Identification, phylogenetic analysis, and genome mining of the tetracycline-resistant Bacillus thuringiensis strain m401 reveal its potential for biotechnological and biocontrol applications.

Bacillus thuringiensis is an entomopathogen belonging to the Bacillus cereus clade. We isolated a tetracycline-resistant strain called m401, recovered it from honey, and identified it as Bacillus thuringiensis sv. kumamotoensis based on the average nucleotide identity calculations (ANIb) comparison and the analysis of the gyrB gene sequences of different B. thuringiensis serovars. Sequences with homology to virulence factors [cytK, nheA, nheB, nheC, hblA, hblB, hblC, hblD, entFM, and inhA] and tetracycline resistance genes [tet(45), tet(V), and tet(M)/tet(W)/tet(O)/tet(S) family] were identified in the bacterial chromosome. The prediction of plasmid-coding regions revealed homolog sequences to the MarR and TetR/AcrR family of transcriptional regulators, toxins, and lantipeptides. The genome mining analysis revealed 12 regions of biosynthetic gene clusters responsible for synthesizing secondary metabolites. We identified biosynthetic gene clusters coding for bacteriocins, siderophores, ribosomally synthesized post-translationally modified peptide products, and non-ribosomal peptide synthetase clusters that provide evidence for the possible use of Bt m401 as a biocontrol agent. Furthermore, Bt m401 showed high inhibition against all Paenibacillus larvae genotypes tested in vitro. In conclusion, Bt m401 owns various genes involved in different biological processes, such as transductional regulators associated with antibiotic resistance, toxins, and antimicrobial peptides with potential biotechnological and biocontrol applications.

A Stringent-Response-Defective Bradyrhizobium diazoefficiens Strain Does Not Activate the Type 3 Secretion System, Elicits an Early Plant Defense Response, and Circumvents NH4NO3-Induced Inhibition of Nodulation.

When subjected to nutritional stress, bacteria modify their amino acid metabolism and cell division activities by means of the stringent response, which is controlled by the Rsh protein in alphaproteobacteria. An important group of alphaproteobacteria are the rhizobia, which fix atmospheric N2 in symbiosis with legume plants. Although nutritional stress is common for rhizobia while infecting legume roots, the stringent response has scarcely been studied in this group of soil bacteria. In this report, we obtained a mutant with a kanamycin resistance insertion in the rsh gene of Bradyrhizobium diazoefficiens, the N2-fixing symbiont of soybean. This mutant was defective for type 3 secretion system induction, plant defense suppression at early root infection, and nodulation competition. Furthermore, the mutant produced smaller nodules, although with normal morphology, which led to lower plant biomass production. Soybean (Glycine max) genes GmRIC1 and GmRIC2, involved in autoregulation of nodulation, were upregulated in plants inoculated with the mutant under the N-free condition. In addition, when plants were inoculated in the presence of 10 mM NH4NO3, the mutant produced nodules containing bacteroids, and GmRIC1 and GmRIC2 were downregulated. The rsh mutant released more auxin to the culture supernatant than the wild type, which might in part explain its symbiotic behavior in the presence of combined N. These results indicate that the B. diazoefficiens stringent response integrates into the plant defense suppression and regulation of nodulation circuits in soybean, perhaps mediated by the type 3 secretion system.IMPORTANCE The symbiotic N2 fixation carried out between prokaryotic rhizobia and legume plants performs a substantial contribution to the N cycle in the biosphere. This symbiotic association is initiated when rhizobia infect and penetrate the root hairs, which is followed by the growth and development of root nodules, within which the infective rhizobia are established and protected. Thus, the nodule environment allows the expression and function of the enzyme complex that catalyzes N2 fixation. However, during early infection, the rhizobia find a harsh environment while penetrating the root hairs. To cope with this nuisance, the rhizobia mount a stress response known as the stringent response. In turn, the plant regulates nodulation in response to the presence of alternative sources of combined N in the surrounding medium. Control of these processes is crucial for a successful symbiosis, and here we show how the rhizobial stringent response may modulate plant defense suppression and the networks of regulation of nodulation.

Polyphasic characterization and identification of the bioremediation agent Bacillus sp. SFC 500-1E.

Bacillus sp. SFC 500-1E is used for the effective treatment of tannery effluents since it consistently removes hexavalent chromium from diverse contaminated matrices. The aim of the present study was to complete identification of the strain through a polyphasic characterization, which included the pattern of carbohydrate utilization, fatty acids profile, multilocus sequence analysis, multiplex PCR profile and the analysis of the complete genome sequence. Morpho-physiological and biochemical characterization results and analysis of 16S rRNA sequences were not conclusive. The strain formed a monophyletic clade with B. toyonensis BCT-7112, B. thuringiensis MC28 and B. cereus Rock 1-3. However, genomic comparisons with type strains of B. cereus and B. thuringiensis showed that the isolated belonged to a different species. Results of this study highlight the relevance of the genome sequence of this strain, identified as Bacillus toyonensis SFC 500-1E, to expand knowledge of its bioremediation potential and to explore unknown decontamination activities.

Proteomic Analysis of Rhizobium favelukesii LPU83 in Response to Acid Stress.

Acid soils constitute a severe problem for leguminous crops mainly through a disturbance in rhizobium-legume interactions. Rhizobium favelukesii-an acid-tolerant rhizobium able to nodulate alfalfa-is highly competitive for nodule occupation under acid conditions but inefficient for biologic nitrogen fixation. In this work, we obtained a general description of the acid-stress response of R. favelukesii LPU83 by means of proteomics by comparing the total proteome profiles in the presence or absence of acid stress by nanoflow ultrahigh-performance liquid chromatography coupled to mass spectrometry. Thus, a total of 336 proteins were identified with a significant differential expression, 136 of which species were significantly overexpressed and 200 underexpressed in acidity. An in silico functional characterization with those respective proteins revealed a complex and pleiotropic response by these rhizobia involving components of oxidative phosphorylation, glutamate metabolism, and peptidoglycan biosynthesis, among other pathways. Furthermore, a lower permeability was evidenced in the acid-stressed cells along with several overexpressed proteins related to γ-aminobutyric acid metabolism, such as the gene product of livK, which gene was mutated. This mutant exhibited an acid-sensitive phenotype in agreement with the proteomics results. We conclude that both the γ-aminobutyric acid metabolism and a modified cellular envelope could be relevant to acid tolerance in R. favelukesii.

Conjugative transfer between Rhizobium etli endosymbionts inside the root nodule.

Since the discovery that biological nitrogen fixation ensues in nodules resulting from the interaction of rhizobia with legumes, nodules were thought to be exclusive for hosting nitrogen-fixing and plant growth promoting bacteria. In this work, we uncover a novel function of nodules, as a niche permissive to acquisition of plasmids via conjugative transfer. We used Rhizobium etli CFN42, which nodulates Phaseolus vulgaris. The genome of R. etli CFN42 contains a chromosome and six plasmids. pRet42a is a conjugative plasmid regulated by Quorum-Sensing (QS), and pRet42d is the symbiotic plasmid. Here, using confocal microscopy and flow cytometry, we show that pRet42a transfers on the root's surface, and unexpectedly, inside the nodules. Conjugation still took place inside nodules, even when it was restricted on the plant surface by placing the QS traI regulator under the promoter of the nitrogenase gene, which is only expressed inside the nodules, or by inhibiting the QS transcriptional induction of transfer genes with a traM antiactivator on an unstable vector maintained on the plant surface and lost inside the nodules. These results conclusively confirm the occurrence of conjugation in these structures, defining them as a protected environment for bacterial diversification.

Plasmid pSfr64a and the symbiotic plasmid pSfr64b of Sinorhizobium fredii GR64 control each other's conjugative transfer through quorum-sensing elements.

Rhizobia are nitrogen-fixing symbionts of plants. Their genomes frequently contain large plasmids, some of which are able to perform conjugative transfer. Plasmid pSfr64a from Sinorhizobium fredii GR64 is a conjugative plasmid, whose transfer is regulated by quorum sensing genes encoded by itself (traR64a, traI64a), in the symbiotic plasmid pSfr64b (traR64b, traI64b), and in the chromosome (ngrI). Also, transfer of pSfr64b requires quorum sensing elements encoded in this plasmid (traR64b, traI64b), in pSfr64a (traR64a), and in the chromosome (ngrI). These results demonstrate that pSfr64a and the symbiotic plasmid depend on each other for conjugative transfer. Plasmid pSfr64a from S. fredii GR64 is unable to transfer from the genomic background of Rhizobium etli CFN42. Our results show that the relaxase of pRet42a is able to process the oriT of pSfr64a, and viceversa, underlining their functional similarity and suggesting that in addition to the external signals, the "cytoplasmic environment" may pose a barrier to plasmid dissemination, even if the plasmids are functional in other aspects.

Insight into the structure, function and conjugative transfer of pLPU83a, an accessory plasmid of Rhizobium favelukesii LPU83.

Plasmids are widely distributed in rhizobia, a group of bacteria able to establish symbiotic relationships with the roots of legume plants. Two types of conjugative transfer (CT) regulation of these elements have been described in more detail. The most prevalent is through Quorum-Sensing (QS), mediated by the interaction of the TraR regulator protein and its cognate acyl-homoserine lactone (AHL) synthesized by TraI. In this study, we analyzed rhizobial plasmids classified according to their TraR regulators into four different groups. Each group has a particular genomic architecture. In one of the groups (I-C), represented by pLPU83a from Rhizobium favelukesii LPU83, CT induction requires TraR. With manual annotation, a traI was located in the plasmid distant to the traR gene. These features make pLPU83a an interesting plasmid for studying novel mechanisms of CT regulation. We mutagenized the traI gene, and found that it does not participate in CT regulation. Furthermore, we studied whether pLPU83a is subject to QS regulation by determining CT at different growth stages (cell densities). Our results showed no positive correlation between increase in culture densities and CT induction, on the contrary a slight decrease in CT was found at higher culture densities, unlike other TraR-depending plasmids. Our results show that transfer of pLPU83a is not regulated in a QS-dependent manner, and suggest that molecules not yet identified may activate its CT. Also, accumulation of a putative inhibitor cannot be disregarded.

Specificity traits consistent with legume-rhizobia coevolution displayed by Ensifer meliloti rhizosphere colonization.

Rhizobia are α- and ß-proteobacteria that associate with legumes in symbiosis to fix atmospheric nitrogen. The chemical communication between roots and rhizobia begins in the rhizosphere. Using signature-tagged-Tn5 mutagenesis (STM) we performed a genome-wide screening for Ensifer meliloti genes that participate in colonizing the rhizospheres of alfalfa and other legumes. The analysis of ca. 6,000 mutants indicated that genes relevant for rhizosphere colonization account for nearly 2% of the rhizobial genome and that most (ca. 80%) are chromosomally located, pointing to the relevance and ancestral origin of the bacterial ability to colonize plant roots. The identified genes were related to metabolic functions, transcription, signal transduction, and motility/chemotaxis among other categories; with several ORFs of yet-unknown function. Most remarkably, we identified a subset of genes that impacted more severely the colonization of the roots of alfalfa than of pea. Further analyses using other plant species revealed that such early differential phenotype could be extended to other members of the Trifoliae tribe (Trigonella, Trifolium), but not the Fabeae and Phaseoleae tribes. The results suggest that consolidation of E. meliloti into its current symbiotic state should have occurred in a rhizobacterium that had already been adapted to rhizospheres of the Trifoliae tribe.

Regulation of conjugative transfer of plasmids and integrative conjugative elements.

Horizontal gene transfer has been recognized as one of the principal contributors to bacterial evolution and diversification. One of the mechanisms involved in this process is conjugative transfer of plasmids and Integrative Conjugative Elements (ICEs). Plasmids and ICEs often encode traits beneficial for bacterial survival in specific environments, or for the establishment of symbiosis or pathogenesis, in addition to genes allowing conjugative transfer. In this review, we analyze the mechanisms that regulate the expression of conjugative transfer genes. For traits such as antibiotic or metal resistance, the compounds involved may induce conjugative transfer directly, while symbiosis and pathogenesis are modulated by quorum-sensing and/or signal molecules released by the host. However, multiple layers of regulation are usually involved in modulating transfer. In addition to the plasmid-encoded regulatory elements, conjugation seems to be regulated by what we have labeled as the "internal environment", defined by the interaction between the host chromosome and the plasmids or ICEs. Another regulatory level depends on the "external environment", which affects conjugative transfer due to the composition and conditions of the community.

Site-specific bacterial chromosome engineering mediated by IntA integrase from Rhizobium etli.

BACKGROUND: The bacterial chromosome may be used to stably maintain foreign DNA in the mega-base range. Integration into the chromosome circumvents issues such as plasmid replication, stability, incompatibility, and copy number variance. The site-specific integrase IntA from Rhizobium etli CFN42 catalyzes a direct recombination between two specific DNA sites: attA and attD (23 bp). This recombination is stable. The aim of this work was to develop a R. etli derivative that may be used as recipient for the integration of foreign DNA in the chromosome, adapting the IntA catalyzed site-specific recombination system. RESULTS: To fulfill our aim, we designed a Rhizobium etli CFN42 derivative, containing a "landing pad" (LP) integrated into the chromosome. The LP sector consists of a green fluorescent protein gene under the control of the lacZ promoter and a spectinomycin resistance gene. Between the lacZ promoter and the GFP gene we inserted an IntA attachment site, which does not affect transcription from the lac promoter. Also, a mobilizable donor vector was generated, containing an attA site and a kanamycin resistance gene; to facilitate insertion of foreign DNA, this vector also contains a multicloning site. There are no promoters flanking the multicloning site. A biparental mating protocol was used to transfer the donor vector into the landing pad strain; insertion of the donor vector into the landing pad sector via IntA-mediated attA X attA recombination thereby interrupted the expression of the green fluorescent protein, generating site-specific cointegrants. Cointegrants were easily recognized by screening for antibiotic sensitivity and lack of GFP expression, and were obtained with an efficiency of 6.18 %. CONCLUSIONS: Integration of foreign DNA in Rhizobium, lacking any similarity with the genome, can be easily achieved by IntA-mediated recombination. This protocol contains the mating and selection procedures for creating and isolating integrants.

Rhizobium favelukesii sp. nov., isolated from the root nodules of alfalfa (Medicago sativa L).

Strains LPU83T and Or191 of the genus Rhizobium were isolated from the root nodules of alfalfa, grown in acid soils from Argentina and the USA. These two strains, which shared the same plasmid pattern, lipopolysaccharide profile, insertion-sequence fingerprint, 16S rRNA gene sequence and PCR-fingerprinting pattern, were different from reference strains representing species of the genus Rhizobium with validly published names. On the basis of previously reported data and from new DNA-DNA hybridization results, phenotypic characterization and phylogenetic analyses, strains LPU83T and Or191 can be considered to be representatives of a novel species of the genus Rhizobium, for which the name Rhizobium favelukesii sp. nov. is proposed. The type strain of this species is LPU83T (=CECT 9014T=LMG 29160T), for which an improved draft-genome sequence is available.

Characterization of Rhizobium grahamii extrachromosomal replicons and their transfer among rhizobia.

BACKGROUND: Rhizobium grahamii belongs to a new phylogenetic group of rhizobia together with Rhizobium mesoamericanum and other species. R. grahamii has a broad-host-range that includes Leucaena leucocephala and Phaseolus vulgaris, although it is a poor competitor for P. vulgaris nodulation in the presence of Rhizobium etli or Rhizobium phaseoli strains. This work analyzed the genome sequence and transfer properties of R. grahamii plasmids. RESULTS: Genome sequence was obtained from R. grahamii CCGE502 type strain isolated from Dalea leporina in Mexico. The CCGE502 genome comprises one chromosome and two extrachromosomal replicons (ERs), pRgrCCGE502a and pRgrCCGE502b. Additionally, a plasmid integrated in the CCGE502 chromosome was found. The genomic comparison of ERs from this group showed that gene content is more variable than average nucleotide identity (ANI). Well conserved nod and nif genes were found in R. grahamii and R. mesoamericanum with some differences. R. phaseoli Ch24-10 genes expressed in bacterial cells in roots were found to be conserved in pRgrCCGE502b. Regarding conjugative transfer we were unable to transfer the R. grahamii CCGE502 symbiotic plasmid and its megaplasmid to other rhizobial hosts but we could transfer the symbiotic plasmid to Agrobacterium tumefaciens with transfer dependent on homoserine lactones. CONCLUSION: Variable degrees of nucleotide identity and gene content conservation were found among the different R. grahamii CCGE502 replicons in comparison to R. mesoamericanum genomes. The extrachromosomal replicons from R. grahamii were more similar to those found in phylogenetically related Rhizobium species. However, limited similarities of R. grahamii CCGE502 symbiotic plasmid and megaplasmid were observed in other more distant Rhizobium species. The set of conserved genes in R. grahamii comprises some of those that are highly expressed in R. phaseoli on plant roots, suggesting that they play an important role in root colonization.

Characterization of an accessory plasmid of Sinorhizobium meliloti and its two replication-modules.

Rhizobia are Gram-negative bacteria known for their ability to fix atmospheric N2 in symbiosis with leguminous plants. Current evidence shows that rhizobia carry in most cases a variable number of plasmids, containing genes necessary for symbiosis or free-living, a common feature being the presence of several plasmid replicons within the same strain. For many years, we have been studying the mobilization properties of pSmeLPU88b from the strain Sinorhizobium meliloti LPU88, an isolate from Argentina. To advance in the characterization of pSmeLPU88b plasmid, the full sequence was obtained. pSmeLPU88b is 35.9 kb in size, had an average GC % of 58.6 and 31 CDS. Two replication modules were identified in silico: one belonging to the repABC type, and the other to the repC. The replication modules presented high DNA identity to the replication modules from plasmid pMBA9a present in an S. meliloti isolate from Canada. In addition, three CDS presenting identity with recombinases and with toxin-antitoxin systems were found downstream of the repABC system. It is noteworthy that these CDS present the same genetic structure in pSmeLPU88b and in other rhizobial plasmids. Moreover, in all cases they are found downstream of the repABC operon. By cloning each replication system in suicide plasmids, we demonstrated that each of them can support plasmid replication in the S. meliloti genetic background, but with different stability behavior. Interestingly, while incompatibility analysis of the cloned rep systems results in the loss of the parental module, both obtained plasmids can coexist together.

Unraveling the genome of Bacillus velezensis MEP218, a strain producing fengycin homologs with broad antibacterial activity: comprehensive comparative genome analysis.

Bacillus sp. MEP218, a soil bacterium with high potential as a source of bioactive molecules, produces mostly C16-C17 fengycin and other cyclic lipopeptides (CLP) when growing under previously optimized culture conditions. This work addressed the elucidation of the genome sequence of MEP218 and its taxonomic classification. The genome comprises 3,944,892 bp, with a total of 3474 coding sequences and a G + C content of 46.59%. Our phylogenetic analysis to determine the taxonomic position demonstrated that the assignment of the MEP218 strain to Bacillus velezensis species provides insights into its evolutionary context and potential functional attributes. The in silico genome analysis revealed eleven gene clusters involved in the synthesis of secondary metabolites, including non-ribosomal CLP (fengycins and surfactin), polyketides, terpenes, and bacteriocins. Furthermore, genes encoding phytase, involved in the release of phytic phosphate for plant and animal nutrition, or other enzymes such as cellulase, xylanase, and alpha 1-4 glucanase were detected. In vitro antagonistic assays against Salmonella typhimurium, Acinetobacter baumanii, Escherichia coli, among others, demonstrated a broad spectrum of C16-C17 fengycin produced by MEP218. MEP218 genome sequence analysis expanded our understanding of the diversity and genetic relationships within the Bacillus genus and updated the Bacillus databases with its unique trait to produce antibacterial fengycins and its potential as a resource of biotechnologically useful enzymes.

Draft genome sequence of the bean-nodulating Sinorhizobium fredii strain GR64.

Sinorhizobium fredii GR64 is a peculiar strain that is able to effectively nodulate bean but not soybean, the common host of S. fredii. Here we present the draft genome of S. fredii GR64. This information will contribute to a better understanding of the symbiotic rhizobium-plant interaction and of rhizobial evolution.

Genome sequence of Rhizobium grahamii CCGE502, a broad-host-range symbiont with low nodulation competitiveness in Phaseolus vulgaris.

Here we present the genome sequence of Rhizobium grahamii CCGE502. R. grahamii groups with other newly described broad-host-range species, which are not very efficient Phaseolus vulgaris symbionts, with a wide geographic distribution and which constitutes a novel Rhizobium clade.

ubiF is involved in acid stress tolerance and symbiotic competitiveness in Rhizobium favelukesii LPU83.

The acidity of soils significantly reduces the productivity of legumes mainly because of the detrimental effects of hydrogen ions on the legume plants, leading to the establishment of an inefficient symbiosis and poor biological nitrogen fixation. We recently reported the analysis of the fully sequenced genome of Rhizobium favelukesii LPU83, an alfalfa-nodulating rhizobium with a remarkable ability to grow, nodulate and compete in acidic conditions. To gain more insight into the genetic mechanisms leading to acid tolerance in R. favelukesii LPU83, we constructed a transposon mutant library and screened for mutants displaying a more acid-sensitive phenotype than the parental strain. We identified mutant Tn833 carrying a single-transposon insertion within LPU83_2531, an uncharacterized short ORF located immediately upstream from ubiF homolog. This gene encodes a protein with an enzymatic activity involved in the biosynthesis of ubiquinone. As the transposon was inserted near the 3' end of LPU83_2531 and these genes are cotranscribed as a part of the same operon, we hypothesized that the phenotype in Tn833 is most likely due to a polar effect on ubiF transcription.We found that a mutant in ubiF was impaired to grow at low pH and other abiotic stresses including 5 mM ascorbate and 0.500 mM Zn2+. Although the ubiF mutant retained the ability to nodulate alfalfa and Phaseolus vulgaris, it was unable to compete with the R. favelukesii LPU83 wild-type strain for nodulation in Medicago sativa and P. vulgaris, suggesting that ubiF is important for competitiveness. Here, we report for the first time an ubiF homolog being essential for nodulation competitiveness and tolerance to specific stresses in rhizobia.

RcgA and RcgR, Two Novel Proteins Involved in the Conjugative Transfer of Rhizobial Plasmids.

Rhizobia are Gram-negative bacteria that are able to establish a nitrogen-fixing symbiotic interaction with leguminous plants. Rhizobia genomes usually harbor several plasmids which can be transferred to other organisms by conjugation. Two main mechanisms of the regulation of rhizobial plasmid transfer have been described: quorum sensing (QS) and the rctA/rctB system. Nevertheless, new genes and molecules that modulate conjugative transfer have recently been described, demonstrating that new actors can tightly regulate the process. In this work, by means of bioinformatics tools and molecular biology approaches, two hypothetical genes are identified as playing key roles in conjugative transfer. These genes are located between conjugative genes of plasmid pRfaLPU83a from Rhizobium favelukesii LPU83, a plasmid that shows a conjugative transfer behavior depending on the genomic background. One of the two mentioned genes, rcgA, is essential for conjugation, while the other, rcgR, acts as an inhibitor of the process. In addition to introducing this new regulatory system, we show evidence of the functions of these genes in different genomic backgrounds and confirm that homologous proteins from non-closely related organisms have the same functions. These findings set up the basis for a new regulatory circuit of the conjugative transfer of plasmids. IMPORTANCE Extrachromosomal DNA elements, such as plasmids, allow for the adaptation of bacteria to new environments by conferring new determinants. Via conjugation, plasmids can be transferred between members of the same bacterial species, different species, or even to organisms belonging to a different kingdom. Knowledge about the regulatory systems of plasmid conjugative transfer is key in understanding the dynamics of their dissemination in the environment. As the increasing availability of genomes raises the number of predicted proteins with unknown functions, deeper experimental procedures help to elucidate the roles of these determinants. In this work, two uncharacterized proteins that constitute a new regulatory circuit with a key role in the conjugative transfer of rhizobial plasmids were discovered.

The nodulation of alfalfa by the acid-tolerant Rhizobium sp. strain LPU83 does not require sulfated forms of lipochitooligosaccharide nodulation signals.

The induction of root nodules by the majority of rhizobia has a strict requirement for the secretion of symbiosis-specific lipochitooligosaccharides (nodulation factors [NFs]). The nature of the chemical substitution on the NFs depends on the particular rhizobium and contributes to the host specificity imparted by the NFs. We present here a description of the genetic organization of the nod gene cluster and the characterization of the chemical structure of the NFs associated with the broad-host-range Rhizobium sp. strain LPU83, a bacterium capable of nodulating at least alfalfa, bean, and Leucena leucocephala. The nod gene cluster was located on the plasmid pLPU83b. The organization of the cluster showed synteny with those of the alfalfa-nodulating rhizobia, Sinorhizobium meliloti and Sinorhizobium medicae. Interestingly, the strongest sequence similarity observed was between the partial nod sequences of Rhizobium mongolense USDA 1844 and the corresponding LPU83 nod genes sequences. The phylogenetic analysis of the intergenic region nodEG positions strain LPU83 and the type strain R. mongolense 1844 in the same branch, which indicates that Rhizobium sp. strain LPU83 might represent an early alfalfa-nodulating genotype. The NF chemical structures obtained for the wild-type strain consist of a trimeric, tetrameric, and pentameric chitin backbone that shares some substitutions with both alfalfa- and bean-nodulating rhizobia. Remarkably, while in strain LPU83 most of the NFs were sulfated in their reducing terminal residue, none of the NFs isolated from the nodH mutant LPU83-H were sulfated. The evidence obtained supports the notion that the sulfate decoration of NFs in LPU83 is not necessary for alfalfa nodulation.

Exopolysaccharide Characterization of Rhizobium favelukesii LPU83 and Its Role in the Symbiosis With Alfalfa.

One of the greatest inputs of available nitrogen into the biosphere occurs through the biological N2-fixation to ammonium as result of the symbiosis between rhizobia and leguminous plants. These interactions allow increased crop yields on nitrogen-poor soils. Exopolysaccharides (EPS) are key components for the establishment of an effective symbiosis between alfalfa and Ensifer meliloti, as bacteria that lack EPS are unable to infect the host plants. Rhizobium favelukesii LPU83 is an acid-tolerant rhizobia strain capable of nodulating alfalfa but inefficient to fix nitrogen. Aiming to identify the molecular determinants that allow R. favelukesii to infect plants, we studied its EPS biosynthesis. LPU83 produces an EPS I identical to the one present in E. meliloti, but the organization of the genes involved in its synthesis is different. The main gene cluster needed for the synthesis of EPS I in E. meliloti, is split into three different sections in R. favelukesii, which probably arose by a recent event of horizontal gene transfer. A R. favelukesii strain devoided of all the genes needed for the synthesis of EPS I is still able to infect and nodulate alfalfa, suggesting that attention should be directed to other molecules involved in the development of the symbiosis.