Regions of mgpB sequence repetition in the M. genitalium G-37^T genome. The computer-based algorithm of Rocha and Blanchard (44) was modified and used to probe the G-37^T genome for sequences with 100% identity to overlapping 23-bp segments of the mgpB gene. Sequences of the mgpB gene found in multiple copies are concentrated within three regions that overlap with repeat regions B (bp 575 to 1016), EF (bp 2292 to 2876), and G (bp 3305 to 3550). Small arrows illustrate the locations of primers listed in Table 2.

Repeat region B mgpB sequences identified in cervical specimens from participant no. 10139, a woman persistently infected with M. genitalium. A total of 40 sequences (10 per cervical sample) were analyzed, and 17 unique variants (A to Q) were detected in the four time points examined. (A) Schematic representation of the 17 sequences identified. White regions are homologous to the published G-37^T sequence, while areas shaded as indicated by the key diverge from the mgpB sequence and instead have homology to G-37^T MgPar sequences or have no homology to any sequences in the G-37^T genome (designated “novel”). Block sizes shown in the key represent five nucleotides each. Sequence I contained an extra adenosine base (shown with an A) at bp 878, which is predicted to alter the reading frame and introduce three in-frame stop codons, indicated with red arrows. The schematically depicted sequences are presented in more detail in panels B and C. (B) Sequence alignment of mgpB bp 620 to 709 of G-37^T; patient no. 10139 sequences A, B, D, E, and I; and the G-37^T MgPar sequences showing the chimeric nature of mgpB sequences and possible explanations for their generation. Sequence A or B could have initially recombined with MgPar 4 to generate sequence D, which then could have recombined with novel sequence 1 to generate sequence E or I. (C) The shift in mgpB sequences over the course of a persistent infection exemplified by an alignment between sequences A through Q and the published G-37^T mgpB sequence between bp 767 to 816. Bases with identity to the G-37^T mgpB sequence are highlighted in gray, while bases identified as novel sequences 3 and 4 are shaded in red and yellow, respectively. The horizontal arrows at the top and bottom of panels A and C, respectively, indicate the conserved sequence targeted by primer 2R (Fig. 2; Table 2) used in the strain typing experiments. Sequences A through Q have been deposited in GenBank under accession numbers DQ248070 through DQ248086, respectively.

Schematic drawing of M. genitalium G-37^T MgPar sequences showing their genome location, architecture, and the location of homologous sequences in the mgpB gene. (A) Location of the full-length mgpB expression site and nine MgPar sequences in the M. genitalium G-37^T genome. (B) Homology between the mgpB gene and the nine MgPar sequences in M. genitalium G-37^T, as determined by pairwise alignment. Vertical dotted lines within mgpB define fragments A through J described by Dallo and Baseman (10). Shaded black boxes (labeled B, EF, and G) represent mgpB repeat regions identified in the analysis presented in Fig. 2. Below, each of the MgPar sequences (MgPar 1 to 9) is listed on the left with their corresponding size. Sequences within each MgPar site with homology to mgpB are shown diagrammatically by a colored line; the location of each line corresponds to the location in the mgpB gene in which homology was observed, numbered according to the GenBank entry (Table 1). For example, MgPar 1 has homology to repeat regions B, EF, and G between bp 12 to 445, 445 to 1038, and 2010 to 2170, respectively. The remainder of MgPar 1 is not homologous to sequences found within the mgpB gene. Sequence alignments for the mgpB and MgPar sequences diagrammatically pictured in panel B are presented in Fig. S1 to S3 in the supplemental material.

Heterogeneity of the mgpB gene, determined by PCR amplification, cloning, and sequencing of M. genitalium G37-S. (A) Diagram of the mgpB gene showing the locations of repeat regions B, EF, and G. (B) Sequence heterogeneity identified in repeat region B. Sequence 1-1 was identical to the mgpB gene of the published M. genitalium G-37^T sequence (17), while alternative sequences 1-2 and 1-3 contained 294 and 209 bases that were identical to MgPar 7 (103 to 391 bp) and MgPar 8 (65 to 263 bp), respectively, in place of the published mgpB sequence. The region B sequence for G37-DK (accession number X91075) (42) contains 45 bases that have identity to MgPar 8 (326 to 370 bp). (C) Sequence heterogeneity identified in the clones analyzed for repeat region G. Sequence 3-1 was identical to the M. genitalium G-37^T published mgpB sequence, while sequence 3-2 contains 115 bp identical to MgPar 3 (474 to 592 bp) in place of the published mgpB sequence. (D) Nucleotide sequence alignment for repeat region B between the published sequences for G-37^T mgpB, 1-1, 1-2, 1-3, G37-DK, and MgPars 7 and 8. The sequence heterogeneity is flanked by regions of sequence identity, and the conserved sequence at bp 838 to 900 is presumably where the recombination event between region B and MgPar 8 that generated sequence 1-3 and G37-DK took place. Bases identical to the G-37^T sequence are highlighted in gray, while sequences identical to MgPars 7 and 8 are highlighted in aqua and pink, respectively. GenBank accession numbers for alternative G37-S sequences are as follows: 1-2, DQ248067; 1-3, DQ248068; 3-2, DQ248069. The accession numbers for MgPars 7 and 8 are listed in Table 1.

Strain typing of M. genitalium sequences derived from cervical samples from women persistently infected with M. genitalium, suggesting that participant no. 10139 was infected with a single strain that can be distinguished from other strains present in this population during the same time period. Participant identification numbers and dates of sample collection are shown to the left. Dashed lines indicate sequence identity to the published G-37^T sequence, between bp 221 and 380 of mgpB. Bases shaded in black are those previously shown to distinguish M. genitalium strains, which therefore serve as the basis of the strain typing system (29). For participant no. 10139, five clones were sequenced per cervical sample, while a minimum of three clones were sequenced from other persistently infected women; all sequences obtained from a given woman were identical. Sequences (with GenBank accession numbers) are as follows: 10139, DQ248092; 10046, DQ248087; 10081, DQ248088; 10089, DQ248089; 10090, DQ248090; 10114, DQ248091; 10163, DQ248093; 10177, DQ248094; 10274, DQ248095.