Genetic Underpinnings of Carotenogenesis and Light-Induced Transcriptome Remodeling in the Opportunistic Pathogen Mycobacterium kansasii

Abstract

Mycobacterium kansasii (Mk) causes opportunistic pulmonary infections with tuberculosis-like features. The bacterium is well known for its photochromogenicity, i.e., the production of carotenoid pigments in response to light. The genetics defining the photochromogenic phenotype of Mk has not been investigated and defined pigmentation mutants to facilitate studies on the role of carotenes in the bacterium’s biology are not available thus far. In this study, we set out to identify genetic determinants involved in Mk photochromogenicity. We screened a library of ~150,000 transposon mutants for colonies with pigmentation abnormalities. The screen rendered a collection of ~200 mutants. Each of these mutants could be assigned to one of four distinct phenotypic groups. The insertion sites in the mutant collection clustered in three chromosomal regions. A combination of phenotypic analysis, sequence bioinformatics, and gene expression studies linked these regions to carotene biosynthesis, carotene degradation, and monounsaturated fatty acid biosynthesis. Furthermore, introduction of the identified carotenoid biosynthetic gene cluster into non-pigmented Mycobacterium smegmatis endowed the bacterium with photochromogenicity. The studies also led to identification of MarR-type and TetR/AcrR-type regulators controlling photochromogenicity and carotenoid breakdown, respectively. Lastly, the work presented also provides a first insight into the Mk transcriptome changes in response to light.

Comprehensive essentiality analysis of the Mycobacterium kansasii genome by saturation transposon mutagenesis and deep sequencing

“…In particular, genome-wide studies aimed at dissecting Mk gene essentiality will significantly expand our understanding of Mk biology and bring to light new potential drug target candidates. With these considerations in mind, we recently investigated and validated the applicability of the ϕMycoMarT7 phage-based specialized transduction method for Himar1 transposon (Tn) mutagenesis in Mk and provided proof-of-principle for its use in combination with Tn insertion sequencing (TnSeq) ( 26 , 32 ). This earlier work set the stage for the use of a saturation Tn mutagenesis–TnSeq approach to study gene essentiality in Mk , as done in several other species of mycobacteria ( 33 - 36 ).…”