Biomedical subjects

John D Helmann

Publications and source records attributed to John D Helmann.

At least 19 recordsLinked to original sources

PubMed2007

Functional specialization within the Fur family of metalloregulators.

The ferric uptake regulator (Fur) protein, as originally described in Escherichia coli, is an iron-sensing repressor that controls the expression of genes for siderophore biosynthesis and iron transport. Although Fur is commonly thought of as a metal-dependent repressor, Fur also activates the expression of many genes by either indirect or direct mechanisms. In the best studied model systems, Fur functions as a global regulator of iron homeostasis controlling both the induction of iron uptake functions (under iron limitation) and the expression of iron storage proteins and iron-utilizing enzymes (under iron sufficiency). We now appreciate that there is a tremendous diversity in metal selectivity and biological function within the Fur family which includes sensors of iron (Fur), zinc (Zur), manganese (Mur), and nickel (Nur). Despite numerous studies, the mechanism of metal ion sensing by Fur family proteins is still controversial. Other family members use metal catalyzed oxidation reactions to sense peroxide-stress (PerR) or the availability of heme (Irr).

Bacterial Proteins↗

PubMed2006

The conformations of the manganese transport regulator of Bacillus subtilis in its metal-free state.

The manganese transport regulator (MntR) from Bacillus subtilis binds cognate DNA sequences in response to elevated manganese concentrations. MntR functions as a homodimer that binds two manganese ions per subunit. Metal binding takes place at the interface of the two domains that comprise each MntR subunit: an N-terminal DNA-binding domain and a C-terminal dimerization domain. In order to elucidate the link between metal binding and activation, a crystallographic study of MntR in its metal-free state has been undertaken. Here we describe the structures of the native protein and a selenomethionine-containing variant, solved to 2.8 A. The two structures contain five crystallographically unique subunits of MntR, providing diverse views of the metal-free protein. In apo-MntR, as in the manganese complex, the dimer is formed by dyad-related C-terminal domains that provide a conserved structural core. Similarly, each DNA-binding domain largely retains the folded conformation found in metal bound forms of MntR. However, compared to metal-activated MntR, the DNA-binding domains move substantially with respect to the dimer interface in apo-MntR. Overlays of multiple apo-MntR structures indicate that there is a greater range of positioning allowed between N and C-terminal domains in the metal-free state and that the DNA-binding domains of the dimer are farther apart than in the activated complex. To further investigate the conformation of the DNA-binding domain of apo-MntR, a site-directed spin labeling experiment was performed on a mutant of MntR containing cysteine at residue 6. Consistent with the crystallographic results, EPR spectra of the spin-labeled mutant indicate that tertiary structure is conserved in the presence or absence of bound metals, though slightly greater flexibility is present in inactive forms of MntR.

Apoproteins↗

PubMed2006

Biochemical characterization of the structural Zn2+ site in the Bacillus subtilis peroxide sensor PerR.

In Bacillus subtilis most peroxide-inducible oxidative stress genes are regulated by a metal-dependent repressor, PerR. PerR is a dimeric, Zn2+-containing metalloprotein with a regulatory metal-binding site that binds Fe2+ (PerR:Zn,Fe) or Mn2+ (PerR: Zn,Mn). Reaction of PerR:Zn,Fe with low levels of hydrogen peroxide (H2O2) leads to oxidation of two His residues thereby leading to derepression. When bound to Mn2+, the resulting PerR:Zn,Mn is much less sensitive to oxidative inactivation. Here we demonstrate that the structural Zn2+ is coordinated in a highly stable, intrasubunit Cys4:Zn2+ site. Oxidation of this Cys4:Zn2+ site by H2O2 leads to the formation of intrasubunit disulfide bonds. The rate of oxidation is too slow to account for induction of the peroxide stress response by micromolar levels of H2O2 but could contribute to induction under severe oxidative stress conditions. In vivo studies demonstrated that inactivation of PerR:Zn,Mn required 10 mM H2O2, a level at least 1000 times greater than that needed for inactivation of PerR:Zn,Fe. Surprisingly even under these severe oxidation conditions there was little if any detectable oxidation of cysteine residues in vivo: derepression was correlated with oxidation of the regulatory site. Because oxidation at this site required bound Fe2+ in vitro, we suggest that treatment of cells with 10 mM H2O2 released sufficient Fe2+ into the cytosol to effect a transition of PerR from the PerR:Zn,Mn form to the peroxide-sensitive PerR: Zn,Fe form. This model is supported by metal ion affinity measurements demonstrating that PerR bound Fe2+ with higher affinity than Mn2+.

Amino Acid Sequence↗

PubMed2006

Structural basis for the metal-selective activation of the manganese transport regulator of Bacillus subtilis.

The manganese transport regulator (MntR) of Bacillus subtilis is activated by Mn(2+) to repress transcription of genes encoding transporters involved in the uptake of manganese. MntR is also strongly activated by cadmium, both in vivo and in vitro, but it is poorly activated by other metal cations, including calcium and zinc. The previously published MntR.Mn(2+) structure revealed a binuclear complex of manganese ions with a metal-metal separation of 3.3 A (herein designated the AB conformer). Analysis of four additional crystal forms of MntR.Mn(2+) reveals that the AB conformer is only observed in monoclinic crystals at 100 K, suggesting that this conformation may be stabilized by crystal packing forces. In contrast, monoclinic crystals analyzed at room temperature (at either pH 6.5 or pH 8.5), and a second hexagonal crystal form (analyzed at 100 K), all reveal the shift of one manganese ion by 2.5 A, thereby leading to a newly identified conformation (the AC conformer) with an internuclear distance of 4.4 A. Significantly, the cadmium and calcium complexes of MntR also contain binuclear complexes with a 4.4 A internuclear separation. In contrast, the zinc complex of MntR contains only one metal ion per subunit, in the A site. Isothermal titration calorimetry confirms the stoichiometry of Mn(2+), Cd(2+), and Zn(2+) binding to MntR. We propose that the specificity of MntR activation is tied to productive binding of metal ions at two sites; the A site appears to act as a selectivity filter, determining whether the B or C site will be occupied and thereby fully activate MntR.

Bacillus subtilis↗

PubMed2006

The PerR transcription factor senses H2O2 by metal-catalysed histidine oxidation.

The sensing of reactive oxygen species is essential for cellular responses to oxidative stress. The sensing of peroxides is typically mediated by redox-active cysteines in sensors such as the bacterial OxyR, OhrR, and Hsp33 proteins. Bacillus subtilis PerR is the prototype for a widespread family of metal-dependent peroxide sensors that regulate inducible peroxide-defence genes. Here we show that PerR senses peroxides by metal-catalysed oxidation. PerR contains two metal-binding sites: a structural Zn2+ site and a regulatory divalent metal ion site that preferentially binds Fe2+ or Mn2+ (ref. 5). Protein oxidation, catalysed by a bound ferrous ion, leads to the rapid and direct incorporation of one oxygen atom into histidine 37 (H37) or H91, two of the residues that coordinate the bound Fe2+. This mechanism accounts for the ability of PerR to sense low levels of hydrogen peroxide in vivo. The reduction of hydrogen peroxide by metal ions to generate highly reactive hydroxyl radicals underlies the genotoxic effects of peroxides, and has been shown to contribute to enzyme inactivation, but has not previously been shown to provide a regulatory mechanism for peroxide sensing.

Bacillus subtilis↗

PubMed2006

Identification of Bacillus subtilis sigma-dependent genes that provide intrinsic resistance to antimicrobial compounds produced by Bacilli.

Bacillus subtilis produces many antibiotics of varying structures and specificity. Here we identify a prominent role for sigma(W), an extracytoplasmic function (ECF) sigma factor, in providing intrinsic resistance to antimicrobial compounds produced by other Bacilli. By using a panel of B. subtilis mutants disrupted for each of the 30 known sigma(W)-dependent operons we identified resistance genes for at least three different antimicrobial compounds. The ydbST and fosB genes contribute to resistance to antimicrobial compound(s) produced by B. amyloliquefaciens FZB42, the yqeZyqfAB operon provides resistance to the SPbeta prophage-encoded bacteriocin sublancin, and the yknWXYZ operon and yfhL provide resistance to the antimicrobial peptide SdpC. YfhL encodes a paralogue of SdpI, a membrane protein that provides immunity to SdpC. In competition experiments, we identify sigma(W) as a key factor in allowing B. subtilis to resist antibiotic killing and encroachment by competing strains. Together with the previous observation that sigma(W) provides inducible resistance against the Streptomyces antibiotic fosfomycin, these studies support the notion that sigma(W) controls an antibiosis regulon important in the microbial ecology of soil bacteria.

Amino Acid Sequence↗

PubMed2006

Assessing the impact of denitrifier-produced nitric oxide on other bacteria.

A series of experiments was undertaken to learn more about the impact on other bacteria of nitric oxide (NO) produced during denitrification. The denitrifier Rhodobacter sphaeroides 2.4.3 was chosen as a denitrifier for these experiments. To learn more about NO production by this bacterium, NO levels during denitrification were measured by using differential mass spectrometry. This revealed that NO levels produced during nitrate respiration by this bacterium were in the low muM range. This concentration of NO is higher than that previously measured in denitrifiers, including Achromobacter cycloclastes and Paracoccus denitrificans. Therefore, both 2.4.3 and A. cycloclastes were used in this work to compare the effects of various NO levels on nondenitrifying bacteria. By use of bacterial overlays, it was found that the NO generated by A. cycloclastes and 2.4.3 cells during denitrification inhibited the growth of both Bacillus subtilis and R. sphaeroides 2.4.1 but that R. sphaeroides 2.4.3 caused larger zones of inhibition in the overlays than A. cycloclastes. Both R. sphaeroides 2.4.3 and A. cycloclastes induced the expression of the NO stress response gene hmp in B. subtilis. Taken together, these results indicate that there is variability in the NO concentrations produced by denitrifiers, but, irrespective of the NO levels produced, microbes in the surrounding environment were responsive to the NO produced during denitrification.

Achromobacter cycloclastes↗

PubMed2006

Regulation of LiaRS-dependent gene expression in bacillus subtilis: identification of inhibitor proteins, regulator binding sites, and target genes of a conserved cell envelope stress-sensing two-component system.

The regulatory network of the cell envelope stress response in Bacillus subtilis involves both extracytoplasmic function sigma-factors and two-component signal transducing systems. One such system, LiaRS, responds to cell wall antibiotics that interfere with the undecaprenol cycle and to perturbation of the cytoplasmic membrane. It is encoded by the last two genes of the liaIHGFSR locus. Here, we analyzed the expression of two LiaR-dependent operons, liaIHGFSR and yhcYZ-yhdA, and characterized a palindromic sequence required for LiaR-dependent activation. Since induction of the strong liaI promoter leads to both liaIH and liaIHGFRS transcripts, LiaR is positively autoregulated. Systematic deletion analysis of the liaI operon revealed that LiaF is a potent negative regulator of LiaR-dependent gene expression: a nonpolar liaF deletion led to constitutive activation of both characterized LiaR-dependent promoters. The liaF gene is conserved in both sequence and genomic context in the Firmicutes group of gram-positive bacteria, located directly upstream of liaSR orthologs. LiaH, a homolog of Escherichia coli phage shock protein A, also plays a more subtle role in negatively modulating the bacitracin-inducible expression from LiaR-dependent promoters. Our results support a model in which the LiaFRS module integrates both positive and negative feedback loops to transduce cell envelope stress signals.

Amino Acid Sequence↗

PubMed2006

Role of the Fur regulon in iron transport in Bacillus subtilis.

The Bacillus subtilis ferric uptake regulator (Fur) protein mediates the iron-dependent repression of at least 20 operons encoding approximately 40 genes. We investigated the physiological roles of Fur-regulated genes by the construction of null mutations in 14 transcription units known or predicted to function in siderophore biosynthesis or iron uptake. We demonstrate that ywbLMN, encoding an elemental iron uptake system orthologous to the copper oxidase-dependent Fe(III) uptake system of Saccharomyces cerevisiae, is essential for growth in low iron minimal medium lacking citric acid. 2,3-Dihydroxybenzoyl-glycine (Itoic acid), the siderophore precursor produced by laboratory strains of B. subtilis, is of secondary importance. In the presence of citrate, the YfmCDEF ABC transporter is required for optimal growth. B. subtilis is unable to grow in minimal medium containing the iron chelator EDDHA unless the ability to synthesize the intact bacillibactin siderophore is restored (by the introduction of a functional sfp gene) or exogenous siderophores are provided. Utilization of the catecholate siderophores bacillibactin and enterobactin requires the FeuABC importer and the YusV ATPase. Utilization of hydroxamate siderophores requires the FhuBGC ABC transporter together with the FhuD (ferrichrome) or YxeB (ferrioxamine) substrate-binding proteins. Growth with schizokinen or arthrobactin is at least partially dependent on the YfhA YfiYZ importer and the YusV ATPase. We have also investigated the effects of a fur mutation on the proteome and documented the derepression of 11 Fur-regulated proteins, including a newly identified thioredoxin reductase homolog, YcgT.

Bacillus subtilis↗

PubMed2006

Stimulus perception in bacterial signal-transducing histidine kinases.

Two-component signal-transducing systems are ubiquitously distributed communication interfaces in bacteria. They consist of a histidine kinase that senses a specific environmental stimulus and a cognate response regulator that mediates the cellular response, mostly through differential expression of target genes. Histidine kinases are typically transmembrane proteins harboring at least two domains: an input (or sensor) domain and a cytoplasmic transmitter (or kinase) domain. They can be identified and classified by virtue of their conserved cytoplasmic kinase domains. In contrast, the sensor domains are highly variable, reflecting the plethora of different signals and modes of sensing. In order to gain insight into the mechanisms of stimulus perception by bacterial histidine kinases, we here survey sensor domain architecture and topology within the bacterial membrane, functional aspects related to this topology, and sequence and phylogenetic conservation. Based on these criteria, three groups of histidine kinases can be differentiated. (i) Periplasmic-sensing histidine kinases detect their stimuli (often small solutes) through an extracellular input domain. (ii) Histidine kinases with sensing mechanisms linked to the transmembrane regions detect stimuli (usually membrane-associated stimuli, such as ionic strength, osmolarity, turgor, or functional state of the cell envelope) via their membrane-spanning segments and sometimes via additional short extracellular loops. (iii) Cytoplasmic-sensing histidine kinases (either membrane anchored or soluble) detect cellular or diffusible signals reporting the metabolic or developmental state of the cell. This review provides an overview of mechanisms of stimulus perception for members of all three groups of bacterial signal-transducing histidine kinases.

Bacterial Physiological Phenomena↗

PubMed2005

Structure of an OhrR-ohrA operator complex reveals the DNA binding mechanism of the MarR family.

The mechanisms by which Bacillus subtilis OhrR, a member of the MarR family of transcription regulators, binds the ohrA operator and is induced by oxidation of its lone cysteine residue by organic hydroperoxides to sulphenic acid are unknown. Here, we describe the crystal structures of reduced OhrR and an OhrR-ohrA operator complex. To bind DNA, OhrR employs a chimeric winged helix-turn-helix DNA binding motif, which is composed of extended eukaryotic-like wings, prokaryotic helix-turn-helix motifs, and helix-helix elements. The reactivity of the peroxide-sensing cysteine is not modulated by proximal basic residues but largely by the positive dipole of helix alpha1. Induction originates from the alleviation of intersubunit steric clash between the sulphenic acid moieties of the oxidized sensor cysteines and nearby tyrosines and methionines. The structure of the OhrR-ohrA operator complex reveals the DNA binding mechanism of the entire MarR family and suggests a common inducer binding pocket.

Amino Acid Motifs↗

PubMed2005

Metal-induced structural organization and stabilization of the metalloregulatory protein MntR.

MntR is a metalloregulatory protein that helps to modulate the level of manganese in Bacillus subtilis. MntR shows a metal-response profile distinct from other members of the DtxR family of metalloregulatory proteins, which are generally considered to be iron(II)-activated. As part of an ongoing effort to elucidate the mechanism and metal-selectivity of MntR, several biophysical studies on wild-type MntR and two active site mutants, MntR E99C and MntR D8M, have been performed. Using circular dichroism (CD) spectroscopy, the thermal stability of these proteins has been examined in the presence of various divalent metal ions. Fluorescence intensity measurements of 8-anilino-1-naphthalenesulfonic acid (ANS) were monitored to examine the folding of these proteins in the presence of different metal ions. These experiments indicate that MntR undergoes a significant conformational change upon metal binding that results in stabilization of the protein structure. These studies also show that the MntR D8M active site mutation causes a detrimental effect on the metal-responsiveness of this protein. Fluorescence anisotropy experiments have been performed to quantify the extent of metal-activated DNA binding by these proteins to two different cognate recognition sequences. Binding of MntR and MntR E99C to the mntA cognate sequence closely parallels that of the mntH operator, confirming that the proteins bind both sequences with comparable affinity depending on the activating metal ion. Fluorescence anisotropy experiments on MntR D8M indicate significantly impaired DNA binding, providing additional evidence that MntR D8M is a dysfunctional regulator.

Anilino Naphthalenesulfonates↗

PubMed2005

Metal ion homeostasis in Bacillus subtilis.

Bacillus subtilis, a Gram-positive soil bacterium, provides a model system for the study of metal ion homeostasis. Metalloregulatory proteins serve as the arbiters of metal ion sufficiency and regulate the expression of metal homeostasis pathways. In B. subtilis, uptake systems are regulated by the highly selective metal-sensing repressors Fur (iron), Zur (zinc), and MntR (manganese). Metal efflux systems are regulated by MerR and ArsR family homologs which, by contrast, can be rather non-specific with regard to metal selectivity. A Fur homolog, PerR, functions as an Fe(II)-dependent peroxide stress sensor and regulates putative metal transport and storage functions.

Bacillus subtilis↗

PubMed2005

Genetic and physiological responses of Bacillus subtilis to metal ion stress.

Metal ion homeostasis is regulated principally by metalloregulatory proteins that control metal ion uptake, storage and efflux genes. We have used transcriptional profiling to survey Bacillus subtilis for genes that are rapidly induced by exposure to high levels of metal ions including Ag(I), Cd(II), Cu(II), Ni(II) and Zn(II) and the metalloid As(V). Many of the genes affected by metal stress were controlled by known metalloregulatory proteins (Fur, MntR, PerR, ArsR and CueR). Additional metal-induced genes are regulated by two newly defined metal-sensing ArsR/SmtB family repressors: CzrA and AseR. CzrA represses the CadA efflux ATPase and the cation diffusion facilitator CzcD and this repression is alleviated by Zn(II), Cd(II), Co(II), Ni(II) and Cu. CadA is the major determinant for Cd(II) resistance, while CzcD protects the cell against elevated levels of Zn(II), Cu, Co(II) and Ni(II). AseR negatively regulates itself and AseA, an As(III) efflux pump which contributes to arsenite resistance in cells lacking a functional ars operon. Our results extend the range of identified effectors for the As(III)-sensor ArsR to include Cd(II) and Ag(I) and for the Cu-sensor CueR to include Ag(I) and, weakly, Cd(II) and Zn(II). In addition to systems dedicated to metal homeostasis, specific metal stresses also strongly induced pathways related to cysteine, histidine and arginine metabolism.

Adenosine Triphosphatases↗

PubMed2005

A novel DNA modification by sulphur.

Streptomyces lividans has a novel DNA modification, which sensitises its DNA to degradation during electrophoresis (the Dnd phenotype). The entire gene cluster (dnd) involved in this modification was localized on an 8 kb DNA fragment and was expressed in a S. lividans deletion mutant (dnd) and in several heterologous hosts. Disruption of the dnd locus abolishes the Dnd phenotype, and gain of the dnd locus conferred the Dnd phenotype respectively. Extensive analysis of the dnd gene cluster revealed five open reading frames, whose hypothetic functions suggested an incorporation of sulphur or a sulphur-containing substance into S. lividans genome, yet in an unknown manner. The Dnd phenotype was also discovered to exist in DNA of widespread bacterial species of variable origin and diverse habitat. Similarly organized gene clusters were found in several bacterial genomes representing different genera and in eDNA of marine organisms, suggesting such modification as a widespread phenomenon. A coincidence between the Dnd phenotype and DNA modification by sulphur was demonstrated to occur in several representative bacterial genomes by the in vivo(35)S-labelling experiments.

Amino Acid Sequence↗

PubMed2005

Bacillus subtilis paraquat resistance is directed by sigmaM, an extracytoplasmic function sigma factor, and is conferred by YqjL and BcrC.

A Bacillus subtilis sigM null mutant, lacking the extracytoplasmic function sigma(M) protein, was sensitive to paraquat (PQ), a superoxide-generating reagent, but not to the redox stress-inducing compounds hydrogen peroxide, cumene hydroperoxide, t-butyl hydroperoxide, or diamide. Surprisingly, a sigM mutant was only sensitive to superoxide-generating compounds with a dipyridyl ring such as PQ, ethyl viologen, benzyl viologen, and diquat but not to menadione, plumbagin, pyrogallol, or nitrofurantoin. Mutational analysis of candidate sigma(M)-regulated genes revealed that both YqjL, a putative hydrolase, and BcrC, a bacitracin resistance protein, were involved in PQ resistance. Expression of yqjL, but not bcrC, from a xylose-inducible promoter restored PQ resistance to the sigM mutant.

ATP-Binding Cassette Transporters↗

PubMed2004

Antibiotic-inducible promoter regulated by the cell envelope stress-sensing two-component system LiaRS of Bacillus subtilis.

Soil bacteria are among the most prodigious producers of antibiotics. The Bacillus subtilis LiaRS (formerly YvqCE) two-component system is one of several antibiotic-sensing systems that coordinate the genetic response to cell wall-active antibiotics. Upon the addition of vancomycin or bacitracin, LiaRS autoregulates the liaIHGFSR operon. We have characterized the promoter of the lia operon and defined the cis-acting sequences necessary for antibiotic-inducible gene expression. A survey for compounds that act as inducers of the lia promoter revealed that it responds strongly to a subset of cell wall-active antibiotics that interfere with the lipid II cycle in the cytoplasmic membrane (bacitracin, nisin, ramoplanin, and vancomycin). Chemicals that perturb the cytoplasmic membrane, such as organic solvents, are also weak inducers. Thus, the reporter derived from P(liaI) (the liaI promoter) provides a tool for the detection and classification of antimicrobial compounds.

Anti-Bacterial Agents↗

PubMed2004

Response of Bacillus subtilis to nitric oxide and the nitrosating agent sodium nitroprusside.

We examined the effects of nitric oxide (NO) and sodium nitroprusside (SNP) on Bacillus subtilis physiology and gene expression. In aerobically growing cultures, cell death was most pronounced when NO gas was added incrementally rather than as a single bolus, suggesting that the length of exposure was important in determining cell survival. DNA microarrays, Northern hybridizations, and RNA slot blot analyses were employed to characterize the global transcriptional response of B. subtilis to NO and SNP. Under both aerobic and anaerobic conditions the gene most highly induced by NO was hmp, a flavohemoglobin known to protect bacteria from NO stress. Anaerobically, NO also induced genes repressed by the Fe(II)-containing metalloregulators, Fur and PerR, consistent with the known ability of NO to nitrosylate the Fe(II) center in Fur. In support of this model, we demonstrate that NO fails to induce PerR-regulated genes under growth conditions that favor the formation of PerR:Mn(II) rather than PerR:Fe(II). Aerobically, NO gas induced hmp, the sigmaB general stress regulon, and, to a lesser extent, the Fur and PerR regulons. Surprisingly, NO gas induced the sigmaB regulon via the energy branch of the sigmaB regulatory cascade while induction by SNP was mediated by the environmental stress branch. This emphasizes that NO and SNP elicit genetically distinct stress responses.

Adaptation, Physiological↗