Transposon Mutagenesis in Chlamydia trachomatis 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 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.

Sigma 54-Regulated Transcription Is Associated with Membrane Reorganization and Type III Secretion Effectors during Conversion to Infectious Forms of Chlamydia trachomatis

“…Purified ctcC expression vector plasmid was transformed into a clonal isolate of L2 C. trachomatis (LGV2 434/Bu; GenBank accession no. CP019386.1 [79]). Briefly, 15 g of vector plasmid was mixed with 100 l 2ϫ CaCl 2 buffer (20 mM Tris [pH 7.5], 100 mM CaCl 2 ) and 25 l of C. trachomatis EBs and diluted with water to reach a final volume of 200 l. This CaCl 2 mixture was incubated at room temperature for 30 min before being diluted into 1ϫ CaCl 2 buffer, added onto a confluent monolayer of L292 cells, and then centrifuged for 30 min at 550 ϫ g and 20°C.…”

“…Purified ctcC expression vector plasmid was transformed into a clonal isolate of L2 C. trachomatis (LGV2 434/Bu; GenBank accession no. CP019386.1 [ 79 ]). Briefly, 15 μg of vector plasmid was mixed with 100 μl 2× CaCl 2 buffer (20 mM Tris [pH 7.5], 100 mM CaCl 2 ) and 25 μl of C. trachomatis EBs and diluted with water to reach a final volume of 200 μl.…”

Chromosomal Recombination Targets in Chlamydia Interspecies Lateral Gene Transfer

“…The present study builds on the transposition system of Wang and colleagues (10), who describe the development of a library of independent transposon mutant C. muridarum strains that are marked with a Himar-generated transposon (chloramphenicol resistant [Cam r ]) at random positions around the chromosome. A similar system for C. trachomatis has been recently described by LaBrie et al (11). The C. muridarum transposon library allows the production of a wide variety of recombinant C. trachomatis strains carrying syntenic C. muridarum chromosomal fragments.…”

An Ancient Molecular Arms Race: Chlamydia vs. Membrane Attack Complex/Perforin (MACPF) Domain Proteins

“…It was originally postulated that the MACPF domain protein may be essential for Chlamydia since a saturating screen for chemically-induced mutations in C. muridarum failed to reveal nonsense mutations in tc0431 ( 81 ). However, inactivating transposon insertions were subsequently observed in both C. muridarum and C. trachomatis ( 82 , 83 ). None of the mutations abolished intracellular survival, indicating that this protein is not essential for cultivation in tissue culture.…”