Iron homeostasis in Bacillus subtilis relies on three differentially expressed efflux systems.

In Bacillus subtilis, iron homeostasis is maintained by the ferric uptake regulator (Fur) and manganese homeostasis relies on the manganese transport regulator (MntR). Both Fur and MntR function as bi-functional metalloregulators that repress import and activate metal ion efflux systems. The ferrous iron efflux ATPase, PfeT, is derepressed by hydrogen peroxide (H2O2) as sensed by PerR and induced by iron as sensed by Fur. Mutants lacking PfeT are sensitive to iron intoxication. Here, we show that mntR mutants are also iron-sensitive, largely due to decreased expression of the MntR-activated MneP and MneS cation diffusion facilitator (CDF) proteins previously defined for their role in Mn2+ export. The ability of MneP and MneS to export iron is apparent even when their expression is not induced by Mn2+. Our results demonstrate that PfeT, MneP and MneS each contribute to iron homeostasis, and a triple mutant lacking all three is more iron-sensitive than any single mutant. We further show that sensitivity to H2O2 does not correlate with iron sensitivity. For example, an mntR mutant is H2O2-sensitive due to elevated Mn(II) that increases PerR-mediated repression of peroxide resistance genes, and this repression is antagonized by elevated Fe2+ in an mntR pfeT mutant. Thus, H2O2-sensitivity reflects the relative levels of Mn2+ and Fe2+ as sensed by the PerR regulatory protein. These results underscore the complex interplay between manganese, iron and oxidative stress in B. subtilis.

The Bacillus subtilis monothiol bacilliredoxin BrxC (YtxJ) and the Bdr (YpdA) disulfide reductase reduce S-bacillithiolated proteins.

The bacterial cytosol is generally a reducing environment with protein cysteine residues maintained in their thiol form. The low molecular weight thiol bacillithiol (BSH) serves as a general thiol reductant, analogous to glutathione, in a wide range of bacterial species. Proteins modified by disulfide bond formation with BSH (S-bacillithiolation) are reduced by the action of bacilliredoxins, BrxA and BrxB. Here, the YtxJ protein is identified as a monothiol bacilliredoxin, renamed BrxC, and is implicated in BSH removal from oxidized cytosolic proteins, including the glyceraldehyde 3-phosphate dehydrogenases GapA and GapB. BrxC can also debacillithiolate the mixed disulfide form of the bacilliredoxin BrxB. Bdr is a thioredoxin reductase-like flavoprotein with bacillithiol-disulfide (BSSB) reductase activity. Here, Bdr is shown to additionally function as a bacilliredoxin reductase. Bdr and BrxB function cooperatively to debacillithiolate OhrR, a transcription factor regulated by S-bacillithiolation on its sole cysteine residue. Collectively, these results expand our understanding of the BSH redox network comprised of three bacilliredoxins and a BSSB reductase that serve to counter the widespread protein S-bacillithiolation that results from conditions of disulfide stress.

A Central Role for Magnesium Homeostasis during Adaptation to Osmotic Stress.

Osmotic stress is a significant physical challenge for free-living cells. Cells from all three domains of life maintain viability during osmotic stress by tightly regulating the major cellular osmolyte potassium (K+) and by import or synthesis of compatible solutes. It has been widely established that in response to high salt stress, many bacteria transiently accumulate high levels of K+, leading to bacteriostasis, with growth resuming only when compatible solutes accumulate and K+ levels are restored to biocompatible levels. Using Bacillus subtilis as a model system, we provide evidence that K+ fluxes perturb Mg2+ homeostasis: import of K+ upon osmotic upshift is correlated with Mg2+ efflux, and Mg2+ reimport is critical for adaptation. The transient growth inhibition resulting from hyperosmotic stress is coincident with loss of Mg2+ and a decrease in protein translation. Conversely, the reimport of Mg2+ is a limiting factor during resumption of growth. Furthermore, we show the essential signaling dinucleotide cyclic di-AMP fluctuates dynamically in coordination with Mg2+ and K+ levels, consistent with the proposal that cyclic di-AMP orchestrates the cellular response to osmotic stress. IMPORTANCE Environments with high concentrations of salt or other solutes impose an osmotic stress on cells, ultimately limiting viability by dehydration of the cytosol. A very common cellular response to high osmolarity is to immediately import high levels of potassium ion (K+), which helps prevent dehydration and allows time for the import or synthesis of biocompatible solutes that allow a resumption of growth. Here, using Bacillus subtilis as a model, we demonstrate that concomitant with K+ import there is a large reduction in intracellular magnesium (Mg2+) mediated by specific efflux pumps. Further, it is the reimport of Mg2+ that is rate-limiting for the resumption of growth. These coordinated fluxes of K+ and Mg2+ are orchestrated by cyclic-di-AMP, an essential second messenger in Firmicutes. These findings amend the conventional model for osmoadaptation and reveal that Mg2+ limitation is the proximal cause of the bacteriostasis that precedes resumption of growth.

Bacillus subtilis Fur Is a Transcriptional Activator for the PerR-Repressed pfeT Gene, Encoding an Iron Efflux Pump.

The physiological relevance of bacterial iron efflux has only recently been appreciated. The Bacillus subtilis P1B4-type ATPase PfeT (peroxide-induced ferrous efflux transporter) was one of the first iron efflux pumps to be characterized, and cells lacking pfeT accumulate high levels of intracellular iron. The pfeT promoter region has binding sites for both PerR, a peroxide-sensing Fur-family metalloregulator, and the ferric uptake repressor Fur. Both Fur and PerR bind DNA with Fe(II) as a cofactor. While reaction of PerR-Fe(II) with peroxide can account for the induction of pfeT under oxidative stress, binding of Fur-Fe(II) would be expected to lead to repression, which is inconsistent with the known role of PfeT as an iron efflux protein. Here, we show that expression of pfeT is repressed by PerR, as anticipated, and induced by Fur in response to Fe(II). Activation by Fur is mediated both by antagonism of the PerR repressor and by direct transcriptional activation, as confirmed using in vitro transcription assays. A similar mechanism of regulation can explain the iron induction of the Listeria monocytogenes PfeT ortholog and virulence factor, FrvA. Mutational studies support a model in which Fur activation involves regions both upstream and downstream of the pfeT promoter, and Fur and PerR have overlapping recognition of a shared regulatory element in this complex promoter region. This work demonstrates that B. subtilis Fur can function as an iron-dependent activator of transcription.IMPORTANCE Iron homeostasis plays a key role at the host-pathogen interface during the process of infection. Bacterial growth restriction resulting from host-imposed iron starvation (nutritional immunity) highlights the importance of iron import during pathogenesis. Conversely, bacterial iron efflux pumps function as virulence factors in several systems. The requirement for iron efflux in pathogens such as Listeria monocytogenes, Streptococcus pyogenes, and Mycobacterium tuberculosis suggests that both import and efflux are needed for cells to successfully navigate rapidly changing levels of iron availability in the host. Here, we provide insight into how iron efflux genes are controlled, an aspect of bacterial iron homeostasis relevant to infectious disease processes.

Aspartate deficiency limits peptidoglycan synthesis and sensitizes cells to antibiotics targeting cell wall synthesis in Bacillus subtilis.

Peptidoglycan synthesis is an important target for antibiotics and relies on intermediates derived from central metabolism. As a result, alterations of metabolism may affect antibiotic sensitivity. An aspB mutant is auxotrophic for aspartate (Asp) and asparagine (Asn) and lyses when grown in Difco sporulation medium (DSM), but not in LB medium. Genetic and physiological studies, supported by amino acid analysis, reveal that cell lysis in DSM results from Asp limitation due to a relatively low Asp and high glutamate (Glu) concentrations, with Glu functioning as a competitive inhibitor of Asp uptake by the major Glu/Asp transporter GltT. Lysis can be specifically suppressed by supplementation with 2,6-diaminopimelate (DAP), which is imported by two different cystine uptake systems. These studies suggest that aspartate limitation depletes the peptidoglycan precursor meso-2,6-diaminopimelate (mDAP), inhibits peptidoglycan synthesis, upregulates the cell envelope stress response mediated by σM and eventually leads to cell lysis. Aspartate limitation sensitizes cells to antibiotics targeting late steps of PG synthesis, but not steps prior to the addition of mDAP into the pentapeptide sidechain. This work highlights the ability of perturbations of central metabolism to sensitize cells to peptidoglycan synthesis inhibitors.

The Role of Bacillithiol in Gram-Positive Firmicutes.

SIGNIFICANCE: Since the discovery and structural characterization of bacillithiol (BSH), the biochemical functions of BSH-biosynthesis enzymes (BshA/B/C) and BSH-dependent detoxification enzymes (FosB, Bst, GlxA/B) have been explored in Bacillus and Staphylococcus species. It was shown that BSH plays an important role in detoxification of reactive oxygen and electrophilic species, alkylating agents, toxins, and antibiotics. Recent Advances: More recently, new functions of BSH were discovered in metal homeostasis (Zn buffering, Fe-sulfur cluster, and copper homeostasis) and virulence control in Staphylococcus aureus. Unexpectedly, strains of the S. aureus NCTC8325 lineage were identified as natural BSH-deficient mutants. Modern mass spectrometry-based approaches have revealed the global reach of protein S-bacillithiolation in Firmicutes as an important regulatory redox modification under hypochlorite stress. S-bacillithiolation of OhrR, MetE, and glyceraldehyde-3-phosphate dehydrogenase (Gap) functions, analogous to S-glutathionylation, as both a redox-regulatory device and in thiol protection under oxidative stress. CRITICAL ISSUES: Although the functions of the bacilliredoxin (Brx) pathways in the reversal of S-bacillithiolations have been recently addressed, significantly more work is needed to establish the complete Brx reduction pathway, including the major enzyme(s), for reduction of oxidized BSH (BSSB) and the targets of Brx action in vivo. FUTURE DIRECTIONS: Despite the large number of identified S-bacillithiolated proteins, the physiological relevance of this redox modification was shown for only selected targets and should be a subject of future studies. In addition, many more BSH-dependent detoxification enzymes are evident from previous studies, although their roles and biochemical mechanisms require further study. This review of BSH research also pin-points these missing gaps for future research. Antioxid. Redox Signal. 28, 445-462.

Sequential induction of Fur-regulated genes in response to iron limitation in Bacillus subtilis.

Bacterial cells modulate transcription in response to changes in iron availability. The ferric uptake regulator (Fur) senses intracellular iron availability and plays a central role in maintaining iron homeostasis in Bacillus subtilis Here we utilized FrvA, a high-affinity Fe2+ efflux transporter from Listeria monocytogenes, as an inducible genetic tool to deplete intracellular iron. We then characterized the responses of the Fur, FsrA, and PerR regulons as cells transition from iron sufficiency to deficiency. Our results indicate that the Fur regulon is derepressed in three distinct waves. First, uptake systems for elemental iron (efeUOB), ferric citrate (fecCDEF), and petrobactin (fpbNOPQ) are induced to prevent iron deficiency. Second, B. subtilis synthesizes its own siderophore bacillibactin (dhbACEBF) and turns on bacillibactin (feuABC) and hydroxamate siderophore (fhuBCGD) uptake systems to scavenge iron from the environment and flavodoxins (ykuNOP) to replace ferredoxins. Third, as iron levels decline further, an "iron-sparing" response (fsrA, fbpAB, and fbpC) is induced to block the translation of abundant iron-utilizing proteins and thereby permit the most essential iron-dependent enzymes access to the limited iron pools. ChIP experiments demonstrate that in vivo occupancy of Fur correlates with derepression of each operon, and the graded response observed here results, at least in part, from higher-affinity binding of Fur to the "late"-induced genes.

Ferrous iron efflux systems in bacteria.

Bacteria require iron for growth, with only a few reported exceptions. In many environments, iron is a limiting nutrient for growth and high affinity uptake systems play a central role in iron homeostasis. However, iron can also be detrimental to cells when it is present in excess, particularly under aerobic conditions where its participation in Fenton chemistry generates highly reactive hydroxyl radicals. Recent results have revealed a critical role for iron efflux transporters in protecting bacteria from iron intoxication. Systems that efflux iron are widely distributed amongst bacteria and fall into several categories: P1B-type ATPases, cation diffusion facilitator (CDF) proteins, major facilitator superfamily (MFS) proteins, and membrane bound ferritin-like proteins. Here, we review the emerging role of iron export in both iron homeostasis and as part of the adaptive response to oxidative stress.

Iron Efflux by PmtA Is Critical for Oxidative Stress Resistance and Contributes Significantly to Group A Streptococcus Virulence.

Group A Streptococcus (GAS) is a human-only pathogen that causes a spectrum of disease conditions. Given its survival in inflamed lesions, the ability to sense and overcome oxidative stress is critical for GAS pathogenesis. PerR senses oxidative stress and coordinates the regulation of genes involved in GAS antioxidant defenses. In this study, we investigated the role of PerR-controlled metal transporter A (PmtA) in GAS pathogenesis. Previously, PmtA was implicated in GAS antioxidant defenses and suggested to protect against zinc toxicity. Here, we report that PmtA is a P1B4-type ATPase that functions as an Fe(II) exporter and aids GAS defenses against iron intoxication and oxidative stress. The expression of pmtA is specifically induced by excess iron, and this induction requires PerR. Furthermore, a pmtA mutant exhibited increased sensitivity to iron toxicity and oxidative stress due to an elevated intracellular accumulation of iron. RNA-sequencing analysis revealed that GAS undergoes significant alterations in gene expression to adapt to iron toxicity. Finally, using two mouse models of invasive infection, we demonstrated that iron efflux by PmtA is critical for bacterial survival during infection and GAS virulence. Together, these data demonstrate that PmtA is a key component of GAS antioxidant defenses and contributes significantly to GAS virulence.

The Listeria monocytogenes Fur-regulated virulence protein FrvA is an Fe(II) efflux P1B4 -type ATPase.

Listeria monocytogenes FrvA (Lmo0641) is critical for virulence in the mouse model and is an ortholog of the Bacillus subtilis Fur- and PerR-regulated Fe(II) efflux P1B4 -type ATPase PfeT. Previously, FrvA was suggested to protect against heme toxicity. Here, we demonstrate that an frvA mutant is sensitive to iron intoxication, but not to other metals. Expression of frvA is induced by high iron and this induction requires Fur. FrvA functions in vitro as a divalent cation specific ATPase most strongly activated by ferrous iron. When expressed in B. subtilis, FrvA increases resistance to iron both in wild-type and in a pfeT null strain. FrvA is a high affinity Fe(II) exporter and its induction imposes severe iron limitation in B. subtilis resulting in derepression of both Fur- and PerR-regulated genes. FrvA also recognizes Co(II) and Zn(II) as substrates and can complement B. subtilis strains defective in the endogenous export systems for these cations. Building on these results, we conclude that FrvA functions in the efflux of Fe(II), and not heme during listerial infection.

Metalloregulator CueR biases RNA polymerase's kinetic sampling of dead-end or open complex to repress or activate transcription.

Metalloregulators respond to metal ions to regulate transcription of metal homeostasis genes. MerR-family metalloregulators act on σ(70)-dependent suboptimal promoters and operate via a unique DNA distortion mechanism in which both the apo and holo forms of the regulators bind tightly to their operator sequence, distorting DNA structure and leading to transcription repression or activation, respectively. It remains unclear how these metalloregulator-DNA interactions are coupled dynamically to RNA polymerase (RNAP) interactions with DNA for transcription regulation. Using single-molecule FRET, we study how the copper efflux regulator (CueR)--a Cu(+)-responsive MerR-family metalloregulator--modulates RNAP interactions with CueR's cognate suboptimal promoter PcopA, and how RNAP affects CueR-PcopA interactions. We find that RNAP can form two noninterconverting complexes at PcopA in the absence of nucleotides: a dead-end complex and an open complex, constituting a branched interaction pathway that is distinct from the linear pathway prevalent for transcription initiation at optimal promoters. Capitalizing on this branched pathway, CueR operates via a "biased sampling" instead of "dynamic equilibrium shifting" mechanism in regulating transcription initiation; it modulates RNAP's binding-unbinding kinetics, without allowing interconversions between the dead-end and open complexes. Instead, the apo-repressor form reinforces the dominance of the dead-end complex to repress transcription, and the holo-activator form shifts the interactions toward the open complex to activate transcription. RNAP, in turn, locks CueR binding at PcopA into its specific binding mode, likely helping amplify the differences between apo- and holo-CueR in imposing DNA structural changes. Therefore, RNAP and CueR work synergistically in regulating transcription.

PfeT, a P1B4 -type ATPase, effluxes ferrous iron and protects Bacillus subtilis against iron intoxication.

Iron is an essential element for nearly all cells and limited iron availability often restricts growth. However, excess iron can also be deleterious, particularly when cells expressing high affinity iron uptake systems transition to iron rich environments. Bacillus subtilis expresses numerous iron importers, but iron efflux has not been reported. Here, we describe the B. subtilis PfeT protein (formerly YkvW/ZosA) as a P1B4 -type ATPase in the PerR regulon that serves as an Fe(II) efflux pump and protects cells against iron intoxication. Iron and manganese homeostasis in B. subtilis are closely intertwined: a pfeT mutant is iron sensitive, and this sensitivity can be suppressed by low levels of Mn(II). Conversely, a pfeT mutant is more resistant to Mn(II) overload. In vitro, the PfeT ATPase is activated by both Fe(II) and Co(II), although only Fe(II) efflux is physiologically relevant in wild-type cells, and null mutants accumulate elevated levels of intracellular iron. Genetic studies indicate that PfeT together with the ferric uptake repressor (Fur) cooperate to prevent iron intoxication, with iron sequestration by the MrgA mini-ferritin playing a secondary role. Protection against iron toxicity may also be a key role for related P1B4 -type ATPases previously implicated in bacterial pathogenesis.

Staphylococcus aureus PerR Is a Hypersensitive Hydrogen Peroxide Sensor using Iron-mediated Histidine Oxidation.

In many Gram-positive bacteria PerR is a major peroxide sensor whose repressor activity is dependent on a bound metal cofactor. The prototype for PerR sensors, the Bacillus subtilis PerRBS protein, represses target genes when bound to either Mn(2+) or Fe(2+) as corepressor, but only the Fe(2+)-bound form responds to H2O2. The orthologous protein in the human pathogen Staphylococcus aureus, PerRSA, plays important roles in H2O2 resistance and virulence. However, PerRSA is reported to only respond to Mn(2+) as corepressor, which suggests that it might rely on a distinct, iron-independent mechanism for H2O2 sensing. Here we demonstrate that PerRSA uses either Fe(2+) or Mn(2+) as corepressor, and that, like PerRBS, the Fe(2+)-bound form of PerRSA senses physiological levels of H2O2 by iron-mediated histidine oxidation. Moreover, we show that PerRSA is poised to sense very low levels of endogenous H2O2, which normally cannot be sensed by B. subtilis PerRBS. This hypersensitivity of PerRSA accounts for the apparent lack of Fe(2+)-dependent repressor activity and consequent Mn(2+)-specific repressor activity under aerobic conditions. We also provide evidence that the activity of PerRSA is directly correlated with virulence, whereas it is inversely correlated with H2O2 resistance, suggesting that PerRSA may be an attractive target for the control of S. aureus pathogenesis.

Mn(2+)-sensing mechanisms of yybP-ykoY orphan riboswitches.

Gene regulation in cis by riboswitches is prevalent in bacteria. The yybP-ykoY riboswitch family is quite widespread, yet its ligand and function remained unknown. Here, we characterize the Lactococcus lactis yybP-ykoY orphan riboswitch as a Mn(2+)-dependent transcription-ON riboswitch, with a ∼30-40 µM affinity for Mn(2+). We further determined its crystal structure at 2.7 Å to elucidate the metal sensing mechanism. The riboswitch resembles a hairpin, with two coaxially stacked helices tethered by a four-way junction and a tertiary docking interface. The Mn(2+)-sensing region, strategically located at the highly conserved docking interface, has two metal binding sites. Whereas one site tolerates the binding of either Mg(2+) or Mn(2+), the other site strongly prefers Mn(2+) due to a direct contact from the N7 of an invariable adenosine. Mutagenesis and a Mn(2+)-free E. coli yybP-ykoY structure further reveal that Mn(2+) binding is coupled with stabilization of the Mn(2+)-sensing region and the aptamer domain.

Bacillithiol is a major buffer of the labile zinc pool in Bacillus subtilis.

Intracellular zinc levels are tightly regulated since zinc is an essential cofactor for numerous enzymes, yet can be toxic when present in excess. The majority of intracellular zinc is tightly associated with proteins and is incorporated during synthesis from a poorly defined pool of kinetically labile zinc. In Bacillus subtilis, this labile pool is sensed by equilibration with the metalloregulator Zur, as an indication of zinc sufficiency, and by CzrA, as an indication of zinc excess. Here, we demonstrate that the low-molecular-weight thiol bacillithiol (BSH) serves as a major buffer of the labile zinc pool. Upon shift to conditions of zinc excess, cells transiently accumulate zinc in a low-molecular-weight pool, and this accumulation is largely dependent on BSH. Cells lacking BSH are more sensitive to zinc stress, and they induce zinc efflux at lower external zinc concentrations. Thiol reactive agents such as diamide and cadmium induce zinc efflux by interfering with the Zn-buffering function of BSH. Our data provide new insights into intracellular zinc buffering and may have broad relevance given the presence of BSH in pathogens and the proposed role of zinc sequestration in innate immunity.

Specificity of metal sensing: iron and manganese homeostasis in Bacillus subtilis.

Metalloregulatory proteins allow cells to sense metal ions and appropriately adjust the expression of metal uptake, storage, and efflux pathways. Bacillus subtilis provides a model for the coordinate regulation of iron and manganese homeostasis that involves three key regulators: Fur senses iron sufficiency, MntR senses manganese sufficiency, and PerR senses the intracellular Fe/Mn ratio. Here, I review the structural and physiological bases of selective metal perception, the effects of non-cognate metals, and mechanisms that may serve to coordinate iron and manganese homeostasis.

Redox regulation in Bacillus subtilis: The bacilliredoxins BrxA(YphP) and BrxB(YqiW) function in de-bacillithiolation of S-bacillithiolated OhrR and MetE.

AIMS: In bacillithiol (BSH)-utilizing organisms, protein S-bacillithiolation functions as a redox switch in response to oxidative stress and protects critical Cys residues against overoxidation. In Bacillus subtilis, both the redox-sensing repressor OhrR and the methionine synthase MetE are redox controlled by S-bacillithiolation in vivo. Here, we identify pathways of protein de-bacillithiolation and test the hypothesis that YphP(BrxA) and YqiW(BrxB) act as bacilliredoxins (Brx) to remove BSH from OhrR and MetE mixed disulfides. RESULTS: We present evidence that the BrxA and BrxB paralogs have de-bacillithiolation activity. This Brx activity results from attack of the amino-terminal Cys residue in a CGC motif on protein BSH-mixed disulfides. B. subtilis OhrR DNA-binding activity is eliminated by S-thiolation on its sole Cys residue. Both the BrxA and BrxB bacilliredoxins mediate de-bacillithiolation of OhrR accompanied by the transfer of BSH to the amino-terminal cysteine of their CGC active site motif. In vitro studies demonstrate that BrxB can restore DNA-binding activity to OhrR which is S-bacillithiolated, but not to OhrR that is S-cysteinylated. MetE is most strongly S-bacillithiolated at Cys719 in vitro and can be efficiently de-bacillithiolated by both BrxA and BrxB. INNOVATION AND CONCLUSION: We demonstrate that BrxA and BrxB function in the reduction of BSH mixed protein disulfides with two natural substrates (MetE, OhrR). These results provide biochemical evidence for a new class of bacterial redox-regulatory proteins, the bacilliredoxins, which function analogously to glutaredoxins. Bacilliredoxins function in concert with other thiol-disulfide oxidoreductases to maintain redox homeostasis in response to disulfide stress conditions.

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

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.

Regulation of Bacillus subtilis bacillithiol biosynthesis operons by Spx.

Bacillithiol is the major low molecular mass thiol produced by many firmicutes bacteria, including the model organism Bacillus subtilis and pathogens such as Bacillus anthracis and Staphylococcus aureus. We have previously shown that four genes (bshA, bshB1, bshB2 and bshC) are involved in bacillithiol biosynthesis. Here, we report that these four genes are encoded within three, unlinked operons all expressed from canonical σ(A)-dependent promoters as determined by 5'RACE (rapid amplification of cDNA ends). The bshA and bshB1 genes are embedded within a seven-gene operon additionally including mgsA, encoding methylglyoxal synthase, and the essential genes cca and birA, encoding tRNA nucleotidyltransferase (CCA transferase) and biotin-protein ligase, respectively. The bshB2 gene is co-transcribed with unknown function genes, while bshC is expressed both as part of a two-gene operon (with the upstream putative pantothenate biosynthesis gene ylbQ) and from its own promoter. All three operons are expressed at a reduced level in an spx null mutant, consistent with a direct role of Spx as a transcriptional activator for these operons, and all three operons are induced by the thiol oxidant diamide. In contrast with other Spx-regulated genes characterized to date, the effects of Spx on basal expression and diamide-stimulated expression appear to be independent of Cys10 in the redox centre of Spx. Consistent with the role of Spx as an activator of bacillithiol biosynthetic genes, cellular levels of bacillithiol are reduced several-fold in an spx null mutant.