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The near‐minimal bacterium Mesoplasma florum is an interesting model for synthetic genomics and systems biology due to its small genome (~ 800 kb), fast growth rate, and lack of pathogenic potential. However, fundamental aspects of its biology remain largely unexplored. Here, we report a broad yet remarkably detailed characterization of M. florum by combining a wide variety of experimental approaches. We investigated several physical and physiological parameters of this bacterium, including cell size, growth kinetics, and biomass composition of the cell. We also performed the first genome‐wide analysis of its transcriptome and proteome, notably revealing a conserved promoter motif, the organization of transcription units, and the transcription and protein expression levels of all protein‐coding sequences. We converted gene transcription and expression levels into absolute molecular abundances using biomass quantification results, generating an unprecedented view of the M. florum cellular composition and functions. These characterization efforts provide a strong experimental foundation for the development of a genome‐scale model for M. florum and will guide future genome engineering endeavors in this simple organism., A deep characterization of the near‐minimal bacterium M. florum reveals important features of this emerging model organism for systems and synthetic biology.

Mycoplasma genitalium is an important etiologic agent of non-gonococcal urethritis (NGU), known for chronicity and multidrug resistance, in which biofilms may play an integral role. In some bacterial species capable of forming biofilms, extracellular polymeric substances (EPS) composed of poly-N-acetylglucosamine (PNAG) are a crucial component of the matrix. Monosaccharide analysis of M. genitalium strains revealed high abundance of GlcNAc, suggesting a biofilm-specific EPS. Chromatograms also showed high concentrations of galactose and glucose as observed in other mycoplasma species. Fluorescence microscopy of M. genitalium biofilms utilizing fluor-coupled lectins revealed differential staining of biofilm structures. Scanning electron microscopy (SEM) showed increasing maturation over time of bacterial “towers” seen in biofilm development. As seen with Mycoplasma pneumoniae, organisms within fully mature M. genitalium biofilms exhibited loss of cell polarization. Bacteria associated with disrupted biofilms exhibited decreased dose-dependent viability after treatment with antibiotics compared to bacteria with intact biofilms. In addition, growth index analysis demonstrated decreases in metabolism in cultures with disrupted biofilms with antibiotic treatment. Taken together, these data suggest that M. genitalium biofilms are a contributing factor in antibiotic resistance.

Aim: To characterize inter- and intra-strain variability of variable-number tandem repeats (VNTRs) in Mycoplasma pneumoniae to determine the optimal multilocus VNTR analysis scheme for improved strain typing. Methods: Whole genome assemblies and next-generation sequencing data from diverse M. pneumoniae isolates were used to characterize VNTRs and their variability, and to compare the strain discriminability of new VNTR and existing markers. Results: We identified 13 VNTRs including five reported previously. These VNTRs displayed different levels of inter- and intra-strain copy number variations. All new markers showed similar or higher discriminability compared with existing VNTR markers and the P1 typing system. Conclusion: Our study provides novel insights into VNTR variations and potential new multilocus VNTR analysis schemes for improved genotyping of M. pneumoniae.

Background Mycoplasma pneumoniae (Mpn), one of the smallest self-replicating prokaryotes, is known to readily adhere to host cells and to form aggregates in suspension. Having only one cell membrane and no cell wall, mycoplasmas present questions as to optimal aggregate disruption method while minimizing cell death in vitro. We compared conventional vortex mixing with other methods for disruption of bacterial aggregates and for its effect on cell viability. Methods Strain UAB PO1, a clinical Mpn isolate, was dispersed using a conventional vortex mixer with or without nonionic detergent (0.1% and 0.01% Tween-20), a probe-type ultrasonicator, or repeated passage through a 27-gauge needle. The resulting suspensions were assayed for recoverable colony-forming units (CFU). Flow cytometric assays were carried out to examine particle size and membrane integrity with the transmembrane potential dye DiBAC4. Wet Scanning Transmission Electron Microscopy (Wet-STEM) was performed for high resolution imaging of the resultant cell suspensions. Additional Mpn strains and other human mollicute species were assayed in a similar manner. Mice were infected with either vortexed or sonicated UAB PO1 and bacterial persistence was examined via Mpn-specific 16S qPCR. Results Comparison between dispersion methods showed a 10-fold enrichment of recoverable Mpn CFU with sonication compared to other methods. Time-course analysis showed significantly lower bacterial CFU with vortexing compared to sonication at all time points. Flow cytometric analysis showed increased cellular membrane damage via DiBAC4 staining in sonicated suspensions, but a decreased particle size. Wet-STEM imaging showed markedly improved dispersion with sonication compared to conventional vortex treatment, and surprisingly vortexing for 30 s produced up to a 100-fold drop in CFU. Results similar to UAB PO1 were obtained with three additional Mpn strains and other Mollicutes species, although they exhibited differential susceptibilities to disaggregation by sonication. Finally, increased persistence of the organism in a mouse model of infection was observed using sonicated suspensions for initial infection. Conclusions Sonication is superior to vortexing with or without nonionic detergent or repeated 27-gauge needle passage for dispersion of Mpn aggregates while preserving cell viability. Preparation of Mpn suspensions for in vivo experiments is best accomplished using brief sonication due to the dramatic increase in CFU produced by sonication. Dispersion methods may affect the final experimental results and should be an important consideration for future research involving mycoplasma species.

Summary Although mycoplasmas have a paucity of glycosyltransferases and nucleotidyltransferases recognizable by bioinformatics, these bacteria are known to produce polysaccharides and glycolipids. We show here that mycoplasmas also produce glycoproteins and hence have glycomes more complex than previously realized. Proteins from several species of Mycoplasma reacted with a glycoprotein stain, and the murine pathogen Mycoplasma arthritidis was chosen for further study. The presence of M. arthritidis glycoproteins was confirmed by high-resolution mass spectrometry. O-linked glycosylation was clearly identified at both serine and threonine residues. No consensus amino acid sequence was evident for the glycosylation sites of the glycoproteins. A single hexose was identified as the O-linked modification, and glucose was inferred by 13C-labelling to be the hexose at several of the glycosylation sites. This is the first study to conclusively identify sites of protein glycosylation in any of the mollicutes.

We investigated the mode of action underlying the anti-mycoplasma activity of cationic antimicrobial peptides (AMPs) using four known AMPs and Mycoplasma pulmonis as a model mycoplasma. Scanning electron microscopy revealed that the integrity of the M. pulmonis membrane was significantly damaged within 30min of AMPs exposure, which was confirmed by measuring the uptake of propidium iodine into the mycoplasma cells. The anti-mycoplasma activity of AMPs was found to depend on the binding affinity for phosphatidylcholine, which was incorporated into the mycoplasma membrane from the growth medium and preferentially distributed in the outer leaflet of the lipid bilayer.

Despite the apparent absence of genes coding for the known pathways for biosynthesis, the monosaccharide rhamnose was detected in the d configuration in Mycoplasma pneumoniae and Mycoplasma pulmonis, and in both the d and l configurations in Mycoplasma arthritidis. Surprisingly, the monosaccharide glucose was not a precursor for rhamnose biosynthesis and was not incorporated at detectable levels in glucose-containing polysaccharides or glycoconjugates. In contrast, carbon atoms from starch, a polymer of glucose, were incorporated into rhamnose in each of the three species examined. When grown in a serum-free medium supplemented with starch, M. arthritidis synthesized higher levels of rhamnose, with a shift in the relative amounts of the d and l configurations. Our findings suggest the presence of a novel pathway for rhamnose synthesis that is widespread in the genus Mycoplasma.

Many proteins that have a primary function as a cytoplasmic protein are known to have the ability to moonlight on the surface of nearly all organisms. An example is the glycolytic enzyme enolase, which can be found on the surface of many types of cells from bacteria to human. Surface enolase is not enzymatic because it is monomeric and oligomerization is required for glycolytic activity. It can bind various molecules and activate plasminogen. Enolase lacks a signal peptide and the mechanism by which it attaches to the surface is unknown. We found that treatment of whole cells of the murine pathogen Mycoplasma pulmonis with phospholipase D released enolase and other common moonlighting proteins. Glycostaining suggested that the released proteins were glycosylated. Cytoplasmic and membrane-bound enolase was isolated by immunoprecipitation. No post-translational modification was detected on cytoplasmic enolase, but membrane enolase was associated with lipid, phosphate and rhamnose. Treatment with phospholipase released the lipid and phosphate from enolase but not the rhamnose. The site of rhamnosylation was identified as a glutamine residue near the C-terminus of the protein. Rhamnose has been found in all species of mycoplasma examined but its function was previously unknown. Mycoplasmas are small bacteria with have no peptidoglycan, and rhamnose in these organisms is also not associated with polysaccharide. We suggest that rhamnose has a central role in anchoring proteins to the membrane by linkage to phospholipid, which may be a general mechanism for the membrane association of moonlighting proteins in mycoplasmas and perhaps other bacteria.

Although they lack a cell wall, mycoplasmas do possess a glycocalyx. The interactions between the glycocalyx, mycoplasmal surface proteins and host complement were explored using the murine pathogen Mycoplasma pulmonis as a model. It was previously shown that the length of the tandem repeat region of the surface lipoprotein Vsa is associated with susceptibility to complement-mediated killing. Cells producing a long Vsa containing about 40 repeats are resistant to complement, whereas strains that produce a short Vsa of five or fewer repeats are susceptible. We show here that the length of the Vsa protein modulates the affinity of the M. pulmonis EPS-I polysaccharide for the mycoplasma cell surface, with more EPS-I being associated with mycoplasmas producing a short Vsa protein. An examination of mutants that lack EPS-I revealed that planktonic mycoplasmas were highly susceptible to complement killing even when the Vsa protein was long, demonstrating that both EPS-I and Vsa length contribute to resistance. In contrast, the mycoplasmas were resistant to complement even in the absence of EPS-I when the cells were encased in a biofilm.

Here, we describe mutants of Mycoplasma pulmonis that were obtained using a minitransposon, Tn4001TF1, which actively transposes but is then unable to undergo subsequent excision events. Using Tn4001TF1, we disrupted 39 genes previously thought to be essential for growth. Thus, the number of genes required for growth has been overestimated. This study also revealed evidence of gene duplications in M. pulmonis and identified chromosome segregation proteins that are dispensable in mycoplasmas but essential in Bacillus subtilis.

Ichthyophthirius multi¢liis(Ich), a ciliated protozoanparasite of ¢sh, expresses surface antigens (i-anti-gens), which react with host antibodies that renderthemimmobile.Thenucleotidesequenceofani-anti-gen gene of I. multi¢liis strain ARS-6 was deduced.The predicted protein of 47493Da is comprised of460 amino acids (aa’s) arranged into ¢ve imperfectrepeats with periodic cysteine residues with thestructure: CX (19)20 CX 2 CX 16 27 CX 2 CX 20(21) CX 3 . TheN-terminal aa’s typify a signal peptide motif whilea stretch of C-terminal aa’s resemble a glycosyl^phosphatidyl^inositol (GPI)-anchor addition site.Thedegreeofdeducedi-antigenaasequenceidentityof strain ARS-6 (GenBank accession # ACH87654and # ACH95659) with other I. multi¢liis i-antigensequences present in GenBank ranges from 99% to36% identity with 52kDa i-antigens of I. multi¢liisstrain G5 (accession #s AAK94941and AAK01661respectively). Immunoblot analysis of i-antigens fol-lowing exposure of I. multi¢liis theronts to cat¢shanti-I. multi¢liis immune serum did not show anyappreciable alteration in i-antigen expression. Themechanism that regulates i-antigen expression inI.multi¢liisremainsapuzzlingquestion.Keywords: immobilization antigen, gene, nucleo-tidesequence,Ichthyophthiriusmulti¢liisIntroductionIchthyophthiriusmulti¢liis(Ich) is a hymenostomatidparasitic ciliate that causes ‘white spot’ disease inmany species of freshwater cultured and/or free liv-ing ¢sh. Fish that recover from I. multi¢liis infectionareknowntopossessbothserumandcutaneousmu-cus antibodies that are capable of immobilizing theparasite in vitro (Clark & Dickerson 1997). The anti-gens on the parasite surface that are involved in theantibody-mediatedimmobilizationarecomprisedofaclass of abundant glycosyl^phosphatidyl^inositol(GPI)-anchoredmembraneproteinsthatareaptlyde-signated as immobilization antigens (i-antigens)(Clark, Gao, Gaertig, Wang & Cheng 2001; Clark FHatanka,Umeda,YamashitaH Swennes, Findly DLee & Kim 2008).Tetrahymena and Paramecium spe-cies are free-living ciliated protists that have servedas model systems, in which i-antigens have been ex-tensively studied (Doerder 2000; Simon & Schmidt2005). Both Tetrahymena and Paramecium possess arepertoire of paralogous genes that encode alterna-tive forms of i-antigens, the expression of which isunder the in£uence of certain environmental cuessuch as temperature and medium, in which they

Summary The presence of capsular exopolysaccharide (EPS) in Mollicutes has been inferred from electron micrographs for over 50 years without conclusive data to support the production of complex carbohydrates by the organism. Mycoplasma pulmonis binds the lectin Griffonia simplicifolia I (GS-I), which is specific for terminal β-linked galactose residues. Mutants that failed to produce the EPS bound by GS-I were isolated from a transposon library. All of the mutants had the transposon located in open reading frame MYPU_7410 or MYPU_7420. These overlapping genes are predicted to code for a heterodimeric pair of ABC transporter permeases and may code for part of a new pathway for synthesis of EPS. Analysis by lectin-affinity chromatography in conjunction with gas chromatography demonstrated that the wild-type mycoplasma produced an EPS (EPS-I) composed of equimolar amounts of glucose and galactose that was lacking in the mutants. Phenotypic analysis revealed that the mutants had an increased propensity to form a biofilm on glass surfaces, colonized mouse lung and trachea efficiently, but had a decreased association with the A549 lung cell line. Confounding the interpretation of these results is the observation that the mutants missing EPS-I had an eightfold overproduction of an apparent second EPS (EPS-II) containing N-acetylglucosamine.

Biofilms are communities of microorganisms that are encased in polymeric matrixes and grow attached to biotic or abiotic surfaces. Despite their enhanced ability to resist antimicrobials and components of the immune system in vitro, few studies have addressed the interactions of biofilms with the host at the organ level. Although mycoplasmas have been shown to form biofilms on glass and plastic surfaces, it has not been determined whether they form biofilms on the tracheal epithelium. We developed a tracheal organ-mounting system that allowed the entire surface of the tracheal lumen to be scanned using fluorescence microscopy. We observed the biofilms formed by the murine respiratory pathogen Mycoplasma pulmonis on the epithelium of trachea in tracheal organ culture and in experimentally infected mice and found similar structure and biological characteristics as biofilms formed in vitro. This tracheal organ-mounting system can be used to study interactions between biofilms formed by respiratory pathogens and the host epithelium and to identify the factors that contribute to biofilm formation in vivo.

Even though free-living, mycoplasmas depend on their hosts for key nutrients, including purines, pyrimidines, several amino acids, and sterols, which are incorporated into the membranes of these wall-less bacteria. These requirements reflect their relative genomic simplicity. The 580-kb genome of Mycoplasma genitalium, for example, is comparable in size to that of a large virus.

The length of the tandem repeat region of the Vsa protein of Mycoplasma pulmonis has previously been shown to modulate the susceptibility of mycoplasmas to killing by complement: cells that produce a short form of the Vsa protein are highly sensitive, and cells producing the long Vsa protein are resistant. In contrast to their differing susceptibilities to complement, the mycoplasmas were highly sensitive to gramicidin irrespective of the length of the Vsa protein produced. We show here that when encased within a biofilm, cells of M. pulmonis producing a short form of the Vsa protein were more resistant to complement and gramicidin than mycoplasmas that were dispersed. The resistance appeared to be localized to those mycoplasmas within tower structures of the biofilms. Biofilm formation may be a mechanism that protects mycoplasmas from host immunity.

Bacterial biofilms are communities of bacteria that are enclosed in an extracellular matrix. Within a biofilm the bacteria are protected from antimicrobials, environmental stresses, and immune responses from the host. Biofilms are often believed to have a highly developed organization that is derived from differential regulation of the genes that direct the synthesis of the extracellular matrix and the attachment to surfaces. The mycoplasmas have the smallest of the prokaryotic genomes and apparently lack complex gene-regulatory systems. We examined biofilm formation by Mycoplasma pulmonis and found it to be dependent on the length of the tandem repeat region of the variable surface antigen (Vsa) protein. Mycoplasmas that produced a short Vsa protein with few tandem repeats formed biofilms that attached to polystyrene and glass. Mycoplasmas that produced a long Vsa protein with many tandem repeats formed microcolonies that floated freely in the medium. The biofilms and the microcolonies contained an extracellular matrix which contained Vsa protein, lipid, DNA, and saccharide. As variation in the number of Vsa tandem repeats occurs by slipped-strand mispairing, the ability of the mycoplasmas to form a biofilm switches stochastically.

Phase-variable lipoproteins are commonly found in Mycoplasma species. Mycoplasma pulmonis contains a family of extensively studied phase- and size-variable lipoproteins encoded by the vsa locus. The Vsa surface proteins vary at a high frequency, the in vivo significance of which has yet to be determined. We investigated the role of Vsa phase variation in respect to tissue tropism and avoidance of the immune system in the mouse host. Mycoplasmas were cultured 3, 14, and 21 days postinoculation from the nose, lung, trachea, liver, and spleen of experimentally infected C57BL/6 (wild-type), C57BL/6-RAG-1 −/− (RAG −/− ), and C57BL/6-inducible nitric oxide synthase (iNOS) −/− (iNOS −/− ) mice. In wild-type and iNOS −/− mice, a large number of Vsa variants were seen by 21 days postinoculation. In contrast, little Vsa variation occurred in all tissues of RAG −/− mice. Analysis of isolates from 14 days postinoculation revealed less variation of the Vsa proteins in iNOS −/− mice than in the wild type. Western blot analysis of isolates from each strain of mouse demonstrated that Vsa phase variation occurred independently of size variation, indicating no obvious selection pressure for size variants. Additionally, these experiments provided no evidence that mycoplasmas producing particular Vsa proteins adhered only to specific tissues. The data strongly indicate that Vsa phase variation is a mechanism for avoidance of the immune system with no obvious contribution to tissue tropism.

Mycoplasma arthritidis causes acute polyarthritis in rats and chronic proliferative arthritis in mice. M. arthritidis -induced arthritis serves as a model for arthritis caused by infectious agents and as a model for examining the role of the superantigen MAM ( M. arthritidis T-cell mitogen) in the development of autoimmunity. M. arthritidis strain 158-1 is a spontaneous mutant of strain 158 that has a drastic reduction in virulence. We show that the mutant is missing a major antigen of 47 kDa (P47) and has acquired a protein of 67 kDa (P67). P47 and P67 partitioned into the detergent phase by extraction with Triton X-114. Coomassie blue staining of sodium dodecyl sulfate-polyacrylamide gels show that P67 is produced in abundance. Analysis of gel-purified P67 by mass spectrometry led to its identification as a lipoprotein (the open reading frame [ORF] 619 gene product) predicted from the genome sequence of M. arthritidis . PCR analysis of genomic DNA from 158 and 158-1 indicates that P47 and P67 are encoded by the same ORF 619 gene and differ only in the number of repeats in a tandem repeat region. By two-dimensional polyacrylamide gel analysis, no protein differences were detectable between 158 and 158-1 other than P47 and P67. Collectively, the data suggest that the tandem repeat region of P47 and P67 influences disease outcome.

The variable surface antigens (Vsa) of the murine respiratory pathogen Mycoplasma pulmonis are associated with the virulence of the microorganism in the lung. In strain UAB CT, the antigens consist of an N-terminal region that is combined with one of seven different C-terminal variable regions comprised of tandem repeats. M. pulmonis producing a VsaA protein with about 40 tandem repeats (R40) does not adhere to red blood cells or polystyrene. Strains that produce VsaH contain a short C-terminal region that lacks tandem repeats and adhere to red blood cells and plastic. We isolated and analyzed M. pulmonis strain CT variants (CT182 and derivatives) that produced a VsaA protein with only three tandem repeats (R3). These variants adhered to plastic and red blood cells similarly to the VsaH-producing strain. When the R3-producing CT182 strain or the VsaH-producing strains were incubated with normal guinea pig serum, they were efficiently killed. Killing was abolished when the serum was heat inactivated. In contrast, the M. pulmonis strains that produced VsaA R40 were highly resistant to complement killing. CT182R3 variants that survived the complement killing reactions all produced the R40 form of VsaA and were resistant to complement killing. VsaA R40 is the first mycoplasmal protein shown to be associated with resistance to complement. As both VsaH and VsaA can mediate adherence to plastic, cytadherence, and susceptibility to complement, we propose that Vsa modulates these phenotypes by nonspecific interactions.

Background Mycoplasma pneumoniae is a common pathogen that causes upper and lower respiratory tract infections in people of all ages, responsible for up to 40 % of community-acquired pneumonias. It also causes a wide array of extrapulmonary infections and autoimmune phenomena. Phylogenetic studies of the organism have been generally restricted to specific genes or regions of the genome, because whole genome sequencing has been completed for only 4 strains. To better understand the physiology and pathogenicity of this important human pathogen, we performed comparative genomic analysis of 15 strains of M. pneumoniae that were isolated between the 1940s to 2009 from respiratory specimens and cerebrospinal fluid originating from the USA, China and England. Results Illumina MiSeq whole genome sequencing was performed on the 15 strains and all genome sequences were completed. Results from the comparative genomic analysis indicate that although about 1500 SNP and indel variants exist between type1 and type 2 strains, there is an overall high degree of sequence similarity among the strains (>99 % identical to each other). Within the two subtypes, conservation of most genes, including the CARDS toxin gene and arginine deiminase genes, was observed. The major variation occurs in the P1 and ORF6 genes associated with the adhesin complex. Multiple hsdS genes (encodes S subunit of type I restriction enzyme) with variable tandem repeat copy numbers were found in all 15 genomes. Conclusions These data indicate that despite conclusions drawn from 16S rRNA sequences suggesting rapid evolution, the M. pneumoniae genome is extraordinarily stable over time and geographic distance across the globe with a striking lack of evidence of horizontal gene transfer. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-1801-0) contains supplementary material, which is available to authorized users.

Mycoplasma pneumoniae causes chronic respiratory disease in humans. Factors thought to be important for colonization include the ability of the mycoplasma to form a biofilm on epithelial surfaces and the production of hydrogen peroxide to damage host tissue. Almost all of the mycoplasmas, including M. pneumoniae, lack superoxide dismutase and catalase and a balance should exist between peroxide production and growth. We show here that the addition of catalase to cultures enhanced the formation of biofilms and altered the structure. The incorporation of catalase in agar increased the number of colony-forming units detected and hence could improve the clinical diagnosis of mycoplasmal diseases.

The lack of a cell wall, flagella, fimbria, and other extracellular appendages and the possession of only a single membrane render the mycoplasmas structurally simplistic and ideal model organisms for the study of glycoconjugates. Most species have genomes of about 800 kb and code for few proteins predicted to have a role in glycobiology. The murine pathogens Mycoplasma arthritidis and Mycoplasma pulmonis have only a single gene annotated as coding for a glycosyltransferase but synthesize glycolipid, polysaccharide and glycoproteins. Previously, it was shown that M. arthritidis glycosylated surface lipoproteins through O-linkage. In the current study, O-linked glycoproteins were similarly found in M. pulmonis and both species of mycoplasma were found to also possess N-linked glycans at residues of asparagine and glutamine. Protein glycosylation occurred at numerous sites on surface-exposed lipoproteins with no apparent amino acid sequence specificity. The lipoproteins of Mycoplasma pneumoniae also are glycosylated. Glycosylation was dependent on the glycosidic linkages from host oligosaccharides. As far as we are aware, N-linked glycoproteins have not been previously described in Gram-positive bacteria, the organisms to which the mycoplasmas are phylogenetically related. The findings indicate that the mycoplasma cell surface is heavily glycosylated with implications for the modulation of mycoplasma-host interactions.

Site-specific DNA invertible elements often control the production of bacterial surface proteins that are subject to phase variation (ON/OFF switching). Inversion of the DNA element occurs as a result of the reciprocal exchange of DNA catalysed by a specialized enzyme (recombinase) that acts at specific sites. By continually switching the orientation of the invertible element in the chromosome, and consequently the production of the variable protein(s), the cell population remains continually responsive to environmental change such as immunological challenge. In addition to phase-variable surface proteins, Mycoplasma pulmonis has a family of phase-variable restriction-modification enzymes. We report here that a single recombinase in M. pulmonis, HvsR, catalyses independent DNA inversions at non-homologous loci, causing variations in surface lipoproteins and in the DNA recognition sequence specificity of restriction enzymes. Thus, HvsR is a site-specific DNA recombinase with dual substrate specificity.

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.

Three highly mutable loci of the wall-less pathogens Mycoplasma bovis , Mycoplasma pulmonis and Mycoplasma agalactiae undergo high-frequency genomic rearrangements and generate extensive antigenic variation of major surface lipoproteins. Adjacent to each locus, an open reading frame exists as a single chromosomal copy and is predicted to encode a site-specific DNA recombinase exhibiting high homology to the recombinases XerD of Escherichia coli and CodV of Bacillus subtilis . Each of the mycoplasmal proteins are members of the λ integrase family of tyrosine site-specific recombinases and likely mediates site-specific DNA inversions observed within the adjacent, variable loci.

Restriction and modification (R-M) systems are generally thought to protect bacteria from invasion by foreign DNA. This paper proposes the existence of an alternative role for the phase-variable R-M systems encoded by the hsd loci of Mycoplasma pulmonis. Populations of M. pulmonis cells that arose during growth in different environments were compared with respect to R-M activity and surface antigen production. When M. pulmonis strain X1048 was propagated in laboratory culture medium, > 95% of colony-forming units (cfu) lacked R-M activity and produced the variable surface protein VsaA. Mycoplasmas isolated from the nose of experimentally infected rats also lacked R-M activity and produced VsaA. In contrast, the cell population of mycoplasmas isolated from the lower respiratory tract of the infected rats was more complex. The most dramatic results were obtained for mycoplasmas isolated from the trachea. At 14 days postinfection, 38% of mycoplasma isolates produced a Vsa protein other than VsaA, and 34% of isolates had active restriction systems. These data suggest that differences in selection pressures in animal tissues affect the surface proteins and the R-M activity of the mycoplasmal cell population. We propose that variations in the production of R-M activity and cell surface proteins are important for the survival of the mycoplasma within the host.

Mycoplasma virus P1 is one of only four viruses isolated from the genus Mycoplasma. The host for P1, Mycoplasma pulmonis, possesses complex, phase-variable restriction and modification enzymes and the Vsa family of phase-variable surface proteins. The ability of P1 virus to infect host cells is influenced by these phase-variable systems, rendering P1 a valuable tool for assessing host properties. The double-stranded P1 DNA genome was sequenced (11,660 bp) and 11 ORFs were identified. The predicted P1 DNA polymerase is similar to that of phages that are known to have terminal protein (TP) attached to the 5' end of their genome, consistent with previous studies indicating that P1 DNA has covalently attached TP. Most of the other predicted P1 proteins have little sequence similarity to known proteins, and P1 virus is unrelated to the other mycoplasma virus, MAV1, for which the genome sequence is known. One of the predicted P1 proteins, the ORF 8 gene product, contains a repetitive collagen-like motif characteristic of some bacteriophage tail fiber proteins and is a candidate for interacting with the Vsa proteins.

Previous attempts to introduce transposon Tn 4001 into Mycoplasma pulmonis and Mycoplasma arthritidis have not been successful, possibly due to functional failure of the transposon's gentamicin resistance determinant. Tn 4001C and Tn 4001T were constructed, respectively, by insertion of a chloramphenicol acetyltransferase gene and the tetM tetracycline resistance determinant into Tn 4001 . Both Tn 4001C and Tn 4001T transposed in M. pulmonis , and Tn 4001T transposed in M. arthritidis . The incorporation of a Tn 4001T derivative that contained lacZ into either Mycoplasma species resulted in transformants with readily detectable LacZ activity. Tn 4001T may be of general utility for use as a mycoplasma cloning vehicle because tetM functions in all species of Mycoplasma examined thus far.

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.

The lysogenic bacteriophage MAV1, which is associated with the arthritogenicity of Mycoplasma arthritidis , was characterized. Several strains of M. arthritidis were examined for their ability to support growth of MAV1. A PFU assay was developed, and the sensitivity of phage to various chemical treatments was assayed. The most notable result was the resistance of MAV1 to proteinase K. The MAV1 genome is a double-stranded, linear DNA molecule of about 16 kb. The site of MAV1 DNA integration in the host chromosome was investigated. The ends of MAV1 DNA were cloned from three independent lysogens shown to have MAV1 DNA inserted at different sites in the host. The nucleotide sequences of the ends of the MAV1 genome and of the MAV1 DNA-chromosomal DNA junctions from each of three lysogens were determined. Sequences flanking the integrated prophage and the ends of native MAV1 DNA were determined, allowing the identification of the phage DNA ( attP ) and bacterial DNA ( attB ) recombination sites. Analysis of the left MAV1 DNA-chromosomal DNA junction sites showed a single-base heterogeneity located within MAV1 DNA sequences immediately adjacent to the attB sequence. A model for MAV1 integration-excision is proposed.

To improve the detection of Mycoplasma pulmonis contamination of isolates of cilia-associated respiratory (CAR) bacillus, we developed a nested PCR method using primers for 16S rRNA gene sequences. Of 140 samples of 16 different CAR bacillus isolates, 73 (52%) were inhibitory in the first PCR, as indicated by the absence of amplicons of the internal control, but only 11 of 140 (7.9%) were inhibitory in the second PCR. Of 27 samples known to contain M. pulmonis, only 12 (44%) were positive in the first PCR, but 25 of 27 (93%) were positive in the second PCR. Nested PCR also detected M. pulmonis in 21 of 61 (34%) CAR bacillus samples from which M. pulmonis could not be cultured and identified 2 additional M. pulmonis-contaminated CAR bacillus isolates. Of 359 respiratory and reproductive tract lavage samples from rats and mice, 35 (9.8%) were inhibitory in the first PCR, but only 15 (4.2%) were inhibitory in the second PCR. Of 72 lavage specimens from rats inoculated with an avirulent, poorly infective M. pulmonis strain, 14 (19%) were positive by nested PCR, but only 2 of 72 (2.8%) were positive by culture. Nested PCR also detected M. pulmonis in 14 of 20 (70%) paraffin sections of lung and trachea from rats and mice inoculated with CAR bacillus isolates known to contain M. pulmonis, whereas single PCR gave no positive results. We conclude that nested PCR is superior to single PCR or culture for detecting M. pulmonis, and that M. pulmonis is present in all but four CAR bacillus isolates in our collection that were from naturally infected rats; the four isolates that were exceptions were obtained from rats from a single colony.

Although mycoplasmas have a paucity of glycosyltransferases and nucleotidyltransferases recognizable by bioinformatics, these bacteria are known to produce polysaccharides and glycolipids. We show here that mycoplasmas also produce glycoproteins and hence have glycomes more complex than previously realized. Proteins from several species of Mycoplasma reacted with a glycoprotein stain, and the murine pathogen Mycoplasma arthritidis was chosen for further study. The presence of M. arthritidis glycoproteins was confirmed by high-resolution mass spectrometry. O-linked glycosylation was clearly identified at both serine and threonine residues. No consensus amino acid sequence was evident for the glycosylation sites of the glycoproteins. A single hexose was identified as the O-linked modification, and glucose was inferred by (13) C-labelling to be the hexose at several of the glycosylation sites. This is the first study to conclusively identify sites of protein glycosylation in any of the mollicutes.

Mycoplasma arthritidis is a rat pathogen causing a severe polyarthritis. The study of its pathogenic mechanisms has been hampered by the lack of genetic systems for use with M. arthritidis. Described here are procedures for genetic transformation of M. arthritidis and conjugal transfer of Tn916 from an enterococcal donor to M. arthritidis. The location of Tn916 insertion sites in the mycoplasmal chromosome was random, suggesting that Tn916 may be useful as an insertional mutagen in this organism. Additionally, a restriction and modification system was identified which presented a strong barrier to gene transfer. For transformation, the restriction system was circumvented by using DNA that was modified in vitro with the appropriate site-specific methylase (AluI).

Several mycoplasma species have been shown to form biofilms that confer resistance to antimicrobials and which may affect the host immune system, thus making treatment and eradication of the pathogens difficult. The present study shows that the biofilms formed by two strains of the human pathogen Mycoplasma pneumoniae differ quantitatively and qualitatively. Compared with strain UAB PO1, strain M129 grows well but forms biofilms that are less robust, with towers that are less smooth at the margins. A polysaccharide containing N-acetylglucosamine is secreted by M129 into the culture medium but found in tight association with the cells of UAB PO1. The polysaccharide may have a role in biofilm formation, contributing to differences in virulence, chronicity and treatment outcome between strains of M. pneumoniae. The UAB PO1 genome was found to be that of a type 2 strain of M. pneumoniae, whereas M129 is type 1. Examination of other M. pneumoniae isolates suggests that the robustness of the biofilm correlates with the strain type.

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.

Mycoplasma virus P1 is a tailed, polyhedral virus isolated from Mycoplasma pulmonis . To characterize the P1 genome, stocks of virus were prepared free of host cell nucleic acids. A single DNA species of 11.3 kb that was shown by plaque hybridization to be of P1 origin was extracted from the virus. Efficient isolation of P1 DNA required digestion with proteolytic enzymes prior to phenol extraction. Although P1 DNA was double-stranded and linear following such treatment, it was resistant to digestion with the 5′-specific λ exonuclease. Electron microscopic analysis indicated that globular material is complexed to the ends of P1 DNA. The globular material was not observed on protease-treated P1 DNA molecules, suggesting that it is composed of protein. Removal of the putative terminal protein by chemical treatment allowed the cloning of the P1 DNA ends, and nucleotide sequence analysis revealed that these ends contain a 350-bp inverted terminal repeat.

Few mycoplasmal polysaccharides have been described and little is known about their role in pathogenesis. The infection of mice with Mycoplasma pulmonis has been utilized in many in vivo and in vitro studies to gain a better understanding of host–pathogen interactions during chronic respiratory infection. Although alveolar macrophages have a primary role in host defence, M. pulmonis is killed inefficiently in vitro. One antiphagocytic factor produced by the mycoplasma is the family of phase- and size-variable Vsa lipoproteins. However, bacteria generally employ multiple strategies for combating host defences, with capsular polysaccharide often having a key role. We show here that mutants lacking the EPS-I polysaccharide of M. pulmonis exhibit increased susceptibility to binding and subsequent killing by alveolar macrophages. These results give further insight into how mycoplasmas are able to avoid the host immune system and sustain a chronic infection.

Summary 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 rigorousiy 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.

Summary An invertible DNA element of 6.8 kb, designated the hsd1 locus, was identified in the chromosome of Mycoplasma pulmonis. Infection of host cells with mycoplasma virus P1 revealed that the organism's restriction and modification (R-M) properties are controlled by inversion of hsd1. The nucleotide sequence of hsd1 revealed several genes, the predicted amino acids of which bear striking similarity to the subunits of the type I R-M enzymes previously found only in enteric bacteria.

The RecA protein has a central role in DNA repair and is essential for homologous recombination in most eubacteria. Little is known about these critical processes in mycoplasmas. By using standard and inverse polymerase chain reactions (PCR) coupled with conventional cloning techniques, a series of overlapping fragments comprising the entire recA genes of Mycoplasma mycoides subsp. mycoides (Mm) and Mycoplasma pulmonis (Mp) were generated. Each gene was sequenced in its entirety. The recA genes of Mm and Mp would encode proteins of 345 amino acids (aa) and 339 aa, respectively. The mycoplasmal RecA proteins revealed strong conservation when compared with RecA sequences from other bacterial species.

Only two plasmids have been isolated and characterized from the entire genus Mycoplasma, which includes over 90 recognized species. Both of these plasmids were obtained from the same species, Mycoplasma mycoides subsp. mycoides. We have previously characterized one of these plasmids, pKMK1, as a preliminary step in developing mycoplasmal cloning vectors. In the present study, we have separately combined pKMK1 with two different Escherichia coli replicons and a tetracycline resistance (tetM) gene. One of the constructs, plasmid p2D4, was shuttled from E. coli to M. mycoides subsp. mycoides and back to E. coli with no deletions or rearrangements occurring in the plasmid. In the second construct, the E. coli replicon was deleted when the plasmid was transformed into M. mycoides subsp. mycoides. This derivative, designated plasmid pIK delta, is noteworthy in that it could be transformed into M. mycoides subsp. mycoides at a much higher frequency than the parental plasmid. A gram-positive bacterial erythromycin resistance determinant (erm) was cloned into both p2D4 and pIK delta. Resistance to erythromycin was stably maintained using both constructs, even in the absence of erythromycin selection, indicating that these plasmids will be useful mycoplasmal cloning vectors.

The infection of mice with Mycoplasma pulmonis is a model for studying chronic mycoplasmal respiratory disease. Many in vivo and in vitro studies have used the organism to gain a better understanding of host-pathogen interactions in chronic respiratory infection. The organism's Vsa proteins contain an extensive tandem repeat region. The length of the tandem repeat unit varies from as few as 11 amino acids to as many as 19. The number of tandem repeats can be as high as 60. The number of repeats varies at a high frequency due to slipped-strand mispairing events that occur during DNA replication. When the number of repeats is high, e.g., 40, the mycoplasma is resistant to lysis by complement but does not form a robust biofilm. When the number of repeats is low, e.g., 5, the mycoplasma is killed by complement when the cells are dispersed but has the capacity to form a biofilm that resists complement. Here, we examine the role of the Vsa proteins in the avoidance of phagocytosis and find that cells producing a protein with many tandem repeats are relatively resistant to killing by macrophages. These results may be pertinent to understanding the functions of similar proteins that have extensive repeat regions in other microbes.

Summary Microorganisms have numerous strategies for coping with environmental changes. In many systems, a single cell has the capacity to generate a seemingly infinite array of phenotypic variants in just a few generations of growth. The resulting heterogeneous population is well equipped for sudden environmental change; even if only a few cells in the population possess a phenotype needed for survival, these cells have the capacity to regenerate a similarly diverse population. Phenotypic switching in these systems usually results from high-frequency DNA rearrangements which are the subject of this review.

Cilia-associated respiratory (CAR) bacillus, an unclassified gliding bacterium associated with respiratory disease in rats, mice, and rabbits, has previously been cultivated only in embryonated chicken eggs, cell culture, or cell culture medium supplemented with conditioned medium from cultured tracheas. A reference strain of CAR bacillus, originally isolated in eggs, grew in cell culture flasks as adherent individual bacilli and ropy, whorled fascicles in cell culture media supplemented only with fetal calf serum. Using Dulbecco's minimal essential medium, we isolated CAR bacillus from naturally infected rats and a naturally infected rabbit and from experimentally inoculated mice and rats. Isolates were maintained for up to 20 passages. Isolates from rats were similar in morphology to the reference strain, but most were more actively motile and formed pincushion-like aggregates. The rabbit bacilli were smaller and formed fewer aggregates. DNAs of rat isolates differed only slightly in restriction fragment patterns from that of the reference strain, whereas that of the rabbit isolate was distinctly different. Cultures of CAR bacilli of all strains from rats contained Mycoplasma fermentans, Mycoplasma pulmonis, or both, and cultures of the CAR bacillus from the rabbit contained an unidentified arginine-utilizing mycoplasma. The sequence of the 16S rRNA gene of the reference strain was determined by amplification by polymerase chain reaction, cloning of the product, and sequencing by the dideoxynucleotide chain termination method. Comparison of the sequence with sequences in the GenBank data base indicated that CAR bacillus is a unique organism most closely related to Flavobacterium ferrugineum and Flexibacter sancti.