title: Gene replacement in Mycobacterium chelonae: application to the construction of porin knock-out mutants.
abstract: Mycobacterium chelonae is a rapidly growing mycobacterial opportunistic pathogen closely related to Mycobacterium abscessus that causes cornea, skin and soft tissue infections in humans. Although M. chelonae and the emerging mycobacterial pathogen M. abscessus 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 M. chelonae and M. abscessus 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 M. abscessus, genetic tools are lacking for M. chelonae. 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 sacB, can be used to efficiently disrupt genes in this species. Knock-out mutants for each of the three porin genes of M. chelonae ATCC 35752 were constructed using both methodologies, one of which displays a significantly reduced glucose uptake rate consistent with decreased porin expression.
authors.affiliation: Mycobacteria Research Laboratories, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado, United States of America.
authors.forename: Vinicius
authors.initials: V
authors.lastname: Calado Nogueira de Moura
authors.affiliation: Mycobacteria Research Laboratories, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado, United States of America.
authors.forename: Sara
authors.initials: S
authors.lastname: Gibbs
authors.affiliation: Mycobacteria Research Laboratories, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado, United States of America.
authors.forename: Mary
authors.initials: M
authors.lastname: Jackson
mesh_terms: D000483:Alleles; D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001692:Biological Transport; D055786:Gene Knockout Techniques / Q000379:methods*; D005818:Genetic Engineering / Q000379:methods*; D005947:Glucose / Q000378:metabolism / Q000493:pharmacokinetics; D008826:Microbial Sensitivity Tests; D050296:Microbial Viability / Q000187:drug effects; D008969:Molecular Sequence Data; D009154:Mutation; D016926:Mycobacterium chelonae / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D018272:Porins / Q000235:genetics* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D014170:Transformation, Genetic
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D018272:Porins; D005947:Glucose
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