Cold Shock of Germinating Bacillus subtilis Spores

Susceptibility of germinating spores of Bacillus subtilis to a rapid chilling was examined by viable countings. Dormant spores were quite resistant to the cold shock but the spores, immediately upon germination, lose viability almost completely by the same treatment. The presence of divalent cation, magnesium, calcium or manganese, in a buffer to which the germinating spores were suspended, markedly protected the cells from the death by the cold shock effect. When the shocked cells were incubated in the buffer containing casein acid hydrolyzate, glucose and magnesium ion for short period of time, a remarkable increase in viable counts was observed. The existence of two critical temperature zones, which were determined by the initial temperature of cell suspension, was confirmed in the cold shock of germinating spores.     

The lytE Gene of Bacillus subtilis 168 Encodes a Cell Wall Hydrolase

Bacillus subtilis cell wall-bound protein CWBP33 is encoded by lytE, a gene expressed during the exponential growth phase. Sequence analysis of LytE, a 33-kDa protein, reveals two domains. The N-terminal domain contains a threefold-repeated motif common to several peptidoglycan binding proteins, while the C-terminal domain, probably carrying the catalytic activity, has homology with certain exoproteins. Zymographs unambiguously reveal that the absence of CWBP33, due to inactivation of lytE, is accompanied by the loss of a lytic activity. In lytE mutants, the cell autolysis rate is significantly decreased, although autolysis of corresponding, purified cell walls does not seem to be affected.     

Phospholipid Composition of Bacillus subtilis

Bacillus subtilis contained at least five phospholipids, four of which have been isolated and identified as a polyglycerol phospholipid, probably cardiolipin, phosphatidylglycerol, phosphatidylethanolamine, and lysylphosphatidylglycerol. Further purification of the latter phosphoglyceride was obtained by high-voltage electrophoresis, and it was shown that this treatment removed amino acid-containing, nonlipidic material from the phosphoglyceride. This associated material, which is not covalently linked to the lipid, gave rise to minor amounts of a number of amino acids, other than lysine, in acid hydrolysates of the lysylphosphatidylglycerol. The phospholipid composition of B. subtilis appeared to depend on the growth conditions. Addition of glucose to the medium lowered the pH during growth; this was accompanied by an increase in the amount of lysylphosphatidylglycerol and a decrease in the phosphatidylglycerol content, when compared with growth at neutral pH. The amount of the other phospholipids and the total amount of phospholipid remained constant under the different conditions. The shape and the osmotic susceptibility of the protoplasts of this organism appeared to depend on the growth conditions. Cells harvested from a neutral growth medium gave spherical protoplasts which lysed rapidly, whereas cells grown in an acidic medium maintained their rod-shaped form to a great extent after the cell wall had been removed, even after being suspended in a hypotonic medium. The latter observation suggests the presence of a more rigid membranous structure in cells which have been exposed to a low environmental pH during growth.     

recE-Dependent Gene Amplification Induced by a Tunicamycin-resistant Mutation (tmr A7) in Bacillus subtilis

A recombinant Bacillus subtilis phage, ρ11-AA248, contains the tmr A7-amy R2-amy E⁺-tmr B⁺-aroI⁺ region of the B. subtilis N7 chromosome on a 22.4 kb DNA fragment. The amy E⁺-tmr B⁺ gene region in the phage genome of the B. subtilis 207-21 transductants by ρ11-AA248 was amplified to approximately 10 copies after cultivation in the presence of tunicamycin (10 μg/ml) and to two copies without tunicamycin. The amplification of the gene region caused hyper-production of extracellular α-amylase. In contrast, no amplification of the gene region was detected in the transductants of B. subtilis 207-25, a recE-deficient derivative of 207-21 strain.     

The Bacillus subtilis ydjL (bdhA) Gene Encodes Acetoin Reductase/2,3-Butanediol Dehydrogenase

Bacillus subtilis is capable of producing 2,3-butanediol from acetoin by fermentation, but to date, the gene encoding the enzyme responsible, acetoin reductase/2,3-butanediol dehydrogenase (AR/BDH), has remained unknown. A search of the B. subtilis genome database with the amino acid sequences of functional AR/BDHs from Saccharomyces cerevisiae and Bacillus cereus resulted in the identification of a highly similar protein encoded by the B. subtilis ydjL gene. A knockout strain carrying a ydjL::cat insertion mutation was constructed, which (i) abolished 2,3-butanediol production in early stationary phase, (ii) produced no detectable AR or BDH activity in vitro, and (iii) accumulated the precursor acetoin in early stationary phase. The ydjL::cat mutation also affected the kinetics of lactate but not acetate production during stationary-phase cultivation with glucose under oxygen limitation. A very small amount of 2,3-butanediol was detected in very-late-stationary-phase (96-hour) cultures of the ydjL::cat mutant, suggesting the existence of a second gene encoding a minor AR activity. From the data, it is proposed that the major AR/BDH-encoding gene ydjL be renamed bdhA.     

Involvement of Membrane Lipids in Cold Shock-induced Autolysis of Bacillus subtilis Cells

Exponentially growing Bacillus subtilis cells autolysed when exposed to cold shock treatment in minimal medium followed by incubation at 37°C. From characteristics of the lysis, it was suggested that the cold-shock-induced cell lysis resulted from the perturbation of membrane organization that is initiated by rapid changes in temperature, lipid phase transitions. For maximum lysis induction to occur, in addition to rapid cooling to 5°C or lower, retention at temperatures lower than 10°C for at least 20 min is required. The cell sensitivity to the autolysis induction by cold shock was different between cells grown at 25°C and cells grown at 37°C. Analyses of the fatty acid composition and the phase transition temperature of membrane lipids suggested that the membrane fluidity may affect the autolysis induction. Experiments to discover the effects of cerulenin treatment and lipid addition on autolysis induction and the autolysin activity level support the hypothesis that membrane lipids are involved in cold-shock-induced cell autolysis.     

Cold shock response in Bacillus subtilis

Following a rapid decrease in temperature, the physiology of Bacillus subtilis cells changes profoundly. Cold shock adaptation has been monitored at the level of membrane composition, adjustment in DNA topology, and change in cytosolic protein synthesis/composition. Some major players in these processes (cold-stress induced proteins and cold acclimatization proteins, CIPs and CAPs) have been identified and mechanisms in cold shock acclimatization begin to emerge; however, important questions regarding their cellular function still need to be answered.     

Phospholipid Metabolism in the Absence of Net Phospholipid Synthesis in a Glycerol-Requiring Mutant of Bacillus subtilis

A glycerol-requiring auxotroph of Bacillus subtilis showed no net synthesis of phospholipid when deprived of glycerol. Although there was no net synthesis of phospholipid, we found that: (i) fatty acids and (32)P were slowly incorporated into phospholipid; (ii) in pulse-chase experiments, both (32)P and (14)C in the glycerol portion of the phospholipids were lost from phosphatidlyglycerol (PG) and lysylphosphatidylglycerol and accumulated in cardiolipin (CL); (iii) the proportions of the phospholipids in the membrane changed with a loss of PG and an accumulation of CL. The addition of glycerol to the glycerol-deprived cells resulted in a rapid incorporation of glycerol and restoration to the predeprivation metabolism and PG to CL ratio.     

氮离子诱变筛选内切葡聚糖酶高产菌株及其基因突变研究

目的：离子注入枯草芽孢杆菌筛选高产内切葡聚糖酶突变菌株,同时进行其酶活性研究,并克隆该基因,研究离子注入对其诱变效应。方法：低能氮离子重复注入枯草芽孢杆菌,筛选获得1株高产内切葡聚糖酶突变菌株Bac11。DNS法测定酶活性。PCR扩增获得出发菌株Bac01和突变菌株Bac11内切葡聚糖酶基因,并对核酸序列及预测氨基酸序列进行多重比对。结果：突变菌株Bac11内切葡聚糖酶活性从93.33IU提高到381.89IU。多重比对Bac01和Bac11内切葡聚糖酶基因编码区1500bp序列,当中有10个碱基发生突变,预测氨基酸序列中有5个氨基酸残基发生变化,且都在其基因纤维素结合域部分。结论：低能氮离子重复注入对枯草芽孢杆菌内切葡聚糖酶活性及其基因有明显的诱变累加效应。     

Gene amplification in Bacillus subtilis

A strain of Bacillus subtilis that carries in its genome a staphylococcal chloramphenicol acetyltransferase gene (from pC194) responds to growth at different concentrations of chloramphenicol by an alteration in the number of copies per genome of the sequences encoding the gene. Growth at 20 micrograms chloramphenicol ml-1 results in a 15-fold amplification of the sequences, whereas growth in the absence of chloramphenicol results in their loss. The mechanism of in situ amplification probably has much in common with that involved in 'R factor transitioning'. The hybridization procedures that have been used for accurately determining the number of copies of the amplified DNA sequences are potentially useful for plasmid copy number determination. The findings reported here also provide a potentially useful alternative to more conventional cloning strategies that are based on autonomous plasmids in B. subtilis. The particular advantages that can be envisaged include enhanced stability of the cloned sequences and control of the number of copies that are present.     

Molecular Characterization of a Barley Gene Induced by Cold Treatment

A cDNA library was made from low positive temperature (6 ?C/2?C) grown barley shoot meristems. Several genes which are differentiallyexpressed, as measured by mRNA abundance, were selected fromthe library using a differential screen. This paper reportsan analysis of in vivo expression in several cultivars, theDNA sequence, copy number and chromosomal location of one gene(BLT14). In addition, genomic restriction fragment length polymorphismfor this gene in the 10 most widely UK-grown spring and 9 mostwidely UK-grown winter barley cultivars is analysed. Key words: Hordeum vulgare, low temperature, RFLP, differential expression, cDNA sequence     

Membrane fluidization by alcohols inhibits DesK-DesR signalling in Bacillus subtilis

After cold shock, the Bacillus subtilis desaturase Des introduces double bonds into the fatty acids of existing membrane phospholipids. The synthesis of Des is regulated exclusively by the two-component system DesK/DesR; DesK serves as a sensor of the state of the membrane and triggers Des synthesis after a decrease in membrane fluidity. The aim of our work is to investigate the biophysical changes in the membrane that are able to affect the DesK signalling state. Using linear alcohols (ethanol, propanol, butanol, hexanol, octanol) and benzyl alcohol, we were able to suppress Des synthesis after a temperature downshift. The changes in the biophysical properties of the membrane caused by alcohol addition were followed using membrane fluorescent probes and differential scanning calorimetry.We found that the membrane fluidization induced by alcohols was reflected in an increased hydration at the lipid-water interface. This is associated with a decrease in DesK activity. The addition of alcohol mimics a temperature increase, which can be measured isothermically by fluorescence anisotropy. The effect of alcohols on the membrane periphery is in line with the concept of the mechanism by which two hydrophilic motifs located at opposite ends of the transmembrane region of DesK, which work as a molecular caliper, sense temperature-dependent variations in membrane properties.     

The ripX Locus of Bacillus subtilis Encodes a Site-Specific Recombinase Involved in Proper Chromosome Partitioning

The Bacillus subtilis ripX gene encodes a protein that has 37 and 44% identity with the XerC and XerD site-specific recombinases of Escherichia coli. XerC and XerD are hypothesized to act in concert at the dif site to resolve dimeric chromosomes formed by recombination during replication. Cultures of ripX mutants contained a subpopulation of unequal-size cells held together in long chains. The chains included anucleate cells and cells with aberrantly dense or diffuse nucleoids, indicating a chromosome partitioning failure. This result is consistent with RipX having a role in the resolution of chromosome dimers in B. subtilis. Spores contain a single uninitiated chromosome, and analysis of germinated, outgrowing spores showed that the placement of FtsZ rings and septa is affected in ripX strains by the first division after the initiation of germination. The introduction of a recA mutation into ripX strains resulted in only slight modifications of the ripX phenotype, suggesting that chromosome dimers can form in a RecA-independent manner in B. subtilis. In addition to RipX, the CodV protein of B. subtilis shows extensive similarity to XerC and XerD. The RipX and CodV proteins were shown to bind in vitro to DNA containing the E. coli dif site. Together they functioned efficiently in vitro to catalyze site-specific cleavage of an artificial Holliday junction containing a dif site. Inactivation of codV alone did not cause a discernible change in phenotype, and it is speculated that RipX can substitute for CodV in vivo.     

Nucleotide Sequence of the Gene That Encodes a Neopullulanase from an Alkalophilic Bacillus

A spore cortex-lytic enzyme was purified in an active form from the exudate of fully germinated spores of Clostridium perfringens S40. The enzyme caused attenuation of absorbance in coatless spore suspensions and phase-darkening of the spores, but had minimal activity on isolated peptidoglycan fragments. The enzyme was identified as a 31 kDa protein which is probably an N-acetylmuramyl-L-alanine amidase. The amino-terminal 15 residues of the enzyme were: VLPEPVVPEYIVVHN.     

Phospholipid Metabolism and Membrane Synthesis During Sporulation in Bacillus megaterium

In view of previously published reports of localized membrane growth in exponentially growing Bacillus megaterium and in sporulating Bacillus cereus, an attempt was made to describe phospholipid metabolism and the topology of membrane synthesis during sporulation in B. megaterium. The cells were pulsed with radioactive glycerol or acetate at the time of septum formation, and the specific activity of the lipid fraction was measured at various times through the free spore stage. The bulk of the material labeled during septation could not be recovered in the spore. Rather, it was found that the labeled lipid fraction underwent considerable turnover during spore development. Additionally, other experiments revealed that the lipid made before the initiation of sporulation was also subject to extensive turnover. In order to minimize both the confounding effects of lipid turnover and the possible presence of lateral diffusion of labeled lipid in the membrane, autoradiography of cells pulse labeled with radioactive glycerol at the time of septation was performed; a symmetrical grain distribution resulted. Thus, despite previously published suggestions to the contrary, the current experimental techniques could not demonstrate the existence of localized membrane synthesis in B. megaterium during sporulation.     

枯草芽孢杆菌碱性蛋白酶基因的克隆和表达

目的:获得碱性蛋白酶基因。方法:用PCR的方法从枯草芽孢杆菌A-109中扩增碱性蛋白酶基因(apr),并进行测序分析,构建表达载体,最后转化大肠杆菌BL21,SDS-聚丙烯酰胺凝胶电泳检测该基因的表达情况。结果:apr基因片段含1092个碱基对。该基因片段核苷酸序列与Bacillus amyloliquefaciens subtilisin DFE precursor有99%的同源性,对应的氨基酸序列与Bacillussp.DJ-4有99%的同源性。apr基因在大肠杆菌BL21中获得表达,并表现出蛋白酶活性。结论:获得了具有活性的新的碱性蛋白酶基因。