Gene Replacement in <i>Mycobacterium chelonae</i>: Application to the Construction of Porin Knock-Out Mutants Calado Nogueira de MouraVinicius GibbsSara JacksonMary 2014 <div><p><i>Mycobacterium chelonae</i> is a rapidly growing mycobacterial opportunistic pathogen closely related to <i>Mycobacterium abscessus</i> that causes cornea, skin and soft tissue infections in humans. Although <i>M. chelonae</i> and the emerging mycobacterial pathogen <i>M. abscessus</i> have long been considered to belong to the same species, these two microorganisms considerably differ in terms of optimum growth temperature, drug susceptibility, pathogenicity and the types of infection they cause. The whole genome sequencing of clinical isolates of <i>M. chelonae</i> and <i>M. abscessus</i> is opening the way to comparative studies aimed at understanding the biology of these pathogens and elucidating the molecular bases of their pathogenicity and biocide resistance. Key to the validation of the numerous hypotheses that this approach will raise, however, is the availability of genetic tools allowing for the expression and targeted mutagenesis of genes in these species. While homologous recombination systems have recently been described for <i>M. abscessus</i>, genetic tools are lacking for <i>M. chelonae</i>. We here show that two different allelic replacement methods, one based on mycobacteriophage-encoded recombinases and the other on a temperature-sensitive plasmid harboring the counterselectable marker <i>sacB</i>, can be used to efficiently disrupt genes in this species. Knock-out mutants for each of the three porin genes of <i>M. chelonae</i> ATCC 35752 were constructed using both methodologies, one of which displays a significantly reduced glucose uptake rate consistent with decreased porin expression.</p></div>