Proximal region of the gene encoding cytadherence-related protein permits molecular typing of Mycoplasma genitalium clinical strains by PCR-restriction fragment length polymorphism

Abstract

Restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified proximal region of the gene encoding cytadherence accessory protein P110 (MG192) revealed DNA sequence divergences among 54 Mycoplasma genitalium clinical strains isolated from the genitourinary tracts of women attending a sexually transmitted disease-related health clinic, plus one from the respiratory tract and one from synovial fluid. Seven of 56 (12.5%) strains exhibited RFLPs following digestion of the proximal region with restriction endonuclease MboI or RsaI, or both. No sequence variability was detected in the distal portion of the gene.

FIG. 1.

Positions of multiple p110-related repetitive elements within the M. genitalium genome. Shaded bars represent positions within the proximal region of the p110 gene of p110-related multicopy elements, which are dispersed throughout the chromosome (BLAST analysis). The length of the highly homologous sequences (solid line, top) and the percentages of homology (values within open boxes) are also indicated, as are the intergenic regions (IGRs) and their locations in the chromosome (*, http://www.stdgen.lanl.gov). The proximal (MG192AB) and distal (MG192CD) regions amplified for RFLP analyses are presented at the bottom (see Table 2 for the primer sequences). **, actual positions of repetitive sequence elements mgp-r2, mgp-r4, and mgp-r7 (28) on the chromosome. nt, nucleotides.

FIG. 2.

PCR-RFLP analyses of p110 proximal regions in M. genitalium strains G37 and TW10-5. (A) The specific product, MG192AB (Fig. 1, bottom), was amplified from both strains and electrophoresed in 1% agarose (a 1-kb DNA ladder is indicated at the far left, with the numbers on both the left and the right being in base pairs). (B) The restriction patterns of amplified MG192AB regions obtained with DdeI, MboI, MnlI, and RsaI were separated and compared in 2% agarose alongside a 100-bp DNA ladder. (C) Predicted region of divergent sequence (cross-hatched box) within the MG192AB proximal region of p110 is indicated, along with the positions of the DdeI (D), MboI (B), MnlI (M), and RsaI (R) restriction sites, in the sequence generated from strain G37.

FIG. 3.

PCR-RFLP analysis of p110 distal regions in M. genitalium strains G37 and TW10-5. The specific product, MG192CD (Fig. 1, bottom), was amplified from both strains. The restriction patterns obtained by using DdeI, HinfI, HpaII, HphI, MboI, MnlI, and RsaI were analyzed as described in the Fig. 2 legend. The numbers on the left are in base pairs.

FIG. 4.

PCR-RFLPs detected in p110 proximal regions of seven M. genitalium clinical strains. PCR products of the expected size (panel PCR) were generated for all strains by using primers MG192A and MG192B (Table 2). The remaining panels present the restriction patterns of the designated strains (Table 1) by using the enzymes DdeI (panel DdeI), MboI (panel MboI), and RsaI (panel RsaI). The products were analyzed as described in the Fig. 2 legend. The numbers on the left are in base pairs.