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

Abstract

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.

Comparative genome analysis of 19 Ureaplasma urealyticum and Ureaplasma parvumstrains

“…The proposed mechanism for variation of the ureaplasma mba locus resembles the previously reported variable loci of Mycoplasma bovis : vsp , Mycoplasma pulmonis: vsa and Mycoplasma agalactiae: vpma [56]. The involvement of a site-specific Xer-like recombinase and inverted repeats was experimentally proven for the M. pulmonis vsa locus [57] and the vpma locus of M. agalactiae [58] , and suggested for the phase variation of the vsp locus in M. bovis [56] . We believe that a Xer-like recombinase is likely to be involved in the phase variation of the mba locus of Ureaplasma spp and a putative recombinase recognition site has been determined.…”

Interaction of the putative tyrosine recombinases RipX (UU145), XerC (UU222), and CodV (UU529) ofUreaplasma parvumserovar 3 with specific DNA

“…The fact that only xerC is present in some Ureaplasma strains that showed high-frequency phase variation in the two loci supports the idea that only one tyrosine recombinase is involved in the site-specific recombination event of these loci. Recombination mechanisms in mycoplasmas, where only a single recombinase mediates sitespecific recombination, have been described for the hsd and vsr systems of M. pulmonis (Sitaraman et al, 2002), the mpl system of M. penetrans (Horino et al, 2009), and the vpma system of M. agalactiae (Czurda et al, 2010). Because all analyzed Ureaplasma strains showed highfrequency phase variation in both the 'mba locus' and the (a) Colony immunoblot of MPmba trunc that had been transformed with pCT::UU222, grown in medium containing 80 lg mL À1 gentamicin and 25 lg mL À1 chloramphenicol, plated on agar, and transferred onto nitrocellulose membrane.…”

“…The first member of this family that was described is the k-Int protein, which promotes integration and excision of the phage genome from that of the host (Nash, 1981). Other family members related to the k-Int, such as the Flp from the yeast 2l plasmid, the XerC/D of Escherichia (E.) coli, the Cre recombinase of phage P1, the HvsR of Mycoplasma (M.) pulmonis, and the Xer1 of M. agalactiae, function in the amplification/maintenance of plasmid copy number (Hoess et al, 1984), the elimination of chromosome dimers from replicated chromosomes (Hayes & Sherratt, 1997), the cyclization of virion DNA and the life cycle of temperate phages (Sternberg et al, 1986), the alteration of the type I restriction modification system and of cell-surface components (Sitaraman et al, 2002), and in phase variation of membrane proteins (Czurda et al, 2010), respectively. In E. coli, the proteins XerC and XerD (CodV and RipX in Bacillus subtilis) act in concert at a sequence designated dif 'deletion-induced filamentation' to resolve dimeric chromosomes after chromosome replication (Blakely et al, 1991(Blakely et al, , 1993Sciochetti et al, 1999Sciochetti et al, , 2001.…”

Role of Vpma phase variation inMycoplasma agalactiaepathogenesis

“…Xer1 recombinase encoded in the vpma pathogenicity island-like locus causes sitespecific vpma gene inversions that place a previously silent gene in front of the single vpma promoter and thus cause Vpma switching (Glew et al, 2002;Chopra-Dewasthaly et al, 2008). Site-specific cleavage and strand exchange occur within a minimal region of 21 bp located within the 5′ untranslated region of all six vpma genes (Czurda et al, 2010). Interestingly, this region is also highly conserved in the vsp lipoprotein genes that form a similar site-specific phase-variable system in Mycoplasma bovis, a very close phylogenetic relative of M. agalactiae (Askaa & Erno, 1976;Pettersson et al, 1996) that causes even more serious economic losses to the cattle and dairy industry (Nicholas & Ayling, 2003;Fox et al, 2005) owing to mastitis and calf pneumonia.…”