The effect of amino acids on the motile behavior of Bacillus subtilis

Constant levels of amino acids enhanced the velocity of Bacillus subtilis 60015 cells about 2-fold and stimulated the response in motility assays. The stimulation of velocity did not occur via the receptors for chemotaxis. Cysteine and methionine, general inhibitors of chemotaxis, both completely inhibited the smooth response in a temporal gradient of attractant. After methionine starvation B. subtilis 60015 showed no measurable response in a temporal gradient of attractant, this in contrast to the effect observed with some other bacteria. Addition of methionine to starved cells restored the response toward attractant. Revertants of B. subtilis 60015 for methionine requirement could not be starved and showed a normal behavior toward temporal gradients of attractant.Abbreviation O.D.₆₀₀ optical density measured at 600 nm     

Control of the chemotactic behavior of Bacillus subtilis cells

The effects of nigericin, valinomycin and some lipophilic cations on the motile behavior of non-starved and methionine-starved Bacillus subtilis cells were studied. For valinomycin and nigericin a quantitative relationship between the flux in the proton-motive force and the duration of the twiddle response was found. Lipophilic cations bind to the ion gate controlling the twiddle frequency and thereby cause the cells to swim smoothly. To explain the transmission of the chemotactic signal a model is given in which receptors, a hyperpolarizing wave, an ion gate and two methylation sites, viz. methyl-accepting chemotaxis proteins and a further methylation site (MT), play a role. For the transmission of the signal caused by an attractant both the hyperpolarizing wave and an interaction between receptor and methylation site (MT) are needed. The methyl-accepting chemotaxis proteins are involved in the adaptation/deadaptation to altered levels of attractant. Artificial changes in the proton-motive force act directly on the ion gate, which finally controlls the twiddle frequency of the cells.Abbreviations KT medium potassium taxis medium - NAT medium sodium taxis medium - HT medium acidic taxis medium - OHT medium alkaline taxis medium - ImT medium imidazole taxis medium - GT medium glycylgycine taxis medium - Di-S-C₃(5) 3,3-dipropyl-2,2-thiacarbocyanine iodide - TPAs⁺ tetraphenylarsonium ion - TPMP⁺ triphenylmethylphosphonium ion - DDA⁺ dibenzyldimethylammonium ion - TPB^- tetraphenylboron ion - pmf proton-motive force - MCP methyl-accepting chemotaxis protein - MT methylation site - membrane potential     

Genetic mapping of regulatory mutations of Bacillus subtilis riboflavin operon

Summary Seven mutations leading to riboflavin overproduction inBacillus subtilis were found to be linked to the markerdnaF133 (145° on theB. subtilis genetic map) by transformation. Cotransfer indexes (42.5%–61.7%) suggest that theribC mutations are alleles of the same locus. Results of transduction and transformation crosses suggest the following order of markers:pyrD26 –ts-6 –dnaF133 –ribC –recA1.     

Interplasmidic illegitimate recombination in Bacillus subtilis

Summary The illegitimate recombination between Staphylococcus aureus plasmids pE194 (or pGG20, the hybrid between pE194 and Escherichia coli plasmid pBR322) and pBD17 (plasmid pUB110 without HpaII C-fragment) was studied in Bacillus subtilis. Cointegrates were generated with the frequency of 1–3x10^-8. Among 22 hybrids analysed 9 types of recombinants were found. Nucleotide sequences of all three parental plasmids were involved in intermolecular recombination. Nucleotide sequencing of recombinant DNA junctions revealed that in 8 cases recombination occurred between short homologous regions (9–15 bp). One recombinant was formed using nonhomologous sites. The similarity was demonstrated between nucleotide sequences of the recombination sites of two types of cointegrates and those used for pE194 integration into the B. subtilis chromosome. Possible mechanisms of illegitimate recombination are discussed.     

Expression of sfp gene and hydrocarbon degradation by Bacillus subtilis

Bacillus subtilis C9 produces a lipopeptide-type biosurfactant, surfactin, and rapidly degrades alkanes up to a chain length of C₁₉. The nucleotide sequence of the sfp gene cloned from B. subtilis C9 was determined and its deduced amino acid sequence showed 100% homology with the sfp gene reported before [Nakano et al. (1992) Mol. Gen. Genet. 232: 313–321]. To transform a non-surfactin producer, B. subtilis 168, to a surfactin producer, the sfp gene cloned from B. subtilis C9 was expressed in B. subtilis 168. The transformed B. subtilis SB103 derivative of the strain 168 was shown to produce surfactin measured by its decrease in surface tension, emulsification activity, and TLC analysis of the surface active compound isolated from the culture broth. Like B. subtilis C9, B. subtilis SB103 containing sfp gene readily degraded aliphatic hydrocarbons (C_10–19), though its original strain did not. The addition of surfactin (0.5%, w/v) to the culture of B. subtilis 168 significantly stimulated the biodegradation of hydrocarbons of the chain lengths of 10–19; over 98% of the hydrocarbons tested were degraded within 24 h of incubation. These results indicate that the lipopeptide-type biosurfactant, surfactin produced from B. subtilis enhances the bioavailability of hydrophobic hydrocarbons.     

Pyrimidine ribonucleoside monophosphokinase and the mode of RNA turnover in Bacillus subtilis

A protein catalyzing the phosphorylation of CMP to CDP was purified and characterized. Kinase activity for UMP copurified during ammonium sulfate fractionation, DEAE-cellulose and hydroxylapatite chromatography, and gel filtration on Sephadex G-75, the ratios of activities for the two substrates remaining constant. The purified product, possessing both activities was homogeneous as judged by the single band following polyacrylamide gel electrophoresis. The protein showed no kinase activity against purine nucleoside monophosphates or the other pyrimidine nucleoside monophosphates: dCMP, dUMP, and dTMP. Thus unlike the enteric bacteria, Escherichia coli and Salmonella typhimurium which have distinct enzymes which phosphorylate UMP and CMP, Bacillus subtilis produces a single pyrimidine ribonucleoside monophosphokinase. The K _mvalues of this enzyme from B. subtilis are 0.04 and 0.25 mM for CMP and UMP, respectively, and 0.04 and 0.4 mM for ATP at saturating concentrations of CMP and UMP, respectively. The properties of this enzyme and the differences between enteric bacteria and B. subtilis with respect to the enzymes which phosphorylate CMP are consistent with the measurements which indicate that turnover of messenger RNA is largely hydrolytic in E. coli but largely phosphorolytic in B. subtilis.Non-Standard Abbreviations PRMK Pyridine ribonucleoside monophosphokinase This paper is affectionately dedicated to Professor R. Y. Stanier     

FT-IR spectroscopic characteristics of differently cultivated Bacillus subtilis

Bacillus subtilis is an aerobic endospore forming bacterium widely spread in different environments. Because it represents a biological agent of some health relevance, its rapid detection and identification is highly desirable. By using FT-IR spectroscopy for this purpose slightly different characteristics were obtained from cell mass grown in differently composed cultural media, and harvested in different phases of bacterial growth. If cultivated uniformly, i.e., 24h at 30 degrees C in a minimum-strength nutrient broth, cell mass of B. subtilis delivered a well differentiated spectrum with major absorption bands of nucleic acid structures at 3300cm(-1), cell wall constituents at 3000-2800cm(-1), proteinaceous structures at 1660, 1544 and 1235cm(-1), and some aliphatic structural units at 1080cm(-1). Attenuated total reflectance, and absorption/transmission scanning techniques, delivered structurally identical spectra but those obtained by the former technique were more expressed.     

Subspecies-specific distribution of intervening sequences in the Bacillus subtilis prophage ribonucleotide reductase genes

A collection of 212 gram-positive bacilli isolated from natural habitats was screened for the presence of intervening sequences (introns and intein-coding sequences) in the SPbeta prophage-related ribonucleotide reductase genes bnrdE and bnrdF. Three novel configurations were identified on the basis of the presence of (i) intervening sequences in bnrdE and bnrdF, and (ii) an ORF in the bnrdE-bnrdF spacer. Analysis of the cell wall genetic determinants as well as of the incorporation of radio-labelled glycerol into cell wall allowed newly and previously identified B. subtilis strains with different configurations of bnrdE/bnrdF intervening sequences to be assigned to one of two subspecies. Strains apparently belonging to the subsp. subtilis contain three intervening sequences many of which are associated with the putative homing endonuclease activity. Strains of the subsp. spizizenii contain only one or two ORF-less group I introns. Introns occupying bnrdF are confined to the subspecies subtilis.     

Conjugal Plasmid Transfer in Bacillus subtilis under Conditions of Soil Microcosms

Conjugal transfer of the small plasmid pUB110 betweenBacillus subtilis strains was studied under conditions of microcosms with sterile and nonsterile soil. Plasmid transfer proved to be possible after soil inoculation with vegetative partner cells or with their spores. Plasmid transfer occurred at temperatures of 30 and 22–23°C.     

The Replication System of Plasmids from Bacillus subtilis Environmental Isolates

Restriction enzyme analysis, cloning, and sequencing showed that large (more than 90 kb) plasmids isolated from different Bacillus subtilisstrains are identical in structure of the region ensuring stable inheritance of plasmid replicons and are widespread in Belarussian environmental strains of B. subtilis.     

On the appearance of Bacillus subtilis intracellular serine protease in the cell membrane and culture medium

While about 80% of the cell-bound intracellular serine protease of Bacillus subtilis A-50 have been recovered in the soluble fraction upon disruption of cells, the rest of the enzyme was found to be associated with the membrane fraction. Soluble cytoplasmic intracellular serine protease, as well as membrane-bound serine protease liberated by nonionic detergent treatment, have been isolated in a pure state and shown to be identical. The same protease might also be found extracellularly, due presumably to cell lysis or altered membrane permeability. Intracellular serine protease of Bacillus subtilis A-50 was clearly related to Bacillus subtilis serine proteases W1 and bacillopeptidase F described as extracellular enzymes.Abbreviations ISP intracellular serine protease - ISP-A-Bsu A-50 and ISP-B-Bsu A-50 molecular forms A and B of B. subtilis A-50 intracellular serine protease, respectively - SDS sodium dodecyl sulfate - PMSF phenylmethyl sulfonylfluoride - pNA p-nitroanilide - Buffer A 50 mM Tris-(hydroxymethyl)aminomethane-1 mM CaCl₂ adjusted to pH 8.5 with HCl     

Extracellular production of human cystatin S and cystatin SA by Bacillus subtilis

We herein describe the development of a Bacillus subtilis system that can be used to produce large quantities of recombinant (r-) human salivary cystatins, a cysteine protease inhibitor of family 2 in the cystatin superfamily. The B. subtilis that lacked the alkaline protease E gene (DeltaaprE type mutant strain) was prepared by homologous recombination. The cDNA fragments coding for mature cystatins (S and SA) were ligated in frame to the DNA segment for the signal peptide of endoglucanase in the pHSP-US plasmid vector that was then use to transform the DeltaaprE type mutant strain of B. subtilis. The transformants carrying the expression vectors were cultivated in 5-L jar fermenters for 3 days at 30 degrees C. Both r-cystatin S and r-cystatin SA were successfully expressed and secreted into the culture broth, and were purified using a fast performance liquid chromatography system. The first use of DeltaaprE type mutant strain of B. subtilis made it possible to obtain a high yield of secreted protein, which makes this system an improvement over expression in Escherichia coli. We conclude that this system has high utility for expression of commercial quantities of secreted proteins.     

Mn2 + improves surfactin production by Bacillus subtilis

Surfactin productivity by Bacillus subtilis was increased from 0.33 g l^–1 to 2.6 g l^–1 by adding 0.01 mM Mn²⁺ to a defined glucose medium. The final yield exceeded that of most reported values for genetically improved strains.     

The mmgA gene from Bacillus subtilis encodes a degradative acetoacetyl-CoA thiolase

Early in sporulation, the mother cell compartment of Bacillus subtilis transcribes the mother cell metabolic gene (mmg) operon. The gene mmgA was assigned by other workers using sequence homology as an acetyl-CoA acetyltransferase [E.C. 2.3.1.9]. The gene was overexpressed in Escherichia coli, and the protein was purified by Ni²⁺-affinity chromatography. However, the expected MmgA-catalyzed biosynthesis of acetoacetyl-CoA from acetyl-CoA was undetectable by a standard UV assay, HPLC, and mass spectrometry. These methods indicated a preference for the reverse degradative thiolytic reaction, with a k _cat of 80 s⁻¹, and a K _m of 70 and 50 μM for CoA and acetoacetyl-CoA, respectively.     

Molecular cloning of Bacillus subtilis genes involved in DNA metabolism

Summary Different clones carrying a chromosomal DNA fragment able to transform Bacillus subtilis mutants dnaA13, dnaB19, dnaG5, recG40 and polA42 to a wild-type phenotype were isolated from a library constructed in plasmid pJH101. A recombinant clone carrying a chromosomal fragment able to transform dnaC mutants was obtained from a Charon 4A library. A restriction map of the cloned DNA fragments was constructed. The 11.3 kb cloned DNA fragment of plasmid pMP60-13 containing the wild-type sequence of dnaG5 was shown to transform a recF33 mutant as well.     

Main physicochemical features of monofunctional flavokinase from Bacillus subtilis

The main properties of a monofunctional riboflavin kinase from B. subtilis have been studied for the first time; the enzyme is responsible for a key reaction in flavin biosynthesis—the ATP-dependent phosphorylation of riboflavin with production of flavin mononucleotide. The active form of the enzyme is a monomer with molecular weight of about 26 kD with a strict specificity for reduced riboflavin. To display its maximum activity, the enzyme needs ATP and Mg²⁺. During the phosphorylation of riboflavin, Mg²⁺ could be partially replaced by ions of other bivalent metals, the efficiencies of which decreased in the series Mg²⁺ > Mn²⁺ > Zn²⁺, whereas Co²⁺ and Ca²⁺ had inhibiting effects. The flavokinase activity was maximal at pH 8.5 and 52°C. ATP could be partially replaced by other triphosphates, their donor activity decreasing in the series: ATP > dATP > CTP > UTP. The Michaelis constants for riboflavin and ATP were 0.15 and 112 M, respectively. As compared to riboflavin, a tenfold excess of its analog 7,8-dimethyl-10-(O-methylacetoxime)-isoalloxazine decreased the enzyme activity by 30%. Other analogs of riboflavin failed to markedly affect the enzyme activity.     

Optimization of inactivation of endospores of Bacillus cereus by antimicrobial lipopeptides from Bacillus subtilis fmbj strains using a response surface method

Bacillus subtilis fmbj can produce a lipopeptide antimicrobial substance, the main components of which are surfactin and fengycin. In this paper, the sensitivity of Bacillus cereus to antimicrobial lipopeptides from B. subtilis fmbj was observed, and the effect of the microstructure of antimicrobial lipopeptide on spores of B. cereus was investigated. At the same time, the optimization of the inactivation of antimicrobial lipopeptides to spores of B. cereus by a response surface methodology was studied. Results showed that B. cereus had high sensitivity to it, whose minimal inhibitory concentration was 156.25 μg/ml. It could result in the death of spores by destroying the structure of resting spores and sprouting spores, as was observed by transmission electron microscopy. The optimization result indicated that spores of B. cereus could be inactivated by 2 orders of magnitude when the temperature was 29.6°C, the action time was 7.6 h, and the concentration was 3.46 mg·ml⁻¹.     

Genetic mapping by means of protoplast fusion in Bacillus subtilis

Summary A new mapping method involving protoplast fusion in Bacillus subtilis is described. Protoplasts from an isogenic standard marker strain containing purA and from a strain containing both purB and the marker, x, to be mapped were fused with polyethylene glycol, and purA ⁺ purB ⁺ fusants were selected. After isolation of single colonies and determination of unselected markers, marker x was mapped between two standard markers. This method was fully applicable to PBS1-resistant strains (e.g., lyt strains). The results obtained by protoplast fusion, conventional transformation and/or lysed protoplast transformation indicated that a lyt strain, Ni15, contained two new autolysin-minus mutations (lyt-151 and lyt-152). The properties of lyt-15 are also discussed.Abbreviations NTG N-methyl-N-nitro-N-nitrosoguanidine - SMM 0.5 M sucrose, 0.02 M MgCl₂, 0.02 M maleate buffer, pH 6.5