PoolSeq analysis of the selection of the Rhizobium genotypes by the legume host plant

Rhizobium leguminosarum establishes highly specific nitrogen-fixing symbioses. We have applied a Pool-Seq approach to study plant host selection of genotypes. Our results confirm, at the genomic level, previous observations regarding plant selection of specific genotypes

Genomics of host specificity in the Rhizobium-legume simbiosis

Most Rhizobium leguminosarum bv. viciae isolates are able to specifically nodulate plants of any of four different legume genera: Pisum, Lens, Vicia, and Lathyrus. However, previous evidence suggests that some genotypes are more adapted to a given plant host than others, and that the plant host can select specific genotypes among those present in a given soil population. We have used a population genomics approach to confirm that this is indeed the case, and to analyze the specific genotypic characteristics that each plant host selects

Population genomics of host specificity in Rhizobium leguminosarum bv. viciae

Legumes establish a root-nodule symbiosis with soil bacteria collectively known as rhizobia. This symbiosis allows legumes to benefit from the nitrogen fixation capabilities of rhizobia and thus to grow in the absence of any fixed nitrogen source. This is especially relevant for Agriculture, where intensive plant growth depletes soils of useable, fixed nitrogen sources. One of the main features of the root nodule symbiosis is its specificity. Different rhizobia are able to nodulate different legumes. Rhizobium leguminosarum bv. viciae is able to establish an effective symbiosis with four different plant genera (Pisum, Lens, Vicia, Lathyrus), and any given isolate will nodulate any of the four plant genera. A population genomics study with rhizobia isolated from P. sativum, L. culinaris, V. sativa or V. faba, all originating in the same soil, showed that plants select specific genotypes from those available in that soil. This was demonstrated at the genome-wide level, but also for specific genes. Accelerated mesocosm studies with successive plant cultures provided additional evidence on this plant selection and on the nature of the genotypes selected. Finally, representatives from the major rhizobial genotypes isolated from these plants allowed characterization of the size and nature of the respective pangenome and specific genome compartments. These were compared to the different genotypes ?symbiotic and non-symbiotic?present in rhizobial populations isolated directly from the soil without plant intervention.

The Pangenome of the Plant-Selected Rhizobium leguminosarum bv viciae Sub-popuations

Rhizobium leguminosarum bv viciae (Rlv) is a bacterium able to establish effective symbioses with four different legume genera: Pisum, Lens, Lathyrus and Vicia. Classic studies using trap plants have previously shown that, given a choice, different plants prefer specific genotypes of rhizobia, which are adapted to the host (1, 2). In previous work we have performed a Pool-Seq analysis bases on pooled DNA samples from Rlv nodule isolates obtained from Pisum sativum, Lens culinaris, Vicia fava and V. sativa plants, used as rhizobial traps. This experiment allowed us to test the host preference hypothesis: different plant hosts select specific sub-populations of rhizobia from the available population present in a given soil. We have observed that plant-selected sub-populations are different at the single nucleotide polymorphism (SNP) level. We have selected individual isolates from each sub-population (9 fava-bean isolates, 14 pea isolates 9 vetch isolates and 9 lentil isolates) and sequenced their genomes at draft level (ca. 30x, 90 contigs). Genomic analyses have been carried out using J-species and CMG-Biotools. All the isolates had similar genome size (7.5 Mb) and number of genes (7,300). The resulting Average Nucleotide Identity (ANIm) tree showed that Rhizobium leguminosarum bv viciae is a highly diverse group. Each plant-selected subpopulation showed a closed pangenome and core genomes of similar size (11,500 and 4,800 genes, respectively). The addition of all four sub-population results in a larger, closed pangenome of 19,040 genes and a core genome of similar size (4,392 genes). Each sub-population contains a characteristic set of genes but no universal, plant-specific genes were found. The core genome obtained from all four sub-populations is probably a representative core genome for Rhizobium leguminosarum, given that the reference genome (Rhizobium leguminosarum bv. viciae strain 3841) contains most of the core genome. We have also analyzed the symbiotic cluster (nod), and different nod cluster genotypes were found in each sub-population. Supported by MINECO (Consolider-Ingenio 2010, MICROGEN Project, CSD2009-00006).

Rhizobium leguminosarum HupE is a highly-specific diffusion facilitator for nickel uptake

Bacteria require nickel transporters for the synthesis of Ni-containing metalloenzymes in natural, low nickel habitats. In this work we carry out functional and topological characterization of Rhizobium leguminosarum HupE, a nickel permease required for the provision of this element for [NiFe] hydrogenase synthesis. Expression studies in the Escherichia coli nikABCDE mutant strain HYD723 revealed that HupE is a medium-affinity permease (apparent Km 227 ! 21 nM; Vmax 49 ! 21 pmol Ni2+ min"1 mg"1 bacterial dry weight) that functions as an energy-independent diffusion facilitator for the uptake of Ni(II) ions. This Ni2+ transport is not inhibited by similar cations such as Mn2+, Zn2+, or Co2+, but is blocked by Cu2+. Analysis of site-directed HupE mutants allowed the identification of several residues (H36, D42, H43, F69, E90, H130, and E133) that are essential for HupE-mediated Ni uptake in E. coli cells. By using translational fusions to reporter genes we demonstrated the presence of five transmembrane domains with a periplasmic N-terminal domain and a C-terminal domain buried in the lipid bilayer. The periplasmic N-terminal domain contributes to stability and functionality of the protein

Expression of early nodulin genes in alfalfa mycorrhizae indicates that signal transduction pathways used in forming arbuscular mycorrhizae and Rhizobium-induced nodules may be conserved

Transcripts for two genes expressed early in alfalfa nodule development (MsENOD40 and MsENOD2) are found in mycorrhizal roots, but not in noncolonized roots or in roots infected with the fungal pathogen Rhizoctonia solani. These same two early nodulin genes are expressed in uninoculated roots upon application of the cytokinin 6-benzylaminopurine. Correlated with the expression of the two early nodulin genes, we found that mycorrhizal roots contain higher levels of trans-zeatin riboside than nonmycorrhizal roots. These data suggest that there may be conservation of signal transduction pathways between the two symbioses—nitrogen-fixing nodules and phosphate-acquiring mycorrhizae.

Rhizobium gone native: Unexpected plasmid stability of indigenous Rhizobium leguminosarum

Lateral transfer of bacterial plasmids is thought to play an important role in microbial evolution and population dynamics. However, this assumption is based primarily on investigations of medically or agriculturally important bacterial species. To explore the role of lateral transfer in the evolution of bacterial systems not under intensive, human-mediated selection, we examined the association of genotypes at plasmid-encoded and chromosomal loci of native Rhizobium, the nitrogen-fixing symbiont of legumes. To this end, Rhizobium leguminosarum strains nodulating sympatric species of native Trifolium were characterized genetically at plasmid-encoded symbiotic (sym) regions (nodulation AB and nodulation CIJT loci) and a repeated chromosomal locus not involved in the symbiosis with legumes. Restriction fragment length polymorphism analysis was used to distinguish genetic groups at plasmid and chromosomal loci. The correlation between major sym and chromosomal genotypes and the distribution of genotypes across host plant species and sampling location were determined using χ2 analysis. In contrast to findings of previous studies, a strict association existed between major sym plasmid and chromosomal genetic groups, suggesting a lack of successful sym plasmid transfer between major Rhizobium chromosomal types. These data indicate that previous observations of sym plasmid transfer in agricultural settings may seriously overestimate the rates of successful conjugation in systems not impacted by human activities. In addition, a nonrandom distribution of Rhizobium genotypes across host plant species and sampling site demonstrates the importance of both factors in shaping Rhizobium population dynamics.

Biosynthetic control of molecular weight in the polymerization of the octasaccharide subunits of succinoglycan, a symbiotically important exopolysaccharide of Rhizobium meliloti

Succinoglycan, a symbiotically important exopolysaccharide of Rhizobium meliloti, is composed of polymerized octasaccharide subunits, each of which consists of one galactose and seven glucoses with succinyl, acetyl, and pyruvyl modifications. Production of specific low molecular weight forms of R. meliloti exported and surface polysaccharides, including succinoglycan, appears to be important for nodule invasion. In a previous study of the roles of the various exo gene products in succinoglycan biosynthesis, exoP, exoQ, and exoT mutants were found to synthesize undecaprenol-linked fully modified succinoglycan octasaccharide subunits, suggesting possible roles for their gene products in polymerization or transport. Using improved techniques for analyzing succinoglycan biosynthesis by these mutants, we have obtained evidence indicating that R. meliloti has genetically separable systems for the synthesis of high molecular weight succinoglycan and the synthesis of a specific class of low molecular weight oligosaccharides consisting of dimers and trimers of the octasaccharide subunit. Models to account for our unexpected findings are discussed. Possible roles for the ExoP, ExoQ, and ExoT proteins are compared and contrasted with roles that have been suggested on the basis of homologies to key proteins involved in the biosynthesis of O-antigens and of certain exported or capsular cell surface polysaccharides.

The common nodABC genes of Rhizobium meliloti are host-range determinants

Symbiotic bacteria of the genus Rhizobium synthesize lipo-chitooligosaccharides, called Nod factors (NFs), which act as morphogenic signal molecules on legume hosts. The common nodABC genes, present in all Rhizobium species, are required for the synthesis of the core structure of NFs. NodC is an N-acetylglucosaminyltransferase, and NodB is a chitooligosaccharide deacetylase; NodA is involved in N-acylation of the aminosugar backbone. Specific nod genes are involved in diverse NF substitutions that confer plant specificity. We transferred to R. tropici, a broad host-range tropical symbiont, the ability to nodulate alfalfa, by introducing nod genes of R. meliloti. In addition to the specific nodL and nodFE genes, the common nodABC genes of R. meliloti were required for infection and nodulation of alfalfa. Purified NFs of the R. tropici hybrid strain, which contained chitin tetramers and were partly N-acylated with unsaturated C16 fatty acids, were able to elicit nodule formation on alfalfa. Inactivation of the R. meliloti nodABC genes suppressed the ability of the NFs to nodulate alfalfa. Studies of NFs from nodA, nodB, nodC, and nodI mutants indicate that (i) NodA of R. meliloti, in contrast to NodA of R. tropici, is able to transfer unsaturated C16 fatty acids onto the chitin backbone and (ii) NodC of R. meliloti specifies the synthesis of chitin tetramers. These results show that allelic variation of the common nodABC genes is a genetic mechanism that plays an important role in signaling variation and in the control of host range.

Symbiosis-specific expression of Rhizobium etli casA encoding a secreted calmodulin-related protein

Symbiosis between Rhizobium and its leguminous host requires elaborate communication between the partners throughout the interaction process. A calmodulin-like protein, termed calsymin, was identified in Rhizobium etli; a calmodulin-related protein in a Gram-negative bacterium had not been described previously. Calsymin possesses three repeated homologous domains. Each domain contains two predicted EF-hand Ca2+-binding motifs. Ca2+-binding activity of calsymin was demonstrated on purified protein. R. etli efficiently secretes calsymin without N-terminal cleavage of the protein. The gene encoding calsymin, casA, is exclusively expressed during colonization and infection of R. etli with the host. Expression of casA is controlled by a repressor protein, termed CasR, belonging to the TetR family of regulatory proteins. Mutation of the casA gene affects the development of bacteroids during symbiosis and symbiotic nitrogen fixation.

Rearrangement of Actin Microfilaments in Plant Root Hairs Responding to Rhizobium etli Nodulation Signals1

The response of the actin cytoskeleton to nodulation (Nod) factors secreted by Rhizobium etli has been studied in living root hairs of bean (Phaseolus vulgaris) that were microinjected with fluorescein isothiocyanate-phalloidin. In untreated control cells or cells treated with the inactive chitin oligomer, the actin cytoskeleton was organized into long bundles that were oriented parallel to the long axis of the root hair and extended into the apical zone. Upon exposure to R. etli Nod factors, the filamentous actin became fragmented, as indicated by the appearance of prominent masses of diffuse fluorescence in the apical region of the root hair. These changes in the actin cytoskeleton were rapid, observed as soon as 5 to 10 min after application of the Nod factors. It was interesting that the filamentous actin partially recovered in the continued presence of the Nod factor: by 1 h, long bundles had reformed. However, these cells still contained a significant amount of diffuse fluorescence in the apical zone and in the nuclear area, presumably indicating the presence of short actin filaments. These results indicate that Nod factors alter the organization of actin microfilaments in root hair cells, and this could be a prelude for the formation of infection threads.

Respiratory Elicitors from Rhizobium meliloti Affect Intact Alfalfa Roots1

Molecules produced by Rhizobium meliloti increase respiration of alfalfa (Medicago sativa L.) roots. Maximum respiratory increases, measured either as CO2 evolution or as O2 uptake, were elicited in roots of 3-d-old seedlings by 16 h of exposure to living or dead R. meliloti cells at densities of 107 bacteria/mL. Excising roots after exposure to bacteria and separating them into root-tip- and root-hair-containing segments showed that respiratory increases occurred only in the root-hair region. In such assays, CO2 production by segments with root hairs increased by as much as 100% in the presence of bacteria. Two partially purified compounds from R. meliloti 1021 increased root respiration at very low, possibly picomolar, concentrations. One factor, peak B, resembled known pathogenic elicitors because it produced a rapid (15-min), transitory increase in respiration. A second factor, peak D, was quite different because root respiration increased slowly for 8 h and was maintained at the higher level. These molecules differ from lipo-chitin oligosaccharides active in root nodulation for the following reasons: (a) they do not curl alfalfa root hairs, (b) they are synthesized by bacteria in the absence of known plant inducer molecules, and (c) they are produced by a mutant R. meliloti that does not synthesize known lipo-chitin oligosaccharides. The peak-D compound(s) may benefit both symbionts by increasing CO2, which is required for growth of R. meliloti, and possibly by increasing the energy that is available in the plant to form root nodules.

Low molecular weight EPS II of Rhizobium meliloti allows nodule invasion in Medicago sativa.

Effective invasion of alfalfa by Rhizobium meliloti Rm1021 normally requires the presence of succinoglycan, an exopolysaccharide (EPS) produced by the bacterium. However, Rm1021 has the ability to produce a second EPS (EPS II) that can suppress the symbiotic defects of succinoglycan-deficient strains. EPS II is a polymer of modified glucose-(beta-1,3)-galactose subunits and is produced by Rm1021 derivatives carrying either an expR101 or mucR mutation. If the ability to synthesize succinoglycan is blocked genetically, expR101 derivatives of Rm1021 are nodulation-proficient, whereas mucR derivatives of Rm1021 are not. The difference in nodulation proficiency between these two classes of EPS II-producing strains is due to the specific production of a low molecular weight form of EPS II by expR101 strains. A low molecular weight EPS II fraction consisting of 15-20 EPS II disaccharide subunits efficiently allows nodule invasion by noninfective strains when present in amounts as low as 7 pmol per plant, suggesting that low molecular weight EPS II may act as a symbiotic signal during infection.

Protein crosslinking studies suggest that Rhizobium meliloti C4-dicarboxylic acid transport protein D, a sigma 54-dependent transcriptional activator, interacts with sigma 54 and the beta subunit of RNA polymerase.

Rhizobium meliloti C4-dicarboxylic acid transport protein D (DCTD) activates transcription by a form of RNA polymerase holoenzyme that has sigma 54 as its sigma factor (referred to as E sigma 54). DCTD catalyzes the ATP-dependent isomerization of closed complexes between E sigma 54 and the dctA promoter to transcriptionally productive open complexes. Transcriptional activation probably involves specific protein-protein interactions between DCTD and E sigma 54. Interactions between sigma 54-dependent activators and E sigma 54 are transient, and there has been no report of a biochemical assay for contact between E sigma 54 and any activator to date. Heterobifunctional crosslinking reagents were used to examine protein-protein interactions between the various subunits of E sigma 54 and DCTD. DCTD was crosslinked to Salmonella typhimurium sigma 54 with the crosslinking reagents succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate and N-hydroxysulfosuccinimidyl-4-azidobenzoate. Cys-307 of sigma 54 was identified by site-directed mutagenesis as the residue that was crosslinked to DCTD. DCTD was also crosslinked to the beta subunit of Escherichia coli core RNA polymerase with succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, but not with N-hydroxysulfosuccinimidyl-4-azidobenzoate. These data suggest that interactions of DCTD with sigma 54 and the beta subunit may be important for transcriptional activation and offer evidence for interactions between a sigma 54-dependent activator and sigma 54, as well as the beta subunit of RNA polymerase.

Nodulating strains of Rhizobium loti arise through chromosomal symbiotic gene transfer in the environment.

Rhizobia were isolated from nodules off a stand of Lotus corniculatus established with a single inoculant strain, ICMP3153, 7 years earlier in an area devoid of naturalized Rhizobium loti. The isolates showed diversity in growth rate, Spe I fingerprint of genomic DNA, and hybridization pattern to genomic DNA probes. The 19% of isolates that grew at the same rate as strain ICMP3153 were the only isolates that had the same fingerprint as strain ICMP3153. Sequencing of part of the 16S rRNA gene of several diverse isolates confirmed that they were not derived from the inoculant strain. Nevertheless, all non-ICMP3153 strains gave EcoRI and Spe I hybridization patterns identical to ICMP3153 when hybridized to nodulation gene cosmids. Hybridization of digests generated by the very rare cutting enzyme Swa I revealed that the symbiotic DNA region (at least 105 kb) was chromosomally integrated in the strains. The results suggest that the diverse strains arose by transfer of chromosomal symbiotic genes from ICMP3153 to nonsymbiotic rhizobia in the environment.