Transposon Mutagenesis ofMycobacterium marinumIdentifies a Locus Linking Pigmentation and Intracellular Survival

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Abstract
Pathogenic mycobacteria survive and replicate within host macrophages, but the molecular mechanisms involved in this necessary step in the pathogenesis of infection are not completely understood.Mycobacterium marinumhas recently been used as a model for aspects of the pathogenesis of tuberculosis because of its close genetic relationship toMycobacterium tuberculosisand because of similarities in the pathology and course of infection caused by this organism in its natural hosts, fish and frogs, with tuberculosis in humans. In order to advance the utility of theM. marinummodel, we have developed efficient transposon mutagenesis of the organism by using aDrosophila melanogaster mariner-based transposon. To determine the efficiency of transposition, we have analyzed pigmentation mutants from the transposon mutant library. In addition to insertions in four known genes in the pathway of pigment biosynthesis, two insertions in novel genes were identified in our mutant library. One of these is in a putative inhibitor of the carotenoid biosynthesis pathway. The second unexpected insertion is in an intergenic region between two genes homologous toRv2603candRv2604cofM. tuberculosis. In addition to a pigmentation defect, this mutant showed increased susceptibility to singlet oxygen and grew poorly in murine macrophages. Complementation withM. tuberculosisgenomic DNA encompassingRv2603ctoRv2606ccorrected the pigmentation and growth defects of the mutant. These data demonstrate the utility ofmariner-based transposon mutagenesis ofM. marinumand thatM. marinumcan be used to study the function ofM. tuberculosisgenes involved in intracellular survival and replication.