AhpA is a peroxidase expressed during biofilm formation in Bacillus subtilis.

Organisms growing aerobically generate reactive oxygen species such as hydrogen peroxide. These reactive oxygen molecules damage enzymes and DNA, potentially causing cell death. In response, Bacillus subtilis produces at least nine potential peroxide-scavenging enzymes; two belong to the alkylhydroperoxide reductase (Ahp) class of peroxidases. Here, we explore the role of one of these Ahp homologs, AhpA. While previous studies demonstrated that AhpA can scavenge peroxides and thus defend cells against peroxides, they did not clarify when during growth the cell produces AhpA. The results presented here show that the expression of ahpA is regulated in a manner distinct from that of the other peroxide-scavenging enzymes in B. subtilis. While the primary Ahp, AhpC, is expressed during exponential growth and stationary phase, these studies demonstrate that the expression of ahpA is dependent on the transition-state regulator AbrB and the sporulation and biofilm formation transcription factor Spo0A. Furthermore, these results show that ahpA is specifically expressed during biofilm formation, and not during sporulation or stationary phase, suggesting that derepression of ahpA by AbrB requires a signal other than those present upon entry into stationary phase. Despite this expression pattern, ahpA mutant strains still form and maintain robust biofilms, even in the presence of peroxides. Thus, the role of AhpA with regard to protecting cells within biofilms from environmental stresses is still uncertain. These studies highlight the need to further study the Ahp homologs to better understand how they differ from one another and the unique roles they may play in oxidative stress resistance.

Insights into the Function of a Second, Nonclassical Ahp Peroxidase, AhpA, in Oxidative Stress Resistance in Bacillus subtilis.

UNLABELLED: Organisms growing aerobically generate reactive oxygen-containing molecules, such as hydrogen peroxide (H2O2). These reactive oxygen molecules damage enzymes and DNA and may even cause cell death. In response, Bacillus subtilis produces at least nine potential peroxide-scavenging enzymes, two of which appear to be the primary enzymes responsible for detoxifying peroxides during vegetative growth: a catalase (encoded by katA) and an alkylhydroperoxide reductase (Ahp, encoded by ahpC). AhpC uses two redox-active cysteine residues to reduce peroxides to nontoxic molecules. A specialized thioredoxin-like protein, AhpF, is then required to restore oxidized AhpC back to its reduced state. Curiously, B. subtilis has two genes encoding Ahp: ahpC and ahpA. Although AhpC is well characterized, very little is known about AhpA. In fact, numerous bacterial species have multiple ahp genes; however, these additional Ahp proteins are generally uncharacterized. We seek to understand the role of AhpA in the bacterium's defense against toxic peroxide molecules in relation to the roles previously assigned to AhpC and catalase. Our results demonstrate that AhpA has catalytic activity similar to that of the primary enzyme, AhpC. Furthermore, our results suggest that a unique thioredoxin redox protein, AhpT, may reduce AhpA upon its oxidation by peroxides. However, unlike AhpC, which is expressed well during vegetative growth, our results suggest that AhpA is expressed primarily during postexponential growth. IMPORTANCE: B. subtilis appears to produce nine enzymes designed to protect cells against peroxides; two belong to the Ahp class of peroxidases. These studies provide an initial characterization of one of these Ahp homologs and demonstrate that the two Ahp enzymes are not simply replicates of each other, suggesting that they instead are expressed at different times during growth of the cells. These results highlight the need to further study the Ahp homologs to better understand how they differ from one another and to identify their function, if any, in protection against oxidative stress. Through these studies, we may better understand why bacteria have multiple enzymes designed to scavenge peroxides and thus have a more accurate understanding of oxidative stress resistance.

Origins of specificity and cross-talk in metal ion sensing by Bacillus subtilis Fur.

Fur (ferric uptake regulator) is the master regulator of iron homeostasis in many bacteria, but how it responds specifically to Fe(II) in vivo is not clear. Biochemical analyses of Bacillus subtilis Fur (BsFur) reveal that in addition to Fe(II), both Zn(II) and Mn(II) allosterically activate BsFur-DNA binding. Dimeric BsFur co-purifies with site 1 structural Zn(II) (Fur(2) Zn(2) ) and can bind four additional Zn(II) or Mn(II) ions per dimer. Metal ion binding at previously described site 3 occurs with highest affinity, but the Fur(2) Zn(2) :Me(2) form has only a modest increase in DNA binding affinity (approximately sevenfold). Metallation of site 2 (Fur(2) Zn(2) :Me(4) ) leads to a ~ 150-fold further enhancement in DNA binding affinity. Fe(II) binding studies indicate that BsFur buffers the intracellular Fe(II) concentration at ~ 1 µM. Both Mn(II) and Zn(II) are normally buffered at levels insufficient for metallation of BsFur site 2, thereby accounting for the lack of cross-talk observed in vivo. However, in a perR mutant, where the BsFur concentration is elevated, BsFur may now use Mn(II) as a co-repressor and inappropriately repress iron uptake. Since PerR repression of fur is enhanced by Mn(II), and antagonized by Fe(II), PerR may co-regulate Fe(II) homeostasis by modulating BsFur levels in response to the Mn(II)/Fe(II) ratio.

Rho/ROCK-dependent inhibition of 3T3-L1 adipogenesis by G-protein-deamidating dermonecrotic toxins: differential regulation of Notch1, Pref1/Dlk1, and ß-catenin signaling.

The dermonecrotic toxins from Pasteurella multocida (PMT), Bordetella (DNT), Escherichia coli (CNF1-3), and Yersinia (CNFY) modulate their G-protein targets through deamidation and/or transglutamination of an active site Gln residue, which results in activation of the G protein and its cognate downstream signaling pathways. Whereas DNT and the CNFs act on small Rho GTPases, PMT acts on the α subunit of heterotrimeric G(q), G(i), and G(12/13) proteins. We previously demonstrated that PMT potently blocks adipogenesis and adipocyte differentiation in a calcineurin-independent manner through downregulation of Notch1 and stabilization of ß-catenin and Pref1/Dlk1, key proteins in signaling pathways strongly linked to cell fate decisions, including fat and bone development. Here, we report that similar to PMT, DNT, and CNF1 completely block adipogenesis and adipocyte differentiation by preventing upregulation of adipocyte markers, PPARγ and C/EBPα, while stabilizing the expression of Pref1/Dlk1 and ß-catenin. We show that the Rho/ROCK inhibitor Y-27632 prevented or reversed these toxin-mediated effects, strongly supporting a role for Rho/ROCK signaling in dermonecrotic toxin-mediated inhibition of adipogenesis and adipocyte differentiation. Toxin treatment was also accompanied by downregulation of Notch1 expression, although this inhibition was independent of Rho/ROCK signaling. We further show that PMT-mediated downregulation of Notch1 expression occurs primarily through G(12/13) signaling. Our results reveal new details of the pathways involved in dermonecrotic toxin action on adipocyte differentiation, and the role of Rho/ROCK signaling in mediating toxin effects on Wnt/ß-catenin and Notch1 signaling, and in particular the role of G(q) and G(12/13) in mediating PMT effects on Rho/ROCK and Notch1 signaling.

Derepression of the Bacillus subtilis PerR peroxide stress response leads to iron deficiency.

The Bacillus subtilis PerR repressor regulates the adaptive response to peroxide stress. The PerR regulon includes the major vegetative catalase (katA), an iron storage protein (mrgA), an alkylhydroperoxide reductase (ahpCF), a zinc uptake system (zosA), heme biosynthesis enzymes (hemAXCDBL), the iron uptake repressor (fur), and perR itself. A perR null strain is resistant to hydrogen peroxide, accumulates a porphyrin-like compound, and grows very slowly. The poor growth of the perR mutant can be largely accounted for by the elevated expression of two proteins: the KatA catalase and Fur. Genetic studies support a model in which poor growth of the perR null mutant is due to elevated repression of iron uptake by Fur, exacerbated by heme sequestration by the abundant catalase protein. Analysis of the altered-function allele perR991 further supports a link between PerR and iron homeostasis. Strains containing perR991 are peroxide resistant but grow nearly as well as the wild type. Unlike a perR null allele, the perR991 allele (F51S) derepresses KatA, but not Fur, which likely accounts for its comparatively rapid growth.

Membrane interaction of Pasteurella multocida toxin involves sphingomyelin.

Pasteurella multocida toxin (PMT) is an AB toxin that causes pleiotropic effects in targeted host cells. The N-terminus of PMT (PMT-N) is considered to harbor the membrane receptor binding and translocation domains responsible for mediating cellular entry and delivery of the C-terminal catalytic domain into the host cytosol. Previous studies have implicated gangliosides as the host receptors for PMT binding. To gain further insight into the binding interactions involved in PMT binding to cell membranes, we explored the role of various membrane components in PMT binding, utilizing four different approaches: (a) TLC-overlay binding experiments with (125) I-labeled PMT, PMT-N or the C-terminus of PMT; (b) pull-down experiments using reconstituted membrane liposomes with full-length PMT; (c) surface plasmon resonance analysis of PMT-N binding to reconstituted membrane liposomes; (d) and surface plasmon resonance analysis of PMT-N binding to HEK-293T cell membranes without or with sphingomyelinase, phospholipase D or trypsin treatment. The results obtained revealed that, in our experimental system, full-length PMT and PMT-N did not bind to gangliosides, including monoasialogangliosides GM(1) , GM(2) or GM(3) , but instead bound to membrane phospholipids, primarily the abundant sphingophospholipid sphingomyelin or phosphatidylcholine with other lipid components. Collectively, these studies demonstrate the importance of sphingomyelin for PMT binding to membranes and suggest the involvement of a protein co-receptor.

Peroxide stress elicits adaptive changes in bacterial metal ion homeostasis.

Exposure to hydrogen peroxide (H(2)O(2)) and other reactive oxygen species is a universal feature of life in an aerobic environment. Bacteria express enzymes to detoxify H(2)O(2) and to repair the resulting damage, and their synthesis is typically regulated by redox-sensing transcription factors. The best characterized bacterial peroxide-sensors are Escherichia coli OxyR and Bacillus subtilis PerR. Analysis of their regulons has revealed that, in addition to inducible detoxification enzymes, adaptation to H(2)O(2) is mediated by modifications of metal ion homeostasis. Analogous adaptations appear to be present in other bacteria as here reviewed for Deinococcus radiodurans, Neisseria gonorrhoeae, Streptococcus pyogenes, and Bradyrhizobium japonicum. As a general theme, peroxide stress elicits changes in cytosolic metal distribution with the net effect of reducing the damage caused by reactive ferrous iron. Iron levels are reduced by repression of uptake, sequestration in storage proteins, and incorporation into metalloenzymes. In addition, peroxide-inducible transporters elevate cytosolic levels of Mn(II) and/or Zn(II) that can displace ferrous iron from sensitive targets. Although bacteria differ significantly in the detailed mechanisms employed to modulate cytosolic metal levels, a high Mn:Fe ratio has emerged as one key correlate of reactive oxygen species resistance.

Functional plasticity of a peroxidase allows evolution of diverse disulfide-reducing pathways.

In Escherichia coli, the glutathione/glutaredoxin and thioredoxin pathways are essential for the reduction of cytoplasmic protein disulfide bonds, including those formed in the essential enzyme ribonucleotide reductase during its action on substrates. Double mutants lacking thioredoxin reductase (trxB) and glutathione reductase (gor) or glutathione biosynthesis (gshA) cannot grow. Growth of Deltagor DeltatrxB strains is restored by a mutant (ahpC*) of the peroxiredoxin AhpC, converting it to a disulfide reductase that generates reduced glutathione. Here, we show that ahpC* also restores growth to a DeltagshB DeltatrxB strain, which lacks glutathione and accumulates only its precursor gamma-glutamylcysteine (gamma-GC). It suppresses this strain by allowing accumulation of reduced gamma-GC, which can substitute for glutathione. Surprisingly, new ahpC suppressor mutations arose in a DeltagshA DeltatrxB strain lacking both glutathione and gamma-GC, a strain that ahpC* does not suppress. Some of these mutant AhpC proteins channel electrons into the disulfide-reducing pathways via either the thioredoxins or the glutaredoxins without, evidently, the intermediary of glutathione. Our results provide insights into the physiological functioning of the glutathione pathway and reveal surprising plasticity of a peroxidase because different mutant versions of AhpC can channel electrons into the disulfide-reducing pathways by at least four distinct routes. Despite the reductase activity of mutant AhpCs, these various suppressor strains exhibit an oxidizing cytoplasm and accumulate correctly folded disulfide-bonded proteins in their cytoplasm. Proteins most effectively oxidized vary between strains, potentially providing useful tools for expressing different disulfide-bonded proteins.

Mutant AhpC peroxiredoxins suppress thiol-disulfide redox deficiencies and acquire deglutathionylating activity.

The bacterial peroxiredoxin AhpC, a cysteine-dependent peroxidase, can be converted through a single amino acid insertion to a disulfide reductase, AhpC*, active in the glutathione and glutaredoxin pathway. Here we show that, whereas AhpC* is inactive as a peroxidase, other point mutants in AhpC can confer the in vivo disulfide reductase activity without abrogating peroxidase activity. Moreover, AhpC* and several point mutants tested in vitro exhibit an enhanced reductase activity toward mixed disulfides between glutathione and glutaredoxin (Grx-S-SG), consistent with the in vivo requirements for these components. Remarkably, this Grx-S-SG reductase activity relies not on the peroxidatic cysteine but rather on the resolving cysteine that plays only a secondary role in the peroxidase mechanism. Furthermore, putative conformational changes, which impart this unusual Grx-S-SG reductase activity, are transmissible across subunits. Thus, AhpC and potentially other peroxiredoxins in this widespread family can elaborate a new reductase function that alleviates disulfide stress.

In vivo requirement for glutaredoxins and thioredoxins in the reduction of the ribonucleotide reductases of Escherichia coli.

Escherichia coli expresses three types of ribonucleotide reductases (RNRs) that utilize the redox chemistry of cysteine to catalyze the reduction of ribonucleotides. Upon reduction, the cysteines form a disulfide bond and must be reduced. The authors present in vivo studies that shed light on the mechanism by which these enzymes are regenerated. The class Ia enzyme, NrdAB, can be reduced by either the thioredoxins 1 and 2 or by glutaredoxin 1. The class Ib enzyme, NrdEF, is reduced in vivo by a dedicated glutaredoxin-like protein, NrdH. Despite its similarities to glutaredoxins, this protein is itself reduced by thioredoxin reductase in vivo. However, in the absence of thioredoxin reductase and NrdH, glutaredoxin 1 can partially replace NrdH. Despite their similar structures, the NrdEF and NrdAB RNRs differ in their abilities to function under low oxygen conditions. With only traces of oxygen present, NrdAB can allow some growth in the absence of the anaerobic enzyme NrdDG. NrdEF cannot. Furthermore, in anaerobiosis, E. coli is dependent for growth on class III RNR, NrdDG, and on having at least one of the two reductive systems, thioredoxin reductase or glutathione reductase. These findings indicate a role for these enzymes either for NrdDG reactivation or some other essential anaerobic process.