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.
Publishedin 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 ΔcysM nor the Δcbs 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.
Minimization of deleterious effects of chemical fertilizers on health, ecosystem and economy can only be achieved by finding healthy, ecofriendly and cheap alternatives. Naturally selected symbiotic relationship between the endophytic bacteria and their host plants makes them an ideal candidate as biofertilizer. They can synthesize various plant growth hormones as well as assist their host in uptake of nutrients from soil.The study was designed to compare plant growth promotion of Solanum lycopersicum by Bacillus spp., Pseudomonas spp. and total endophytic community isolated from roots of S. lycopersicum, grown in the soil samples collected from various locations of Kathmandu valley of Nepal. Tomato seeds were inoculated with mixtures of eight endophytic strains of Bacillus spp. and Pseudomonas spp., and crude endophytes obtained from each location separately.Endophytic treatments, except Pseudomonas spp., inhibited seminal root growth during 12-days germination period. However, after plantation, root and shoot biomass was enhanced by the endophytes, with no significant differences among the bacterial treatments. The shoot height was also enhanced, among which Pseudomonas spp. had the strongest effect. In phosphate solubilization assay, out of seventy-two isolates each of Bacillus spp. and Pseudomonas spp. tested, twenty-four isolates of Pseudomonas spp. and sixteen isolates of Bacillus spp. could solubilize phosphate. Higher number of phosphate solubilizing isolates of Pseudomonas spp. might provide a possible explanation for the greater enhancement of shoot height by Pseudomonas spp. as compared to Bacillus spp.
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