Emergence and development of chlorhexidine resistance during sporulation of Bacillus subtilis 168

Abstract During sporulation of Bacillus subtilis strain 168 initiated by step-down conditions, resistance to chlorhexidine diacetate (CHA) developed at about t _3.5, before heat but after toluene resistance. Mutants blocked at stage IV of sporulation were sensitive to all three treatments. Stage V mutants were toluene resistant but moderately sensitive to heat and CHA. A stage VI mutant was resistant to all three treatments. Thus, chlorhexidine resistance is likely to be a result of spore coat, rather than of cortex, development.     

Inducible resistance to zinc ions in Bacillus subtilis 168

Abstract Chromosomally determined resistance to Zn²⁺ ions is a property of Bacillus subtilis 168. Resistance is inducible and is not connected with Cd²⁺ resistance, characteristics of plasmid determined resistance in bacteria. A few proteins were shown to respond to inducible levels of the cation.     

Uptake of l-[14C]-alanine by glutaraldehyde-treated and untreated spores of Bacillus subtilis

The effect of glutaraldehyde on the uptake of L-alanine, and subsequent germination, in spores of Bacillus subtilis NCTC 8236 was examined. Germination was induced by single amino acids, D-glucose and phosphate buffer at 37 degrees C. L-alanine was the best germinant of all amino acids tested. Pretreatment of spores with low concentrations of acid and alkaline glutaraldehyde inhibited subsequent germination, complete inhibition being observed at concentrations of 0.1% (w/v). This concentration also prevented the loss of heat resistance of spores placed in germination medium and exposed to 75 degrees C. Radioactive studies indicated that maximum uptake of L-alanine occurred after ca 30 min at 37 degrees C. Only 1.2% of available L-alanine was taken up during germination. Pretreatment of spores with glutaraldehyde did not interfere with L-alanine uptake at aldehyde concentrations up to 0.5% (w/v). However, this was significantly reduced at a glutaraldehyde concentration of 1.0% (w/v). Minimal differences were observed between acid and alkaline forms of the aldehyde. The results are discussed in terms of the mode of action of glutaraldehyde.     

PspA外源表达对枯草芽孢杆菌168蛋白质分泌的影响

PspA同源物广泛存在于细菌和高等生物的组织中.在本研究中克隆了来源于地衣芽孢杆菌的PspA基因,并将其克隆于用于大肠-芽孢穿梭诱导表达载体pDG-StuI中构建重组质粒pDG-PspA.将构建的诱导表达型的重组质粒转化到Bacillus subtilis 168中,研究PspA的外源表达对该菌的生长,总蛋白分泌,以及Sec分泌途径中α-淀粉酶分泌的影响,结果表明,PspA基因的外源表达,在发酵过程后期能在一定程度上提高总蛋白的分泌量,在发酵过程后期能在一定程度上提高分泌的α-淀粉酶浓度.     

Permeability of dormant spores of Bacillus subtilis to gramicidin S

Abstract Gramicidin S, dissolved in ethanol, penetrated into the inside of the dormant spores of Bacillus subtilis , had a partial inhibitory effect on l-alanine-initiated germination and completely inhibited their outgrowth and vegetative growth. The activity of particulate NADH oxidase of the antibiotic-treated dormant spores was also influenced significantly. Abnormal morphological changes were observed in germinated spores from gramicidin S-treated dormant spores. An immunoelectron microscopy method with colloidal gold-IgG complex showed that the penetration site of gramicidin S inside dormant spores was mainly the core region. These facts suggest that gramicidin S induces the damage of not only the outer membrane-spore coat complex but also the inner membrane surrounding the spore protoplast, and is able to penetrate into the core region of B. subtilis dormant spores.     

途径优化强化枯草芽胞杆菌合成肝素前体

肝素前体是化学酶法合成肝素的起点,肝素前体的微生物高效合成具有重要意义。在已构建的产肝素前体的枯草芽胞杆菌((1.71±0.08)g/L)中,分析了UDP-葡萄糖醛酸(UDP-GlcUA)途径中关键酶基因(pgcA、gtaB、tuaD)以及UDP-乙酰氨基葡糖(UDP-GlcNAc)途径中关键酶基因(glmS、glmM、glmU)的过量表达对肝素前体产量及其分子量的影响。在此基础上,通过共表达tuaD、gtaB、glmU、glmM和glmS基因,摇瓶中肝素前体产量提高至(2.89±0.11)g/L,分子量为(75.90±1.18)kDa。通过在3 L发酵罐中进行补料分批发酵,肝素前体的产量最终积累到(7.25±0.36)g/L,分子量为(46.66±2.71)kDa,为工业化生产肝素奠定了基础。     

Injury and repair in biocide-treated spores of Bacillus subtilis

Abstract Bacillus subtilis NCTC 8236 spores exposed to appropriate concentrations of test biocides (glutaraldehyde, two iodine and two chlorine preparations) were able to repair injury if subsequently held in nutrient broth at 37°C but not in broth at 22°C, sterile filtered water at 4, 22 or 37°C or germination medium at 37°C. Repair appeared to occur primarily during outgrowth and was initiated soonest for iodine-treated spores and latest for glutaraldehyde-treated ones.     

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.     

Permeability of gentamicin into the inside of Bacillus subtilis spores

Abstract The penetration of gentamicin into the inside of Bacillus subtilis spores was examined by an immunoelectron microscopy method with colloidal gold-immunoglobulin G complex. The colloidal gold particles were located mainly in the coat regions of spores and were not observed in the cortex or core regions. This result suggests the existence of an outer membrane inside the coat region as the primary permeability barrier to gentamicin.     

Restriction on Conjugational Transfer of pLS20 in Bacillus subtilis 168

Conjugational transfer of pLS20 in Bacillus subtilis Marburg 168 is restricted by the BsuM restriction-modification system. Restriction efficiency was measured using pLS20 derivatives possessing various numbers of XhoI sites, which are known to be recognized by BsuM. An increase in XhoI sites clearly reduced the conjugational efficiency of pLS20 as compared with that of pUB110 plasmid lacking XhoI.     

枯草芽胞杆菌168不对称转化产生磷霉素的蛋白质组学分析

旨在用蛋白质组学方法揭示枯草芽胞杆菌Bacillus subtilis 168将顺丙烯磷酸转化成磷霉素的机理.B.subtilis 168能够将顺丙烯磷酸不对称转化成磷霉素.气相色谱分析发现在转化培养基发酵液中的磷霉素的含量达816.6 tg/mL,转化率为36.05％.将分别培养在含有底物和不含底物的培养基中的B.subtilis 168的胞质蛋白进行双向凝胶电泳.对两种条件下的电泳图谱进行比较,发现有98个差异表达蛋白.其中在有底物存在时,表达量下调的点有20个,表达量上调的点52个,底物特异性表达的点有26个.对差异表达蛋白进行质谱鉴定,共鉴定到80个蛋白点,其中下调的点17个,上调的点45个,底物特异性表达的点18个.这些蛋白分别参与胁迫反应、氧化还原反应、物质转运、核苷酸代谢、糖代谢、氨基酸和蛋白质代谢等.根据上述对B.subtilis 168蛋白质组学分析结果,推测菌株是通过两步将顺丙烯磷酸转化成磷霉素的.第一步是水化反应,第二步是脱氢反应.     

枯草杆菌的芽胞在肉鸡肠道中的生活状态和分布

目的探讨枯草芽胞杆菌的芽胞在肉鸡肠道中的生活状态和分布。方法以20日龄AA肉鸡为研究对象,饲喂枯草芽胞菌剂,分别测定鸡粪中芽胞数量和鸡肠道不同部位的活菌数量。结果饲喂3h后,鸡粪中开始检测到芽胞的存在,24h达到最高值,直至饲喂120h后,肠道内的芽胞基本排除。排出芽胞总量为饲喂芽胞总数的3．0倍左右,同时研究还表明：芽胞在实验肉鸡的十二指肠2内开始萌发,并进行了繁殖,在小肠的后端,即小肠3和小肠4,活菌数量达到高峰。结论部分芽胞进入小肠后即可开始萌发,并进行生长繁殖,而且在肠道内有短暂滞留。     

Homology modeling of an antifungal metabolite plipastatin synthase from the Bacillus subtilis 168

Lipopeptides have a widespread role in different pathways of Bacillus subtilis; they can act as antagonists, spreader and immunostimulators. Plipastatin, an antifungal antibiotic, is one of the most important lipopeptide nonribosomly produced by Bacillus subtilis. Plipastatin has strong fungitoxic activity and involve in inhibition of phospholipase A2 and biofilm formation. For better understanding of the molecule and pathway by which lipopeptide plipastatin is synthesized, we present a computationally predicted structure of plipastatin using homology modeling. Primary and secondary structure analysis suggested that ppsD is a hydrophilic protein containing a significant proportion of alpha helices, and subcellular localization predictions suggested it is a cytoplasmic protein. The tertiary structure of protein (plipastatin synthase subunit D) was predicted by homology modeling. The results suggest a flexible structure which is also an important characteristic of active enzymes enabling them to bind various cofactors and substrates for proper functioning. Validation of 3D structure was done using Ramachandran plot ProsA-web and QMEAN score.This predicted information will help in better understanding of mechanisms underlying plipastatin synthase subunit D synthesis. Plipastatin can be used as an inhibitor of various fungal diseases in plants.     

Evidence that dipicolinic acid is covalently bound to specific macromolecules in spores of Bacillus subtilis

Abstract Two dipicolinic acid (DPA)-binding macromolecules with molecular masses of about 440 kDa and 230 kDa were detected in the soluble fractions of dormant and germinated spores of Bacillus subtilis using native PAGE and an immunological technique. In SDS-PAGE, only one band with the molecular mass of about 50 kDa was found. Proteinase K partially digested the 440-kDa macromolecule of dormant spores to convert it into a 230-kDa one, and completely digested both the 440-kDa and 230-kDa bands of germinated spores. DNase I did not affect either DPA-binding macromolecules. This suggests that the two DPA-binding macromolecules are of similar origin, their main component is protein and a conformational change may occur during germination. DPA was not dissociated from the DPA-binding macromolecules by extensive dialysis and SDS treatment, suggesting the presence of a covalent bonding.     

YtsCD and YwoA, two independent systems that confer bacitracin resistance to Bacillus subtilis

The Bacillus subtilis yts, yxd and yvc gene clusters encode a putative ABC transporter and a functionally coupled two-component system. When tested for their sensitivity towards a series of antibiotics, null yts mutants were found to be sensitive to bacitracin. Real-time polymerase chain reaction (PCR) experiments demonstrated that the presence of bacitracin in the growth medium strongly stimulates the expression of the ytsCD genes encoding the ABC transporter and that this stimulation strictly depends on the YtsA response regulator. The ywoA gene encodes a protein known to confer some resistance to bacitracin on the bacterium. When it was mutated in a null yts background, the ywoA yts double mutant was found to be five times more sensitive than the yts one. We propose that (i) the YtsCD ABC transporter exports the bacitracin; (ii) YwoA, the protein that contains an acidPPc (PAP2 or PgpB) domain, is not part of an ABC transporter but competes with bacitracin for the dephosphorylation of the C55-isoprenyl pyrophosphate (IPP); (iii) the two resistance mechanisms are independent and complementary.     

Mechanisms of killing of Bacillus subtilis spores by hypochlorite and chlorine dioxide

AIMS: To determine the mechanisms of Bacillus subtilis spore killing by hypochlorite and chlorine dioxide, and its resistance against them. METHODS AND RESULTS: Spores of B. subtilis treated with hypochlorite or chlorine dioxide did not accumulate damage to their DNA, as spores with or without the two major DNA protective alpha/beta-type small, acid soluble spore proteins exhibited similar sensitivity to these chemicals; these agents also did not cause spore mutagenesis and their efficacy in spore killing was not increased by the absence of a major DNA repair pathway. Spore killing by these two chemicals was greatly increased if spores were first chemically decoated or if spores carried a mutation in a gene encoding a protein essential for assembly of many spore coat proteins. Spores prepared at a higher temperature were also much more resistant to these agents. Neither hypochlorite nor chlorine dioxide treatment caused release of the spore core's large depot of dipicolinic acid (DPA), but hypochlorite- and chlorine dioxide-treated spores much more readily released DPA upon a subsequent normally sub-lethal heat treatment than did untreated spores. Hypochlorite-killed spores could not initiate the germination process with either nutrients or a 1 : 1 chelate of Ca2+-DPA, and these spores could not be recovered by lysozyme treatment. Chlorine dioxide-treated spores also did not germinate with Ca2+-DPA and could not be recovered by lysozyme treatment, but did germinate with nutrients. However, while germinated chlorine dioxide-killed spores released DPA and degraded their peptidoglycan cortex, they did not initiate metabolism and many of these germinated spores were dead as determined by a viability stain that discriminates live cells from dead ones on the basis of their permeability properties. CONCLUSIONS: Hypochlorite and chlorine dioxide do not kill B. subtilis spores by DNA damage, and a major factor in spore resistance to these agents appears to be the spore coat. Spore killing by hypochlorite appears to render spores defective in germination, possibly because of severe damage to the spore's inner membrane. While chlorine dioxide-killed spores can undergo the initial steps in spore germination, these germinated spores can go no further in this process probably because of some type of membrane damage. SIGNIFICANCE AND IMPACT OF THE STUDY: These results provide information on the mechanisms of the killing of bacterial spores by hypochlorite and chlorine dioxide.     

Assembly of the CotSA coat protein into spores requires CotS in Bacillus subtilis

The CotSA protein, encoded by cotSA (ytxN) of Bacillus subtilis, was detected from the cells at 5 h after the onset of sporulation (T5) and in the spore coat of wild-type cells, but not in cotE, cotS, gerE, or cotSA mutant spores. CotSA was also detected in the sporangium at T5 to T7 but not in the sporangium at T18 of cotS mutant cells, while the incorporation of CotS into the coat was not dependent upon CotSA. These results suggested that CotSA was synthesized simultaneously with CotS during T5 to T7 of sporulation and assembled into the coat dependent upon CotS.     

Germination and inactivation of Bacillus subtilis spores induced by moderate hydrostatic pressure

In this study, we investigated the mechanisms of spore inactivation by high pressure at moderate temperatures to optimize the sterilization efficiency of high‐pressure treatments. Bacillus subtilis spores were first subjected to different pressure treatments ranging from 90 to 550 MPa at 40°C, with holding times from 10 min to 4 h. These treatments alone caused slight inactivation, which was related to the pressure‐induced germination of the spores. After these pressures treatments, the sensitivity of these processed spores to heat (80°C/10 min) or to high pressure (350 MPa/40°C/10 min) was tested to determine the pressure‐induced germination rate and the advancement of the spores in the germination process. The subsequent heat or pressure treatments were applied immediately after decompression from the first pressure treatment or after a holding time at atmospheric pressure. As already known, the spore germination is more efficient at low pressure level than at high pressure level. Our results show that this low germination efficiency at high pressure seemed not to be related either to a lower induction or a difference in the induction mechanisms but rather to an inhibition of enzyme activities which are involved in germination process. In fact, high pressure was necessary and very efficient in inducing spore germination. However, it seemed to slow the enzymatic digestion of the cortex, which is required for germinated spores to be inactivated by pressure. Although these results indicate that high‐pressure treatments are more efficient when the two treatments are combined, a small spore population still remained dormant and was not inactivated with any holding time or pressure level. Biotechnol. Bioeng. 2010;107: 876–883. © 2010 Wiley Periodicals, Inc.     

Formaldehyde gas inactivation of Bacillus anthracis, Bacillus subtilis, and Geobacillus stearothermophilus spores on indoor surface materials

AIMS: To evaluate the decontamination of Bacillus anthracis, Bacillus subtilis, and Geobacillus stearothermophilus spores on indoor surface materials using formaldehyde gas. METHODS AND RESULTS: B. anthracis, B. subtilis, and G. stearothermophilus spores were dried on seven types of indoor surfaces and exposed to approx. 1100 ppm formaldehyde gas for 10 h. Formaldehyde exposure significantly decreased viable B. anthracis, B. subtilis, and G. stearothermophilus spores on all test materials. Significant differences were observed when comparing the reduction in viable spores of B. anthracis with B. subtilis (galvanized metal and painted wallboard paper) and G. stearothermophilus (industrial carpet and painted wallboard paper). Formaldehyde gas inactivated>or=50% of the biological indicators and spore strips (approx. 1x10(6) CFU) when analyzed after 1 and 7 days. CONCLUSIONS: Formaldehyde gas significantly reduced the number of viable spores on both porous and nonporous materials in which the two surrogates exhibited similar log reductions to that of B. anthracis on most test materials. SIGNIFICANCE AND IMPACT OF THE STUDY: These results provide new comparative information for the decontamination of B. anthracis spores with surrogates on indoor surfaces using formaldehyde gas.