Gene Families Encoding Phase- and Size-Variable Surface Lipoproteins of Mycoplasma hyorhinis

LR-PCR strategy to amplify and characterize completevlp gene families in the M. hyorhinischromosome.Our previous studies (21, 22) identified the vlp families in two cloned isolates shown in Fig. 1A. One clonal isolate from strain SK76 (SK76[3]) contained three genes,vlpA to -C, whereas a clonal isolate (47.1.2) of type strain GDL contained six genes, including additional genesvlpD to -F (Fig. 1, GDL[6]).

In both strains, all vlp genes occurred in single copy, were clustered as shown in association with IS1221 elements (23), and were flanked by consistent chromosomal boundary sequences. Further analysis of early-passage SK76 revealed a subpopulation that contained more than three vlp genes (10, 22). The presence of this SK76 variant population was compatible with the possibility that the original pathogenic strain (cloned at the time of isolation) contained a more extensive repertoire of vlp genes that might differ from that in the extensively passaged GDL strain derived from tissue culture. To analyze the SK76 population containing the expanded repertoire, a variant (termed SK76[7]; clonal isolate 8II) was selected, and a general method to more directly characterize the composition and organization ofvlp gene families was developed. This was needed as an alternative to complement the traditional chromosomal cloning protocols, which were confounded by redundancy and size variation in intergenic and intragenic sequences throughout this region.

Based on the premise (confirmed below) that the vlp family in SK76[7] would be flanked in the chromosome by the distinctive sequences at each boundary of the two known families, we designed primers (shown as opposing arrows in Fig. 1A) and developed conditions for LR-PCR amplification of complete vlp gene clusters from chromosomal DNA templates derived from mycoplasmal clonal isolates. Examples of the products obtained with these flanking primers are shown in Fig. 1B. LR-PCR amplimers from the three-gene SK76[3] isolate and six-gene GDL[6] isolate showed the expected 4.8- and 8.8-kbp sizes, respectively, as predicted from previous cloning, restriction analysis, and sequencing of these vlp gene families (21, 22). The authenticity of LR-PCR amplimers from thesevlp gene families was confirmed by diagnostic Southern hybridization of ClaI and XbaI fragments as previously described (21, 22). Amplification products were reproducibly obtained and, based on subsequent sequencing of various regions and comparison to known vlp gene sequences, appeared to be free of PCR-derived errors in amplification (data not shown).

The vlp family of the SK76[7] clonal subpopulation was analyzed using LR-PCR in conjunction with conventional mapping techniques. First, an LR-PCR amplimer of approximately 14.4 kbp was obtained (Fig. 1B, lane 3). This was larger than known vlpfamilies and could reflect highly extended repeat structures in individual vlp genes, additional IS1221 elements, additional vlp genes, or unknown sequences. Southern hybridization with probes specific for each of the known vlpgenes was used to further characterize the LR-PCR amplimers. These probes represented the distinctive repeat regions of each of the genesvlpA to -F as described previously (3, 21, 22). Because vlp genes contain a highly conserved DNA sequence encoding the prolipoprotein signal peptide common to all Vlp products, and because this sequence contains a distinctiveClaI restriction site accounting for all ClaI sites in the known vlp gene clusters (Fig. 1A),ClaI restriction fragments could be used to segregate individual vlp genes (21, 22). ClaI fragments bearing vlp genes could be identified by their hybridization with a conserved signal peptide probe (Sig) immediately downstream of the ClaI site and further distinguished using specific probes for individual vlp genes (Table 1).

ClaI restriction of chromosomal DNA from the SK76[7] isolate, or of the LR-PCR amplimer generated from this isolate, enabled assignment of known vlp genes to ClaI fragments and a comparison of the genomic organization with that found in the LR-PCR product. The pattern of ClaI restriction fragments hybridizing with vlp gene probes was completely concordant between chromosomal DNA and the LR-PCR product, with the anticipated exception of a 1.5-kbp ClaI fragment at the 3′ end of the LR-PCR product bearing vlpC. Figure 1C (lanes 1 and 2) compares these patterns, using the conserved vlp signal peptide probe to elucidate all ClaI fragments bearingvlp genes. Probes specific to known vlp genes (and later to a new gene, vlpG, described below) further assigned specific genes to these fragments and additionally confirmed that known vlpA to -F gene sequences were present in SK76[7]. Higher-resolution gels revealed two pairs of nearly comigrating bands (data not shown), indicated in Fig. 1C asvlpD/E and vlpB/G. To complete the restriction analysis of the LR-PCR product, the distal ClaI fragments at the 5′ and 3′ ends of the amplimer were identified with probes representing the chromosomal boundary primers used to generate the LR-PCR product. The 1.5-kbp 3′ ClaI fragment is shown in lane 3 (this fragment also bears vlpC), and the 0.8-kbp 5′ClaI fragment is shown in lane 4 (this fragment bears no target for vlp probes). XbaI fragments of the LR-PCR amplimer were also subjected to hybridization withvlp-specific probes, in order to map specific genes onto these larger fragments. The distribution of individual vlpgenes on ClaI and XbaI fragments is summarized in Table 2. With the exception of an unusually large XbaI fragment bearing vlpA, the pattern was generally comparable to that for the vlp family previously identified (22) in GDL[6]. As in earlier studies, no evidence for vlp-related sequences outside this cluster was found in strain SK76[7].

Due to similarity in the sizes of some ClaI fragments bearing vlp genes, an independent method was used to determine the ordered positions of ClaI sites in the LR-PCR product. This was accomplished by partial ClaI restriction of the amplimer followed by Southern hybridization of the resulting fragments with the 3′ or 5′ primers used to generate the LR-PCR amplimer (Fig. 1D, lanes 1 and 2, respectively). As seen in the examples shown, the size interval between fragments could be used to calculate the distance between successive ClaI sites, from the 3′ or 5′ end. Results from ClaI site mapping, along with mapping of vlp genes to limit fragments shown in Table 2, are consistent with the map shown in Fig. 1A (SK76[7]).

Another independent approach was used to localize specificvlp genes and determine their order and orientation. This employed specific primers to generate nested PCR products amplified from the LR-PCR product as template. Unique sequences in particularvlp genes were used to design forward and reverse primer pairs that encompass subsets of vlp genes (Table 1). These were used to generate a nested set of amplimers (Fig. 1A, numbered 1 through 6) that could be positioned on the restriction map shown (SK76[7]). The specificities of primer sequences and sizes of discrete nested PCR products confirmed the positions and relative orientations of most vlp genes in this complex. Finally, to further confirm the predicted map shown, specific vlp probes were hybridized to the nested PCR products 1 through 6, in a dot blot matrix summarized in Table 2. All of these approaches confirmed the map shown in Fig. 1A for SK76[7]. The one abnormality in SK76[7] compared to previously reported vlp families was the presence of an exceptionally large (4.6-kbp) XbaI fragment bearing vlpA.

Identification and positional analysis of vlpG, a structurally distinctive and phase-variable vlp gene.To examine the genomic region adjoining vlpA, chromosomal DNA from isolate SK76[7] was digested to completion withXbaI, and a library was generated by ligating these fragments with XbaI-digested vector pGEM-7Z, as described in Materials and Methods. This vector also places inserts under the inducible T7 promoter for overexpression in E. coli. Clones from this library that hybridized with the Sig probe (Table 1) were collected, and the insert of one of these (PT1) was subcloned and sequenced.

The sequence of the 4.6-kbp XbaI DNA fragment in PT1 revealed two adjacent open reading frames (ORFs) typical ofvlp gene structures (Fig. 1A and2A) described previously (17, 20-22). Comparisons with known vlp gene sequences identified one ORF as vlpA (also confirmed by independent serologic means; see below). The second ORF was not previously known and was designated vlpG. As illustrated in Fig. 1A,vlpG and vlpA are similarly oriented and are flanked by portions of two IS1221 elements, positioned in divergent orientations. A vlpG-specific oligonucleotide probe was synthesized and used to localize vlpG by Southern hybridization (Fig. 1C; Table 2) to ClaI or XbaI restriction fragments generated from genomic DNA and the LR-PCR amplimer from SK76[7]. The orientation of the 4.6-kbp XbaI fragment within the family was further established. First, specific nested PCR products from the LR-PCR amplimer template were generated. As indicated in Fig. 1A, products 3 and 4 were generated using a reverse primer representing the specific 3′ end of vlpA, and product 1 was generated using a forward primer in a specific portion (region II) of vlpA. Second, dot hybridization patterns established the distribution of all seven vlp genes on these nested PCR products (Table 2). Collectively, these data confirmed the positions and orientations of vlpG and vlpA.

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Fig. 2.

Structural features and phase-variable expression of thevlpG gene. (A) Schematic structure of a prototypevlp 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 genomicXbaI fragment from SK76[7] bearing genes vlpGand vlpA (** in Fig. 1A) was cloned and expressed inE. 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.)

vlpG contained the typical promoter and poly(A) tract characteristic of vlp genes and a stem-loop structure (ΔG = −10.5 kcal) 3′ of the translation stop (3, 20, 21). The VlpG ORF shown in Fig. 2A extends from a GTG start codon (typical of Vlp proteins) through structural motifs consistent with Vlp prolipoprotein processing and repeated regions. Like ORFs encoding other Vlps, VlpG contains no UGA (Trp) codons and is overlapped on both strands by additional ORFs (17, 20-22). To directly evaluate the presence and expression features of a putativevlpG gene translation product, a specific PAb to a synthetic peptide (pepG) comprising the predicted C-terminal tip and repeat structure of VlpG was prepared and used to detect the gene product in two ways. First, as shown in Fig. 2B, T7 induction of the recombinant PT1 plasmid, containing the XbaI insert bearingvlpG and vlpA, resulted in overexpression inE. coli of two products identified, respectively, by antibody to pepG and a previously described MAb to VlpA (2). The PAb to VlpG recognized a product of similar size in Western blots of mycoplasmal proteins (Fig. 2B, lane 3) prepared from isolate SK76[7]. This result confirmed the translation and the reading frame (defined by the Ab) of the VlpG product. Because the Ab to VlpG was determined not to immunostain other known Vlp products (A to F), using an array of variants of SK76[3] and GDL which lack thevlpG gene (data not shown), it was further used in colony immunoblotting (18) to determine whether the VlpG product expressed in SK76[7] was subject to phase variation. As indicated in Fig. 2C, the marked sectoring of single colonies revealed by Ab to VlpG was consistent with the notion that this gene product shared with other Vlps the feature of phase variation. This was very likely due to mutations in the poly(A) tract of vlpG (Fig. 2A), analogous to other vlp genes, that would affect transcription (3). Finally, the accessibility of the VlpG repeat region III to the Ab directed to pepG was consistent with the predicted orientations and surface locations of all other known products ofvlp genes (11, 12).

A noteworthy feature of the VlpG structure is the distinctive (22-residue) length of the tandem protein repeats in region III of the product (Fig. 2A). Despite this longer unit, compared to 12 or 13 residues for analogous units in VlpA-F (21, 22), VlpG shares the charge distribution and composition found in repeat units of all Vlps, representing a net negative charge and high content of Gly, Ser, and Thr (17, 20, 22). An unusual feature of VlpG, like all Vlps, is the discordance between the mass of the protein predicted from the deduced sequence and the predicted migration of the product in SDS-PAGE. This feature has recently been attributed to properties of region III repeats in these proteins (6). The entire sequence of the vlpG gene could not be precisely determined due to its extensive repetitive region. However, sequence data clearly revealed 13 full repeats, all of which were identical. This number was also consistent with the number of repeats determined by probing fragments generated from a recombinant insert bearing vlpG, after partial restriction with XmnI, which recognizes a site in each repeat unit of vlpG (data not shown). Consequently, the calculated mass of the mature VlpG protein from the SK76[7] strain with 13 full repeats was 30.3 kDa, predicting a much faster migration in SDS-PAGE than the 82-kDa relative molecular mass observed for both the recombinant and authentic mycoplasmal VlpG products shown in Fig. 2B.

Overall, the vlpG gene found in SK76[7] encodes a unique repetitive structure within a product otherwise typical of the Vlp family of lipoproteins. Moreover, the seven-gene repertoire in this isolate is likely to represent the complete vlp gene family in the original pathogenic SK76 strain (11, 12, 22).

VlpA contains a complex repetitive structure.While VlpB to -G contain a region III comprised of nearly identical, tandemly repeated units of 12, 13, or (in VlpG) 22 amino acids, comparative analysis ofvlpA genes from clonal variants of SK76[3], SK76[7], and GDL[6] available from this or previous studies revealed a more complex repeat structure. The repeat structure of the shortest known VlpA variant, A39 from SK76[3] (Fig.3A), contains all known repeat elements of region III in the VlpA product. These include a sequence of 39 amino acids (rep), a version of rep with a deletion of 60 bp, a small repeat of 13 amino acids, and a conserved distal C-terminal sequence (Tip). A39 is the smallest of four spontaneous deletion size variants emerging in broth culture from a clonal population of SK76[3] expressing the largest known VlpA, A87 (Fig. 3A). The vlpA sequence from this A87 clonal variant revealed a much more complex repeat structure in region III, containing composites of the same basic elements arranged in alternative configurations as shown in Fig. 3A (including, for example, rep* and rep**). From the A87 variant of SK76[3], three other spontaneous deletion variants, in addition to A39, have been obtained by screening for colony opacity variants that were known to be correlated with altered length of the VlpA products expressed (12). Sequences from these variants (A46, A50, and A57), like that of A39, showed a variety of in-frame deletions within thevlpA gene. These deletions could be explained by the elimination of various internal repeated elements from the A87 configuration through intragenic deletion mechanisms (Fig. 3A).

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Fig. 3.

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 vlpAgenes encoding the mature lipoprotein regions are represented by arrows and are described in Materials and Methods. (B) The structure ofvlpA 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.)

As the vlpA gene of the SK76[7] isolate was sequenced, we were able to confirm that it too contained the specific A87 configuration. This might be expected since the SK76[7] and SK76[3] isolates had the same origin prior to culture passage. Finally, sequencing of vlpA from the long-term tissue culture passaged GDL strain showed an intermediate size VlpA, with a repeat structure having the same basic elements but in yet another unique configuration (Fig. 3B). Interestingly, this structure could also have been generated in principle by intragenic deletions from a configuration such as that in SK76 A87. In light of analysis of these deletion variants and our earlier demonstration that thevlpA gene is also subject to expansion in length from small and intermediate forms (21), these results indicate thatvlpA has a more complex and distinctive repeat structure than other vlp genes and that diverse variants can occur spontaneously during propagation in culture conditions.

The natural vlp repertoire.We propose that the enlarged vlp gene family in the pathogenic SK76 strain, containing seven distinct vlp genes, may be a prototype ofvlp families in the natural host environment. Simple deletions from this configuration could yield the SK76[3] family, derived from the same clonal stock of SK76 after passage in broth medium, or the six-gene family in the tissue culture contaminant GDL, isolated on a different continent over an interval spanning several years (22). Further examination of field and pathogenic isolates should allow us to test the hypothesis that the naturalvlp family contains seven functional vlp genes. Horizontal sampling may also reveal the conservation of the genomic context of these arrays, as well as the stability of genetic signatures linked to IS1221 elements within vlp families (Fig. 1). Finally, such studies will determine whether or not the entire family resides on a larger element, or can be located at diverse chromosomal locations.

The range and nature of functions associated with a possibly fixed array of structurally diverse vlp genes remains an intriguing enigma. Considering the nearly limitless diversity (17, 20) embodied in the Vlp system (e.g., through intergenic recombination, frameshift mutations in overlapping reading frames, or specific composites created by multiple reiterated sequences in region III of vlpA or the extended repeat unit in region III ofvlpG), selection and maintenance of a specific array of Vlp products will be a critical point to establish, since it would argue strongly for specific functions associated with each product, presumably selected in the natural host niche. This study establishes the hypothesis and tools to address these questions and identifies additional forms of diversity in two Vlp products that may be useful in understanding Vlp structure and function.