List of oligonucleotide primer sequences.

<div><p>The ability to efficiently target loci in the <i>Chlamydia trachomatis</i> genome for deletion remains a desirable goal in the field and new strategies need to be developed and refined. Here we describe the development and application of a lambda red recombineering system for <i>Chlamydia</i>. Using a non-replicative plasmid encoding key lambda Red components and targeting sequences, we demonstrate the efficient deletion of numerous gene targets in the model chlamydial strains <i>C</i>. <i>trachomatis</i> L2/434 and <i>C</i>. <i>muridarum</i>. For initial development of the system, we targeted the <i>incA</i> gene of <i>C</i>. <i>trachomatis</i> for deletion. Deletion mutants containing a chloramphenicol resistance marker were recovered within 3 rounds of selection, or 2 rounds of passaging, in McCoy cells and the resulting clones (CTΔ<i>incA</i>) were verified by PCR-based genotyping and whole genome sequencing. Phenotypic assessment of host cells infected with CTΔ<i>incA</i> was performed by immunofluorescence microscopy and confirmed the lack of IncA expression and the uniform presence of nonfusogenic vacuoles (inclusions) across CTΔ<i>incA</i>-infected monolayers. To explore the utility of this system, we deleted 5 additional candidate virulence factors in <i>C</i>. <i>trachomatis</i> and <i>C</i>. <i>muridarum</i>, including deletions of single and multiple genes. We expect lambda Red recombineering to offer a powerful new strategy for making gene deletion and/or replacement mutants in <i>Chlamydia</i>.</p></div>