Characterization of a New Family of Bacillithiol Transferases Encoded by Bacillus subtilis and Staphylococcus aureus

Bacteria have evolved an arsenal of molecular weaponry to combat environmental stressors. Some of the oldest and most widespread protective systems are the low molecular weight thiols, which react readily with a number of molecules through their nucleophilic sulfur atom. Low molecular weight thiols are found in aerobic organisms and are involved in maintaining the redox status of the cytoplasm, detoxifying xenobiotics, and protecting cells against oxidative stress. Bacteria produce a diverse selection of low molecular weight thiols, which include glutathione, mycothiol, ergothionine, and bacillithiol. Glutathione was discovered in 1914 and since then, has drawn the attention of several fields, ranging from the cosmetics industry to the pharmaceutical industry. Very little is known about bacillithiol, the newest protective thiol identified, and the enzymes that catalyze bacillithiol-dependent reactions, such as bacillithiol transferases. While there are a number of parallels between bacillithiol and glutathione, it is unclear whether the bacillithiol system partakes in additional reactions and/or developmental pathways. The focus of this work has been to characterize bacillithiol and the predicted bacillithiol transferases that are members of the S-transferase like (STL) superfamily, a new superfamily of thiol transferases that are related by structural rather than sequence similarity. We first selected the single predicted STL bacillithiol transferase, BstA, encoded in Staphylococcus to characterize the enzyme using saturation kinetics, identify natural product inhibitors, and identify one in vitro antibiotic substrate. Using S. aureus BstA as a model, we then confirm the activity of the seven remaining uncharacterized STL bacillithiol transferases encoded by Bacillus subtilis. To better understand the physiological roles and natural substrates of the enzymes, we studied the expression patterns of all eight bacillithiol transferases encoded in B. subtilis during different stages of growth and development. Finally, we begin to explore S-bacillithiolation of cysteine residues during non-stress conditions. Together, our studies will aid in understanding the significance of the bacillithiol system and its contribution to the physiological fitness of bacteria