Bacitracin sensing in Bacillus subtilis

The extracellular presence of antibiotics is a common threat in microbial life. Their sensitive detection and subsequent induction of appropriate resistance mechanisms is therefore a prerequisite for survival. The bacitracin stress response network of Bacillus subtilis consists of four signal-transducing systems, the two-component systems (TCS) BceRS, YvcPQ and LiaRS, and the extracytoplasmic function (ECF) σ factor σ^M. Here, we investigated the mechanism of bacitracin perception and the response hierarchy within this network. The BceRS–BceAB TCS/ABC transporter module is the most sensitive and efficient bacitracin resistance determinant. The ABC transporter BceAB not only acts as a bacitracin detoxification pump, but is also crucial for bacitracin sensing, indicative of a novel mechanism of stimulus perception, conserved in Firmicutes bacteria. The Bce system seems to respond to bacitracin directly (drug sensing), whereas the LiaRS TCS and σ^M respond only at higher concentrations and indirectly to bacitracin action (damage sensing). The YvcPQ–YvcRS system is subject to cross-activation via the paralogous Bce system, and is therefore only indirectly induced by bacitracin. The bacitracin stress response network is optimized to respond to antibiotic gradients in a way that maximizes the gain and minimizes the costs of this stress response.     

Transport and binding of riboflavin by Bacillus subtilis.

Riboflavine uptake and membrane-associated riboflavin-binding activity has been investigated in Bacillus subtilis. Riboflavin uptake proceeds via a system whose general properties are indicative of a carrier-mediated process: it is inhibited by substrate analogues, exhibits saturation kinetics, and is temperature-dependent. The organism concentrates riboflavin primarily as the phosphorylated cofactors FMN and FAD. Energy is required for uptake but whether the energy demand is required for both uptake and phosphorylation or only for the phosphorylation step is not known. Membrane-associated binding activity for riboflavin has also been demonstrated in membrane vesicles prepared from B. subtilis, and the binding component can be "solubilized" with Triton X-100. Evidence supporting the function of the binding component in riboflavin uptake by the intact cells includes the following. (i) Riboflavin analogues inhibit binding and uptake to nearly the same extent and with similar specificity of action. (ii) The KD for riboflavin-binding and the Km for uptake are in the same range. Similarly the Ki determined for the inhibitory analogue 5-deazariboflavin in the uptake assay and the KD for its interaction with the riboflavin-binding component of membrane vesicles are in the same range. (iii) Uptake in cells and binding in vesicles vary in the same direction with differences in growth conditions.     

Sequential binding of cobalt(II) to metallo-beta-lactamase CcrA

In an effort to probe Co(II) binding to metallo-beta-lactamase CcrA, EPR, EXAFS, and (1)H NMR studies were conducted on CcrA containing 1 equiv (1-Co(II)-CcrA) and 2 equiv (Co(II)Co(II)-CcrA) of Co(II). The EPR spectra of 1-Co(II)-CcrA and Co(II)Co(II)-CcrA are distinct and indicate 5/6-coordinate Co(II) ions. The EPR spectra also reveal the absence of significant spin-exchange coupling between the Co(II) ions in Co(II)Co(II)-CcrA. EXAFS spectra of 1-Co(II)-CcrA suggest 5/6-coordinate Co(II) with two or more histidine ligands. EXAFS spectra of Co(II)Co(II)-CcrA also indicate 5/6 ligands at a similar average distance to 1-Co(II)-CcrA, including an average of about two histidines per Co(II). (1)H NMR spectra for 1-Co(II)-CcrA revealed seven paramagnetically shifted resonances, three of which were solvent-exchangeable, while the NMR spectra for Co(II)Co(II)-CcrA showed at least 16 shifted resonances, including an additional solvent-exchangeable resonance and a resonance at 208 ppm. The data indicate sequential binding of Co(II) to CcrA and that the first Co(II) binds to the consensus Zn(1) site in the enzyme.     

Cell wall binding properties of the Bacillus subtilis autolysin(s)

Cell walls isolated from exponentially growing Bacillus subtilis have autolysin(s) attached to them. An autolysin can be released from the walls by incubation at 0 C with 3 m LiCl. The enzyme can reattach to walls when the salt concentration is reduced. The bound enzyme cannot be removed or destroyed by washing the walls with 8 m urea at 0 C. The binding of free enzyme to walls at 0 C can take place normally in the presence of 2 m urea.     

真菌吸附铅锌的研究

正随着采矿业的迅速发展,越来越多的重金属通过多种途径进入土壤环境中,对生态环境造成了不可估量的破坏并严重威胁人类健康。铅锌在工业上具有非常重要的作用且其应用极为广泛,而他们具有的难去除、难迁移和生物累积等特性使得铅锌在环境中的污染尤为突出。通过微生物的生长代谢,有效降低土壤重金属毒性,是促进植物生长的重要步骤之一。同时也要求微生物自身具有抵抗重金属的功能,根际微生     

Functional analysis of the Bacillus subtilis Zur regulon

The Bacillus subtilis zinc uptake repressor (Zur) regulates genes involved in zinc uptake. We have used DNA microarrays to identify genes that are derepressed in a zur mutant. In addition to members of the two previously identified Zur-regulated operons (yciC and ycdHI-yceA), we identified two other genes, yciA and yciB, as targets of Zur regulation. Electrophoretic mobility shift experiments demonstrated that all three operons are direct targets of Zur regulation. Zur binds to an approximately 28-bp operator upstream of the yciA gene, as judged by DNase I footprinting, and similar operator sites are found preceding each of the previously described target operons, yciC and ycdHI-yceA. Analysis of a yciA-lacZ fusion indicates that this operon is induced under zinc starvation conditions and derepressed in the zur mutant. Phenotypic analyses suggest that the YciA, YciB, and YciC proteins may function as part of the same Zn(II) transport pathway. Mutation of yciA or yciC, singly or in combination, had little effect on growth of the wild-type strain but significantly impaired the growth of the ycdH mutant under conditions of zinc limitation. Since the YciA, YciB, and YciC proteins are not obviously related to any known transporter family, they may define a new class of metal ion uptake system. Mutant strains lacking all three identified zinc uptake systems (yciABC, ycdHI-yceA, and zosA) are dependent on micromolar levels of added zinc for optimal growth.     

Metal binding by melanins: studies of colloidal dihydroxyindole-melanin, and its complexation by Cu(II) and Zn(II) ions

Melanins are colloidal pigments known to have a high affinity for metal ions. In this work, the nature of the metal-binding sites are determined and the binding affinities are quantified. Initial potentiometric titrations have been performed on synthetic dihydroxyindole (DHI) melanin solutions to determine the chemical speciation of quinole/quinone subunits. Two types of acidic functionalities are assignable: catechol groups, with pK(a) between 9 and 13, and quinone imines (QI), with pK(a) of 6.3. The presence of the quinone-imine tautomer has, to our knowledge, never been assessed in polymeric melanins. Melanin solutions obtained from N-methylated DHI lack the pK(a) 6.3 buffer, consistent with its inability to form the quinone-imine tautomer. EPR spectroscopy of the DHI-melanin samples demonstrates that the semiquinone radical is in too low a concentration to contribute to the bulk binding of metals. Changes in the titration curves after addition of Cu(II) and Zn(II) ions were analyzed to obtain the binding constants and stoichiometry of the metal-melanin complexes, using the BEST7 program. UV-Vis spectra at neutral and high pH are used to identify absorbances due to Cu-bound quinone imine and catechol groups. The derived binding constants were used to determine speciation of the Cu(II) and Zn(II) ions coordinated to the quinone imine and catechol groups at various pH. The mixed complexes, Zn(QI)(Cat)(-) and Cu(QI)(Cat)(-) are shown to dominate at physiological pH.     

The dimanganese(II) site of Bacillus subtilis class Ib ribonucleotide reductase

Class Ib ribonucleotide reductases (RNRs) use a dimanganese-tyrosyl radical cofactor, Mn(III)(2)-Y(?), in their homodimeric NrdF (β2) subunit to initiate reduction of ribonucleotides to deoxyribonucleotides. The structure of the Mn(II)(2) form of NrdF is an important component in understanding O(2)-mediated formation of the active metallocofactor, a subject of much interest because a unique flavodoxin, NrdI, is required for cofactor assembly. Biochemical studies and sequence alignments suggest that NrdF and NrdI proteins diverge into three phylogenetically distinct groups. The only crystal structure to date of a NrdF with a fully ordered and occupied dimanganese site is that of Escherichia coli Mn(II)(2)-NrdF, prototypical of the enzymes from actinobacteria and proteobacteria. Here we report the 1.9 ? resolution crystal structure of Bacillus subtilis Mn(II)(2)-NrdF, representative of the enzymes from a second group, from Bacillus and Staphylococcus. The structures of the metal clusters in the β2 dimer are distinct from those observed in E. coli Mn(II)(2)-NrdF. These differences illustrate the key role that solvent molecules and protein residues in the second coordination sphere of the Mn(II)(2) cluster play in determining conformations of carboxylate residues at the metal sites and demonstrate that diverse coordination geometries are capable of serving as starting points for Mn(III)(2)-Y(?) cofactor assembly in class Ib RNRs.     

In vitro type II binding of chromosomal DNA to membrane in Bacillus subtilis

DNA-membrane association critical for initiation of DNA replication in Bacillus subtilis can be classified into two types. Type I is salt resistant and dependent on the initiation gene, dnaB, and type II is salt sensitive and independent of the dnaB gene. We found and sequenced two adjacent areas of type II binding within 1% of oriC on the B. subtilis chromosome.     

Zn(II) binding to Escherichia coli 70S ribosomes

Escherichia coli 70S ribosomes tightly bind 8 equiv of Zn(II), and EXAFS spectra indicate that Zn(II) may be protein-bound. Ribosomes were incubated with EDTA and Zn(II), and after dialysis, the resulting ribosomes bound 5 and 11 equiv of Zn(II), respectively. EXAFS studies show that the additional Zn(II) in the zinc-supplemented ribosomes binds in part to the phosphate backbone of the ribosome. Lastly, in vitro translation studies demonstrate that EDTA-treated ribosomes do not synthesize an active Zn(II)-bound metalloenzyme, while the as-isolated ribosomes do. These studies demonstrate that the majority of intracellular Zn(II) resides in the ribosome.     

Degradation of guaiacol glyceryl ether (GGE) by Bacillus subtilis

Summary Bacillus subtilis utilized guaiacol glyceryl ether (GGE) as sole carbon source and catabolized it via guaiacol and catechol. Cell free extracts of GGE grown cells contained high levels of catechol 1,2-dioxygenase and cleaved catechol via the ortho pathway. Nutrients such as sugars, organic acids, methanol, nitrogen and phosphate influenced the catabolism of GGE by B. subtilis.     

Bacteria-shaped Gymnoplasts (Protoplasts) of Bacillus subtilis

Addition of glucose to the medium in which Bacillus subtilis was grown lowered the pH and increased the amount of lysylphosphatidylglycerol relative to the phosphatidylglycerol content of the membrane fraction. This change in phospholipid composition was accompanied by changes in the shape and behavior of the gymnoplasts obtained by cell wall removal with lysozyme. These gymnoplasts appeared to retain most of their original cell shape and internal organization, often with preservation of the mesosomes. Cells harvested from neutral growth medium gave the usual spherical gymnoplasts. In a hypotonic medium, the spherical gymnoplasts lysed rapidly, whereas the rod-like gymnoplasts lost only part of their cell content while showing a tendency to preserve the original shape. This type of gymnoplast could not be produced from cells grown in neutral medium by simply raising the magnesium concentration. When this was done the gymnoplasts assumed bizarre shapes; they became compact and susceptible to the tonicity of the medium. Gymnoplasts or protoplasts, produced from bacilli exposed to low pH values, were found not to conform to the formulations on "protoplasts" given in 1958 by 13 authors. Cells exposed to a low environmental pH during growth seemed to possess a more rigid membrane structure than cells grown at neutral pH.     

Physiological levels of glutathione enhance Zn(II) binding by a Cys4 zinc finger

Using fluorescence and UV-vis spectroscopies and mass spectrometry, we demonstrated that the presence of physiological levels of reduced glutathione enhances the binding of Zn(II) to XPAzf, a Cys4 zinc finger peptide derived from the XPA protein, by means of formation of a ternary complex of a general formula ZnXPAzf[GSH]. Similar complexes were also indicated by ESI-MS for isostructural Co(II)- and Cd(II)-substituted XPAzf. The observed enhancement of the Zn(II) binding to XPAzf by a factor of 50 over the physiological range of GSH concentrations of 1-20 mM corresponds to a dissociation constant of GSH from the ZnXPAzf[GSH] complex of 0.05 μM. This effect may account for an apparent discrepancy between relatively low Zn(II) binding constants measured in vitro for many zinc fingers, and the requirement of tight Zn(II) binding enforced by intracellular zinc buffering by the thionein/metallothionein couple.