Chlamydia trachomatis Transformation and Allelic Exchange Mutagenesis

Abstract

Gene inactivation is essential for forward and reverse genetic approaches to establish protein function. Techniques such as insertion or chemical mutagenesis have been developed to mutagenize chlamydiae via targeted or random mutagenesis, respectively. Both of these approaches require transformation of chlamydiae to either introduce insertion elements or complement mutants. We have recently developed a targeted mutagenesis strategy, fluorescence-reported allelic exchange mutagenesis (FRAEM), to delete Chlamydia trachomatis L2 genes. This approach overcomes several barriers for genetically manipulating intracellular bacteria. Perhaps most significantly, FRAEM employs fluorescence reporting to indicate successful transformation and subsequent recombination events. Three protocols are provided that detail methods to construct gene-specific suicide vectors, transform C. trachomatis L2 to select for recombinants, and isolate clonal populations via limiting dilution. In aggregate, these protocols will allow investigators to engineer C. trachomatis L2 strains carrying complete deletions of desired gene(s).

Initial Characterization of the Two ClpP Paralogs of Chlamydia trachomatis Suggests Unique Functionality for Each

“…Chlamydial transformation. The protocol followed was a modification of the method developed by Mueller et al (70). For transformation, 10 6 C. trachomatis serovar L2 EBs (25667R) naturally lacking the endogenous plasmid were incubated with 2 g of unmethylated plasmid in a volume of 50 l CaCl 2 at room temperature for 30 min.…”

Floxed-Cassette Allelic Exchange Mutagenesis Enables Markerless Gene Deletion in Chlamydia trachomatis and Can Reverse Cassette-Induced Polar Effects

“…Our approach to create a markerless tmeA strain began by creating a new tmeA mutant. WT C. trachomatis L2 was transformed with pSUmC-tmeA-lox-gfp-bla, and FRAEM was performed as previously described (15,30). Isolates were then subjected to cultivation in the presence of rifampin (Rif) to select Rif-resistant chlamydiae, and a clonal strain was derived by limiting dilution.…”

“…Our strategy relies on the expression of Cre in Chlamydia spp. using the conditionally replicating plasmid pSUmC (30). Cre is constitutively expressed from pSU-CRE via a blaM promoter, and the plasmid is maintained via selection with Spec and aTC to induce the expression of pgp6.…”

“…Rifampin (Rif)-resistant strains were generated as described previously (45) by cultivation for 4 passages in 2.5 ng/ml Rif, followed by 4 passages in 5 ng/ml Rif. Clonal isolates for all final Chlamydia strains were obtained as described by 2 sequential limiting dilution passages in 384 plates (30). Chlamydial fitness was assessed by enumeration of infectious progeny or quantitative assessment of inclusion area as described by McKuen et al (26).…”

The ClpX and ClpP2 Orthologs of Chlamydia trachomatis Perform Discrete and Essential Functions in Organism Growth and Development

“…The protocol followed was a modification of the method developed by Mueller and Fields (88) and as previously described (17). For transformation, 10 6 C. trachomatis serovar L2 EBs (25667R) naturally lacking the endogenous plasmid were incubated with 2 μg of unmethylated plasmid in a volume of 50 μL CaCl 2 at room temperature for 30 minutes.…”