The chromosome of the murine pathogen Mycoplasma pulmonis undergoes rearrangements at a high frequency. We show that some of these rearrangements regulate the phase-variable expression of a cluster of genes (the vsa locus) that encode the variable V-1 surface antigens. Only one vsa gene is associated with an expression site; the other vsa genes are transcriptionally silent. The silent genes lack the 5' end region (promoter and ribosome-binding site) that is present in the expressed gene, and DNA rearrangements regulate gene expression by reassorting the 5' end region from an expressed gene with the 3' end region from a previously silent gene. All vsa rearrangements identified so far are site-specific DNA inversions that occur between copies of a specific 34 bp sequence that is conserved in each vsa gene. Interestingly, DNA inversions within the vsa locus apparently occur in concert with inversion of the hsd1 element, which regulates restriction and modification activity in M. pulmonis.

The cDNA for human erythrocyte ankyrin has been isolated from a series of overlapping clones obtained from a reticulocyte cDNA library. The composite cDNA sequence has a large open reading frame of 5636 base pairs (bp) with the complete coding sequence for a polypeptide of 1879 amino acids with a predicted molecular mass of 206 kDa. The derived amino acid sequence contained 194 residues that were identical to those obtained by direct amino acid sequencing of 11 ankyrin proteolytic peptides. The primary sequence contained 23 highly homologous repeat units of 33 amino acids within the 90-kDa band 3 binding domain. Two cDNA clones showed evidence of apparent mRNA processing, resulting in the deletions of 486 bp and 135 bp, respectively. The 486-bp deletion resulted in the removal of a 16-kDa highly acidic peptide, and the smaller deletion had the effect of altering the COOH terminus of the molecule. Radiolabeled ankyrin cDNAs recognized two erythroid message sizes by RNA blot analysis, one of which was predominantly associated with early erythroid cell types. An ankyrin message was also observed in RNA from the human cerebellum by the same method. The ankyrin gene is assigned to chromosome 8 using genomic DNA from a panel of sorted human chromosomes.

SummaryTo obtain mutants for the study of the basic biology and pathogenic mechanisms of mycoplasmas, the insertion site of transposon Tn4001T was determined for 1700 members of a library of Mycoplasma pulmonis mutants. After evaluating several criteria for gene disruption, we concluded that 321 of the 782 protein coding regions were inactivated. The dispensable and essential genes of M. pulmonis were compared with those reported for Mycoplasma genitalium and Bacillus subtilis. Perhaps the most surprising result of the current study was that unlike other bacteria, ribosomal proteins S18 and L28 were dispensable. Carbohydrate transport and the susceptibility of selected mutants to UV irradiation were examined to assess whether active transposition of Tn4001T within the genome would confound phenotypic analysis. In contrast to earlier reports suggesting that mycoplasmas were limited in their DNA repair machinery, mutations in recA, uvrA, uvrB and uvrC resulted in a DNA-repair deficient phenotype. A mutant with a defect in transport of N-acetylglucosamine was identified.

The vsa genes of Mycoplasma pulmonis encode the V-1 lipoproteins. Most V-1 proteins contain repetitive domains and are thought to be involved in mycoplasma-host cell interactions. Previously, we have reported the isolation and characterization of six vsa genes comprising a 10-kb region of the genome of M. pulmonis strain KD735-15. In the current study, vsa-specific probes were used to clone several fragments from a genomic library of KD735-15 DNA and assemble a single 20-kb contig containing 11 vsa genes. The middle region of the vsa locus contains a large open reading frame (ORF) that is not a vsa gene and has undergone an internal deletion in some strains. The ORF is predicted to encode a membrane protein that may have a role in disease pathogenesis. To examine vsa genes in a strain of M. pulmonis that is unrelated to KD735-15, strain CT was studied. Through Southern hybridization and genomic cloning analyses, CT was found to possess homologs of the KD735-15 vsaA, -C, -E, and -F genes and two unique genes (vsaG and vsaH) that were not found in KD735-15. High-frequency, site-specific DNA inversions serve to regulate the phase-variable production of individual V-1 proteins. As a result of the sequence analysis of vsa recombination products, a model in which DNA inversion arises from strand exchange involving at least six nucleotides of the vrs box is proposed.Mycoplasmas cause slowly progressive, chronic diseases in human and animals. The mechanisms of mycoplasmal disease pathogenesis are poorly understood, and there are no effective control measures. Mycoplasma pulmonis is the etiologic agent of murine respiratory mycoplasmosis and can also cause genital disease and arthritis in rats and mice (31). Thus, M. pulmonis can colonize a variety of epithelial surfaces. Rat isolates of M. pulmonis such as strains UAB 5782 and UAB 6510 are generally more virulent in rats than in mice (10,11,24). In the mouse, UAB 5782 and UAB 6510 colonize the respiratory tract without usually causing lesions (10). In contrast, the mouse isolate strain CT causes severe respiratory disease in the mouse (6,7,10,12). Mycoplasma factors that contribute to the host specificity of disease are unknown. A comparison of the proteins produced by 18 strains of M. pulmonis revealed mostly conserved proteins that were invariant among strains (38). An exception was the V-1 family of surface proteins that are encoded by the vsa (variable surface antigen) genes (4,21,33,35,39). Variation in the V-1 proteins may contribute to the host specificity of the mycoplasma and to the chronicity and severity of disease.The chronic nature of mycoplasmal diseases indicates that mycoplasmas can adapt to the rapidly changing conditions in the host. Previous studies had shown that phenotypic variation and genetic recombination occur at high frequencies in M. pulmonis (3). The vsa genes comprise one of the highly recombinogenic loci in this species. Recombination between vsa genes involves site-specific DNA inversions occurring at a 34-bp sequence that defines the vsa ...

Experiments were undertaken to examine gene transfer in Mycoplasma pulmonis. Parent strains containing transposon-based tetracycline and chloramphenicol resistance markers were combined to allow transfer of markers. Two mating protocols were developed. The first consisted of coincubating the strains in broth culture for extended periods of time. The second protocol consisted of a brief incubation of the combined strains in a 50% solution of polyethylene glycol. Using either protocol, progeny that had acquired antibiotic resistance markers from both parents were obtained. Analysis of the progeny indicated that only the transposon and not flanking genomic DNA was transferred to the recipient cell. Gene transfer was DNase resistant and probably the result of conjugation or cell fusion.Transformation, transduction, and conjugation are the three primary mechanisms of horizontal gene exchange in bacteria. Because mollicutes lack a cell wall and are bound by a single membrane, a process involving membrane fusion is a fourth potential mechanism for gene exchange between these organisms. The first reports of chromosomal gene transfer in the class Mollicutes was in Spiroplasma citri (2, 3). Gene exchange has also been reported in Mycoplasma pulmonis (26), but it was later shown that the organism used for this study was actually Acholeplasma (11).Exchange of chromosomal DNA between cells of the genus Mycoplasma has not been previously demonstrated. Mycoplasmas probably do not acquire DNA by natural transformation (12, 17). Although mycoplasma phages are known to exist, transduction has not been described. Several mycoplasmas can acquire the conjugative transposon Tn916 by mating with an enterococcal donor, but conjugal transfer of any genetic element, including Tn916, from a mycoplasmal donor has not been described (12,17). However, circumstantial evidence suggests that horizontal gene exchange has occurred between species of Mycoplasma. The IS1221 insertion sequence element is found in M. hyorhinis, M. hyopneumoniae, and M. flocculare (19). These three species are all parasites of swine, and IS1221 could have spread by horizontal gene exchange in the animal host. The pMGA genes of M. gallisepticum have closely related genes in M. imitans and M. synoviae but not in phylogenetically related mycoplasmas from humans (27, 28). These three species are avian pathogens, leading again to the suggestion of horizontal gene exchange in animal hosts.Genetic markers have recently become available to study gene exchange in the murine pathogen M. pulmonis. The tetM determinant of transposon Tn916 has been a widely used antibiotic resistance marker in many mycoplasmas, including M. pulmonis (12,17). We constructed a chloramphenicol acetyltransferase gene (cat) that functions in M. pulmonis and described the use of transposon Tn4001 as a delivery vehicle to insert the cat and tetM genes into the mycoplasma chromosome (15a). Two parent strains, one with tetM and the other with cat, were mixed in broth and incubated together in the absence ...

Mycoplasmas have originated from Gram-positive bacteria via rapid degenerative evolution. The results of previous investigations of mycoplasmal DNA polymerases suggest that the process of evolution has wrought a major simplification of the typical Gram-positive bacterial DNA polymerase profile, reducing it from three exonuclease (exo)-positive enzymes to a single exo-negative species. The objective of this work was to rigorously investigate this suggestion, focusing on the evolutionary fate of DNA polymerase III (Pol III), the enzyme which Gram-positive bacteria specifically require for replicative DNA synthesis. The approach used Mycoplasma pulmonis as the model organism and exploited structural gene cloning, enzymology, and Pol III-specific inhibitors of the HPUra class as investigative tools. Our results indicate that M. pulmonis has strongly conserved a single copy of a structural gene homologous to polC, the Gram-positive bacterial gene encoding Pol III. M. pulmonis was found to possess a DNA polymerase that displays the size, primary structure, exonuclease activity, and level of HPUra sensitivity expected of a prototypical Gram-positive Pol III. The high level of sensitivity of M. pulmonis growth to Gram-positive Pol III-selective inhibitors of the HPUra type strongly suggests that Mycoplasma has conserved not only the basic structure of Pol III, but also its essential replicative function. Evidence for a second, HPUra-resistant polymerase activity in M. pulmonis is also described, indicating that the DNA polymerase composition of Mycoplasma is complex and closer to that of Gram-positive bacteria than previously thought.

Mycoplasma arthritidis induces an acute to chronic arthritis in rodents. Arthritis induced in mice histologically resembles human rheumatoid arthritis and can be associated with lethal toxicity following systemic injection. The M. arthritidis mitogen (MAM) superantigen has long been implicated as having a role in pathogenesis, but its significance with respect to toxicity and arthritogenicity in mycoplasma-induced disease is unclear. To study the pathogenic significance of MAM, M. arthritidis mutants that overproduced or failed to produce MAM were developed. MAM overproduction and knockout mutants were more and less mitogenic, respectively, than the wild-type strain. The degree of mitogenic activity correlated with lethal toxicity in DBA/2J mice. In contrast, histopathological studies detected no correlation between MAM production and the severity of arthritis induced in DBA/2J and CBA/J mice.

The lysogenic bacteriophage MAV1 has been shown to be a virulence factor for the development of arthritis in rats infected with Mycoplasma arthritidis. In the present study, arthritis was evaluated by histopathologic examination to demonstrate that MAV1 is a virulence factor not only in the rat but also in the mouse. Specifically, the MAV1 lysogen 158L3-1 was more virulent than the nonlysogen strain 158 in DBA/2NCr, C3H/HeNCr, C3H/HeJ, and C3Smn.CB17-Prkdc(scid)/J mice, as well as in LEW rats.