Genetic Manipulation of Chlamydia trachomatis: Chromosomal Deletions

Abstract

Progress in understanding molecular mechanisms contributing to chlamydial pathogenesis has been greatly facilitated by recent advances in genetic manipulation of C. trachomatis. Valuable approaches such as random, chemically induced mutagenesis or targeted, insertion-based gene disruption have led to significant discoveries. We describe herein a technique for generating definitive null strains via complete deletion of chromosomal genes in C. trachomatis. Fluorescence-reported allelic exchange mutagenesis (FRAEM), using the suicide vector pSUmC, enables targeted deletion of desired chromosomal DNA. The protocol provided here describes steps required to produce transformation competent chlamydiae, generate a specific allelic exchange plasmid construct, carry out mutagenesis, and isolate clonal populations of resulting mutant strains.

Keywords: Allelic exchange; FRAEM; Mutagenesis.

Figure 1-

Schematic of overall cloning strategy used for constructing allelic exchange plasmid. (1) The 3 kb left flank (upstream) of gene of interest (ct00X) is PCR-amplified from C. trachomatis L2 genomic DNA and cloned into linearized (SalI) pSuMc-sal-sbf by Gibson Assembly to yield pSUmC-sal-sbf + LF. (2) The 3 kb right flank (downstream) of gene of interest (ctl00X) is then mobilized into linearized (SalI) pSuMc-sal-sbf + LF ct00X to yield the final construct pSU-Δ00X.