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J D Helmann

Publications and source records attributed to J D Helmann.

At least 19 recordsLinked to original sources
PubMed2001

Roles of metal ions and hydrogen peroxide in modulating the interaction of the Bacillus subtilis PerR peroxide regulon repressor with operator DNA.

The inducible response to H(2)O(2) stress in Bacillus subtilis is under the control of PerR, one of three Fur homologues in this organism. PerR was purified in both an inactive, metal-dependent form and an active, metal-containing form as determined using DNA-binding assays. Active PerR contains both zinc and iron and is designated PerR:Zn,Fe. Added manganous ion competes for binding to the iron site and can restore DNA-binding activity to the metal-dependent form of PerR, presumably generating PerR:Zn,Mn. The DNA-binding activity of PerR:Zn,Fe is eliminated by exposure to H(2)O(2) whereas PerR:Zn,Mn is comparatively resistant. DNA-binding activity can be restored by a thiol-reducing agent, suggesting that redox-active cysteines are involved in peroxide sensing. Experiments using reporter fusions demonstrate that elevated levels of manganese repress PerR regulon genes and prevent their full induction by H(2)O(2). In contrast, in cells grown with iron supplementation, a PerR-repressed gene is completely derepressed by H(2)O(2). These results are consistent with the idea that the intracellular form of the PerR metalloprotein, and therefore its hydrogen peroxide sensitivity, can be altered by growth conditions.

Bacillus subtilis↗
PubMed2001

OhrR is a repressor of ohrA, a key organic hydroperoxide resistance determinant in Bacillus subtilis.

Bacillus subtilis displays a complex adaptive response to the presence of reactive oxygen species. To date, most proteins that protect against reactive oxygen species are members of the peroxide-inducible PerR and sigma(B) regulons. We investigated the function of two B. subtilis homologs of the Xanthomonas campestris organic hydroperoxide resistance (ohr) gene. Mutational analyses indicate that both ohrA and ohrB contribute to organic peroxide resistance in B. subtilis, with the OhrA protein playing the more important role in growing cells. Expression of ohrA, but not ohrB, is strongly and specifically induced by organic peroxides. Regulation of ohrA requires the convergently transcribed gene, ohrR, which encodes a member of the MarR family of transcriptional repressors. In an ohrR mutant, ohrA expression is constitutive, whereas expression of the neighboring ohrB gene is unaffected. Selection for mutant strains that are derepressed for ohrA transcription identifies a perfect inverted repeat sequence that is required for OhrR-mediated regulation and likely defines an OhrR binding site. Thus, B. subtilis contains at least three regulons (sigma(B), PerR, and OhrR) that contribute to peroxide stress responses.

Amino Acid Sequence↗
PubMed2001

Global transcriptional response of Bacillus subtilis to heat shock.

In response to heat stress, Bacillus subtilis activates the transcription of well over 100 different genes. Many of these genes are members of a general stress response regulon controlled by the secondary sigma factor, sigma(B), while others are under control of the HrcA or CtsR heat shock regulators. We have used DNA microarrays to monitor the global transcriptional response to heat shock. We find strong induction of known sigma(B)-dependent genes with a characteristic rapid induction followed by a return to near prestimulus levels. The HrcA and CtsR regulons are also induced, but with somewhat slower kinetics. Analysis of DNA sequences proximal to newly identified heat-induced genes leads us to propose ~70 additional members of the sigma(B) regulon. We have also identified numerous heat-induced genes that are not members of known heat shock regulons. Notably, we observe very strong induction of arginine biosynthesis and transport operons. Induction of several genes was confirmed by quantitative reverse transcriptase PCR. In addition, the transcriptional responses measured by microarray hybridization compare favorably with the numerous previous studies of heat shock in this organism. Since many different conditions elicit both specific and general stress responses, knowledge of the heat-induced general stress response reported here will be helpful for interpreting future microarray studies of other stress responses.

Arginine↗
PubMed2001

The -10 region is a key promoter specificity determinant for the Bacillus subtilis extracytoplasmic-function sigma factors sigma(X) and sigma(W).

Transcriptional selectivity derives, in large part, from the sequence-specific DNA-binding properties of the sigma subunit of RNA polymerase. There are 17 sigma factors in Bacillus subtilis which, in general, recognize distinct sets of promoters. However, some sigma factors have overlapping promoter selectivity. We hypothesize that the overlap between the regulons activated by the sigma(X) and sigma(W) factors can be explained by overlapping specificity for the -10 region: sigma(X) recognizes -10 elements with the sequence CGAC and sigma(W) recognizes CGTA, while both can potentially recognize CGTC. To test this model, we mutated the sigma(X)-specific autoregulatory site (P(X)), containing the -10 element CGAC, to either CGTC or GCTA. Conversely, the sigma(W) autoregulatory site (P(W)) was altered from CGTA to CGTC or CGAC. Transcriptional analyses, both in vitro and in vivo, indicate that changes to the -10 element are sufficient to switch a promoter from the sigma(X) to the sigma(W) regulon or, conversely, from the sigma(W) to the sigma(X) regulon, but context effects clearly play an important role in determining promoter strength. It seems likely that these subtle differences in promoter selectivity derive from amino acid differences in conserved region 2 of sigma, which contacts the -10 element. However, we were unable to alter promoter selectivity by replacements of two candidate recognition residues in sigma(W).

Amino Acid Sequence↗
PubMed2001

FosB, a cysteine-dependent fosfomycin resistance protein under the control of sigma(W), an extracytoplasmic-function sigma factor in Bacillus subtilis.

We demonstrate that the Bacillus subtilis fosB(yndN) gene encodes a fosfomycin resistance protein. Expression of fosB requires sigma(W), and both fosB and sigW mutants are fosfomycin sensitive. FosB is a metallothiol transferase related to the FosA class of Mn(2+)-dependent glutathione transferases but with a preference for Mg(2+) and L-cysteine as cofactors.

Amino Acid Sequence↗
PubMed2000

Identification of the Escherichia coli K-12 Nramp orthologue (MntH) as a selective divalent metal ion transporter.

The Escherichia coli mntH (formerly yfeP) gene encodes a putative membrane protein (MntH) highly similar to members of the eukaryotic Nramp family of divalent metal ion transporters. To determine the function of E. coli MntH, a null mutant was created and MntH was overexpressed both in wild-type E. coli and in the metal-dependent mutant hflB1(Ts). At the restrictive temperature 42 degrees C, the mntH null mutation reduces the suppression of hflB1(Ts) thermosensitivity by exogenous divalent metals. Conversely, overexpression of MntH restores growth at 42 degrees C, increases suppression of the ts phenotype by Fe(II) and Ni(II) and renders hflB1(Ts) cells hypersensitive to Mn(II). Transport studies in intact cells show that MntH selectively facilitates uptake of 54Mn(II) and 55Fe(II) in a temperature-, time- and proton-dependent manner. Competition studies in uptake assays and growth inhibition experiments in hflB1(Ts) mutants together indicate that MntH is a divalent metal cation transporter of broad substrate specificity. The functional characteristics of MntH suggest that it corresponds to the previously described manganese transporter of E. coli. This study indicates that proton-dependent divalent metal ion uptake has been preserved in the Nramp family from bacteria to humans.

Base Sequence↗
PubMed2000

Manganese homeostasis in Bacillus subtilis is regulated by MntR, a bifunctional regulator related to the diphtheria toxin repressor family of proteins.

The Bacillus subtilis yqhN gene encodes a metalloregulatory protein distantly related to the Corynebacterium diphtheriae diphtheria toxin repressor (DtxR). While DtxR mediates the iron-dependent repression of iron uptake, we demonstrate that yqhN (herein renamed mntR) encodes a manganese modulated regulator of manganese transport. An mntR mutant strain is sensitive to both manganese and cadmium, suggesting that the transport of these metals is derepressed. We selected Tn10 insertions that suppress the Mn(II) sensitivity of the mntR mutant or that increase the Cd(II) tolerance of wild-type cells, and in both cases we recovered insertions in mntH (formerly ydaR). MntH is a member of the NRAMP family of proton-coupled, metal ion transporters. MntR also regulates expression of a Mn(II) ABC transporter (MntABCD). The MntH and MntABCD transporters are both selectively repressed by Mn(II) and this regulation requires MntR. In high Mn(II) conditions, MntR functions as a Mn(II)-dependent repressor of mntH transcription. In contrast, MntR acts as a positive regulator of the mntABCD operon under low Mn(II) growth conditions. Biochemical studies demonstrate that MntR binding to the mntH control region requires Mn(II), while interaction with the mntABCD control region does not depend on Mn(II).

ATP-Binding Cassette Transporters↗
PubMed2000

Mutations in multidrug efflux homologs, sugar isomerases, and antimicrobial biosynthesis genes differentially elevate activity of the sigma(X) and sigma(W) factors in Bacillus subtilis.

The sigma(X) and sigma(W) extracytoplasmic function sigma factors regulate more than 40 genes in Bacillus subtilis. sigma(W) activates genes which function in detoxification and the production of antimicrobial compounds, while sigma(X) activates functions that modify the cell envelope. Transposon mutagenesis was used to identify loci which negatively regulate sigma(W) or sigma(X) as judged by up-regulation from the autoregulatory promoter site P(W) or P(X). Fourteen insertions that activate P(W) were identified. The largest class of insertions are likely to affect transport. These include insertions in genes encoding two multidrug efflux protein homologs (yqgE and yulE), a component of the oligopeptide uptake system (oppA), and two transmembrane proteins with weak similarity to transporters (yhdP and yueF). Expression from P(W) is also elevated as a result of inactivation of at least one member of the sigma(W) regulon (ysdB), an ArsR homolog (yvbA), a predicted rhamnose isomerase (yulE), and a gene (pksR) implicated in synthesis of difficidin, a polyketide antibiotic. In a parallel screen, we identified seven insertions that up-regulate P(X). Remarkably, these insertions were in functionally similar genes, including a multidrug efflux homolog (yitG), a mannose-6-phosphate isomerase gene (yjdE), and loci involved in antibiotic synthesis (srfAB and possibly yogA and yngK). Significantly, most insertions that activate P(W) have little or no effect on P(X), and conversely, insertions that activate P(X) have no effect on P(W). This suggests that these two regulons respond to distinct sets of molecular signals which may include toxic molecules which are exported, cell density signals, and antimicrobial compounds.

Anti-Bacterial Agents↗
PubMed1999

Adenines at -11, -9 and -8 play a key role in the binding of Bacillus subtilis Esigma(A) RNA polymerase to -10 region single-stranded DNA.

The sigma subunit of RNA polymerase interacts with the promoter DNA in at least two regions: the -35 and the -10 consensus elements. The latter contacts are involved both in recognition and in melting of the promoter DNA to form the transcriptionally-competent open complex. RNA polymerase holoenzyme, but neither core nor sigma alone, binds with high selectivity to single-stranded DNA (ssDNA) containing the non-template -10 consensus sequence. We have used equilibrium competition to assess the specificity of holoenzyme binding to a 19 base oligonucleotide containing a -10 consensus element, TATAAT. Analysis of all 18 possible single point mutations in the -10 consensus sequence reveals that binding by Bacillus subtilis Esigma(A)holoenzyme depends critically upon adenine at position -11 and, unexpectedly, is strongly affected by substitutions of the poorly conserved adenines at -9 and -8. Similarly, ssDNA binding by Escherichia coli Esigma(70)holoenzyme is most strongly affected by substitutions of adenines within the -10 region consensus. The critical role of -11A in binding ssDNA supports a key role for this base in the nucleation of DNA melting. A novel role for -9A and -8A is proposed in the context of recent models of promoter melting.

Adenine↗
PubMed1999

Expression, abundance, and RNA polymerase binding properties of the delta factor of Bacillus subtilis.

The delta protein is a dispensable subunit of Bacillus subtilis RNA polymerase (RNAP) that has major effects on the biochemical properties of the purified enzyme. In the presence of delta, RNAP displays an increased specificity of transcription, a decreased affinity for nucleic acids, and an increased efficiency of RNA synthesis because of enhanced recycling. Despite these profound effects, a strain containing a deletion of the delta gene (rpoE) is viable and shows no major alterations in gene expression. Quantitative immunoblotting experiments demonstrate that delta is present in molar excess relative to RNAP in both vegetative cells and spores. Expression of rpoE initiates from a single, sigmaA-dependent promoter and is maximal in transition phase. A rpoE mutant strain has an altered morphology and is delayed in the exit from stationary phase. For biochemical analyses we have created derivatives of delta and sigmaA that can be radiolabeled with protein kinase A. Using electrophoretic mobility shift assays, we demonstrate that delta binds core RNAP with an apparent affinity of 2.5 x 10(6) M-1, but we are unable to demonstrate the formation of a ternary complex containing core enzyme, delta, and sigmaA.

Bacillus subtilis↗
PubMed1999

Anti-sigma factors.

Anti-sigma factors modulate the expression of numerous regulons controlled by alternative sigma factors. Anti-sigma factors are themselves regulated by either secretion from the cell (i.e. FlgM export through the hook-basal body), sequestration by an anti-anti-sigma (i.e. phosphorylation regulated partner-switching modules), or interaction with extracytoplasmic proteins or small molecule effectors (i.e. transmembrane regulators of extracytoplasmic function sigma factors). Recent highlights include the genetic description of the opposed sigma/anti-sigma binding surfaces; the unexpected role of FlgM in holoenzyme destabilization and the finding that folding of FlgM is coupled to sigma28 binding; the first structure determination for an anti-sigma antagonist; and the detailed dissection of two complex partner-switching modules in Bacillus subtilis.

Bacterial Proteins↗
PubMed1999

Identification of target promoters for the Bacillus subtilis extracytoplasmic function sigma factor, sigma W.

The Bacillus subtilis sigW gene encodes an extracytoplasmic function (ECF) sigma factor that is expressed in early stationary phase from a sigW-dependent autoregulatory promoter, PW. Using a consensus-based search procedure, we have identified 15 operons preceded by promoters similar in sequence to PW. At least 14 of these promoters are dependent on sigma W both in vivo and in vitro as judged by lacZ reporter fusions, run-off transcription assays and nucleotide resolution start site mapping. We conclude that sigma W controls a regulon of more than 30 genes, many of which encode membrane proteins of unknown function. The sigma W regulon includes a penicillin binding protein (PBP4*) and a co-transcribed amino acid racemase (RacX), homologues of signal peptide peptidase (YteI), flotillin (YuaG), ABC transporters (YknXYZ), non-haem bromoperoxidase (YdjP), epoxide hydrolase (YfhM) and three small peptides with structural similarities to bacteriocin precursor polypeptides. We suggest that sigma W activates a large stationary-phase regulon that functions in detoxification, production of anti-microbial compounds or both.

Bacillus subtilis↗
PubMed1999

Interaction of Bacillus subtilis Fur (ferric uptake repressor) with the dhb operator in vitro and in vivo.

Bacillus subtilis contains three metalloregulatory proteins belonging to the ferric uptake repressor (Fur) family: Fur, Zur, and PerR. We have overproduced and purified Fur protein and analyzed its interaction with the operator region controlling the expression of the dihydroxybenzoate siderophore biosynthesis (dhb) operon. The purified protein binds with high affinity and selectivity to the dhb regulatory region. DNA binding does not require added iron, nor is binding reduced by dialysis of Fur against EDTA or treatment with Chelex. Fur selectively inhibits transcription from the dhb promoter by sigmaA RNA polymerase, even if Fur is added after RNA polymerase holoenzyme. Since neither DNA binding nor inhibition of transcription requires the addition of ferrous ion in vitro, the mechanism by which iron regulates Fur function in vivo is not obvious. Mutagenesis of the fur gene reveals that in vivo repression of the dhb operon by iron requires His97, a residue thought to be involved in iron sensing in other Fur homologs. Moreover, we identify His96 as a second likely iron ligand, since a His96Ala mutant mediates repression at 50 microM but not at 5 microM iron. Our data lead us to suggest that Fur is able to bind DNA independently of bound iron and that the in vivo role of iron is to counteract the effect of an inhibitory factor, perhaps another metal ion, that antagonizes this DNA-binding activity.

Bacillus subtilis↗
PubMed1998

Identification of target promoters for the Bacillus subtilis sigma X factor using a consensus-directed search.

The promoter selectivity of RNA polymerase (RNAP) can be altered by the association with alternative sigma subunits. Bacillus subtilis hosts a multitude of sigma factors, several of which coordinate the complex developmental program culminating in endospore formation. Genome sequencing has revealed an unanticipated seven new sigma factors of the highly divergent extracytoplasmic function (ECF) sub-family. Virtually nothing is known regarding either the promoter selectivity or the target genes for these newly identified sigma factors. We have used saturation mutagenesis to define a promoter consensus for recognition by one such ECF sigma factor, sigma X. The resulting consensus sequence was used to identify candidate sigma X target sites. Three newly identified sigma X-dependent promoters precede genes encoding regulatory proteins: an AbrB homolog (Abh), a putative response regulator aspartate phosphatase (RapD), and a regulator of autolysin expression (LytR). sigma X also contributes to the expression of CsbB, a putative membrane-bound glucosyl transferase that is partially controlled by the sigma B stress response sigma factor. Since LytR modulates the expression of the major autolytic amidase and CsbB may function in peptidoglycan synthesis or modification, we suggest that sigma X participates in the regulation of peptidoglycan synthesis and turnover.

Bacillus subtilis↗
PubMed1998

Bacillus subtilis contains multiple Fur homologues: identification of the iron uptake (Fur) and peroxide regulon (PerR) repressors.

Fur (ferric uptake regulator) proteins control iron uptake in many Gram-negative bacteria. Although Fur homologues have been identified in Gram-positive bacteria, their roles in gene regulation are unknown. Genome sequencing has revealed three fur homologues in Bacillus subtilis: yqkL, yqfV and ygaG. We demonstrate that yqkL encodes an iron uptake repressor: both siderophore biosynthesis and transcription of ferri-siderophore uptake genes is constitutive in the yqkL mutant. Thus, yqkL encodes a repressor that is functionally as well as structurally related to Fur. B. subtilis peroxide stress genes are induced by either H2O2 or by metal ion limitation. Previous genetic studies defined a regulatory locus, perR, postulated to encode the peroxide regulon repressor. We demonstrate that a ygaG mutant has the perR phenotype: It is highly resistant to peroxides and overexpresses catalase, alkyl hydroperoxide reductase and the DNA binding protein MrgA. Nine spontaneous perR mutations, isolated by virtue of their ability to derepress mrgA transcription in the presence of managanous ion, all contain sequence changes in the ygaG locus and can be complemented by the cloned ygaG gene. Thus, ygaG encodes the peroxide regulon repressor and is allelic with perR.

Amino Acid Sequence↗
PubMed1998

Interactions between the promoter regions of nitrogenase structural genes (nifHDK2) and DNA-binding proteins from N2- and ammonium-grown cells of the archaeon Methanosarcina barkeri 227.

Transcription initiation in Archaea (archaebacteria) resembles the eucaryotic process, having been shown to involve TATA box-like promoter regions as well as TATA-binding protein and TFIIB homologs. However, little is known about transcription regulation in archaea. We have previously demonstrated that transcripts of nifHDK2 genes, encoding Methanosarcina barkeri nitrogenase, are present in N2-grown cells but not in ammonium-grown cells, indicating that nif transcription is regulated by the nitrogen source. In this study, we detected proteins in M. barkeri cell extracts that bind specifically to DNA containing the putative promoter region of nifHDK2. No binding was found when the promoter region was deleted from the DNA. A competition assay showed that the methyl coenzyme M reductase (mcr) promoter region DNA and the nifH2 promoter region DNA competed for a common factor(s). There was no binding to the nifH2 promoter region by extracts of ammonium-grown cells, but there was binding by these extracts to promoter regions for mcr genes, which are presumably constitutively expressed. Interestingly, extracts of ammonium-grown cells inhibited binding to the nif promoter region by extracts of N2-grown cells. Fractionation of extracts of ammonium-grown cells with a heparin-Sepharose column resolved them into a fraction eluting at 0 M NaCl, which inhibited binding by extracts of N2-grown cells, and a fraction eluting at 0.5 to 0.75 M NaCl, which showed binding to the promoter region. These results are congruent with a model for regulation of nif gene expression in M. barkeri in which a substance present in ammonium-grown cells inhibits DNA binding by a transcription-associated protein or proteins.

Archaeal Proteins↗
PubMed1998

Promoter recognition by Bacillus subtilis sigmaW: autoregulation and partial overlap with the sigmaX regulon.

The Bacillus subtilis genome encodes at least 17 distinct sigma factors, including seven members of the extracytoplasmic function (ECF) subfamily. We have investigated the expression and regulation of the ECF sigma factor encoded by the sigW gene. A sigmaW-dependent promoter (PW) precedes sigW, demonstrating that this transcription factor is positively autoregulated. Expression of sigW is regulated by both growth phase and medium composition. Maximal expression is attained in early-stationary-phase cells grown in rich medium. We previously reported that sigW mutants have elevated transcription of some sigmaX-controlled genes, and we now report that the converse is also true: in a sigX mutant, PW is derepressed during logarithmic growth. Thus, these two regulons are mutually antagonistic. Reconstituted sigmaW holoenzyme faithfully recognizes the PW preceding sigW but does not recognize the PX promoter preceding the sigX gene. Autoregulation of sigX is also highly specific: sigmaX holoenzyme initiates transcription from PX but recognizes PW poorly if at all. In contrast, several promoters that are at least partially under sigmaX control are active with both the sigmaX and sigmaW holoenzymes in vitro. This finding supports the suggestion that the sigmaW and sigmaX regulons overlap. Sequence comparisons suggest that promoters recognized by these two sigma factors have similar -35 elements but are distinguished by different base preferences at two key positions within the -10 element.

Bacillus↗