This project is exploring the biological mechanisms that allow cells to protect cytosolic proteins against chemical modification. These biological mechanisms are critical for cells to avoid damage, for example, due to oxidative stress or exposure to toxic heavy metals. Cytosolic proteins often contain reduced cysteine residues which contain a thiol functional group (RSH). Such groups are susceptible to modification by reactive oxygen and nitrogen species, electrophiles, and toxic metals and metalloids. Cells contain abundant, low molecular mass thiol compounds that serve to buffer the harmful impact of these reactive chemicals and may also participate in the repair of damaged proteins. In many cells, this role is played by glutathione (GSH), a cysteine-containing tripeptide. Many Gram positive bacteria (phylum Firmicutes) lack GSH and this protective role is assumed by bacillithiol (BSH), a recently characterized compound containing cysteine, glucosamine, and malic acid. This research explores the diverse roles of BSH in the model organism Bacillus subtilis. The role of BSH in protection against reactive oxygen compounds (including peroxides and hypochlorous acid), electrophiles (methylglyoxal, formaldehyde), and toxic metalloids are being investigated using genetic, physiological, and biochemical methods. BSH is a required co-substrate for some detoxification enzymes and differs from GSH in its chemical properties and affinity for metal ions. The presence of BSH in diverse bacteria argues that the paradigms established by a detailed characterization of this novel thiol are having far-reaching implications.
Broader impacts
This project involves a combination of biochemical, genetic, genomic, and physiological approaches. The results will have general implications for biological systems including those pertaining to medical and agriculture problems. The experimental work is conducted by undergraduate, graduate, and post-doctoral students who will receive broad training in multidisciplinary approaches to investigating bacterial physiology. Students employ state-of-the-art transcriptional profiling, proteomics, chemical and biophysical techniques and gain experience in analyzing and interpreting the resulting data. Students at all levels present their work locally during group meetings and in department seminars and at regional, national, and international conferences as either posters or oral presentations. Students prepare drafts of manuscripts and are involved in all stages of the publishing process. In addition, students are being expected to be active as mentors to new members of the laboratory. Finally, students have the opportunity to interact with collaborating laboratories with similar research interests and learn about ongoing, complementary projects in related organisms. Previous students who have worked on this project have gone on to successful careers in academics and industry.
The ability to sense and respond to reactive oxygen species (ROS) is critical for survival in an aerobic environment and is a key component of the defensive arsenal of bacterial pathogens that must resist the oxidative attack orchestrated by macrophages (in mammals) or plant cells. Previously, we defined two key regulators of inducible oxidative-stress responses in the model Gram-positive bacterium Bacillus subtilis: PerR and OhrR. Our previous NSF-funded studies of OhrR revealed that this transcription factor is modified under oxidative stress conditions by a novel low molecular weight (LMW) thiol designated bacillithiol (BSH) [Fig. 1]. BSH is now appreciated as the dominant LMW thiol in B. subtilis (and most other low GC Gram positive bacteria and some from other phyla). This project explores the biological functions of BSH as a functional replacement for glutathione (GSH), a prototypic LMW thiol. BSH is proposed to (i) maintain the reducing environment of the cytosol, (ii) help prevent protein oxidation, (iii) serve as a metal ion chelator and (iv) function as a cofactor for numerous thiol-dependent enzymes. The structure of BSH has been determined and the genes for BSH biosynthesis have been identified. During this project period we demonstrated that BSH plays a central role in resistance to reactive electrophilic species (RES) including methylglyoxal. We have also discovered that BSH functions as a major intracellular buffer for Zn(II) ions. Zinc is essential for the growth of all cells and tremendous progress has been made in understanding how zinc ions are imported into cells, but the molecules that engage with Zn(II) in the cytosol and participate in Zn(II) trafficking were poorly understood. Our results indicate that BSH has an important role as an intracellular buffer for Zn(II) and possibly other metal ions. We have also made substantial progress in defining the role of BSH as an intracellular redox buffer. Under oxidative stress conditions, proteins are oxidized on exposed cysteine thiols and these can then modified by reaction with BSH in a reaction called S-bacillithiolation (as originally discovered with OhrR). We have shown that several thioredoxin-like proteins found specifically in those organisms that produce BSH function as de-bacillithiolating enzymes that we have designated as bacilliredoxins. In addition, we have identified a thioredoxin-reductase family protein as an enzyme that harnesses the reducing power of NAD(P)H to reduce oxidized BSH (BSSB) and convert it back to the reduced form. Collectively, our studies have revealed several new roles for BSH and BSH-utilizing proteins in the redox biology of Bacillus subtilis, the model organism for the Gram positive bacteria. Since Bacilli are widely used in biotechnology and in agriculture (as plant-growth promoting bacteria), insights into the physiology of this important class of organisms have broad implications for improving their utility in these and other applications.
