B-assembler: a circular bacterial genome assembler.

BACKGROUND: Accurate bacteria genome de novo assembly is fundamental to understand the evolution and pathogenesis of new bacteria species. The advent and popularity of Third-Generation Sequencing (TGS) enables assembly of bacteria genomes at an unprecedented speed. However, most current TGS assemblers were specifically designed for human or other species that do not have a circular genome. Besides, the repetitive DNA fragments in many bacterial genomes plus the high error rate of long sequencing data make it still very challenging to accurately assemble their genomes even with a relatively small genome size. Therefore, there is an urgent need for the development of an optimized method to address these issues. RESULTS: We developed B-assembler, which is capable of assembling bacterial genomes when there are only long reads or a combination of short and long reads. B-assembler takes advantage of the structural resolving power of long reads and the accuracy of short reads if applicable. It first selects and corrects the ultra-long reads to get an initial contig. Then, it collects the reads overlapping with the ends of the initial contig. This two-round assembling procedure along with optimized error correction enables a high-confidence and circularized genome assembly. Benchmarked on both synthetic and real sequencing data of several species of bacterium, the results show that both long-read-only and hybrid-read modes can accurately assemble circular bacterial genomes free of structural errors and have fewer small errors compared to other assemblers. CONCLUSIONS: B-assembler provides a better solution to bacterial genome assembly, which will facilitate downstream bacterial genome analysis.

Integrative characterization of the near-minimal bacterium Mesoplasma florum.

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.

Mycoplasma genitalium Biofilms Contain Poly-GlcNAc and Contribute to Antibiotic Resistance.

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.

Inter- and intra-strain variability of tandem repeats in Mycoplasma pneumoniae based on next-generation sequencing data.

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.

Shaken or stirred?: Comparison of methods for dispersion of Mycoplasma pneumoniae aggregates for persistence in vivo.

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 30s 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.

Rhamnose Links Moonlighting Proteins to Membrane Phospholipid in Mycoplasmas.

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.

General N-and O-Linked Glycosylation of Lipoproteins in Mycoplasmas and Role of Exogenous Oligosaccharide.

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.

Comparative genome analysis of Mycoplasma pneumoniae.

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.

Catalase Enhances Growth and Biofilm Production of Mycoplasma pneumoniae.

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.

Housing conditions modulate the severity of Mycoplasma pulmonis infection in mice deficient in class A scavenger receptor.

Mycoplasmosis is a frequent causative microbial agent of community-acquired pneumonia and has been linked to exacerbation of chronic obstructive pulmonary disease. The macrophage class A scavenger receptor (SRA) facilitates the clearance of noxious particles, oxidants, and infectious organisms by alveolar macrophages. We examined wildtype and SRA(-/-) mice, housed in either individually ventilated or static filter-top cages that were cycled with fresh bedding every 14 d, as a model of gene-environment interaction on the outcome of pulmonary Mycoplasma pulmonis infection. Intracage NH3 gas measurements were recorded daily prior to infection. Mice were intranasally infected with 1 × 10(7) cfu M. pulmonis UAB CT and evaluated at 3, 7, and 14 d after inoculation. Wildtype mice cleared 99.5% of pulmonary M. pulmonis by 3 d after infection but remained chronically infected through the study. SRA (-/-) mice were chronically infected with 40-fold higher mycoplasma numbers than were wildtype mice. M. pulmonis caused a chronic mixed inflammatory response that was accompanied with high levels of IL1ß, KC, MCP1, and TNFα in SRA(-/-) mice, whereas pulmonary inflammation in WT mice was represented by a monocytosis with elevation of IL1ß. Housing had a prominent influence on the severity and persistence of mycoplasmosis in SRA(-/-) mice. SRA(-/-) mice housed in static cages had an improved recovery and significant changes in surfactant proteins SPA and SPD compared with baseline levels. These results indicate that SRA is required to prevent chronic mycoplasma infection of the lung. Furthermore, environmental conditions may exacerbate chronic inflammation in M. pulmonis-infected SRA(-/-) mice.

O-linked protein glycosylation in Mycoplasma.

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.

Interaction of cationic antimicrobial peptides with Mycoplasma pulmonis.

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 30 min 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.

Rhamnose biosynthesis in mycoplasmas requires precursor glycans larger than monosaccharide.

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.

Type 1 and type 2 strains of Mycoplasma pneumoniae form different biofilms.

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.

EPS-I polysaccharide protects Mycoplasma pulmonis from phagocytosis.

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.

Mycoplasma polysaccharide protects against complement.

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.

Mycoplasma pulmonis Vsa proteins and polysaccharide modulate adherence to pulmonary epithelial cells.

The Mycoplasma pulmonis Vsa proteins are a family of size- and phase-variable lipoproteins that shield the mycoplasmas from complement and modulate attachment to abiotic surfaces. Mycoplasmas producing a long Vsa protein hemadsorb poorly and yet are proficient at colonizing rats and mice. The effect of the length of the Vsa protein on the attachment of mycoplasmas to epithelial cells has not been previously explored. We find that independent of Vsa isotype, mycoplasmas producing a long Vsa protein with many tandem repeats adhere poorly to murine MLE-12 cells compared with mycoplasmas producing a short Vsa. We also find that mutants lacking the EPS-I polysaccharide of M. pulmonis exhibited decreased adherence to MLE-12 cells, even though it has been shown previously that such mutants have an enhanced ability to form a biofilm.

The Vsa shield of Mycoplasma pulmonis is antiphagocytic.

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.

Fewer essential genes in mycoplasmas than previous studies suggest.

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.

Identification of exopolysaccharide-deficient mutants of Mycoplasma pulmonis.

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 beta-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.