Analysis of mutants disrupted in bacillithiol metabolism in Staphylococcus aureus

Abstract

Bacillithiol (BSH), an α-anomeric glycoside of L-cysteinyl-D-glucosaminyl-L-malate, is a major low molecular weight thiol found in low GC Gram-positive bacteria, such as Staphylococcus aureus. Like other low molecular weight thiols, BSH is likely involved in protection against a number of stresses. We examined S. aureus transposon mutants disrupted in each of the three genes associated with BSH biosynthesis. These mutants are sensitive to alkylating stress, oxidative stress, and metal stress indicating that BSH and BSH-dependent enzymes are involved in protection of S. aureus. We further demonstrate that BshB, a deacetylase involved in the second step of BSH biosynthesis, also acts as a BSH conjugate amidase and identify S. aureus USA 300 LAC 2626 as a BSH-S-transferase, which is able to conjugate chlorodinitrobenzene, cerulenin, and rifamycin to BSH.

Importance of Bacillithiol in the Oxidative Stress Response of Staphylococcus aureus

“…This discrepancy can be explained by the use of a nonstandard protocol for MIC assays in the prior study (e.g., the use of TSB instead of Mueller-Hinton broth, as suggested in the CLSI protocol [27]). In addition, the previous BSH studies have utilized transposon mutants (18,38) constructed in a strain cured of its plasmids (52); in addition, these studies did not complement the mutation to exclude any insertional polar effects that may have been caused by the transposon's insertion. We have confirmed here the contribution of both BSH and FosB to fosfomycin resistance in S. aureus using cis and trans complementation of our mutations.…”

“…In previous studies, FosB was shown to mediate fosfomycin resistance in S. aureus through the use of BSH to disrupt the epoxide ring of fosfomycin (18,19,38). We investigated here the role of BSH in fosfomycin resistance mediated by FosB in a wide variety of methicillin-sensitive and methicillin-resistant S. aureus strains, including clinical isolates.…”

“…In Gram-negative bacteria, oxidative stress has been shown to affect the cellular NADPH levels (47), as NADPH-dependent disulfide reductases reduce oxidized GSH (48,49). There are four putative oxidoreductases (YpdA, YqiW, YphP, and YtxJ) that have been postulated to reduce oxidized BSH (BSSB) (10,11,38). Given the presence of these genes in S. aureus (e.g., a ypdA homolog [GenBank accession number YP_008485371]), we explored whether mutation of fosB or bshA led to changes in NADPH levels in the mutants relative to the wild type.…”

Purification and characterization of the Staphylococcus aureus bacillithiol transferase BstA

“…Previously, it was postulated that detoxification of rifamycin S, a bacterial RNA polymerase inhibitor, might be bacillithiol transferase dependent in S. aureus [21, 26]. We found that BSH reacted directly with rifamycin S, primarily forming the reduced conjugate, BS-rifamycin SV, in a BstA-independent manner at pH 7.0 and pH 6.0 with a rate of >35 μM min −1 (Supplementary Table S1).…”

“…Thiol analysis of the bshB Tn-mutant has shown that unlike B. subtilis bshB1 and bshB2 single mutants, the S. aureus bshB mutant does not produce BSH, indicating that there is no compensating BshB activity (data not shown and [26]). Indeed, the wild type S. aureus USA300 (JE2) dialyzed cell-free extract showed robust BCA activity (670 ± 70 nmoles min −1 mg −1 ) similar to that of the bstA mutant extract (570 ± 10 nmoles min −1 mg −1 ), whereas the bshB mutant extract showed BCA activity at just 10% of the wild type level (70 ± 13 nmoles min −1 mg −1 , Figure 6B).…”