Analysis of mutants disrupted in bacillithiol metabolism in Staphylococcus aureus

Rajkarnikar, Arishma; Strankman, Andrew; Duran, Shayla; Vargas, Derek; Roberts, A. W.; Barretto, Kathryn; Upton, Heather; Hamilton, Christopher; Rawat, Mamta

doi:10.1016/j.bbrc.2013.04.027

Cited by 32 publications

(61 citation statements)

References 25 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…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.…”

Section: Discussionmentioning

confidence: 99%

“…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.…”

Section: Fosb Utilizes Bsh As a Substrate To Confer Fosfomycin Resistmentioning

confidence: 99%

“…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.…”

Section: Fosb Utilizes Bsh As a Substrate To Confer Fosfomycin Resistmentioning

confidence: 99%

See 2 more Smart Citations

Importance of Bacillithiol in the Oxidative Stress Response of Staphylococcus aureus

et al. 2014

Self Cite

View full text Add to dashboard Cite

In Staphylococcus aureus, the low-molecular-weight thiol called bacillithiol (BSH), together with cognate S-transferases, is believed to be the counterpart to the glutathione system of other organisms. To explore the physiological role of BSH in S. aureus, we constructed mutants with the deletion of bshA (sa1291), which encodes the glycosyltransferase that catalyzes the first step of BSH biosynthesis, and fosB (sa2124), which encodes a BSH-S-transferase that confers fosfomycin resistance, in several S. aureus strains, including clinical isolates. Mutation of fosB or bshA caused a 16-to 60-fold reduction in fosfomycin resistance in these S. aureus strains. High-pressure liquid chromatography analysis, which quantified thiol extracts, revealed some variability in the amounts of BSH present across S. aureus strains. Deletion of fosB led to a decrease in BSH levels. The fosB and bshA mutants of strain COL and a USA300 isolate, upon further characterization, were found to be sensitive to H 2 O 2 and exhibited decreased NADPH levels compared with those in the isogenic parents. Microarray analyses of COL and the isogenic bshA mutant revealed increased expression of genes involved in staphyloxanthin synthesis in the bshA mutant relative to that in COL under thiol stress conditions. However, the bshA mutant of COL demonstrated decreased survival compared to that of the parent in human wholeblood survival assays; likewise, the naturally BSH-deficient strain SH1000 survived less well than its BSH-producing isogenic counterpart. Thus, the survival of S. aureus under oxidative stress is facilitated by BSH, possibly via a FosB-mediated mechanism, independently of its capability to produce staphyloxanthin.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Fosb Utilizes Bsh As a Substrate To Confer Fosfomycin Resistmentioning

confidence: 99%

Section: Fosb Utilizes Bsh As a Substrate To Confer Fosfomycin Resistmentioning

confidence: 99%

See 1 more Smart Citation

Importance of Bacillithiol in the Oxidative Stress Response of Staphylococcus aureus

et al. 2014

Self Cite

View full text Add to dashboard Cite

show abstract

“…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).…”

Section: Resultsmentioning

confidence: 72%

“…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).…”

Section: Resultsmentioning

confidence: 99%

Purification and characterization of the Staphylococcus aureus bacillithiol transferase BstA

Perera

Newton

Parnell

et al. 2014

Biochimica et Biophysica Acta (BBA) - General Subjects

View full text Add to dashboard Cite

Background Gram-positive bacteria in the phylum Firmicutes synthesize the low molecular weight thiol bacillithiol rather than glutathione or mycothiol. The bacillithiol transferase YfiT from Bacillus subtilis was identified as a new member of the recently discovered DinB/YfiT-like Superfamily. Based on structural similarity using the Superfamily program, we have determined 30 of 31 Staphylococcus aureus strains encode a single bacillithiol transferase from the DinB/YfiT-like Superfamily, while the remaining strain encodes two proteins. Methods We have cloned, purified, and confirmed the activity of a recombinant bacillithiol transferase (henceforth called BstA) encoded by the S. aureus Newman ORF NWMN_2591. Moreover, we have studied the saturation kinetics and substrate specificity of this enzyme using in vitro biochemical assays. Results BstA was found to be active with the co-substrate bacillithiol, but not with other low molecular weight thiols tested. BstA catalyzed bacillithiol conjugation to the model substrates monochlorobimane, 1-chloro-2,4-dinitrobenzene, and the antibiotic cerulenin. Several other molecules, including the antibiotic rifamycin S, were found to react directly with bacillithiol, but the addition of BstA did not enhance the rate of reaction. Furthermore, cells growing in nutrient rich medium exhibited low BstA activity. Conclusions BstA is a bacillithiol transferase from Staphylococcus aureus that catalyzes the detoxification of cerulenin. Additionally, we have determined that bacillithiol itself might be capable of directly detoxifying electrophilic molecules. General Significance BstA is an active bacillithiol transferase from Staphylococcus aureus Newman and is the first DinB/YfiT-like Superfamily member identified from this organism. Interestingly, BstA is highly divergent from Bacillus subtilis YfiT.

show abstract

Biophysical Features of Bacillithiol, the Glutathione Surrogate of Bacillus subtilis and other Firmicutes

et al. 2013

Self Cite

View full text Add to dashboard Cite

Bacillithiol (BSH) is the major low-molecular-weight (LMW) thiol in many low-G+C Gram-positive bacteria (Firmicutes). Evidence now emerging suggests that BSH functions as an important LMW thiol in redox regulation and xenobiotic detoxification, analogous to what is already known for glutathione and mycothiol in other microorganisms. The biophysical properties and cellular concentrations of such LMW thiols are important determinants of their biochemical efficiency both as biochemical nucleophiles and as redox buffers. Here, BSH has been characterised and compared with other LMW thiols in terms of its thiol pKa, redox potential and thiol–disulfide exchange reactivity. Both the thiol pKa and the standard thiol redox potential of BSH are shown to be significantly lower than those of glutathione whereas the reactivities of the two compounds in thiol–disulfide reactions are comparable. The cellular concentration of BSH in Bacillus subtilis varied over different growth phases and reached up to 5 mm, which is significantly greater than previously observed from single measurements taken during mid-exponential growth. These results demonstrate that the biophysical characteristics of BSH are distinctively different from those of GSH and that its cellular concentrations can reach levels much higher than previously reported.

show abstract

Analysis of mutants disrupted in bacillithiol metabolism in Staphylococcus aureus

Cited by 32 publications

References 25 publications

Importance of Bacillithiol in the Oxidative Stress Response of Staphylococcus aureus

Importance of Bacillithiol in the Oxidative Stress Response of Staphylococcus aureus

Purification and characterization of the Staphylococcus aureus bacillithiol transferase BstA

Biophysical Features of Bacillithiol, the Glutathione Surrogate of Bacillus subtilis and other Firmicutes

Contact Info

Product

Resources

About