identity with HupE. Expression of both genes in the Escherichia coli nikABCDE mutant strain HYD723 restored hydrogenase activity and nickel transport. However, nickel transport assays revealed that HupE and HupE2 displayed different levels of nickel uptake. Site-directed mutagenesis of histidine residues in HupE revealed two motifs (HX(5)DH and FHGX[AV]HGXE) that are required for HupE functionality. An R. leguminosarum double mutant, SPF22A (hupE hupE2), exhibited reduced levels of hydrogenase activity in free-living cells, and this phenotype was complemented by nickel supplementation. Low levels of symbiotic hydrogenase activity were also observed in SPF22A bacteroid cells from lentil (Lens culinaris L.) root nodules but not in pea (Pisum sativum L.) bacteroids. Moreover, heterologous expression of the R. leguminosarum hup system in bacteroid cells of Rhizobium tropici and Mesorhizobium loti displayed reduced levels of hydrogen uptake in the absence of hupE. These data support the role of R. leguminosarum HupE as a nickel permease required for hydrogen uptake under both free-living and symbiotic conditions.

Microvirga tunisiensis sp. nov., a root nodule symbiotic bacterium isolated from Lupinus micranthus and L. luteus grown in Northern Tunisia.

Three bacterial strains, LmiM8T, LmiE10 and LluTb3, isolated from nitrogen-fixing nodules of Lupinus micranthus (Lmi strains) and L. luteus (Llu strain) growing in Northern Tunisia were analysed using genetic, phenotypic and symbiotic approaches. Phylogenetic analyses based on rrs and concatenated gyrB and dnaK genes suggested that these Lupinus strains constitute a new Microvirga species with identities ranging from 95 to 83% to its closest relatives Microvirga makkahensis, M. vignae, M. zambiensis, M. ossetica, and M. lotononidis. The genome sequences of strains LmiM8T and LmiE10 exhibited pairwise Average Nucleotide Identities (ANIb) above 99.5%, significantly distant (73-89% pairwise ANIb) from other Microvirga species sequenced (M. zambiensis and M. ossetica). A phylogenetic analysis based on the symbiosis-related gene nodA placed the sequences of the new species in a divergent clade close to Mesorhizobium, Microvirga and Bradyrhizobium strains, suggesting that the M. tunisiensis strains represent a new symbiovar different from the Bradyrhizobium symbiovars defined to date. In contrast, the phylogeny derived from another symbiosis-related gene, nifH, reproduced the housekeeping genes phylogenies. The study of morphological, phenotypical and physiological features, including cellular fatty acid composition of the novel isolates demonstrated their unique profile regarding close reference Microvirga strains. Strains LmiM8T, LmiE10 and LluTb3 were able to nodulate several Lupinus spp. Based on genetic, genomic and phenotypic data presented in this study, these strains should be grouped within a new species for which the name Microvirga tunisiensis sp. nov. is proposed (type strain LmiM8T=CECT 9163T, LMG 29689T).

Novel arrangement of enhancer sequences for NifA-dependent activation of the hydrogenase gene promoter in Rhizobium leguminosarum bv. viciae.

The transcriptional activation of the NifA-dependent sigma(54) promoter of the Rhizobium leguminosarum hydrogenase structural genes hupSL (P(1)) has been studied through gel retardation analysis and detailed mutagenesis. Gel retardation analysis indicated the existence of a physical interaction between NifA and the promoter. Extensive mutagenesis followed by in vivo expression analysis showed that three sequences of 4 bases each (-170 ACAA -167, -161 ACAA -158, and -145 TTGT -142) are required for maximal stimulation of in vivo transcription of the P(1) promoter. The arrangement of these upstream activating sequences (ACAA N(5) ACAA N(12) TTGT) differs from the canonical 5'ACA N(10) TGT 3' UAS structure involved in NifA-dependent activation of nif/fix genes. Mutant promoter analysis indicated that the relative contribution of each of these sequences to P(1) promoter activity increases with its proximity to the transcription start site. Analysis of double mutants altered in two out of the three enhancer sequences suggests that each of these sequences functions in NifA-dependent activation of the P(1) promoter in an independent but cooperative mode. The similarities and differences between cis elements of hup and nif/fix promoters suggest that the structure of the P(1) promoter has adapted to activation by NifA in order to coexpress hydrogenase and nitrogenase activities in legume nodules.

Host-dependent expression of Rhizobium leguminosarum bv. viciae hydrogenase is controlled at transcriptional and post-transcriptional levels in legume nodules.

The legume host affects the expression of Rhizobium leguminosarum hydrogenase activity in root nodules. High levels of symbiotic hydrogenase activity were detected in R. leguminosarum bacteroids from different hosts, with the exception of lentil (Lens culinaris). Transcription analysis showed that the NifA-regulated R. leguminosarum hydrogenase structural gene promoter (P(1)) is poorly induced in lentil root nodules. Replacement of the P(1) promoter by the FnrN-dependent promoter of the fixN gene restored transcription of hup genes in lentil bacteroids, but not hydrogenase activity. In the P(fixN)-hupSL strain, additional copies of the hup gene cluster and nickel supplementation to lentil plants increased bacteroid hydrogenase activity. However, the level of activity in lentil still was significantly lower than in pea bacteroids, indicating that an additional factor is impairing hydrogenase expression inside lentil nodules. Immunological analysis revealed that lentil bacteroids contain reduced levels of both hydrogenase structural subunit HupL and nickel-binding protein HypB. Altogether, results indicate that hydrogenase expression is affected by the legume host at the level of both transcription of hydrogenase structural genes and biosynthesis or stability of nickel-related proteins HypB and HupL, and suggest the existence of a plant-dependent mechanism that affects hydrogenase activity during the symbiosis by limiting nickel availability to the bacteroid.

Characterization of a novel MIIA domain-containing protein (MdcE) in Bradyrhizobium spp.

Several genes coding for proteins with metal ion-inducible autocleavage (MIIA) domains were identified in type III secretion system tts gene clusters from draft genomes of recently isolated Bradyrhizobium spp. MIIA domains have been first described in the effectors NopE1 and NopE2 of Bradyrhizobium diazoefficiens USDA 110. All identified genes are preceded by tts box promoter motifs. The identified proteins contain one or two MIIA domains. A phylogenetic analysis of 35 MIIA domain sequences from 16 Bradyrhizobium strains revealed four groups. The protein from Bradyrhizobium sp. LmjC strain contains a single MIIA domain and was designated MdcE (MdcELmjC). It was expressed as a fusion to maltose-binding protein (MalE) in Escherichia coli and subsequently purified by affinity chromatography. Recombinant MalE-MdcELmjC-Strep protein exhibited autocleavage in the presence of Ca2+, Cu2+, Cd2+ and Mn2+, but not in the presence of Mg2+, Ni2+ or Co2+. Site-directed mutagenesis at the predicted cleavage site abolished autocleavage activity of MdcELmjC. An LmjC mdcE- mutant was impaired in the ability to nodulate Lupinus angustifolius and Macroptilium atropurpureum.

Definition of two new symbiovars, sv. lupini and sv. mediterranense, within the genera Bradyrhizobium and Phyllobacterium efficiently nodulating Lupinus micranthus in Tunisia.

In this study, a polyphasic approach was used to analyze three representative strains (LmiH4, LmiM2 and LmiT21) from a collection of six previously described strains isolated in Tunisia from root nodules of Lupinus micranthus. The phylogenetic analysis of the concatenated rrs, recA and glnII genes showed that strain LmiH4 had 100% concatenated gene sequence identity with the type strain Bradyrhizobium retamae Ro19T. Similarly, strain LmiM2 shared 100% concatenated gene sequence identity with the species Bradyrhizobium valentinum LmjM3T. However, strain LmiT21 showed an identical concatenated gene sequence with reference strain Phyllobacterium sophorae CCBAU03422T. The recA-glnII concatenated protein-coding genes used produced incongruent phylogenies compared with 16S rDNA phylogeny. The nodC gene analysis showed that the strains were phylogenetically divergent to the Bradyrhizobium symbiovars defined to date, and represented two new symbiovars. Plant infection analysis revealed that the three strains showed moderate host range and symbiotic specificities. Based on their symbiotic characteristics, we propose that the three strains isolated from Lupinus micranthus nodules belong to two new symbiovars, with the first denominated lupini within the two species Bradyrhizobium valentinum (type strain LmiM2) and B. retamae (type strain LmiH4), and the second denominated mediterranense within the species P. sophorae (type strain LmiT21).

Bradyrhizobium algeriense sp. nov., a novel species isolated from effective nodules of Retama sphaerocarpa from Northeastern Algeria.

We have characterized genetic, phenotypic and symbiotic properties of bacterial strains previously isolated from nitrogen-fixing nodules of Retama sphaerocarpa from Northern Algeria. Phylogenetic analyses of 16S rRNA genes and three concatenated housekeeping genes, recA, atpD and glnII, placed them in a new divergent group that is proposed to form a new Bradyrhizobium species, Bradyrhizobium algeriense sp. nov. (type strain RST89T, LMG 27618 and CECT 8363). Based on these phylogenetic markers and on genomic identity data derived from draft genomic sequences, Bradyrhizobium valentinum LmjM3T, Bradyrhizobium lablabi CCBAU 23086T, Bradyrhizobium retamae Ro19T, and Bradyrhizobium jicamae PAC68T are the closest relatives of B. algeriense RST89T, with sequence identities of 92-94% and Average Nucleotide Identities (ANIm) under 90%, well below the 95-96% species circumscription threshold. Likewise, a comparison of whole-cell proteomic patterns, estimated by Matrix-Assisted Laser Desorption/Ionization-Time-of-Flight (MALDI-TOF) mass spectrometric analysis, yielded almost identical spectra between B. algeriense strains but significant differences with B. valentinum, Bradyrhizobium paxllaeri, Bradyrhizobium icense, B. lablabi, B. jicamae and B. retamae. A phylogenetic tree based on symbiotic gene nodC revealed that the B. algeriense sequences cluster with sequences from the Bradyrhizobium symbiovar retamae, previously defined with B. retamae strains isolated from Retama monosperma. B. algeriense strains were able to establish effective symbioses with Retama raetam, Lupinus micranthus, Lupinus albus and Genista numidica, but not with Lupinus angustifolius or Glycine max.

Genomic Diversity in the Endosymbiotic Bacterium Rhizobium leguminosarum.

Rhizobium leguminosarum bv. viciae is a soil α-proteobacterium that establishes a diazotrophic symbiosis with different legumes of the Fabeae tribe. The number of genome sequences from rhizobial strains available in public databases is constantly increasing, although complete, fully annotated genome structures from rhizobial genomes are scarce. In this work, we report and analyse the complete genome of R. leguminosarum bv. viciae UPM791. Whole genome sequencing can provide new insights into the genetic features contributing to symbiotically relevant processes such as bacterial adaptation to the rhizosphere, mechanisms for efficient competition with other bacteria, and the ability to establish a complex signalling dialogue with legumes, to enter the root without triggering plant defenses, and, ultimately, to fix nitrogen within the host. Comparison of the complete genome sequences of two strains of R. leguminosarum bv. viciae, 3841 and UPM791, highlights the existence of different symbiotic plasmids and a common core chromosome. Specific genomic traits, such as plasmid content or a distinctive regulation, define differential physiological capabilities of these endosymbionts. Among them, strain UPM791 presents unique adaptations for recycling the hydrogen generated in the nitrogen fixation process.

Members of Microvirga and Bradyrhizobium genera are native endosymbiotic bacteria nodulating Lupinus luteus in Northern Tunisian soils.

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.

Genome sequence of Bradyrhizobium sp. LMTR 3, a diazotrophic symbiont of Lima bean (Phaseolus lunatus).

Bradyrhizobium sp. LMTR 3 is a representative strain of one of the geno(species) of diazotrophic symbionts associated with Lima bean (Phaseolus lunatus) in Peru. Its 7.83 Mb genome was sequenced using the Illumina technology and found to encode a complete set of genes required for nodulation and nitrogen fixation, and additional genes putatively involved in root colonization. Its draft genome sequence and annotation have been deposited at GenBank under the accession number MAXC00000000.

Draft genome sequence of Bradyrhizobium paxllaeri LMTR 21T isolated from Lima bean (Phaseolus lunatus) in Peru.

Bradyrhizobium paxllaeri is a prevalent species in root nodules of the Lima bean (Phaseolus lunatus) in Peru. LMTR 21T is the type strain of the species and was isolated from a root nodule collected in an agricultural field in the Peruvian central coast. Its 8.29 Mbp genome encoded 7635 CDS, 71 tRNAs and 3 rRNAs genes. All genes required to stablish a nitrogen-fixing symbiosis with its host were present. The draft genome sequence and annotation have been deposited at GenBank under the accession number MAXB00000000.

Proteomic analysis of quorum sensing in Rhizobium leguminosarum biovar viciae UPM791.

Production of quorum-sensing signal molecules of the acyl-homoserine lactone (AHL) type by Rhizobium leguminosarum bv. viciae UPM791 is dependent on its plasmid content. Curing of two of its four native plasmids, pUPM791d and pSym, resulted in loss of production of the largest (C(14)) and the three smaller (C(6)-C(8)) AHLs, respectively. Introduction of a lactonase-containing plasmid resulted in AHL signal degradation and quorum quenching. The quorum-dependent proteome was studied in these strains by DIGE. Quorum quenching affected a small (1.7%) fraction of the detected spots in the wild-type and a smaller (0.6%) fraction in the pSym-cured strain. Unexpectedly, quorum quenching affected up to 3.3% of the detected spots in the pUPM791d-cured strain, suggesting that C(14)-AHL normally interferes with the quorum response mediated by other AHLs. This, together with the observation that ca. 50% of the quorum-regulated proteins in strain UPM791 showed AHL-mediated repression, suggests that an important part of their functionality can be exerted through repression, although AHLs are usually considered as gene expression inducers. The three main quorum-induced polypeptides were identified by MALDI-MS as charge isoforms of the rhizospheric RhiA protein. Another major quorum-induced polypeptide was only present in the pUPM791d-cured strain and could not be identified.