Multilocus Hybridization Typing in Pediococcus acidilactici Strains

In previous a study we demonstrated the presence of several genomic subpopulations within a collection of Pediococcus acidilactici strains isolated from different environments, through a multilocus typing analysis taking into consideration housekeeping conserved loci and protein coding genes of the primary metabolism. In this study, representative strains of five genomic subpopulations previously described (I, II, III, V, VII) were analyzed by restriction analysis of chromosomal DNA and subsequent hybridization assays using as probes amplified fragments obtained from five housekeeping genes (16S rDNA, rpoC, ldhD, ldhL, and metS). A computer similarity and clustering analysis of hybridization data showed the subdivision of P. acidilactici strains in five distinct genotypes according to the grouping previously obtained confirming that pediocin AcH/PA-1 producer strains represent one genomic lineage within the species P. acidilactici. Received: 30 January 2001 / Accepted: 16 May 2001     

rhs Genes Are Potential Markers for Multilocus Sequence Typing of Escherichia coli O157:H7 Strains

DNA sequence-based molecular subtyping methods such as multilocus sequence typing (MLST) are commonly used to generate phylogenetic inferences for monomorphic pathogens. The development of an effective MLST scheme for subtyping Escherichia coli O157:H7 has been hindered in the past due to the lack of sequence variation found within analyzed housekeeping and virulence genes. A recent study suggested that rhs genes are under strong positive selection pressure, and therefore in this study we analyzed these genes within a diverse collection of E. coli O157:H7 strains for sequence variability. Eighteen O157:H7 strains from lineages I and II and 15 O157:H7 strains from eight clades were included. Examination of these rhs genes revealed 44 polymorphic loci (PL) and 10 sequence types (STs) among the 18 lineage strains and 280 PL and 12 STs among the 15 clade strains. Phylogenetic analysis using rhs genes generally grouped strains according to their known lineage and clade classifications. These findings also suggested that O157:H7 strains from clades 6 and 8 fall into lineage I/II and that strains of clades 1, 2, 3, and 4 fall into lineage I. Additionally, unique markers were found in rhsA and rhsJ that might be used to define clade 8 and clade 6. Therefore, rhs genes may be useful markers for phylogenetic analysis of E. coli O157:H7.Escherichia coli O157:H7 was first described in 1983 as the causative agent of a food-borne outbreak attributed to contaminated ground beef patties (35), and it has subsequently emerged as a very important food-borne pathogen. Diseases caused by E. coli O157:H7, such as hemorrhagic colitis and hemolytic uremic syndrome, can be very severe or even life-threatening. Cattle are believed to be the main reservoir for E. coli O157:H7 (5, 15, 41), although other animals may also carry this organism (6, 21). Outbreaks are commonly associated with the consumption of beef and fresh produce that come into contact with bovine feces or feces-contaminated environments, such as food contact surfaces, animal hides, or irrigation water (12, 21, 30, 38).It is well-established that strains of E. coli O157:H7 vary in terms of virulence and transmissibility to humans and that strains differing in these characteristics can be distinguished using DNA-based methods (22, 29, 42). For example, octamer-based genome scanning, which is a PCR approach using 8-bp primers, provided the first evidence that there are at least two lineages of O157:H7, termed lineage I and lineage II (22). Strains classified as lineage I are more frequently isolated from humans than are lineage II strains (42). A later refinement of this classification system was coined the lineage-specific polymorphism assay (LSPA), which classified strains based upon the amplicon size obtained using PCRs targeting six chromosomal regions of E. coli O157:H7 and assigned a six-digit code based upon the pattern obtained (42). Most strains of lineage I grouped into LSPA type 111111, while the majority of lineage II strains fell into LSPA types 211111, 212111, and 222222. More recently, it was suggested that LSPA type 211111 strains comprise a separate group called lineage I/II (45).To gain greater insight into the recent evolution of E. coli O157:H7, a method that is more discriminatory than the LSPA method is desirable. Multilocus sequence typing (MLST) is a method that discriminates between strains of a bacterial species by identifying DNA sequence differences in six to eight targeted genes. Satisfactory MLST schemes exist for other bacterial pathogens (28, 43); however, due to the lack of sequence variations in previously targeted gene markers in E. coli O157:H7 (13, 33), MLST approaches for subtyping this pathogen have been more difficult to develop. More recently, high-throughput microarray and sequencing platforms have been used to identify hundreds of single nucleotide polymorphisms (SNPs) that are useful for discriminating between strains of E. coli O157:H7 during epidemiologic investigations and for drawing phylogenetic inferences (11, 20, 29, 44). Particularly noteworthy, Manning et al. (29) developed a subtyping scheme based upon the interrogation of 32 putative SNP loci. This method separated 528 strains into 39 distinct SNP genotypes, which were grouped into nine statistically supported phylogenetic groups called clade 1 through clade 9. By analyzing the rates of hemolytic uremic syndrome observed in patients infected with strains of clades 2, 7, and 8, it was also concluded that clade 8 strains are more virulent to humans than other strains (29).One drawback of current DNA sequence-based subtyping schemes for E. coli O157:H7 is that they require screening of at least 32 SNP loci. We were interested in asking whether a simpler approach that targets a few informative gene markers could be developed for rapid strain discrimination and phylogenetic determination. A recent analysis of E. coli genomes predicted that rearrangement hot spot (rhs) genes are under the strongest positive selection of all coding sequences analyzed (34). Therefore, we hypothesized that these genes would display significant sequence variations for subtyping O157:H7 strains. The rhs genes were first discovered as elements mediating tandem duplication of the glyS locus in E. coli K-12 (26); however, their function remains unknown. There are nine rhs genes within the genome of the prototypical E. coli O157:H7 strain Sakai, and these genes are designated rhsA, -C, -D, -E, -F, -G, -I, -J, and -K (see Table S1 in the supplemental material) (16). Three of these nine rhs genes, rhsF, -J, and -K, were previously studied by Zhang et al. (44), and a number of SNPs were identified among these genes. However, no studies have been conducted to comprehensively investigate rhs genes as markers in an MLST scheme for subtyping E. coli O157:H7.The primary purpose of the present study was to investigate whether there are sufficient DNA sequence variations among rhs genes to develop an MLST approach for subtyping E. coli O157:H7. In this study, a greater level of DNA sequence variation was observed among rhs genes than in gene markers targeted in previous studies (13, 33). Furthermore, phylogenetic analysis using these rhs genes generally agreed with the established lineage and clade classifications of O157:H7 strains defined previously. We also wanted to determine whether there is a correlation between the lineage classification of O157:H7 strains (42) and the recently proposed clade classification (29). The present study reports evidence that O157:H7 strains from clade 8 are classified as lineage I/II, which is a different lineage from well-studied E. coli O157:H7 outbreak strains, such as EDL933 and Sakai. Therefore, we suggest that outbreaks of O157:H7 are caused by two lineages of this pathogen, lineage I and lineage I/II.     

An Expanded Multilocus Sequence Typing Scheme for Propionibacterium acnes: Investigation of 'Pathogenic', 'Commensal' and Antibiotic Resistant Strains

The Gram-positive bacterium Propionibacterium acnes is a member of the normal human skin microbiota and is associated with various infections and clinical conditions. There is tentative evidence to suggest that certain lineages may be associated with disease and others with health. We recently described a multilocus sequence typing scheme (MLST) for P. acnes based on seven housekeeping genes (http://pubmlst.org/pacnes). We now describe an expanded eight gene version based on six housekeeping genes and two 'putative virulence' genes (eMLST) that provides improved high resolution typing (91eSTs from 285 isolates), and generates phylogenies congruent with those based on whole genome analysis. When compared with the nine gene MLST scheme developed at the University of Bath, UK, and utilised by researchers at Aarhus University, Denmark, the eMLST method offers greater resolution. Using the scheme, we examined 208 isolates from disparate clinical sources, and 77 isolates from healthy skin. Acne was predominately associated with type IA(1) clonal complexes CC1, CC3 and CC4; with eST1 and eST3 lineages being highly represented. In contrast, type IA(2) strains were recovered at a rate similar to type IB and II organisms. Ophthalmic infections were predominately associated with type IA(1) and IA(2) strains, while type IB and II were more frequently recovered from soft tissue and retrieved medical devices. Strains with rRNA mutations conferring resistance to antibiotics used in acne treatment were dominated by eST3, with some evidence for intercontinental spread. In contrast, despite its high association with acne, only a small number of resistant CC1 eSTs were identified. A number of eSTs were only recovered from healthy skin, particularly eSTs representing CC72 (type II) and CC77 (type III). Collectively our data lends support to the view that pathogenic versus truly commensal lineages of P. acnes may exist. This is likely to have important therapeutic and diagnostic implications.     

Multilocus Sequence Typing of Clinical Isolates of Cryptococcus from India

Mycopathologia - Cryptococcosis is a life-threatening infection caused by Cryptococcus neoformans and C. gattii species complex. In the present study, to understand the molecular epidemiology of...     

海南岛光滑假丝酵母菌多位点序列分型研究

目的:对海南省光滑假丝酵母菌临床分离株进行基因分型研究,了解菌株的遗传特征及遗传进化关系.方法:采用多位点序列分型(Multilocus sequence typing,MLST)技术,对临床分离的25株光滑假丝酵母菌6个管家基因序列进行测定;并将各个基因的序列与MLST数据库中储存的序列比对,确定其等位基因谱型及菌株序列型(STs).结果:25株临床分离的光滑假丝酵母菌通过MLST产生ST7、ST19、ST15、ST26、ST45共5个不同的序列型,其中ST7为主要序列型.结论:海南省光滑假丝酵母菌感染型别丰富,具有多样性;MLST分型具有较高的分辨力,可用于流行病学和菌群多态性的研究.     

Multilocus Sequence Typing as a Replacement for Serotyping in Salmonella enterica

Salmonella enterica subspecies enterica is traditionally subdivided into serovars by serological and nutritional characteristics. We used Multilocus Sequence Typing (MLST) to assign 4,257 isolates from 554 serovars to 1092 sequence types (STs). The majority of the isolates and many STs were grouped into 138 genetically closely related clusters called eBurstGroups (eBGs). Many eBGs correspond to a serovar, for example most Typhimurium are in eBG1 and most Enteritidis are in eBG4, but many eBGs contained more than one serovar. Furthermore, most serovars were polyphyletic and are distributed across multiple unrelated eBGs. Thus, serovar designations confounded genetically unrelated isolates and failed to recognize natural evolutionary groupings. An inability of serotyping to correctly group isolates was most apparent for Paratyphi B and its variant Java. Most Paratyphi B were included within a sub-cluster of STs belonging to eBG5, which also encompasses a separate sub-cluster of Java STs. However, diphasic Java variants were also found in two other eBGs and monophasic Java variants were in four other eBGs or STs, one of which is in subspecies salamae and a second of which includes isolates assigned to Enteritidis, Dublin and monophasic Paratyphi B. Similarly, Choleraesuis was found in eBG6 and is closely related to Paratyphi C, which is in eBG20. However, Choleraesuis var. Decatur consists of isolates from seven other, unrelated eBGs or STs. The serological assignment of these Decatur isolates to Choleraesuis likely reflects lateral gene transfer of flagellar genes between unrelated bacteria plus purifying selection. By confounding multiple evolutionary groups, serotyping can be misleading about the disease potential of S. enterica. Unlike serotyping, MLST recognizes evolutionary groupings and we recommend that Salmonella classification by serotyping should be replaced by MLST or its equivalents.     

Wolbachia Infection and Cytoplasmic Incompatibility in Drosophila Species

Forty-one stocks from 30 Drosophila species were surveyed for Wolbachia infection using PCR technology. D. sechellia and two strains of D. auraria were found to be infected and were tested for the expression of cytoplasmic incompatibility, along with D. ananassae and D. melanogaster strains, which are already known to be infected. D. ananassae and D. melanogaster show levels of incompatibility up to 25%, while D. auraria and D. sechellia exhibit levels of egg mortality ~60%. A dot-blot assay using the dnaA sequence as probe was developed to assess the infection levels in individual males that were used in incompatibility crosses. A positive correlation between bacterial density and cytoplasmic incompatibility was observed. The stocks examined can be clustered into at least two groups, depending on the levels of infection relative to the degree of cytoplasmic incompatibility exhibited. One group, containing D. simulans Hawaii, D. sechellia, and D. auraria, exhibits high levels of cytoplasmic incompatibility relative to levels of infection; all the other species and D. simulans Riverside exhibit significantly lower levels of cytoplasmic incompatibility relative to levels of infection. These data show that, in addition to bacterial density, bacterial and/or host factors also affect the expression of cytoplasmic incompatibility.     

Multilocus Sequence Typing Identifies Epidemic Clones of Flavobacterium psychrophilum in Nordic Countries

Flavobacterium psychrophilum is the causative agent of bacterial cold water disease (BCWD), which affects a variety of freshwater-reared salmonid species. A large-scale study was performed to investigate the genetic diversity of F. psychrophilum in the four Nordic countries: Denmark, Finland, Norway, and Sweden. Multilocus sequence typing of 560 geographically and temporally disparate F. psychrophilum isolates collected from various sources between 1983 and 2012 revealed 81 different sequence types (STs) belonging to 12 clonal complexes (CCs) and 30 singleton STs. The largest CC, CC-ST10, which represented almost exclusively isolates from rainbow trout and included the most predominant genotype, ST2, comprised 65% of all isolates examined. In Norway, with a shorter history (<10 years) of BCWD in rainbow trout, ST2 was the only isolated CC-ST10 genotype, suggesting a recent introduction of an epidemic clone. The study identified five additional CCs shared between countries and five country-specific CCs, some with apparent host specificity. Almost 80% of the singleton STs were isolated from non-rainbow trout species or the environment. The present study reveals a simultaneous presence of genetically distinct CCs in the Nordic countries and points out specific F. psychrophilum STs posing a threat to the salmonid production. The study provides a significant contribution toward mapping the genetic diversity of F. psychrophilum globally and support for the existence of an epidemic population structure where recombination is a significant driver in F. psychrophilum evolution. Evidence indicating dissemination of a putatively virulent clonal complex (CC-ST10) with commercial movement of fish or fish products is strengthened.     

Multilocus Sequence Typing Scheme for Bacteria of the Bacillus cereus Group

In this study we developed a multilocus sequence typing (MLST) scheme for bacteria of the Bacillus cereus group. This group, which includes the species B. cereus, B. thuringiensis, B. weihenstephanensis, and B. anthracis, is known to be genetically very diverse. It is also very important because it comprises pathogenic organisms as well as bacteria with industrial applications. The MLST system was established by using 77 strains having various origins, including humans, animals, food, and soil. A total of 67 of these strains had been analyzed previously by multilocus enzyme electrophoresis, and they were selected to represent the genetic diversity of this group of bacteria. Primers were designed for conserved regions of housekeeping genes, and 330- to 504-bp internal fragments of seven such genes, adk, ccpA, ftsA, glpT, pyrE, recF, and sucC, were sequenced for all strains. The number of alleles at individual loci ranged from 25 to 40, and a total of 53 allelic profiles or sequence types (STs) were distinguished. Analysis of the sequence data showed that the population structure of the B. cereus group is weakly clonal. In particular, all five B. anthracis isolates analyzed had the same ST. The MLST scheme which we developed has a high level of resolution and should be an excellent tool for studying the population structure and epidemiology of the B. cereus group.     

Analysis of the Population Structure of Anaplasma phagocytophilum Using Multilocus Sequence Typing

Anaplasma phagocytophilum is a Gram-negative obligate intracellular bacterium that replicates in neutrophils. It is transmitted via tick-bite and causes febrile disease in humans and animals. Human granulocytic anaplasmosis is regarded as an emerging infectious disease in North America, Europe and Asia. However, although increasingly detected, it is still rare in Europe. Clinically apparent A. phagocytophilum infections in animals are mainly found in horses, dogs, cats, sheep and cattle. Evidence from cross-infection experiments that A. phagocytophilum isolates of distinct host origin are not uniformly infectious for heterologous hosts has led to several approaches of molecular strain characterization. Unfortunately, the results of these studies are not always easily comparable, because different gene regions and fragment lengths were investigated. Multilocus sequence typing is a widely accepted method for molecular characterization of bacteria. We here provide for the first time a universal typing method that is easily transferable between different laboratories. We validated our approach on an unprecedented large data set of almost 400 A. phagocytophilum strains from humans and animals mostly from Europe. The typability was 74% (284/383). One major clonal complex containing 177 strains was detected. However, 54% (49/90) of the sequence types were not part of a clonal complex indicating that the population structure of A. phagocytophilum is probably semiclonal. All strains from humans, dogs and horses from Europe belonged to the same clonal complex. As canine and equine granulocytic anaplasmosis occurs frequently in Europe, human granulocytic anaplasmosis is likely to be underdiagnosed in Europe. Further, wild boars and hedgehogs may serve as reservoir hosts of the disease in humans and domestic animals in Europe, because their strains belonged to the same clonal complex. In contrast, as they were only distantly related, roe deer, voles and shrews are unlikely to harbor A. phagocytophilum strains infectious for humans, domestic or farm animals.     

Identification of Brevibacteriaceae by Multilocus Sequence Typing and Comparative Genomic Hybridization Analyses

Multilocus sequence typing with nine selected genes is shown to be a promising new tool for accurate identifications of Brevibacteriaceae at the species level. A developed microarray also allows intraspecific diversity investigations of Brevibacterium aurantiacum showing that 13% to 15% of the genes of strain ATCC 9174 were absent or divergent in strain BL2 or ATCC 9175.Brevibacteriaceae play a major part in the cheese smear community (6, 11). The classification and typing of cheese-related Brevibacteriaceae have been based mainly on molecular methods such as amplified ribosomal DNA restriction enzyme analysis, pulsed-field gel electrophoresis, and ribotyping (8, 10, 12). Recently, the original Brevibacterium linens group was split into two species on the basis of their physiological and biochemical characteristics, the sugar and polyol composition of their teichoic acids, and their 16S rRNA sequence and DNA-DNA hybridization levels. One species remains B. linens and is represented by type strain ATCC 9172. The other, represented by type strain ATCC 9175, has been renamed Brevibacterium aurantiacum. Regarding this new classification, the taxonomic position of cheese-related isolates has to be revisited and potential relationships between phylogenetic affiliation and the potential occurrence of given metabolic characteristics redefined (7). The unfinished genome sequence of B. aurantiacum ATCC 9174 has recently been released by the Joint Genome Institute (http://genome.jgi-psf.org/draft_microbes/breli/breli.home.html). The development of focused phylogenetic approaches using multiple markers in conjunction with whole-genome screening techniques such as comparative genomic hybridization (CGH) has proven to be useful for the detailed characterization of pathogenic species, including food pathogens (3, 5, 9). However, only a few technological species have been investigated at an intraspecies level (2). Our intention was thus to develop modern tools to facilitate the typing of strains of technological interest, for which Brevibacteriaceae could be used as a case study.     

Phylogenetic Analysis of Burkholderia Species by Multilocus Sequence Analysis

Burkholderia comprises more than 60 species of environmental, clinical, and agro-biotechnological relevance. Previous phylogenetic analyses of 16S rRNA, recA, gyrB, rpoB, and acdS gene sequences as well as genome sequence comparisons of different Burkholderia species have revealed two major species clusters. In this study, we undertook a multilocus sequence analysis of 77 type and reference strains of Burkholderia using atpD, gltB, lepA, and recA genes in combination with the 16S rRNA gene sequence and employed maximum likelihood and neighbor-joining criteria to test this further. The phylogenetic analysis revealed, with high supporting values, distinct lineages within the genus Burkholderia. The two large groups were named A and B, whereas the B. rhizoxinica/B. endofungorum, and B. andropogonis groups consisted of two and one species, respectively. The group A encompasses several plant-associated and saprophytic bacterial species. The group B comprises the B. cepacia complex (opportunistic human pathogens), the B. pseudomallei subgroup, which includes both human and animal pathogens, and an assemblage of plant pathogenic species. The distinct lineages present in Burkholderia suggest that each group might represent a different genus. However, it will be necessary to analyze the full set of Burkholderia species and explore whether enough phenotypic features exist among the different clusters to propose that these groups should be considered separate genera.     

High Genetic Diversity among Strains of the Unindustrialized Lactic Acid Bacterium Carnobacterium maltaromaticum in Dairy Products as Revealed by Multilocus Sequence Typing

Dairy products are colonized with three main classes of lactic acid bacteria (LAB): opportunistic bacteria, traditional starters, and industrial starters. Most of the population structure studies were previously performed with LAB species belonging to these three classes and give interesting knowledge about the population structure of LAB at the stage where they are already industrialized. However, these studies give little information about the population structure of LAB prior their use as an industrial starter. Carnobacterium maltaromaticum is a LAB colonizing diverse environments, including dairy products. Since this bacterium was discovered relatively recently, it is not yet commercialized as an industrial starter, which makes C. maltaromaticum an interesting model for the study of unindustrialized LAB population structure in dairy products. A multilocus sequence typing scheme based on an analysis of fragments of the genes dapE, ddlA, glpQ, ilvE, pyc, pyrE, and leuS was applied to a collection of 47 strains, including 28 strains isolated from dairy products. The scheme allowed detecting 36 sequence types with a discriminatory index of 0.98. The whole population was clustered in four deeply branched lineages, in which the dairy strains were spread. Moreover, the dairy strains could exhibit a high diversity within these lineages, leading to an overall dairy population with a diversity level as high as that of the nondairy population. These results are in agreement with the hypothesis according to which the industrialization of LAB leads to a diversity reduction in dairy products.     

Multilocus Sequence Typing for Comparison of Veterinary and Human Isolates of Campylobacter jejuni

Multilocus sequence typing (MLST) has been applied to 266 Campylobacter jejuni isolates, mainly from veterinary sources, including cattle, sheep, poultry, pigs, pets, and the environment, as well as isolates from human cases of campylobacteriosis. The populations of veterinary and human isolates overlap, suggesting that most veterinary sources should be considered reservoirs of pathogenic campylobacters. There were some associations between source and sequence type complex, indicating that host or source adaptation may exist. The pig isolates formed a distinct group by MLST and may well represent a potential pig-adapted clone of C. jejuni. A subset (n = 82) of isolates was reanalyzed with a second MLST scheme which provided a unique set of isolates that had been analyzed at a total of 12 loci. The distribution of isolates among the complexes in each of the two schemes was similar but not identical. In addition to isolates from human outbreaks, one group of isolates that were not epidemiologically linked was also identical at all 12 loci. This group of isolates is believed to represent another stable strain of C. jejuni.     

Multilocus Sequence Typing and Phylogenetic Analyses of Pseudomonas aeruginosa Isolates from the Ocean

Recent isolation of Pseudomonas aeruginosa strains from the open ocean and subsequent pulsed-field gel electrophoresis analyses indicate that these strains have a unique genotype (N. H. Khan, Y. Ishii, N. Kimata-Kino, H. Esaki, T. Nishino, M. Nishimura, and K. Kogure, Microb. Ecol. 53:173-186, 2007). We hypothesized that ocean P. aeruginosa strains have a unique phylogenetic position relative to other strains. The objective of this study was to clarify the intraspecies phylogenetic relationship between marine strains and other strains from various geographical locations. Considering the advantages of using databases, multilocus sequence typing (MLST) was chosen for the typing and discrimination of ocean P. aeruginosa strains. Seven housekeeping genes (acsA, aroE, guaA, mutL, nuoD, ppsA, and trpE) were analyzed, and the results were compared with data on the MLST website. These genes were also used for phylogenetic analysis of P. aeruginosa. Rooted and unrooted phylogenetic trees were generated for each gene locus and the concatenated gene fragments. MLST data showed that all the ocean strains were new. Trees constructed for individual and concatenated genes revealed that ocean P. aeruginosa strains have clusters distinct from those of other P. aeruginosa strains. These clusters roughly reflected the geographical locations of the isolates. These data support our previous findings that P. aeruginosa strains are present in the ocean. It can be concluded that the ocean P. aeruginosa strains have diverged from other isolates and form a distinct cluster based on MLST and phylogenetic analyses of seven housekeeping genes.     

Multilocus Sequence Typing (MLST) for Characterization of Enterobacter cloacae

Enterobacter cloacae is an important emerging pathogen, which sometime causes respiratory infection, surgical site infection, urinary infection, sepsis, and outbreaks at neonatal units. We have developed a multilocus sequence typing (MLST) scheme utilizing seven housekeeping genes and evaluated the performance in 101 clinical isolates. The MLST scheme yielded 83 sequence types (ST) including 78 novel STs found in the clinical isolates. These findings supported the robustness of the MLST scheme developed in this study.     

Application of Multilocus Sequence Typing To Study the Genetic Structure of Megaplasmids in Medicago-Nodulating Rhizobia

A multilocus sequence typing (MLST) analysis was used to examine the genetic structure and diversity within the two large extrachromosomal replicons in Medicago-nodulating rhizobia (Sinorhizobium meliloti and Sinorhizobium medicae). The allelic diversity within these replicons was high compared to the reported diversity within the corresponding chromosomes of the same strains (P. van Berkum et al., J. Bacteriol. 188:5570-5577, 2006). Also, there was strong localized linkage disequilibrium (LD) between certain pSymA loci: e.g., nodC and nifD. Although both of these observations could be explained by positive (or diversifying) selection by plant hosts, results of tests for positive selection did not provide consistent support for this hypothesis. The strong LD observed between the nodC and nifD genes could also be explained by their close proximity on the pSymA replicon. Evidence was obtained that some nodC alleles had a history of intragenic recombination, while other alleles of this locus had a history of intergenic recombination. Both types of recombination were associated with a decline in symbiotic competence with Medicago sativa as the host plant. The combined observations of LD between the nodC and nifD genes and intragenic recombination within one of these loci indicate that the symbiotic gene region on the pSymA plasmid has evolved as a clonal segment, which has been laterally transferred within the natural populations.Plants of the genus Medicago are legumes that often benefit from a mutualistic symbiosis with rhizobia. The most agriculturally significant species of rhizobia that nodulate these plants are Sinorhizobium meliloti (9) and Sinorhizobium medicae (22). Previously reported population genetic analyses of these bacteria have focused on the study of how allelic variants at multiple loci are distributed within and among natural populations (2, 3, 10, 26, 31, 32). This was also the focus of the present study, but it was extended by examining more loci in many more strains of both species of Sinorhizobium coupled with an analysis having a range of symbiotic genotypes. One goal was to determine if there were any obvious correlations between the megaplasmid genotypes observed and their symbiotic competence. A second goal was to determine if selection by their host plants may have influenced the evolution of their symbiotic relationships.The genes for symbiosis reside on the extrachromosomal replicons pSymA (1,354,226 nucleotides [nt]) and pSMED02 (1,245,408 nt) in the genomes of S. meliloti Rm1021 and S. medicae WSM419, respectively (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AE006469","term_id":"25168258","term_text":"AE006469"}}AE006469 and {"type":"entrez-nucleotide","attrs":{"text":"CP000740","term_id":"150031715","term_text":"CP000740"}}CP000740, respectively). Besides these two plasmids, these two strains each harbor one other large extrachromosomal replicon, pSymB (1,683,333 nt) and pSMED01 (1,570,951 nt), respectively (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AL591985","term_id":"30407151","term_text":"AL591985"}}AL591985 and {"type":"entrez-nucleotide","attrs":{"text":"CP000739","term_id":"150030273","term_text":"CP000739"}}CP000739, respectively).Multilocus sequence typing (MLST) (16) is a form of genomic indexing that is commonly used to study the population genetic structure and phylogenetic relatedness within diverse groups of bacteria. In this method, nucleotide sequences of a fixed set of common loci are obtained from a collection of strains, and polymorphic sites among these sequences are used to derive an allelic profile or sequence type (ST) for each genome. Comparisons of the resulting data can be used to infer phylogenetic relationships among the organisms in the sample population, and they also can be used to infer how evolutionary processes, such as recombination and selection, have shaped the genetic structure of the population. For example, levels of intergenic recombination among chromosomal genes in natural populations of Neisseria meningitidis reportedly are relatively high, while corresponding levels within populations of Staphylococcus aureus were low (28). Depending on the specific pairs of loci examined, the levels of linkage disequilibrium (LD) (a lack of intergenic recombination) among several chromosomally carried core genes of S. meliloti were reported to be generally moderate to high (26).The MLST approach has been used to confirm that the chromosomes of S. meliloti and S. medicae are sexually isolated (2, 3, 31) and to provide evidence that horizontal gene transfer (HGT) does occur between the symbiotic megaplasmids of these species (3, 32). It has also been used to demonstrate that levels of intergenic recombination, as indicated by linkage disequilibrium, differ between the three replicons of S. meliloti (26). Levels of intergenic recombination within the pSymB replicons of these strains are generally high, unlike the chromosomes and pSymA replicons within the same strains (26). Bailly et al. (3) hypothesized that the region of the pSymA plasmid that contains the nodulation (nod) genes is frequently transferred in natural populations. They also suggested that selective pressures from the host plant may have influenced both nod gene diversity and patterns of polymorphism across the entire nod gene region.In the present study, multilocus allelic variation of the two megaplasmids was examined among 231 Medicago-nodulating rhizobia that originated primarily from southwest Asia (10). Previously, 91 different chromosomal sequence types (STs) were identified among the same strains from sequence variation in 10 loci (31). This collection of strains had earlier been divided into two closely related groups based on results of multilocus enzyme electrophoresis (10), and this result was subsequently cited in support of separating the Medicago-nodulating rhizobia into the two species S. meliloti and S. medicae (22).The objectives of this study were (i) to use MLST to examine the genetic relationships within and among the large extrachromosomal replicons in S. meliloti and S. medicae, (ii) to estimate levels of intergenic and intragenic recombination in these replicons, (iii) to evaluate the nitrogen-fixing competence of representative symbiotic genotypes with Medicago sativa, and (iv) to determine whether positive (or diversifying) selection may have influenced the genetic structure of the megaplasmids.