Date of Degree

9-2022

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biology

Advisor

Luis E. N. Quadri

Committee Members

Amy E. Ikui

Anjana D. Saxena

Louis M.T. Bradbury

Kathleen A. McDonough

Subject Categories

Bacteriology | Biology | Microbiology | Pathogenic Microbiology

Keywords

Mycobacterium kansasii, Mycobacterium tuberculosis, carotenoid pigmentation, photolyase, oleic acid, transposon mutagenesis

Abstract

Mycobacterium kansasii (Mk) is an opportunistic pulmonary pathogen causing infections, predominantly in immunocompromised people, with symptoms similar to those caused by its close relative Mycobacterium tuberculosis (Mt). In contrast to the latter, little work has been done on Mk genetics and even less on its defining characteristic of photochromogenicity, the controlled expression of carotenoid pigments in response to light exposure.

This work represents major progress in the investigation of pigmentation-related genetic factors in Mk. We report the creation of a large-scale, random transposon-insertion mutant library of Mk and its subsequent screening for mutants with an aberration in the pigmentation phenotype. As an outcome of the screen, we show the identification of several genetic loci never before connected to pigmentation in Mk. For one of these loci, which we have termed the Mk carotenoid biosynthesis locus, we provide mutational and transcriptional evidence of its regulation by a MarR-family transcriptional regulator, encoded as part of the locus. For a second locus we demonstrate the regulation of the encoded putative carotenoid cleavage oxygenase by its neighbor, a TetR/AcrR-family transcriptional regulator. In addition, we demonstrate the functional homology of the Mt ortholog of the regulator in Mk. A third identified locus contains putative ferredoxin reductase and a fatty acid desaturase genes, which we show to be involved in oleic acid synthesis.

Through transcriptomics analyses, we show the light-dependent regulation of the carotenoid biosynthesis locus genes, as well as several other previously unidentified loci, including one containing a putative deoxyribodipyrimidine photolyase.

Lastly, we present the creation of a novel system for the investigation of light-dependent mycobacterial pigmentation regulation using the non-pathogenic and non-pigmented rapid‑grower Mycobacterium smegmatis as a host.

Together, this work represents a significant body of previously unavailable evidence for the genetic basis for pigment production, regulation and degradation in Mk, including factors never before shown to be connected to a light-dependent physiological response in any mycobacterium. Furthermore, the presented research sets the stage for further investigations into these light-response- and pigmentation-related processes and their clinical significance in mycobacteria.

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