INTRODUCTION

Top Abstract Introduction Materials and Methods Results and Discussion References

Several species of the wall-less eubacteria Mycoplasma spp. contain in their small genomes sets of related genes encoding alternative surface lipoproteins (recently reviewed in reference 9). Studies of some mycoplasma gene families have revealed that these genes are subject to high-frequency, reversible mutations that generate alternate or composite expression patterns of corresponding gene products, as well as structural variation of the products reflecting changes in intragenic repetitive regions. Whereas specific functions associated with mycoplasmal systems of surface variation are not clear, conservation of such multigenic systems per se suggests that these variable arrays are pivotal elements in the adaptive strategies of mycoplasmas as chronic infectious pathogens of various host species. Determining the range of alternative genes in these families, and the degree and nature of structural variation in the corresponding gene products, can provide initial insight into possible functions. This study examines gene families encoding the variable lipoprotein (Vlp) system of Mycoplasma hyorhinis.

M. hyorhinis chronically infects the swine host, where it causes rhinitis as well as severe serositis and arthritis (13, 14). This agent has also been recently linked to the occurrence of otitis media (8). An experimental swine model of arthritis has been developed using the arthritogenic SK76 strain of M. hyorhinis, a prototype pathogenic strain derived as a cloned isolate from a diseased site in a natural infection (11, 12, 14). Subclones of SK76 propagated by passage in broth culture have been used to characterize the rudimentary Vlp system (11, 12, 21). In addition to its natural host, M. hyorhinis occupies an important artificial niche as one of the most prevalent mycoplasmal tissue culture contaminants worldwide (7). Although the origins and initial pathway for transmitting these cell culture-adapted contaminants are not known, one such isolate, type strain GDL (1), has been characterized and shown to contain additional genes of the Vlp family (10, 22).

Genetic mechanisms underlying mycoplasma surface variation were first elucidated during a study of M. hyorhinis membrane lipoproteins of strains SK76 and GDL (3, 12, 20, 21), which contain repertoires of distinct, single-copy genes encoding surface-exposed Vlps. Reversible, high-frequency mutations govern the phase-variable expression of each Vlp product (12, 21). Insertion or deletion of deoxyadenosine residues in a characteristic promoter region [poly(A) tract] linked to each vlp gene results in drastically altered transcription of the individual genes (3, 21). In addition, size variation of Vlp products results from in-frame insertion or deletion of tandemly repeated intragenic sequences within the 3' region of individual vlp genes, thereby contracting or expanding the surface-exposed C-terminal region of the corresponding Vlp (12, 21). This structural variation affects the abundance (3) and functional consequences (2) of Vlps on the mycoplasma surface, but the full consequences of this variation are yet to be fully understood. The two independent forms of reversible mutation affect expression and size of the product from each vlp gene. In a propagating population, a single organism carrying multiple vlp genes has the capacity to generate a large number of variants expressing antigenically distinct vlp gene products, either alone or in combinatorial mosaics on the cell surface.

The functions of the Vlp system are likely to be complex. Structural variation in vlp genes of M. hyorhinis was recently shown to govern susceptibility to growth inhibition by host antibody (Ab) produced during infection of swine (2). Nevertheless, this property may represent only one aspect of a system which might mediate diverse additional surface interactions. Knowing the size and extent of diversity embodied in the combinatorial repertoire of vlp genes, particularly in a known pathogenic isolate, will be highly informative in this regard. Defining new or consistent features of the Vlp system, including additional vlp genes, their comparative organization in families, and their common structural features, is an essential step in predicting the functions of the Vlp system in adaptive variation. Comparison of cloned isolates of M. hyorhinis strains SK76 and GDL showed that the vlp repertoires in these two strains comprised three and six vlp genes, respectively (21, 22). However, evidence raising the possibility of additional vlp genes in other isolates of strain SK76 has been reported (22).

A long-range PCR (LR-PCR) procedure capable of amplifying and analyzing complete clusters of contiguous vlp genes was therefore developed and used to characterize the vlp family in strain SK76. This report (i) identifies an expanded prototype vlp gene family in the pathogenic SK76 strain that includes a new vlp gene with an atypical intragenic repeat structure, (ii) shows one vlp gene to have a repeat structure subject to complex spontaneous intragenic variation, and (iii) provides a mechanistic basis for differences observed in the size of vlp gene families in cultured organisms through deletions of vlp genes or gene blocks from a larger prototype vlp family.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results and Discussion References

Bacterial strains, plasmids, and culture conditions. M. hyorhinis SK76 was provided by R. F. Ross, Iowa State University, Ames, as a filter-cloned culture. Isogenic clonal variants derived from M. hyorhinis SK76 and GDL have been previously described (3, 12, 21) and were propagated at 37°C in modified Hayflick broth or agar medium (19). Cloning procedures were performed using the vector pGEM-7Z (Promega Corporation, Madison, Wis.) or the pT7Blue T-Vector (Novagen, Madison, Wis.). Competent Escherichia coli DH5 MCR or DH10B (Life Technologies, Inc., Gaithersburg, Md.) was used as a host to clone recombinant products and grown at 37°C in Luria-Bertani broth supplemented with 100 µg of ampicillin per ml for plasmid preparation.

DNA manipulations and sequencing. Mycoplasma cells were harvested and subsequently lysed in a buffer containing 1% sodium dodecyl sulfate (SDS), 45 mM Tris (pH 8.0), and 9 mM EDTA for 10 min at 50°C and then for 10 min at 37°C. Genomic DNA was extracted by standard methods (15) or by commercial kits as described below. Agarose gel electrophoresis, restriction endonuclease digestion, ligation, chemical transformation, and electroporation were performed as described elsewhere (15). 3'-Digoxigenin-11-ddUTP (DIG)-labeled oligonucleotides and Southern hybridization were prepared according to the Genius system user's guide for membrane hybridization, version 3.0 (Roche Molecular Biochemicals). For the partial ClaI restriction digestion, 150 ng of the purified (Qiaex II gel extraction kit; Qiagen, Inc., Valencia, Calif.) LR-PCR product of SK76[7] was incubated with 1 U of ClaI (Promega) for 30 or 60 min, and the reaction was stopped by heat inactivation at 65°C for 20 min.

Cloning and DNA sequence analysis. Genomic DNA from M. hyorhinis SK76[7] clonal variant was digested to completion with XbaI. The resulting fragments were inserted into XbaI-restricted, dephosphorylated pGEM-7Z vector. The ligation mixture was used to transform competent E. coli DH5 cells by electroporation (Life Technologies). Recombinant clones carrying vlp genes were detected by hybridization on colonies, using the DIG-labeled oligonucleotide Sig (Table 1). One recombinant plasmid (PT1) contained a 4.6-kbp XbaI insert. This was subcloned by additional ClaI restriction and fully sequenced, revealing a new vlp gene, vlpG, located upstream of the vlpA gene and downstream of an IS1221 element. To identify other vlp genes, the LR-PCR product generated from the SK76[7] genomic template was digested by ClaI, and fragments were cloned into pGEM-7Z. Internal ClaI fragments shown in Fig. 1A (SK76[7]), except the fragment bearing vlpG, were analyzed by this approach.

View larger version (54K): [in this window] [in a new window]   FIG. 1.   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.)

Identification and sequence comparison of vlpA genes. To identify and characterize vlpA genes from assorted clonal isolates, PCR was first used to amplify the vlpA gene from chromosomal DNA. The amplimers were purified and sequenced either directly or after cloning into pT7Blue T-Vector. PCR was performed in a volume of 100 µl with 5 U of AmpliTaq DNA polymerase (PE Corp., Norwalk, Conn.), 0.3 mM each primer, 0.35 mM deoxynucleoside triphosphate mix (10 mM each; Life Technologies), 1.75 mM MgCl₂, 1× AmpliTaq DNA polymerase buffer II, and 20 ng of genomic DNA. Thermocycling was performed in a Perkin-Elmer DNA Thermo Cycler 480 with the following conditions: 1 cycle at 94°C for 2 min; 40 cycles at 94°C for 1 min, 57°C for 1 min, 72°C for 2 min; and a final extension cycle of 72°C for 7 min. Ten microliters of the PCR product was analyzed on a 0.8% agarose gel (FMC BioProducts, Rockland, Maine). The resulting PCR products were purified with a QIAquick gel extraction kit (Qiagen), and 375 fmol was sequenced directly. In some instances, indicated below, the purified PCR product was ligated into pT7Blue T-Vector and sequenced using T7 and U19 primers provided by Novagen. Additional internal primers were used to obtain and confirm sequences.

LR-PCR and nested PCR. LR-PCR and nested PCR products were produced using the protocol of the Expand Long Template PCR system (Roche Molecular Biochemicals). The LR-PCR DNA template was generated using primers LR-F and LR-R (Table 1) from genomic DNA. Nested PCR products were generated using 20 ng of the LR-PCR product as template and the nested primer sets listed in Table 1. The annealing temperatures for the primer sets varied and were typically 6 to 9°C below the predicted primer melting temperature. Identification of vlp genes was determined by direct sequencing of corresponding PCR fragments purified with a Qiaex II or QIAquick gel extraction kit (Qiagen) or by sequencing subcloned restriction fragments of the PCR products.

Antibodies to synthetic Vlp peptides and immunoblotting procedures. The synthetic peptide pepG was prepared on a model 432A peptide synthesizer (Applied Biosystems) by standard 9-fluorenylmethylcarbonyl protection chemistries and purified as previously described (4, 22). The amino acid sequence of pepG, C-SGTSSTTETGSTTESSGQA)DSTSGTSTSI, corresponds to the C-terminal 29 amino acid residues of VlpG deduced from the 3' region of the vlpG gene sequence determined in this study. The parenthesis indicates the end of a 22-residue repeat unit in the VlpG sequence, and the underlined residues indicate an adjoining partial repeat comprising part of the tip structure. The Cys residue was incorporated at the N terminus in order to couple the peptide to maleimide-activated keyhole limpet hemocyanin, using a hapten carrier conjugate kit (Pierce Chemical Co., Rockford, Ill.). A BALB/c mouse was immunized with three weekly intraperitoneal injections of approximately 15 µg of conjugated peptide emulsified with incomplete Freund adjuvant. The presence of specific anti-pepG polyclonal Abs (PAbs) in the hyperimmune serum was monitored by Western blot analysis of defined clonal variants SK76[3] or GDL[6] that lacked the vlpG gene or of the clonal variant SK76[7] containing the vlpG gene. A monoclonal Ab (MAb) directed to a synthetic peptide representative of the C-terminal repeat and tip structure of VlpA has been described elsewhere (2) and was used as a specific probe for the VlpA product.

Nucleotide sequence accession numbers. The following sequences (with corresponding accession numbers) were deposited in the GenBank sequence database: PCR-derived sequences of A39 (AF193874), A46 (AF193875), A50 (AF193876), A57 (AF193877), and A87 (AF193878), IS1221-vlpG-vlpA-IS1221 from SK76[7] (AF193880), and vlpA from GDL[6] (AF193879).

	RESULTS AND DISCUSSION

Top Abstract Introduction Materials and Methods Results and Discussion References

LR-PCR strategy to amplify and characterize complete vlp gene families in the M. hyorhinis chromosome. 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 genes vlpD to -F (Fig. 1, GDL[6]).

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 with XbaI, 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.

View larger version (19K): [in this window] [in a new window]   FIG. 2.   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.)

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 of vlpA 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 the vlpA gene. These deletions could be explained by the elimination of various internal repeated elements from the A87 configuration through intragenic deletion mechanisms (Fig. 3A).

View larger version (16K): [in this window] [in a new window]   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 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.)

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 of vlp 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 natural vlp 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.