Mechanisms of Bacillus subtilis spore resistance to and killing by aqueous ozone

AIMS: To determine the mechanisms of Bacillus subtilis spore killing by and resistance to aqueous ozone. METHODS AND RESULTS: Killing of B. subtilis spores by aqueous ozone was not due to damage to the spore's DNA, as wild-type spores were not mutagenized by ozone and wild-type and recA spores exhibited very similar ozone sensitivity. Spores (termed alpha-beta-) lacking the two major DNA protective alpha/beta-type small, acid-soluble spore proteins exhibited decreased ozone resistance but were also not mutagenized by ozone, and alpha-beta- and alpha-beta-recA spores exhibited identical ozone sensitivity. Killing of spores by ozone was greatly increased if spores were chemically decoated or carried a mutation in a gene encoding a protein essential for assembly of the spore coat. Ozone killing did not cause release of the spore core's large depot of dipicolinic acid (DPA), but these killed spores released all of their DPA after a subsequent normally sublethal heat treatment and also released DPA much more readily when germinated in dodecylamine than did untreated spores. However, ozone-killed spores did not germinate with either nutrients or Ca(2+)-DPA and could not be recovered by lysozyme treatment. CONCLUSIONS: Ozone does not kill spores by DNA damage, and the major factor in spore resistance to this agent appears to be the spore coat. Spore killing by ozone seems to render the spores defective in germination, perhaps because of damage to the spore's inner membrane. SIGNIFICANCE AND IMPACT OF THE STUDY: These results provide information on the mechanisms of spore killing by and resistance to ozone.     

Construction of spore mutants of Bacillus subtilis for the development as a host for foreign protein production

For the development of Bacillus subtilis as a host for foreign protein synthesis, three types of sigma factor deleted mutants (spoIIAC, spoIIIG and spoIIIC) were constructed by antibiotic marker insertion using plasmid vector-mediated method or LFH (Long Flanking Homology)-PCR. Mother cell specific sigma factor mutants of B. subtilis (^K), B. subtilis DB104 spoIIIC (km ^r)::pMK101, had two to three times higher subtilisin activity than the wild type DB104::pMK101. Subtilisin expression by the other two mutants, B. subtilis DB104 spoIIAC (km ^r)::pMK101 and DB104 spoIIIG (km ^r)::pMK101, which are pre-spore specific sigma factor (^F and ^G) deleted strains, was similar to, or less than that of the wild type.     

Interactions between Bacillus subtilis early spore coat morphogenetic proteins

When challenged by stresses such as starvation, the soil bacterium Bacillus subtilis produces an endospore surrounded by a proteinaceous coat composed of >70 proteins that are organized into three main layers: an amorphous undercoat, lightly staining lamellar inner coat and electron-dense outer coat. This coat protects the spore against a variety of chemicals or lysozyme. Mutual interactions of the coat's building blocks are responsible for the formation of this structurally complex and extraordinarily resistant shell. However, the assembly process of spore coat proteins is still poorly understood. In the present work, the main focus is on the three spore coat morphogenetic proteins: SpoIVA, SpoVID and SafA. Direct interaction between SpoIVA and SpoVID proteins was observed using a yeast two-hybrid assay and verified by coexpression experiment followed by Western blot analysis. Coexpression experiments also confirmed previous findings that SpoVID and SafA directly interact, and revealed a novel interaction between SpoIVA and SafA. Moreover, gel filtration analysis revealed that both SpoIVA and SpoVID proteins form large oligomers.     

Enhanced production of alpha-amylase in fed-batch cultures of Bacillus subtilis TN106[pAT5

Growth of Bacillus subtilis TN106[pAT5] and synthesis of plasmid-encoded protein (alpha-amylase) are investigated in batch, continuous, and fed-batch cultures using a defined medium containing glucose and/or starch as the carbohydrate source. The batch culture studies reveal that reduced availability of arginine hampers growth of recombinant cells (which lack an arginine synthesis gene) but promotes production of alpha-amylase and substitution of glucose by starch as the carbohydrate source leads to slower growth of recombinant cells and increased production of alpha-amylase per unit cell mass. Retention of recombinant cells over prolonged periods in continuous cultures is not possible without continuous application of antibiotic selection pressure owing to segregational plasmid instability. Fed-batch experiments with constant volumetric feed rate demonstrate that alpha-amylase production is enhanced at lower feed concentration of starch (sole carbohydrate source) and lower volumetric feed rate. Such slow addition of starch is however not conducive for growth of recombinant cells. The expression of the thermostable alpha-amylase gene carried on the recombinant plasmid pAT5 (derived from a plasmid isolated from a thermophilic bacterium) is promoted at higher temperatures, while growth of recombinant cells is depressed. In all batch and fed-batch experiments, production of alpha-amylase is observed to be inversely related to growth of recombinant cells. The efficacy of two-stage bioreactor operations, with growth of recombinant cells being promoted in the first stage and alpha-amylase production in the second stage, in attaining increased bulk alpha-amylase activity is demonstrated. (c) 1993 John Wiley & Sons, Inc.     

Immunological detection of the GerA spore germination proteins in the spore integuments of Bacillus subtilis using scanning electron microscopy

Abstract To clarify the molecular mechanisms that trigger spore germination of Bacillus subtilis , the location of GerA proteins (GerAA, GerAB and GerAC), which were reported to be putative gene products of a receptor for one of the germinants, l-alanine, was investigated by immunological techniques using anti-GerA peptide antibodies. Four antibodies were raised against the corresponding epitopes, two in GerAA, one in GerAB and the other in GerAC molecules. The binding of all four antibodies to the inner surface of the cortex-less spore coat fragments could be seen by scanning immunoelectron microscopy with colloidal gold particles. The result agreed with the fact, previously reported, that the colloidal gold particles were visualized just inside the spore coat layer by transmission immunoelectron microscopy using another anti-GerAB peptide antibody.     

四溴甘脲对枯草杆菌黑色变种芽胞损伤作用的电镜观察

为探索四溴甘脲消毒剂杀灭细菌的机理,采用透射电镜技术对四溴甘脲消毒剂处理过的枯草杆菌黑色变种芽胞的超微结构进行了分析和比较.结果显示,以含有效溴274mg/L的四溴甘脲消毒剂作用30min,可使枯草杆菌黑色变种芽胞杀灭率达到100%.在透射电镜下观察到,经该消毒剂作用的枯草杆菌黑色变种芽胞壳质破损断裂明显,壳内结构模糊,核心溶解,有的芽胞近似空壳.结果显示,四溴甘脲消毒剂杀灭芽胞效果优于普通含氯消毒剂,对细菌芽胞超微结构破坏明显.     

Mechanisms of Bacillus subtilis spore killing by and resistance to an acidic Fe-EDTA-iodide-ethanol formulation

AIMS: To determine the mechanisms of Bacillus subtilis spore killing by and resistance to an acidic solution containing Fe(3+), EDTA, KI and ethanol termed the KMT reagent. METHODS AND RESULTS: Wild-type B. subtilis spores were not mutagenized by the KMT reagent but the wild-type and recA spores were killed at the same rate. Spores (alpha(-)beta(-)) lacking most DNA-protective alpha/beta-type small, acid-soluble spore proteins were less resistant to the KMT reagent than wild-type spores but were also not mutagenized, and alpha(-)beta(-) and alpha(-)beta(-)recA spores exhibited nearly identical resistance. Spore resistance to the KMT reagent was greatly decreased if spores had defective coats. However, the level of unsaturated fatty acids in the inner membrane did not determine spore sensitivity to the KMT reagent. Survivors in spore populations killed by the KMT reagent were sensitized to killing by wet heat or nitrous acid and to high salt in plating medium. KMT reagent-killed spores had not released their dipicolinic acid (DPA), although these killed spores released their DPA more readily when germinated with dodecylamine than did untreated spores. However, KMT reagent-killed spores did not germinate with nutrients or Ca(2+)-DPA and were recovered only poorly by lysozyme treatment in a hypertonic medium. CONCLUSIONS: The KMT reagent does not kill spores by DNA damage and a major factor in spore resistance to this reagent is the spore coat. KMT reagent treatment damages the spore's ability to germinate, perhaps by damaging the spore's inner membrane. However, this damage is not oxidation of unsaturated fatty acids. SIGNIFICANCE AND IMPACT OF THE STUDY: These results provide information on the mechanism of spore resistance to and killing by the KMT reagent developed for killing Bacillus spores.     

The safety of Bacillus subtilis and Bacillus indicus as food probiotics

Aims:  To conduct in vitro and in vivo assessments of the safety of two species of Bacillus , one of which, Bacillus subtilis , is in current use as a food supplement.
Methods and Results:  Cultured cell lines, Caco-2, HEp-2 and the mucus-producing HT29-16E cell line, were used to evaluate adhesion, invasion and cytotoxicity. The Natto strain of B. subtilis was shown to be able to invade and lyse cells. Neither species was able to adhere significantly to any cell line. The Natto strain was also shown to form biofilms. No strain produced any of the known Bacillus enterotoxins. Disc-diffusion assays using a panel of antibiotics listed by the European Food Safety Authority (EFSA) showed that only Bacillus indicus carried resistance to clindamycin at a level above the minimum inhibitory concentration breakpoints set by the EFSA. In vivo assessments of acute and chronic dosing in guinea pigs and rabbits were made. No toxicity was observed in animals under these conditions.
Conclusions:  Bacillus indicus and B. subtilis should be considered safe for oral use although the resistance of B. indicus to clindamycin requires further study.
Significance and Impact of the Study:  The results support the use of B. subtilis and B. indicus strains as food supplements.     

枯草芽孢杆菌C1-B941的D-核糖发酵中间试验

使用转酮醇酶变异株—枯草芽孢杆菌C1－B941进行了D－核糖发酵中间试验。3000L发酵罐试验结果表明,发酵培养基中的葡萄糖浓度为18％时,发酵周期约为64h,发酵转化率达36．84％。发酵液经离子交换树脂纯化后,可以直接用于生产VBZ合成的中间体—N－D－核糖醇基－3,4－二甲苯胶。     

Characterization of Bacillus subtilis spore inactivation in low‐pressure,low‐temperature gas plasma sterilization processes

Aims: To identify structural components of Bacillus subtilis spores serving as targets for sterilization with microwave induced low‐pressure, low‐temperature nitrogen‐oxygen plasma. Methods and Results: The inactivation of spores followed a biphasic kinetics consisting of a log‐linear phase with rapid inactivation followed by a slow inactivation phase. In the course of plasma treatment, damage to DNA, proteins and spore membranes were observed by monitoring the occurrence of auxotrophic mutants, inactivation of catalase (KatX) activity and the leakage of dipicolinic acid, respectively. Spores of the wild‐type strain showed the highest resistance to plasma treatment. Spores of mutants defective in nucleotide excision repair (uvrA) and small acid‐soluble proteins (ΔsspA ΔsspB) were more sensitive than those defective in the coat protein CotE or spore photoproduct repair (splB). Exclusion of reactive particles and spectral fractions of UV radiation from access to the spores revealed that UV‐C radiation is the most effective inactivation agent in the plasma, whereby the splB and ΔcotE mutant spores were equally and slightly less sensitive, respectively, than the wild‐type spores. Finally, the extent of damages in the spore DNA determined by quantitative PCR correlated with the spore inactivation. Conclusions: Spore inactivation was efficiently mediated by a combination of DNA damage and protein inactivation. DNA was identified to be the primary target for spore inactivation by UV radiation emitted by the plasma. Coat proteins were found to constitute a protective layer against the action of the plasma. Significance and Impact of the Study: The results provide new evidence to the understanding of plasma sterilization processes. This knowledge supports the identification of useful parameters for novel plasma sterilization equipment to control process safety.     

Studies on the mechanism of the osmoresistance of spores of Bacillus subtilis

AIMS: To determine the reason that spores of Bacillus species, in particular Bacillus subtilis, are able to form colonies with high efficiency on media with very high salt concentrations. METHODS AND RESULTS: Spores of various Bacillus species have a significantly higher plating efficiency on media with high salt concentration (termed osmoresistance) than do log or stationary phase cells. This spore osmoresistance is higher on richer media. Bacillus subtilis spores lacking various small, acid-soluble spore proteins (SASP) were generally significantly less osmoresistant than were wild-type spores, as shown previously (Ruzal et al. 1994). Other results included: (a) spore osmoresistance varied significantly between species; (b) the osmoresistance of spores lacking SASP was not restored well by amino acid osmolytes added to plating media, but was completely restored by glucose; (c) the osmoresistance of spores lacking SASP was restored upon brief germination in the absence of salt in a process that did not require protein synthesis; (d) significant amounts of amino acids generated by SASP degradation were retained within spores upon germination in a medium with high but not low salt; (e) slowing but not abolishing SASP degradation by loss of the SASP-specific germination protease (GPR) did not affect spore osmoresistance; (f) sporulation at higher temperatures produced less osmoresistant spores; and (g) spore osmoresistance was not decreased markedly by the absence of the stress sigma factor for RNA polymerase, sigmaB. CONCLUSIONS: Spore osmoresistance appears as a result of three major factors: (1) specific characteristics of spores and cells of individual species; (2) the precise sporulation conditions that produce the spores; and (3) sufficient energy generation by the germinating and outgrowing spore to allow the spore to adapt to conditions of high osmotic strength; the substrates for this energy generation can come from either the endogenous generation of amino acids by SASP degradation or from the spore's environment, in the form of a readily taken up and metabolized energy source such as glucose. SIGNFICANCE AND IMPACT OF STUDY: These results provide information on the mechanisms of spore osmoresistance, a spore property that can be of major applied significance given the use of high osmotic strength with or without high salt as a means of food preservation.     

Screening for Bacillus subtilis mutants deficient in pressure induced spore germination: identification of ykvU as a novel germination gene

Exposure to high pressure induces germination in spores of Bacillus subtilis. To investigate the mechanisms of this process and to compare the pressure and nutrient induced germination pathways, a random transposon knock-out library of B. subtilis was constructed and screened for clones with a compromised pressure induced germination at 100 MPa. Two mutants were isolated and their transposon insertion was mapped to gerAC and ykvU respectively. While GerAC is required for production of the l-alanine receptor which has been implicated in pressure-induced germination before, YkvU is shown here to be a novel germination determinant in B. subtilis, affecting germination by high (100 MPa) and very high (600 MPa) pressure, by nutrients and by dodecylamine, but not by Ca(2+)-dipicolinic acid.     

大蒜汁对枯草芽孢杆菌抑制作用的研究

通过测定菌体浓度、抑菌圈直径和2,6-吡啶二羧酸(DPA)含量,研究大蒜汁对枯草芽孢杆菌(BS)的营养体及芽孢生长、发芽的影响,并采用响应面分析法优化确定大蒜汁抑菌适宜处理条件.结果表明:(1)大蒜汁对BS的最低抑菌浓度(MIC)和最低杀菌浓度(MBC)分别为0.4%和1%;(2)大蒜汁抑制作用主要是延长了BS的生长延缓期,0.3%的大蒜汁可使BS延缓期增加12 h;(3)大蒜汁对BS的芽孢和DPA形成有明显的抑制作用,但对芽孢的发芽无抑制作用;(4)加热温度超过35℃、时间大于5 h时处理的大蒜汁,对BS的抑制作用明显降低.在pH 3～8范围的大蒜汁都有很好的抑菌活性,但pH＞8.5时抑菌活性急剧下降;(5)响应面试验分析法优化确立了大蒜汁对BS抑制的二次回归方程和适宜处理条件,即在pH 4.5、温度45℃加热处理5 h的大蒜汁抑菌效果最好.     

Effects of modification of membrane lipid composition on Bacillus subtilis sporulation and spore properties

Aims: To determine effects of inner membrane lipid composition on Bacillus subtilis sporulation and spore properties. Methods and Results: The absence of genes encoding lipid biosynthetic enzymes had no effect on B. subtilis sporulation, although the expected lipids were absent from spores’ inner membrane. The rate of spore germination with nutrients was decreased c. 50% with mutants that lacked the major cardiolipin (CL) synthase and another enzyme for synthesis of a major phospholipid. Spores lacking the minor CL synthase or an enzyme essential for glycolipid synthesis exhibited 50–150% increases in rates of dodecylamine germination, while spores lacking enzymes for phosphatidylethanolamine (PE), phosphatidylserine (PS) and lysylphosphatidylglycerol (l‐PG) synthesis exhibited a 30–50% decrease. Spore sensitivity to H₂O₂ and tert‐butylhydroperoxide was increased 30–60% in the absence of the major CL synthase, but these spores’ sensitivity to NaOCl or Oxone^? was unaffected. Spores of lipid synthesis mutants were less resistant to wet heat, with spores lacking enzymes for PE, PS or l‐PG synthesis exhibiting a two to threefold decrease and spores of other strains exhibiting a four to 10‐fold decrease. The decrease in spore wet heat resistance correlated with an increase in core water content. Conclusions: Changing the lipid composition of the B. subtilis inner membrane did not affect sporulation, although modest effects on spore germination and wet heat and oxidizing agent sensitivity were observed, especially when multiple lipids were absent. The increases in rates of dodecylamine germination were likely due to increased ability of this compound to interact with the spore’s inner membrane in the absence of some CL and glycolipids. The effects on spore wet heat sensitivity are likely indirect, because they were correlated with changes in core water content. Significance and Impact of the Study: The results of this study provide insight into roles of inner membrane lipids in spore properties.     

Approximation of continuous growth of Cephalotaxus harringtonia plant cell cultures using fed-batch operation

In Cephalotaxus harringtonia plant cell cultures, periods of batch growth that are limited by hexose uptake are too short to make an accurate estimate of the Monod saturation constant. Continuous cultures are infeasible on a laboratory scale, and semicontinuous cultures require too frequent sampling. Fed-batch operation, consisting of intermittent removal from a culture that is fed continuously, was investigated as a possible solution to these problems. For a constant feed rate, computer simulations showed that a steady state can be achieved which is useful for studying growth at different specific growth rates. In terms of the dilution rate it was confirmed that the operation is essentially equivalent to continuous culture when the samples represent a small fraction of the total culture volume. Experiments with glucose or fructose as the carbon source were carried out in shake flasks fed by a multichannel syringe pump. Results indicate that Monod kinetics based on medium glucose levels cannot adequately describe growth under these conditions. Monod's expression for specific growth rate using internal glucose concentration gives an improved correlation.     

Germination of spores of Bacillus subtilis with dodecylamine

AIMS: To determine the properties of Bacillus subtilis spores germinated with the alkylamine dodecylamine, and the mechanism of dodecylamine-induced spore germination. METHODS AND RESULTS: Spores of B. subtilis prepared in liquid medium were germinated efficiently by dodecylamine, while spores prepared on solid medium germinated more poorly with this agent. Dodecylamine germination of spores was accompanied by release of almost all spore dipicolinic acid (DPA), degradation of the spore's peptidoglycan cortex, release of the spore's pool of free adenine nucleotides and the killing of the spores. The dodecylamine-germinated spores did not initiate metabolism, did not degrade their pool of small, acid-soluble spore proteins efficiently and had a significantly lower level of core water than did spores germinated by nutrients. As measured by DPA release, dodecylamine readily induced germination of B. subtilis spores that: (a) were decoated, (b) lacked all the receptors for nutrient germinants, (c) lacked both the lytic enzymes either of which is essential for cortex degradation, or (d) had a cortex that could not be attacked by the spore's cortex-lytic enzymes. The DNA in dodecylamine-germinated wild-type spores was readily stained, while the DNA in dodecylamine-germinated spores of strains that were incapable of spore cortex degradation was not. These latter germinated spores also did not release their pool of free adenine nucleotides. CONCLUSIONS: These results indicate that: (a) the spore preparation method is very important in determining the rate of spore germination with dodecylamine, (b) wild-type spores germinated by dodecylamine progress only part way through the germination process, (c) dodecylamine may trigger spore germination by a novel mechanism involving the activation of neither the spore's nutrient germinant receptors nor the cortex-lytic enzymes, and (d) dodecylamine may trigger spore germination by directly or indirectly activating release of DPA from the spore core, through the opening of channels for DPA in the spore's inner membrane. SIGNIFICANCE AND IMPACT OF THE STUDY: These results provide new insight into the mechanism of spore germination with the cationic surfactant dodecylamine, and also into the mechanism of spore germination in general. New knowledge of mechanisms to stimulate spore germination may have applied utility, as germinated spores are much more sensitive to processing treatments than are dormant spores.     

Enhanced phenylpyruvic acid production with Proteus vulgaris in fed-batch and continuous fermentation

Phenylpyruvic acid is a deaminated form of phenylalanine and is used in various areas such as development of cheese and wine flavors, diagnosis of phenylketonuria, and to decrease excessive nitrogen accumulation in the manure of farm animals. However, reported phenylpyruvic acid fermentation studies in the literature have been usually performed at shake-flask scale with low production. In this study, phenylpyruvic acid production was evaluated in bench-top bioreactors by conducting fed-batch and continuous fermentation for the first time. As a result, maximum phenylpyruvic acid concentrations increased from 1350 mg/L (batch fermentation) to 2958 mg/L utilizing fed-batch fermentation. Furthermore, phenylpyruvic acid productivity was increased from 48 mg/L/hr (batch fermentation) to 104 and 259 mg/L/hr by conducting fed-batch and continuous fermentation, respectively. Overall, this study demonstrated that fed-batch and continuous fermentation significantly improved phenylpyruvic acid production in bench-scale bioreactor production.     

Properties of regeneration mutants of Bacillus subtilis

Abstract Higher regeneration mutants were isolated from Bacillus subtilis . Protoplasts from two out of four mutants regenerated at a 100% frequency on a semi-synthetic hypertonic medium. They conferred less autolytic productivity, and a revertant regained the parental levels of regeneration frequency and autolytic activities. This mutation ( rgn -1) expressed the other pleiotropic properties, i.e., nonmotility, phage PBS1 resistance and different cell morphology.