Generation of targeted chlamydia trachomatis null mutants

Laszlo Kari, Morgan M. Goheen, Linnell B. Randall, Lacey D. Taylor, John H. Carlson, William M. Whitmire, D. Virók, Krithika Rajaram, V. Endrész, Grant McClarty, David E. Nelson, Harlan D. Caldwell

Research output: Contribution to journalArticle

100 Citations (Scopus)

Abstract

Chlamydia trachomatis is an obligate intracellular bacterial pathogen that infects hundreds of millions of individuals globally, causing blinding trachoma and sexually transmitted disease. More effective chlamydial control measures are needed, but progress toward this end has been severely hampered by the lack of a tenable chlamydial genetic system. Here, we describe a reverse-genetic approach to create isogenic C. trachomatis mutants. C. trachomatis was subjected to low-level ethyl methanesulfonate mutagenesis to generate chlamydiae that contained less then one mutation per genome. Mutagenized organisms were expanded in small subpopulations that were screened for mutations by digesting denatured and reannealed PCR amplicons of the target gene with the mismatch specific endonuclease CEL I. Subpopulations with mutations were then sequenced for the target region and plaque-cloned if the desired mutation was detected. We demonstrate the utility of this approach by isolating a tryptophan synthase gene (trpB) null mutant that was otherwise isogenic to its parental clone as shown by de novo genome sequencing. The mutant was incapable of avoiding the anti-microbial effect of IFN-γ-induced tryptophan starvation. The ability to genetically manipulate chlamydiae is a major advancement that will enhance our understanding of chlamydial pathogenesis and accelerate the development of new anti-chlamydial therapeutic control measures. Additionally, this strategy could be applied to other medically important bacterial pathogens with no or difficult genetic systems.

Original languageEnglish
Pages (from-to)7189-7193
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume108
Issue number17
DOIs
Publication statusPublished - Apr 26 2011

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Chlamydia trachomatis
Mutation
Chlamydia
Tryptophan Synthase
Genome
Ethyl Methanesulfonate
Trachoma
Reverse Genetics
Deoxyribonuclease I
Sexually Transmitted Diseases
Starvation
Tryptophan
Mutagenesis
Genes
Clone Cells
Polymerase Chain Reaction
Therapeutics

Keywords

  • Genetics
  • Mutation screen

ASJC Scopus subject areas

  • General

Cite this

Kari, L., Goheen, M. M., Randall, L. B., Taylor, L. D., Carlson, J. H., Whitmire, W. M., ... Caldwell, H. D. (2011). Generation of targeted chlamydia trachomatis null mutants. Proceedings of the National Academy of Sciences of the United States of America, 108(17), 7189-7193. https://doi.org/10.1073/pnas.1102229108

Generation of targeted chlamydia trachomatis null mutants. / Kari, Laszlo; Goheen, Morgan M.; Randall, Linnell B.; Taylor, Lacey D.; Carlson, John H.; Whitmire, William M.; Virók, D.; Rajaram, Krithika; Endrész, V.; McClarty, Grant; Nelson, David E.; Caldwell, Harlan D.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 108, No. 17, 26.04.2011, p. 7189-7193.

Research output: Contribution to journalArticle

Kari, L, Goheen, MM, Randall, LB, Taylor, LD, Carlson, JH, Whitmire, WM, Virók, D, Rajaram, K, Endrész, V, McClarty, G, Nelson, DE & Caldwell, HD 2011, 'Generation of targeted chlamydia trachomatis null mutants', Proceedings of the National Academy of Sciences of the United States of America, vol. 108, no. 17, pp. 7189-7193. https://doi.org/10.1073/pnas.1102229108
Kari, Laszlo ; Goheen, Morgan M. ; Randall, Linnell B. ; Taylor, Lacey D. ; Carlson, John H. ; Whitmire, William M. ; Virók, D. ; Rajaram, Krithika ; Endrész, V. ; McClarty, Grant ; Nelson, David E. ; Caldwell, Harlan D. / Generation of targeted chlamydia trachomatis null mutants. In: Proceedings of the National Academy of Sciences of the United States of America. 2011 ; Vol. 108, No. 17. pp. 7189-7193.
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