Date of Degree


Document Type


Degree Name





Luis E. N. Quadri

Committee Members

Peter Lipke

Nicolas Biais

Anuradha Janakiraman

Edward Chan

Subject Categories

Bacteriology | Life Sciences | Microbial Physiology


Mycobacteria, Colony morphology, sRNA, Galleria mellonella, virulence, biofilm


Mycobacterium kansasii (Mk) is a nontuberculous mycobacterium (NTM) and medically relevant opportunistic human pathogen. Mk causes dangerous disease pathologies ranging from tuberculosis-like chronic pulmonary disease (CPD) to non-pulmonary focal or disseminated infections that are exacerbated by comorbidities such as chronic obstructive pulmonary disease (COPD), co-infection with HIV, or cancer. Among the most frequently identified cause of NTM-linked CPD, Mk infections contribute to a globally increasing NTM disease burden and are difficult to treat, requiring a long-term, multi-drug regimen. Although a less virulent pathogen than Mycobacterium tuberculosis (Mtb), Mk elicits similar disease features and shares in vitro growth characteristics with the ‘tubercle bacillus’. Due to a high level of genetic similarity and lower biosafety concern, Mk also has potential as an attractive model organism for the study of Mtb. Despite this, research into Mk gene function is limited and no genome-wide studies have been reported. Here, we provide evidence that demonstrates the functionality of a phage-based transposon (Tn) mutagenesis system in Mk by generating and screening a library of random insertion mutants for altered macrocolony morphology. In this screen, we identified 41 mutants exhibiting unique phenotypes, among them one carrying disruption to a gene encoding a conserved mycobacterial small regulatory RNA for which we established a previously unrecognized role in (macro)colony morphology and biofilm formation. Additionally, we showed Galleria mellonella larva are susceptible to Mk infection, demonstrating the potential of this increasingly popular infection model for the study of Mk pathogenicity and efficacy of antimicrobial compounds. Finally, we combined Tn mutagenesis with next generation sequencing to identify 12,071 unique insertion sites that do not impact Mk viability. A comparison of our results to published transposon sequencing data for Mtb identified 11 Mk orthologs of essential Mtb genes tolerant of transposon insertion that potentially reflect significant differences in genetic requirements between the two species. Together, this work demonstrates Tn mutagenesis is an effective tool to study gene function in Mk and sets the stage for more comprehensive comparative genetic studies of Mk and Mtb.

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