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Journal of Bacteriology, March 2000, p. 1356-1363, Vol. 182, No. 5
Department of Molecular Microbiology and
Immunology, University of Missouri School of Medicine, Columbia,
Missouri 65212
Received 30 August 1999/Accepted 29 November 1999
A prototype family of seven genes encoding the variable surface
lipoproteins (Vlps) of Mycoplasma hyorhinis is
characterized in the pathogenic SK76 strain, using long-range PCR to
amplify and analyze the single chromosomal region containing expressed genes vlpA to -G, each of which is subject to
phase and size variation. Smaller families of vlp genes in
subclones of SK76 or in another strain of M. hyorhinis,
GDL, can be attributed to deletions of specific vlp genes
from the prototype array described here. Two genes, vlpA
and the newly revealed vlpG, contain repeat motifs in their
3' coding regions that differ from the short tandem repeats in other
vlp genes yet retain structural features common to all vlp gene products. SK76 and GDL vlp gene
families are similarly organized and show sequence similarity between
corresponding individual vlp genes. In light of the
extensive potential for diversity within the vlp gene
system, such conservation provides a provisional basis to hypothesize
that vlp genes may exist in specific arrays that endow
selected functions while retaining common structural features required
during phase-variable expression of this set of gene products.
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.
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 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.
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Gene Families Encoding Phase- and Size-Variable
Surface Lipoproteins of Mycoplasma hyorhinis
and
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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.
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.
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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.
The vlpA gene of the M. hyorhinis strain GDL[6] clonal isolate (see Fig. 3B) was amplified from genomic DNA, as described above, using the forward primer vlpFtip-F 5'-CAAGTCTCAAAATAACAACTATTC-3', located at the 3' end of vlpF (Fig. 1A), and the reverse primer p9vlpA-R 5'-TAATAGGAACTAAGATGTGTGTTG-3', complementary to the 3' end of vlpA of GDL[6]. This sequence includes a portion of the vlpA tip structure, conserved between SK76[3] and GDL[6]. PCR products containing vlpA were purified and cloned into pT7Blue T-Vector (Novagen). Sequences obtained directly from the PCR product and from the cloned insert were identical. The vlpA genes from spontaneous size variants of SK76[3] were amplified from genomic DNA extracted using a Wizard genomic DNA purification kit (Promega). Primers used for amplification were N1-F (Table 1) and p9vlpA-R. The PCR products were purified and sequenced directly using these same primers. The vlpA amplimer from the clonal variant A87 was also cloned into pT7Blue T-Vector to confirm the sequence. As a result of this analysis, the sequence of the A39 size variant, described in a previous study (21), has been corrected in the GenBank database (accession no. X62936).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.
Western blotting of SDS-polyacrylamide gel electrophoresis (PAGE)-separated proteins and colony blotting were performed as described elsewhere (12), using PAb to pepG diluted 1:300 or MAb to VlpA (ascites) diluted 1:1,000 as primary Ab, followed by peroxidase-conjugated goat antiserum to mouse immunoglobulin G for detection. Abs were diluted in phosphate-buffered saline containing 10% calf serum.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).
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RESULTS AND DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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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]).
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 of vlp 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 these vlp 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 vlp families 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 vlp genes was used to further characterize the LR-PCR amplimers. These probes represented the distinctive repeat regions of each of the genes vlpA 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 distinctive ClaI 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 bearing vlp 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 as vlpD/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 with vlp-specific probes, in order to map specific genes onto these larger fragments. The distribution of individual vlp genes 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].
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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.
The sequence of the 4.6-kbp XbaI DNA fragment in PT1 revealed two adjacent open reading frames (ORFs) typical of vlp gene structures (Fig. 1A and 2A) 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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G = 
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 putative
vlpG 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 bearing vlpG and vlpA, resulted in overexpression in
E. 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 the
vlpG 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 of
vlp 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 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).
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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.
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 of vlpG), 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.| |
ACKNOWLEDGMENTS |
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We thank Mary F. Kim and Jeong Im for preparation and analysis of key peptide and serologic reagents used in this study and Matthew Mouw for assistance in chromosomal analyses.
This work was supported in part by USPHS grant AI31656 from the
National Institute of Allergy and Infectious Diseases and by a
University of Missouri
Columbia Molecular Biology Program fellowship
(C.C.).
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Molecular Microbiology and Immunology, University of Missouri School of Medicine, Columbia, MO 65212. Phone: (573) 882-5644. Fax: (573) 882-4287. E-mail: wisek{at}health.missouri.edu.
Present address: Institute of Bacteriology, Mycology and Hygiene,
University of Veterinary Medicine, A-1210 Vienna, Austria.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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| 1. |
Butler, M., and R. H. Leach.
1964.
A mycoplasma which induces acidity and cytopathic effect in tissue culture.
J. Gen. Microbiol.
34:285-294 |
| 2. | Citti, C., M. F. Kim, and K. S. Wise. 1997. Elongated versions of Vlp surface lipoproteins protect Mycoplasma hyorhinis escape variants from growth-inhibiting host antibodies. Infect. Immun. 65:1773-1785[Abstract]. |
| 3. | Citti, C., and K. S. Wise. 1995. Mycoplasma hyorhinis vlp gene transcription: critical role in phase variation and expression of surface lipoproteins. Mol. Microbiol. 18:649-660[CrossRef][Medline]. |
| 4. | Cleavinger, C. M., M. F. Kim, J. H. Im, and K. S. Wise. 1995. Identification of mycoplasma membrane proteins by systematic TnphoA mutagenesis of a recombinant library. Mol. Microbiol. 18:283-293[CrossRef][Medline]. |
| 5. | Devereux, J., P. Haeberli, and O. Smithies. 1984. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12:387-395. |
| 6. | Leigh, S., C. Citti, J. Im, and K. Wise. 1996. Aberrant structural features and instability of internal sequences in Vlp lipoproteins defined by analysis of recombinant Vlp fusion proteins. IOM Lett. 4:386-387. |
| 7. | McGarrity, G. J., H. Kotani, and G. H. Butler. 1992. Mycoplasmas and tissue culture cells, p. 445-454. In J. Maniloff, R. N. McElhaney, L. R. Finch, and J. B. Baseman (ed.), Mycoplasmas: molecular biology and pathogenesis. American Society for Microbiology, Washington, D.C. |
| 8. | Morita, T., H. Fukuda, T. Awakura, A. Shimada, T. Umemura, S. Kazama, and T. Yagihashi. 1995. Demonstration of Mycoplasma hyorhinis as a possible primary pathogen for porcine otitis media. Vet. Pathol. 32:107-111[Abstract]. |
| 9. |
Razin, S.,
D. Yogev, and Y. Naot.
1998.
Molecular biology and pathogenicity of mycoplasmas.
Microbiol. Mol. Biol. Rev.
62:1094-1156 |
| 10. |
Rosengarten, R.,
P. M. Theiss,
D. Yogev, and K. S. Wise.
1993.
Antigenic variation in Mycoplasma hyorhinis: increased repertoire of variable lipoproteins expanding surface diversity and structural complexity.
Infect. Immun.
61:2224-2228 |
| 11. |
Rosengarten, R., and K. S. Wise.
1990.
Phenotypic switching in mycoplasmas: phase variation of diverse surface lipoproteins.
Science
247:315-318 |
| 12. |
Rosengarten, R., and K. S. Wise.
1991.
The Vlp system of Mycoplasma hyorhinis: combinatorial expression of distinct size variant lipoproteins generating high-frequency surface antigenic variation.
J. Bacteriol.
173:4782-4793 |
| 13. | Ross, R. F. 1990. Mycoplasmal pneumonia of swine, p. 537-543. In A. D. Leman, B. E. Straw, W. L. Mengeling, S. D'Allaire, and D. J. Taylor (ed.), Diseases of swine. Iowa State University Press, Ames, Iowa. |
| 14. | Ross, R. F., S. E. Dale, and J. R. Duncan. 1973. Experimentally induced Mycoplasma hyorhinis arthritis in swine: immune response to 26th postinoculation week. Am. J. Vet. Res. 34:367-372[Medline]. |
| 15. | Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. |
| 16. |
Tabor, S., and C. C. Richardson.
1985.
A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes.
Proc. Natl. Acad. Sci. USA
82:1074-1078 |
| 17. | Wise, K. S. 1993. Adaptive surface variation in mycoplasmas. Trends Microbiol. 1:59-63[CrossRef][Medline]. |
| 18. | Wise, K. S., M. F. Kim, and R. Watson-McKown. 1995. Variant membrane proteins, p. 227-241. In S. Razin, and J. G. Tully (ed.), Molecular and diagnostic procedures in mycoplasmology. Academic Press, New York, N.Y. |
| 19. |
Wise, K. S., and R. K. Watson.
1983.
Mycoplasma hyorhinis GDL surface protein antigen p120 defined by monoclonal antibody.
Infect. Immun.
41:1332-1339 |
| 20. | Wise, K. S., D. Yogev, and R. Rosengarten. 1992. Antigenic variation, p. 473-490. In J. Maniloff, R. N. McElhaney, L. R. Finch, and J. B. Baseman (ed.), Mycoplasmas: molecular biology and pathogenesis. American Society for Microbiology, Washington, D.C. |
| 21. | Yogev, D., R. Rosengarten, R. Watson-McKown, and K. S. Wise. 1991. Molecular basis of Mycoplasma surface antigenic variation: a novel set of divergent genes undergo spontaneous mutation of periodic coding regions and 5' regulatory sequences. EMBO J. 10:4069-4079[Medline]. |
| 22. |
Yogev, D.,
R. Watson-McKown,
R. Rosengarten,
J. H. Im, and K. S. Wise.
1995.
Increased structural and combinatorial diversity in an extended family of genes encoding Vlp surface proteins of Mycoplasma hyorhinis.
J. Bacteriol.
177:5636-5643 |
| 23. | Zheng, J., and M. A. McIntosh. 1995. Characterization of IS1221 from Mycoplasma hyorhinis: expression of its putative transposase in Escherichia coli incorporates a ribosomal frameshift mechanism. Mol. Microbiol. 16:669-685[Medline]. |
Journal of Bacteriology, March 2000, p. 1356-1363, Vol. 182, No. 5
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