Occurrence,Plasticity, and Evolution of the vpma Gene Family,a Genetic System Devoted to High-Frequency Surface Variation in Mycoplasma agalactiae

Mycoplasma agalactiae, an important pathogen of small ruminants, exhibits a very versatile surface architecture by switching multiple, related lipoproteins (Vpmas) on and off. In the type strain, PG2, Vpma phase variation is generated by a cluster of six vpma genes that undergo frequent DNA rearrangements via site-specific recombination. To further comprehend the degree of diversity that can be generated at the M. agalactiae surface, the vpma gene repertoire of a field strain, 5632, was analyzed and shown to contain an extended repertoire of 23 vpma genes distributed between two loci located 250 kbp apart. Loci I and II include 16 and 7 vpma genes, respectively, with all vpma genes of locus II being duplicated at locus I. Several Vpmas displayed a chimeric structure suggestive of homologous recombination, and a global proteomic analysis further indicated that at least 13 of the 16 Vpmas can be expressed by the 5632 strain. Because a single promoter is present in each vpma locus, concomitant Vpma expression can occur in a strain with duplicated loci. Consequently, the number of possible surface combinations is much higher for strain 5632 than for the type strain. Finally, our data suggested that insertion sequences are likely to be involved in 5632 vpma locus duplication at a remote chromosomal position. The role of such mobile genetic elements in chromosomal shuffling of genes encoding major surface components may have important evolutionary and epidemiological consequences for pathogens, such as mycoplasmas, that have a reduced genome and no cell wall.Bacteria of the Mycoplasma genus belong to the class Mollicutes and represent a remarkable group of organisms that derived from the Firmicutes lineage by massive genome reduction (41, 51). Consequent to this regressive evolution, modern mycoplasmas have been left with small genomes (580 to 1,400 kb), a limited number of metabolic pathways, and no cell wall. Due to these particularities, members of the Mycoplasma genus have often been portrayed as “minimal self-replicating organisms.” Despite this apparent simplicity, a large number of mycoplasma species are successful pathogens of humans and a wide range of animals, in which they are known to cause diseases that are often chronic and debilitating (1, 33). The surface of their single membrane is considered a key interface in mediating adaptation and survival in the context of a complex, immunocompetent host (10, 13, 34, 40). Indeed, mycoplasmas possess a highly versatile surface architecture due to a number of sophisticated genetic systems that promote intraclonal variation in the expression and structure of abundant surface lipoproteins (9, 50). Usually, these systems combine a set of contingency genes with a molecular switch for turning expression on or off that is based on either (i) spontaneous mutation (slipped-strand mispairing), (ii) gene conversion, or (iii) specific DNA rearrangements (9). While high-frequency phenotypic variation using the two first mechanisms has been described thoroughly for other bacteria (47), switching of surface components by shuffling of silent genes at a particular single expression locus has been studied mainly in mycoplasmas (3, 8, 14, 16, 23, 39, 43).Mycoplasma agalactiae, an important pathogen responsible for contagious agalactia in small ruminants (listed by the World Organisation for Animal Health), possesses a family of lipoproteins encoded by the vpma genes for which phase variation in expression is driven by a “cut-and-paste” mechanism involving a tyrosine site-specific recombinase designated Xer1 (16). Data previously gathered with the PG2 type strain identified a single vpma cluster (42) composed of six vpma genes adjacent to one xer1 gene (Fig. ​(Fig.1A).1A). Based on fine genetic analyses, Xer1 was further shown to mediate frequent site-specific DNA rearrangements by targeting short DNA sequences located upstream of each vpma gene (8, 16). While some vpma rearrangements can be phenotypically silent, others result in Vpma on-off switching by linking a silent vpma gene sequence immediately downstream of the unique vpma promoter. Because site-specific recombination can be reciprocal, the initial vpma configuration can be restored without a loss of genetic information.Open in a separate windowFIG. 1.Comparison of M. agalactiae vpma loci between the PG2 type strain and strain 5632. Schematics represent the organization of the vpma loci in clonal variant 55.5 derived from PG2 (16, 42) (A) and in clonal variant c1 derived from strain 5632 (B). (C) Counterpart of locus II₅₆₃₂ in PG2 showing the absence of vpma genes in this region. (D) The presence of two distinct loci in 5632 was confirmed by PCR, using the primer pair xerF-phydR or xerF-agpR, and the resulting amplicons are shown. The locations of the primers are indicated by arrowheads in panels A, B, and C. Large white arrows labeled with letters represent Vpma CDSs. The positions of the promoters are represented by black arrowheads labeled “P.” The two non-Vpma-related CDSs (abiG_I and abiG_II) are indicated by large arrows filled with a dotted pattern. ISMag1 elements are indicated by hatched boxes. Recombination sites downstream of each vpma gene are indicated by black dots. An asterisk indicates that the corresponding vpma gene is present at two distinct loci. Schematics were drawn approximately to scale. HP, hypothetical protein; CHP, conserved hypothetical protein. Small letters and bars indicate the positions of short particular sequences mentioned in the text and in Fig. ​Fig.33 and ​and4.4. The pictures on the left side of panels A and B illustrate the variable surface expression of Vpma, as previously described (8, 17). These correspond to colony immunoblots using Vpma-specific polyclonal antibodies recognizing PG2 VpmaW (α W) and VpmaY (α Y) epitopes.The vsa family of the murine pathogen M. pulmonis (3, 39), the vsp family of the bovine pathogen M. bovis (2, 24), and the vpma family of M. agalactiae all generate intraclonal surface diversity by using very similar molecular switches (23), although their overall coding sequences seem to be specific to the Mycoplasma species. DNA rearrangements also govern phase variation of the 38 mpl genes of the human pathogen M. penetrans (27, 35, 38). However, in this mycoplasma species the molecular switch is slightly different, since each mpl gene possesses its own invertible promoter (19). In M. penetrans, the individual expression of each mpl gene can then be switched on and off in a combinatory manner, resulting in a large number of possible Mpl surface configurations. Since M. pulmonis, M. bovis, and M. agalactiae all belong to the Mycoplasma hominis phylogenetic cluster (48) and are relatively closely related, while M. penetrans belongs to the distinct Mycoplasma pneumoniae phylogenetic cluster (30, 48), it is tempting to speculate that the vsa, vsp, and vpma systems were all inherited from a common ancestor and that the bulk of their coding sequences evolved independently in their respective hosts while the molecular switch mechanism was retained.In so-called “minimal” bacteria, the occurrence of relatively large genomic portions dedicated to multigene families, with genes encoding phase-variable, related surface proteins, suggests that they serve an important function(s). Data accumulated over the years for several mycoplasma species tend to indicate that one general purpose of these systems is to provide the mycoplasma with a variable shield that modulates surface accessibility in order to escape the host response and to adapt to rapidly changing environments (10, 11, 13, 40, 50). On the other hand, the sequences of phase-variable proteins are relatively conserved within one species but divergent between species, suggesting a more specific role for these molecules.The role of the Vpma family of M. agalactiae has yet to be elucidated, but it was recently shown that Vpma switches in expression occur at a remarkably high rate in vitro (10⁻² to 10⁻³ per cell per generation) (8, 17). The vpma systems described for PG2 (16) and another M. agalactiae strain, isolated in Israel (in which Vpmas were designated Avg proteins [14]), both revealed a repertoire of six vpma genes and only one promoter, suggesting that in M. agalactiae the number of Vpma configurations is limited to six. This contrasts with the situation commonly found in other Mycoplasma variable systems, which can offer a larger mosaic of surface architecture because of the concomitant switches of several related surface proteins and/or because of a larger number of phase-variable genes.To further understand the degree of diversity that can be generated at the surface of M. agalactiae, we analyzed the vpma gene content of a field strain, 5632, whose genome was recently sequenced by our group (unpublished data). The present study shows that 5632 contains a total of 23 vpma genes distributed in two distinct loci that both contain a recombinase gene. Further genomic and proteomic analyses indicated that the capacity of 5632 to vary its Vpma surface architecture is far more complex than that described for the type strain. Unlike the case for PG2, both 5632 vpma loci are associated with several mobile genetic insertion elements (IS) that could play an evolutionary role in the dynamics of vpma repertoires, as suggested by data presented here. One 5632 vpma locus contains open reading frames (ORFs) that are highly conserved in both M. bovis, a closely related bovine mycoplasma, and the phylogenetically distant mycoplasmas of the M. mycoides cluster, which are also important ruminant pathogens. Whether these were acquired through evolution or through horizontal transfer is discussed. The present study reveals an additional degree of complexity for the Vpma system and further suggests that some field strains might have more dynamic genomes and a more variable surface than was first estimated (42).