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PubMed2001

Effect of depletion of FtsY on spore morphology and the protein composition of the spore coat layer in Bacillus subtilis.

Bacillus subtilis FtsY is a homolog of the alpha-subunit of mammalian signal recognition particle (SRP) receptor, and is essential for protein translocation and vegetative cell growth. An FtsY conditional null mutant (strain ISR39) can express ftsY during the vegetative stage but not during spore formation. Spores of ISR39 have the same resistance to heat and chloroform as the wild-type, while their resistance to lysozyme is reduced. Electron microscopy showed that the outer coat of spores was incompletely assembled. The coat protein profile of the ftsY mutant spores was different from that of wild-type spores. The amounts of CotA, and CotE were reduced in spore coat proteins of ftsY mutant spores and the molecular mass of CotB was reduced. In addition, CotA, CotB, and CotE are present in normal form at T(8) of sporulation in ftsY mutant cells. These results suggest that FtsY has a pivotal role in assembling coat proteins onto the coat layer during spore morphogenesis.

Bacillus subtilis↗

PubMed2006

Maltose and maltodextrin utilization by Bacillus subtilis.

Bacillus subtilis can utilize maltose and maltodextrins that are derived from polysaccharides, like starch or glycogen. In this work, we show that maltose is taken up by a member of the phosphoenolpyruvate-dependent phosphotransferase system and maltodextrins are taken up by a maltodextrin-specific ABC transporter. Uptake of maltose by the phosphoenolpyruvate-dependent phosphotransferase system is mediated by maltose-specific enzyme IICB (MalP; synonym, GlvC), with an apparent K(m) of 5 microM and a V(max) of 91 nmol . min(-1) . (10(10) CFU)(-1). The maltodextrin-specific ABC transporter is composed of the maltodextrin binding protein MdxE (formerly YvdG), with affinities in the low micromolar range for maltodextrins, and the membrane-spanning components MdxF and MdxG (formerly YvdH and YvdI, respectively), as well as the energizing ATPase MsmX. Maltotriose transport occurs with an apparent K(m) of 1.4 microM and a V(max) of 4.7 nmol . min(-1) . (10(10) CFU)(-1).

Bacillus subtilis↗

PubMed2002

The products of the spoVA operon are involved in dipicolinic acid uptake into developing spores of Bacillus subtilis.

Bacillus subtilis cells with mutations in the spoVA operon do not complete sporulation. However, a spoVA strain with mutations that remove all three of the spore's functional nutrient germinant receptors (termed the ger3 mutations) or the cortex lytic enzyme SleB (but not CwlJ) did complete sporulation. ger3 spoVA and sleB spoVA spores lack dipicolinic acid (DPA) and have lower core wet densities and levels of wet heat resistance than wild-type or ger3 spores. These properties of ger3 spoVA and sleB spoVA spores are identical to those of ger3 spoVF and sleB spoVF spores that lack DPA due to deletion of the spoVF operon coding for DPA synthetase. Sporulation in the presence of exogenous DPA restored DPA levels in ger3 spoVF spores to 53% of the wild-type spore levels, but there was no incorporation of exogenous DPA into ger3 spoVA spores. These data indicate that one or more products of the spoVA operon are involved in DPA transport into the developing forespore during sporulation.

Bacillus subtilis↗

PubMed2004

Effect of loss of CheC and other adaptational proteins on chemotactic behaviour in Bacillus subtilis.

Bacillus subtilis has a more complex mechanism of chemotaxis than does the paradigm organism, Escherichia coli. In order to understand better the role of the novel chemotaxis proteins--CheC, CheD and CheV--mutants in which increasing numbers of the corresponding genes had been deleted were studied as tethered cells and their biases and sometimes durations of counterclockwise (CCW) and clockwise (CW) flagellar rotations in response to addition and removal of the attractant asparagine were observed. The cheC mutant was found to have considerably reduced switching frequency (that is, prolonged CCW and CW rotations) without a significantly different prestimulus CCW bias, compared with wild-type. This result may indicate that in absence of CheC the switch might be in a conformation less resembling the transition state than in presence of CheC. Conversely, the cheB (methylesterase) mutant showed considerably increased switching frequency without affecting CCW bias, compared with wild-type. Removal of all known adaptation systems--the methylation, CheC and CheV systems--resulted in a mutant (cheRBCDV) that still retained some adaptation following the addition of attractant.

Bacillus subtilis↗

PubMed1984

SP3: a flagellotropic bacteriophage of Bacillus subtilis.

Bacillus subtilis bacteriophage SP3 was shown to be flagellotropic. Mechanical deflagellation of host strain 168wt prevented phage adsorption but adsorption was normal when deflagellated 186wt was allowed sufficient time to regenerate flagella. Two host strains lacking flagella (130 fla- and 19 fla-) did not adsorb SP3. Two host strains with paralysed flagella (38mot- and 13mot-) adsorbed SP3 inefficiently. Revertants of fla- and mot- strains to the motile phenotype adsorbed SP3 normally. A temperature-sensitive host mutant, ts46, with reduced numbers of flagella at 46 degrees C was incapable of efficient SP3 adsorption at this temperature. However, transfection of ts46 by SP3 DNA occurred with virtually identical efficiency at either temperature. Adsorption of a non-flagellotropic phage, SP82, to all bacterial strains used was unaffected by absence of flagella. Isolated flagella adsorbed SP3 inefficiently. Spontaneously non-motile strains of 168wt had lost the ability to support growth of SP3 but SP82 grew normally. Revertants of these strains to the motile phenotype regained the ability to support SP3 growth. Strains derived from surviving bacteria in SP3 lysates of 168wt were non-motile.

Bacillus subtilis↗

PubMed1998

[Purification and properties of antifungal protein X98III from Bacillus subtilis].

Bacillus subtilis BS-98 is one of the antagonistic strains strongly against plant fungal pathogens such as Physalospora piricola Nose. The antifungal protein was purified by ammonium sulphate precipitation and column chromatography on Sephadex G-100 and DEAE-cellulose, and it was named X98III. Molecular weight of X98III is 59,000 by SDS-PAGE and PI value is 4.50 by PAG-IEF, respectively. X98III was demonstrated as glycoprotein and lipoprotein by CAM (cellulose acetate membrane) electrophoresis and special staining. We estimated it contains 6% saccharides by using DNS methods. This protein was also found to be thermostabale and partially sensitive to proteinases. The amino acid analysis of the protein X98III showed that it comprises of 11 different amino acids and Glu, Tyr, Cys are the abundant amino acids. No Asp, Phe and Met were found. Purified X98III has strong inhibiting activity against the pathogens of Physalospora piricola, Phoma asparagi, etc. The antifungal mechanism of X98III was mainly disintegration of the cell wall to make the hyphae abnormal and the spores germinate abnormally or can not germinate at all.

Amino Acids↗

PubMed1987

Kinetic characterization of cytochrome c oxidase from Bacillus subtilis.

Bacillus subtilis aa3-type cytochrome c oxidase is capable of oxidizing cytochrome c from different origins. The kinetic properties of the enzyme are influenced by ionic strength. The affinity for Saccharomyces cerevisiae cytochrome c declines with increasing ionic strength whereas the Vmax remains almost constant. An increase of Vmax is observed when the enzyme is incorporated in artificial membranes. Negatively charged phospholipids allow high turnover rates of the aa3-type oxidase. The effect of ionic strength on oxidation of horse heart cytochrome c results in significant changes of both Km and Vmax. These effects can be explained by disturbances of enzyme-substrate interactions and are not related to changes in the aggregation state of the enzyme. The respiration control index of the enzyme reconstituted in artificial membranes appeared to be dependent on phospholipid composition, protein/lipid ratios and also on the external pH. The action of the ionophores nigericin and valinomycin, at various pH values, on the enzyme activity and proton-permeability measurements of the membranes indicate that both components of the proton-motive force, the membrane potential and the pH gradient, can in principle regulate enzyme activity in the reconstituted state.

Animals↗

PubMed1994

Physiological and genetic characterization of the osmotic stress response in Bacillus subtilis.

Bacillus subtilis cultures submitted to an osmotic upshock (1.5 M NaCl) lysed unless stationary phase had been reached. Several physiological variations were observed, such as delayed growth (adaptation), a filamentous bacterial appearance, RecA-dependent osmoresistance (SOS), and cross-induction by a previous stress (heat shock). Osmoresistance and sporulation seem to share pathways of regulation such as inhibition in the presence of glucose and glutamine and derepression in a catabolite-resistant mutant such as degUh. However, spores were not obtained on hypertonic media. Mutants of later sporulation stages (spoII, spoIII) presented a response similar to that of the wild-type parent, indicating that both processes probably shared early controls. Null mutations in any of the known key modulators of sporulation (spoOA or degU) resulted in similar levels of osmosensitivity. Sensor mutations in kinA and degS also led to strains with altered responses, the kinA mutant being even more osmosensitive than the degS mutant. Several spoOA mutant phenotypes are due to this gene's control of abrB, a regulator of stationary-phase events, and an abrB mutation relieved the osmosensitivity of the spoOA-containing mutant but had no effect on a wild-type strain.

Adaptation, Physiological↗

PubMed1987

Plasmid marker rescue transformation proceeds by breakage-reunion in Bacillus subtilis.

Bacillus subtilis carrying a plasmid which replicates with a copy number of about 1 was transformed with linearized homologous plasmid DNA labeled with the heavy isotopes 2H and 15N, in the presence of 32Pi and 6-(p-hydroxyphenylazo)-uracil to inhibit DNA replication. Plasmid DNA was isolated from the transformed culture and fractionated in cesium chloride density gradients. The distribution of total and donor plasmid DNA was examined, using specific hybridization probes. The synthesis of new DNA, associated with the integration of donor moiety, was also monitored. Donor-specific sequences were present at a density intermediate between that of light and hybrid DNA. This recombinant DNA represented 1.4% of total plasmid DNA. The latter value corresponded well with the transforming activity (1.7%) obtained for the donor marker. Newly synthesized material associated with plasmid DNA at the recombinant density amounted to a minor portion of the recombinant plasmid DNA. These data suggest that, like chromosomal transformation, plasmid marker rescue transformation does not require replication for the integration of donor markers and, also like chromosomal transformation, proceeds by a breakage-reunion mechanism. The extent of donor DNA replacement of recipient DNA per plasmid molecule of 54 kilobases (27 kilobase pairs) was estimated as 16 kilobases.

Bacillus subtilis↗

PubMed2004

Hpr (ScoC) and the phosphorelay couple cell cycle and sporulation in Bacillus subtilis.

Bacillus subtilis sporulation is a developmental process that culminates in the formation of a highly resistant and persistent endospore. Inhibiting DNA synthesis prior to the completion of the final round of DNA replication blocks sporulation at an early stage. Conditions that prevent compartmentalization of gene expression, i.e. inhibition of asymmetric septum formation or chromosome partitioning, also block sporulation at an early stage. Multiple mechanisms including a RecA-dependent, a RecA-independent, and the soj-spo0J operon have been implicated in signal transduction, connecting DNA replication and chromosome partitioning to the onset of sporulation in B. subtilis. We suggest that a single mechanism involving Hpr (ScoC) and Sda couple cell cycle signaling to sporulation initiation. We show that transcription of phosphorelay sensory chain genes is adversely affected by post-exponential perturbation of the cell cycle. DNA replication arrest by chemical treatments, such as hydroxyphenylazouracil, hydroxyurea, nalidixic acid, and through genetic means using dnaA1ts and dnaB19ts temperature-sensitive mutants caused substantial down-regulation of spo0F and kinA expression and elevated the expression of spo0A and spo0H (sigH). Despite the elevation in spo0A expression, Spo0A approximately P-dependent sinI expression was substantially down-regulated indicating that in vivo Spo0A approximately P levels may be diminished. Similar alterations in gene expression patterns were observed in an ftsA279ts mutant background, indicating that cytokinesis and sporulation may also be coupled by a similar mechanism. Loss of function mutation in hpr (scoC) restored sporulation in a dnaA1ts mutant, blocked the DNA replication arrest induction of spo0A expression and restored expression of spo0F, kinA and sinI. Moreover, hpr expression was up-regulated in response to DNA replication arrest. The increase in hpr expression required Sda. These results suggest a role for Hpr (ScoC) in mediating the coupling of cell cycle events to the onset of sporulation.

Bacillus subtilis↗

PubMed1980

Extracellular and membrane-bound proteases from Bacillus subtilis.

Bacillus subtilis YY88 synthesizes increased amounts of extracellular and membrane-bound proteases. More than 99% of the extracellular protease activity is accounted for by an alkaline serine protease and a neutral metalloprotease. An esterase having low protease activity accounts for less than 1% of the secreted protease. These enzymes were purified to homogeneity. Molecular weights of approximately 28,500 and 39,500 were determined for the alkaline and neutral proteases, respectively. The esterase had a molecular weight of approximately 35,000. Amino-terminal amino acid sequences were determined, and the actions of a number of inhibitors were examined. Membrane vesicles contained bound forms of alkaline and neutral proteases and a group of previously undetected proteases (M proteases). Membrane-bound proteases were extracted with Triton X-100. Membrane-bound alkaline and neutral proteases were indistinguishable from the extracellular enzymes by the criteria of molecular weight, immunoprecipitation, and sensitivity to inhibitors. The M protease fraction accounted for approximately 7% of the total activity in Triton X-100 extracts of membrane vesicles. The M protease fraction was partially fractionated into four species (M1 through M4) by ion-exchange chromatography. Immunoprecipitation and sensitivity to inhibitors distinguished membrane-bound alkaline and neutral proteases from M proteases. In contrast to alkaline and neutral proteases, proteases M2 and M3 exhibited exopeptidase activity.

Amino Acid Sequence↗

PubMed1993

Expression, purification, and characterization of CTP:glycerol-3-phosphate cytidylyltransferase from Bacillus subtilis.

Bacillus subtilis contains the gene for CTP:glycerol-3-phosphate cytidylyltransferase, which is involved in biosynthesis of the major teichoic acid of the B. subtilis cell wall. When this gene was expressed in Escherichia coli under the control of the T7 promoter, the glycerol-3-phosphate cytidylyltransferase accumulated to a level of about 15% of cellular protein. The expressed glycerol-3-phosphate cytidylyltransferase was purified to homogeneity by ion-exchange chromatography, gel filtration, and affinity chromatography on blue Sepharose. Approximately 47 mg of pure enzyme was obtained from a 660-ml culture. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the subunit molecular weight of the purified enzyme was about 15,000. The molecular weight of the native enzyme was found to be 30,900 by gel filtration analysis, suggesting that the native enzyme is a homodimer. The pH optimum was very broad, from 6.5 to 9.5, and the enzyme was stable at alkaline conditions. A divalent cation, either Co2+, Mg2+, Mn2+, or Fe2+, was required for enzyme activity. Km values for CTP and glycerol 3-phosphate were 3.85 and 3.23 mM, respectively, and the Vmax was 185 units/mg of protein. Initial rate studies and product inhibition patterns indicated that the enzyme catalyzes the reaction by means of a rapid eqilibrium random order mechanism. The availability of large amounts of glycerol-3-phosphate cytidylyltransferase will facilitate enzymological and structural studies on this model cytidylyltransferase.

Amino Acid Sequence↗

PubMed1994

Multiple copies of the proB gene enhance degS-dependent extracellular protease production in Bacillus subtilis.

Bacillus subtilis secretes extracellular proteases whose production is positively regulated by a two-component regulatory system, DegS-DegU, and other regulatory factors including DegR. To identify an additional regulatory gene(s) for exoprotease production, we performed a shotgun cloning in the cell carrying multiple copies of degR and found a transformant producing large amounts of the exoproteases. The plasmid in this transformant, pLC1, showed a synergistic effect with multiple copies of degR on the production of the extracellular proteases, and it required degS for its enhancing effect. The DNA region responsible for the enhancement contained the proB gene, as shown by restriction analyses and sequence determination. The proB gene encoding gamma-glutamyl kinase was followed by the proA gene encoding glutamyl-gamma-semialdehyde dehydrogenase at an interval of 39 nucleotides, suggesting that the genes constitute an operon. pLC1 contained the complete proB gene and a part of proA lacking the proA C-terminal region. It was also found that proB on the chromosome showed a synergistic effect with multiple copies of degR. We consider on the basis of these results that the metabolic intermediate, gamma-glutamyl phosphate, would transmit a signal to DegS, resulting in a higher level of phosphorylated DegU. Possible involvement of DegR in this process is discussed.

1-Pyrroline-5-Carboxylate Dehydrogenase↗

PubMed1988

Gene encoding sigma E is transcribed from a sigma A-like promoter in Bacillus subtilis.

Bacillus subtilis produces several RNA polymerase sigma factors. At least two of these factors are essential for endospore formation, sigma H, which is present in vegetative cells, and sigma E, which is produced exclusively after the start of endospore formation. The structural gene that encodes sigma E is part of the spoIIG operon, which is transcribed after the onset of sporulation. We have determined the starting point of transcription and the nucleotide sequence of the spoIIG promoter. This promoter contains sequences that are similar to those found at the -10 and -35 regions of promoters that are used by E sigma A, the primary form of RNA polymerase in vegetative cells. The unusual feature of this promoter is that these putative sigma A contact sites are separated by 22 base pairs, rather than the typical 17 or 18 base pairs. Single-base substitutions in the -10-like sequence reduced utilization of the spoIIG promoter in vivo. Furthermore, E sigma A, but not E sigma H and other secondary forms of RNA polymerase, accurately initiated transcription from the spoIIG promoter in an in vitro assay; therefore, we suggest that E sigma A transcribes the spoIIG operon in vivo. A base substitution in the -35-like sequence caused constitutive transcription from the promoter in vegetative cells; therefore, regulation of this sporulation-specific transcription may involve a novel mechanism.

Bacillus subtilis↗

PubMed2004

DegU-P represses expression of the motility fla-che operon in Bacillus subtilis.

Bacillus subtilis implements several adaptive strategies to cope with nutrient limitation experienced at the end of exponential growth. The DegS-DegU two-component system is part of the network involved in the regulation of postexponential responses, such as competence development, the production of exoenzymes, and motility. The degU32(Hy) mutation extends the half-life of the phosphorylated form of DegU (DegU-P); this in turn increases the production of alkaline protease, levan-sucrase, and other exoenzymes and inhibits motility and the production of flagella. The expression of the flagellum-specific sigma factor SigD, of the flagellin gene hag, and of the fla-che operon is strongly reduced in a degU32(Hy) genetic background. To investigate the mechanism of action of DegU-P on motility, we isolated mutants of degU32(Hy) that completely suppressed the motility deficiency. The mutations were genetically mapped and characterized by PCR and sequencing. Most of the mutations were found to delete a transcriptional termination signal upstream of the main flagellar operon, fla-che, thus allowing transcriptional readthrough from the cod operon. Two additional mutations improved the sigmaA-dependent promoter sequence of the fla-che operon. Using an electrophoretic mobility shift assay, we have demonstrated that purified DegU binds specifically to the PA promoter region of the fla-che operon. The data suggest that DegU represses transcription of the fla-che operon, and they indicate a central role of the operon in regulating the synthesis and assembly of flagella.

Adaptation, Physiological↗

PubMed1987

Spectral and potentiometric analysis of cytochromes from Bacillus subtilis.

Bacillus subtilis cytoplasmic membranes contain several cytochromes which are linked to the respiratory chain. At least six different cytochromes have been separated and identified by ammonium sulphate fractionation and ion-exchange chromatography. They include two terminal oxidases with CO-binding properties and cyanide sensitivity. One of these is an aa3-type cytochrome c oxidase which has characteristic absorption maxima in the reduced-oxidized difference spectrum at 601 nm in the alpha-band and at 443 nm in the Soret band regions. In the alpha-band two separate electron transitions with Em = +205 mV and Em = +335 mV can be discriminated by redox potentiometric titration. The other CO-binding cytochrome c oxidase contains two cytochrome b components with alpha-band maxima at 556 nm and 559 nm. Cytochrome b556 can be reduced by ascorbate and has an Em + +215 mV, whereas cytochrome b559 has an Em = +140 mV. Furthermore a complex consisting of a cytochrome b564 (Em = +140 mV) associated with a cytochrome c554 (Em = +250 mV) was found. This cytochrome c554, which can be reduced by ascorbate, appears to have an asymmetrical alpha-peak and stains for heme-catalyzed peroxidase activity on SDS-containing polyacrylamide gels. A protein with a molecular mass of about 30 kDa is responsible for this activity. A cytochrome b559 (Em = +65 mV) appears to be an essential part of succinate dehydrogenase. Finally a cytochrome c550 component with an apparent mid-point potential of Em = +195 mV has been detected.

Ammonium Sulfate↗

PubMed1976

Purification and some properties of an extracellular maltase from Bacillus subtilis.

Bacillus subtilis P-11, capable of producing extracellular maltase, was isolated from soil. Maximum enzyme production was obtained on a medium containing 2.0% methyl-alpha-D-glucose, 0.5% phytone, and 0.2% yeast extract. After the removal of cells, extracellular maltase was precipitated by ammonium sulfate (85% saturation). The enzyme was purified by using the following procedures: Sephadex G-200 column chromatography, diethylaminoethyl-Sephadex A-50 ion-exchange column chromatography, and a second Sephadex G-200 column chromatography. A highly purified maltase without amylase or proteinase activities was obtained. Some properties of the extracellular maltase were determined: optimum pH, 6.0; optimum temperature, 45 C, when the incubation time was 30 min; pH stability, within 5.5 to 6.5; heat stability, stable up to 45 C; isoelectric point, pH 6.0 (by gel-isoelectric focusing); molecular weight, 33,000 (by gel filtration with Sephadex G-200); substrate specificity: the relative rates of hydrolysis of maltose, maltotriose, isomaltose, and maltotetraose were 100:15:14:4, respectively, and there was no activity toward alkyl or aryl-alpha-D-glucosides, amylose, or other higher polymers. Transglucosylase activity was present. Glucose and tris(hydroxymethyl)aminomethane were competitive inhibitors with Ki values of 4.54 and 75.08 mM, respectively; cysteine was a noncompetitive inhibitor. Michaelis constants were 5 mM for maltose, 1 mM for maltoriose, and 10 mM for isomaltose. A plot of pKm (-log Km) versus pH revealed two deflection points, one each at 5.5 and 6.5; these probably corresponded to an imidazole group of a histidine residue in or near the active center; this assumption was supported by the strong inhibition of enzyme activity by rose bengal.

Ammonium Sulfate↗

PubMed2004

Transcriptional regulation of genes encoding arabinan-degrading enzymes in Bacillus subtilis.

Bacillus subtilis produces hemicellulases capable of releasing arabinosyl oligomers and arabinose from plant cell walls. In this work, we characterize the transcriptional regulation of three genes encoding arabinan-degrading enzymes that are clustered with genes encoding enzymes that further catabolize arabinose. The abfA gene comprised in the metabolic operon araABDLMNPQ-abfA and the xsa gene located 23 kb downstream most probably encode alpha-L-arabinofuranosidases (EC 3.2.1.55). Here, we show that the abnA gene, positioned immediately upstream from the metabolic operon, encodes an endo-alpha-1,5-arabinanase (EC 3.2.1.99). Furthermore, by in vivo RNA studies, we inferred that abnA and xsa are monocistronic and are transcribed from sigma(A)-like promoters. Transcriptional fusion analysis revealed that the expression of the three arabinases is induced by arabinose and arabinan and is repressed by glucose. The levels of induction by arabinose and arabinan are higher during early postexponential growth, suggesting a temporal regulation. Moreover, the induction mechanism of these genes is mediated through negative control by the key regulator of arabinose metabolism, AraR. Thus, we analyzed AraR-DNA interactions by in vitro quantitative DNase I footprinting and in vivo analysis of single-base-pair substitutions within the promoter regions of xsa and abnA. The results indicate that transcriptional repression of the abfA and xsa genes is achieved by a tightly controlled mechanism but that the regulation of abnA is more flexible. We suggest that the expression of genes encoding extracellular degrading enzymes of arabinose-containing polysaccharides, transport systems, and intracellular enzymes involved in further catabolism is regulated by a coordinate mechanism triggered by arabinose via AraR.

AraC Transcription Factor↗