Mycoplasma Genomes Research Articles

Phylogenetic Relationships Among Mycoplasmas Based on the Whole Genomic Information

With the rapid increase in available bacterial whole-genome information, comparison of bacteria at the whole-genome level has proven to be highly useful in microbial phylogenetic research. Here we constructed a phylogenetic tree based on 15 whole genomes of Mycoplasma and the related bacteria. First, 143 orthologous gene families that are shared by all of the 15 bacteria were selected and 143 multiple alignments were generated. Next, a concatenated multiple alignment inferred from the 143 multiple alignments was generated. A total of 43,370 amino acid sites were considered in the neighbor-joining analysis. The phylogenetic tree based on the whole-genomic information indicated that the 15 bacteria were divided into four major groups with 100% bootstrap support, i.e., the M. hyopneumoniae (Mhy) group, the M. mycoides (Mmy) group, the M. pneumoniae (Mpn) group, and the Bacillus-Phytoplasma (BP) group. In the phylogenetic tree, the Mhy group was more closely related to the Mpn group than the Mmy group. The relationships among the Mhy, Mmy, Mpn, and BP groups were supported with 100% in bootstrap analysis. The phylogenetic tree based on the whole-genome comparison is different from the 16S rRNA tree. Thirty-nine of the 143 phylogenetic trees had the same type of the topology based on the whole-genome comparison. However, we could not identify a gene family contributing or solely belonging to the topology of the 39 proteins. In this study, we showed that some proteins, such as RpoA, RpoB, RpoC, and RpoD, are not suitable for evolutionary studies on relationships among major groups of mycoplasmas. We also showed that glycolysis-related genes of Ureaplasma have a higher substitution rate than the other bacteria. The phylogenetic approaches at the whole-genome level are very important and will be essential for microbial evolutionary studies.

Being Pathogenic, Plastic, and Sexual while Living with a Nearly Minimal Bacterial Genome

Mycoplasmas are commonly described as the simplest self-replicating organisms, whose evolution was mainly characterized by genome downsizing with a proposed evolutionary scenario similar to that of obligate intracellular bacteria such as insect endosymbionts. Thus far, analysis of mycoplasma genomes indicates a low level of horizontal gene transfer (HGT) implying that DNA acquisition is strongly limited in these minimal bacteria. In this study, the genome of the ruminant pathogen Mycoplasma agalactiae was sequenced. Comparative genomic data and phylogenetic tree reconstruction revealed that ∼18% of its small genome (877,438 bp) has undergone HGT with the phylogenetically distinct mycoides cluster, which is composed of significant ruminant pathogens. HGT involves genes often found as clusters, several of which encode lipoproteins that usually play an important role in mycoplasma–host interaction. A decayed form of a conjugative element also described in a member of the mycoides cluster was found in the M. agalactiae genome, suggesting that HGT may have occurred by mobilizing a related genetic element. The possibility of HGT events among other mycoplasmas was evaluated with the available sequenced genomes. Our data indicate marginal levels of HGT among Mycoplasma species except for those described above and, to a lesser extent, for those observed in between the two bird pathogens, M. gallisepticum and M. synoviae. This first description of large-scale HGT among mycoplasmas sharing the same ecological niche challenges the generally accepted evolutionary scenario in which gene loss is the main driving force of mycoplasma evolution. The latter clearly differs from that of other bacteria with small genomes, particularly obligate intracellular bacteria that are isolated within host cells. Consequently, mycoplasmas are not only able to subvert complex hosts but presumably have retained sexual competence, a trait that may prevent them from genome stasis and contribute to adaptation to new hosts.

Use of pathway analysis and genome context methods for functional genomics of Mycoplasma pneumoniae nucleotide metabolism

Elementary modes analysis allows one to reveal whether a set of known enzymes is sufficient to sustain functionality of the cell. Moreover, it is helpful in detecting missing reactions and predicting which enzymes could fill these gaps. Here, we perform a comprehensive elementary modes analysis and a genomic context analysis of Mycoplasma pneumoniae nucleotide metabolism, and search for new enzyme activities. The purine and pyrimidine networks are reconstructed by assembling enzymes annotated in the genome or found experimentally. We show that these reaction sets are sufficient for enabling synthesis of DNA and RNA in M. pneumoniae. Special focus is on the key modes for growth. Moreover, we make an educated guess on the nutritional requirements of this micro-organism. For the case that M. pneumoniae does not require adenine as a substrate, we suggest adenylosuccinate synthetase (EC 6.3.4.4), adenylosuccinate lyase (EC 4.3.2.2) and GMP reductase (EC 1.7.1.7) to be operative . GMP reductase activity is putatively assigned to the NRDI_MYCPN gene on the basis of the genomic context analysis. For the pyrimidine network, we suggest CTP synthase (EC 6.3.4.2) to be active. Further experiments on the nutritional requirements are needed to make a decision. Pyrimidine metabolism appears to be more appropriate as a drug target than purine metabolism since it shows lower plasticity.

Tools for the genetic analysis of Mycoplasma

The mycoplasmas have attracted much scientific attention since they contain the smallest known genomes of any independently viable bacterial species. However, the detailed genetic analysis of these bacteria has lagged behind the well-analyzed bacterial model organisms for a long time. This is due to the use of the UGA codon for tryptophan rather than as stop codon, which had often prevented the expression of full-length Mycoplasma proteins in heterologous hosts. Additionally, insufficient efficiency of homologous recombination prevented the targeted disruption of genes in some species such as M. pneumoniae whereas homologous recombination is operative in other mycoplasmas. Only recently, efficient screening systems for the use of transposon-based mutagenesis have been developed to circumvent this problem and to allow the targeted isolation of desired transposon insertion mutants. With the availability of several Mycoplasma genome sequences, artificial plasmids based on the chromosomal origin of replication were constructed that can now be used for complementation studies and for the stable introduction of foreign genetic material. In this review, we give an overview on recent developments in Mycoplasma genetics that facilitate the genetic manipulation of these interesting organisms.

Mycoplasma genomics: tailoring the genome for minimal life requirements through reductive evolution

Prokaryotic organisms of the genus Mycoplasma are characterized by their small body and genome size containing a 0.6-1.35 M bp genome. The genome is noted for its low G+C frequency ranging from 8-40 mol%. The Mycoplasma genus stems from the class Mollicutes (for soft skin), which lacks the cell walls and external motility appendages often present in other bacteria. To date, there are more than 100 known species of Mycoplasma. 34 species have been partially or completely sequenced. Widely known pathogenic species of Mycoplasma include: M. pneumoniae, causing pneumonia and other respiratory disorders, and M. genitalium, which are involved in pelvic inflammatory disease. Because of their small genome size, Mycoplasmas provide researchers a unique model of the minimal genomic requirements to maintain life. As the number of complete Mycoplasma genomes increase, these organisms become more established, thus laying the foundation for mapping evolutionary development. This manuscript provides an overview and update on Mycoplasma research, with particular focus on current genomics.

Regulation of gene expression in Mycoplasmas: contribution from Mycoplasma hyopneumoniae and Mycoplasma synoviae genome sequences

This report describes the transcription apparatus of Mycoplasma hyopneumoniae (strains J and 7448) and Mycoplasma synoviae, using a comparative genomics approach to summarize the main features related to transcription and control of gene expression in mycoplasmas. Most of the transcription-related genes present in the three strains are well conserved among mycoplasmas. Some unique aspects of transcription in mycoplasmas and the scarcity of regulatory proteins in mycoplasma genomes are discussed.

Mycoplasma Grasps and Releases Solid Surfaces

Mycoplasma, a group of parasitic bacteria, forms a membrane protrusion at a cell pole. They bind to solid surfaces such as animal cell and glass, and glide. The mycoplasma genomes do not have any genes involved in the bio-motility systems ever known, suggesting that they move with a mysterious mechanism. We have studied about the properties of motility, structures of machinery, and enzymatic reaction coupled to gliding, and suggested a putative molecular mechanism. “Many filamentous legs on the base of membrane protrusion repeat binding and release from sialic acid fixed on the solid surface and pull the cell body.”

Genes involved in cell division in mycoplasmas

Bacterial cell division has been studied mainly in model systems such as Escherichia coli and Bacillus subtilis, where it is described as a complex process with the participation of a group of proteins which assemble into a multiprotein complex called the septal ring. Mycoplasmas are cell wall-less bacteria presenting a reduced genome. Thus, it was important to compare their genomes to analyze putative genes involved in cell division processes. The division and cell wall (dcw) cluster, which in E. coli and B. subtilis is composed of 16 and 17 genes, respectively, is represented by only three to four genes in mycoplasmas. Even the most conserved protein, FtsZ, is not present in all mycoplasma genomes analyzed so far. A model for the FtsZ protein from Mycoplasma hyopneumoniae and Mycoplasma synoviae has been constructed. The conserved residues, essential for GTP/GDP binding, are present in FtsZ from both species. A strong conservation of hydrophobic amino acid patterns is observed, and is probably necessary for the structural stability of the protein when active. M. synoviae FtsZ presents an extended amino acid sequence at the C-terminal portion of the protein, which may participate in interactions with other still unknown proteins crucial for the cell division process.

Analysis of Distribution Indicates Diverse Functions of Simple Sequence Repeats in Mycoplasma Genomes

Simple sequence repeats (SSRs) composed of extensive tandem iterations of a single nucleotide or a short oligonucleotide are rare in most bacterial genomes, but they are common among Mycoplasma. Some of these repeats act as contingency loci in association with families of surface antigens. By contraction or expansion during replication, these SSRs increase genetic variance of the population and facilitate avoidance of the immune response of the host. Occurrence and distribution of SSRs are analyzed in complete genomes of 11 Mycoplasma and 3 related Mollicutes in order to gain insights into functional and evolutionary diversity of the SSRs in Mycoplasma. The results revealed an unexpected variety of SSRs with respect to their distribution and composition and suggest that it is unlikely that all SSRs function as contingency loci or recombination hot spots. Various types of SSRs are most abundant in Mycoplasma hyopneumoniae, whereas Mycoplasma penetrans, Mycoplasma mobile, and Mycoplasma synoviae do not contain unusually long SSRs. Mycoplasma hyopneumoniae and Mycoplasma pulmonis feature abundant short adenine and thymine runs periodically spaced at 11 and 12 bp, respectively, which likely affect the supercoiling propensities of the DNA molecule. Physiological roles of long adenine and thymine runs in M. hyopneumoniae appear independent of location upstream or downstream of genes, unlike contingency loci that are typically located in protein-coding regions or upstream regulatory regions. Comparisons among 3 M. hyopneumoniae strains suggest that the adenine and thymine runs are rarely involved in genome rearrangements. The results indicate that the SSRs in the Mycoplasma genomes play diverse roles, including modulating gene expression as contingency loci, facilitating genome rearrangements via recombination, affecting protein structure and possibly protein-protein interactions, and contributing to the organization of the DNA molecule in the cell.

The evolving saga of Mycoplasma genitalium

Abstract Mycoplasma genitalium has the smallest genome of any organism capable of independent growth. Sequence comparisons among completed bacterial genomes indicate that the 580-kb genome of M. genitalium arose by minimization of the 816-kb genome of the human respiratory pathogen Mycoplasma pneumoniae . Thus, the selective pressure that has led to genome minimization renders M. genitalium an ideal starting organism to define determinants essential for life. Further, the advent of molecular and serological diagnostic approaches has established M. genitalium as a significant cause of human infection, although its highly fastidious nature has made the study of its clinical significance and pathogenesis very challenging. As such, the virulence determinants that allow this minimal organism to establish human infections, circumvent host responses, and potentially contribute to chronic infections are just beginning to be elucidated. The following review will focus on recent advances in our understanding of the biology and pathogenesis of this smallest of all bacterial pathogens.

DNA repair in reduced genome: The Mycoplasma model

The occurrence of bacteria with a reduced genome, such as that found in Mycoplasmas, raises the question as to which genes should be enough to guarantee the genomic stability indispensable for the maintenance of life. The aim of this work was to compare nine Mycoplasma genomes in regard to DNA repair genes. An in silico analysis was done using six Mycoplasma species, whose genomes are accessible at GenBank, and M. synoviae, and two strains of M. hyopneumoniae, whose genomes were recently sequenced by The Brazilian National Genome Project Consortium and Southern Genome Investigation Program (Brazil) respectively. Considering this reduced genome model, our comparative analysis suggests that the DNA integrity necessary for life can be primarily maintained by nucleotide excision repair (NER), which is the only complete repair pathway. Furthermore, some enzymes involved with base excision repair (BER) and recombination are also present and can complement the NER activity. The absence of RecR and RecO-like ORFs was observed only in M. genitalium and M. pneumoniae, which can be involved with the conservation of gene order observed between these two species. We also obtained phylogenetic evidence for the recent acquisition of the ogt gene in M. pulmonis and M. penetrans by a lateral transference event. In general, the presence or nonexistence of repair genes is shared by all species analyzed, suggesting that the loss of the majority of repair genes was an ancestral event, which occurred before the divergence of the Mycoplasma species.

Mollicutes

Mollicutes

The Mycoplasma fermentans prophage φMFV1: genome organization, mobility and variable expression of an encoded surface protein

The approximately 16 kb genome of the Mycoplasma fermentans phiMFV1 prophage is described, and its mobility, replication and effect on the mycoplasma surface phenotype are demonstrated. In various M. fermentans strains, phiMFV1 was either absent or integrated at diverse (and sometimes multiple) chromosomal sites, each marked by a conserved TTTTTA target sequence that is duplicated upon integration. Precise excision, replication of an extrachromosomal form and loss of phiMFV1 from the mycoplasmal genome were documented in a series of clonal derivatives of M. fermentans propagated in culture. Of 18 open reading frames (ORFs) encoded by phiMFV1, most can be ascribed functions related to phage biology, whereas one encodes a unique coiled-coil membrane surface protein, Mem, that was confirmed to be expressed in propagating populations of M. fermentans. With the exception of Mem and other minor ORFs, the striking similarity between the deduced proteomes of phiMFV1 and the recently described phiMAV1 of arthritogenic strains of Mycoplasma arthritidis, along with the prominent gene synteny between these elements, provides the taxonomic basis for a new family of prophage. Their coding features are consistent with long-term residence in mycoplasma genomes and the divergence of species within a phylogenetic clade of mycoplasmas. The unique Mem protein expressed from phiMFV1 and the unique hypothetical surface lipoproteins encoded by phiMAV1 and phiMFV1 also suggest that prophage-associated genes may provide specific, selectable phenotypic traits during co-evolution of mycoplasma species with their respective mammalian hosts. Retention of these labile prophage elements in organisms with such drastically reduced genome sizes implies a significant role in adaptation and survival.

Inactivation of mycoplasma species in blood by INACTINE PEN110 process.

Mycoplasmas have been associated with multiple acute and chronic diseases. Mycoplasma genome is found in the blood of 10 to 15 percent of subjectively healthy individuals. If blood borne and viable in donated blood, mycoplasmas could potentially be transfusion transmissible. The INACTINE PEN110 technology is a pathogen reduction process that is in Phase 3 clinical studies. The present study investigated the ability of this process to eradicate mycoplasmas in human blood. Identical whole blood or RBC units inoculated with Mycoplasma arthritidis or M. pneumoniae were incubated with PEN110 (inactivating agent) for 24 hours at 23 degrees C. Sham controls were treated with buffer under the same conditions. 4 degrees C controls were put on storage immediately after the spike. No viable microorganisms were detected in PEN110-treated units after 24 hours of incubation. Sham controls showed no changes to mycoplasma titers during the incubation. In 4 degrees C controls, minor decrease of mycoplasma titers was observed during the storage. The INACTINE process inactivates more than 107 mycoplasma CFU per mL in whole blood and RBCs. This study is the first demonstration of susceptibility of mycoplasmas to pathogen reduction. The data provide further support for the ability of INACTINE technology to address microbial safety issues that are not well characterized.

Reductive evolution suggested from the complete genome sequence of a plant-pathogenic phytoplasma.

The minimal gene set essential for life has long been sought. We report the 860-kb genome of the obligate intracellular plant pathogen phytoplasma (Candidatus Phytoplasma asteris, OY strain). The phytoplasma genome encodes even fewer metabolic functions than do mycoplasma genomes. It lacks the pentose phosphate cycle and, more unexpectedly, ATP-synthase subunits, which are thought to be essential for life. This may be the result of reductive evolution as a consequence of life as an intracellular parasite in a nutrient-rich environment.

DNA methylation and Mycoplasma genomes.

DNA methylation is one of the many hypotheses proposed to explain the observed deficiency in CpG dinucleotides in a variety of genomes covering a wide taxonomic distribution. Recent studies challenged the methylation hypothesis on empirical grounds. First, it cannot explain why the Mycoplasma genitalium genome exhibits strong CpG deficiency without DNA methylation. Second, it cannot explain the great variation in CpG deficiency between M. genitalium and M. pneumoniae that also does not have CpG-specific methyltransferase genes. I analyzed the genomic sequences of these Mycoplasma species together with the recently sequenced genomes of M. pulmonis, Ureaplasma urealyticum, and Staphylococcus aureus, and found the results fully compatible with the methylation hypothesis. In particular, I present compelling empirical evidence to support the following scenario. The common ancestor of the three Mycoplasma species has CpG-specific methyltransferases, and has evolved strong CpG deficiency as a result of the specific DNA methylation. Subsequently, this ancestral genome diverged into M. pulmonis and the common ancestor of M. pneumoniae and M. genitalium. M. pulmonis has retained methyltransferases and exhibits the strongest CpG deficiency. The common ancestor lost the methyltransferase gene and then diverged into M. genitalium and M. pneumoniae. M. genitalium and M. pneumoniae, after losing methylation activities, began to regain CpG dinucleotides through random mutation. M. genitalium evolved more slowly than M. pneumoniae, gained relatively fewer CpG dinucleotides, and is more CpG-deficient.

Using homolog groups to create a whole-genomic tree of free-living organisms: an update.

Genomic trees have been constructed based on the presence and absence of families of protein-encoding genes observed in 27 complete genomes, including genomes of 15 free-living organisms. This method does not rely on the identification of suspected orthologs in each genome, nor the specific alignment used to compare gene sequences because the protein-encoding gene families are formed by grouping any protein with a pairwise similarity score greater than a preset value. Because of this all inclusive grouping, this method is resilient to some effects of lateral gene transfer because transfers of genes are masked when the recipient genome already has a homolog (not necessarily an ortholog) of the incoming gene. Of 71 genes suspected to have been laterally transferred to the genome of Aeropyrum pernix, only approximately 7 to 15 represent genes where a lateral gene transfer appears to have generated homoplasy in our character dataset. The genomic tree of the 15 free-living taxa includes six different bacterial orders, six different archaeal orders, and two different eukaryotic kingdoms. The results are remarkably similar to results obtained by analysis of rRNA. Inclusion of the other 12 genomes resulted in a tree only broadly similar to that suggested by rRNA with at least some of the differences due to artifacts caused by the small genome size of many of these species. Very small genomes, such as those of the two Mycoplasma genomes included, fall to the base of the Bacterial domain, a result expected due to the substantial gene loss inherent to these lineages. Finally, artificial "partial genomes" were generated by randomly selecting ORFs from the complete genomes in order to test our ability to recover the tree generated by the whole genome sequences when only partial data are available. The results indicated that partial genomic data, when sampled randomly, could robustly recover the tree generated by the whole genome sequences.

Novel deoxynucleoside-phosphorylating enzymes in mycoplasmas: evidence for efficient utilization of deoxynucleosides.

Mycoplasmas are unable to synthesize purine and pyrimidine bases de novo. Therefore, salvage of existing nucleosides and bases is essential for their survival. Four mycoplasma species were studied with regard to their ability to phosphorylate deoxynucleosides. High levels of thymidine kinase (TK), deoxycytidine kinase (dCK), deoxyguanosine kinase (dGK) and deoxyadenosine kinase (dAK) activities were detected in extracts from Mycoplasma pneumoniae, Mycoplasma mycoides subsp. mycoides SC (M. mymySC), Acholeplasma laidlawii (A. laidlawii) and Mycoplasma arginini (M. arginini). Nucleoside phosphotransferase activities were found at high levels in A. laidlawii and low levels in M. arginini. Pyrophosphate-dependent deoxynucleoside kinase activities were detected mainly in A. laidlawii and M. mymySC extracts. Two open reading frames were identified in the M. mymySC genome; one showed 25% sequence identity to human dGK and the other one had about 26% sequence identity to human TK1. The M. mymySC dGK-like enzyme was cloned, expressed in Escherichia coli and affinity-purified. This enzyme phosphorylated dAdo, dGuo and dCyd, and the highest catalytic rate was with dAdo as substrate. Therefore, we suggest that this enzyme should be named deoxyadenosine kinase. The physiological role of mycoplasma dAK and TK may be to support the unusually large dATP and dTTP pools required for replication of mycoplasma genomes.

The complete genome sequence of the murine respiratory pathogen Mycoplasma pulmonis.

Mycoplasma pulmonis is a wall-less eubacterium belonging to the Mollicutes (trivial name, mycoplasmas) and responsible for murine respiratory diseases. The genome of strain UAB CTIP is composed of a single circular 963 879 bp chromosome with a G + C content of 26.6 mol%, i.e. the lowest reported among bacteria, Ureaplasma urealyticum apart. This genome contains 782 putative coding sequences (CDSs) covering 91.4% of its length and a function could be assigned to 486 CDSs whilst 92 matched the gene sequences of hypothetical proteins, leaving 204 CDSs without significant database match. The genome contains a single set of rRNA genes and only 29 tRNAs genes. The replication origin oriC was localized by sequence analysis and by using the G + C skew method. Sequence polymorphisms within stretches of repeated nucleotides generate phase-variable protein antigens whilst a recombinase gene is likely to catalyse the site-specific DNA inversions in major M.pulmonis surface antigens. Furthermore, a hemolysin, secreted nucleases and a glyco-protease are predicted virulence factors. Surprisingly, several of the genes previously reported to be essential for a self-replicating minimal cell are missing in the M.pulmonis genome although this one is larger than the other mycoplasma genomes fully sequenced until now.

Identification of novel Mycoplasma hyorhinis gene fragments by differential display analysis of co-cultures

Mycoplasmas are a diverse group of wall-less prokaryotes that have evolved an unusually small genome by adopting a parasitic mode of life. Recently, intense efforts have been made to sequence mycoplasma genomes and to define a minimal genome using mycoplasma as a model. Due to their parasitic nature, mycoplasma species are often difficult to cultivate, making it challenging to identify and sequence mycoplasma genes. In this report, we describe a method for identifying mycoplasma gene fragments from co-cultures using differential display analysis. Using this technique, we have identified fragments of seven putative genes from Mycoplasma hyorhinis. Sequence similarities suggest that four of these genes are members of the proposed minimal mycoplasma genome. The application of differential display analysis to co-cultures should be useful in the identification of genes from a variety of pathogenic organisms that are difficult to cultivate without a host.