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Interstrain gene transfer in Chlamydia trachomatis in vitro: mechanism and significance.

ABSTRACT: The high frequency of between-strain genetic recombinants of Chlamydia trachomatis among isolates obtained from human sexually transmitted infections suggests that lateral gene transfer (LGT) is an important means by which C. trachomatis generates variants that have enhanced relative fitness. A mechanism for LGT in C. trachomatis has not been described, and investigation of this phenomenon by experimentation has been hampered by the obligate intracellular development of this pathogen. We describe here experiments that readily detected LGT between strains of C. trachomatis in vitro. Host cells were simultaneously infected with an ofloxacin-resistant (Ofx(r)) mutant of a serovar L1 strain (L1:Ofx(r)-1) and a rifampin-resistant (Rif(r)) mutant of a serovar D strain (D:Rif(r)-1). Development occurred in the absence of antibiotics, and the progeny were subjected to selection for Ofx(r) Rif(r) recombinants. The parental strains differed at many polymorphic nucleotide sites, and DNA sequencing was used to map genetic crossovers and to determine the parental sources of DNA segments in 14 recombinants. Depending on the assumed DNA donor, the estimated minimal length of the transferred DNA was > or = 123 kb in one recombinant but was > or = 336 to > or = 790 kb in all other recombinants. Such trans-DNA lengths have been associated only with conjugation in known microbial LGT systems, but natural DNA transformation remains a conceivable mechanism. LGT studies can now be performed with diverse combinations of C. trachomatis strains, and they could have evolutionary interest and yield useful recombinants for functional analysis of allelic differences between strains.

SUBMITTER: DeMars R

PROVIDER: S-EPMC2258673 | biostudies-literature | 2008 Mar

REPOSITORIES: biostudies-literature

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Interstrain gene transfer in Chlamydia trachomatis in vitro: mechanism and significance.

DeMars Robert R Weinfurter Jason J

Journal of bacteriology 20071214 5

The high frequency of between-strain genetic recombinants of Chlamydia trachomatis among isolates obtained from human sexually transmitted infections suggests that lateral gene transfer (LGT) is an important means by which C. trachomatis generates variants that have enhanced relative fitness. A mechanism for LGT in C. trachomatis has not been described, and investigation of this phenomenon by experimentation has been hampered by the obligate intracellular development of this pathogen. We describ ...[more]

PMID: 18083799

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Project description:Pre-genomic and post-genomic studies demonstrate that chlamydiae actively recombine in vitro and in vivo, although the molecular and cellular biology of this process is not well understood. In this study, we determined the genome sequence of twelve Chlamydia trachomatis recombinants that were generated in vitro under antibiotic selection. These strains were used to explore the process of recombination in Chlamydia spp., including analysis of candidate recombination hotspots, and to correlate known C. trachomatis in vitro phenotypes with parental phenotypes and genotypes.Each of the 190 examined recombination events was the product of homologous recombination, and no candidate targeting motifs were identified at recombination sites. There was a single deletion event in one recombinant progeny that resulted in the removal of 17.1 kilobases between two rRNA operons. There was no evidence for preference for any specific region of the chromosome for recombination, and analyses of a total of over 200 individual recombination events do not provide any support for recombination hotspots in vitro. Two measurable phenotypes were analyzed in these studies. First, the efficiency of attachment to host cells in the absence of centrifugation was examined, and this property segregated to regions of the chromosome that carry the polymorphic membrane protein (Pmp) genes. Second, the formation of secondary inclusions within cells varied among recombinant progeny, but this did not cleanly segregate to specific regions of the chromosome.These experiments examined the process of recombination in C. trachomatis and identified tools that can be used to associate phenotype with genotype in recombinant progeny. There were no data supporting the hypothesis that particular nucleotide sequences are preferentially used for recombination in vitro. Selected phenotypes can be segregated by analysis of recombination, and this technology may be useful in preliminary analysis of the relationship of genetic variation to phenotypic variation in the chlamydiae.

Project description:Transposon mutagenesis is a widely applied and powerful genetic tool for the discovery of genes associated with selected phenotypes. Chlamydia trachomatis is a clinically significant, obligate intracellular bacterium for which many conventional genetic tools and capabilities have been developed only recently. This report describes the successful development and application of a Himar transposon mutagenesis system for generating single-insertion mutant clones of C. trachomatis This system was used to generate a pool of 105 transposon mutant clones that included insertions in genes encoding flavin adenine dinucleotide (FAD)-dependent monooxygenase (C. trachomatis148 [ct148]), deubiquitinase (ct868), and competence-associated (ct339) proteins. A subset of Tn mutant clones was evaluated for growth differences under cell culture conditions, revealing that most phenocopied the parental strain; however, some strains displayed subtle and yet significant differences in infectious progeny production and inclusion sizes. Bacterial burden studies in mice also supported the idea that a FAD-dependent monooxygenase (ct148) and a deubiquitinase (ct868) were important for these infections. The ct339 gene encodes a hypothetical protein with limited sequence similarity to the DNA-uptake protein ComEC. A transposon insertion in ct339 rendered the mutant incapable of DNA acquisition during recombination experiments. This observation, along with in situ structural analysis, supports the idea that this protein is playing a role in the fundamental process of lateral gene transfer similar to that of ComEC. In all, the development of the Himar transposon system for Chlamydia provides an effective genetic tool for further discovery of genes that are important for basic biology and pathogenesis aspects.IMPORTANCEChlamydia trachomatis infections have an immense impact on public health; however, understanding the basic biology and pathogenesis of this organism has been stalled by the limited repertoire of genetic tools. This report describes the successful adaptation of an important tool that has been lacking in Chlamydia studies: transposon mutagenesis. This advance enabled the generation of 105 insertional mutants, demonstrating that numerous gene products are not essential for in vitro growth. Mammalian infections using these mutants revealed that several gene products are important for infections in vivo Moreover, this tool enabled the investigation and discovery of a gene critical for lateral gene transfer; a process fundamental to the evolution of bacteria and likely for Chlamydia as well. The development of transposon mutagenesis for Chlamydia has broad impact for the field and for the discovery of genes associated with selected phenotypes, providing an additional avenue for the discovery of molecular mechanisms used for pathogenesis and for a more thorough understanding of this important pathogen.

Dataset Information

Interstrain gene transfer in Chlamydia trachomatis in vitro: mechanism and significance.

Publications

Interstrain gene transfer in Chlamydia trachomatis in vitro: mechanism and significance.

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