Mycoplasma Gallisepticum strain S6 genome contains three regions hybridizing with 16 S rRNA and two regions hybridizing with 23 S and 5 S rRNA

Scamrov, A.; Beabealashvilli, Robert Sh.

doi:10.1016/0014-5793(91)81106-i

Cited by 8 publications

(4 citation statements)

References 8 publications

(8 reference statements)

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“…These data suggest that two copies of the 23S rRNA gene exist within the genome, located within the Sm2 and Sm3 fragments. These data are consistent with previous reports (3,31) that two copies of the rRNA operon exist in M. gallisepticum S6: one complete copy of the 16S-23S-5S rRNA genes and one copy of the 16S rRNA gene separated from the 23S-5S rRNA genes. Scamrov and Beabealashvilli (31) also suggest the existence of a third 16S rRNA gene.…”

supporting

confidence: 83%

“…The Mycoplasma capricolum rrn genes hybridized, under conditions of moderate stringency, with fragments Sm2, Sm3, Sm7, Ea1, Ea3, Ea4, and Ea5. These hybridization data, the I-CeuI restriction enzyme digest data, and a previous report of the tuf gene being located adjacent to the complete rRNA operon in M. gallisepticum S6 (31) suggest that the complete 16S-23S-5S rRNA operon exists within the Sm2 fragment, that the separated 16S and 23S-5S rRNA genes exist within the Sm3 fragment, and that the third 16S rRNA gene lies within the Sm7 fragment.…”

mentioning

confidence: 78%

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

1995

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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....

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confidence: 83%

mentioning

confidence: 78%

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

1995

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“…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.…”

Section: General Features Of the Genomementioning

confidence: 66%

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

et al. 2003

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The complete genome of Mycoplasma gallisepticum strain R low has been sequenced. The genome is composed of 996 422 bp with an overall G+C content of 31 mol%. It contains 742 putative coding DNA sequences (CDSs), representing a 91 % coding density. Function has been assigned to 469 of the CDSs, while 150 encode conserved hypothetical proteins and 123 remain as unique hypothetical proteins. The genome contains two copies of the rRNA genes and 33 tRNA genes. The origin of replication has been localized based on sequence analysis in the region of the dnaA gene. The vlhA family (previously termed pMGA) contains 43 genes distributed among five loci containing 8, 2, 9, 12 and 12 genes. This family of genes constitutes 10?4 % (103 kb) of the total genome. Two CDSs were identified immediately downstream of gapA and crmA encoding proteins that share homology to cytadhesins GapA and CrmA. Based on motif analysis it is predicted that 80 genes encode lipoproteins and 149 proteins contain multiple transmembrane domains. The authors have identified 75 proteins putatively involved in transport of biomolecules, 12 transposases, and a number of potential virulence factors. The completion of this sequence has spawned multiple projects directed at defining the biological basis of M. gallisepticum. INTRODUCTIONPhylogenetic analyses indicate that mycoplasmas (class Mollicutes) have undergone a degenerative evolution from related, low G+C content, Gram-positive eubacteria (Rogers et al., 1985;Woese et al., 1980). The reduction of the mycoplasma genome has resulted in the loss of the cell wall and has limited the biosynthetic capabilities of these organisms. As a consequence of this loss of biosynthetic machinery, mycoplasmas are obligate parasites and rely on the uptake of many essential molecules from their environment.Mycoplasmas have long been considered model systems for defining the minimal set of genes required for a living cell (Morowitz, 1984). For this reason, it was not surprising when Mycoplasma genitalium (580 kb) was selected as one of the first targets for complete genome sequencing (Fraser et al., 1995). Since this initial report, the genomes of four additional mycoplasmas have been sequenced, Mycoplasma pneumoniae (816 kb; Dandekar et al., 2000;Himmelreich et al., 1996), Ureaplasma urealyticum (752 kb; Glass et al., 2000), Mycoplasma pulmonis (964 kb; Chambaud et al., 2001) and Mycoplasma penetrans (1358 kb; Sasaki et al., 2000). Theoretical and experimental approaches have estimated the minimum number of essential mycoplasma genes to be between 265 and 350 (Hutchison et al., 1999;Mushegian & Koonin, 1996).Abbreviations: CDS, coding DNA sequence; COGs, conserved orthologous groups.The GenBank accession number for the sequence reported in this paper is AE015450. Mycoplasma gallisepticum is an avian pathogen involved in chronic respiratory disease in chickens resulting in considerable economic losses in poultry production. Infection with this bacterium is spread by aerosol exposure and egg transmission. Outbreaks spread...

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“…Restriction endonuclease digestion and Southern hybridization analysis were performed in order to separate the different copies of 23S rRNA genes in the M. gallisepticum genome [27]. Intact M. gallisepticum genomic DNA was extracted as previously described [28] and digested with restriction endonuclease Bgl II (Promega).…”

Section: Methodsmentioning

confidence: 99%

Induction of macrolide resistance inMycoplasma gallisepticumin vitro and its resistance-related mutations within domain V of 23S rRNA

Yi-bao

et al. 2005

FEMS Microbiology Letters

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Antibiotic-resistant mutants of Mycoplasma gallisepticum were selected in vitro from the susceptible strains S6 and BG44T by serial passages in stepwise concentrations of erythromycin, tylosin, or tilmicosin. High resistance to erythromycin or tilmicosin developed readily, whereas resistance to tylosin developed only after greater numbers of passages. Three mutants selected by each selector antibiotic were cloned and detected, and all cloned mutants exhibited cross-resistance to the three selector antibiotics as well as to lincomycin. Portions of the genes encoding domain V of 23S rRNA of the cloned mutants were amplified by PCR, and their nucleotide sequences were compared to those of the susceptible parent strains. Five of the six mutants selected by erythromycin harbored an A2058G (Escherichia coli numbering) mutation in one of the two 23S rRNA. One of the six mutants selected by erythromycin harbored a G2057A mutation and an A2059G mutation in the other 23S rRNA. In tilmicosin-selected mutants, two mutations, A2058G and A2503U, occurred in one of the two 23S rRNA. No mutation was detected in the two 23S rRNA of tylosin-selected mutants with low-level resistance. Mutations at homologous locations in the 23S rRNA of other macrolide-resistant bacteria indicate that the phenotype of macrolide resistance occurring in M. gallisepticum is strongly associated with point mutations in domain V of 23S rRNA.

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Mycoplasma Gallisepticum strain S6 genome contains three regions hybridizing with 16 S rRNA and two regions hybridizing with 23 S and 5 S rRNA

Cited by 8 publications

References 8 publications

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

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

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

Induction of macrolide resistance inMycoplasma gallisepticumin vitro and its resistance-related mutations within domain V of 23S rRNA

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