Genetic Diversity among Rhizobium leguminosarum bv. Trifolii Strains Revealed by Allozyme and Restriction Fragment Length Polymorphism Analyses

Allozyme electrophoresis and restriction fragment length polymorphism (RFLP) analyses were used to examine the genetic diversity of a collection of 18 Rhizobium leguminosarum bv. trifolii, 1 R. leguminosarum bv. viciae, and 2 R. meliloti strains. Allozyme analysis at 28 loci revealed 16 electrophoretic types. The mean genetic distance between electrophoretic types of R. leguminosarum and R. meliloti was 0.83. Within R. leguminosarum, the single strain of bv. viciae differed at an average of 0.65 from strains of bv. trifolii, while electrophoretic types of bv. trifolii differed at a range of 0.23 to 0.62. Analysis of RFLPs around two chromosomal DNA probes also delineated 16 unique RFLP patterns and yielded genetic diversity similar to that revealed by the allozyme data. Analysis of RFLPs around three Sym (symbiotic) plasmid-derived probes demonstrated that the Sym plasmids reflect genetic divergence similar to that of their bacterial hosts. The large genetic distances between many strains precluded reliable estimates of their genetic relationships.     

Analysis of genetic structure in soil populations of Rhizobium leguminosarum recovered from the USA and the UK

Although many studies have shown that animal-associated bacterial species exhibit linkage disequilibrium at chromosomal loci, recent studies indicate that both animal-associated and soil-borne bacterial species can display a nonclonal genetic structure in which alleles at chromosomal loci are in linkage equilibrium. To examine the situation in soil-borne species further, we compared genetic structure in two soil populations of Rhizobium leguminosarum bv. trifolii and two populations of R. leguminosarum bv. viciae from two sites in Oregon, with genetic structure in R. leguminosarum bv. viciae populations recovered from peas grown at a site in Washington, USA, and at a site in Norfolk, UK. A total of 234 chromosomal types (ET) were identified among 682 strains analysed for allelic variation at 13 enzyme-encoding chromosomal loci by multilocus enzyme electrophoresis (MLEE). Chi-square tests for heterogeneity of allele frequencies showed that the populations were not genetically uniform. A comparison of the genetic diversity within combined and individual populations confirmed that the Washington population was the primary cause of genetic differentiation between the populations. Each individual population exhibited linkage disequilibrium, with the magnitude of the disequilibrium being greatest in the Washington population and least in the UK population of R. leguminosarum bv. viciae. Linkage disequilibrium in the UK population was created between two clusters of 9 and 23 ETs, which, individually, were in linkage equilibrium. Strong linkage disequilibrium between the two major clusters of 8 and 12 ETs in the Washington population was caused by the low genetic diversity of the ETs within each cluster relative to the inter-cluster genetic distance. Because neither the magnitude of genetic diversity nor of linkage disequilibrium increased as hierarchical combinations of the six local populations were analysed, we conclude that the populations have not been isolated from each other for sufficient time, nor have they been exposed to enough selective pressure to develop unique multilocus genetic structure.     

Arctic and subarctic soil populations of Rhizobium leguminosarum biovar trifolii nodulating three different clover species: Characterisation by diversity at chromosomal and symbiosis loci

Indigenous soil populations of Rhizobium leguminosarum biovar trifolii from Arctic and subarctic regions have been characterised with emphasis on chromosomal and symbiotic genes. Three clover species were used to trap rhizobia from soils along a latitudinal gradient from 78°N to 60°N in Norway. For the first time R. l. bv. trifolii was isolated from Svalbard at 78°N. Under the extreme conditions in the Arctic, rhizobia have survived as saprophytes and in symbiosis with clover legumes. The chromosomal diversity of the soil populations was mapped by rep-PCR. Separation of chromosomal types were strongly influenced by geographic origin. Symbiotic genes, the nodEF and nifDK IGS gene regions, were investigated by PCR-RFLP. The nifDK IGS were more conserved than the nodEF genes. Sym plasmids were widely distributed in different chromosomal types and across the latitudinal gradient.     

Genetic Structure of Rhizobium leguminosarum biovar trifolii and viciae Populations Found in Two Oregon Soils under Different Plant Communities

An investigation was carried out to determine the genetic structure in soil populations of Rhizobium leguminosarum bv. trifolii and viciae at each of two Oregon sites (A and C) that were 1 km apart. Although the soils were similar, the plant communities were quite different because grazing by domestic animals had been allowed (site A) or prevented (site C). Analysis of allelic variation at 13 enzyme-encoding loci by multilocus enzyme electrophoresis delineated 202 chromosomal types (ETs) among a total of 456 isolates representing two populations of R. leguminosarum bv. trifolii (AT and CT) and two populations of R. leguminosarum bv. viciae (AV and CV). Regardless of their site of origin or biovar affiliation, isolates of the same ET were confirmed to be more closely related to each other than to isolates of other ETs by repetitive extragenic palindromic and enterobacterial repetitive intergeneric consensus sequences and the PCR technique. Despite the wide range in densities of the Rhizobium populations (<10² to >10⁵/g of soil), their overall genetic diversities were similar (mean genetic diversity, 0.45 to 0.51), indicating that low-density populations of soil-borne bacterial species are not necessarily of little genetic diversity. Linkage disequilibrium analysis revealed significant multilocus structure (nonrandom associations of alleles) within each of the four populations. From a combination of cluster and linkage disequilibrium analyses, a total of eight distinct groups of ETs were defined in the four populations. Two groups (I and III) contributed significant numbers of ETs and isolates to each population. The two populations of R. leguminosarum bv. viciae (AV and CV) exhibited similar genetic structures despite existing at different densities, in different plant communities, and in the presence (CV) or absence (AV) of their local Vicia hosts. In contrast, total linkage disequilibrium was partitioned differently in two biovar populations occupying the same soil (AV and AT), with disequilibrium in the latter being due entirely to the presence of group V.     

Population level processes in Rhizobium leguminosarum bv. trifolii: the role of founder effects

The importance of genotype-specific selection between host and symbiont, founder effect, and clonal reproduction in Rhizobia leguminosarum biovar trifolii populations is relatively unknown. A field experiment was conducted to sample 1268 isolates of R. l. bv. trifolii from four genotypically distinct Trifolium pratense plants for allozyme variation at nine loci. Genetic and genotypic variation, population genetic substructure, and linkage disequilibrium were estimated. Of the 1268 isolates 188 genotypically distinct strains (electrophoretic types or ETs) were identified with an average of 11.04 different ETs per plant. Total genetic diversity in the plot was 0.346 and most of the variation was found within plants (= 80%). Our data suggests that genotype-specific selection between the rhizobia and the four host-plant genotypes tested does not influence local population structure, but evidence of founder effect was present. Significant linkage disequilibrium was observed and is most likely due to the clonal reproduction of R. l. bv. trifolii.     

Analysis of N-acyl homoserine-lactone quorum-sensing molecules made by different strains and biovars of Rhizobium leguminosarum containing different symbiotic plasmids

Strains of Rhizobium leguminosarum use a cell density-dependent gene regulatory system to assess their population density. This is achieved by the accumulation of N-acyl-homoserine lactones (AHLs) in the environment during growth of the bacteria and these AHLs stimulate the induction of various bacterial genes that are up-regulated in the late-exponential and stationary phases of growth. A genetically well-characterised strain of R. leguminosarum biovar viciae was found to have four genes, whose products synthesise different AHLs. We have analysed AHL production by four genetically distinct isolates of R. leguminosarum, three of bv. viciae and one of bv. phaseoli. Distinct differences were seen in the pattern of AHLs produced by the bv. viciae strains compared with bv. phaseoli and the increased levels and diversity of AHLs found in bv. viciae strains can be attributed to the rhiI gene, which is located on the symbiotic (Sym) plasmid and is up-regulated when the bacteria are grown in the rhizosphere. Additional complexity to the profile of AHLs is found to be associated with highly transmissible plasmid pRL1JI of R. leguminosarum bv. viciae, but this is not observed with some other strains, including those carrying different transmissible plasmids. In addition to AHLs produced by the products of genes on the symbiotic plasmid, there is clear evidence for the presence of other AHL production loci. Expression levels and patterns of AHLs can change markedly in different growth media. These results indicate that there is a network of quorum-sensing loci in different strains of R. leguminosarum and these loci may play a role in adapting to rhizosphere growth and plasmid transfer.     

Parallel variation in isoenzyme and nitrogen fixation markers in a Rhizobium population

Twenty isolates of Rhizobium leguminosarum bv. viceae were isolated at random from one field and examined for symbiotic plasmid fragment length polymorphisms and for isoenzyme patterns. The latter are most probably chromosome markers. With one exception both methods separated the isolates into the same 13 different groups. The largest group was represented 7 times according to isoenzymes and 8 times according to RFLP. This fixed non-random association of plasmid and chromosomal genotypes is consistent with a clonal population structure; it indicates limited exchange of plasmids under natural conditions. Seventeen isolates of 11 groups were highly effective and 2 isolates in one group almost ineffective.     

Genotypic and Phenotypic Comparisons of Chromosomal Types within an Indigenous Soil Population of Rhizobium leguminosarum bv. trifolii

The relative genetic similarities of 200 isolates of Rhizobium leguminosarum bv. trifolii recovered from an Oregon soil were determined at 13 enzyme loci by multilocus enzyme electrophoresis (MLEE). These isolates represented 13 antigenically distinct serotypes recovered from nodules formed on various clover species. The MLEE-derived levels of relatedness among isolates of R. leguminosarum bv. trifolii were found to be in good agreement with the levels of relatedness established by using repetitive (repetitive extragenic palindromic and enterobacterial repetitive intergeneric consensus) sequences and the PCR technique and with levels of relatedness from previously published DNA reassociation studies. BIOLOG substrate utilization patterns showed that isolates within an electrophoretic type (ET) were phenotypically more similar to each other than to isolates of other ETs. The soil isolates were represented by 53 ETs which could be clustered into seven groups (groups B, E, G, H1, H2, I, and J). Evidence for multilocus structure within the population was obtained, and group B was identified as the primary creator of the disequilibrium. Of 75 isolates belonging to the nodule-dominant serotype AS6 complex, 72 were found in group B. Isolates WS2-01 and WS2-02 representing nodule-dominant serotypes recovered from subclover grown at another Oregon site were also found in group B. Isolates representing the most numerous ETs in group B (ETs 2 and 3) were either suboptimally effective or completely ineffective at fixing nitrogen on six different clover species. Another four groups of isolates (groups A, C, D, and F) were identified when 32 strains of diverse origins were analyzed by MLEE and incorporated into the cluster analysis. Group A was most dissimilar in comparisons with other groups and contained strain USDA 2124 (T24), which produces trifolitoxin and has unique symbiotic characteristics.     

Construction of an Acid-Tolerant Rhizobium leguminosarum Biovar Trifolii Strain with Enhanced Capacity for Nitrogen Fixation

Strain ANU1173 is an acid-tolerant Rhizobium leguminosarum biovar trifolii strain that is able to nodulate subterranean clover plants growing in agar culture at pH 4.4 At pH 6.5, its symbiotic effectiveness in association with Trifolium subterraneum cv. Mt. Barker was 80% relative to that of strain ANU794, a Sm^r derivative of the commercial inoculant R. leguminosarum bv. trifolii TA1. Strain ANU1173 contained four indigenous megaplasmids, the smallest of these being the symbiotic (Sym) plasmid. The critical pH requirement for growth of strain ANU1173 in laboratory media was shown not to be associated with this plasmid. When the Sym plasmid of strain ANU1173(pSym-1173) was mobilized into a Nod^- strain of R. leguminosarum bv. viciae, the plasmid conferred to the transconjugant a level of symbiotic effectiveness in association with T. subterraneum that was similar to that observed with ANU1173. The symbiotic effectiveness of strain ANU1173 was improved by first curing pSym-1173 (generating strain ANU1184) and replacing it with another R. leguminosarum bv. trifolii Sym plasmid, pBR1AN. Subterranean clover plants inoculated with strain ANU1184 (pBR1AN) exhibited a 35 or 53% increase in acetylene reduction activity and a 20 or 17% increase in dry weight when grown at pH 6.5 and pH 4.4, respectively, compared with plants inoculated with strain ANU1173 and grown under the same pH conditions. It was further shown that pBR1AN was stably maintained in strain ANU1184 under free-living and symbiotic conditions. These results indicate that it is possible to construct an acid-tolerant strain of R. leguminosarum bv. trifolii with an enhanced capacity for nitrogen fixation.     

Evidence for genetic exchange and recombination of Rhizobium symbiotic plasmids in a soil population

A soil population of 16 Rhizobium leguminosarum bv. trifolii isolates was characterized by using three Sym (for symbiotic) plasmid-specific DNA hybridization probes: (i) an R. leguminosarum bv. trifolii-specific, repeated-sequence probe; (ii) a nifHDK gene probe, and (iii) a nod gene probe. A predominant Sym plasmid family was identified among the isolates. Three other unrelated Sym plasmid families were also identified. The isolates were also classified either by using a chromosomal DNA hybridization probe or by serological relatedness to 25 different R. leguminosarum bv. trifolii antisera. With either method, it was possible to group the 16 soil isolates into identical or related families. However, the correlation between the two techniques was not high. Irrespective of the means used to classify the bacterial host strain, it was possible to identify the same Sym plasmids in unrelated strains, as well as unrelated Sym plasmids in identical host strains. These data indicate that, within this soil population, there has been genetic exchange of Sym plasmids, and in one instance the hybridization pattern indicates that in vivo recombination of two different Sym plasmids may have occurred. Symbiotic effectiveness tests on red, strawberry, and subterranean clovers clearly differentiated the isolates. In general, the pattern of response was similar within groupings on the basis of Sym plasmid and chromosomal profiles but different between such groups.     

Genetic structure of the introduced and local populations of <Emphasis Type="Italic">Rhizobioum leguminosarum</Emphasis> in plant-soil systems

Comparative study of Rhizobium leguminosarum populations formed under the conditions of the Srednii Island (White Sea) demonstrated the introduced clover rhizobia (R. l. bv. trifolii) to be more variable than the aboriginal vetch/vetchling rhizobia (R. l. bv. viceae) in the chromosomal IGS locus, while being less variable in the plasmid-located symbiotic genes nodD and nifH. The analysis of these genes revealed the most pronounced differences between the clover and vetch/vetchling rhizobia populations. These differences, together with the results of ERIC-fingerprinting, indicated that the evolution of the clover rhizobia was mainly linked with the adaptation to local soil environment, and the evolution of the vetch/vetchling rhizobia, to the adaptation to various species of the host plants. High panmixia of R. leguminosarum population suggests its evolution to be based on the combinatory variability associated with the transfer of Sym-plasmids between R. l. bv. trifolii and R. l. bv. viceae, as well as with genomic rearrangements in the resulting recombinants.     

<Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> bv. <Emphasis Type="Italic">trifolii rosR</Emphasis> is required for interaction with clover,biofilm formation and adaptation to the environment

Background  

Rhizobium leguminosarum bv. trifolii is a symbiotic nitrogen-fixing bacterium that elicits nodules on roots of host plants Trifolium spp. Bacterial surface polysaccharides are crucial for establishment of a successful symbiosis with legumes that form indeterminate-type nodules, such as Trifolium, Pisum, Vicia, and Medicago spp. and aid the bacterium in withstanding osmotic and other environmental stresses. Recently, the R. leguminosarum bv. trifolii RosR regulatory protein which controls exopolysaccharide production has been identified and characterized.     

Increased Bean (Phaseolus vulgaris L.) Nodulation Competitiveness of Genetically Modified Rhizobium Strains

Rhizobium leguminosarum bv. phaseoli strain collections harbor heterogeneous groups of bacteria in which two main types of strains may be distinguished, differing both in the symbiotic plasmid and in the chromosome. We have analyzed under laboratory conditions the competitive abilities of the different types of Rhizobium strains capable of nodulating Phaseolus vulgaris L. bean. R. leguminosarum bv. phaseoli type I strains (characterized by nif gene reiterations and a narrow host range) are more competitive than type II strains (that have a broad host range), and both types are more competitive than the promiscuous rhizobia isolated from other tropical legumes able to nodulate beans. Type I strains become even more competitive by the transfer of a non-Sym, 225-kilobase plasmid from type II strain CFN299. This plasmid has been previously shown to enhance the nodulation and nitrogen fixation capabilities of Agrobacterium tumefaciens transconjugants carrying the Sym plasmid of strain CFN299. Other type I R. leguminosarum bv. phaseoli transconjugants carrying two symbiotic plasmids (type I and type II) have been constructed. These strains have a diminished competitive ability. The increase of competitiveness obtained in some transconjugants seems to be a transient property.     

Identification and cloning of nodulation genes and host specificity determinants of the broad host-range Rhizobium leguminosarum biovar phaseoli strain CIAT899

Rhizobium Ieguminosarum biovar phaseoli type II strain CIAT899 nodulates a wide range of hosts: Phaseolus vulgaris (beans), Leucaena esculenta (leucaena) and Macroptilium atropurpureum (siratro). A nodulation region from the symbiotic plasmid has been isolated and characterized. This region, which is contained in the overlapping cosmid clones pCV38 and pCV117, is able to induce nodutes in beans, leucaena and siratro roots when introduced in strains cured for the symbiotic plasmid, pSym. In addition, this cloned region extends the host range of Rhizobium meliloti and R. leguminosarum biovar (bv.) trifolii wild-type strains to nodulate beans. Analysis of constructed subclones indicates that a 6.4 kb Hin dlll fragment contains the essential genes required for nodule induction on all three hosts. Rhizobium leguminosarum bv. phaseoli type I strain CE3 nodulates only beans. However, CE3 transconjugants harbouring plasmid pCV3802 (which hybridized to a nodD heterologous probe), were capable of eliciting nodules on leucaena and siratro roots. Our results suggest that the CIAT899 DNA region hybridizing with the R. meliloti nodD detector is involved in the extension of host specificity to promote nodule formation in P. vulgaris, L. esculenta and M. atropurpureum.     

Expression by Soil Bacteria of Nodulation Genes from Rhizobium leguminosarum biovar trifolii

Gram-negative, rod-shaped bacteria from the soil of white clover-ryegrass pastures were screened for their ability to nodulate white clover (Trifolium repens) cultivar Grasslands Huia and for DNA homology with genomic DNA from Rhizobium leguminosarum biovar trifolii ICMP2668 (NZP582). Of these strains, 3.2% were able to hybridize with strain ICMP2668 and nodulate white clover and approximately 19% hybridized but were unable to nodulate. Strains which nodulated but did not hybridize with strain ICMP2668 were not detected. DNA from R. leguminosarum biovar trifolii (strain PN165) cured of its symbiotic (Sym) plasmid and a specific nod probe were used to show that the relationship observed was usually due to chromosomal homology. Plasmid pPN1, a cointegrate of the broad-host-range plasmid R68.45 and a symbiotic plasmid pRtr514a, was transferred by conjugation to representative strains of nonnodulating, gram-negative, rod-shaped soil bacteria. Transconjugants which formed nodules were obtained from 6 of 18 (33%) strains whose DNA hybridized with that of PN165 and 1 of 9 (11%) strains containing DNA which did not hybridize with that of PN165. The presence and location of R68.45 and nod genes was confirmed in transconjugants from three of the strains which formed nodules. Similarly, a pLAFR1 cosmid containing nod genes from a derivative of R. leguminosarum biovar trifolii NZP514 formed nodules when transferred to soil bacteria.     

repABC-based replication systems of Rhizobium leguminosarum bv. trifolii TA1 plasmids: incompatibility and evolutionary analyses

Soil bacteria of the genus Rhizobium possess complex genomes consisting of a chromosome and in addition, often, multiple extrachromosomal replicons, which are usually equipped with repABC genes that control their replication and partition. The replication regions of four plasmids of Rhizobium leguminosarum bv. trifolii TA1 (RtTA1) were identified and characterized. They all contained a complete set of repABC genes. The structural diversity of the rep regions of RtTA1 plasmids was demonstrated for parS and incα elements, and this was especially apparent in the case of symbiotic plasmid (pSym). Incompatibility assays with recombinant constructs containing parS or incα demonstrated that RtTA1 plasmids belong to different incompatibility groups. Horizontal acquisition was plausibly the main contributor to the origin of RtTA1 plasmids and pSym is probably the newest plasmid of this strain. Phylogenetic and incompatibility analyses of repABC regions of three closely related strains: RtTA1, R. leguminosarum bv. viciae 3841 and Rhizobium etli CFN42, provided data on coexistence of their replicons in a common genomic framework.     

Host-specific nodulation is encoded on a 14kb DNA fragment in Rhizobium trifolii

Summary The Rhizobium trifolii genes necessary for nodule induction and development have been isolated on a 14.0kb fragment of symbiotic (Sym) plasmid DNA. When cloned into a broad-host-range plasmid vector, these sequences confer a clover nodulation phenotype on a derivative of R. trifolii which has been cured of its endogenous Sym plasmid. Furthermore, these sequences encode both host specificity and nodulation functions since they confer the ability to recognize and nodulate clover plants on Agrobacterium and a fast-growing cowpea Rhizobium. This indicates that the bacterial genes essential for the initial, highly-specific interaction with plants are closely linked.     

Rhizobium pisi sv. trifolii K3.22 harboring nod genes of the Rhizobium leguminosarum sv. trifolii cluster

The taxonomic status of the Rhizobium sp. K3.22 clover nodule isolate was studied by multilocus sequence analysis (MLSA) of 16S rRNA and six housekeeping chromosomal genes, as well as by a subsequent phylogenic analysis. The results revealed full congruence with the Rhizobium pisi DSM 30132^T core genes, thus supporting the same taxonomic position for both strains. However, the K3.22 plasmid symbiosis nod genes demonstrated high sequence similarity to Rhizobium leguminosarum sv. trifolii, whereas the R. pisi DSM 30132^Tnod genes were most similar to R. leguminosarum sv. viciae. The strains differed in the host range nodulation specificity, since strain K3.22 effectively nodulated red and white clover but not vetch, in contrast to R. pisi DSM 30132^T, which effectively nodulated vetch but was not able to nodulate clover. Both strains had the ability to form nodules on pea and bean but they differed in bean cultivar specificity. The R. pisi K3.22 and DSM 30132^T strains might provide evidence for the transfer of R. leguminosarum sv. trifolii and sv. viciae symbiotic plasmids occurring in natural soil populations.     

Sequence Analysis of Hypothetical Lysine Exporter Genes of Rhizobium leguminosarum bv. trifolii from Calamine Old Waste Heaps and Their Evolutionary History

The aim of this study was to identify heavy metal detoxification system in Rhizobium leguminosarum bv. trifolii isolated from Trifolium repens inhabiting old (70–100 years) Zn–Pb waste heaps in Poland by PCR reaction with czcD1 and czcD2 primers. By sequence analysis, four different genotypes of obtained amplicons were identified among eight examined isolates. Their sequence similarity ranged 91–99 %. They indicated the highest sequence identity to the hypothetical lysine exporter gene of R. leguminosarum bv. trifolii WSM1325 (91–97 %) and 76–81 % sequence similarity to hypothetical lysine exporter genes of R. leguminosarum bv. trifolii WSM2304 and R. etli CFN42 and CIAT652. On phylogenetic tree of obtained amplicons, all four studied R. leguminosarum bv. trifolii genotypes formed common monophyletic cluster with R. leguminosarum bv. trifolii WSM1325 at 100 % bootstrap support showing that all four amplicons obtained in PCR with czcD1 and czcD2 primers are fragments of hypothetical lysine exporter gene (lysE). We also suggest that Lys efflux exporter may participate in heavy metal transport out of R. leguminosarum bv. trifolii cells.