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Phylogeny of the Mycoplasma mycoides cluster based on analysis of five conserved protein-coding sequences and possible implications for the taxonomy of the group

¹ CIRAD BIOS, UPR15, Control of Exotic and Emerging Animal Diseases, TA A-15/G, Campus International Baillarguet, 34398 Montpellier Cedex 5, France
² CIRAD BIOS, UPR75, Biomathematics, Genetic Improvement of Vegetatively Propagated Crops, TA 71/09, Campus Lavalette, 34398 Montpellier Cedex 5, France

Correspondence
Lucía Manso-Silván
lucia.manso-silvan{at}cirad.fr

	ABSTRACT

TOP ABSTRACT INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
A phylogenetic tree of the Mycoplasma mycoides cluster was inferred from a set of concatenated sequences from five housekeeping genes (fusA, glpQ, gyrB, lepA and rpoB). The relevance of this phylogeny was reinforced by detailed analysis of the congruence of the phylogenies derived from each of the five individual gene sequences. Two subclusters were distinguished. The M. mycoides subcluster comprised M. mycoides subsp. mycoides biotypes Small Colony (SC) and Large Colony (LC) and M. mycoides subsp. capri. The latter two groups could not be clearly separated, which supports previous proposals that they be united into a single taxonomic entity. The Mycoplasma capricolum subcluster included M. capricolum subsp. capricolum, M. capricolum subsp. capripneumoniae and Mycoplasma sp. bovine group 7 of Leach, a group of strains that remains unassigned. This group constituted a distinct branch within this cluster, supporting its classification as a subspecies of M. capricolum. Mycoplasma cottewii and Mycoplasma yeatsii clustered in a group that was distinct from Mycoplasma putrefaciens and they were all clearly separated from the M. mycoides cluster. In conclusion, this approach has allowed us to assign phylogenetic positions to all members of the M. mycoides cluster and related species and has proved the need to adjust the existing taxonomy. Furthermore, this method may be used as a reference technique to assign an unequivocal position to any particular strain related to this cluster and may lead to the development of new techniques for rapid species identification.

The GenBank/EMBL/DDBJ accession numbers of the partial fusA, glpQ, gyrB, lepA and rpoB gene sequences of 26 strains analysed here are listed in Supplementary Table S1.

Accession numbers of the sequences used in this study are available with the online version of this paper.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
The Mycoplasma mycoides cluster, as described by Cottew et al. (1987), represents a group of six closely related mycoplasmas that are pathogenic for ruminants and which are currently referred to as Mycoplasma mycoides subsp. mycoides Small Colony biotype (MmmSC) and Large Colony biotype (MmmLC), M. mycoides subsp. capri (Mmc), Mycoplasma capricolum subsp. capricolum (Mcc), M. capricolum subsp. capripneumoniae (Mccp) and Mycoplasma sp. bovine group 7 of Leach (MBG7). In addition, on the basis of 16S rRNA gene sequence similarity, other species such as the pathogen Mycoplasma putrefaciens (Weisburg et al., 1989) and the related saprophytic species Mycoplasma cottewii and Mycoplasma yeatsii (Heldtander et al., 1998) have been included in the phylogenetic M. mycoides cluster, within the Spiroplasma group.

MmmSC and Mccp are the causative agents of contagious bovine and contagious caprine pleuropneumonia (CBPP and CCPP), respectively, which are diseases of major concern, classed by the World Organization for Animal Health (OIE) as notifiable animal diseases. Other members of the cluster (MmmLC, Mmc and Mcc) and M. putrefaciens are involved in the ‘MAKePS’ syndrome, characterized by mastitis, arthritis, keratoconjunctivitis, pneumonia and septicaemia in small ruminants (Thiaucourt & Bölske, 1996). Finally, MBG7 represents a group of strains that are serologically distinct from other bovine mycoplasmas (Leach, 1967) and which cause mastitis and polyarthritis in cattle.

Because the different members of the M. mycoides cluster share many genotypic and phenotypic traits, their classification and evolutionary relationships have been difficult to establish. This may explain why the group of strains known as MBG7 remains unassigned, while Mccp was named only relatively recently (Leach et al., 1993). Initial classification within this cluster was attempted based on DNA–DNA hybridization and biochemical and serological methods as well as 2D PAGE patterns (Cottew et al., 1987), which resulted in differentiation of the M. mycoides subspecies on one side and the M. capricolum subspecies on the other. The position of MBG7 was controversial due to conflicting data obtained from the various methods applied and remains unresolved, though recent studies concur in favour of classification of this group of strains within a subspecies of M. capricolum (Harasawa et al., 2000; Pettersson et al., 1996; Thiaucourt et al., 2000).

Another controversial issue is the distinction of MmmLC and Mmc as separate subspecies: numerous investigations, based on both genetic and antigenic analyses, suggest that they may be classed together as a single subspecies within the M. mycoides subcluster (Costas et al., 1987; Leach et al., 1989; Monnerat et al., 1999; Olsson et al., 1990; Rodwell, 1982; Salih et al., 1983; Taylor et al., 1992; Vilei et al., 2006).

The phylogeny of the M. mycoides cluster inferred from 16S rRNA gene sequences (Pettersson et al., 1996) relied on too few nucleotide variations to construct a reliable phylogenetic tree. It was then proposed that further studies be based on sequence analysis of alternative genes. To this aim, five housekeeping genes have been identified (fusA, glpQ, gyrB, lepA and rpoB), taking into consideration the requirements for phylogenetic studies described by Woese (1987). Phylogenetic analysis based on concatenated sequences from these five genes and reinforced by separate analysis of each of the five genes has enabled us to determine evolutionary relationships between all the members of the M. mycoides cluster.

	METHODS

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Determination of partial gene sequences.
A sample of strains from various geographical origins was chosen carefully, including representatives of all members of the M. mycoides cluster and related species and adapting the number of strains to the known variability within each group. The 28 strains analysed are listed in Table 1, with reference to their origin. The strains were characterized according to results obtained using two different techniques: growth inhibition with hyperimmune sera (Clyde, 1983) and dot immunobinding (Poumarat et al., 1992).

The selection of primer pairs for amplification of fusA, glpQ, gyrB, lepA and rpoB sequences (Table 2) was based on initial alignments of sequences obtained from published mycoplasma genomes (http://cbi.labri.fr/outils/molligen/home.php) as well as from ongoing genome sequencing projects (MmmLC strain 95010-C1 and MmmSC strain 8740-Rita). Amplification of each of the target sequences was performed in 50 µl reactions containing 1x Taq buffer (Qiagen) (giving a final MgCl₂ concentration of 1.5 mM), 150 µM dCTP and dGTP, 300 µM dATP and TTP (dNTPs from Roche), 0.4 µM each of the corresponding forward and reverse primer, 1 U Taq polymerase (Qiagen) and 1 µl sample. The PCR thermal program consisted of an initial denaturation step of 2 min at 94 °C followed by 35 cycles of 30 s at 94 °C, 30 s at 52 °C and 45 s at 72 °C and a final extension step of 5 min at 72 °C. An exception was made for the PCR with primers GlpQ-L-F/-R, for which the annealing temperature was 45 °C, with the reaction conditions otherwise remaining the same.

Phylogenetic analyses.
Sequence analyses were conducted using MEGA 3.1 (Kumar et al., 2004; http://www.megasoftware.net), whereas Darwin 5.0 (Perrier et al., 2003; http://darwin.cirad.fr/darwin) was used for diversity analyses. Phylogenetic trees were constructed using the neighbour-joining algorithm (Saitou & Nei, 1987) based on pairwise dissimilarities between strains. Since other methods such as minimum evolution and maximum parsimony by close-neighbour interchange (MEGA 3.1) generated nearly identical topologies, the neighbour-joining method was retained for its properties of convergence and consistency, as well as low computational complexity.

Pairwise distances were calculated as the proportion of non-matching sites between pairs of sequences. The three positions corresponding to an extra codon in the glpQ sequences of the M. putrefaciens strains were deleted to avoid gaps. Since the sequences were highly similar, the effect of multiple substitutions was considered negligible and no correction was applied to dissimilarities. The mean net distance between groups was also calculated as the arithmetic mean of the pairwise distances between two groups corrected by the corresponding mean distance within groups (MEGA 3.1).

A bootstrap analysis with 500 replicates was performed to test the stability on randomly chosen sets of positions. To analyse the stability through the sample of strains, the influence of each sequence on the tree structure was estimated using a tool available in Darwin 5.0. In a jackknife-like procedure, each unit is successively removed and a partial tree is inferred from all units except the removed one. This tree is compared to the initial tree using a topological criterion, the quartet tree distance (Qd), which measures among all quartets (subsets of four sequences) the proportion of those that do not share the same topology. This distance is regarded as a measure of the influence that the removed sequence has on the tree structure. A high Qd value for a given unit means that this sequence conveys a strong and atypical signal, largely incompatible with the common signal of the other units, and is highly influential on the tree structure. On the other hand, a low Qd value means that the removed unit conveys a signal similar to that of other units and is not particularly influential on the structure. Similar and low Qd values are expected for a coherent set of units. The detection of particularly high Qd values warns of the presence of atypical units. Since tree construction methods are known to be very sensitive to atypical units, sequences with unusual strong influence were excluded in order to infer more reliable trees.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
Phylogenetic approach
Five conserved protein-coding genes, presenting greater variability than 16S rRNA gene sequences, were selected for phylogenetic analysis of the M. mycoides cluster. These genes were located at distant positions, distributed along the genome of sequenced mycoplasmas belonging to this cluster. Four of them are informational genes: fusA encodes the elongation factor G, gyrB encodes the DNA gyrase subunit B, lepA encodes the GTP-binding membrane protein or elongation factor LepA and rpoB encodes the DNA-dependent RNA polymerase -subunit. These universally conserved genes have been selected as complementary to 16S rRNA gene analysis for bacterial identification and phylogenetic studies (Santos & Ochman, 2004) and rpoB has already been used for phylogenetic analysis of Mycoplasma species (Kim et al., 2003; Vilei et al., 2006). An operational gene, glpQ, was also included. It encodes glycerophosphoryl diester phosphodiesterase and has also been used to define prokaryote taxa (Schwan et al., 2005).

Assuming that the evolutionary history of organismal lineages in the M. mycoides cluster can be properly estimated in the form of a phylogenetic tree, it was proposed that this ‘species tree’ may be inferred from the phylogenies based on the five selected genes, the ‘gene trees’. However, it is now well established that different genes sampled from the same set of taxa can produce markedly different phylogenies because of limited variability, inadequate sampling or random convergence, but also because of biological phenomena related to evolutionary events such as horizontal transfer or selection pressure. The problem is then to reconcile the trees derived from different genes to approximate the species tree. Two approaches are generally proposed: consensus methods and total evidence. The former (strict consensus, relaxed consensus or, more recently, consensus networks) considers discordance as uncertainty and produces trees that are often largely unresolved. In contrast, the total evidence approach infers a tree from a set of concatenated gene sequences assuming that each gene tree contains, at least in part, a common phylogenetic signal that will emerge with sufficient sampling. It has been shown that total evidence is often efficient in overcoming the incongruences present in single-gene analyses (Rokas et al., 2003). Therefore, the approach chosen in this study was to infer the phylogeny of the M. mycoides cluster from a sequence generated by concatenation head-to-tail of the five selected gene sequences. However, since the expected statistical smoothing is not guaranteed with only five genes, the confidence attached to the inferred phylogeny would be strengthened if the common structure could be retrieved, at least at the level of the main subclusters, in each of the individual gene phylogenies or if the biological causes for strong incongruence on a particular gene could be recognized and explained. For this reason, each of the five gene trees was analysed separately and compared to the total evidence tree.

The M. mycoides cluster tree as inferred from a set of five concatenated protein-coding sequences
Primer pairs for amplification of fusA, glpQ, gyrB, lepA and rpoB sequences were selected on the basis of homologous regions detected by alignment of the available sequences of the type strains MmmSC PG1^T and Mcc California kid^T and sequences obtained through ongoing genome sequencing projects (MmmLC strain 95010-C1 and MmmSC strain 8740-Rita). The primers listed in Table 2 yielded the expected PCR products from all of the 24 additional strains belonging to the M. mycoides cluster and related species analysed in this study (Table 1), whereas unrelated mycoplasmas such as Mycoplasma auris UIA^T failed to yield a PCR product. It must be noted that, as seen by analysis of available, unrelated mycoplasma gene sequences, it is unlikely that these primer pairs will be adequate for PCR amplification from other taxa. In all, 28 sequences were obtained for each of the five genes, which were aligned and trimmed to the same size, providing gene fragments ranging from 533 to 657 bp (Table 3). The five gene fragments corresponding to each of the 28 strains were concatenated head-to-tail, providing a set of sequences of 2914 bp for phylogenetic analysis.

View larger version (39K): [in this window] [in a new window]   Fig. 1. Pairwise distance analysis of concatenated protein-coding sequences from 28 strains calculated as the proportion of non-matching sites between pairs of sequences. The mean net distances between groups are displayed at the top right. Boxes group the different subspecies, with a bold box grouping M. mycoides subsp. mycoides LC and M. mycoides subsp. capri. For details on strains including abbreviations refer to Table 1.

View larger version (17K): [in this window] [in a new window]   Fig. 2. Phylogenetic tree derived from distance analysis of five concatenated protein-coding sequences (fusA, glpQ, gyrB, lepA, rpoB). The tree was constructed using the neighbour-joining algorithm. Bootstrap percentage values were calculated from 500 resamplings and values over 70 % are displayed. Bar, distance equivalent to 1 substitution per 100 nucleotide positions. The location of subspecies and groups of strains is indicated. See Table 1 for strain details and Supplementary Table S1 for sequence accession numbers.

The structure of each of the trees based on individual gene sequences was compared to the topology of the tree based on concatenated sequences, focusing comparisons on the distribution of the different subspecies according to the main branches supported by high bootstrap values.

Tree based on fusA sequences.
The phylogenetic tree constructed from fusA sequences (Fig. 3a) was in general agreement with the tree based on concatenated sequences. The two subclusters where clearly distinguished: M. mycoides subspecies on one side, M. capricolum subspecies and MBG7 on the other. Within the M. mycoides subcluster, the two MmmSC strains clustered together, though they were not neatly separated from the rest of the subcluster, where MmmLC and Mmc remained indistinguishable. As for the M. capricolum subgroup, Mccp strains could not be clearly separated from Mcc strains, whereas MBG7 constituted a distinct branch. All these main branches were supported by high bootstrap values (91–100 %).

View larger version (18K): [in this window] [in a new window]   Fig. 3. Evolutionary distance trees based on fusA sequences of 561 bp (a) and glpQ sequences of 538 bp (b). The trees were constructed using the neighbour-joining algorithm on a set of 23 strains belonging to the M. mycoides cluster. They are rooted on the grafting position of the uncharacterized strain 8756-C13 (labelled ‘M sp.’), used as an outgroup. Bootstrap percentage values were calculated from 500 replications and values over 70 % are displayed. Bars, distance equivalent to 1 substitution per 100 nucleotide positions. See Table 1 for strain details and Supplementary Table S1 for sequence accession numbers.

Tree based on gyrB sequences.
The tree inferred from gyrB sequences was incongruent with the phylogeny based on concatenated sequences (Fig. 4a). It differed from the fusA and glpQ trees in the position of the MmmSC strains, which left the M. mycoides subcluster to join the M. capricolum subcluster (98 % bootstrap). The MBG7 strains were attached to the M. capricolum subcluster, where Mccp constituted a separate branch. Independent analyses on each codon position revealed the same incongruent topology, and no influential units were identified among the 23 sequences. However, closer examination of variable nucleotide positions showed that the MmmSC strains shared several variant nucleotides with MBG7 strains, which attracted them towards the M. capricolum subcluster. These nucleotides were concentrated (7 of 10) in a short subsequence between positions 140 and 240. Therefore, an analysis restricted to the second half of the sequence (Fig. 4b) agreed globally with the concatenated sequence phylogeny, with MmmSC placed in the M. mycoides subcluster.

View larger version (22K): [in this window] [in a new window]   Fig. 4. Evolutionary distance trees based on gyrB sequences of 533 bp (a) and based on the second half of these sequences from nucleotide position 267 (b). For further details refer to Fig. 3. See Table 1 for strain details and Supplementary Table S1 for sequence accession numbers.

View larger version (19K): [in this window] [in a new window]   Fig. 5. Evolutionary distance trees based on lepA sequences of 657 bp (a) and based on the second half of these lepA sequences, from nucleotide position 328 (b), and a tree inferred from the first codon positions of the 657 bp lepA sequences (c). For further details refer to Fig. 3. See Table 1 for strain details and Supplementary Table S1 for sequence accession numbers.

View larger version (24K): [in this window] [in a new window]   Fig. 6. Evolutionary distance trees based on rpoB sequences of 625 bp constructed from the set of 23 strains belonging to the M. mycoides cluster (a) and constructed as above but excluding strain MmmLC-1 (b). For further details refer to Fig. 3. See Table 1 for strain details and Supplementary Table S1 for sequence accession numbers.

This approach has allowed us to construct a reliable tree to assign phylogenetic positions to all members of the M. mycoides cluster, showing once more that the existing taxonomy needs to be reviewed.

Phylogenetic position of Mycoplasma sp. bovine group 7 of Leach.
The greatest incongruences, found in two of the five gene sequences analysed in this study, resulted from the attraction between MmmSC and MBG7. For gyrB, this proximity was limited to a subsequence and, in the case of lepA, it essentially concerned the least-constrained codon positions. The common structure corresponding to fusA and glpQ (and rpoB when excluding the influential unit MmmLC-1), which places MmmSC in the M. mycoides subcluster and MBG7 in the M. capricolum subcluster, is not strongly contradicted by analyses of the other two genes and can therefore be retained. Furthermore, the partial attraction observed between MBG7 and MmmSC explains the ambiguity that persists in the present classification and why, unlike the other members of the M. mycoides cluster, the group of strains referred to as MBG7 has not yet been assigned a species name. Investigations based on DNA–DNA hybridization (Christiansen & Ernø, 1982), identification with a DNA probe (Taylor et al., 1992) and electrophoretic analysis of isoenzymes and serology (Salih et al., 1983) have suggested the classification of MBG7 as a distinct species. DNA–DNA hybridization studies by Askaa et al. (1978), lipoprotein sequence similarity and immunological cross-reactions (Frey et al., 1998), as well as sequence similarity of the glycerol transport locus gtsABC (Djordjevic et al., 2003), have revealed a close relatedness to MmmSC. In contrast, serological cross-reactions instead suggested that MBG7 was closer to the M. capricolum subspecies (Cottew et al., 1987; Ernø et al., 1983), which was confirmed by 1D and 2D SDS-PAGE profile comparisons (Costas et al., 1987; Olsson et al., 1990). In addition, MSC4, a small stable RNA, was found to be specific to the species M. capricolum and MBG7 (Ushida et al., 2003), and recent phylogenetic studies based on 16S rRNA gene sequences (Pettersson et al., 1996), 16S–23S rRNA intergenic spacer sequences (Harasawa et al., 2000) and a putative membrane protein gene (Thiaucourt et al., 2000) all grouped MBG7 in the M. capricolum subcluster. The results presented here provide additional evidence regarding the genetic identification of this group of strains and support its classification as a subspecies of M. capricolum.

Interestingly, all strains of MBG7 provided identical sequences for each of the five genes analysed. This seems to indicate a very limited intraspecies variability for this group of strains and, presumably, a recent differentiation within the species M. capricolum.

All members of the M. mycoides cluster have been isolated from small ruminants (Atalaia et al., 1987; Kusiluka et al., 2001), where multiple infections are frequent (Cottew & Yeats, 1982). These conditions may well promote horizontal gene transfer between the different species. Genomic exchanges were suspected for several strains analysed in this study. This is the case for strain MmmLC-5, for which the first half of the lepA sequence was characteristic of M. capricolum while the second half corresponded to a typical M. mycoides sequence. A lateral transfer of genes or gene fragments may explain the local proximity that exists between MBG7 and MmmSC that has been observed previously and is again shown in two of the five genes analysed in this work.

Relatedness of M. mycoides subsp. mycoides LC and M. mycoides subsp. capri.
The distinction of MmmLC and Mmc as separate subspecies has been questioned for some time. Numerous investigations have provided evidence to suggest that they may be grouped as a single subspecies within the M. mycoides subcluster. Although early DNA–DNA hybridization studies by Askaa et al. (1978) suggested that Mmc was closer to MmmSC than to MmmLC, analyses of 1D and 2D PAGE protein patterns proved the opposite (Costas et al., 1987; Leach et al., 1989; Olsson et al., 1990; Rodwell, 1982; Salih et al., 1983). Identification using a DNA probe (Taylor et al., 1992) and analysis of LppA sequence similarity and serological cross-reactions (Monnerat et al., 1999) also suggested a very close relatedness between MmmLC and Mmc, and two phylogenetic studies of the M. mycoides cluster concluded that they should be grouped into a single entity (Pettersson et al., 1996; Thiaucourt et al., 2000). However, the International Committee on Systematics of Prokaryotes Subcommittee on the taxonomy of Mollicutes has indicated that additional comparative analysis is required in order to clarify this matter (Johansson & Bradbury, 2007).

Additional evidence has recently been presented by Vilei et al. (2006) based on rpoB sequence comparisons. In the present work, comparisons have been extended to five genes using a similar sample of strains, though strain WK354, included as an Mmc strain in previous studies (Monnerat et al., 1999; Thiaucourt et al., 2000), was characterized in this work as MmmLC by both growth inhibition and dot immunobinding techniques. As in previous studies, the data obtained in this work show that MmmLC is closer to Mmc than to the biotype MmmSC and that MmmLC and Mmc are not sufficiently distant to constitute separate entities. Furthermore, neither of these two organisms represents the aetiological agent of a characteristic disease, which may have been a justification for their separation. For example, this had been the rationale for the establishment of Mccp as a separate subspecies of M. capricolum.

In conclusion, MmmLC and Mmc appear to constitute a single entity, which could justify their combination into the same taxonomic subspecies.

Position of M. putrefaciens and other related species.
M. putrefaciens is not included in the classical M. mycoides cluster and may be easily differentiated from the members of this cluster by biochemical and serological methods (Tully et al., 1974) as well as by a specific PCR test (Peyraud et al., 2003). However, on the basis of 16S rRNA gene sequence similarity, it was included in the phylogenetic M. mycoides cluster, within the Spiroplasma group (Weisburg et al., 1989). Also based on 16S rRNA sequence data, the closely related species M. cottewii and M. yeatsii were included in this phylogenetic cluster, branching with M. putrefaciens (Heldtander et al., 1998). Although the latter authors suggested that these three species should actually constitute a separate cluster, their precise taxonomic position remains uncertain. In this study, M. putrefaciens, M. cottewii and M. yeatsii were distant from the members of the classical cluster, with an important number of strictly specific nucleotide variations, confirming that they should not be included in the M. mycoides cluster.

Application to identification and classification of strains
The great relevance of diseases such as CBPP and CCPP and the fact that several members of the M. mycoides cluster produce similar clinical signs in small ruminants with different virulence mean that precise identification is of paramount importance. However, some of these mycoplasmas are difficult to differentiate by conventional techniques (Cottew et al., 1987). Growth inhibition, which is often used for species identification, is frequently hampered both by interspecies cross-reactions and by intraspecies variability. Cross-reactions are frequent between biotypes MmmSC and MmmLC (Cottew & Yeats, 1978; Thigpen et al., 1983) and between MBG7 and Mccp (Cottew et al., 1987; Ernø et al., 1983; Kibe et al., 1985), hindering confirmation of CBPP and CCPP, respectively. A high degree of intraspecies heterogeneity has also been shown using several techniques, particularly within MmmLC/Mmc and also Mcc to a certain extent, whereas MmmSC and Mccp appear rather homogeneous (Abu-Groun et al., 1994; Christiansen & Ernø, 1990; Costas et al., 1987; Kusiluka et al., 2001). Our data correlate with these observations and emphasize once more the limitations encountered in the classification of certain strains and, furthermore, in the definition and delimitation of species and subspecies within the M. mycoides cluster.

Given the increased resolving power of the strategy presented in this work, it was hypothesized that it might allow strains that have proved difficult to affiliate to be classified. This is the case for strain 8756-C13, isolated from a Rocky Mountain goat, for which identification by classical methods is challenging. Cultural parameters indicated that it might be an MmmLC strain, though growth-inhibition tests were negative for both MmmLC and Mmc. It was finally classed as MmmLC on the basis of immunofluorescence results, although electrophoretic patterns were inconclusive. The data from this study show that this is not a typical strain of the M. mycoides cluster, but a nearby relative, located closer to this cluster than to other related species such as M. putrefaciens, M. cottewii and M. yeatsii. Presumably, study of new isolates originating from diverse geographical and host origins will result in the identification of further ‘atypical’ strains related to this cluster and, until whole-genome sequence comparisons become accessible to diagnostic laboratories, protein gene sequence data may prove to be a good addition to 16S rRNA genes for fine strain characterization. However, analyses based on multiple gene sequences are still difficult to use for routine diagnostic purposes. On the other hand, fusA may be an interesting alternative for rapid identification and phylogenetic positioning by PCR and sequencing, for which the set of sequences presented in this work may serve as a reference: although variability was low among the fusA sequences from M. mycoides strains, the tree inferred from this gene was very similar to that based on the concatenated sequences.

Concluding remarks
Conserved protein-coding gene sequences may be used to complement and extend 16S rRNA gene sequence data to allow differentiation of closely related groups of organisms, such as the members of the M. mycoides cluster. However, a single gene tree can be largely incongruent with the species tree, which represents its true evolutionary history. Therefore, several genes must be analysed simultaneously. It is proposed here to consider the tree inferred from five concatenated gene sequences as a reliable estimation of the species phylogeny, since this ‘mean structure’ can be detected in each individual gene tree, even if it is partially masked by specific gene evolution.

This approach has resulted in the construction of a reliable phylogenetic tree, which will be a universal tool for the classification of strains of the M. mycoides cluster, including those difficult to identify by classical methods. Furthermore, it may lead to the development of new techniques for rapid species identification and molecular epidemiology.

	ACKNOWLEDGEMENTS

 
We are grateful to Valerie Barbe from Genoscope (Evry, France) for the results of MmmLC strain 95010-C1 whole-genome sequencing and to François Poumarat and Florence Tardy from AFSSA (Lyon, France) for characterization of the strains by dot immunobinding. We also thank Alan Blanchard and Pascal Sirand-Pugnet (INRA-Bordeaux, France) for invaluable discussions and are indebted to Janet Bradbury for critical reading of the manuscript and for her precious suggestions.

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