- Record: found
- Abstract: found
- Article: found
Unexpected genetic diversity of Mycoplasma agalactiae caprine isolates from an endemic geographically restricted area of Spain
research-article
Author(s):
Christian De la Fe
1
,
,
Joaquín Amores
1 ,
Florence Tardy
2 ,
Eveline Sagne
4
,
3 ,
Laurent-Xavier Nouvel
4
,
3 ,
Christine Citti
3
,
4
Publication date (Electronic):
27 August 2012
Journal:
BMC Veterinary Research
Publisher:
BioMed Central
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Abstract
Background
The genetic diversity of Mycoplasma agalactiae (MA) isolates collected in Spain from goats in an area with contagious agalactia (CA) was assessed using a set of validated and new molecular typing methods. Validated methods included pulsed field gel electrophoresis (PFGE), variable number of tandem repeats (VNTR) typing, and Southern blot hybridization using a set of MA DNA probes, including those for typing the vpma genes repertoire. New approaches were based on PCR and targeted genomic regions that diverged between strains as defined by in silico genomic comparisons of sequenced MA genomes.
Results
Overall, the data showed that all typing tools yielded consistent results, with the VNTR analyses being the most rapid method to differentiate the MA isolates with a discriminatory ability comparable to that of PFGE and of a set of new PCR assays. All molecular typing approaches indicated that the Spanish isolates from the endemic area in Murcia were very diverse, with different clonal isolates probably restricted to separate, but geographically close, local areas.
Conclusions
The important genetic diversity of MA observed in infected goats from Spain contrasts with the overall homogeneity of the genomic background encountered in MA from sheep with CA in Southern France or Italy, suggesting that assessment of the disease status in endemic areas may require different approaches in sheep and in goats. A number of congruent sub-typing tools are now available for the differentiation of caprine isolates with comparable discriminatory powers.
Related collections
Most cited references27
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Genomic sequencing.
Unique DNA sequences can be determined directly from mouse genomic DNA. A denaturing gel separates by size mixtures of unlabeled DNA fragments from complete restriction and partial chemical cleavages of the entire genome. These lanes of DNA are transferred and UV-crosslinked to nylon membranes. Hybridization with a short 32P-labeled single-stranded probe produces the image of a DNA sequence "ladder" extending from the 3' or 5' end of one restriction site in the genome. Numerous different sequences can be obtained from a single membrane by reprobing. Each band in these sequences represents 3 fg of DNA complementary to the probe. Sequence data from mouse immunoglobulin heavy chain genes from several cell types are presented. The genomic sequencing procedures are applicable to the analysis of genetic polymorphisms, DNA methylation at deoxycytidines, and nucleic acid-protein interactions at single nucleotide resolution.
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- Abstract: not found
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A simple and rapid method for the preparation of gram-negative bacterial genomic DNA.
W. P. Chen, T. Kuo (1993)
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- Abstract: found
- Article: not found
Being Pathogenic, Plastic, and Sexual while Living with a Nearly Minimal Bacterial Genome
Pascal Sirand-Pugnet, Carole Lartigue, Marc Marenda … (2007)
Introduction Organisms belonging to the Mycoplasma genus (class Mollicutes) are commonly described as the simplest and smallest self-replicating bacteria because of their total lack of cell wall, the paucity of their metabolic pathways, and the small size of their genome [1,2]. In the 1980s, they were shown to have evolved from more classical bacteria of the firmicutes taxon by a so-called regressive evolution that resulted in massive genome reduction [3,4]. One of the models attempting to improve understanding of the evolution of bacteria with small genomes proposes that erosion of bacterial genomes is more prone to occur in bacterial populations that are spatially isolated and sexually deficient [5]. In restricted habitats, the environment is rather steady and natural selection tends to be reduced, resulting in the inactivation of many genes by genetic drift [5,6]. In this scenario, DNA acquisition would be strongly limited, resulting, after losses of large genomic regions and accumulation of mutations, in genome stasis [7]. This evolution scheme is relevant for a number of obligate intracellular bacteria, including insect endosymbionts (e.g., Buchnera and Wigglesworthia spp.), and arguably for Chlamydia, and Rickettsia spp. The recent findings of a putative conjugative plasmid in Rickettsia felis [8] and of a substantial number of prophage, transposase and mobile-DNA genes in the insect endosymbiont Wolbachia pipientis challenged this model and it was proposed that gene inflow by horizontal gene transfer (HGT) may occur in some obligate intracellular species depending on their lifestyles [9]. Mycoplasmas share with obligate intracellular bacteria a small genome size with marked AT nucleotide bias and a low number of genes involved in recombination and repair, but forces driving their evolution may not be quite the same, as they do have a very different lifestyle. Indeed, mycoplasmas mainly occur as extracellular parasites [10] and are often restricted to a living host, with some species having the ability to invade host cells [11]. They have a predilection for the mucosal surfaces of the respiratory and urogenital tracts, where they successfully compete for nutrients with many other organisms, establishing chronic infections (Table S1). Therefore, mycoplasma populations are far from being isolated and inhabit niches where exchange of genetic material may take place. The none-to-rare occurrence of HGT reported so far for mycoplasmas [12] is therefore surprising and seems to conflict with their lifestyle. On the other hand, HGT may depend on several other factors [9] that were described as limited or lacking in most mycoplasma species and that include an efficient machinery for recombination, genetic mobile elements such as prophages or conjugative plasmids, and a means for DNA uptake. However, this view of mycoplasma biology is changing, since homologous recombination has been demonstrated in these bacteria [13,14] and some new means of exchanging DNA are being discovered [15,16]. Indeed, several pathogenic mycoplasma species relevant to the veterinary field and the murine pathogen M. pulmonis were recently shown to form biofilms [17,18], structures that have been proposed to promote DNA exchange among bacteria. This finding, together with previous evidence for DNA transfer under laboratory conditions in M. pulmonis via conjugation [19], raises the exciting question of whether some mycoplasmas species are sexually competent. Subsequently, this would suggest that mycoplasma species which co-infect the same host niches might exchange genetic material. Remarkably, biofilm formation and the occurrence of an integrative conjugative element (ICE) have both been newly described in the M. agalactiae species [16,18]. This pathogen is responsible for contagious agalactia in small ruminants [20], a syndrome that includes mastitis, pneumonia, and arthritis and that is also caused by some members of the so-called mycoides cluster, such as M. capricolum subsp. capricolum and M. mycoides subsp. mycoides Large Colony. Although producing similar symptoms in the same host, these species belong to two distinct and distant branches of the mollicute phylogenetic tree (Figure 1). Their relative phylogenetic positions are irrespective of whether the tree is constructed from aligned 16S rDNA (Figure 1A) or from 30 aligned proteins shared by all living organisms [21] (Figure 1B). M. agalactiae belongs to the hominis phylogenetic branch, together with a closely related ruminant pathogen, M. bovis, while the six members that comprise the “mycoides cluster” belong to the spiroplasma phylogenetic branch [22]. Whole-genome sequences are available for two members of the mycoides cluster; M. mycoides subsp. mycoides SC [23], which is responsible for contagious bovine pleuropneumonia [24], and M. capricolum subsp. capricolum [25]. In contrast, there is a limited amount of sequence data available for M. agalactiae and M. bovis. Mycoplasmas that have been fully sequenced in the hominis phylogenetic group are a murine pathogen M. pulmonis [26], a swine pathogen M. hyopneumoniae (strain 232 [27]; strains 7748 and J [12]), an avian pathogen M. synoviae [12], and a mycoplasma isolated from fish, M. mobile [28] (Figure 1B). Figure 1 Phylogenetic Tree of Selected Mollicutes of the Spiroplasma/Mycoplasma Branch Inferred from 16S rDNA Sequences [3] or from Concatenated Shared Proteins (A) The tree was constructed using the distance (neighbor joining) method and the gaps complete deletion option of the MEGA2 software. A bootstrap of 500 replicates was performed; the number on each node indicates the percentage with which each branch topology was supported. The phylogenetic groups spiroplasma, pneumoniae, and hominis are indicated by S, P, and H, respectively. M, mycoides cluster. Candidatus phytoplasma asteris (Onion Yellows strain) and Aster Yellows phytoplasma were chosen as outgroup species. (B) 30 COGs shared by all sequenced mollicute genomes were extracted from the MolliGen database (see Materials and Methods). After alignment of each COG, the aligned sequences were concatenated. The tree was constructed using the maximum likelihood method (PhyML). A bootstrap of 100 replicates was performed; the number on each node indicates the percentage with which each branch topology was supported. The phylogenetic groups spiroplasma, pneumoniae, and hominis are indicated by S, P and H, respectively. Candidatus phytoplasma asteris (Onion Yellows strain) and Aster Yellows phytoplasma were chosen as outgroup species. Mechanisms underlying ruminant mycoplasma diseases have yet to be elucidated and very little is known regarding the mycoplasma factors that are involved in virulence and host interaction. Genes thus far identified in M. agalactiae and for which a function in relation to virulence has been predicted are (i) a family of phase-variable related surface proteins, designated as Vpma, which are encoded by a locus subjected to high-frequency DNA rearrangements and could be involved in adhesion [29,30], (ii) the P40 protein, which is involved in host–cell adhesion in vitro but is not expressed in all field isolates [31], and (iii) the P48 protein, which has homology to an M. fermentans product with a macrophage-stimulatory activity [32]. Several of these gene products have homologs in M. bovis but not in mycoplasmas of the mycoides cluster. Whole-genome comparison between phylogenetically distant mycoplasmas that colonize the same host could provide a basis from which to comprehend the factors involved in mycoplasma host adaptation. With this initial goal, we sequenced the M. agalactiae genome of the pathogenic type strain PG2. Results revealed a classical mollicute genome with a coding capacity of 751 CDSs, half of which are annotated as encoding hypothetical products. Unexpectedly, comparative analysis of the M. agalactiae genome with that of other mollicutes and bacteria suggests that a significant amount of genes (∼18 %) has been horizontally transferred to or acquired from mycoplasmas of the mycoides cluster that are phylogenetically distant while sharing common ruminant hosts. In light of these data, we re-examined mollicute genomes for HGT events with a particular focus on those that occurred after mycoplasmas branched into three phylogenetic groups (see Figure 1 for the hominis, pneumoniae, and spiroplasma phylogenetic groups). Our analyses confirm data so far reported regarding the low incidence of HGT between Mycoplasma species with the exception of that described in this study, between M. agalactiae and members of the mycoides cluster and, to a lesser extent, between M. gallisepticum and M. synoviae. To our knowledge, this is the first description of large-scale horizontal gene transfer between mycoplasmas. Results M. agalactiae: Overall Features of a Small Genome The genome of the M. agalactiae type strain PG2 consists of a single, circular chromosome; general features are summarized in Table 1. The genome sequence was numbered clockwise starting from the first nucleotide of the dnaA gene, which was designated as the first CDS (MAG0010). This gene is involved in the early steps of the replication initiation process [33] and is typically located near mycoplasma origins of replication. Indeed, dnaA boxes flanking the dnaA gene were shown in M. agalactiae to promote free replication of the ColE1-based E. coli vectors in which they were cloned [34]. Although these experiments clearly localized the M. agalactiae oriC in the vicinity of the dnaA gene, whole-genome analysis did not indicate a significant GC-skew inversion [35] in this region (unpublished data). In contrast to other mycoplasma genomes [36], a high level of gene-strand bias was not observed, even when restricting the analysis to the dnaA vicinity. Table 1 General Features of the M. agalactiae (MA) Genome Compared to Those of Mycoplasma Species of the Same Phylogenetic Group (MYPU, MMOB, MHP) and Other Phylogenetically Remote Ruminant Mycoplasmas (MCAP and MmmSC) Overall, M. agalactiae strain PG2 possesses a typical mollicute genome, with a small size (877,438 bp), a low GC content (29.7 moles %), a high gene compaction (88% of coding sequence), and UGA preferentially used as a tryptophan codon over UGG (Table 1). Its GC% value is slightly higher than that observed for some other mycoplasma species but is close to the average GC content (28%) calculated from the 16 available mollicute genomes. Using the CAAT-box software package, 751 CDSs were identified, 404 (53.8%) of which had a predicted function. The genome also contains 34 tRNA genes and two nearly identical sets of rRNA genes with two 16S–23S rRNA operons (MAG16S1-MAG23S1 and MAG16S2-MAG;23S2) and the two 5S rRNA genes (MAG5S1 and MAG5S2) clustered in two loci separated from each other by ∼400 kb (Figure 2). Figure 2 Schematic Presenting the Circular M. agalactiae Genome and the Location of Genes Inherited by Horizontal Transfer from the Mycoides Cluster The 136 genes potentially inherited from the mycoides cluster are shown on the outer circle as yellow bars, with the inner circles representing the positive (green) or the negative (red) strand. The two adjacent 16S-23S rRNA operons and the two 5S rRNA genes are represented by a star and a dot, respectively. The genomic organization of some transferred genes (yellow boxes above and below the line according to their relative orientation on the genome) is illustrated around the circular map, with pseudogenes indicated by a red cross and tRNAs by black arrowheads. Gene clusters presenting the same organization in M. agalactiae, M. mycoides subsp. mycoides SC, and M. capricolum subsp. capricolum are underlined by red bars. The ICE region, homologous to that of M. capricolum subsp. capricolum but missing in M. mycoides subsp. mycoides SC type strain, is noted by a cross. CHP, conserved hypothetical protein; HP, hypothetical protein; Lipo, predicted lipoprotein; and TMB, predicted transmembrane protein. HGT among Distant Mycoplasma Species Sharing the Same Host Prediction of M. agalactiae CDS function was based on BLAST searches against SwissProt, trembl, and MolliGen databases. For CDSs showing significant similarities with database entries, most best BLAST hits (BBH) were found with M. synoviae and M. pulmonis, which belong, together with M. agalactiae, to the hominis phylogenetic group (Figure 1). Unexpectedly, a large number of BBH were also obtained with M. mycoides subsp. mycoides SC or M. capricolum subsp. capricolum, which both belong to the mycoides cluster (Figure S1). Since this cluster is exclusively composed of ruminant pathogens and is relatively distant from M. agalactiae in the mollicute phylogenetic tree (Figure 1), this prompted us to closely examine the corresponding CDSs. A total of 136 M. agalactiae CDSs were then identified as having their BBH with organisms from the mycoides cluster, with 50 having no significant similarity outside of this cluster (Table S2). Of the remaining 86, 73 also had a homolog in at least one in the four available genomes of the hominis group (M. pulmonis, M. mobile, M. synoviae, and M. hyopneumoniae) (Table S2) and 13 in other mollicutes or bacteria (Tables S2 and S3). Further phylogenetic tree reconstruction showed that 75 out of 86 CDSs display highly significant bootstrap values (≥ 90%) supporting HGT with homologs of the mycoides cluster. Among the 11 CDSs with low bootstrap values, six belong to gene clusters in which synteny is conserved in the mycoides cluster, three belong to an ICE element (see below) found in M. agalactiae and M. capricolum subsp. capricolum and two others were not further considered, suggesting that ∼134 CDS have undergone horizontal gene transfer in between mycoplasma(s) of the mycoides cluster and M. agalactiae or its ancestor. Of the predicted transferred CDSs, nine and 22 have a homolog either in M. mycoides subsp. mycoides SC or in M. capricolum subsp. capricolum, respectively, while 102 have homologs in both species. Phylogenetic analysis and similarity comparisons of the 102 CDSs did not allow us to conclude whether they were more similar to M. mycoides subsp. mycoides SC or to M. capricolum subsp. capricolum (Figure S2). Additionally, one CDS (MAG4270) had a homolog only in M. mycoides subsp. capri, for which only a limited number of sequences are available. The occurrence of HGT was further supported by the genomic organization in M. agalactiae of 115 of the predicted transferred genes that occur as clusters containing two to 12 elements with approximately half of them displaying the same organization as in M. mycoides subsp. mycoides SC and M. capricolum subsp. capricolum genomes. Eleven of these clusters, which are distributed all over the M. agalactiae genome, are shown in Figure 2. As previously mentioned, 73 of the predicted transferred CDSs have an ortholog in genomes of the hominis group. In a hypothesis regarding transfer from the mycoides cluster to M. agalactiae, one might expect to detect pseudo-paralogs [37] in the M. agalactiae genome, with one inherited from an ancestor of the hominis group, while the other was acquired by HGT. Indeed, in 17 unambiguous cases, vertically and horizontally inherited pseudo-paralogs were found. As an example, the gene encoding the glucose-inhibited division protein is present as a single copy in the genomes of M. pulmonis, M. synoviae, M. mobile, and M. hyopneumoniae. In M. agalactiae, two copies of this gene were found; one, MAG2970, has a BBH in M. pulmonis, while the other, MAG1470, has a BBH in M. mycoides subsp. mycoides SC. The oligopeptide ABC transporter locus (opp genes) is another interesting example, since opp genes occur twice in M. agalactiae, at two distinct loci. As shown in Figure 3, one opp locus (designated as the type 1) is composed of four opp genes (B–D and F), the sequences of which are highly similar to those of one of the two M. pulmonis opp loci. The other opp locus of M. agalactiae (type 3, Figure 3) is composed of five opp genes (A–D and F), the sequences and organization of which are closer to one of the two opp gene loci of M. capricolum subsp. capricolum and M. mycoides subsp. mycoides SC. Phylogenetic analyses of the oppB genes of types 1 and 3 with homologous sequences of other mycoplasma species suggest different origins for the two M. agalactiae opp loci. While the type 1 was inherited from a common ancestor of the hominis branch, the type 3 was laterally acquired from the mycoides cluster. A third, isolated, copy of the oppB gene (MAG4700) was predicted in the M. agalactiae genome, and might represent a relic of a displaced opp operon, as its best orthologs were found in mycoplasmas of the hominis group. Figure 3 Genomic Organization and Phylogenetic Relationship of the Genes Encoding the Oligopeptide Transporter in Mycoplasmas (A) Comparison of the genomic organization of the opp loci in M. agalactiae, M. pulmonis, M. capricolum subsp. capricolum, and M. mycoides subsp. mycoides SC. Genes are represented by boxes positioned above or below the main line according to their relative orientation on the genome. Homologous genes are indicated by identical color; closest orthologs are connected by dashed lines. A 1,357-aa insertion in the orange-colored CHP of M. agalactiae is represented by a hatched box. CHP, conserved hypothetical protein; lipo, predicted lipoprotein; M.aga., M. agalactiae; M. cap., M. capricolum subsp. capricolum; MmmSC, M. mycoides subsp. mycoides SC; and M.pul., M. pulmonis. (B) Phylogenetic tree inferred from the amino acid sequence of OppB proteins. Bootstrap support percentages (based on 500 replicates) are indicated near each node of the tree. The two copies present in M. agalactiae are indicated by red arrows. Sequences are designated according to their mnemonic followed by their identification number as indicated in public databases. MAG, M. agalactiae; MCAP, M. capricolum subsp. capricolum; Mfl, Me. florum; mhp, M. hyopneumoniae; MMOB, M. mobile; MSC, M. mycoides subsp. mycoides SC; and MYPU, M. pulmonis. For CDSs found only once in the genome of M. agalactiae, the situation might be more complex, as illustrated by the glycerol kinase/glycerol uptake facilitator operon, glpK–glpF (MAG4470–MAG4480), which was unambiguously found to originate from a mycoides ancestor (Figure S3). This operon occurs as a single copy in all mycoplasma genomes of the hominis group but is absent from M. synoviae. Because of the relative phylogenetic closeness of M. agalactiae and M. synoviae (Figure 1B), the question arises as to whether glpK–glpF was lost in their common ancestor and acquired later on by M. agalactiae from the mycoides cluster. While examining M. agalactiae candidates for HGT, sequence alignments showed that 38 are truncated versions of their homologs in M. capricolum subsp. capricolum and M. mycoides subsp. mycoides SC, or were annotated as pseudogenes (Table S2 and Figure S2). Additionally, only 14 CDSs were suspected to have undergone HGT between M. agalactiae and species of the pneumoniae phylogenetic group or non-mollicute bacteria (Table S3). Putative Barrier to Gene Transfer: Hyper-Variable Restriction-Modification Systems Since restriction–modification (RM) systems serve in bacteria as a tool against invading DNA [38], it was of interest to specifically search for these systems in light of the high level of HGT in M. agalactiae. One locus encoding a putative RM system is composed of six genes with homology to type I RM systems (Figure S4) and was designated hsd. It contains (i) two hsdM genes (MAG5650 and MAG5730), coding for two almost identical modification (methylase) proteins (94% identity), which would methylate specific adenine residues; (ii) three hsdS genes (MAG5640, MAG5680, and MAG5720), each coding for a distinct RM specificity subunit (HsdS) that shares homology with the others (between 50% to 97% similarities); and (iii) one hsdR pseudo-gene (MAG5700/MAG5710), which is interrupted in the middle by a stop codon and would otherwise encode a site-specific endonuclease (HsdR). Finally, the hsd locus contains two hypothetical CDSs (MAG5660 and MAG5670) and one gene (MAG5690), whose product displays 76.9 % similarity to a phage family integrase of Bifidobacterium longum [39] and motifs found in molecules involved in DNA recombination and integration. In M. pulmonis, the hsd locus has been shown to undergo frequent DNA rearrangements but the gene encoding the putatively involved recombinase is located elsewhere on the genome [26,40]. Apart from this locus, only three other unrelated M. agalactiae CDSs display similarities with the restriction–modification system, one of which was annotated as a pseudogene. M. agalactiae Lipoproteins: An Extended Repertoire Including Several Lipoprotein Genes Involved in HGT with the Mycoides Cluster Mycoplasma lipoproteins are of particular interest because they have been proposed to play a role in the colonization of specific niches and in interaction with the host [11,41]. In order to identify the putative lipoproteins encoded by the M. agalactiae genome, we combined results obtained by PS-SCAN analysis with the detection of a signature that was defined by using MEME/MAST software and a set of previously identified mycoplasma lipoproteins (see Material and Methods). This strategy resulted in the prediction of 66 lipoproteins, 85% of which were annotated as hypothetical proteins. The remaining 15% correspond to the previously characterized Vpmas, P40, P30, and P48; and to two CDSs homologous to the substrate-binding protein of an oligopeptide (OppA, MAG0380) and to an Alkylphosphonate ABC (MAG5030) transporter, respectively. Among the genes encoding the 66 predicted lipoproteins, our analyses indicated that the corresponding genes of 19 have undergone HGT with the mycoides cluster (see Tables S2, S5, and S6). These 19 CDSs were annotated as hypothetical proteins, however, four (MAG2430, MAG3260, MAG6480, and MAG7270) share a high level of similarity, and constitute, with nine other polypeptides (MAG0210, MAG0230, MAG1330, MAG1340, MAG3270, MAG4220, MAG4310, MAG6460, and MAG6490), a protein family. A MEME/MAST analysis indicated that the 13 proteins of this family shared one to ten repeats of a 25 amino-acid motif A ([KN]W[DN][TV]SNVT[ND]MSSMFxGAK[KS]FNQ[DN][IL]S) (Figure S5). This motif is highly similar to the DUF285 domain of unknown function predicted in a large number of mycoplasma lipoproteins and found only in the mycoides cluster and in some non-mollicute bacteria (i.e., Listeria monocytogenes, Enterococcus faecalis, Lactobacillus plantarum, and Helicobacter hepaticus). A second motif B ([FM]PKN[VT][KV]KVPKELP[EL][EK][IV]TSLEKAFK[GN]) was also found in most of the family proteins. Of the 13 members of the family, whose corresponding genes are distributed all over the chromosome, five were predicted to be lipoproteins; the others may constitute a reservoir of sequence to generate surface variability. Altogether, these data suggest that M. agalactiae has inherited a family of genes encoding potentially variable lipoproteins that are otherwise specific to the mycoides cluster. Another remarkable lipoprotein family is found in the portion of the genome (MAG7050–MAG7100; Figure S4) that encodes the phase-variable, related Vpma products. The Vpma family has been extensively described [29,30] and was previously shown to present typical elements of mobile pathogenicity islands [29]. However, comparison of the Vpmas coding sequences with other mycoplasma genomes indicate that they are specific of the M. agalactiae species, although their variation in expression and genetic organization closely resembles the Vsp system found in the close relative M. bovis [42–44]. No similar system or coding sequences was found in the mycoides cluster. ICE as Vehicles for HGT in Mycoplasmas? To our knowledge, attempts to naturally transform M. agalactiae or other mycoplasma species have failed, suggesting that HGT, if it occurs, is mediated via another mechanism. Only a limited number of viruses or natural plasmids have been described so far in mycoplasmas that could account as vehicles for HGT, apart from a new ICE that has been described in a few Mycoplasma spp. [12,15]. In a recent study, we documented the occurrence of such an element in M. agalactiae strain 5632 (ICEA5632) as chromosomal multiple copies and as a free circular form [16]. One copy, ICEA5632-I, was fully sequenced and Southern blot analyses suggested that it occurs in a minority of strains that did not include the PG2 type strain [16, 45]. However, detailed sequence analyses performed in this study revealed that 17 CDSs of the M. agalactiae PG2 genome display different levels of similarities to CDSs present in ICEA5632-I and in other ICEs (Table S4) found in M. capricolum subsp. capricolum (ICEC), M. fermentans (ICEF-I and –II) [15], and M. hyopneumoniae strain 7448 (ICEH) [12]. These seventeen CDSs are clustered in the PG2 genome within a unique 20-kb locus, ICEAPG2 (Figure 4), and those with an ortholog in M. fermentans ICEF and/or M. agalactiae ICEA5632-I were designated as in previous reports [15,16]. Surprisingly, best alignments for ICEA products of the PG2 strain were consistently obtained with M. capricolum subsp. capricolum ICEC counterparts, with an average of 40% identity and 75% similarity, whereas alignments with ICEA5632-I or ICEF gave lower values. This close relationship between ICEAPG2 and ICEC was confirmed by bootstrap values of the phylogenetic trees inferred from the amino acid sequence of TraG, TraE, ORF19, and ORF22 (Figure S6). Moreover, ICEAPG2 and ICEC share three homologous CDSs (noted as x, y, and z in Figure 4) lacking in ICEA5632-I and other ICEs. All these results indicate a close relationship between ICEAPG2 and ICEC, and suggest that the ICEs found in strains PG2 and 5632 have a different history. Figure 4 Genomic Organization of ICEs and ICE-Related Genes in Mollicutes Arrows represent CDSs, hatched arrows represent pseudogenes. Homologous CDSs are identified by the same colour and same number or letter underneath. Numbering is using the nomenclature of M. fermentans ICEs (ICEFI and II) as a reference [15], whereas letters refer to the CDSs without any ortholog in ICEF. Locus tags for genes from the ICEs of M. agalactiae strain PG2, M. capricolum subsp. capricolum strain California Kid, and M. hyopneumoniae strain 7448 (MAG_4060–3860, MCAP_0554–0571, and MHP7448_424– 412, respectively) are indicated above the arrows. Other ICE-related genes present in M. hyopneumoniae strain 232, M. pulmonis, M. mycoides subsp. mycoides SC, and S. citri are not shown on the figure. ICEAPG2 , ICE of M. agalactiae strain PG2; ICEC, ICE of M. capricolum subsp. capricolum strain California Kid; ICEA5632-I, ICE of M. agalactiae strain 5632; ICEH7448, ICE of M. hyopneumoniae strain 7448; and ICEH232, ICE of M. hyopneumoniae strain 232. In strain PG2, the gene encoding TraE (MAG3910/MAG3920), a major actor in DNA transport across the conjugative pore, was found to be disrupted. In addition, a total of 11 out of the 20 ICEAPG2-CDSs might represent pseudogenes (hatched arrows in Figure 4), due to the presence of stop codons and/or frameshifts. Finally, regions directly flanking ICEAPG2 do not display the typical motifs found on each side of integrated ICEF and ICEA5632. These data strongly suggest that ICEAPG2 is unlikely to be functional. In M. agalactiae strain 5632, ICEA5632-I excision leads to a chromosomal site that is reorganized into an “empty” locus carrying remnant motifs that cover a 476-bp sequence [16]. Interestingly, in the PG2 chromosome, a 476-bp sequence located ∼ 270 kb upstream from ICEAPG2 was found that is 94% identical to the sequenced “empty” ICEA5632-I locus, and includes the putative remnant motifs in the same order and spacing (Figure S7). Unfinished sequence data from the strain 5632 reveals that this 476-bp sequence is actually part of a larger (∼40 kb) synthenic region between PG2 and 5632. HGT among Phylogenetically Remote Mycoplasma Species with Sequenced Genomes The high number of CDSs predicted to have undergone HGT between M. agalactiae and organisms of the mycoides cluster prompted us to examine possible HGT events among other mycoplasma species whose genomes have been sequenced. For each mycoplasma genome, the CDSs with a BBH in a phylogenetic group different from that of the query were then identified (see Materials and Methods). Phylogenetic analyses, when possible, were applied to detect which, among the identified CDSs, were candidates for HGT (Table 2). Overall, this analysis clearly pointed out two cases of significant HGT levels, between the mycoides cluster and M. agalactiae and between M. gallisepticum and M. synoviae. Detailed examination of the data revealed a clear picture for M. synoviae, in which all identified CDSs but one designate M. gallisepticum as the HGT partner (Tables 2, S8, and S9). This is confirmed by the reciprocal data in M. gallisepticum, although in several cases the phylogeny was not strong enough to support with certainty a direct association with M. synoviae. These data are consistent with a previous study in which HGT between those two species was suspected [12]. No significant HGT was detected among other mycoplasma species across phylogenetic groups apart from that described above between M. agalactiae and mycoplasmas of the mycoides cluster (see also Tables S5 and S6). Table 2 Number of Candidates for HGT among Mollicutes across the Spiroplasma, Pneumoniae, and Hominis Phylogenetic Groups For the human mycoplasma M. penetrans, which has the largest genome of the dataset, a fairly large number of CDSs had BBH in a phylogenetic group other than the pneumoniae group. However, none of these candidates for HGT were confirmed by further phylogenetic analysis. Discussion Mycoplasma agalactiae Genome Has Evolved by Substantial Gene Gain Sixteen genome sequences from different mycoplasma species are now available in public databases and provide comprehensive data for comparative genomic studies that will, for instance, contribute to the understanding of their intriguing regressive evolution (by loss of genetic material) from Gram-positive bacteria with low GC content. Indeed, mycoplasmas are thought to be fast-evolving bacteria, as supported by their positioning on some of the longest branches of the bacterial phylogenetic tree [21]. This observation is in agreement with their small genome size, and hence with their limited DNA-repair capabilities [46]. Consequently, mycoplasma genomes would be prone to accumulate mutations that would contribute to further downsizing. In this scenario, acquisition of new genes by HGT was not considered to play a major role in mycoplasma evolution. Indeed, statistical analyses predicted that the smallest proportion of HGT occurred among bacteria in symbiotic or in parasitic species, including mycoplasmas [47]. Nonetheless, a few remarkable cases of HGT involving mycoplasmas have been described that include the independent displacements of the rpsR and ruvB genes with orthologs from ɛ–Proteobacteria [48,49] and the horizontal transfer of the surface-protein VlhA encoding gene among three phylogenetically distant mycoplasmas (M. gallisepticum, M. imitans, and M. synoviae), which are respiratory pathogens of gallinaceous birds [50,51]. More recently, sequencing of the M. synoviae genome suggested that ∼3% of the total genome length has undergone HGT in between M. gallisepticum and M. synoviae [12]. Analyses performed in this study confirmed this trend using a different approach, which estimated that ∼3%–8 % of their CDS have been involved in HGT in between the two avian species. However, these values are much lower than the ones found for M. agalactiae, in which 10%–18% of its coding genome was predicted to have undergone HGT with mycoplasmas belonging to the mycoides cluster. This proportion represents, to our knowledge, the highest extent of HGT for a bacterium with a small genome size ( 80%) and conservation of gene synteny. Supporting Information Figure S1 Similarity of the CDSs from M. agalactiae and M. hyopneumoniae with Their BBH in M. pulmonis and M. capricolum subsp. capricolum (54 KB PDF) Click here for additional data file. Figure S2 Similarity of the 136 M. agalactiae CDSs with their BBH in M. mycoides subsp. mycoides SC and M. capricolum subsp. capricolum (18 KB PDF) Click here for additional data file. Figure S3 Phylogenetic Tree Inferred from the Amino Acid Sequence of GlpK Proteins (4 KB PDF) Click here for additional data file. Figure S4 Schematic Representing the Genetic Organization of Two Remarkable Loci of M. agalactiae (13 KB PDF) Click here for additional data file. Figure S5 Schematic Representation of the 13 M. agalactiae Proteins Containing the DUF285 Domain (3 KB PDF) Click here for additional data file. Figure S6 Phylogenetic Tree Inferred from the Amino Acid Sequence of TraG, TraE, ORF19, and ORF 22 Proteins (27 KB PDF) Click here for additional data file. Figure S7 Alignment of the ICEA5632 Locus from M. agalactiae Strain 5632 with a Synthenic Region from M. agalactiae PG2 (6 KB PDF) Click here for additional data file. Table S1 Common Hosts and Tissue Tropisms for Mycoplasma, Ureaplasma Species, and Phytoplasmas with sequenced genomes (35 KB DOC) Click here for additional data file. Table S2 CDS Candidates for HGT among M. agalactiae and mycoplasmas of the mycoides cluster, M. mycoides subsp. mycoides and M. capricolum subsp. capricolum MAG, M. agalactiae; MCAP, M. capricolum subsp. capricolum; MSC, M. mycoides subsp. mycoides SC. (259 KB DOC) Click here for additional data file. Table S3 CDS Candidates for HGT among M. agalactiae and Mycoplasmas of the Pneumoniae Group or Non-mollicute Bacteria MAG, M. agalactiae. (39 KB DOC) Click here for additional data file. Table S4 Fastap Alignments of ICEAPG2 CDS Products with their Homologs in ICEC, ICEA5632-I, and ICEF (84 KB DOC) Click here for additional data file. Table S5 CDS Candidates for HGT between M. mycoides subsp. mycoides SC and M. agalactiae MAG, M. agalactiae; MSC, M. mycoides subsp. mycoides SC. (250 KB DOC) Click here for additional data file. Table S6 CDS Candidates for HGT between M. capricolum subsp. capricolum and M. agalactiae MAG, M. agalactiae; MCAP, M. capricolum subsp. capricolum. (191 KB DOC) Click here for additional data file. Table S7 CDS Candidates for HGT between M. penetrans and Other Mollicutes MYPE, M. penetrans. (80 KB DOC) Click here for additional data file. Table S8 CDS Candidates for HGT between M. gallisepticum and M. synoviae or Other Mollicutes MGA, M. gallisepticum; MS, M. synoviae. (102 KB DOC) Click here for additional data file. Table S9 CDS Candidates for HGT between M. synoviae and M. galliepticum MGA, M. gallisepticum; MS, M. synoviae. (149 KB DOC) Click here for additional data file. Table S10 List of the 30 Shared Proteins Used for Supertree Construction of Figure 1B (36 KB DOC) Click here for additional data file. Accession Numbers The genome sequence from M. agalactiae PG2 strain, as well as related features, were submitted to the EMBL (http://www.ebi.ac.uk/embl), GenBank (http://www.ncbi.nih.gov/Genbank/index.html), and DDBJ databases (http://www.ddbj.nig.ac.jp) under accession number CU179680. All data are also available from the MolliGen database (http://cbi.labri.fr/outils/molligen). The National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov) accession numbers of other genomes mentioned in this manuscript are: M. capricolum subsp. capricolum, NC_007633; M. gallisepticum, NC_004829; M. genitalium, NC_000908; M. hyopneumoniae 232, NC_006360; M. hyopneumoniae 7448, NC_007332; M. hyopneumoniae J, NC_007295; M. mobile, NC_006908; M. mycoides subsp. mycoides SC, NC_005364; M. penetrans, NC_004432; M. pneumoniae, NC _000912; M. pulmonis, NC_002771; M. synoviae, NC_007294; Me. florum, NC_006055; and U. urealyticum/parvum, NC_002162. The NCBI locus tags of the genes and gene products mentioned in this manuscript are M. capricolum subsp. capricolum OppA, MCAP_0116; M. hyopneumoniae gidA, mhp003; M. mobile gidA, MMOB1540; M. mycoides subsp. mycoides SC OppA, MSC_0964; M. mycoides subsp. mycoides SC GlpF, MSC_0257; M. mycoides subsp. mycoides SC GlpK, MSC_0258; M. mycoides subsp. mycoides SC GlpO, MSC_0259; M. mycoides subsp. mycoides SC GtsABC transporter components, MSC_0516/MSC_0517/ MSC_0518; M. pulmonis gidA, MYPU_2530; and M. synoviae gidA, MS53_0515. The Pfam database (http://www.sanger.ac.uk/Software/Pfam) accession numbers for the protein motifs/domains mentioned in this paper are phage integrase motif, PF00589; and DUF285 domain of unknown function, PF03382. The PROSITE database (http://www.expasy.ch/prosite) accession number for the prokaryotic membrane lipoprotein lipid attachment site motif is PS51257.
Author and article information
Journal
Journal ID (nlm-ta): BMC Vet Res
Journal ID (iso-abbrev): BMC Vet. Res
Title:
BMC Veterinary Research
Publisher:
BioMed Central
ISSN
(Electronic):
1746-6148
Publication date Collection: 2012
Publication date
(Electronic):
27
August
2012
Volume: 8
Page: 146
Affiliations
[1
]Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad de Murcia, Campus
de Espinardo s/n, 30100, Murcia, Spain
[2
]UMR Mycoplasmoses des Ruminants, Anses, Laboratoire de Lyon, 31 Avenue Tony Garnier,
69364, Lyon Cedex 07, France
Article
Publisher ID: 1746-6148-8-146DOI: 10.1186/1746-6148-8-146
PMC ID: 3514313
PubMed ID: 22920649
SO-VID: ff9d9757-10ef-4919-a689-9bd6496a769d
Copyright © Copyright ©2012 De la Fe et al.; licensee BioMed Central Ltd.
License:
This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
History
Date
received
: 9
September
2011
Date
accepted
: 14
August
2012
Categories
Subject:
Research Article
ScienceOpen disciplines: Veterinary medicine