Gene Deletion by Fluorescence-Reported Allelic Exchange Mutagenesis in Chlamydia trachomatis

Abstract

Although progress in Chlamydia genetics has been rapid, genomic modification has previously been limited to point mutations and group II intron insertions which truncate protein products. The bacterium has thus far been intractable to gene deletion or more-complex genomic integrations such as allelic exchange. Herein, we present a novel suicide vector dependent on inducible expression of a chlamydial gene that renders Chlamydia trachomatis fully genetically tractable and permits rapid reverse genetics by fluorescence-reported allelic exchange mutagenesis (FRAEM). We describe the first available system of targeting chlamydial genes for deletion or allelic exchange as well as curing plasmids from C. trachomatis serovar L2. Furthermore, this approach permits the monitoring of mutagenesis by fluorescence microscopy without disturbing bacterial growth, a significant asset when manipulating obligate intracellular organisms. As proof of principle, trpA was successfully deleted and replaced with a sequence encoding both green fluorescent protein (GFP) and β-lactamase. The trpA-deficient strain was unable to grow in indole-containing medium, and this phenotype was reversed by complementation with trpA expressed in trans. To assess reproducibility at alternate sites, FRAEM was repeated for genes encoding type III secretion effectors CTL0063, CTL0064, and CTL0065. In all four cases, stable mutants were recovered one passage after the observation of transformants, and allelic exchange was limited to the specific target gene, as confirmed by whole-genome sequencing. Deleted sequences were not detected by quantitative real-time PCR (qPCR) from isogenic mutant populations. We demonstrate that utilization of the chlamydial suicide vector with FRAEM renders C. trachomatis highly amenable to versatile and efficient genetic manipulation.

A Functional Core of IncA Is Required for Chlamydia trachomatis Inclusion Fusion

“…A robust means to complement mutants using a second, selectable marker such as chloramphenicol resistance adds a valuable tool for confirming the function of genes in chlamydia. As an example of how rapidly the field of chlamydia genetics is expanding, a means for allelic exchange by homologous recombination under conditions of beta-lactamase selection was recently described and with it a means for complementation using a separate plasmid expressing spectinomycin resistance (41). The availability of multiple selectable markers can only help accelerate the development of chlamydial genetic systems.…”

Chlamydial Plasmid-Dependent Pathogenicity

“…Furthermore, immunization of mice with pgp3 gene or pGP3 protein has been shown to induce protective immunity [69–71]. However, assessing the role of the plasmid-encoded pGP3 in chlamydial pathogenesis was made possible only after the first report on the successful transformation of C. trachomatis L2 [72], which has permitted the detailed characterization of the plasmid-encoded ORFs in cell culture [17, 73, 74] and animal models [21] and encouraged the development of various genetic engineering tools for investigating chlamydial biology and pathogenesis [51, 75–78]. The success in transforming C. muridarum [19] further enabled the evaluation of the plasmid-encoded pGPs in a female mouse hydrosalpinx/infertility induction model [79–81].…”

Interrogating Genes That Mediate Chlamydia trachomatis Survival in Cell Culture Using Conditional Mutants and Recombination

“…Tools for genetic modification of Chlamydia spp., including mutagenesis coupled with forward and reverse genetic analyses (10,11,13), intraspecies and interspecies lateral gene transfer (LGT) (14)(15)(16), transformation (17), targeted disruption of genes using type II introns (18,19), and deletion of genes by allelic exchange (20), have been reported, but all have limitations for the study of genes that impact chlamydial fitness in cell culture (3,21). Another group attempted to identify the lethal temperature-sensitive (TS) allele in a C. abortus mutant isolated from a chemically mutagenized library by comparing the genome of that mutant to that of its parent (22,23).…”