Date of Graduation


Document Type


Degree Name

Master of Science in Cell & Molecular Biology (MS)

Degree Level



Biological Sciences


Young Min Kwon

Committee Member

Suresh Thallapuranam

Second Committee Member

Inés Pinto


Cystathionine beta-synthase, Cysteine biosynthesis, Cysteine synthase, Knockout mutation, Mycobacteria, Mycolicibacterium smegmatis


Mycobacteria include serious pathogens of humans and animals. Mycolicibacterium smegmatis is a non-pathogenic model that is widely used to study core mycobacterial metabolism. This thesis explores mycobacterial pathways of cysteine biosynthesis by generating and study of genetic mutants of M. smegmatis. Published in vitro biochemical studies had revealed three independent routes to cysteine synthesis in mycobacteria involving separate homologs of cysteine synthase, namely CysK1, CysK2, and CysM. However, in vivo data were lacking. The M. smegmatis genome encodes only a CysM homolog and lacks orthologs for CysK1 or CysK2. The gene that codes for CysM is a part of an operon, mec+cysOM whose products are involved in the cysteine biosynthesis pathway. The M. smegmatis genome also encodes a putative cystathionine beta-synthase (CBS) protein that has two domain – an N-terminal domain that shares a weak sequence similarity with CysK1 and a C-terminal domain that is specific to CBS enzymes. CBS is a metabolic enzyme that catalyzes the conversion of homocysteine to cystathionine in all three domains of life (Bacteria, Archaea, and Eukarya). To dissect the roles of CysM and CBS proteins in cysteine biosynthesis in vivo, a series of unmarked knockout mutants and complementation strains of M. smegmatis were generated and analyzed phenotypically. Neither the Δmec+cysOM nor the Δcbs mutants of M. smegmatis were auxotrophic for cysteine. However, a Δmec+cysOM_cbs double mutant of M. smegmatis was auxotrophic for cysteine. Genetic complementation of the double mutant using either cbs gene or mec+cysOM operon rescued cysteine auxotrophy. Furthermore, the N-terminal CysK1-like domain of the putative CBS was sufficient to rescue cysteine auxotrophy. Thus, these in vivo data implicate a role for the putative CBS in cysteine biosynthesis and also suggest that the protein may have dual functions in mycobacteria. Multidrug-resistant (MDR) strains of M. tuberculosis, the causative agent of Tuberculosis (TB), are becoming a global crisis. Mycobacterial sulfur metabolism has emerged as a vital target for developing novel drugs to treat MDR-TB. Our findings reveal a potentially new target in mycobacterial sulfur metabolism relevant to strategic development of novel TB drugs.