Physical mapping of the Mycoplasma gallisepticum S6 genome with localization of selected genes

Abstract

We report the construction of a physical map of the Mycoplasma gallisepticum S6 genome by field-inversion gel electrophoresis of DNA fragments generated by digestion of genomic DNA with rare-cutting restriction endonucleases. The size of the M. gallisepticum S6 genome was calculated to be approximately 1,054 kb. The loci of several genes have been assigned to the map by Southern hybridization utilizing specific gene probes.Phylogenetic studies indicate that mycoplasmas have undergone degenerative evolution arising from a clostridial ancestry (38). The reduced genome of mycoplasmas results in a loss of cellular structures, the most notable of which being the absence of a cell wall. Although their apparent simplicity makes the mycoplasmas attractive subjects for genetic analysis, classical approaches have proven relatively difficult to apply. Their complex nutritional requirements and microscopic growth on solid medium have hindered isolation of auxotrophic mutants useful in establishing metabolic pathways. Their use of the universal termination codon UGA as a tryptophan codon (15, 39) has complicated the utility of Escherichia coli for expression of cloned mycoplasma DNA sequences. For these reasons, a more guided approach to the examination of the mycoplasma genome is appropriate.Pulsed-field electrophoretic techniques have proven useful in constructing physical maps of several mycoplasmas (2,4,6,16,18,(25)(26)(27)35). In an organism in which the isolation and characterization of induced mutants is difficult, physical mapping establishes points of reference to analyze sites of insertional mutagenesis (8,9,24,35,37) and enables the identification of genes involved in pathogenesis.Several studies comparing DNA cleavage patterns and random amplified polymorphic DNA and fingerprint analysis have demonstrated that a genotypic heterogeneity exists among strains of Mycoplasma gallisepticum (13,28,30). We have chosen M. gallisepticum S6 for our studies on the basis of the premise that it is widely studied throughout the world. Here we describe the construction of a physical genomic map of M. gallisepticum S6 which provides a foundation for continued genetic analysis of this organism.M. gallisepticum S6 was cultured at 37ЊC in Frey's medium (12). The cells (2 ϫ 10 9 /ml) were pelleted by centrifugation at 12,000 ϫ g for 5 min at 4ЊC and washed twice with an equal volume of phosphate-buffered saline (130 mM NaCl-10 mM Na 2 HPO 4 and 130 mM NaCl-10 mM NaH 2 PO 4 ). DNA was prepared in agarose plugs according to a modification of the procedure described by Krause and Mawn (16). Three milliliters of pelleted mycoplasma culture (6 ϫ 10 9 cells) was suspended in 125 l of TE buffer (10 mM Tris base, 1 mM EDTA, pH 8.0). Then 125 l of degrading solution (1 mg of proteinase K per ml in 100 mM EDTA, pH 8.0) and 250 l of 1.5% low-melting-point agarose (Gibco BRL, Gaithersburg, Md.), melted and equilibrated at 42ЊC, were added. This mixture was transferred to a plastic mold to form 60-l agarose plugs. The plugs were transferred to 3....

The complete genome sequence of the avian pathogen Mycoplasma gallisepticum strain Rlow

“…The mean occurrence of the amino acid tryptophan was 3?6 per CDS and the usage of the codons TGA and TGG was 2?8 and 0?8 per CDS, respectively. As previously reported (Chen & Finch, 1989;Gorton et al, 1995;Scamrov & Beabealashvilli, 1991), the M. gallisepticum genome contains two copies of the rRNA genes. One set is organized as an operon, with adjacent 16S, 23S and 5S genes; and a second copy of the 16S rRNA gene lies 221 kb upstream of the 23S and 5S rRNA genes.…”

Size and genomic location of the pMGA multigene family of Mycoplasma gallisepticum

“…It was of interest to locate the pMGA genes on the S6 chromosome as the identity of the expressed pMGA gene was known and its sequence has been determined (Markham et al ., 1994). In addition, there were two restriction maps available for the S6 chromosome to aid mapping the multigene family (Chen, 1992; Gorton et al ., 1995). The fragments of the strain S6 genome which hybridized to the pMGA gene probes were determined by comparison of photographs of the ethidium bromide stained gels to the autoradiographs of the blot of the gel after hybridization.…”

“…The work described in this paper has determined that the pMGA multigene family is second only in size to the tRNA family amongst prokaryotes and that the pMGA genes are clustered in four regions on the chromosome of S6. The genomic sizes of M. gallisepticum strains S6, PG31 and R range from 998 to 1054 kb (Chen, 1992; Gorton et al ., 1995; Tigges & Minion, 1994). It is remarkable that such a large multigene family should be present within one of the most genetically simple bacteria.…”

Molecular Biology and Pathogenicity of Mycoplasmas

“…The 5S rRNA gene is separated from the 16S-23S genes in M. hyopneumoniae (445a) and M. flocculare (429). M. gallisepticum S6 carries two rRNA operons, one with the complete set of 16S-23S-5S genes and the other with the 16S rRNA genes separated from the 23S-5S rRNA genes (162). The existence in M. gallisepticum A5969, but not in S6, of a third truncated 16S rRNA pseudogene was reported, but it is unclear whether this truncated gene is expressed (424).…”