Transposon Mutagenesis in <named-content content-type="genus-species">Chlamydia trachomatis</named-content> Identifies CT339 as a ComEC Homolog Important for DNA Uptake and Lateral Gene Transfer

ABSTRACT 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 devel...

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Autores principales:	Scott D. LaBrie, Zoë E. Dimond, Kelly S. Harrison, Srishti Baid, Jason Wickstrum, Robert J. Suchland, P. Scott Hefty
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2019
Materias:	Chlamydia trachomatis genetic competence mutagenesis transposons Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/b54a999996b046d1b1b1a981f120fac1
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spelling	oai:doaj.org-article:b54a999996b046d1b1b1a981f120fac12021-11-15T16:22:11ZTransposon Mutagenesis in <named-content content-type="genus-species">Chlamydia trachomatis</named-content> Identifies CT339 as a ComEC Homolog Important for DNA Uptake and Lateral Gene Transfer10.1128/mBio.01343-192150-7511https://doaj.org/article/b54a999996b046d1b1b1a981f120fac12019-08-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.01343-19https://doaj.org/toc/2150-7511ABSTRACT 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. trachomatis 148 [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. IMPORTANCE Chlamydia 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.Scott D. LaBrieZoë E. DimondKelly S. HarrisonSrishti BaidJason WickstrumRobert J. SuchlandP. Scott HeftyAmerican Society for MicrobiologyarticleChlamydia trachomatisgenetic competencemutagenesistransposonsMicrobiologyQR1-502ENmBio, Vol 10, Iss 4 (2019)
institution	DOAJ
collection	DOAJ
language	EN
topic	Chlamydia trachomatis genetic competence mutagenesis transposons Microbiology QR1-502
spellingShingle	Chlamydia trachomatis genetic competence mutagenesis transposons Microbiology QR1-502 Scott D. LaBrie Zoë E. Dimond Kelly S. Harrison Srishti Baid Jason Wickstrum Robert J. Suchland P. Scott Hefty Transposon Mutagenesis in <named-content content-type="genus-species">Chlamydia trachomatis</named-content> Identifies CT339 as a ComEC Homolog Important for DNA Uptake and Lateral Gene Transfer
description	ABSTRACT 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. trachomatis 148 [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. IMPORTANCE Chlamydia 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.
format	article
author	Scott D. LaBrie Zoë E. Dimond Kelly S. Harrison Srishti Baid Jason Wickstrum Robert J. Suchland P. Scott Hefty
author_facet	Scott D. LaBrie Zoë E. Dimond Kelly S. Harrison Srishti Baid Jason Wickstrum Robert J. Suchland P. Scott Hefty
author_sort	Scott D. LaBrie
title	Transposon Mutagenesis in <named-content content-type="genus-species">Chlamydia trachomatis</named-content> Identifies CT339 as a ComEC Homolog Important for DNA Uptake and Lateral Gene Transfer
title_short	Transposon Mutagenesis in <named-content content-type="genus-species">Chlamydia trachomatis</named-content> Identifies CT339 as a ComEC Homolog Important for DNA Uptake and Lateral Gene Transfer
title_full	Transposon Mutagenesis in <named-content content-type="genus-species">Chlamydia trachomatis</named-content> Identifies CT339 as a ComEC Homolog Important for DNA Uptake and Lateral Gene Transfer
title_fullStr	Transposon Mutagenesis in <named-content content-type="genus-species">Chlamydia trachomatis</named-content> Identifies CT339 as a ComEC Homolog Important for DNA Uptake and Lateral Gene Transfer
title_full_unstemmed	Transposon Mutagenesis in <named-content content-type="genus-species">Chlamydia trachomatis</named-content> Identifies CT339 as a ComEC Homolog Important for DNA Uptake and Lateral Gene Transfer
title_sort	transposon mutagenesis in <named-content content-type="genus-species">chlamydia trachomatis</named-content> identifies ct339 as a comec homolog important for dna uptake and lateral gene transfer
publisher	American Society for Microbiology
publishDate	2019
url	https://doaj.org/article/b54a999996b046d1b1b1a981f120fac1
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Transposon Mutagenesis in <named-content content-type="genus-species">Chlamydia trachomatis</named-content> Identifies CT339 as a ComEC Homolog Important for DNA Uptake and Lateral Gene Transfer

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