Ionic channels induced by surfactin in planar lipid bilayer membranes.

Surfactin is a lipopeptide produced by certain strains of Bacillus subtilis and has potent surface activity. Here, we present the first results showing that ion-conducting pores can be formed by surfactin in artificial lipid membranes. With a low aqueous concentration of surfactin (1 microM) and a restricted membrane area (5.10(-5) cm2) we observed conductance jumps that indicate the formation of individual ionic channels in the presence of K+, Rb+, Cs+, Na+ or Li+ chlorides. Although for every salt concentration (Ci), the distribution in amplitude of the conductance steps (lambda i) may be rather broad, there is always a step amplitude which is more frequent than the others. In addition, the channels corresponding to this most frequent step amplitude are the longest in duration. For Ci = 1 M, the cationic selectivity sequence deduced from these most frequent events is K+ greater than Rb+ greater than Na+ greater than Cs+ = Li+ with respective values for lambda Mi: 130, 110, 80 and 30 pS. In KCl solutions lambda MKCl increases as a function of Ci for low Ci, and shows a plateau for Ci greater than 0.5 M. When measured on larger area membranes (10(-2)cm2) with 1 M solutions of the monovalent salts KCl, NaCl, RbCl and CsCl or the divalent salt CaCl2, the macroscopic low voltage conductance (G0) increases with a slope of 2 on a log-log plot as a function of surfactin concentration. These results demonstrate that surfactin produces selective cationic channels in lipid bilayer membranes and suggest that at higher salt concentration, a dimer is involved in this functional channel-forming process.

A new and rapid method for the assay of bacterial fatty acids using high resolution capillary gas chromatography and trimethylsulfonium hydroxide.

Gas-liquid chromatography of cellular fatty acids is a useful tool for the identification of bacteria. Derivatization of bacterial fatty acids to methyl esters by conventional techniques is usually time-consuming and complicated. A new one-step technique using trimethyl-sulfonium hydroxide allows the direct formation of fatty acid methyl esters within 1-2 min. Some random examples of profiles demonstrate that straight, branched, saturated, unsaturated, hydroxy and cyclopropyl fatty acids match conventional preparations well. The method is a very sensitive one, since only a few colonies are sufficient for preparation of fatty acid methyl esters.

The thermostability of DNA-binding protein HU from bacilli.

The primary and tertiary structures of DNA-binding protein HU from Bacillus stearothermophilus are already known. The primary structure has been previously determined for HU from the closely related B. globigii and the determinations of the sequences from B. caldolyticus and B. subtilis are described here. These bacteria have optimum growth temperatures of greater than 70 degrees C (B. caldolyticus), 65 degrees C (B. stearothermophilus), 37 degrees C (B. subtilis) and 30 degrees C (B. globigii). In vitro measurements from circular dichroic spectra described here give Tm values reflecting these growth temperatures, of 68, 64, 43 and 41 degrees C respectively. We discuss here the relative thermostability of the four proteins in terms of the amino acid differences between the sequences and the three-dimensional model of the B. stearothermophilus HU. The current model for the interaction of the protein with DNA is only discussed in terms of its relevance with regard to thermostability.

Overproduction, purification, and characterization of Bacillus subtilis RNA polymerase sigma A factor.

By use of a T7 expression system, large amounts of active Bacillus subtilis RNA polymerase sigma A factor were produced in Escherichia coli cells. This overproduced protein was found in the form of inclusion bodies and constituted 40% of the total cellular protein. Because of the ease of isolation of the inclusion bodies and the acidic properties of sigma A, the protein was purified to more than 99% purity and the yield was about 90 mg/liter of culture. Gel mobility, antigenicity, specificity of promoter recognition, and N-terminal amino acid sequence of the overproduced sigma were found to be the same as those of native sigma A. Partial proteolysis analysis of sigma A protein suggested the presence of a protease-sensitive surface region in the C-terminal part of the sigma A protein. The promoter -10 binding region of sigma A was less sensitive to proteases and was probably involved in a hydrophobic, tightly folded domain of sigma A protein.

High-performance liquid chromatography of transfer ribonucleic acids on spherical hydroxyapatite beads.

High-performance liquid chromatography (HPLC) on newly developed spherical beads of hydroxyapatite was applied to the analysis of purified E. coli tRNAs (Val, Met, Tyr and Phe). tRNAs were eluted from the column separately with appreciable differences in retention time by a 45-min gradient of phosphate buffer (pH 6.8) of concentration from 64 to 123 mM; both the retention times and peak areas of respective tRNAs were highly reproducible. Total tRNA (tRNA(Total)) preparations obtained from E. coli and B. subtilis were also analysed on the column. It is possible even to elute tRNA(Total) which may, in general, contain 60 or more tRNA species with a relatively shallow gradient such as 75-132 mM. The recovery of tRNA from the column was as high as 90%. Owing to the complicated composition, the elution profile of tRNA(Total) had a wide spread irregular shape but, with a 1-h gradient more than ten peaks were easily detected. When the amino acid-accepting activity of tRNA in the eluate of tRNA(Total) was determined, for ten specific tRNAs, each activity peak was eluted sharply from the column. In addition, several tRNA activities were eluted in different fractions. This indicates that isoacceptors were separated by the column. The results show that HPLC on hydroxyapatite beads is useful for the purification and characterization of tRNA.

Isolation of a secY homologue from Bacillus subtilis: evidence for a common protein export pathway in eubacteria.

Genetic and biochemical studies have shown that the product of the Escherichia coli secY gene is an integral membrane protein with a central role in protein secretion. We found the Bacillus subtilis secY homologue within the spc-alpha ribosomal protein operon at the same position occupied by E. coli secY. B. subtilis secY coded for a hypothetical product 41% identical to E. coli SecY, a protein thought to contain 10 membrane-spanning segments and 11 hydrophilic regions, six of which are exposed to the cytoplasm and five to the periplasm. We predicted similar segments in B. subtilis SecY, and the primary sequences of the second and third cytoplasmic regions and the first, second, fourth, fifth, seventh, and tenth membrane segments were particularly conserved, sharing greater than 50% identity with E. coli SecY. We propose that the conserved cytoplasmic regions interact with similar cytoplasmic secretion factors in both organisms and that the conserved membrane-spanning segments actively participate in protein export. Our results suggest that despite the evolutionary differences reflected in cell wall architecture, Gram-negative and Gram-positive bacteria possess a similar protein export apparatus.

The use of multiple mass spectral line pairs for enhanced precision in isotope enrichment studies of 15N-labeled amino acids.

A method for enhancing the precision in the calculation of isotope enrichment for 15N-labeled amino acids is presented. The method utilizes multiple line pairs for the calculation of isotope enrichment. Using multiple line pairs allows the evaluation of calibration curves for nonlinear behavior and permits differentiation among sites containing more than one labeled nitrogen. The increase in precision is related to the number of isotopically shifted line pairs used in calculating the isotopic enrichment and varies with the amino acid of interest.

Purification and properties of the DNA-binding protein HPB12 from Bacillus subtilis nucleoid.

We report the purification to homogeneity of a 12 KDa protein (HPB12) present in the nucleoids of Bacillus subtilis. From the purification data the abundance of the protein was estimated to about 20,000 monomers per cell. The HPB12 protein is heat-stable and acid-soluble and binds preferentially to supercoiled and linearized double-stranded DNAs. The binding of the protein to the supercoiled DNA occurs very rapidly and appears to be cooperative. Moreover, the complexes are extremely stable and do not dissociate after 90 min. These properties are consistent with a role of the HPB12 protein in the structure of the B. subtilis chromosome.

Mechanical properties of peptidoglycan as determined from bacterial thread.

Experiments are described in which the tensile strength, the extensibility and the initial Young's modulus of bacterial cell wall have been determined as functions of relative humidity in the range 11-98%. Data on stress relaxation and recovery are also given. Standard fibre-measuring technique has been used on 'bacterial thread', made from a cell-separation-suppressed mutant of Bacillus subtilis. The data show that peptidoglycan, the load bearing polymer in the cell wall, behaves very much like other viscoelastic polymers. Its mechanical behaviour when dry is that of a glassy polymer with tensile strength about 300 MPa and modulus about 20 GPa. When wet, it is weaker and much less stiff with tensile strength about 3 M Pa and modulus 10 M Pa. The relaxation data indicate a wide spectrum of relaxation times. The results are discussed in terms of the structure of peptidoglycan and its orientation in the bacterial cell wall. The way in which mechanical behaviour depends strongly on humidity is compared with that of other biopolymers in terms of possible hydrogen-bond density and the ordering of water molecules. The possibility of a well-defined glass transition is briefly examined.

Modification of Bacillus subtilis elongation factor Tu by N-tosyl-L-phenylalanyl chloromethane abolishes its ability to interact with the 3'-terminal polynucleotide structure but not with the acyl bond in aminoacyl-tRNA.

Modification of B. subtilis EF-Tu by N-tosyl-L-phenylalanyl chloromethane destroyed its ability to promote protein synthesis and resulted in selective dissociation of the two binding activities of the protein for aminoacyl-tRNA. The modified EF-Tu was completely ineffective in the protection of the 3'-terminal CCA structure of tRNA against pancreatic ribonuclease, while remaining almost fully active in the protection of the ester bond between the 3'-terminal adenosine and the amino acid residue in aminoacyl-tRNA.

A procedure for the preparation of bacterial DNA that employs dimethyl sulfoxide to induce the lysis of cells.

A protocol for the preparation of DNA from Escherichia coli and Bacillus subtilis without the use of lysozyme as a permeabilizing agent is described. This preliminary step is carried out by treating the cells with dimethyl sulfoxide. A 5-min incubation of the cell pellet in the pure solvent, followed by the treatment with sodium dodecyl sulfate, is sufficient to induce cell lysis. The plasmid DNAs obtained by this method were equivalent in purity and quantity to the material prepared from lysozyme-digested cells and amenable to restriction and ligation. Transformation by plasmid and genomic DNAs prepared from dimethyl sulfoxide-treated cells was demonstrated.

Presence of proteins derived from the vegetative cell membrane in the dormant spore coat of Bacillus subtilis.

To confirm the presence of the outer spore membrane in dormant spore coats of Bacillus subtilis, the proteins from vegetative cell membrane and dormant spore coat fractions were compared by immunoblot assay with antibodies prepared against both preparations. The spore coat fraction contained at least 11 proteins antigenically identical to those in the vegetative cell membranes. Further, the cytochemical localization of the proteins derived from vegetative cell membrane in dormant spores was examined by an immunoelectron microscopy method with a colloidal gold-immunoglobulin G complex. The colloidal gold particles were observed in the coat region and around the core region of dormant spore. These results have provided evidence that some proteins from vegetative cell membrane remain in the dormant spore coat region of B. subtilis, although it is not clear whether the outer membrane persists as an intact functional entity or not.

Structural studies on the neutral polysaccharide of Bacillus subtilis AHU 1219 cell walls.

A neutral and an acidic polysaccharide with molecular masses of about 22 kDa and 45 kDa, respectively, were isolated from the N-acetylated cell walls of Bacillus subtilis AHU 1219 by heating at pH 2.5, followed by separation of the water-soluble product by ion-exchange chromatography and gel chromatography. The neutral polysaccharide, accounting for 40% of the mass of the cell walls, contained glucose, N-acetylglucosamine, N-acetylgalactosamine and N-acetylmannosamine in a molar ratio of 1:2:1:1. The minor, acidic polysaccharide contained glucuronic acid, glucose, galactose, L-serine and L-threonine in an approximate molar ratio of 1:1:1:0.5:0.5. Lysozyme digestion of the N-acetylated cell walls gave a polymer containing the neutral polysaccharide and glycopeptide components and another polymer which contained the acidic polysaccharide components together with small proportions of the neutral polysaccharide and glycopeptide components. Thus, the neutral and acidic polysaccharide chains seem to be attached to peptidoglycan through acid-labile linkages in the cell walls of this strain. Structural analysis of the neutral-polysaccharide preparation, involving 1H-NMR and 13C-NMR measurement, methylation and Smith degradation, led to the most likely structure, ----6)[Glc(beta 1----3)]GalNAc(alpha 1----4)-[GlcNAc(beta 1----3)]ManNAc(beta 1----4)GlcNAc(beta 1----, for the repeating units of this polysaccharide.

Structural studies on the amino-acid-linked teichuronic acid of Bacillus subtilis AHU 1219 cell walls.

Structural studies were carried out on a teichuronic acid isolated from a mild acid extract of Bacillus subtilis AHU 1219 cell walls. The teichuronic acid contained D-glucuronic acid, D-glucose, D-galactose, L-serine and L-threonine in a molar ratio of 1:1:1:0.5:0.5. Results of analyses of the polysaccharide by Smith degradation, methylation and 1H-NMR and 13C-NMR spectroscopy, in combination with data on analyses of oligosaccharides obtained by partial acid hydrolysis and alkaline hydrolysis of the polymer, led to the most likely structure for the repeating unit, ----4)(L-Ser/L-Thr)-D-GlcA(beta 1----3)-D-Glc(beta 1----4)-D-Gal(alpha 1----. In each unit, either amino acid is linked to the glucuronic acid residue through an amide bond.

[Changes in the redox potential and the number of accessible sulfhydryl groups in Escherichia coli and Bacillus subtilis cultures during transient processes]. / Izmeneniia redoks-potentsiala i kolichestva dostupnykh SH-grupp v kul'turakh Escherichia coli i Bacillus subtilis pri perekhodnykh protsessakh.

It was shown that changes in the redox potential can be due to the influence of compounds which alter the intracellular pH (acetate or propionate, protonophore carbonyl-cyanide-m-chlorophenyl hydrazone, permeate cation TPP+). A correlation was found between the redox potential changes and the number of SH-groups in the medium and on cell surface. It was shown also that the previously reported redox potential shifts during the transition of E. coli and B. subtilis cultures to the stationary phase under glucose or ammonium exhaustion are due to the increase in the number of SH-groups in the medium and on cell surface. A hypothesis is put forward, according to which the changes in intracellular pH play a trigger role, whereas those in the thiol: disulfide ratio inside and outside the cells are thought to amplify regulatory signals.

A novel role for cAMP in the control of the activity of the E. coli chromosome replication initiator protein, DnaA.

DnaA protein interacts with cAMP with a KD of 1 microM. This interaction stimulates DnaA protein binding to the chromosome replication origin (oriC) and the mioC promoter region, protects DnaA protein from thermal inactivation, releases ADP but not ATP bound to DnaA protein, and restores normal DNA replication activity and ATPase activity in inactive ADP-DnaA protein preparations. A model is proposed in which cellular cAMP levels govern the replication activity of DnaA protein by promoting the recycling of the inactive ADP-DnaA protein form into the active ATP form.

Precipitation and recovery of proteins from culture supernatants using zinc.

A convenient method for the concentration of proteins from culture supernatants is described. Heterologous gene products expressed and secreted into the culture medium by Saccharomyces cerevisiae, Chinese hamster ovary cells (CHO), and Bacillus subtilis were precipitated out of solution by the addition of millimolar concentrations of zinc chloride. Porcine urokinase secreted by S. cerevisiae, the tissue-type plasminogen activator analog FK2P secreted by CHO cells, and human interleukin-1 beta secreted by B. subtilis were all precipitated by zinc ions. Both urokinase and FK2P were precipitated with as low as 2 mM zinc, with recovery of activity in the precipitate approaching 100%.

Multiple isoelectric forms of bacterial penicillin-binding proteins: artefacts or genuine cellular components?

Some bacterial penicillin-binding proteins (PBPs) are reported to exist as multiple isoelectric forms. Other PBPs were examined by two-dimensional electrophoresis to establish whether multiple isoelectric forms are widespread amongst PBPs. Bacillus subtilis PBPs 3 and 5 and Escherichia coli PBPs 1b alpha and 1b gamma focussed as discrete spots with no evidence of multiple isoforms. However, E. coli PBPs 1b beta and 4 displayed isoelectric variants. The results are discussed in relation to current models of bacterial peptidoglycan structure.

Specific extraction of bacterial cardiolipin from sporulating Bacillus subtilis.

A method for rapid purification of bacterial cardiolipin is presented. The cardiolipin level was first increased by suspending Bacillus subtilis cells in a buffer containing an uncoupling agent. At least 90% of the phosphatidylglycerol molecules were rapidly converted into cardiolipin. In sporulating strains, the accumulated cardiolipin appeared to be unextractable by conventional phospholipid extraction procedures. Sporulating bacteria were therefore treated first by a classical technique in order to eliminate lipids other than cardiolipin; a second extraction in a highly acidic medium then allowed us to quantitatively extract the remaining cardiolipin. Besides simplicity and rapidity, this method has the advantage of yielding cardiolipin in a nearly pure form from a relatively low number of bacteria.