Dissertations, Theses, and Capstone Projects

Date of Degree


Document Type


Degree Name





Luis E.N. Quadri

Committee Members

Shaneen Singh

Anjana Saxena

Weigang Qiu

Dean Crick

Subject Categories

Bacteriology | Molecular Genetics | Pathogenic Microbiology


mycobacteria, antibiotic resistance, gene essentiality, forward genetics


Mycobacterium kansasii (Mk) is an opportunistic pathogen capable of causing tuberculosis-like pulmonary disease in immunocompromised individuals and those with other risk factors including chronic obstructive pulmonary disease or malignancy. Mk is frequently isolated from man-made water sources where it forms resilient biofilms, posing a health risk to these susceptible individuals. Despite its medical relevance as an environmental pathogen, and a close phylogenetic relationship with the obligate pathogen Mycobacterium tuberculosis (Mtb), few studies to date have probed the molecular biology and genetics of Mk. Here, we sought to apply a transposon (Tn) mutagenesis tool to dissect gene function in Mk using both deep sequencing and forward genetics approaches. Through deep sequencing of 2.5 million Mk Tn mutants, we defined the in vitro essential gene set of Mk and then compared these results to essentiality data available for several other species of mycobacteria. Importantly, this analysis shows that several orthologs of essential Mtb genes encoding drug target candidates were identified as nonessential in Mk, suggesting they may not be viable drug targets in Mk. Next, we carried out a forward genetic screen of 10,000 individually arrayed Mk Tn mutants and identified those displaying increased susceptibility to the antitubercular agent p-aminosalicylic acid (PAS), to which Mk is naturally resistant. Interestingly, most of the PAS-susceptible Mk Tn mutants analyzed carried insertions in genes sharing orthologs in Mtb. This finding suggests that the natural resistance of Mk to PAS may not be due to the presence of dedicated Mk-specific PAS resistance mechanisms, but rather it is a result of fundamental differences in physiology between Mk and Mtb. Finally, we performed preliminary characterization and complementation analysis of select PAS-susceptible Mk Tn mutants to further explore these genotype-phenotype associations. Altogether, our findings from both approaches represent valuable resources to assist in the process of identifying and prioritizing potential Mk drug target candidates and to guide future studies on Mk biology.

Text S1 Detailed TnSeq Protocol.docx (49 kB)
Text S1. Detailed TnSeq protocol

Excel S1 Newly Annotated H37Rv ORFs.xlsx (12 kB)
Excel S1. Newly annotated H37Rv ORFs

Excel S2 Newly Annotated MAH ORFs.xlsx (12 kB)
Excel S2. Newly annotated MAH ORFs

Excel S3 TnSeq Library Statistics.xlsx (12 kB)
Excel S3. TnSeq library statistics

Excel S4 Chr TA Site Essentiality Data.xlsx (4407 kB)
Excel S4. Chromosome TA site essentiality data

Excel S5 Chr Gene Essentiality Data.xlsx (325 kB)
Excel S5. Chromosome gene essentiality data

Excel S6 Comparative Genomics Analysis.xlsx (435 kB)
Excel S6. Comparative genomics analysis

Excel S7 Genes Essential in All Species.xlsx (22 kB)
Excel S7. Genes essential in all species

Excel S8 Essentiality of Non-ORF Genomic Annotated.xlsx (12 kB)
Excel S8. Essentiality of non-ORF genomic features

Excel S9 IGR Containing CUTA Segments.xlsx (44 kB)
Excel S9. Intergenic regions containing CUTA segments

Excel S10 Plasmid TA Site Essentiality Data.xlsx (117 kB)
Excel S10. Plasmid TA site essentiality data

Excel S11 Plasmid Gene Essentiality Data.xlsx (25 kB)
Excel S11. Plasmid gene essentiality data

Excel S12 pMK12478 vs pMD2.xlsx (11 kB)
Excel S12. pMK12478 vs pMD2

Excel S13 ESX Loci Essentiality.xlsx (13 kB)
Excel S13. ESX loci essentially

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