Cystine and Antibiotic Treatment Alters Low Molecular Weight Thiol Levels in Mycobacterium smegmatis

Endogenous and exogenous H₂S can influence the virulence of bacteria and their susceptibility to antibiotics and oxidative stress. Escherichia coli and Bacillus subtilis, when grown in minimal medium with sulfate as the sole sulfur source, produce H₂S when treated with cystine or under stress conditions, including exposure to chloramphenicol and ciprofloxacin. However, it is unknown whether Mycobacterium smegmatis is capable of producing sulfide under these conditions and how this production affects cell physiology.

Real-time monitoring of dissolved oxygen (dO₂), pH, extracellular K⁺, and sulfide was performed directly in culture flasks using selective electrodes. Changes in the level of low molecular weight (LMW) thiols were recorded using spectrophotometric methods and high performance liquid chromatography (HPLC).

Sudden addition of cystine or chloramphenicol to growing M. smegmatis cultures increased the intracellular level of cysteine and induced its homeostasis mechanisms, which include the export of excess cysteine from cells and its incorporation into mycothiol (MSH), along with desulfurization with H₂S formation. Ciprofloxacin also increased intracellular cysteine concentration and sulfide production but did not induce cysteine release. Both antibiotics inhibited growth and respiration, whereas cystine transiently increased respiration and glucose uptake in M. smegmatis, in contrast to E. coli, which showed a transient inhibition of these processes.

The mechanisms of cysteine homeostasis under the action of antibiotics in M. smegmatis are similar to those in E. coli and B. subtilis, indicating the universal nature of stress response. The opposing effects of cystine-derived H₂S on physiological parameters in M. smegmatis and E. coli may be important factors contributing to their susceptibility to antibiotics.