The Relative Contributions of Recombination and Mutation to the Divergence of Clones of Neisseria meningitidis

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1 The Relative Contributions of Recombination and Mutation to the Divergence of Clones of Neisseria meningitidis Edward J. Feil,* Martin C. J. Maiden,* Mark Achtman, and Brian G. Spratt* *Wellcome Trust Centre for the Epidemiology of Infectious Disease, Department of Zoology, University of Oxford, Oxford, England; and Max-Planck-Institut für Molekulare Genetik, Berlin, Germany Multilocus sequence typing (MLST) is a recently developed nucleotide sequence-based method for the definitive assignment of isolates within bacterial populations to specific clones. MLST uses the same principles as multilocus enzyme electrophoresis and provides data that can be used to investigate aspects of the population genetics and evolution of bacterial species. We used an MLST data set consisting of the sequences of 4-bp fragments from seven housekeeping loci from a large strain collection of Neisseria meningitidis to estimate the relative impact of recombination compared with point mutation in the diversification of N. meningitidis clonal complexes. 26 meningococcal isolates were assigned to clonal complexes, 9 of which contained minor clonal variants. The allelic variation within each complex was classified as a recombinational exchange or a putative point mutation through a comparison of the sequences of each variant allele with that of the allele typically found in the clonal complex. The nine clonal complexes contained a total of 23 allelic variants, and analysis of the sequences of these variant alleles revealed that a single nucleotide site in a meningococcal housekeeping gene is at least 8-fold more likely to change as a result of recombination than as a result of mutation. This value is estimated to be -fold for Escherichia coli and -fold for Streptococcus pneumoniae. Introduction The rate and evolutionary impact of horizontal genetic exchange within bacterial populations is a matter of continuing debate and probably varies considerably between species (Maynard Smith et al. 993). Much of the debate has focused on evidence from multilocus enzyme electrophoresis (MLEE; Selander et al. 986), which has been used to demonstrate significant linkage disequilibrium between alleles in populations of many bacterial species and the presence of clusters of isolates that share identical, or very similar, multilocus allelic profiles (Ochman and Selander 984; Caugant et al. 987; Musser et al. 988; Selander and Musser 99). The presence of a limited number of clonal complexes within a population, and a correspondingly high level of linkage disequilibrium, has been interpreted as strong evidence for low rates of horizontal genetic exchange within the population relative to mutation (Selander and Musser 99). However, linkage disequilibrium can be introduced by a number of mechanisms into a population in which recombination is frequent (Maynard Smith 99; Reeves 992; Maynard Smith et al. 993; Guttman 997), and the assumption that linkage disequilibrium reflects a low rate of recombination relative to mutation is not always justified. Examination of the sequences of housekeeping genes can also provide evidence for the significance of recombination, as the variation within these genes is likely to be selectively neutral. The assumption of neutrality in housekeeping genes is central to the utility of MLEE (Selander et al. 986), which is the method upon which most of our knowledge of bacterial population Key words: Neisseria meningitidis, recombination, mutation, clonal divergence, multilocus sequence typing. Address for correspondence and reprints: Edward Feil, Wellcome Trust Centre for the Epidemiology of Infectious Disease, Department of Zoology, University of Oxford, South Parks Road, Oxford OX 3FY, U.K. ed.feil@ceid.ox.ac.uk. Mol. Biol. Evol. 6(): by the Society for Molecular Biology and Evolution. ISSN: structures is based. Recombination within housekeeping genes can be detected by either the noncongruence of gene trees (Dykhuizen and Green 99; Feil et al. 996; Zhou, Bowler, and Spratt 997), the presence of mosaic structure (Zhou and Spratt 992; Feil, Carpenter, and Spratt 99; Spratt et al. 99), an excess of homoplasies within maximum-parsimony trees (Maynard Smith and Smith 998; Suerbaum et al. 998), or the appearance of a network of relationships between sequences, rather than a bifurcating tree-like phylogeny, using split decomposition (Holmes, Urwin, and Maiden 999). The analysis of MLEE data sets and the detection of recombination in gene sequences can provide evidence for recombination within bacterial populations. However, a method for quantifying the rate of recombination, relative to mutation, using nucleotide sequence data has proved elusive owing to difficulties in estimating the relevant population genetics parameters, as well as the problem of ancient events being obscured by more recent ones. A simple way of bypassing these problems is to focus on the variation occurring within clonal complexes, rather than between them, as this variation must be recent. This approach was first suggested by Guttman and Dykhuizen (994), who, by specifically focusing on the nucleotide sequence divergence within the Escherichia coli group A lineage, estimated that recombination is up to -fold more effective in driving clonal divergence in E. coli than mutation. As they put it: [This approach] avoids the problems associated with the obscuring of past events by those occurring more recently and is independent of population dynamics and structure. MLEE has proved invaluable in defining clonal complexes within microbial populations and also in detecting the variation within these clonal complexes. However, it is not possible to quantify the relative contributions of recombination and mutation to the generation of variation within clonal complexes from MLEE data sets, because the molecular basis underlying the ob- 496 Downloaded from on April 28

2 Impact of Recombination in Meningococci 497 served electrophoretic variation cannot be ascertained. The molecular basis of the allelic variation within clonal complexes could, however, be discerned if the alleles at multiple housekeeping loci were assigned directly through nucleotide sequencing. These data are becoming available for several species as a consequence of the introduction of a new molecular typing scheme (multilocus sequence typing [MLST]), a development of MLEE which identifies clonal complexes within bacterial populations by analyzing the sequences of 4-bp internal fragments of seven housekeeping loci (Enright and Spratt 998; Maiden et al. 998). For each locus, every unique sequence is defined as a different allele, and for each isolate, the alleles at the seven loci define an allelic profile or sequence type. The clustering of isolates into discrete clonal complexes can be visualized by constructing a dendrogram from the matrix of pairwise differences between the allelic profiles. MLST indexes variation within housekeeping genes encoding essential metabolic enzymes, as these are likely to be under strong stabilizing selection, and the variation detected by MLST is, for the most part, likely to be selectively neutral. The selective constraints that housekeeping genes are under mean that it is unlikely that large indels or other genomic rearrangements will be tolerated. Here, we demonstrate the utility of MLST data sets for obtaining a quantitative measure of recombination using Neisseria meningitidis as an example. This organism commonly colonizes the human nasopharynx but occasionally enters the bloodstream and cerebrospinal fluid to cause invasive disease (Peltola 983). MLEE has identified four hypervirulent clonal complexes within serogroup B and C meningococci (ET-, ET-37, A4, and Lineage 3; Caugant et al. 986, 987, 99) and has resolved serogroup A isolates, which are associated with epidemic disease (Achtman 99a, 99b), into subgroups (clonal complexes within serogroup A are called subgroups, for historical reasons) (Wang et al. 992; Bart et al. 998). Most isolates of a clonal complex, or subgroup, have identical nucleotide sequences at each of the seven loci (the typical allelic profile), but a few isolates differ from the typical profile at one or two loci. These variant alleles can be either putative point mutations (single nucleotide changes) or recombinational imports (multiple nucleotide changes). There are, however, some ambiguities in these assignments, most notably due to the fact that a single nucleotide change could be due to mutation or recombination. However, as multiple nucleotide changes are unlikely to be due to independent mutations, the analysis provides a lower bound of the ratio of recombinational exchanges to point mutations in the diversification of the meningococcal lineages. Materials and Methods Bacterial Strains The 7 N. meningitidis strains studied previously are described in Maiden et al. (998). These were selected from a larger sample of N. meningitidis isolates Table Properties of the Additional Meningococcal Isolates Assigned to Clonal Complexes or Subgroups Clonal Groupings A:I... A:II... A:VI... A:VII... A:V... A:IV-2... A:VII... A:IX... ET No. of Strains Sequence Types a ST ST, ST7 ST2, ST8 ST3 ST3, ST9 ST4 ST ST6, ST6 ST, ST, ST a Sequence types were assigned using seven loci. The sequence types (seven loci) of all strains can be found at DDBJ/EMBL/ GenBank accession numbers are AF3773 AF3798, AF86737 AF867, and AF63-AF6322. that had been characterized by MLEE to provide multiple isolates of the known meningococcal serogroup B and C clonal complexes and serogroup A subgroups. Thirty-two of the isolates from this collection (mostly of serogroups other than A, B, or C) are not members of these clonal complexes and were excluded from this analysis. However, a supplemental collection of isolates (table ) have since been unambiguously assigned as members of specific clonal complexes using MLST, and these were also included in the analysis. Therefore, a total of 26 strains were used, the allelic profiles and properties of which are available on the meningococcal MLST web site ( Nucleotide Sequencing PCR amplification and nucleotide sequencing of 4-bp internal fragments of abcz (encoding a putative ABC transporter), adk (adenylate kinase), aroe (shikimate dehydrogenase), gdh (glucose-6-phosphate dehydrogenase), pdhc (part of the pyruvate dehydrogenase multienzyme complex), and pgm (phosphoglucomutase) were carried out as previously described (Maiden et al. 998). A seventh housekeeping gene, fumc, has subsequently been added to the meningococcal MLST scheme to provide increased discriminatory power, so that unrelated isolates are extremely unlikely to have identical or even similar allelic profiles. A 46-bp internal fragment of fumc was amplified by PCR using the primers fumc-p ( -CACCGAACACGACACGATGG-3 ) and fumc-p2 ( -ACGACCAGTTCGTCAAACTC-3 ) and an annealing temperature of C. The fumc fragments were sequenced on an ABI377 automated sequencer with drhodamine-labeled terminators (Applied Biosystems, Foster City, Calif.) using the primers fumc-s ( - TCGGCACGGGTTTGAACAGC-3 ) and fumc-s2 ( - CAACGGCGGTTTCGCGCAAC-3 ). The sequences of all allelic variants which differed from the allele typically present in the clonal complexes at a single nucleotide site were verified by resequencing from a new PCR amplicon. Visualization of the Clonal Complexes Each of the different sequences at a particular locus was assigned an allele number, and the alleles at the Downloaded from on April 28

3 498 Feil et al. FIG.. Unweighted pair grouping method with arithmetic means (UPGMA) dendrogram of 26 strains of Neisseria meningitidis resolved into clonal complexes. The dendrogram was produced by the UPGMA method from the matrix of pairwise differences between the allelic profiles of the 26 strains. The serogroup B and C clonal complexes (light shading) and the serogroup A subgroups (dark shading) are shown. The allelic profiles included in the clonal clusters and subgroups are shown by the asterisks at the nodes. seven loci defined the allelic profile of each strain. The relationships among isolates were visualized by constructing a dendrogram using Statistica (StatSoft) from the matrix of pairwise differences between the allelic profiles of the strains by the unweighted pair grouping method with arithmetic means (UPGMA) (fig. ). Results and Discussion Molecular Characterization of Allelic Variants The 26 isolates defined clonal complexes, which correspond to the four serogroup B and C clonal complexes and six serogroup A subgroups (fig. ). Although serogroup A subgroups have been previously resolved by MLEE, only 6 are resolved by MLST (the closely related pairs of subgroups I and II, V and VII, III and VIII, and IV- and IV-2 are not distinguished). Within each of the clonal complexes, all strains either possessed identical allelic profiles or differed at one or two loci from the typical allelic profile. Nine of the clonal complexes or subgroups contained strains which differed at one or two loci, and a total of 23 different variant alleles were noted within these groups (table 2). All 6 isolates in the combined subgroup IV- IV-2 had identical allelic profiles. One variant allele was detected in two different ET- isolates and was only counted once because these two strains might reflect siblings that inherited the allele from a common ancestor. Table 2 shows the number of nucleotide sites at which each variant allele differs from the allele typically present within the clonal complex, and figure 2 shows the differences in the nucleotide sequences of these normal and variant alleles. Of the 23 allelic variants, differed from the allele typical of the clonal complex at a single nucleotide site, which is indicative of a point mutation. All of the other variant alleles differed from the typical clonal alleles by at least five polymorphisms and were therefore considered recombinational imports. This gives an estimate of the ratio of recombinational events to mutational events per 4-bp gene fragment of 8: (3.6:). In other words, recombination has generated new alleles at a frequency 3.6 times as high as that of mutation. The assignment of each variant allele as the result of a point mutation or a recombinational import is complicated by the fact that these two processes are not mutually exclusive. For example, it is possible that a point mutation may arise within an allele which itself has been imported by recombination. Alternatively, imported alleles may represent multiple recombinational events, and this would cause a bias in favor of mutation, which would increase according to the age of the clone. However, because we are looking at the initial stages of clonal diversification, and because the clones themselves are thought to have emerged recently, perhaps within the last 2 years (Morelli et al. 997), the likelihood that a single variant allele represents multiple independent genetic events is low. This assumption is supported by the finding that no point mutations or recombinational replacements have occurred in the vast majority (88/ 882; 97.3%) of the alleles within the 26 isolates in the time since the emergence of the common ancestors of each of the clonal complexes. This strongly suggests that most of those variant alleles that were detected represent single recombinational or mutational events. A more serious source of error is the possibility that some of the single nucleotide changes may have arisen through recombination with a donor allele that differs at only a single site. Errors of this type will underestimate the impact of recombination, and the ratio of 8: should therefore be regarded as a lower bound of the frequency of recombinational imports compared with point mutations. A point mutation is very likely to generate a novel allele, whereas imported alleles may be found in other strains in the data set. Evidence that sin- Downloaded from on April 28

4 Impact of Recombination in Meningococci 499 Table 2 Allelic Variants Within each Clonal Complex or Subgroup NOTE. Variant alleles within each clonal complex or subgroup are highlighted. Recombinational imports are shown as white text on a black background, and putative mutations are shown as black text on a white background. Superscripts are the numbers of polymorphic nucleotide sites between the variant allele and the typical allele of the clonal group (e.g., abcz allele 7 in strain 2846 differs from allele 2, which is the typical ET-37 allele, at 6 nucleotide sites). The 6 strains corresponding to the combined subgroup IV- and IV-2 were all identical and thus are not given in this table. gle nucleotide differences may have arisen by recombination, rather than mutation, can therefore be found by showing the existence of the variant allele elsewhere in the meningococcal population. One of the five putative mutations, the abcz allele of the Lineage 3 strain Z6426, corresponds to an allele which is found in an unrelated strain excluded from this analysis, which suggests that this single base change may represent a recombinational import. The Frequency of Allelic Variants Within Serogroup A, B, and C Isolates Table 3 gives the total number of isolates and the total number of allelic variants within each clonal complex. A 2 2 chi-square test showed that the number of allelic variants per isolate within serogroup A strains was significantly less than those within serogroup B and C strains (P.). This could be due to a difference in the ages of the clones, bias in the sampling of the strains, or the epidemiology of serogroup A isolates, which cause epidemic disease and may meet other lineages relatively rarely. Every effort was made to eliminate sampling bias from this analysis by using isolates from the most diverse temporal and geographical ranges possible. For example, there were 72 serogroup A isolates included in this analysis, and these were collected from 27 countries between 97 and 996. The 4 strains representing serogroups B and C were isolated from 2 countries between 964 and 997. Therefore, although the effects of sampling bias can never be ruled out completely, it is unlikely that they can account for the relative uniformity of serogroup A isolates compared with those from serogroups B and C. The relative uniformity of serogroup A subgroups has previously been noted (Morelli et al. 997) and is thought to reflect the homogenizing effect of sequential bottlenecks, where epidemic spread is initiated through the introduction of a very limited number of different genotypes (Achtman 99b). It is also possible that recombinational exchanges between different lineages are less common relative to mutation in the serogroup A strains (Spratt et al. 99). There is some support for this suggestion in the data set, as of the imports and 2 of the putative mutations were found in the serogroup A strains, whereas 3 imports and 3 putative mutations were detected in the serogroup B and C strains. The reason behind this apparent difference is at present unclear, although it may reflect the epidemiology of the serogroup A strains, which are known to spread very quickly, or ecological factors, which result in serogroup A strains meeting other lineages relatively rarely. Estimating the Per-Site Ratio of Recombination to Point Mutation in N. meningitidis As recombinational imports in relatively diverse species are very likely to introduce multiple polymor- Downloaded from on April 28

5 Feil et al. FIG. 2. Polymorphic sites within each of the variant alleles. For each gene, the allele numbers are shown on the left, and the clonal complex or subgroup is shown on the right. For each clonal complex or subgroup, the typical allele is the upper sequence (in bold), and the variant allele(s) is shown below. Sequences which represent both a variant allele and the typical allele within two different groups (e.g., aroe allele 4 and pdhc allele 6) are shown twice. The polymorphic sites are shown for the uppermost allele, and in the other alleles, only those sites that differ from this sequence are shown. The polymorphic sites are numbered above the sequences in vertical format. Putative point mutations are in bold and underlined. Table 3 Distribution of Allelic Variants Between Clonal Groups No. of Isolates No. of Imports No. of Putative Mutations Serogroup B and C clonal complexes A4... ET-... ET Lineage (3) 2 (8) 4 3 (2) Subtotal... 4 (8) 3 (4) 3 Serogroup A subgroups VI... I,II... V,VII... IV-,IV-2... III, V... IX (2) Subtotal (6) 2 Total (24) 8 (6) NOTE. Figures in parentheses include identical allelic variants in more than one clonal variant. phic nucleotide sites, whereas a point mutation only results in a single polymorphism, the ratio of recombinational events to mutational events per 4-bp gene fragment does not provide a realistic impression of the impact of these two processes on sequence divergence. Guttman and Dykhuizen (994) have suggested that a more suitable approach is to estimate the probability that an individual nucleotide site will change by recombination compared with the probability that it will change by mutation. It is possible to estimate this per-site recombination to mutation (r/m) parameter empirically by counting the number of polymorphisms introduced by recombination and mutation. In this analysis, a minimum of 4 polymorphisms were introduced by the 8 recombinational imports, and a maximum of were introduced by point mutation. Therefore, a particular nucleotide site within a meningococcal housekeeping gene is at least 8 times as likely to change by recombination as it is to change by mutation. If we accept that one of the single nucleotide changes arose through recombination (see above), the r/m parameter rises to approximately. The usefulness of this approach can be illustrated by making tentative comparisons with other species. For Downloaded from on April 28

6 Impact of Recombination in Meningococci example, analysis of the MLST data set of 7 strains of Streptococcus pneumoniae (Enright and Spratt 998; unpublished data) suggests that the ratio of recombinational to mutational events per 4-bp may be in the order of :, (unpublished data) which is approximately three times the ratio estimated for N. meningitidis. However, an average recombinational exchange in the pneumococcus introduces approximately fourfold fewer polymorphisms within a 4-bp housekeeping gene fragment compared with the meningococcus. This suggests that the per-site r/m parameter in the pneumococcus is around half that estimated for the meningococcus, although this parameter is more difficult to estimate in this relatively uniform species. In E. coli, the per-site r/m parameter has been estimated to be up to (Guttman and Dykhuizen 994). Using this figure, the present analysis therefore implies that the impact of recombination on sequence evolution in N. meningitidis is up to twice that estimated for E. coli, a conclusion in broad agreement with the analysis by Suerbaum et al. (998), who used the homoplasy test (Maynard Smith and Smith 998) to compare these two species. Conclusions The significance of recombination during the evolution of bacterial populations is controversial and, as Guttman and Dykhuizen (994) proposed, the per-site r/ m parameter provides a general and quantitative measure of the impact of recombination at neutral loci in different species. The current analysis demonstrates how this approach can be applied to large MLST data sets. The seven loci used for MLST in the meningococci are dispersed throughout the genome (Maiden et al. 998), and the calculated per-site r/m ratio should therefore be typical of meningococcal housekeeping genes. A great advantage of utilizing MLST data sets to quantify recombination rates is that MLST is already being used by several laboratories for typing meningococci. The characterization of an increasing number of isolates from disease and from nasopharyngeal carriage will therefore allow continuous improvement of the per site r/m parameter in meningococci and a clearer understanding of whether this parameter varies between clonal complexes associated with differing epidemiological behaviors (e.g., carriage, endemic disease, or epidemics). This approach may also enable comparisons between different regions of the genome, for example, comparisons between housekeeping genes and genes encoding cell surface antigens. MLST is also being used to unambiguously assign isolates of other bacterial pathogens to clonal complexes (Enright and Spratt 998; see and is likely to become the method of choice for the unambiguous molecular typing of many pathogens. Consequently, large and continually expanding MLST data sets will be produced over the next few years for several major pathogens, which will allow estimates of the same per-site r/m parameter in different species. The application of this quantitative approach to other MLST data sets will allow the well-documented high rate of recombination within N. meningitidis to be placed within a proper context through meaningful comparisons with other species. Acknowledgments This work was funded by the Wellcome Trust. B.G.S. is a Wellcome Trust Principal Research Fellow. M.C.J.M. is a Wellcome Trust Senior Fellow in Biodiversity Research. We are extremely grateful to Ian Feavers, Giovanna Morelli, Rachel Urwin, Kerstin Zurth, and Dominique Caugant for providing unpublished data and to John Maynard Smith and Aldert Bart for helpful discussions. LITERATURE CITED ACHTMAN, M. 99a. Global epidemiology of meningococcal disease. Pp. 9 7 in K. CARTWIGHT, ed. Meningococcal disease. 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