Biosynthesis and functions of bacillithiol, a major low-molecular-weight thiol in Bacilli

Abstract

Bacillithiol (BSH), the alpha-anomeric glycoside of L-cysteinyl-D-glucosamine with L-malic acid, is a major low-molecular-weight thiol in Bacillus subtilis and related bacteria. Here, we identify genes required for BSH biosynthesis and provide evidence that the synthetic pathway has similarities to that established for the related thiol (mycothiol) in the Actinobacteria. Consistent with a key role for BSH in detoxification of electrophiles, the BshA glycosyltransferase and BshB1 deacetylase are encoded in an operon with methylglyoxal synthase. BshB1 is partially redundant in function with BshB2, a deacetylase of the LmbE family. Phylogenomic profiling identified a conserved unknown function protein (COG4365) as a candidate cysteine-adding enzyme (BshC) that co-occurs in genomes also encoding BshA, BshB1, and BshB2. Additional evolutionarily linked proteins include a thioredoxin reductase homolog and two thiol:disulfide oxidoreductases of the DUF1094 (CxC motif) family. Mutants lacking BshA, BshC, or both BshB1 and BshB2 are devoid of BSH. BSH is at least partially redundant in function with other low-molecular-weight thiols: redox proteomics indicates that protein thiols are largely reduced even in the absence of BSH. At the transcriptional level, the induction of genes controlled by two thiol-based regulators (OhrR, Spx) occurs normally. However, BSH null cells are significantly altered in acid and salt resistance, sporulation, and resistance to electrophiles and thiol reactive compounds. Moreover, cells lacking BSH are highly sensitive to fosfomycin, an epoxide-containing antibiotic detoxified by FosB, a prototype for bacillithiol-S-transferase enzymes.

Fig. 1.

Parallels between the predicted BSH biosynthetic pathway (Upper) and the pathway for MSH biosynthesis (Lower). BSH synthesis entails three steps catalyzed by BshA (glycosyltransferase), BshB (N-acetylhydrolase), and BshC (cysteine-adding enzyme). MSH biosynthesis follows a parallel logic, with the addition of a dephosphorylation step (MshA2) after the initial glycosyltransferase reaction and a final N-acetylation of cysteine (MshD).

Fig. 2.

Enzymatic activities of BshA and BshB. (A) Production of UDP from 1 mM UDP-GlcNAc with 300 μM D- or L-malic acid in the presence of 6.3 μg/mL of B. subtilis BshA. (B) Analyses for monitoring of the preparative reaction used to generate GlcN-Mal from 70 mM GlcNAc-Mal in the presence of 220 μg/mL of B. anthracis Ba1557 BshB1.

Fig. 3.

Phylogenomic profiling of BSH-related functions. The interaction network (as displayed by EMBL STRING) for genetically interacting proteins possibly related in function with B. subtilis BshC (YllA) is shown. Green lines indicate colocalization in genomes (likely operon structures), and blue lines indicate statistically significant co-occurrence across multiple genomes.

Fig. 4.

Northern blots of gene regulation in wild-type vs. BSH null cells. RNA samples were probed with the ohrA gene (to detect derepression by oxidation of OhrR) and with the trxA and nrfA genes (for regulation by Spx). co, untreated control cells; CHP, 100 μM cumene hydroperoxide; H, 100 μM hydrogen peroxide; D, 1 mM diamide; M, 0.5 mM methylhydroquinone; B, 5 μM benzoquinone.