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.

FIG. 1.

Three-primer PCR inversion assay for detection of inversions within the two 21-bp RS conserved in the 5′ UTRs of all vpma genes. The schematic representation shows inversion of plasmid pBADXerRS21x2 (A), carrying two 21-bp inverted repeat RS (RS21; indicated by bold black arrows) along with the xer1 gene, in comparison with plasmid pBADRS21x2 (B), which carries the same RS but cannot undergo inversions because it lacks the xer1 gene and thus acts as a negative control. Primers P1, P2, and P4, annealing to the araC sequence (regulatory gene of l-arabinose operon), the bla sequence (ampicillin resistance gene), and the region adjacent to the ori (pBR322 origin), respectively, are indicated by thin black arrows. (C) Agarose gel electrophoresis of PCR products at different stages of arabinose induction. The presence of a 1-kb P1-P2 amplicon corresponds to inversion events in E. coli between the two RS21 sequences only upon Xer1 induction at 6 and 20 h (lanes 7 and 8) for pBADXerRS21x2, and this amplicon is absent for the uninduced overnight sample (lane 6). The latter, as well as both the induced (lanes 4 and 5) and uninduced (lane 3) samples of control plasmid pBADRS21x2, show only the 1.4-kb P1-P4 product amplified from the sequences of unrecombined plasmids. Lane 2, no-DNA template control; lanes 1 and 9, molecular size marker (1-kb ladder; Invitrogen).

FIG. 2.

Restriction analysis of inversion events occurring between two 200-bp inverted repeat RS in E. coli. (A) Schematic representation of inversion of plasmid pBADXerIR200Y, carrying two 200-bp inverted repeat RS (RS200Y; indicated by bold black arrows) along with the xer1 gene (white arrow). Induction of xer1 results in inversion of the DNA fragment flanked by the two RS200Y elements, resulting in plasmid pBADXerIR200Y IV, in which the HindIII and EcoRV sites are located close to each other. ori, pBR322 origin; bla, ampicillin resistance gene; araC, regulatory gene of l-arabinose operon. (B) Agarose gel electrophoresis of HindIII- and EcoRV-digested recombination products obtained at different time points. Two fragments, of 3.6 kb (OP1) and 1.9 kb (OP2), corresponding to the original unrecombined plasmid, were present in all samples. An inversion fragment of 4.9 kb (IV) was visible after 2 h (lane 4), 4 h (lane 5), and 6 h (lane 6) of xer1 induction, whereas it was absent in the uninduced cells grown overnight (lane 2) and in the sample taken at the start of induction (lane 3). Lanes 1 and 7, molecular size marker (1-kb ladder; Invitrogen). (C) Sequence of the RS200Y fragment obtained from the vpmaY gene of M. agalactiae. The bold letters represent the 21-bp conserved sequence found in the 5′ UTRs of all vpma genes.

FIG. 3.

Xer1 mediates excisions between two 200-bp RS. (A) Schematic representation of excision of plasmid pBADXerDR200Y, carrying two 200-bp direct repeat RS (RS200Y; indicated by bold black arrows) along with the xer1 gene (white arrow). Induction of xer1 results in excision of the DNA fragment flanked by the two direct repeat RS200Y elements, resulting in two recombination products: a miniplasmid (mp) and a minicircle (mc). ori, pBR322 origin; bla, ampicillin resistance gene; araC, regulatory gene of l-arabinose operon. (B) Inverted image of agarose gel electrophoresis of supercoiled (sc) recombination products. The supercoiled miniplasmid (sc mp) and supercoiled minicircle (sc mc) were visible 2 h (lane 4), 4 h (lane 5), and 6 h (lane 6) after xer1 induction, in addition to the band corresponding to the unrecombined original plasmid (sc op). Recombination products were not visible for the uninduced cells grown overnight (on; lane 2) and the sample taken at the start of induction (0 h; lane 3). Lanes 1 and 7, molecular size marker (1-kb ladder; Invitrogen). (C) Inverted image of agarose gel electrophoresis of HindIII- and XbaI-linearized (lin) recombination products, confirming the sizes of the original unrecombined plasmid pBADXerDR200Y (X/H op; 5.5 kb) and the minicircle and miniplasmid (lin mc [2.3 kb] and lin mp [3.2 kb]) excised out of it upon xer1 induction. Lanes 1 and 7, molecular size marker (1-kb ladder; Invitrogen).

FIG. 4.

Sequences flanking the 21-bp RS at the 5′ end enhance the amounts of Xer1 inversion products. One of the two 200-bp vpmaY RS in pBADXerIR200Y was replaced by shorter versions of different lengths and with different flanking regions, namely, RS21Y (in pBADXerIR21Y; the white box represents the 21-bp conserved 5′ UTR), RS31Y3′ (in pBADXerIR31Y3′), RS56Y3′ (in pBADXerIR56Y3′), RS111Y (in pBADXerIR111Y), and RS57Y5′ (in pBADXerIR57Y5′) (A), to determine the minimal RS which gives detectable amounts of inversion products (IV) during restriction analysis and agarose gel electrophoresis (B). Samples were digested with HindIII and EcoRV after 20 h of xer1 induction. Plasmid pBADXerIR200Y showed a 4.9-kb inversion band between RS200Y and RS200Y after induction (lane 3) and served as a positive control, whereas its uninduced overnight sample was negative for an inversion band (lane 2) and showed only bands that corresponded to the unrecombined parent plasmid (OP1 and OP2). Similarly, no inversion was detectable between RS200Y and RS21Y (lane 4), RS200Y and RS31Y3′ (lane 5), or RS200Y and RS56Y3′ (lane 6), whereas the appearance of 4.8-kb (lane 7) and 4.75-kb (lane 8) bands indicated inversion events between RS200Y and RS111Y and between RS200Y and RS57Y5′, respectively. Lanes 1 and 9, molecular size marker (1-kb ladder; Invitrogen).

FIG. 5.

Inversion events between different vpma RS. Plasmids carrying RS sequences derived from two different vpma genes were constructed to reflect the native recombination events operative in M. agalactiae and were transformed into E. coli to check for inversion events. (A) Schematic representation of the RS elements used in the different plasmid constructs, i.e., RS200Y and RS57Y5′ (from vpmaY), RS184U and RS53U5′ (from vpmaU), and RS56X5′ (from vpmaX). The white box depicts the 21-bp conserved region. (B) Agarose gel electrophoresis of HindIII and EcoRV digests of different samples removed after 20 h of xer1 induction. The inversion bands (IV) and the bands corresponding to the original unrecombined plasmid (OP1 and OP2) are indicated in the left margin. Plasmid pBADXerIR200Y showed a 4.9-kb inversion band between RS200Y and RS200Y after induction (lane 3) and served as a positive control, whereas its uninduced overnight sample was negative for an inversion band (lane 2) and showed only bands that corresponded to the unrecombined parent plasmid (OP1 and OP2). Inversion bands of 4.9 kb (lane 4), 4.75 kb (lane 5), and 4.6 kb (lane 6) were observed for the newly constructed pBADXerIR200Y/184U, pBADXerIR200Y/56X5′, and pBADXerIR53U5′/57Y5′ plasmids, respectively, in which the two RS originated from different vpma genes, as indicated. Lanes 1 and 7, molecular size marker (1-kb ladder; Invitrogen).

FIG. 6.

Demonstration of Xer1-mediated excisions in M. agalactiae. (A) Illustration of plasmid pILDR, used to study Xer1-mediated excisions in M. agalactiae. The lacZ gene is flanked by a 184-bp vpmaU RS and a 200-bp vpmaY RS (RS184U and RS200Y, respectively; black arrowheads), aligned as direct repeats within the left insertion sequence of transposon Tn4001mod (IS256L). ori, pBR322 origin; bla, ampicillin resistance gene; gm, gentamicin resistance gene. (B) Schematic representation of the genomic integration of pILDR in M. agalactiae type strain PG2 (PG2 pILDR) and the xer1 disruptant PLMY (PLMY pILDR). Xer1 recombinase (white ellipse) mediates excision between RS184U and RS200Y, resulting in deletion of the interjacent lacZ sequence in PG2 but not in PLMY. Primers 184Ubfw and ISR-f (thin black arrows), used to detect excisions via PCR, and their corresponding amplicons (thin black lines) are indicated for both PG2 and PLMY. (C) Colony phenotypes of lacZ transformants. When transformed into PG2 (PG2 pIL) and PLMY (PLMY pIL), the parent plasmid pIL, which carries the lacZ gene without direct repeat RS (not shown), resulted in intense blue colonies on SP4 agar plates containing X-Gal. Introduction of plasmid pILDR in PG2 (PG2 pILDR) resulted in white colonies, indicating that the lacZ gene was lost by site-specific excision between the direct repeat RS, mediated by the M. agalactiae-encoded Xer1 recombinase (see panel B). In contrast, excision was absent in the xer1 mutant PLMY, as indicated by blue colony formation (PLMY pILDR). (D) Agarose gel electrophoresis of PCRs verifying excision events in M. agalactiae. Primers 184Ubfw and ISR-f (indicated in panel B), both annealing outside the region of recombination, were used to detect excision of the lacZ gene. Genomic DNA of PLMY/pILDR (lane 3) enabled amplification of only a 3.5-kb fragment, corresponding to the lacZ sequence, whereas PCRs using genomic DNA of PG2/pILDR transformants (lane 4) also displayed a 245-bp excision fragment corresponding to the hybrid RSUY sequence created after excision between RS184U and RS200Y, in addition to the 3.5-kb lacZ fragment. Lane 2, no-DNA template control; lanes 1 and 5, molecular size marker (1-kb ladder; Invitrogen).

FIG. 7.

Dyad symmetry in the 21-bp sequence conserved in the 5′ UTRs of vpma genes. (A) Nucleotide sequence of the conserved 21-bp region. (B) Aligned sequences of 21-bp and 18-bp (where the first three nucleotides of 21 bp are excluded) regions with the respective reverse complemented sequences. The central palindromic sequence is shown in bold, and regions with dyad symmetry are indicated by asterisks.