Project description:The genes encoding the 62-kDa lipoproteins from the Mycoplasma mycoides subsp. mycoides large-colony type (LC) strain Y-goat and the M. mycoides subsp. capri strain PG3 were cloned and analyzed by sequencing. These two lipoproteins have been named LppA[MmymyLC] and LppA[Mmyca], and their corresponding genes have been named lppA[MmymyLC] and lppA[Mmyca], respectively. The nucleotide and deduced amino acid sequences of these two lipoproteins showed a very high degree of similarity between these two mycoplasmas. Given the sequence data, LppA seems to fulfill the same structural functions as the previously described major lipoproteins P72 of M. mycoides subsp. mycoides small-colony type and P67 of the Mycoplasma species bovine group 7. Based on lppA gene sequences of M. mycoides subsp. mycoides LC and M. mycoides subsp. capri type strains, a specific PCR assay was developed so that it amplified this gene in all field strains of the two species analyzed in this study but not in the other members of the M. mycoides cluster. Analysis of the PCR-amplified lppA genes with frequently cutting restriction enzymes showed a certain degree of genetic variability which, however, did not cluster the two subspecies. This PCR therefore allows a rapid identification of M. mycoides subsp. mycoides LC and M. mycoides subsp. capri but does not distinguish between these two closely related subspecies. LppA was expressed in Escherichia coli K-12 and used for the production of polyclonal mouse antiserum. Antibodies against recombinant LppA[MmymyLC] reacted with a 62-kDa protein in all M. mycoides subsp. mycoides LC and M. mycoides subsp. capri type strains and field strains tested but not with the other members of the M. mycoides cluster, thus showing the antigenic specificity of LppA and further supporting the concept that a close relationship exists between these two mycoplasmas.
Project description:The sequenced genome of Mycoplasma mycoides subsp. capri revealed the presence of a Type III restriction-modification system (MmyCI). The methyltransferase (modification) subunit of MmyCI (M.MmyCI) was shown to recognize the sequence 5'-TGAG-3' and methylate the adenine. The coding region of the methyltransferase gene contains 12 consecutive AG dinucleotide repeats that result in a translational termination at a TAA codon immediately beyond the repeat region. This strain does not have MmyCI activity. A clone was found with 10 AG repeats such that the gene is in frame, and this strain has MmyCI activity, suggesting that the expression of the MmyCI methyltransferase may be phase variable.
Project description:Mycoplasma capricolum subsp.subsp. capripneumonia (Mccp) and Mycoplasma mycoides subsp.sbusp. capri (Mmc) cause caprine pleuropneumonia (CCPP) and mycoplasmal pneumonia in goats and sheep (MPGS), respectively. These diseases cannot be identified on clinical symptoms alone and it is laborious to distinguish them using biochemical methods. It is therefore important to establish a simple, rapid identification method for Mccp and Mmc. Here, we report a high-resolution melting (HRM) curve analysis using specific primers based on the Mmc 95010 strain MLC_0560 and Mccp F38 strain MCCPF38_00984 gene sequences. The method was highly specific with intra- and inter-batch coefficients of variation < 1%. The lower limit of detection for Mccp and Mmc was 55 copies/μL and 58 copies/μL, respectively. HRM and fluorescence qPCR results were compared using 106 nasal swabs and 47 lung tissue samples from goats (HRM-qPCR coincidence rate 94.8%; 145/153). Mycoplasma isolation and identification was performed on 30 lung tissue samples and 16 nasal swabs (HRM-culturing coincidence rate 87.0%; 40/46). HRM analysis was more sensitive than fluorescence qPCR and Mycoplasma isolation, indicating the practicality of HRM for accurate and rapid identification of Mccp and Mmc, and diagnosis and epidemiology of CCPP and MPGS.
Project description:A suicide plasmid, pExp1-ctpA::tetM-recAec, employing recA from Escherichia coli and tetM as a selection marker, was used to generate ctpA knockout mutants in Mycoplasma mycoides subsp. capri through targeted gene disruption. Inclusion of E. coli recA greatly enhanced both the consistency and the recovery of mutants generated by homologous recombination.
Project description:BACKGROUND: The Mycoplasma mycoides cluster consists of five species or subspecies that are ruminant pathogens. One subspecies, Mycoplasma mycoides subspecies mycoides Small Colony (MmmSC), is the causative agent of contagious bovine pleuropneumonia. Its very close relative, Mycoplasma mycoides subsp. capri (Mmc), is a more ubiquitous pathogen in small ruminants causing mastitis, arthritis, keratitis, pneumonia and septicaemia and is also found as saprophyte in the ear canal. To understand the genetics underlying these phenotypic differences, we compared the MmmSC PG1 type strain genome, which was already available, with the genome of an Mmc field strain (95010) that was sequenced in this study. We also compared the 95010 genome with the recently published genome of another Mmc strain (GM12) to evaluate Mmc strain diversity. RESULTS: The MmmSC PG1 genome is 1,212 kbp and that of Mmc 95010 is ca. 58 kbp shorter. Most of the sequences present in PG1 but not 95010 are highly repeated Insertion Sequences (three types of IS) and large duplicated DNA fragments. The 95010 genome contains five types of IS, present in fewer copies than in PG1, and two copies of an integrative conjugative element. These mobile genetic elements have played a key role in genome plasticity, leading to inversions of large DNA fragments. Comparison of the two genomes suggested a marked decay of the PG1 genome that seems to be correlated with a greater number of IS. The repertoire of gene families encoding surface proteins is smaller in PG1. Several genes involved in polysaccharide metabolism and protein degradation are also absent from, or degraded in, PG1. CONCLUSIONS: The genome of MmmSC PG1 is larger than that of Mmc 95010, its very close relative, but has less coding capacity. This is the result of large genetic rearrangements due to mobile elements that have also led to marked gene decay. This is consistent with a non-adaptative genomic complexity theory, allowing duplications or pseudogenes to be maintained in the absence of adaptive selection that would lead to purifying selection and genome streamlining over longer evolutionary times. These findings also suggest that MmmSC only recently adapted to its bovine host.