Transposon Mutagenesis in Chlamydia trachomatis Identifies CT339 as a ComEC Homolog Important for DNA Uptake and Lateral Gene Transfer

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.S.D.L., Z.E.D., K.S.H., S.B., R.J.S., and P.S.H. were funded by NIH (AI126785)J.W. and P.S.H. were supported by NIH AI125929. P.S.H. was also supported by P20GM113117Support for genomic sequencing was supplemented by P20GM10363

Inter-species lateral gene transfer focused on the Chlamydia plasticity zone identifies loci associated with immediate cytotoxicity and inclusion stability

This is the peer reviewed version of the following article: Dimond, Z.E., Suchland, R.J., Baid, S., LaBrie, S.D., Soules, K.R., Stanley, J., et al (2021) Inter-species lateral gene transfer focused on the Chlamydia plasticity zone identifies loci associated with immediate cytotoxicity and inclusion stability. Molecular Microbiology, 116, 1433– 1448. https://doi.org/10.1111/mmi.14832, which has been published in final form at https://doi.org/10.1111/mmi.14832. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions. This article may not be enhanced, enriched or otherwise transformed into a derivative work, without express permission from Wiley or by statutory rights under applicable legislation. Copyright notices must not be removed, obscured or modified. The article must be linked to Wiley’s version of record on Wiley Online Library and any embedding, framing or otherwise making available the article or pages thereof by third parties from platforms, services and websites other than Wiley Online Library must be prohibited.Chlamydia muridarum actively grows in murine mucosae and is a representative model of human chlamydial genital tract disease. In contrast, C. trachomatis infections in mice are limited and rarely cause disease. The factors that contribute to these differences in host adaptation and specificity remain elusive. Overall genomic similarity leads to challenges in the understanding of these significant differences in tropism. A region of major genetic divergence termed the plasticity zone (PZ) has been hypothesized to contribute to the host specificity. To evaluate this hypothesis, lateral gene transfer was used to generate multiple hetero-genomic strains that are predominately C. trachomatis but have replaced regions of the PZ with those from C. muridarum. In vitro analysis of these chimeras revealed C. trachomatis-like growth as well as poor mouse infection capabilities. Growth-independent cytotoxicity phenotypes have been ascribed to three large putative cytotoxins (LCT) encoded in the C. muridarum PZ. However, analysis of PZ chimeras supported that gene products other than the LCTs are responsible for cytopathic and cytotoxic phenotypes. Growth analysis of associated chimeras also led to the discovery of an inclusion protein, CTL0402 (CT147), and homolog TC0424, which was critical for the integrity of the inclusion and preventing apoptosis