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.     

Colon cancer associated genes exhibit signatures of positive selection at functionally significant positions

ABSTRACT: BACKGROUND: Cancer, much like most human disease, is routinely studied by utilizing model organisms. Of these model organisms, mice are often dominant. However, our assumptions of functional equivalence fail to consider the opportunity for divergence conferred by ~180 Million Years (MY) of independent evolution between these species. For a given set of human disease related genes, it is therefore important to determine if functional equivalency has been retained between species. In this study we test the hypothesis that cancer associated genes have different patterns of substitution akin to adaptive evolution in different mammal lineages. RESULTS: Our analysis of the current literature and colon cancer databases identified 22 genes exhibiting colon cancer associated germline mutations. We identified orthologs for these 22 genes across a set of high coverage (>6X) vertebrate genomes. Analysis of these orthologous datasets revealed significant levels of positive selection. Evidence of lineage-specific positive selection was identified in 14 genes in both ancestral and extant lineages. Lineage-specific positive selection was detected in the ancestral Euarchontoglires and Hominidae lineages for STK11, in the ancestral primate lineage for CDH1, in the ancestral Murinae lineage for both SDHC and MSH6 genes and the ancestral Muridae lineage for TSC1. CONCLUSION: Identifying positive selection in the primate, Hominidae, Muridae and Murinae lineages suggest an ancestral functional shift in these genes between the rodent and primate lineages. Analyses such as this, combining evolutionary theory and predictions - along with medically relevant data, can thus provide us with important clues for modeling human diseases.     

Life on Arginine for Mycoplasma hominis: Clues from Its Minimal Genome and Comparison with Other Human Urogenital Mycoplasmas

Mycoplasma hominis is an opportunistic human mycoplasma. Two other pathogenic human species, M. genitalium and Ureaplasma parvum, reside within the same natural niche as M. hominis: the urogenital tract. These three species have overlapping, but distinct, pathogenic roles. They have minimal genomes and, thus, reduced metabolic capabilities characterized by distinct energy-generating pathways. Analysis of the M. hominis PG21 genome sequence revealed that it is the second smallest genome among self-replicating free living organisms (665,445 bp, 537 coding sequences (CDSs)). Five clusters of genes were predicted to have undergone horizontal gene transfer (HGT) between M. hominis and the phylogenetically distant U. parvum species. We reconstructed M. hominis metabolic pathways from the predicted genes, with particular emphasis on energy-generating pathways. The Embden–Meyerhoff–Parnas pathway was incomplete, with a single enzyme absent. We identified the three proteins constituting the arginine dihydrolase pathway. This pathway was found essential to promote growth in vivo. The predicted presence of dimethylarginine dimethylaminohydrolase suggested that arginine catabolism is more complex than initially described. This enzyme may have been acquired by HGT from non-mollicute bacteria. Comparison of the three minimal mollicute genomes showed that 247 CDSs were common to all three genomes, whereas 220 CDSs were specific to M. hominis, 172 CDSs were specific to M. genitalium, and 280 CDSs were specific to U. parvum. Within these species-specific genes, two major sets of genes could be identified: one including genes involved in various energy-generating pathways, depending on the energy source used (glucose, urea, or arginine) and another involved in cytadherence and virulence. Therefore, a minimal mycoplasma cell, not including cytadherence and virulence-related genes, could be envisaged containing a core genome (247 genes), plus a set of genes required for providing energy. For M. hominis, this set would include 247+9 genes, resulting in a theoretical minimal genome of 256 genes.     

Cell division in a minimal bacterium in the absence of ftsZ

Mycoplasma genomes exhibit an impressively low amount of genes involved in cell division and some species even lack the ftsZ gene, which is found widespread in the microbial world and is considered essential for cell division by binary fission. We constructed a Mycoplasma genitalium ftsZ null mutant by gene replacement to investigate the role of this gene and the presence of alternative cell division mechanisms in this minimal bacterium. Our results demonstrate that ftsZ is non‐essential for cell growth and reveal that, in the absence of the FtsZ protein, M. genitalium can manage feasible cell divisions and cytokinesis using the force generated by its motile machinery. This is an alternative mechanism, completely independent of the FtsZ protein, to perform cell division by binary fission in a microorganism. We also propose that the mycoplasma cytoskeleton, a complex network of proteins involved in many aspects of the biology of these microorganisms, may have taken over the function of many genes involved in cell division, allowing their loss in the regressive evolution of the streamlined mycoplasma genomes.     

Genome-wide analysis of core cell cycle genes in the unicellular green alga Ostreococcus tauri

The cell cycle has been extensively studied in various organisms, and the recent access to an overwhelming amount of genomic data has given birth to a new integrated approach called comparative genomics. Comparing the cell cycle across species shows that its regulation is evolutionarily conserved; the best-known example is the pivotal role of cyclin-dependent kinases in all the eukaryotic lineages hitherto investigated. Interestingly, the molecular network associated with the activity of the CDK-cyclin complexes is also evolutionarily conserved, thus, defining a core cell cycle set of genes together with lineage-specific adaptations. In this paper, we describe the core cell cycle genes of Ostreococcus tauri, the smallest free-living eukaryotic cell having a minimal cellular organization with a nucleus, a single chloroplast, and only one mitochondrion. This unicellular marine green alga, which has diverged at the base of the green lineage, shows the minimal yet complete set of core cell cycle genes described to date. It has only one homolog of CDKA, CDKB, CDKD, cyclin A, cyclin B, cyclin D, cyclin H, Cks, Rb, E2F, DP, DEL, Cdc25, and Wee1. We have also added the APC and SCF E3 ligases to the core cell cycle gene set. We discuss the potential of genome-wide analysis in the identification of divergent orthologs of cell cycle genes in different lineages by mining the genomes of evolutionarily important and strategic organisms.     

Detection and identification of Mycoplasma infections by DNA hybridization

Infection of cell cultures by mycoplasmas can be detected by hybridization of the DNA of suspected cell cultures with recombinant plasmids containing fragments of the mycoplasma DNA. The test is very sensitive and allows detection of as little as 1 ng of mycoplasmal DNA, roughly equivalent to the DNA amount of 10(6) mycoplasmas. This approach turns out to be effective for detection and identification of mycoplasmas in clinical material, plant and insect tissues. A set of DNA probes for detection of mycoplasmas infecting cell cultures by dot hybridization has been constructed. This set consists of specific DNA probes and universal DNA probe. Recombinant plasmids, pAl32, pMa13, pMh9, containing specific DNA fragments of Acholeplasma-laidlawii, Mycoplasma arginini, Mycoplasma hominis (the prevalent mycoplasma contaminants of home cell cultures) are species-specific DNA probes. Recombinant plasmid pMg16 containing rRNA genes of Mycoplasma gallisepticum is the universal DNA probe for detection of any mycoplasma (or any prokaryote) contaminations. These two classes of DNA probes may be considered as complementing each other. These 32P labeled probes do not hybridize with eukaryotic DNA. The set of DNA probes allows not only to detect infection of cell cultures by mycoplasmas but also to identify the species of mycoplasmas and to evaluate the multiplicity of mycoplasma infection.     

Determination of the core of a minimal bacterial gene set.

The availability of a large number of complete genome sequences raises the question of how many genes are essential for cellular life. Trying to reconstruct the core of the protein-coding gene set for a hypothetical minimal bacterial cell, we have performed a computational comparative analysis of eight bacterial genomes. Six of the analyzed genomes are very small due to a dramatic genome size reduction process, while the other two, corresponding to free-living relatives, are larger. The available data from several systematic experimental approaches to define all the essential genes in some completely sequenced bacterial genomes were also considered, and a reconstruction of a minimal metabolic machinery necessary to sustain life was carried out. The proposed minimal genome contains 206 protein-coding genes with all the genetic information necessary for self-maintenance and reproduction in the presence of a full complement of essential nutrients and in the absence of environmental stress. The main features of such a minimal gene set, as well as the metabolic functions that must be present in the hypothetical minimal cell, are discussed.     

Positive selection during the diversification of class I vomeronasal receptor-like (V1RL) genes,putative pheromone receptor genes,in human and primate evolution

Vomeronasal receptors are the major receptors for pheromones in vertebrates, and five putative type 1 vomeronasal receptors (V1RL) have been identified in humans. The evolution of the V1RL1 gene in non-human primates, and patterns of selection on V1RL genes, were investigated. The presumed ortholog of V1RL1 was sequenced from 13 species of nonhuman primate, and in eight of these species V1RL1 was a pseudogene. Phylogenetic reconstructions reveal that V1RL1 pseudogene formation occurred independently in multiple primate lineages. Using maximum likelihood estimates of dN/dS ratios in PAML, we show that V1RL genes have evolved under neutral evolution in lineages in which they became a pseudogene. In contrast, among lineages in which V1RL genes contain an open reading frame, the majority of sites are under purifying selection and a minority are under significant positive selection. These results provide an interesting case where all three categories of selection can be teased apart in the same data set using maximum likelihood methods. The finding of positive selection on V1RL genes during primate evolution provides indirect support for the hypothesis that V1RL genes have a function in species-specific pheromone detection in primates.     

PCR技术在鼠肺支原体检测中应用的初步研究

目的　探讨PCR技术在鼠肺支原体检测中的应用,希望能建立一种可行、快速、敏感的检测方法。方法　使用支原体通用引物及鼠肺支原体特异性引物对14 份大鼠喉气管拭子洗液和拭子支原体培养液进行PCR扩增,2 % 琼脂糖电泳鉴定。另设M53 和ATCC19612 二株标准鼠肺支原体菌株作阳性对照。结果　通用引物对大鼠喉气管拭子洗液检出率8/14 ,拭子支原体培养液检出率14/14,鼠肺支原体特异引物PCR扩增对大鼠喉气管拭子洗液检出率0/14 ,拭子支原体培养液3/14。通用引物扩增M53 和ATCC19612 二株标准株均呈现阳性,而鼠肺支原体特异引物扩增M53 和ATCC19612,只有M53 呈现阳性。结论　PCR通用引物检测比普通分离培养省时省力,而我们采用国外某学者认为对鼠肺支原体有特异性的引物,是否可用于鼠肺支原体的特异性PCR 检查仍需进一步探讨。     

基层中医院735例女性宫颈分泌物支原体检测及药敏分析

目的了解生殖道支原体感染与年龄、白带清洁度的关系及对抗生素的敏感性,为临床用药提供依据。方法对735例疑似生殖道感染标本采用珠海迪尔生物有限公司生产的试剂盒进行支原体培养和药敏试验,取阴道分泌物作清洁度检查。结果735例标本中检出支原体阳性402例,阳性率为54．7％,解脲脲原体（Uu）、Uu＋Mh混合感染及人型支原体（Mh）分别占75．1％、20．9％、4．0％。Uu、Uu＋Mh阳性患者好发于18～40岁。白带清洁度异常者的Uu、Uu＋Mh、Mh阳性率明显高于白带清洁度正常者。药敏结果显示,支原体对强力霉素、美满霉素敏感性较高,敏感率为85．7％～100．0％。结论白带清洁度异常者为支原体感染的高危人群,诊治时应常规做支原体检测,支原体感染的治疗应根据感染病原体的类型和药敏结果合理应用抗生素。     

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.     

Functional diversification of B MADS-box homeotic regulators of flower development: Adaptive evolution in protein-protein interaction domains after major gene duplication events

B-class MADS-box genes have been shown to be the key regulators of petal and stamen specification in several eudicot model species such as Arabidopsis thaliana, Antirrhinum majus, and Petunia hybrida. Orthologs of these genes have been found across angiosperms and gymnosperms, and it is thought that the basic regulatory function of B proteins is conserved in seed plant lineages. The evolution of B genes is characterized by numerous duplications that might represent key elements fostering the functional diversification of duplicates with a deep impact on their role in the evolution of the floral developmental program. To evaluate this, we performed a rigorous statistical analysis with B gene sequences. Using maximum likelihood and Bayesian methods, we estimated molecular substitution rates and determined the selective regimes operating at each residue of B proteins. We implemented tests that rely on phylogenetic hypotheses and codon substitution models to detect significant differences in substitution rates (DSRs) and sites under positive adaptive selection (PS) in specific lineages before and after duplication events. With these methods, we identified several protein residues fixed by PS shortly after the origin of PISTILLATA-like and APETALA3-like lineages in angiosperms and shortly after the origin of the euAP3-like lineage in core eudicots, the 2 main B gene duplications. The residues inferred to have been fixed by positive selection lie mostly within the K domain of the protein, which is key to promote heterodimerization. Additionally, we used a likelihood method that accommodates DSRs among lineages to estimate duplication dates for AP3-PI and euAP3-TM6, calibrating with data from the fossil record. The dates obtained are consistent with angiosperm origins and diversification of core eudicots. Our results strongly suggest that novel multimer formation with other MADS proteins could have been crucial for the functional divergence of B MADS-box genes. We thus propose a mechanism of functional diversification and persistence of gene duplicates by the appearance of novel multimerization capabilities after duplications. Multimer formation in different combinations of regulatory proteins can be a mechanistic basis for the origin of novel regulatory functions and a gene regulatory mechanism for the appearance of morphological innovations.     

Evidence for positive selection in putative virulence factors within the Paracoccidioides brasiliensis species complex

Paracoccidioides brasiliensis is a dimorphic fungus that is the causative agent of paracoccidioidomycosis, the most important prevalent systemic mycosis in Latin America. Recently, the existence of three genetically isolated groups in P. brasiliensis was demonstrated, enabling comparative studies of molecular evolution among P. brasiliensis lineages. Thirty-two gene sequences coding for putative virulence factors were analyzed to determine whether they were under positive selection. Our maximum likelihood-based approach yielded evidence for selection in 12 genes that are involved in different cellular processes. An in-depth analysis of four of these genes showed them to be either antigenic or involved in pathogenesis. Here, we present evidence indicating that several replacement mutations in gp43 are under positive balancing selection. The other three genes (fks, cdc42 and p27) show very little variation among the P. brasiliensis lineages and appear to be under positive directional selection. Our results are consistent with the more general observations that selective constraints are variable across the genome, and that even in the genes under positive selection, only a few sites are altered. We present our results within an evolutionary framework that may be applicable for studying adaptation and pathogenesis in P. brasiliensis and other pathogenic fungi.     

咽拭子快速培养在肺炎支原体感染中的临床应用价值

目的:探讨咽拭子快速培养在肺炎支原体感染中的临床应用价值。方法:收集2014年2月~2016年2月期间我院收治的呼吸道感染患儿220例,用肺炎支原体专用液体培养基进行肺炎支原体快速培养,用胶体金法检测肺炎支原体MP-Ig M。比较两种方法的阳性率。结果:咽拭子培养快速培养阳性率与血清MP-Ig M检测阳性率比较,差异无统计学意义(P0.05)。MP-Ig检测显示,≤1岁阳性率最低,其阳性率随年龄增加不断增高(P0.05)。肺炎支原体咽拭子培养显示,≤1岁阳性率最高,2~8岁最低(P0.05)。病程≤7 d患者肺炎支原体咽拭子培养阳性率(34.21%)显著高于肺炎支原体MP-Ig检测阳性率(14.04%)(P0.05)。病程7 d患者肺炎支原体咽拭子培养阳性率(11.32%)显著低于肺炎支原体MP-Ig检测阳性率(52.83%)(P0.05)。肺炎支原体咽拭子培养的灵敏度性以及特异性显著高于肺炎支原体MP-Ig检测,差异具有统计学意义(P0.05)。结论:咽拭子快速培养对肺炎支原体感染的早期诊断有一定临床应用价值,方法简单,无创伤,值得临床进一步研究和应用。     

The rate of adaptive evolution in enteric bacteria

Here we estimate the rate of adaptive substitution in a set of 410 genes that are present in 6 Escherichia coli and 6 Salmonella enterica genomes. We estimate that more than 50% of amino acid substitutions in this set of genes have been fixed by positive selection between the E. coli and S. enterica lineages. We also show that the proportion of adaptive substitutions is uncorrelated with the rate of amino acid substitution or gene function but that it may be correlated with levels of synonymous codon usage bias.     

Gene-specific signatures of elevated non-synonymous substitution rates correlate poorly across the Plasmodium genus

Background

Comparative genome analyses of parasites allow large scale investigation of selective pressures shaping their evolution. An acute limitation to such analysis of Plasmodium falciparum is that there is only very partial low-coverage genome sequence of the most closely related species, the chimpanzee parasite P. reichenowi. However, if orthologous genes have been under similar selective pressures throughout the Plasmodium genus then positive selection on the P. falciparum lineage might be predicted to some extent by analysis of other lineages.

Principal Findings

Here, three independent pairs of closely related species in different sub-generic clades (P. falciparum and P. reichenowi; P. vivax and P. knowlesi; P. yoelii and P. berghei) were compared for a set of 43 candidate ligand genes considered likely to be under positive directional selection and a set of 102 control genes for which there was no selective hypothesis. The ratios of non-synonymous to synonymous substitutions (dN/dS) were significantly elevated in the candidate ligand genes compared to control genes in each of the three clades. However, the rank order correlation of dN/dS ratios for individual candidate genes was very low, less than the correlation for the control genes.

Significance

The inability to predict positive selection on a gene in one lineage by identifying elevated dN/dS ratios in the orthologue within another lineage needs to be noted, as it reflects that adaptive mutations are generally rare events that lead to fixation in individual lineages. Thus it is essential to complete the genome sequences of particular species of phylogenetic importance, such as P. reichenowi.     

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.