GapA and CrmA coexpression is essential for Mycoplasma gallisepticum cytadherence and virulence.

It was previously demonstrated that avirulent Mycoplasma gallisepticum strain R(high) (passage 164) is lacking three proteins that are expressed in its virulent progenitor, strain R(low) (passage 15). These proteins were identified as the cytadhesin molecule GapA, the putative cytadhesin-related molecule CrmA, and a component of a high-affinity transporter system, HatA. Complementation of R(high) with wild-type gapA restored expression in the transformant (GT5) but did not restore the cytadherence phenotype and maintained avirulence in chickens. These results suggested that CrmA might play an essential role in the M. gallisepticum cytadherence process. CrmA is encoded by the second gene in the gapA operon and shares significant sequence homology to the ORF6 gene of Mycoplasma pneumoniae, which has been shown to play an accessory role in the cytadherence process. Complementation of R(high) with wild-type crmA resulted in the transformant (SDCA) that lacked the cytadherence and virulence phenotype comparable to that found in R(high) and GT5. In contrast, complementation of R(high) with the entire wild-type gapA operon resulted in the transformant (GCA1) that restored cytadherence to the level found in wild-type R(low). In vivo pathogenesis trials revealed that GCA1 had regained virulence, causing airsacculitis in chickens. These results demonstrate that both GapA and CrmA are required for M. gallisepticum cytadherence and pathogenesis.

Juxtaposition of an active promoter to vsp genes via site-specific DNA inversions generates antigenic variation in Mycoplasma bovis.

Mycoplasma bovis, the most important etiological agent of bovine mycoplasmosis, undergoes extensive antigenic variation of major and highly immunogenic surface lipoprotein antigens (Vsps). A family of 13 related but divergent vsp genes, which occur as single chromosomal copies, was recently found in the chromosome of M. bovis. In the present study, the molecular mechanism mediating the high-frequency phase variation of two Vsps (VspA and VspC) as representatives of the Vsp family was investigated. Analysis of clonal isolates exhibiting phase transitions of VspA or of VspC (i.e., ON-->OFF-->ON) has shown that DNA inversions occur during Vsp phase variation. The upstream region of each vsp gene contains two sequence cassettes. The first (cassette no. 1), a 71-bp region upstream of the ATG initiation codon, exhibits 98% homology among all vsp genes, while the second (cassette no. 2), upstream of cassette no. 1, ranges in size from 50 to 180 bp and is more divergent. Examination of the ends of the inverted fragments during VspA or VspC phase variation revealed that in both cases, a change in the organization of vsp upstream cassettes involving three vsp genes had occurred. Primer extension and Northern blot analysis have shown that a specific cassette no. 2, designated A(2), is an active promoter and that juxtaposition of this regulatory element to a silent vsp gene by DNA inversions allows transcription initiation of the recipient gene. Further genetic analysis revealed that phase variation of VspA or of VspC involves two site-specific DNA inversions occurring between inverted copies of a specific 35-bp sequence present within the conserved cassette no. 1. A model for the control of Vsp phase variation is proposed.

Intrachromosomal recombination within the vsp locus of Mycoplasma bovis generates a chimeric variable surface lipoprotein antigen.

A family of 13 related but divergent vsp genes was recently found in the chromosome of the bovine pathogen Mycoplasma bovis. The vsp genomic locus was shown to undergo high-frequency rearrangements and to mediate phenotypic switching of variable lipoprotein antigens (Vsps) on the mycoplasma cell surface. Here we report that the vsp gene repertoire is subject to changes. Genetic analysis of M. bovis clonal isolates displaying distinct Vsp phenotypes showed that an intergenic recombination event between two closely related members of the vsp gene family, the formerly expressed vspA gene and the vspO gene, led to the formation of a new chimeric and functional vsp gene, vspC. The 5' end of the recombination event was identified within the highly conserved vsp-upstream region, while the 3' end was localized within the first repetitive domain (R(A)1) present in both vspA and vspO structural genes. As a result, the vspC gene is an embodiment of the following domains: an N-terminus-encoding region linked to the highly conserved vsp-upstream region provided by the vspO gene; and a C-terminus-encoding region and the more distal and divergent vsp-upstream region acquired from the vspA gene. The generation of chimeric genes encoding surface antigens may provide an important element of genetic variation and an additional source of antigenic diversification within the mycoplasma population.

Molecular variability of the adhesin-encoding gene pvpA among Mycoplasma gallisepticum strains and its application in diagnosis.

Mycoplasma gallisepticum is an important pathogen of chickens and turkeys that causes considerable economic losses to the poultry industry worldwide. The reemergence of M. gallisepticum outbreaks among poultry, the increased use of live M. gallisepticum vaccines, and the detection of M. gallisepticum in game and free-flying song birds has strengthened the need for molecular diagnostic and strain differentiation tests. Molecular techniques, including restriction fragment length polymorphism of genomic DNA (RFLP) and PCR-based random amplification of polymorphic DNA (RAPD), have already been utilized as powerful tools to detect intraspecies variation. However, certain intrinsic drawbacks constrain the application of these methods. The main goal of this study was to determine the feasibility of using an M. gallisepticum-specific gene encoding a phase-variable putative adhesin protein (PvpA) as the target for molecular typing. This was accomplished using a pvpA PCR-RFLP assay. Size variations among PCR products and nucleotide divergence of the C-terminus-encoding region of the pvpA gene were the basis for strain differentiation. This method can be used for rapid differentiation of vaccine strains from field isolates by amplification directly from clinical samples without the need for isolation by culture. Moreover, molecular epidemiology of M. gallisepticum outbreaks can be performed using RFLP and/or sequence analysis of the pvpA gene.

A chromosomal region of Mycoplasma agalactiae containing vsp-related genes undergoes in vivo rearrangement in naturally infected animals.

A Mycoplasma agalactiae genomic fragment carrying four vsp-related genes (designated avg: agalactiae variable genes) was cloned, sequenced and compared to the vspA gene of Mycoplasma bovis. The following features were revealed: (i) the presence of a highly conserved vsp 5' upstream region; (ii) a highly homologous vsp N-terminal end encoding a putative lipoprotein signal sequence; (iii) sequence divergence of the rest of the mature proteins. By using avg specific probes in Southern blot analysis of genomic DNAs of M. agalactiae strains as well as of isolates from infected animals, marked DNA polymorphism of avg fragments was demonstrated. In addition, the avg genomic fingerprints were monitored for a period of 7 months, in isolates of M. agalactiae from an individual chronically infected animal. The results provided evidence that a chromosomal region of M. agalactiae, carrying vsp-related genes, undergoes rearrangements in vivo in the natural animal host during the course of infection.

Molecular characterization of the Mycoplasma gallisepticum pvpA gene which encodes a putative variable cytadhesin protein.

A putative cytadhesin-related protein (PvpA) undergoing variation in its expression was identified in the avian pathogen Mycoplasma gallisepticum. The pvpA gene was cloned, expressed in Escherichia coli, and sequenced. It exhibits 54 and 52% homology with the P30 and P32 cytadhesin proteins of the human pathogens Mycoplasma pneumoniae and Mycoplasma genitalium, respectively. In addition, 50% homology was found with the MGC2 cytadhesin of M. gallisepticum and 49% homology was found with a stretch of 205 amino acids of the cytadherence accessory protein HMW3 of M. pneumoniae. The PvpA molecule possesses a proline-rich carboxy-terminal region (28%) containing two identical directly repeated sequences of 52 amino acids and a tetrapeptide motif (Pro-Arg-Pro-X) which is repeated 14 times. Genetic analysis of several clonal isolates representing different expression states of the PvpA product ruled out chromosomal rearrangement as the mechanism for PvpA phase variation. The molecular basis of PvpA variation was revealed in a short tract of repeated GAA codons, encoding five successive glutamate resides, located in the N-terminal region and subject to frequent mutation generating an in-frame UAA stop codon. Size variation of the PvpA protein was observed among M. gallisepticum strains, ranging from 48 to 55 kDa and caused by several types of deletions occurring at the PvpA C-terminal end and within the two directly repeated sequences. By immunoelectron microscopy, the PvpA protein was localized on the mycoplasma cell surface, in particular on the terminal tip structure. Collectively, these findings suggest that PvpA is a newly identified variable surface cytadhesin protein of M. gallisepticum.

Epitope mapping of immunogenic and adhesive structures in repetitive domains of Mycoplasma bovis variable surface lipoproteins.

The family of variable surface lipoproteins (Vsps) of the bovine pathogen Mycoplasma bovis includes some of the most immunogenic antigens of this microorganism. Vsps were shown to undergo high-frequency phase and size variations and to possess extensive reiterated coding sequences extending from the N-terminal end to the C-terminal end of the Vsp molecule. In the present study, mapping experiments were conducted to detect regions with immunogenicity and/or adhesion sites in repetitive domains of four Vsp antigens of M. bovis, VspA, VspB, VspE, and VspF. In enzyme-linked immunosorbent assay experiments, sera obtained from naturally infected cattle showed antibodies to different repeating peptide units of the Vsps, particularly to units R(A)1, R(A)2, R(A)4.1, R(B)2.1, R(E)1, and R(F)1, all of which were found to contain immunodominant epitopes of three to seven amino acids. Competitive adherence trials revealed that a number of oligopeptides derived from various repeating units of VspA, VspB, VspE, and VspF partially inhibited cytoadhesion of M. bovis PG45 to embryonic bovine lung cells. Consequently, putative adherence sites were identified in the same repeating units (R(A)1, R(A)2, R(A)4.1, R(B)2.1, R(E)1, and R(F)1) and in R(F)2. The positions and lengths of the antigenic determinants were mostly identical to those of adhesion-mediating sites in all short repeating units, whereas in the considerably longer R(F)1 unit (84 amino acid residues), there was only one case of identity among four immunogenic epitopes and six adherence sites. The identification of epitopes and adhesive structures in repetitive domains of Vsp molecules is consistent with the highly immunogenic nature observed for several members of the Vsp family and suggests a possible function for these Vsp molecules as complex adherence-mediating regions in pathogenesis.

The vsp locus of Mycoplasma bovis: gene organization and structural features.

Major lipoprotein antigens, known as variable membrane surface lipoproteins (Vsps), on the surface of the bovine pathogen Mycoplasma bovis were shown to spontaneously undergo noncoordinate phase variation between ON and OFF expression states. The high rate of Vsp phenotypic switching was also shown to be linked with DNA rearrangements that occur at high frequency in the M. bovis chromosome (I. Lysnyansky, R. Rosengarten, and D. Yogev, J. Bacteriol. 178:5395-5401, 1996). In the present study, 13 single-copy vsp genes organized in a chromosomal cluster were identified and characterized. All vsp genes encode highly conserved N-terminal domains for membrane insertion and lipoprotein processing but divergent mature Vsp proteins. About 80% of each vsp coding region is composed of reiterated coding sequences that create a periodic polypeptide structure. Eighteen distinct repetitive domains of different lengths and amino acid sequences are distributed within the products of the various vsp genes that are subject to size variation due to spontaneous insertions or deletions of these periodic units. Some of these repeats were found to be present in only one Vsp family member, whereas other repeats recurred at variable locations in several Vsps. Each vsp gene is also 5' linked to a highly homologous upstream region composed of two internal cassettes. The findings that rearrangement events are associated with Vsp phenotypic switching and that multiple regions of high sequence similarity are present upstream of the vsp genes and within the vsp coding regions suggest that modulation of the Vsp antigenic repertoire is determined by recombination processes that occur at a high frequency within the vsp locus of M. bovis.

Heat inactivation of fetal calf serum is not required for in vitro measurement of lymphocyte functions.

This study was undertaken to test whether fetal calf serum (FCS) must be heat inactivated before use in tissue culture. We tested various immune functions of lymphocytes growing in medium containing non-treated and heat-inactivated FCS. The data clearly show that heat inactivation of the serum is not mandatory. In some cases, the addition of untreated FCS resulted in elevated response levels, while maintaining immune function specificity.

Intraspecies polymorphism of vsp genes and expression profiles of variable surface protein antigens (Vsps) in field isolates of Mycoplasma bovis.

To assess the extent of interstrain variation, 50 isolates of Mycoplasma (M.) bovis including the type strain PG45 were examined for the presence of a family of variable membrane surface lipoproteins (Vsps) and their genes. Southern hybridization using a genomic fragment carrying three distinct vsp genes (vspAEF) revealed a striking heterogeneity, with only 2/50 strains having identical banding patterns. Cluster analysis of the data showed that most isolates from interrelated herds (groups 1, 2 and 3) were combined in a cluster of 50% homology, while isolates from distinct geographical regions (groups 4, 5 and 6) were linked only at 18% homology. Vsp antigen expression was monitored by Western immunoblotting using four specific monoclonal antibodies (MAbs). Resembling the findings at the DNA level, interstrain variation of Vsp expression among groups 1-3 was less pronounced than among non-interrelated isolates from groups 4-6. Ten out of 50 strains did not hybridize with the vspAEF gene probe at high-stringency conditions, 8/50 failed to react with any of the Vsp-related MAbs, and 6/50 proved negative in both assays. Interestingly, most of these isolates produced hybridization signals at low stringency suggesting major distinctions in their vsp gene structure. The extensive evidence obtained on interstrain vsp gene polymorphism and variation in Vsp expression could provide a basis for a future understanding of the pathogenic potential of individual M. bovis strains.

Molecular biology and pathogenicity of mycoplasmas.

The recent sequencing of the entire genomes of Mycoplasma genitalium and M. pneumoniae has attracted considerable attention to the molecular biology of mycoplasmas, the smallest self-replicating organisms. It appears that we are now much closer to the goal of defining, in molecular terms, the entire machinery of a self-replicating cell. Comparative genomics based on comparison of the genomic makeup of mycoplasmal genomes with those of other bacteria, has opened new ways of looking at the evolutionary history of the mycoplasmas. There is now solid genetic support for the hypothesis that mycoplasmas have evolved as a branch of gram-positive bacteria by a process of reductive evolution. During this process, the mycoplasmas lost considerable portions of their ancestors' chromosomes but retained the genes essential for life. Thus, the mycoplasmal genomes carry a high percentage of conserved genes, greatly facilitating gene annotation. The significant genome compaction that occurred in mycoplasmas was made possible by adopting a parasitic mode of life. The supply of nutrients from their hosts apparently enabled mycoplasmas to lose, during evolution, the genes for many assimilative processes. During their evolution and adaptation to a parasitic mode of life, the mycoplasmas have developed various genetic systems providing a highly plastic set of variable surface proteins to evade the host immune system. The uniqueness of the mycoplasmal systems is manifested by the presence of highly mutable modules combined with an ability to expand the antigenic repertoire by generating structural alternatives, all compressed into limited genomic sequences. In the absence of a cell wall and a periplasmic space, the majority of surface variable antigens in mycoplasmas are lipoproteins. Apart from providing specific antimycoplasmal defense, the host immune system is also involved in the development of pathogenic lesions and exacerbation of mycoplasma induced diseases. Mycoplasmas are able to stimulate as well as suppress lymphocytes in a nonspecific, polyclonal manner, both in vitro and in vivo. As well as to affecting various subsets of lymphocytes, mycoplasmas and mycoplasma-derived cell components modulate the activities of monocytes/macrophages and NK cells and trigger the production of a wide variety of up-regulating and down-regulating cytokines and chemokines. Mycoplasma-mediated secretion of proinflammatory cytokines, such as tumor necrosis factor alpha, interleukin-1 (IL-1), and IL-6, by macrophages and of up-regulating cytokines by mitogenically stimulated lymphocytes plays a major role in mycoplasma-induced immune system modulation and inflammatory responses.

Genetic variation among Mycoplasma agalactiae isolates detected by the variant surface lipoprotein gene (vspA) of Mycoplasma bovis.

Multiple restriction fragments, homologous to the previously described Mycoplasma bovis vspA gene, were identified in the chromosome of Mycoplasma agalactiae. The vspA, a representative variable surface lipoprotein gene of the vsp gene family, and four synthetic oligonucleotides, representing sequences complementary to selected regions of the vsp genes, were used as probes against digested chromosomal DNAs of several M. agalactiae clinical isolates. The resulting Southern blot analysis demonstrated a marked DNA polymorphism of multiple vspA-related fragments among the isolates. An oligonucleotide representing a conserved 5'-region common to all known vsp genes, was found to hybridize to multiple M. agalactiae genomic fragments while the other three oligonucleotides, representing distinct repetitive structures within the coding region of three known vsp genes (vspA, vspE, and vspF), failed to react. These results argue for the possible existence of a gene family in M. agalactiae analogous to the vsp system of M. bovis but comprised of diverse genes.

Adherence of Mycoplasma gallisepticum involves variable surface membrane proteins.

Adherence of Mycoplasma gallisepticum to erythrocytes was examined by colony immunoblotting, detergent phase fractionation, trypsin treatment, comparison of protein profiles, and comparison of erythrocyte-bound mycoplasma protein fractions of hemadsorption-positive and -negative mutants. The binding of M. gallisepticum to chicken or human erythrocytes was found to be mediated via surface-exposed membrane proteins undergoing high-frequency phase variation.

Phenotypic switching of variable surface lipoproteins in Mycoplasma bovis involves high-frequency chromosomal rearrangements.

Mycoplasma bovis, an important pathogen of cattle, was recently shown to possess a family of phase- and size-variable membrane surface lipoprotein antigens (Vsps). These proteins spontaneously undergo noncoordinate phase variation between ON and OFF expression states, generating surface antigenic variation. In the present study, we show that the spontaneously high rate of Vsp phenotypic switching involves DNA rearrangements that occur at high frequency in the M. bovis chromosome. A 1.5-kb HindIII genomic fragment carrying the vspA gene from M. bovis PG45 was cloned and sequenced. The deduced VspA amino acid sequence revealed that 80% of the VspA molecule is composed of reiterated intragenic coding sequences, creating a periodic polypeptide structure. Four distinct internal regions of repetitive sequences in the form of in-tandem blocks extending from the N-terminal to the C-terminal portion of the Vsp product were identified. Southern blot analysis of phenotypically switched isogenic lineages representing ON or OFF phase states of Vsp products suggested that changes in the Vsp expression profile were associated with detectable changes at the DNA level. By using a synthetic oligonucleotide representing a sequence complementary to the repetitive vspA gene region as a probe, we could identify the vspA-bearing restriction fragment undergoing high-frequency reversible rearrangements during oscillating phase transition of vspA. The 1.5-kb HindIII fragment carrying the vspA gene (on state) rearranged and produced a 2.3-kb HindIII fragment (OFF state) and vice versa. Two newly discovered vsp genes (vspE and vspF) were localized on two HindIII fragments flanking the vsp gene upstream and downstream. Southern blot hybridization with vspE- and vspF-specific oligonucleotides as probes against genomic DNA of VspA phase variants showed that the organization and size of the fragments adjacent to the vspA gene remained unchanged during VspA ON-OFF switching. The mechanisms regulating the vsp genes are yet unknown; our findings suggest that a recombinative mechanism possibly involving DNA inversions, DNA insertion, or mobile genetic elements may play a role in generating the observed high-frequency DNA rearrangements.

Variant colony surface antigenic phenotypes within mycoplasma strain populations: implications for species identification and strain standardization.

Immunobinding assays with mycoplasma colonies on agar plates (immunofluorescence and immunoperoxidase techniques) or with imprints of colonies transferred to solid supports (colony immunoblotting) are widely used as standard diagnostic tests for serological species identification of mycoplasma isolates. However, in light of the high rate of variability of surface antigens in many mycoplasmas, diagnostic data obtained with these techniques require a more critical evaluation. In this report, we demonstrate with some examples that mycoplasma surface variability based on alterations in expression, in size, and in surface presentation of integral and peripheral membrane proteins may lead to misinterpretation of colony immunostaining reactions obtained by using specific monoclonal antibodies as well as conventional diagnostic hyperimmune sera. To more easily identify phenotypically mixed isolates or samples which contain more than one species, we have introduced some minor modifications of the colony immunoblot technique which provide sharp signals of positive as well as negative reactions and enable identification of cryptic epitopes. It is further demonstrated that because of the variability in colony surface antigenic phenotype, mycoplasma strains, including well-established reference and other prototype strains which are used under the same designation in many laboratories, can differ markedly in their antigen profiles and their potentially virulence-related surface properties, since they are usually purified by filter cloning and often propagated by subcultivation of randomly selected agar-grown subpopulations. We conclude from this study that because of this surface variability, the establishment of criteria for standardization of mycoplasma strains and diagnostic antisera is urgently required in order to obtain reproducible results in different laboratories.

Increased structural and combinatorial diversity in an extended family of genes encoding Vlp surface proteins of Mycoplasma hyorhinis.

Variable lipoproteins (Vlp) constitute the major coat protein of Mycoplasma hyorhinis. They are products of multiple, divergent, single-copy genes organized in a chromosomal cluster. Three genes, vlpA, vlpB, and vlpC, have been previously identified in clonal isolates of M. hyorhinis SK76. Each is linked to a characteristic promoter region containing a homopolymeric tract of adenine residues [poly(A) tract], subject to hypermutation, that transcriptionally controls phase variation of vlp genes and leads to combinatorial surface mosaics of distinct Vlp products. The size of the natural vlp gene repertoire is unknown but may critically determine the degree of structural and combinatorial diversity available in this species. In this study, the vlp repertoire of M. hyorhinis GDL-1 was characterized and shown to contain three additional genes, vlpD, vlpE, and vlpF, clustered with other known vlp genes in the order 5'-vlpD-vlpE-vlpF-IS-vlpA-IS-vlpB-vlpC+ ++-3', where IS represents copies of the IS1221 element of M. hyorhinis. The 5' boundary of this expanded family was identical to that of the more limited family 5'-vlpA-IS-vlpB-vlpC-3' previously described in a clonal isolate of strain SK76. A recombinant construct containing vlpD, vlpE, and vlpF expressed antigenically distinguishable products corresponding to each gene. These genes encode characteristic C-terminal repetitive regions that are subject to size variation by insertion or deletion of intragenic repeats but maintain an extended, charged structure. Each vlp gene also contained characteristic alternative open reading frames, which provide a potential reservoir of coding sequence for Vlp diversity, possibly recruited through insertion and/or deletion mutations. These findings demonstrate a vastly expanded potential for structural diversity and combinatorial display of surface mosaics on this organism and suggest that modulation of the vlp repertoire, possibly in conjunction with mobile elements, may determine the capacity for surface variation in natural populations and laboratory strains of this mycoplasma species.

In vivo variation of Mycoplasma gallisepticum antigen expression in experimentally infected chickens.

The antigen expression profiles of Mycoplasma gallisepticum isolates obtained from tracheal swabs of chickens after aerosol-inoculation with M. gallisepticum strain R or clonal variant R/E were examined in western immunoblots. A reference anti-M. gallisepticum chicken antiserum and antisera from individual infected chickens as well as monoclonal antibodies (mAbs) specific for surface proteins were used to monitor in vivo antigenic variation. mAbs 1E5 and 12D8, recognizing PvpA and p67a, recently shown to undergo high-frequency in vitro phase variation, were used for consecutive staining of colony and western immunoblots in order to distinguish between the resultant phenotypes with respect to the corresponding epitopes. Marked differences in the expression of major immunogenic proteins, including p67a, were observed between the two inocula as well as among reisolates recovered at different times of infection. Comparative western immunoblot analysis of the rapidly changing chicken serum antibody response and reisolates recovered during the course of an experimental infection with M. gallisepticum R or clonal variant R/E suggest that immune modulation may have a key role in generating surface diversity. In addition, comparison of colony immunoblots of strain R inoculum and of reisolated colonies from tracheas of birds 8 days post infection indicated an in vivo selection of the PvpA+p67a- phenotype. This study established that surface antigens of M. gallisepticum are subjected in vivo to rapid alteration in their expression. This variability may function as a crucial adaptive mechanism, enabling the organism to escape from the host immune defense and to adapt to the changing host environment at different stages of a natural infection.

A surface epitope undergoing high-frequency phase variation is shared by Mycoplasma gallisepticum and Mycoplasma bovis.

We have recently reported that three distinct size- and phase-variable surface lipoproteins (Vsps) of the bovine pathogen Mycoplasma bovis possess a common epitope recognized by monoclonal antibody 1E5. In the present study, we show that this epitope is also present on a size-variant protein (PvpA) of the avian pathogen Mycoplasma gallisepticum. Application of monoclonal antibody 1E5 in Western immunoblot analysis of Triton X-114 phase-fractionated proteins and in colony immunoblots, as well as in trypsin and carboxypeptidase digestion experiments, has demonstrated that (i) PvpA is an integral membrane protein with a free C terminus, (ii) the shared epitope is surface exposed, and (iii) PvpA is subjected to high-frequency phase variation in expression. By using serum antibodies from M. gallisepticum-infected chickens, we were able to demonstrate the immunogenic nature of PvpA and identify three additional highly immunogenic Triton X-114 phase proteins (p67, p72, and p75) also undergoing high-frequency phase variation spontaneously and independently. Metabolic labeling experiments with [14C]palmitate and [14C]oleate revealed that PvpA, in contrast to p67, p72, and p75, is not lipid modified. Southern blot hybridization with restriction fragments carrying the pvpA gene of M. gallisepticum or the vspA gene of M. bovis against digested genomic DNA of the two Mycoplasma species indicated the absence of genetic relatedness between the pvpA and vspA genes. The apparent complexity of the antigenic variation phenomenon in M. gallisepticum is discussed.