Methylglyoxal resistance in Bacillus subtilis: contributions of bacillithiol-dependent and independent pathways

Abstract

Methylglyoxal (MG) is a toxic by-product of glycolysis that damages DNA and proteins ultimately leading to cell death. Protection from MG is often conferred by a glutathione-dependent glyoxalase pathway. However, glutathione is absent from the low-GC Gram-positive Firmicutes, such as Bacillus subtilis. The identification of bacillithiol (BSH) as the major low-molecular-weight thiol in the Firmicutes raises the possibility that BSH is involved in MG detoxification. Here, we demonstrate that MG can rapidly and specifically deplete BSH in cells, and we identify both BSH-dependent and BSH-independent MG resistance pathways. The BSH-dependent pathway utilizes glyoxalase I (GlxA, formerly YwbC) and glyoxalase II (GlxB, formerly YurT) to convert MG to d-lactate. The critical step in this pathway is the activation of the KhtSTU K(+) efflux pump by the S-lactoyl-BSH intermediate, which leads to cytoplasmic acidification. We show that cytoplasmic acidification is both necessary and sufficient for maximal protection from MG. Two additional MG detoxification pathways operate independent of BSH. The first involves three enzymes (YdeA, YraA and YfkM) which are predicted to be homologues of glyoxalase III that converts MG to d-lactate, and the second involves YhdN, previously shown to be a broad specificity aldo-keto reductase that converts MG to acetol.

Figure 1. Summary of BSH-dependent and BSH-independent MG detoxification pathways

Proteins demonstrated as important for MG detoxification in vivo by this study are highlighted in bold. For proteins that have been renamed, the previous name is given below the new name in parentheses.

Figure 2. Determination of BSH-dependent MG detoxification pathway

Susceptibility of wild-type and mutant strains to MG (27.5 mmol) was tested by disk diffusion assay. The zone of inhibition is expressed as the diameter of the clearance zone in millimeters. The mean and standard deviation from at least three biological replicates is shown.

Figure 3. Recovery of BSH after MG challenge requires GlxB

Cellular BSH concentration as a function of time after MG addition in wild-type (A), bshB2 (B), glxA(C) and glxB (D) mutant strains. Cells were challenged with 1 mM MG (time 0) and harvested at the indicated time points. Representative growth curves are shown for cells grown in the absence (grey squares) and presence (grey circles) of MG. Cellular BSH levels in the absence (black squares) and presence (black circles) of MG were quantified by HPLC after derivatization with monobromobimane. The average and standard deviation calculated from three independent experiments are shown.

Figure 4. MG exposure leads to BSH and KhaSTU-dependent cytoplasmic acidification

Intracellular pH values for wild-type (A), bshC (B), glxB (C), and khtU (D) strains in the presence of MG. 1.5 mM MG was added at time 0. The averages and standard deviation calculated from three independent experiments are shown.

Figure 5. Cytoplasmic acidification is sufficient for protection from MG exposure

Wild-type (A), bshC (B), and khaU (C) strains grown in modified M63 media buffered to pH 7.0 with 50 mM MOPS were challenged at time 0 with 3 mM MG. After 5 minutes of MG treatment, 30 mM of sodium benzoate was added. Aliquots were removed at various times after MG addition, then diluted, spread on LB plates, and incubated at 37°C overnight. Percent survival was calculated as the number of colonies that grew after treatment divided by the number of colonies that grew in the absence of treatment multiplied by 100.

Figure 6. Contribution BSH independent pathways to MG resistance

Susceptibility of (A) putative glyoxalase III and (B) aldo-keto reductase mutant strains to MG (27.5 mmol) was tested by disk diffusion assay. The zone of inhibition is expressed as the diameter of the clearance zone in millimeters. The mean and standard deviation from at least three biological replicates is shown.