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.     

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。     

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.     

Revival of biocide-treated spores of Bacillus subtilis

Spores of Bacillus subtilis NCTC 8236 were treated with biocides and then subjected to various revival procedures. Sodium hydroxide (optimum concentration 25 mmol 1⁻¹) revived a small portion of glutaraldehyde-treated spores but not of spores exposed to formaldehyde, polyvinylpyrrolidone-iodine (PVP-I), Lugol's iodine, sodium hypochlorite or sodium dichloroisocyanurate (NaDCC). Post-treatment heat shock (at 70° or 80°C) increased the numbers of colony-forming units (cfu) of formaldehyde-injured spores. Coat-extraction procedures had the greatest effect on iodine-pretreated spores. The uptake of iodine and chlorine was more rapid and occurred to a greater extent with outgrowing, germinating and especially coat-deficient spores than with mature, resting spores.     

Emergence of resistance to glutaraldehyde in spores of Bacillus subtilis 168

Abstract The emergence of resistance to glutaraldehyde in spores of Bacillus subtilis 168 was examined. Resistance to an organic solvent (toluene), heat and lysozyme were included for comparison. A sequential development of resistance was observed, with toluene resistance occuring early on in sporulation (stages III and IV), thermal resistance at early stage V, lysozyme resistance at middle stage V and glutaraldehyde resistance arising late in stage V. Studies with sporulation mutants also indicate that glutaraldehyde resistance is acquired even later than lysozyme resistance and may therefore possibly be considered as a very late marker event for sporulation, characterizing late stages of B. subtilis 168 spore formation.     

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.     

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.     

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.     

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.     

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.     

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.     

Cloning in Bacillus subtilis of temperature-dependent promoter fragments from Bacillus stearothermophilus and Bacillus subtilis

Abstract Using promoter-probe plasmids, more than 200 promoter-containing fragments from Bacillus stearothermophilus and Bacillus subtilis were cloned in B. subtilis . Among these, 15 promoter fragments were highly temperature-dependent in activity compared to the promoter sequence (TTGAAA for the −35 region, TATAAT for the −10 region) of the amylase gene, amyT , from B. stearothermophilus . Some fragments exhibited higher promoter activities at elevated temperature (48°C), others showed higher activities at lower temperature (30°C). Active promoter fragments at higher and lower temperatures were obtained mainly from the thermophile ( B. stearothermophilus ) and the mesophile ( B. subtilis ), respectively. A promoter fragment active at high temperature was sequenced, and the feature of the putative promoter region was discussed.     

Immunomodulatory effects of Bacillus subtilis (natto) B4 spores on murine macrophages

To investigate the immunomodulatory effects of Bacillus subtilis (B. subtilis) (natto) B4 spores on murine macrophage, RAW 264.7 cells were cultured alone or with B subtilis (natto) B4 spores at 37°C for 12 hrs, then both cells and culture supernatants were collected for analyses. Exposure of RAW 264.7 cells to B. subtilis (natto) B4 spores had no significant effects on macrophage viability and amounts of extracellular lactate dehydrogenase (LDH). However, it remarkably increased the activities of acid phosphatase (ACP), lactate dehydrogenase (LDH) and inducible nitric oxide synthase (iNOS) in cells and the amounts of nitric oxide (NO) and cytokines (tumor necrosis factor‐alpha, interferon‐gamma, interleukin [IL]‐1 beta, IL‐6, IL‐12, IL‐10 and macrophage inflammatory protein‐2) in culture supernatants. These results demonstrate that B. subtilis (natto) B4 spores are harmless to murine macrophages and can stimulate their activation through up‐regulation of ACP and LDH activities and enhance their immune function by increasing iNOS activity and stimulating NO and cytokine production. The above findings suggest that B. subtilis (natto) B4 spores have immunomodulatory effects on macrophages.     

Decolorization of indigo carmine by laccase displayed on Bacillus subtilis spores

Blue multicopper oxidases, laccases displayed on the surface of Bacillus spores were used to decolorize a widely used textile dyestuff, indigo carmine. The laccase-encoding gene of Bacillus subtilis, cotA, was cloned and expressed in B. subtilis DB104, and the expressed enzyme was spontaneously localized on Bacillus spores. B. subtilis spores expressing laccase exhibited maximal activity for the oxidation of 2,2′-azino-bis (3-ethylthiazoline-6-sulfonate) (ABTS) at pH 4.0 and 80 °C, and for the decolorization of indigo carmine at pH 8.0 and 60 °C. The displayed enzyme retained 80% of its original activity after pre-treatment with organic solvents such as 50% acetonitrile and n-hexane for 2 h at 37 °C. The apparent K_m of the enzyme displayed on spores was 443 ± 124 μM for ABTS with a V_max of 150 ± 16 U/mg spores. Notably, 1 mg of spores displaying B. subtilis laccase (3.4 × 10² U for ABTS as a substrate) decolorized 44.6 μg indigo carmine in 2 h. The spore reactor (0.5 g of spores corresponding to 1.7 × 10⁵ U in 50 mL) in a consecutive batch recycling mode decolorized 223 mg indigo carmine/L to completion within 42 h at pH 8.0 and 60 °C. These results suggest that laccase displayed on B. subtilis spores can serve as a powerful environmental tool for the treatment of textile dye effluent.     

以CotX为分子载体在枯草芽胞杆菌芽胞表面展示绿色荧光蛋白

芽胞衣壳蛋白CotB、CotC、CotG等可作为芽胞表面展示外源蛋白的分子载体,制备口服重组疫苗或具有催化活性的重组酶。CotX为枯草芽胞杆菌Bacillussubtilis芽胞衣壳中的另一种结构蛋白。为证明CotX能否作为分子载体将外源蛋白展示在芽胞表面,本研究将cotX基因与绿色荧光蛋白基因gfp的编码序列进行基因重组,构建融合表达CotX-GFP的整合型重组质粒,将该质粒转化枯草芽胞杆菌,筛选重组菌株并诱导产生芽胞,观察到重组芽胞表面具有GFP绿色荧光。结果表明枯草芽胞杆菌的芽胞衣壳蛋白CotX位于芽胞衣壳外层,可作为芽胞表面展示外源蛋白的载体分子。     

Immunostimulatory activity of Bacillus spores

Bacillus species, typically Bacillus subtilis, are being used as probiotics and mounting evidence indicates that Bacillus species are important for development of a robust gut-associated lymphoid system (GALT). We used a number of gut isolates of Bacillus incorporating three species, B. subtilis, Bacillus licheniformis and Bacillus flexus to evaluate the nature of interaction between spores and the GALT. In mice orally administered with spores, evidence of cell proliferation was determined in the germinal centers of Peyer's patches. Stimulation of antigen-presenting cells and T lymphocytes was also markedly enhanced. Cytokines were shown to be induced in spleens and mesenteric lymph nodes of mice including the proinflammatory cytokines, tumour necrosis factor-alpha and IL-6. We also demonstrated that vegetative cells of B. subtilis can stimulate expression of the toll-like receptor (TLR) genes for TLR2 and TLR4. However, we were able to show that spores could not stimulate either and must, by default, interact with another TLR and by this mechanism help activate innate immunity.     

Validation of a polynomial regression model: the thermal inactivation of Bacillus subtilis spores in milk

AIMS: The predicted survival of Bacillus subtilis 168 spores from a polynomial regression equation was validated in milk. METHODS AND RESULTS: Bias factor suggested as an index of model performance was used to validate the polynomial model predictions in ultrahigh temperature (UHT) treated and sterilized whole and skim milk. Model predictions were fail safe, predicting higher D-values (decimal reduction times) in buffer than actually noted in milk. CONCLUSIONS: The D-values for spores were lower in milk as compared with those predicted in potassium phosphate buffer contrary to the popular expectation of better spore survival in complex food systems. The Bias factor, a quantitative measure of the model performance, indicated that on average the model predictions exceed the observations by 40% in the case of whole milk and by 60% in the case of skim milk. SIGNIFICANCE AND IMPACT OF THE STUDY: The present work is an attempt to ascertain the extent of reliability that one can safely place in polynomial model predictions, without compromising on the safety or palatability of foods where it is eventually intended to be applied. The work has also highlighted the differences in the thermal inactivation pattern of spores in buffer and in milk with a possible influence of the various constituents of milk. The work will assist the dairy industry to better design thermal processes to ensure longer shelf life of dairy foods.     

The yabG gene of Bacillus subtilis encodes a sporulation specific protease which is involved in the processing of several spore coat proteins

The synthesis and proteolysis of the spore coat proteins, SpoIVA and YrbA, of Bacillus subtilis were analyzed using antisera. Almost no intact full-length proteins of either type were extracted from wild-type spores, while yabG mutant spores contained intact SpoIVA and YrbA proteins. We purified recombinant YrbA and YabG proteins from Escherichia coli transformants and found that YrbA was cleaved to the smaller moiety in the presence of YabG in vitro. These observations indicate that YabG is a protease involved in the proteolysis and maturation of SpoIVA and YrbA proteins, conserved with the cortex and/or coat assembly by B. subtilis.