巨大芽孢杆菌分子伴侣GroES和GroEL新基因的克隆及同源性分析

巨大芽孢杆菌作为革兰氏阳性细菌的一种,是良好的重组蛋白的表达宿主.本研究利用PCR技术从巨大芽孢杆菌基因组克隆出一条1.9Kb的基因片段.核酸序列分析结果表明,该片段全长1984bp,包含2个ORF,分别与芽孢杆菌来源的GroES和GroEL基因有高度的相似性.氨基酸序列比对发现,GroES蛋白与枯草芽孢杆菌来源的GroES蛋白氨基酸序列同源性为91%,GroEL蛋白氨基酸序列同源性为90%.     

Hydroperoxide inactivation of enzymes within spores of Bacillus megaterium ATCC19213

Abstract Hydroperoxide inactivation of the protoplast enzymes enolase, aldolase and glucose-6-phosphate dehydrogenase in intact spores of Bacillus megaterium ATCC19213 was assessed by first treating the cells with lethal levels of H₂O₂, then germinating them in the presence of chloramphenicol prior to permeabilization and enzyme assays. Glucose-6-phosphate dehydrogenase proved to be more sensitive to H₂O₂than enolase or aldolase, in agreement with findings for isolated enzymes. Average D values (time for 90% inactivation) for spores treated with 0.50% H₂O₂ were 173 min for enolase, 67 min for aldolase and 32 min for glucose-6-phosphate dehydrogenase, compared with a D value of 34 min for spore killing. H₂O₂ killing of spores was found to be conditional in that recoveries of survivors were greater on complex medium than on minimal medium. Overall, it appeared that oxidative inactivation of enzymes may be important for hydroperoxide killing of spores.     

Characterization of the germination of Bacillus megaterium spores lacking enzymes that degrade the spore cortex

Aims:  To determine roles of cortex lytic enzymes (CLEs) in Bacillus megaterium spore germination.
Methods and Results:  Genes for B. megaterium CLEs CwlJ and SleB were inactivated and effects of loss of one or both on germination were assessed. Loss of CwlJ or SleB did not prevent completion of germination with agents that activate the spore's germinant receptors, but loss of CwlJ slowed the release of dipicolinic acid (DPA). Loss of both CLEs also did not prevent release of DPA and glutamate during germination with KBr. However, cwlJ sleB spores had decreased viability, and could not complete germination. Loss of CwlJ eliminated spore germination with Ca²⁺ chelated to DPA (Ca-DPA), but loss of CwlJ and SleB did not affect DPA release in dodecylamine germination.
Conclusions:  CwlJ and SleB play redundant roles in cortex degradation during B. megaterium spore germination, and CwlJ accelerates DPA release and is essential for Ca-DPA germination. The roles of these CLEs are similar in germination of B. megaterium and Bacillus subtilis spores.
Significance and Impact of the Study:  These results indicate that redundant roles of CwlJ and SleB in cortex degradation during germination are similar in spores of Bacillus species; consequently, inhibition of these enzymes will prevent germination of Bacillus spores.     

仙鹤藓的孢子萌发及愈伤组织诱导

通过对采自河北雾灵山海拔1500m的仙鹤藓（Atrichum undulatum）的孢子萌发以及原丝体发育的观察,发现仙鹤藓孢子无休眠现象,孢子接种3天左右萌发：其原丝体发育分为绿丝体和轴丝体两个阶段。扩大培养实验结果表明。仙鹤藓茎叶体在添加2％葡萄糖的MS培养基上,置于25℃／20℃、14小时光照/10小时黑暗、36μmol·m^-2·s^-1条件下培养．产生新生茎叶体最多,且茎叶体长势最好,可以获得大量无菌材料。仙鹤藓愈伤组织诱导实验显示,形成愈伤组织的最佳培养基为添加2％葡萄糖和1．0mg·L^-16-BA的MS培养基。     

巨大芽孢杆菌青霉素G酰化酶基因在枯草杆菌中的高表达

用ＰＣＲ方法从巨大芽孢杆菌的基因组ＤＮＡ中扩增到青霉素Ｇ酰化酶基因,并装载到枯草杆菌质粒ｐＰＺＷ１０３中,将其转化到枯草杆菌ＤＢ１０４中进行了分泌表达,重组菌株产酶无需苯乙酸诱导。在３７℃培养２４ｈ,菌液酶活力可达６ｕ／ｍｌ。１０天的连续传代实验表明重组菌株的稳定性很高。     

Analysis of the action of compounds that inhibit the germination of spores of Bacillus species

AIMS: To determine the mechanism of action of inhibitors of the germination of spores of Bacillus species, and where these inhibitors act in the germination process. METHODS AND RESULTS: Spores of various Bacillus species are significant agents of food spoilage and food-borne disease, and inhibition of spore germination is a potential means of reducing such problems. Germination of the following spores was studied: (i) wild-type B. subtilis spores; (ii) B. subtilis spores with a nutrient receptor variant allowing recognition of a novel germinant; (iii) B. subtilis spores with elevated levels of either the variant nutrient receptor or its wild-type allele; (iv) B. subtilis spores lacking all nutrient receptors and (v) wild-type B. megaterium spores. Spores were germinated with a variety of nutrient germinants, Ca2+-dipicolinic acid (DPA) and dodecylamine for B. subtilis spores, and KBr for B. megaterium spores. Compounds tested as inhibitors of germination included alkyl alcohols, a phenol derivative, a fatty acid, ion channel blockers, enzyme inhibitors and several other compounds. Assays used to assess rates of spore germination monitored: (i) the fall in optical density at 600 nm of spore suspensions; (ii) the release of the dormant spore's large depot of DPA; (iii) hydrolysis of the dormant spore's peptidoglycan cortex and (iv) generation of CFU from spores that lacked all nutrient receptors. The results with B. subtilis spores allowed the assignment of inhibitory compounds into two general groups: (i) those that inhibited the action of, or response to, one nutrient receptor and (ii) those that blocked the action of, or response to, several or all of the nutrient receptors. Some of the compounds in groups 1 and 2 also blocked action of at least one cortex lytic enzyme, however, this does not appear to be the primary site of their action in inhibiting spore germination. The inhibitors had rather different effects on germination of B. subtilis spores with nutrients or non-nutrients, consistent with previous work indicating that germination of B. subtilis spores by non-nutrients does not involve the spore's nutrient receptors. In particular, none of the compounds tested inhibited spore germination with dodecylamine, and only three compounds inhibited Ca2+-DPA germination. In contrast, all compounds had very similar effects on the germination of B. megaterium spores with either glucose or KBr. The effects of the inhibitors tested on spores of both Bacillus species were largely reversible. CONCLUSIONS: This work indicates that inhibitors of B. subtilis spore germination fall into two classes: (i) compounds (most alkyl alcohols, N-ethylmaleimide, nifedipine, phenols, potassium sorbate) that inhibit the action of, or response to, primarily one nutrient receptor and (ii) compounds [amiloride, HgCl2, octanoic acid, octanol, phenylmethylsulphonylfluoride (PMSF), quinine, tetracaine, tosyl-l-arginine methyl ester, trifluoperazine] that inhibit the action of, or response to, several nutrient receptors. Action of these inhibitors, is reversible. The similar effects of inhibitors on B. megaterium spore germination by glucose or KBr indicate that inorganic salts likely trigger germination by activating one or more nutrient receptors. The lack of effect of all inhibitors on dodecylamine germination suggests that this compound stimulates germination by creating channels in the spore's inner membrane allowing DPA release. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides new insight into the steps in spore germination that are inhibited by various chemicals, and the mechanism of action of these inhibitors. The work also provides new insights into the process of spore germination itself.     

The Presence of Low Molecular Weight Germinative Substances in Bacillus cereus T Spore Extract

An extract from intact spores of Bacillus cereus T having a germination-inducing activity was studied. Two distinct germinative principles were found through dialysis of the extract. One was diffusible through the dialysis membrane and the other was non-diffusible. The activity of the former fraction was inhibited by the addition of 1 mM glycoletherdiamine-N, N, N', N'-tetraacetic acid (GEDTA), whereas the latter fraction was inactive unless GEDTA was added to the assay system. The diffusible principle maintained the major portion of the activity found in the crude spore extract. By means of high-performance liquid chromatography (HPLC) using a gel permeation chromatography column, 9 fractions were obtained from the deproteinized diffusible fraction. Of those fractions, two fractions (No. 1 and No. 8) were responsible for the germination-inducing activity, but no reconstituted activity was observed unless both fractions No. 1 and No. 8 were added to the assay system. Amino acid analysis of fraction No. 1 revealed that the fraction was rich in free amino acids, especially in alanine. On the other hand, by the use of reverse-phase HPLC and fast atom bombardment mass spectrometry, it was concluded that the effective substance in fraction No. 8 was inosine. Based on these findings, it was suggested that the active substances in fraction No. 1 might be a free amino acid such as L-alanine and/or Ca²⁺ and a Ca²⁺-binding substance.     

Spore Germination and Mycelial Growth of Streptomycetes at Different Humidity Levels

This study is the first to show the ability of streptomycetes to develop at a very low humidity level. All of the streptomycetes studied produced growth at low humidity (a_w 0.86 and 0.67). This capacity was most markedly pronounced in Streptomyces odorifer, whose spores were capable of germinating, and mycelial germs increased in length, at the air humidity a_w 0.50. The formation of lateral branches (mycelium branching) at this humidity was noted only in single S. odorifer germs and only after 72 h of incubation. Study of streptomycete growth on an agarized medium with different osmotic pressures, created by various glycerol concentrations in the medium, showed that, at a_w 0.67, the spores of all the streptomycetes studied germinate, producing mycelial germs but not microcolonies. The ecological significance of mycelial prokaryotes in soil microbial communities that develop and function under conditions of extremely low humidity is discussed.     

Coexpression of the type I signal peptidase gene sipM increases recombinant protein production and export in Bacillus megaterium MS941

The removal of the signal peptide from a precursor protein is a crucial step of protein secretion. In order to improve Bacillus megaterium as protein production and secretion host, the influence of homologous type I signal peptidase SipM overproduction on recombinant Leuconostoc mesenteroides dextransucrase DsrS synthesis and export was investigated. The dsrS gene was integrated as a single copy into the chromosomal bgaM locus encoding beta-galactosidase. Desired clones were identified by blue-white selection. In this strain, the expression of sipM from a multicopy plasmid using its own promoter increased the amount of secreted DsrS 3.7-fold. This increase in protein secretion by SipM overproduction was next transferred to a high level DsrS production strain using a multicopy plasmid encoding sipM with its natural promoter and dsrS under control of a strong xylose-inducible promoter. No further increase in DsrS export were observed when this vector was carrying two sipM copies. Similarly, bicistronic sipM and dsrS high level expression did not enhance DsrS secretion, indicating the natural limitation of the approach. Interestingly, SipM-enhanced DsrS secretion also resulted in an overall increase of DsrS production.     

An Experimental Investigation of the Effect of Bacillus megaterium on Apatite Dissolution

Field observations suggest that some mineral dissolution rates can be enhanced by microbial activity indirectly, without direct contact with the mineral surface. A series of apatite dissolution experiments was performed to better understand this rate enhancement process. Far-from equilibrium abiotic apatite dissolution rates, measured in mixed-flow reactors at 25°C were enhanced by increasing concentration of aqueous organic acids and decreasing aqueous phosphate activity, demonstrating the existence of indirect pathways for microbial rate enhancement. Further apatite dissolution experiments were performed in closed-system reactors in the presence of Bacillus megaterium , a common heterotrophic aerobe. Experiments were designed to allow the bacteria to be either in direct contact or indirect contact with the apatite; in the latter case, the microbes were physically separated from the apatite using dialysis bags. Apatite dissolution in indirect contact with Bacillus megaterium was 50 to 900% faster than abiotic controls. Bacterial rate enhancement was, however, 3 to over 10 times lower when Bacillus megaterium was in direct contract versus indirect contact with the apatite surfaces. These results show that (1) bacteria can accelerate rates without being in physical contact with the dissolving mineral, and (2) microbially mediated dissolution may be less effective when bacteria are in direct contact with mineral surfaces. Supression of mineral dissolution is interpreted to stem from the preferential colonization of reactive sites on the mineral surface.     

电脉冲穿孔法将几丁质酶基因导入巨大芽胞杆菌

为了探索适宜于巨大芽胞杆菌电脉冲转化条件,将带有沙雷氏菌几丁质酶编码基因的穿梭质粒转化到植病生防菌—巨大芽胞杆菌B1301中。以芽胞杆菌B1301对数期细胞为感受态细胞,采用不同的电击转化条件进行转化,通过转化率和几丁质酶活性表达认为巨大芽胞杆菌的最佳电击转化条件为电压1000 V/mm,电容25μF,电阻400Ω,转化率为9.6×104/μg质粒,几丁质酶活性表达的菌株几率为41.67%。     

巨大芽孢杆菌b-淀粉酶基因的克隆、表达和酶学性质分析

从巨大芽孢杆菌(Bacillus megaterium)的全基因组DNA文库中筛选出一个b-淀粉酶基因amyG, 分析测定了其核苷酸序列并进行了诱导表达; 其中amyG编码的蛋白有545个氨基酸、分子量为60.194 kD, 与已报道的巨大芽孢杆菌DSM319的b-淀粉酶序列有着94.5%的同源性。经氨基酸序列比较分析发现, AmyG从N末端到C末端依次由信号肽域、糖基水解酶催化功能域和淀粉结合域3个功能域组成。其中催化功能域里含有第14家族糖基水解酶常见的几个高度保守的酶催化活性区。经多步纯化, 重组酶的比活共提高了7.4倍, 获得凝胶电泳均一的蛋白样品; 经SDS-PAGE电泳测定, 酶AmyG的分子量为57 kD。该酶的最适反应温度为60oC, 最适反应pH为7.0; 在温度不超过60oC时, 酶活较稳定; AmyG能迅速降解淀粉生成麦芽糖, 属于外切b-糖苷酶。     

粗茎鳞毛蕨孢子萌发研究

以经过3年低温储藏的粗茎鳞毛蕨孢子为实验材料,从孢子离心、孢子消毒、培养基种类、光质等4方面对孢子萌发进行研究,结果表明:在离心转数≤14 000 r.min-1、离心时间≤30 min条件下,离心处理对孢子萌发基本无影响;对孢子进行1%NaClO水溶液浸泡处理20～30 min为最佳消毒条件;改良Knop’s培养基为最佳孢子萌发培养基;黑暗条件下孢子不能萌发,但是黑暗处理能够明显提高孢子萌发整齐性;红光比白光能促进孢子提早萌发1 d左右,但对提高萌发率效果不显著。     

巨大芽孢杆菌分子伴侣GroES和GroEL新基因的克隆及同源性分析

巨大芽孢杆菌作为革兰氏阳性细菌的一种,是良好的重组蛋白的表达宿主.本研究利用PCR技术从巨大芽孢杆菌基因组克隆出一条1.9 Kb的基因片段.核酸序列分析结果表明,该片段全长1 984 bp,包含2个ORF,分别与芽孢杆菌来源的GroES和GroEL基因有高度的相似性.氨基酸序列比对发现,GroES蛋白与枯草芽孢杆菌来源的GroES蛋白氨基酸序列同源性为91%,GroEL蛋白氨基酸序列同源性为90%.     

Germination of Bacillus thuringiensis var. israelensis spores in the gut of Aedes larvae (Diptera: Culicidae)

In laboratory experiments, germination and growth of Bacillus thuringiensis israelensis in the gut of Aedes aegypti and A. vexans larvae (Culicidae: Diptera) was observed. The number of spores and vegetative cells in the gut of living larvae and in cadavers was estimated by plaing homogenized larvae on selective agar plates. The number of spores per gut increased in the first 40–140 min of exposure to a maximum, and decreased in the subsequent time, demonstrating spore germination in living larvae, moribunds, and in cadavers. Twenty-four hours after the death of the larvae, a minimal amount of spores, but an increased number of vegetative cells, was found in cadavers. In A. aegypti larvae, germination and growth of B. thuringiensis israelensis in the larval gut was photographically documented.     

Effects of Mn levels on resistance of Bacillus megaterium spores to heat, radiation and hydrogen peroxide

Aims: To determine the effects of Mn levels in Bacillus megaterium sporulation and spores on spore resistance. Methods and Results: Bacillus megaterium was sporulated with no added MnCl₂ and up to 1 mmol l^?1 MnCl₂. The resultant spores were purified and loosely bound Mn removed, and spore Mn levels were found to vary c. 100‐fold. The Mn level had no effect on spore γ‐radiation resistance, but B. megaterium spores with elevated Mn levels had higher resistance to UVC radiation (as did Bacillus subtilis spores), wet and dry heat and H₂O₂. However, levels of dipicolinic acid and the DNA‐protective α/β‐type small, acid‐soluble spore proteins were the same in spores with high and low Mn levels. Conclusions: Mn levels either in sporulation or in spores are important factors in determining levels of B. megaterium spore resistance to many agents, with the exception of γ‐radiation. Significance and Impact of the Study: The Mn level in sporulation is an important factor to consider when resistance properties of B. megaterium spores are examined, and will influence the UV resistance of B. subtilis spores, some of which are used as biological dosimeters.     

Cloning and expression of keratinase gene in Bacillus megaterium and optimization of fermentation conditions for the production of keratinase by recombinant strain

AIMS: Cloning and expression of keratinase gene in Bacillus megaterium and optimization of fermentation conditions for the production of keratinase by recombinant strain. METHODS AND RESULTS: The keratinase gene with and without leader sequence from the chromosomal DNA of Bacillus licheniformis MKU3 was amplified by PCR and cloned into pET30b and transferred into Escherichia coli BL21. The ker gene without leader sequence only expressed in E. coli and the recombinant strain produced an intracellular keratinase activity of 74.3 U ml(-1). The ker gene was further subcloned into E. coli-Bacillus shuttle vector, pWH1520. Bacillus megaterium ATCC 14945 carrying the recombinant plasmid pWHK3 expressed the ker gene placed under xylA promoter and produced an extracellular keratinase activity of 95 U ml(-1). Response surface methodology (RSM) was employed to optimize the fermentation condition and to improve the level of keratinase production by the recombinant strain. A maximum keratinolytic activity of 166.2 U ml(-1) (specific activity, 33.25 U mg(-1)) was obtained in 18 h of the fermentation carried out with an initial inoculum of 0.4 OD600 nm and xylose concentration of 0.75% w/v. CONCLUSIONS: Bacillus licheniformis keratinase was cloned and successfully expressed using T7 promoter in E. coli and xylose inducible expression system in B. megaterium. Response surface methodology was employed to optimize the process parameters, which resulted in a three-fold higher level of keratinase production by the recombinant B. megaterium (pWHK3) than the wild type strain B. licheniformis MKU3. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests that B. megaterium is a suitable host for the expression of cloned genes from heterologous origin. Optimization of fermentation conditions improved the keratinase production by B. megaterium (pWHK3) and suggested that this recombinant strain could be used for the production of keratinase.     

Analysis of broth-cultured Bacillus atrophaeus and Bacillus cereus spores

Aims: To compare physical properties of spores that were produced in broth sporulation media at greater than 10⁸ spores ml⁻¹. Methods and Results: Bacillus atrophaeus reproducibly sporulated in nutrient broth (NB) and sporulation salts. Microscopy measurements showed that the spores were 0·68 ± 0·11 μm wide and 1·21 ± 0·18 μm long. Coulter Multisizer (CM3) measurements revealed the spore volumes and volume-equivalent spherical diameters, which were 0·48 ± 0·38 μm³ and 0·97 ± 0·07 μm, respectively. Bacillus cereus reproducibly sporulated in NB, sporulation salts, 200 mmol l⁻¹ glutamate and antifoam. Spores were 0·95 ± 0·11 μm wide and 1·31 ± 0·17 μm long. Spore volumes were 0·78 ± 0·61 μm³ and volume-equivalent spherical diameters were 1·14 ± 0·11 μm. Bacillus atrophaeus spores were hydrophilic and B. cereus spores were hydrophobic. However, spore hydrophobicity was significantly altered after treatment with pH-adjusted bleach. Conclusions: The utility of a CM3 for both quantifying Bacillus spores and measuring spore sizes was demonstrated, although the volume between spore exosporium and spore coat was not measured. This study showed fundamental differences between spores from a Bacillus subtilis- and B. cereus-group species. Significance and Impact of the Study: This is useful for developing standard methods for broth spore production and physical characterization of both living and decontaminated spores.     

Polyamine Levels During Growth, Sporulation, and Spore Germination of Bacillus megaterium

Spermidine was the major (>95%) polyamine of Bacillus megaterium in all stages of growth, although it could be replaced completely by spermine. Log-phase cells had 40 to 50% as much spermidine, based on ribonucleic acid (RNA) content, as did either stationary-phase cells or dormant spores; similar results were obtained in three other bacilli including an asporogenous mutant. Polyamine levels were essentially the same in B. megaterium grown in rich or poor media, or in media of high or low ionic strength. Polyamine levels were elevated three- to sixfold by exogenous spermidine without a major effect on growth, sporulation, or subsequent spore germination. During germination, the absolute amount of spermidine remained constant for almost 2 h until net RNA synthesis had lowered the polyamine/RNA ratio to a value close to that in log-phase cells. At this time, the spermidine level began to rise, and thereafter spermidine and RNA increased in parallel. This parallel relationship between the spermidine and RNA levels was abolished by actinomycin D, but not by chloramphenicol.     

苏云金杆菌以色列亚种几丁质酶基因的克隆及序列分析

从苏云金芽孢杆菌以色列亚种（Bacillus thuringiensis subsp israelensis)中提取基因组DNA,通过合成1对特异性引物,用touchdown PCR的方法扩增几丁质酶ichi基因序列（GenBank登录号：AF526379）。ichi序列全长为2570bp,含有1个2067bp的开放阅读框（ORF）,编码688个氨基酸,推测分子量为75.79kDa,等电点pI=5.90的几丁质酶前体。序列和结构比较分析表明：Ichi氨基酸序列与蜡状芽孢杆菌（Bacillus cereus)28-9几丁质酶CW、蜡状芽孢杆菌CH几丁质酶B及苏云金芽孢杆菌墨西哥亚种几丁质酶的同源性分别为97.24%、97.18%、97.63%,而与苏云金芽孢杆菌巴基斯坦亚种的同源性只有63.07%。Ichi编码区由分泌信号肽（46AA）、催化区（105AA)、粘蛋白Ⅲ型同源区（74AA）及几丁质结合区（40AA）组成。