| Mycoplasmas are widespread in nature as parasites of humans, mammals, reptiles, fish, arthropods, and plants. As conditional pathogenic organism, they associate with various diseases, including pneumonia, arthritis, meningitis and chronic urogenital tract disease. Since Mycoplasmas are total lack of a cell wall, they are not sensitive to β-lactamase drugs, which increases the difficulty of disease prevention and control. With the rapid development of breeding industry, the economic losses caused by Mycoplasmas are gradually revealed.Mycoplasma hyopneumoniae is the causative agent of porcine enzootic pneumonia, which results in a mild, chronic pneumonia of swine. While progress has been made in understanding the molecular basis of some Mycoplasma diseases, advances in M. hyopneumoniae research have been hampered by its fastidious growth condition and the lack of genetic tools and transformation protocols. To date, few virulence determinants or virulence-associated determinants have been identified. Attachment to the respiratory epithelium is a prerequisite for host colonization and is mediated by the membrane protein P97. This protein is located on the outer membrane surface, and its role in adherence has been firmly established. Previous studies have revealed that P102, P146, P159and P216are also identified as virulence-associated determinants. Whether there is any other virulence factors contributed to M. hyopneumoniae pathogenesis remains largely unknown. In this study, we performed the first comprehensive analysis of M. hyopneumoniae strain168and its attenuated strain and made a preliminary survey of coding sequences （CDSs） that may be related to virulence. M. hyopneumoniae and M. hyorhinis are the causal agents of swine mycoplasmosis. The former causes a mild, chronic pneumonia of swine and results in deactivation of mucociliary functions. This agent is infective for a single host species. M. hyorhinis is generally considered a swine pathogen, yet is most commonly infect laboratory cell lines, implying that it can thrive among different species of cell lines. A strong link between M. hyorhinis and human cancer was reported recently. Interest has therefore shifted to questions of why M. hyorhinis exhibit high levels of functional diversity. The main researches are described as follows.1. Genome Sequencing and bioinformatics analysis of M. hyopneumoniae and M. hyorhinisWhole-genome sequencing was performed by combining GS FLX, ABI3730and Solexa paired-end sequencing technologies. Gaps were filled by local assembly of the Solexa/Roche454reads or sequencing PCR products using an ABI3730capillary sequencer. Open reading frames containing more than30amino acid residues were predicted using Glimmer3.0and verified by comparing with closely related genome sequences. This is the first complete genome sequence of M. hyorhinis, and its availability will provide a better defined genetic background for future studies of gene expression and regulation.We have constructed the metabolic network of M. hyopneumoniae, including eight metabolic pathways:Nucleotide Metabolism, Glycolysis, Glycan Biosynthesis and Metabolism, Lipid Metabolism, Metabolism of Cofactors and Vitamins, Amino Acid Metabolism, and Energy Metabolism。 Further analysis revealed that, the carbon source and energy are mainly produced by the glycolytic pathway. However, M. hyopneumoniae fails to utilize a-D-Glucose-1P, and almost all the carbon source comes from the uptake of exogenous glucose.2. Comparative genomic analyses of Mycoplasma hyopneumoniae pathogenic168strain and its high-passaged attenuated strainTo gain new insight into the components that contribute to virulence and the mechanisms by which M. yopneumoniae causes disease, we sequenced the genomes of strains168and168-L. The168-L genome has a highly similar gene content and order to that of168, but is4,483bp smaller because there are60insertions nd43deletions in168-L. Besides these indels,227single nucleotide variations （SNVs） were identified. We further investigated the variants affected CDSs, and compared them to reported virulence determinants. Notably, almost all the reported virulence determinants are included in these variants affected CDSs, including mycoplasma adhesins （P97, P102, P146, P159, P216, and LppT）, cell envelope roteins （P95）, cell surface antigens （P36）, secreted proteins and chaperone protein （DnaK）, mutations in genes elated to metabolism and growth. Furthermore, many mutations were located in the previously described repeat motif, which may be of primary importance for irulence.As Mycoplasmas are dependent on the exogenous supply of many nutrients, it has been predicted that they may need many transport systems. M. hyopneumoniae has two transport systems, including PTS transporter system and ABC transporter system. No mutations were identified in this PTS transporter family. However, five missense mutations and one synonymous substitution were identified in ABC transporter family. These included an ABC transporter permease protein （MHP168L394） and ABC transporter ATP-binding proteins （MHP168L413）. Interestingly, the expression of MHP168L394and MHP168L413was reported to be up-regulated in vivo during disease relative to in vitro-grown. The variability between strains168and168-L in multi-transport proteins indicates that they may affect growth and survival in different hosts or host tissues.3. Comparative genomics of Mycoplasma: analysis of conserved essential genes and diversity of the pan-GenomeMycoplasma, the smallest self-replicating organism with a minimal metabolism and little genomic redundancy, is expected to be a close approximation to the minimal set of genes needed to sustain bacterial life. This study employs comparative evolutionary analysis of twenty Mycoplasma genomes to gain an improved understanding of essential genes. By analyzing the core genome of mycoplasmas, we finally revealed the conserved essential genes set for mycoplasma survival. Further analysis showed that the core genome set has many characteristics in common with experimentally identified essential genes. Several key genes, which are related to DNA replication and repair and can be disrupted in transposon mutagenesis studies, may be critical for bacteria survival especially over long period natural selection. Phylogenomic reconstructions based on3,355homologous groups allowed robust estimation of phylogenetic relatedness among mycoplasma strains. To obtain deeper insight into the relative roles of molecular evolution in pathogen adaptation to their hosts, we also analyzed the positive selection pressures on particular sites and lineages. The oriC region was identified to be under positive selection in the HUB-1lineage. Previous studies have already demonstrated that replication may contribute to proliferation and efficiency of the colonization of hostile environments. Therefore, we suspected that selection pressure on oriC may be one of the reasons why M. hyorhinis can thrive among different species of cell lines. |
