Dataset Information

Analysis of 0.5-kilobase-pair repeats in the Mycoplasma hominis lmp gene system and identification of gene products.

ABSTRACT: Mycoplasma hominis, an opportunistic pathogenic bacterium of humans, has a small genome of 700 kb. Despite this, multiple copies of gene sequences with similarities to the structural gene (lmp1) of a 135-kDa surface-located membrane protein (Lmp1) have been identified on the genome of M. hominis PG21 (lmp2, lmp3, and lmp4). The distance between the lmp1-lmp2 region and the lmp3-lmp4 region was more than 110 kb. lmp3-lmp4 of M. hominis PG21 was sequenced and found to contain two putative genes. The gene region of 6.5 kb contained a 5' unique region and a 3' unique region separated by 9 0.5-kb repeats with 51 to 90% similarity to 10 similar repeats found in the lmp1-lmp2 region. The 0.5-kb DNA repeats thus comprised about 1% of the entire genome. In both regions, a base change in one of the repeats gave rise to a stop codon, and thereby lmp2 and lmp4 occurred. By PCR amplification of reverse-transcriptase-generated cDNA it was shown that all four genes were transcribed. By use of Lmp-specific antibodies we showed that both lmp1 and lmp3 were translated into proteins (Lmp1 and Lmp3). Each of the four lmp genes represented by their unique cloned segments was used as a probe to analyze the presence, distribution, and organization of the genes within the genome in 13 M. hominis isolates. The repetitive element was detected at one or two locations on the chromosome for all isolates. The lmp3-specific element was present in all isolates, and lmp1- and lmp2-specific elements were present in all but one isolate. The lmp4-specific element was present in about half the isolates tested. For five M. hominis isolates the chromosomal location of the lmp genes was mapped.

SUBMITTER: Ladefoged SA

PROVIDER: S-EPMC178011 | biostudies-other | 1996 May

REPOSITORIES: biostudies-other

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Analysis of 0.5-kilobase-pair repeats in the Mycoplasma hominis lmp gene system and identification of gene products.

Ladefoged S A SA Jensen L T LT Brock B B Birkelund S S Christiansen G G

Journal of bacteriology 19960501 10

Mycoplasma hominis, an opportunistic pathogenic bacterium of humans, has a small genome of 700 kb. Despite this, multiple copies of gene sequences with similarities to the structural gene (lmp1) of a 135-kDa surface-located membrane protein (Lmp1) have been identified on the genome of M. hominis PG21 (lmp2, lmp3, and lmp4). The distance between the lmp1-lmp2 region and the lmp3-lmp4 region was more than 110 kb. lmp3-lmp4 of M. hominis PG21 was sequenced and found to contain two putative genes. T ...[more]

PMID: 8631664

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Project description:Integrative and conjugative elements (ICEs) are modular mobile genetic elements that can disseminate through excision, circularization, and transfer. Mycoplasma ICEs have recently been found distributed among some mycoplasma species and there is accumulating evidence that they play a pivotal role in horizontal gene transfers. The occurrence of ICEs has not been documented in Mycoplasma hominis, a human urogenital pathogen responsible for urogenital infections, neonatal infections and extragenital infections. In this study, we searched for, characterized, and compared ICEs by genome analyses of 12 strains of M. hominis. ICEs of 27-30 kb were found in one or two copies in seven of the 12 M. hominis strains sequenced. Only five of these ICEs seemed to be functional, as assessed by detection of circular forms of extrachromosomal ICE. Moreover, the prevalence of ICEs in M. hominis was estimated to be 45% in a collection of 120 clinical isolates of M. hominis, including 27 tetracycline-resistant tet(M)-positive isolates. The proportion of ICEs was not higher in isolates carrying the tet(M) gene, suggesting that ICEs are not involved in tetracycline resistance. Notably, all M. hominis ICEs had a very similar structure, consisting of a 4.0-5.1 kb unusual module composed of five to six juxtaposed CDSs. All the genes forming this module were specific to M. hominis ICEs as they had no homologs in other mycoplasma ICEs. In each M. hominis ICE, one to three CDSs encode proteins that share common structural features with transcription activator-like (TAL) effectors involved in polynucleotide recognition and signal transduction in symbiotic plant pathogen bacteria. The conserved and specific structure of M. hominis ICEs and the high prevalence in clinical strains suggest that these ICEs may confer a selective advantage for the physiology or pathogenicity of this human pathogenic bacterium. These data open the way for further studies aiming at unraveling horizontal gene transfers and virulence factors in M. hominis.

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

Analysis of 0.5-kilobase-pair repeats in the Mycoplasma hominis lmp gene system and identification of gene products.

Publications

Analysis of 0.5-kilobase-pair repeats in the Mycoplasma hominis lmp gene system and identification of gene products.

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