Xer1-Mediated Site-Specific DNA Inversions and Excisions in Mycoplasma agalactiae

Surface antigen variation in Mycoplasma agalactiae, the etiologic agent of contagious agalactia in sheep and goats, is governed by site-specific recombination within the vpma multigene locus encoding the Vpma family of variable surface lipoproteins. This high-frequency Vpma phase switching was previously shown to be mediated by a Xer1 recombinase encoded adjacent to the vpma locus. In this study, it was demonstrated in Escherichia coli that the Xer1 recombinase is responsible for catalyzing vpma gene inversions between recombination sites (RS) located in the 5′-untranslated region (UTR) in all six vpma genes, causing cleavage and strand exchange within a 21-bp conserved region that serves as a recognition sequence. It was further shown that the outcome of the site-specific recombination event depends on the orientation of the two vpma RS, as direct or inverted repeats. While recombination between inverted vpma RS led to inversions, recombination between direct repeat vpma RS led to excisions. Using a newly developed excision assay based on the lacZ reporter system, we were able to successfully demonstrate under native conditions that such Xer1-mediated excisions can indeed also occur in the M. agalactiae type strain PG2, whereas they were not observed in the control xer1-disrupted VpmaY phase-locked mutant (PLMY), which lacks Xer1 recombinase. Unless there are specific regulatory mechanisms preventing such excisions, this might be the cost that the pathogen has to render at the population level for maintaining this high-frequency phase variation machinery.Members of the bacterial class Mollicutes, which are generally referred to as mycoplasmas, are considered among the simplest self-replicating prokaryotes carrying minimal genomes. Even having lost many biosynthetic pathways during a reductive evolution, mycoplasmas represent important pathogens of humans, animals, and plants, as they are equipped with sophisticated molecular mechanisms allowing them to spontaneously change their cell surface repertoire to persist in immunocompetent hosts (25).The important ruminant pathogen Mycoplasma agalactiae causes contagious agalactia in sheep and goats and exhibits antigenic diversity by site-specific DNA rearrangements within a pathogenicity island-like gene locus (9, 10, 26). The so-called vpma locus constitutes a family of six distinct but related genes that encode major immunodominant membrane lipoproteins, the Vpmas (variable proteins of Mycoplasma agalactiae) (10, 11). These surface-associated proteins vary in expression at an unusually high frequency, and only one vpma gene at a time is transcribed from a single promoter present in that locus, while all other genes are silent (9, 10). An open reading frame (ORF) with homology to the λ-integrase family of site-specific recombinases was found in the vicinity of the vpma locus and was predicted to mediate DNA inversions responsible for switching the promoter from an active vpma gene to a silent one, resulting in alteration of vpma expression (9, 10). This recombinase, designated Xer1, was indeed recently demonstrated to be responsible for phase variation of Vpma proteins (4). Targeted knockouts of the xer1 gene by homologous recombination prevented Vpma switching and produced Vpma phase-locked mutants (PLMs) steadily expressing a single vpma gene without any variation. Complementation of the wild-type xer1 gene in these PLMs restored Vpma phase variation (4). Similar systems generating surface diversity by DNA inversions involving site-specific recombination have been identified in other mycoplasma species (3, 18, 26).Site-specific recombination systems are widespread among bacteria, and the biological functions of these systems depend strongly on the participating recombination sites (RS) (16, 24, 27). Excision events between direct repeat RS usually resolve chromosome or plasmid dimers, which can arise through homologous recombination, ensuring proper segregation of newly replicated genetic material to daughter cells (1). Also, site-specific recombination mediates integration and excision of phage genomes into and out of the host chromosome (13). In contrast, site-specific inversion involving inverted repeat RS generates genetic diversity and often controls the expression of genes that are important for pathogenesis (21).The Xer1 recombinase of M. agalactiae belongs to the λ-integrase family of site-specific recombinases (10). Members of this family share four strongly conserved amino acid residues (R-H-R-Y) within the C-terminal half of the protein. This tetrad includes the active tyrosine residue that is directly involved in the recombination reaction (8). Recombination occurs by formation and resolution of a Holliday junction intermediate involving a covalent linkage between the recombinase and the DNA through the tyrosine residue. Since energy cofactors such as ATP are not required, such recombination events can occur in the absence of replication (16, 24).Sequence alignment of vpma genes identified a conserved 21-bp region within the 5′-untranslated region (UTR) in all vpma genes that was predicted to be involved in Xer1-mediated inversions (10). The present study clearly demonstrates that the Xer1 recombinase recognizes RS located within the 5′ UTR of vpma genes, causing cleavage and strand exchange within a conserved region of 21 bp. By placing two vpma-derived RS on a plasmid along with the xer1 gene, recombination events were demonstrated in Escherichia coli upon Xer1 induction via PCR and restriction analysis. Although the conserved 21-bp region was sufficient for inversions, additional nucleotides flanking it at the 5′ end were found to have a positive influence on the rate of recombination. An interesting outcome of these studies was that Xer1 also mediated excisions between direct repeat vpma RS in E. coli. This raised the intriguing possibility that such Xer1-mediated excisions also occur in the native M. agalactiae system. For further analysis of such excision events in the native system, we tested the feasibility of using the lacZ reporter tool in M. agalactiae, as lacZ is known to be expressed successfully in few other mycoplasma species, to study gene expression by use of promoter probe vectors (15, 19, 22, 23). We developed an excision assay based on blue-white phenotype selection to study Xer1-mediated excisions in M. agalactiae, thus displaying a novel application of the lacZ reporter gene in mycoplasmas. Successful implementation of this reporter system demonstrated Xer1-mediated excisions in the M. agalactiae type strain PG2, based on blue-white selection and PCR analysis. As expected, such excisions were not observed in the control xer1-disrupted VpmaY phase-locked mutant (PLMY), which lacks Xer1. Excisions in the native system imply that genetic material is susceptible to loss, which might be the cost for maintaining the machinery of high-frequency gene shuffling for a greater population advantage, unless there are specific regulatory mechanisms preventing such excisions.