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Sequencing analysis reveals a unique gene organization in the gyrB region of Mycoplasma hominis.

ABSTRACT: The homolog of the gyrB gene, which has been reported to be present in the vicinity of the initiation site of replication in bacteria, was mapped on the Mycoplasma hominis genome, and the region was subsequently sequenced. Five open reading frames were identified flanking the gyrB gene, one of which showed similarity to that which encodes the LicA protein of Haemophilus influenzae. The organization of the genes in the region showed no resemblance to that in the corresponding regions of other bacteria sequenced so far. The gyrA gene was mapped 35 kb downstream from the gyrB gene.

SUBMITTER: Ladefoged SA

PROVIDER: S-EPMC196789 | biostudies-other | 1994 Sep

REPOSITORIES: biostudies-other

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Sequencing analysis reveals a unique gene organization in the gyrB region of Mycoplasma hominis.

Ladefoged S A SA Christiansen G G

Journal of bacteriology 19940901 18

The homolog of the gyrB gene, which has been reported to be present in the vicinity of the initiation site of replication in bacteria, was mapped on the Mycoplasma hominis genome, and the region was subsequently sequenced. Five open reading frames were identified flanking the gyrB gene, one of which showed similarity to that which encodes the LicA protein of Haemophilus influenzae. The organization of the genes in the region showed no resemblance to that in the corresponding regions of other bac ...[more]

PMID: 7521872

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Project description:BackgroundMycoplasma hominis is a human urogenital pathogen involved in gynaecological, neonatal and extra-genital infections. However, no versatile genetic tools are currently available to study the pathogenicity of this bacterium. Targeting-Induced Local Lesions IN Genomes (TILLING) is a reverse-genetic method that combines point mutations induced by chemical mutagenesis with a DNA screening technique. We used ethyl methanesulfonate (EMS) that introduces C-G to T-A transition mutations to generate a library of M. hominis mutants. As a proof of concept, mutagenized organisms were screened for mutations in two target genes previously associated with the mycoplasma pathogenicity, the vaa gene encoding an adhesin lipoprotein and the oppA gene encoding the main ectoATPase of the bacterium. The resulting mutants were evaluated using functional assays, an adhesion to HeLa cell assay for vaa-mutants and an ATPase activity test for oppA-mutants.ResultsA 1200-clone library was generated by exposing M. hominis PG21 to 9 mg/mL EMS for 3 h. To identify mutants of interest, targeted gene fragments were amplified, heat-denatured, slowly reannealed and digested with the mismatch-specific endonuclease ENDO1. If multiple alleles were present in the PCR amplicons, these alleles formed heteroduplexes during reannealing that were specifically cleaved by ENDO1 at mismatching positions. A total of four vaa-mutants and two oppA-mutants harbouring missense mutations were obtained and fully sequenced. Zero to eight additional mutations were identified in the genomes of each mutant. The vaa-mutants were tested for adhesion to immobilized HeLa cells but their adhesion was not significantly different from the adhesion of M. hominis PG21. One of the two oppA-mutants that were tested for ATPase activity presented a higher affinity for its ATP substrate than the parental strain.ConclusionFor the first time, we demonstrated that M. hominis gene-targeted mutants could be successfully obtained using this TILLING strategy. In the absence of robust genetic tools for studying M. hominis, the TILLING strategy that can target any gene of the genome could help to elucidate gene functions and to better understand the pathogenesis of this human pathogenic species.

Dataset Information

Sequencing analysis reveals a unique gene organization in the gyrB region of Mycoplasma hominis.

Publications

Sequencing analysis reveals a unique gene organization in the gyrB region of Mycoplasma hominis.

Similar Datasets

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