A Putative Cystathionine Beta‐Synthase Homolog of Mycobacterium smegmatis is Involved in de novo Cysteine Biosynthesis

Mycobacteria include serious pathogens of humans and animals. Mycobacterium smegmatis is a non‐pathogenic model that is widely used to study core mycobacterial metabolism. Using M. smegmatis , we are genetically investigating mycobacterial pathways for biosynthesis of cysteine, a vital sulfur‐containing amino acid. 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. However, the genome encodes a putative cystathionine beta‐synthase (CBS) protein that has two domains ‐ an N‐terminal domain that shares 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 the three domains of life (Bacteria, Archaea, and Eukarya). To dissect the roles of CysM and CBS proteins in cysteine biosynthesis in vivo , we generated a series of unmarked gene deletion mutants and gene complementation strains of M. smegmatis and analyzed them phenotypically . We found that neither the Δ cysM nor the Δ cbs mutants of M. smegmatis were auxotrophic for cysteine. However, a ΔcbsΔcysM double mutant of M. smegmatis was auxotrophic for cysteine. Genetic complementation of the double mutant using either cbs or cysM genes rescued cysteine auxotrophy. Furthermore, the N‐terminal CysK1‐like domain of the putative CBS was sufficient to rescue cysteine auxotrophy. Thus, our 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. Support or Funding Information This project was supported by the Cell and Molecular Biology program and the Department of Biological Sciences at the University of Arkansas, Fayetteville, AR 72701.