Structural features and phase-variable expression of the vlpG gene. (A) Schematic structure of a prototype vlp gene compared to that of vlpG, which contains a characteristic promoter with a homopolymeric tract of adenine residues (polyA) located between the −35 and −10 sequences and a likely transcription terminator (T) (3, 12, 20). The predicted VlpG protein is initiated with a GTG start codon, followed by a highly conserved signal peptide (region I) with a prolipoprotein signal peptidase recognition site, AISC, characteristic of known Vlp proteins (20, 22). The mature VlpG protein is encoded by region II (divergent among Vlps) and region III (composed of a tandemly repeated sequence, specific for this Vlp). The distal C-terminal portion of VlpG (Tip) is composed of a partial repeat motif. The amino acid sequence of the repeated motif (in single-letter code; hatched) in region III (showing negatively charged residues) and the location of a sequence used to generate synthetic peptide pepG are indicated below the schematic structure of vlpG. Additional repeats in region III (*) are not shown. (B) Expression of Vlp proteins from recombinant vlpG and vlpA genes. The genomic XbaI fragment from SK76[7] bearing genes vlpG and vlpA (** in Fig. 1A) was cloned and expressed in E. coli under the T7 promoter (16) as previously described (22), and Western immunoblots of bacterial lysates (lanes 1 and 2) were stained with PAb to pepG (lane 1) or a combination of this PAb and a MAb (2) to VlpA (lane 2). Lane 3 represents a Western blot of SK76[7] mycoplasmas run on the same (8%) gel and immunostained with PAb to pepG. Relative molecular masses of recombinant VlpA and VlpG were 87 and 82 kDa, respectively. (C) Phase variation of VlpG. A colony immunoblot of SK76[7] stained with PAb to pepG is shown, with a sectored colony indicated by the arrow. (This figure was constructed using Adobe Photoshop version 4.0 and 5.0 for Windows NT, Magicscan V4.1, Umax Power Look III scanner, Hewlett-Packard LaserJet 4000N printer, and Dell OptiPlex GXPro or Gateway E-4100 computer.)

Chromosomal organization of the vlp gene cluster in M. hyorhinis strains SK76 and GDL. (A) vlp gene families from strain SK76 containing three (SK76[3]) or seven (SK76[7]) genes and from strain GDL containing six genes (GDL[6]). The maps shown are not to scale but reflect the positions and features of all genes. Solid lines show the chromosomal location of ClaI (C), XbaI (X), and EcoRI (E) restriction sites within vlp gene families and the distinct, conserved regions flanking vlp families at the 5′ (stippled) and 3′ (open) boundaries. Corresponding oligonucleotide primers (LR-F and LR-R [Table 1]), used to amplify vlp gene clusters by LR-PCR, are indicated by opposing open arrows above the lines. Large open arrows below each line indicate the location and orientation of ORFs encoding Vlp proteins, and shaded boxes (P) indicate the locations of conserved promoter regions upstream of each vlp gene or in one case a distinctive partial promoter structure downstream of vlpF (*). The positions and orientations of IS1221 elements (IS) are shown as large open boxes. The double asterisk indicates a genomic XbaI fragment from SK76[7] that was cloned and analyzed further (Fig. 2B). The LR-PCR product generated from SK76[7] genomic DNA template is indicated by a line below (LR-PCR amplimer). Additional lines represent nested PCR products generated from the LR-PCR product template by using primers described in Table 1. Nested products 1 through 6 were generated with primer sets N1-F–N1-R, N2-F–N2-R, N3-F–N3-R, N4-F–N4-R, N5-F–N5-R, and N6-F–N6-R, respectively. (B) Ethidium bromide-stained amplimers were generated as described in Materials and Methods from genomic DNA template from clonal isolates SK76[3] (lane 1), GDL[6] (lane 2), and SK76[7] (lane 3) and separated by electrophoresis through 0.9% agarose gels. Sizes of the amplimers, determined using lambda HindIII markers (lane 4), are indicated on the left in kilobase pairs. (C) Southern blot analysis of ClaI restriction fragments from SK76[7] genomic DNA (lane 1) or the LR-PCR product from SK76[7] (lanes 2 through 4). Blots were hybridized with oligonucleotide probes (Table 1) representing a highly conserved signal peptide sequence (Sig) located 3′ of the ClaI site in each vlp gene (lanes 1 and 2), the 3′ flanking primer sequence LR-R (lane 3), or the 5′ flanking sequence, LR-F (lane 4). Chromosomal ClaI fragments bearing specific vlp genes are identified to the left of lane 1, based on hybridization patterns (data not shown) obtained with oligonucleotides specific for vlpA to -G. The sizes of fragments are indicated in kilobase pairs. Comigrating 1.7-kbp fragments bearing vlpD and vlpE, respectively (vlpD/E), and 1.6-kbp fragments bearing vlpB and vlpG, respectively (vlpB/G), were not resolved by the gel system shown. vlpC and vlpC* indicate ClaI fragments from genomic (7.0-kbp) and LR-PCR (1.5-kbp) DNA, respectively, bearing the vlpC gene. (D) ClaI site mapping of the LR-PCR amplimer bearing vlp genes. A partial ClaI digest of the LR-PCR product from SK76[7] was subjected to Southern blot analysis with oligonucleotide probes LR-R (lane 1) and LR-F (lane 2) to identify partially and completely digested fragments bearing these 3′- and 5′-terminal flanking sequences, respectively. Sizes of fragments are indicated to the left of each lane. The terminal 1.5-kbp 3′ fragment and 0.8-kbp 5′ fragment correspond to those in the complete digests shown in panel C, lanes 3 and 4. Additional gels resolving larger fragments are not shown. (This figure was constructed using Adobe Photoshop version 4.0 and 5.0 for Windows NT, Magicscan V4.1, Umax Power Look III scanner, Hewlett-Packard LaserJet 4000N printer, and Dell OptiPlex GXPro or Gateway E-4100 computer.)

Comparison of vlpA genes among VlpA size variants. (A) Size variants of vlpA (A39, A46, A50, A57, and A87, expressing VlpA protein products of 39, 46, 50, 57, and 87 kDa, respectively, as determined by SDS-PAGE) were derived from a clonal lineage of spontaneous variants from SK76[3] (12, 21). Regions I, II, and III are indicated as in Fig. 2. In region III, a basic repeat unit (rep; underlined) is indicated by an open box. Filled areas in region III represent a modified repeat comprising a 60-bp deletion of the rep unit. Hatched boxes represent identical repeated sequences of 13 amino acids. Larger composite repeated motifs are indicated as rep* and rep**. Brackets depict tandemly repeated rep** sequences. The C-terminal sequence (Tip) is indicated in all variants. Location of the primers used to amplify portions of vlpA genes encoding the mature lipoprotein regions are represented by arrows and are described in Materials and Methods. (B) The structure of vlpA from a previously described (22) cloned isolate of M. hyorhinis strain GDL is depicted, using symbols described for panel A to indicate the repeated structure within region III. (This figure was constructed using Adobe Photoshop version 4.0 and 5.0 for Windows NT, Magicscan V4.1, Umax Power Look III scanner, Hewlett-Packard LaserJet 4000N printer, and Dell OptiPlex GXPro or Gateway E-4100 computer.)