Organization and nucleotide sequence of the atp genes encoding the ATP synthase from alkaliphilic Bacillus firmus OF4

Summary The atp operon from the extreme alkaliphile Bacillus firmus OF4 was cloned and sequenced, and shown to contain genes for the eight structural subunits of the ATP synthase, preceded by a ninth gene predicted to encode a 14 kDa hydrophobic protein. The arrangement of genes is identical to that of the atp operons from Escherichia coli, Bacillus megaterium, and thermophilic Bacillus PS3. The deduced amino acid sequences of the subunits of the enzyme are also similar to their homologs in other ATP synthases, except for several unusual substitutions, particularly in the a and c subunits. These substitutions are in domains that have been implicated in the mechanism of proton translocation through F₀-ATPase, and therefore could contribute to the gating properties of the alkaliphile ATP synthase or its capacity for proton capture.     

一株地衣芽胞杆菌产碱性蛋白酶条件优化

【背景】碱性蛋白酶是众多芽胞杆菌的发酵产物,是工业上极其重要的一类酶。【目的】利用酪素培养基从环境样品中筛选出一株产碱性蛋白酶的菌株,对传代次数、发酵的碳源、氮源、金属离子、磷酸盐、初始pH、接种量和温度进行优化,提高其产碱性蛋白酶的能力并降低发酵成本。【方法】采用革兰氏染色法、扫描电镜、生理生化试验、16S rRNA基因序列对分离的菌株进行鉴定;采用单因素、Plackett-Burman、最陡爬坡和响应面试验优化碱性蛋白酶的发酵条件,使用Minitab对试验数据进行分析。【结果】经鉴定分离菌株为地衣芽胞杆菌,命名为Bacillus licheniformis NWMCC0046。优化后的发酵培养基组成为（g/L）:豆粕50.00,葡萄糖10.00,酵母浸膏13.46,CaCl₂ 0.50,Na₂HPO₄·12H₂O 4.00,KH₂PO₄ 0.30;优化后的培养条件为:pH 7.5,34.81℃,接种量4.13%。在此条件下,摇瓶发酵48 h时碱性蛋白酶...     

一株拮抗黄单胞菌的贝莱斯芽孢杆菌的分离和鉴定

【目的】为了筛选防治水稻条斑病(bacterial leaf streak,BLS)的生防细菌。【方法】以水稻条斑病菌(Xanthomonas oryzae pv. oryzicola,Xoc)的模式菌株RS105为靶标菌,采用平板稀释和抑菌圈法,从空心菜根际土壤中筛选到一株对RS105具有拮抗作用的细菌菌株504。通过形态学、生理生化特征以及16SrDNA和gyrA序列分析对菌株504进行了鉴定。利用牛津杯法测定504对植物病原黄单胞菌的拮抗活性及其无菌发酵液拮抗活性的稳定性。通过PCR扩增预测504编码合成脂肽类和聚酮类化合物的合成相关基因。采用苗期水稻注射接菌法来评价水稻组织中504对Xoc的拮抗活性。【结果】菌株鉴定结果表明504为贝莱斯芽孢杆菌,命名为Bacillusvelezensis504。抑菌实验显示,B.velezensis504对黄单胞菌属的细菌具有较好的抑菌活性,对水稻白叶枯病菌(X. oryzae pv. oryzae,Xoo)的拮抗效果最显著。基因预测结果显示,B. velezensis 504含有fenA、dhbA、sfrA、bmyA、beaS、dfnA及bacA等编码脂肽类和聚酮糖类抑菌化合物的基因簇。其无菌发酵液的活性物质耐高温和蛋白酶降解,但不耐强酸、强碱,在pH值为5.5–8.9时仍具有稳定的拮抗活性。在高感水稻品种原丰早上,B. velezensis 504对Xoc在水稻叶片中引起的水渍症状具有显著的抑制作用。【结论】B. velezensis 504能够特异性拮抗黄单胞菌,在黄单胞菌引起的细菌性病害的生物防治中将具有较大的应用潜力。     

Hymenopteran specificity of Bacillus thuringiensis strain PS86Q3

The biological activity of Bacillus thuringiensis (Bt) strain PS86Q3 against five Hymenopteran species was determined by means of bioassays adapted to each species. Four species of sawfly that are important pests of conifers (Diprion pini, Gilpinia hercyniae and Pristiphora abietina) or ornamental plants (Arge rosae), as well as the non-target honeybee, Apis mellifera, were studied. Two out of the four sawfly species tested were found to be sensitive to PS86Q3 crystals or spore/crystal suspensions. A sporulated culture of this strain was moderately active on D. pini, and a complete bioassay with solubilized crystals was performed to estimate the LC₅₀ of 4.9 mg/ml. Pristiphora abietina was also found to be sensitive to PS86Q3, with an LC₅₀ of 1.6 mg/ml. By contrast, at the concentrations tested, PS86Q3 did not prove active on the remaining sawflies, G. hercyniae and A. rosae. The strain was administered orally to check its effects on honeybees which were fed sucrose solutions supplemented with a PS86Q3 sporulated suspension, in a field assay using commercial beehives. No significant differences in larval mortality (as deduced by comparing the number of larvae, pupae and empty cells) were found between the Bt and control treatments. On the basis of the results presented here, the suitability of PS86Q3 for the control of Hymenopteran pests, particularly sawflies, in terms of both potency and environmental safety, is discussed.     

Cloning and expression of the gene for neutral protease of Bacillus amyloliquefaciens in Bacillus subtilis

A Bacillus amyloliquefaciens neutral protease gene was cloned and expressed in Bacillus subtilis.The chromosomal DNA of B. amyloliquefaciens strain F was partially digested with restriction endonuclease Sau3AI, and 2 to 9 kb fragments isolated were ligated into the BamHI site of plasmid pUB110. Then, B. subtilis strain 1A289 was transformed with the hybrid plasmids by the method of protoplast transformation and kanamycin-resistant transformants were screened for the formation of large halo on a casein plate. A transformant that produced a large amount of an extracellular neutral protease harbored a plasmid, designated as pNP150, which contained a 1.7 kb insert.The secreted neutral protease of the transformant was found to be indistinguishable from that of DNA donor strain B. amyloliquefaciens by double immunodiffusion test and SDS-polyacrylamide gel electrophoresis.The amount of the neutral protease activity excreted into culture medium by the B. subtilis transformed with pNP150 was about 50-fold higher than that secreted by B. amyloliquefaciens. The production of the neutral protease in the transformant was partially repressed by addition of glucose to the medium.     

苏云金芽胞杆菌rocE基因的转录调控

【目的】rocE基因编码精氨酸降解途径中的精氨酸通透酶,通过分析苏云金芽胞杆菌(Bacillus thuringiensis,Bt) rocE基因的转录活性,明确rocE基因的转录调控机制。【方法】通过RT-PCR确定rocE基因所在基因簇的转录单元;β-半乳糖苷酶活性测定分析rocE基因启动子(ProcE)的转录活性;采用同源重组技术敲除BtHD73菌株的rocE基因;通过融合His标签的方法在大肠杆菌中表达纯化RocR蛋白的HTH结构域;通过凝胶阻滞实验明确RocR与rocE基因启动子的结合作用。【结果】在M9培养基中,精氨酸可诱导ProcE的转录活性;在SSM培养基和精氨酸诱导培养基中,与出发菌株HD73相比,ProcE在sigL (编码Sigma54因子)突变体和rocR突变体中的转录活性显著下降。RocR-HTH蛋白与ProcE有结合作用。rocE基因的缺失对菌体生长和Cry1Ac蛋白产量无显著影响。rocE缺失突变体的芽胞形成率为65.5%,HD73出发菌株为85.7%,显著性分析结果表明差异显著(P0.05)。【结论】rocE基因的转录活性受Sigma54的控制,并受RocR正调控。rocE基因的缺失影响菌株的芽胞形成率。     

一株产高酶活性碱性磷酸酶解淀粉芽孢杆菌的分离及其phoD碱性磷酸酶基因的克隆与表达

[背景]碱性磷酸酶作为工具酶被广泛应用于各个领域,在免疫学检测方面应用较多的是PhoA家族的碱性磷酸酶,尚无关于PhoD家族的碱性磷酸酶在免疫学检测方面的研究。[目的]筛选出一株产高酶活性PhoD家族碱性磷酸酶的细菌,并将其phoD基因进行克隆表达,研究PhoD的酶学性质,为PhoD家族的碱性磷酸酶在免疫学检测方面的应用奠定一定的基础。[方法]采取有机质丰富的土样在有机磷平板中进行细菌分离,以4-硝基苯磷酸二钠盐（4-nitrophenyl phosphate disodium salt hexahydrate,p-NPP）为底物测定有机磷平板中单菌落的酶活性,选取酶活性高的菌株作为目的菌株,克隆其phoD基因。[结果]筛选到一株产碱性磷酸酶酶活性高的菌株S2-4,通过16S rRNA基因序列同源性比较分析,鉴定该菌株为解淀粉芽孢杆菌,克隆了其phoD基因并进行诱导表达。研究了纯化后PhoD的酶学性质,PhoD的最适反应温度为70℃;最适反应pH为9.8;PhoD最适Ca²⁺浓度为3 mmol/L,Mg²⁺对PhoD的酶活性有抑制作用,K     

Characterization of lysine-tagged Bacillus stearothermophilus leucine aminopeptidase II immobilized onto carboxylated gold nanoparticles

Bacillus stearothermophilus leucine aminopeptidase II tagged C-terminally with either tri- or nona-lysine (BsLAPII-Lys_3/9) was constructed and over-expressed in Escherichia coli M15 (pRep4). The recombinant enzymes were purified to homogeneity by nickel-chelate chromatography and their molecular masses were determined to be approximately 45 kDa by SDS/PAGE. Surface modification of colloidal gold with 16-mercaptohexadecanoic acid was employed to generate the carboxylated nanoparticles. BsLAPII-Lys₉ was efficiently immobilized onto the carboxylated gold nanoparticles (AuNP-COOH) and the obtained bioconjugate showed excellent biocatalytic activity in the immobilized form. Additionally, the bioconjugate material exhibited a significant enhancement in temperature stability and could be reused over 5 successive cycles.     

一株自中国大鲵的副炭疽芽孢杆菌分离和鉴定

[目的]对陕西某大鲵养殖场患病的中国大鲵腹水中分离培养得到的一株蜡样芽孢杆菌群细菌疑似菌株进行鉴定,明确该菌生长特性和种类。[方法]无菌解剖患病大鲵,取肠道、腹水、皮肤等各部位的样品均质稀释并分离纯化,从腹水中获得疑似蜡样芽孢杆菌群细菌的纯菌株,命名为SHOU-BC01。对该菌株进行形态与染色特性、培养与生化特性、生物膜形成能力、芽孢形成、药敏检测、全基因组测序等试验鉴定,并根据测序结果进行平均核苷酸相似度(average nucleotide identity,ANI)、数字DNA-DNA杂交(digital DNA-DNA hybridization,dDDH)、多位点序列分型(multilocus sequence typing,MLST)、全基因组SNP聚类和毒力因子分析。[结果]菌株SHOU-BC01为革兰氏阳性杆菌,表面粗糙;具有蛋白酶、卵磷脂酶和溶血酶活性;能够发酵L-阿拉伯糖、D-核糖、D-木糖等多种糖类,能利用色氨酸、丙酮酸盐等;有较强生物膜形成能力;120 h的芽孢形成率达到70.60%;该菌株对青霉素G、头孢噻吩、万古霉素等15种抗生素耐药,对哌拉西林、头孢唑啉、庆大霉素等25种抗生素敏感;根据生物学特性结合ANI、dDDH及全基因组SNP聚类分析,鉴定菌株SHOU-BC01为副炭疽芽孢杆菌(Bacillus paranthracis),经MLST分型,该菌株属于ST205序列型;该菌株含有鞘磷脂酶、CytK和NheC毒素、多糖荚膜、PlcR-PapR群感效应系统及Ⅶ型分泌系统等毒力因子。[结论]成功从中国大鲵腹水中分离出副炭疽芽孢杆菌,丰富了大鲵副炭疽芽孢杆菌数据。     

Development of a cost-effective medium for the large scale production of Bacillus thuringiensis var israelensis

Bacillus thuringiensis var israelensis (B.t.i.) has been widely used in mosquito control programs, but the large scale production of this bacillus is expensive because of the high cost of the medium. In this study, we attempted to develop a cost-effective medium, based on inexpensive, locally available raw materials including soybean flour (Glycine max), groundnut cake powder (Arachis hypogea), and wheat bran extract (Triticum aestivum) by using 100-L fermentor. Sporulation, toxicity, and biomass were satisfactory after B.t.i. was produced on all the three media. Use of the soybean culture medium resulted in maximum toxicity (LC₅₀ 8.89 ng/ml against Culex quinquefasciatus IIIrd instar larvae), highest spore count (0.48 × 10¹¹ c.f.u./ml), and maximum biomass (7.8 g/L) within a short fermentation time of 24 h. Hence, this soybean-based culture medium was considered most economical for the large scale industrial production of B.t.i.     

ccpA基因对蜡样芽胞杆菌氨肽酶生产的影响

【目的】构建蜡样芽胞杆菌(Bacillus cereus)ccp A缺失菌株,并初步探索ccp A基因对其碳代谢及氨肽酶生产的影响。【方法】利用温敏型质粒p KSV7构建蜡样芽胞杆菌CZ ccp A基因缺失突变株CZΔccp A,通过回补菌株对敲除株表型进行验证;不同碳源发酵对比菌株碳代谢的变化,进行氨肽酶发酵优化。【结果】成功构建ccp A缺失菌株CZΔccp A与回补菌株CZ1,三株菌在LB培养基中生长无差异;在柠檬酸钠以及甘露低聚糖为碳源时,菌株的代谢产生明显变化;以D-木糖为单一碳源时,氨肽酶的产量提高48.25%。【结论】CZ ccp A基因对柠檬酸钠、甘露低聚糖、D-木糖为单一碳源时的代谢可能具有调控作用,ccp A基因缺失可以提高蜡样芽胞杆菌CZ的氨肽酶产量。     

Lysis of Aphanizomenon flos-aquae (Cyanobacterium) by a bacterium Bacillus cereus

A phytoplankton-lytic (PL) bacterium, Bacillus cereus, capable of lysing the bloom-forming cyanobacterium Aphanizomenon flos-aquae was isolated from Lake Dianchi of Yunnan province, China. This bacterium showed lytic activities against a wide range of cyanobacteria/algae, including A. flos-aquae, Microcystis viridis, Microcystis wesenbergi, Microcystis aeruginosa, Chlorella ellipsoidea, Oscillatoria tenuis, Nostoc punctiforme, Anabaena flos-aquae, Spirulina maxima, and Selenastrum capricornutum. Chlorophyll a contents, phycocyanin contents, and photosynthetic activities of the A. flos-aquae decreased evidently in an infected culture for a period. Bacterium B. cereus attacked rapidly A. flos-aquae cells by cell-to-cell contact mechanism. It was shown that the lysis of A. flos-aquae began with the breach of the cyanobacterial cell wall, and the cyanobacterial cell appeared abnormal in the presence of the PL bacterium. Moreover, transmission electron microscope examinations revealed that a close contact between the bacterium and the cyanobacterium was necessary for lysis. Some slime extrusions produced from B. cereus assisted the bacterial cells to be in close association with and lyse the cyanobacterial cells. These findings suggested that this bacterium could play an important role in controlling the Aphanizomenon blooms in freshwaters.     

基于表达元件和宿主优化促地衣芽胞杆菌高效表达L-天冬酰胺酶

L-天冬酰胺酶(L-asparaginase, L-ASN)广泛用于恶性肿瘤治疗及低丙烯酰胺食品生产,然而其较低的表达水平限制了应用推广。异源蛋白表达是提高目标酶表达水平的有效策略,芽胞杆菌广泛用于酶蛋白的高效生产,本研究拟通过表达元件及宿主优化提高芽胞杆菌(Bacillus)中L-天冬酰胺酶产量。首先,筛选了5种信号肽(SP_SacC、SP_AmyL、SP_AprE、SP_YwbN、SP_WapA)用于L-天冬酰胺酶的分泌表达,其中SP_SacC介导下L-天冬酰胺酶分泌效果最好,酶活达到157.61 U/mL。随后,选取了4种芽胞杆菌强启动子(P43、P_ykzA-P43、P_Ubay、P_{bac A}),其中串联启动子P_ykzA-P43介导的L-天冬酰胺酶表达量最高,较对照菌株提高了52.94%。最后,筛选了3种芽胞杆菌表达宿主：地衣芽胞杆菌(Bacillus licheniformi...     

一株抑制产气荚膜梭菌的枯草芽孢杆菌BS-2特性

【背景】感染产气荚膜梭菌会引起动物坏死性肠炎,通常使用抗生素进行预防和治疗。随着我国饲料禁抗、养殖减抗的实施,寻找绿色微生态制剂及其代谢产物成为当前研究的热点。【目的】旨在研究前期筛选的一株抑制产气荚膜梭菌的枯草芽孢杆菌BS-2特性。【方法】检测了菌株生长曲线、代谢物质的抑菌特性及细菌素基因簇mRNA表达。【结果】枯草芽孢杆菌BS-2代谢物质对革兰氏阴性菌无抑制作用,而对革兰氏阳性菌具有较强的抑菌性能,并且对产气荚膜梭菌的抑菌性能在2-12 h内迅速增长,在12-24 h内抑菌性能较稳定;该抑菌性能不受胃蛋白酶、胰蛋白酶、蛋白酶K的影响,具有良好的热稳定性;进一步分析抑菌物质基因簇mRNA表达,发现枯草芽孢杆菌BS-2抑制产气荚膜梭菌的活性可能与表面活性素(surfactin)和美杀菌素(mersacidin)表达有关。【结论】枯草芽孢杆菌BS-2对产气荚膜梭菌具有较强的抑制作用,可能通过抑菌物质surfactin和mersacidin表达发挥作用。     

反复冻融对枯草芽孢杆菌St-zn-34抑制链格孢菌活性的影响北大核心CSCD

【目的】以从冬枣分离的内生枯草芽孢杆菌(Bacillus subtilis St-zn-34)为供试菌株,明确反复冻融对菌体形态及其发酵滤液抑制枣缩果病初侵染病菌(Alternaria alternata)活性的变化。【方法】对供试菌株分批发酵后进行反复冻融,采用梯度稀释计数法、滤纸片法分别测定活菌数量和芽孢含量、发酵滤液抑菌活性,对供试菌株的形态变化进行电镜观察。【结果】发酵培养中pH值、活菌量、芽孢量及抑菌活性随发酵时间的增加均呈先上升后下降的趋势,其中发酵60 h发酵滤液抑菌活性最大,对此时的发酵液反复冻融,冻融3次枯草芽孢杆菌活菌量和发酵滤液抑菌活性依次减少,以后再冻融差异不显著(P>0.05),电镜观察发现随冻融次数增加,菌体变小,表面凹陷、扭曲,胶状物流出。抑菌谱检测发现发酵滤液对12种植物病原菌具有抑菌能力。不同温度和蛋白酶处理发酵滤液表明,低于60℃以下,抑菌活性与对照相比差异不显著;80℃以上抑菌活性随温度上升而下降,与对照相比差异显著;蛋白酶K可降低抑菌活性。【结论】反复冻融影响细菌形态并降低发酵滤液的抑菌活性。     

苏云金芽胞杆菌BkdR和CcpA对bkd基因簇的转录调控

【目的】通过分析苏云金芽胞杆菌(Bacillus thuringiensis,Bt)转录调控因子BkdR和多效调控因子CcpA对亮氨酸、异亮氨酸、缬氨酸代谢基因簇bkd的转录调控,明确bkd基因簇的转录调控机制。【方法】通过β-半乳糖苷酶活性测定分析bkd基因簇启动子的诱导转录活性,采用同源重组技术敲除Bt HD73菌株的ccpA基因,通过融合His标签的方法在大肠杆菌中表达纯化BkdR和CcpA蛋白,通过凝胶阻滞实验明确BkdR和CcpA蛋白与bkd基因簇启动子的结合作用。【结果】亮氨酸、异亮氨酸、缬氨酸可诱导bkd基因簇启动子Pptb的转录活性。Pptb的诱导活性在bkdR突变体中明显降低,而在ccpA突变体中明显上升。BkdR和CcpA蛋白与Pptb均有结合作用。【结论】bkd基因簇的转录活性受BkdR正调控,而受CcpA负调控。     

苏云金芽胞杆菌G03 spoIVF操纵子敲除对芽胞和晶体形成的影响

spoIVF是一个普遍存在于芽胞杆菌中的操纵子。在枯草芽胞杆菌中,它编码的两个蛋白是芽胞形成所必需的。采用基因重组技术敲除了苏云金芽胞杆菌G03菌株中的spoIVF操纵子,构建了spoIVF缺失株G03(spoIVF-)。研究表明:该突变株丧失了形成芽胞和晶体的能力。lacZ基因与cry1Aa基因的启动子融合表达分析发现:突变株中的cry1Aa基因的活性严重降低。利用载体pSTK携带spoIVF操纵子在突变株中的表达,使突变株部分恢复了产胞和形成杀虫晶体蛋白的能力。这说明spoIVF操纵子是所必需的,同时该操纵子还影响σ^E因子控制的cry1Aa基因表达。     

Characterization of melanin produced by a wild-type strain of Bacillus cereus

Bacillus cereus 58 (Bc58) is a UV-resistant wild type strain that has an ability to produce a sorrel pigment induced by L-tyrosine. The Fourier-transform infrared (FT-IR) spectra and chemical tests of its pigment are similar to that of the standard melanin (Sigma). A bioassay shows that the LC₅₀ of a Bacillus thuringiensis (Bt) formulation added with the melanin of Bc58 and exposed to UV for 5 h is 16.1 μg/ml, which is similar to that of the Bt formulation without UV treatment, however, it is almost double that of the Bt formulation exposed to UV without the melanin of Bc58. The result of SDS-PAGE indicates that the melanin of Bc58 can protect the insecticidal crystal proteins from degradation. This suggests that it is an excellent UV protective agent for the insecticidal crystal proteins of the Bt formulation. Translated from Microbiology, 2006, 33(1): 42–45 [译自: 微生物学通报]     

枯草芽胞杆菌降解毒死蜱特性研究

研究生物量、pH、毒死蜱浓度和温度对枯草芽胞杆菌3374菌株(编号为GU086422)在水溶液中降解毒死蜱特性,考察该菌株对白菜上毒死蜱残留的降解特性。结果表明,在毒死蜱质量浓度为240 mg/L、pH7.0、温度30℃的适宜条件下,枯草芽胞杆菌3374菌株对毒死蜱的降解率达到92.48%。该菌株能够有效提高白菜叶面上毒死蜱残留的降解速度,表明其在白菜上具有有效降解毒死蜱的能力,在无公害农产品生产中具有广阔的应用潜力。     

植物根际益生细菌代表性菌株贝莱斯芽孢杆菌FZB42对松材线虫的抑杀性

[目的]由松材线虫导致的松树萎蔫病是松树的毁灭性病害,也是我国最主要的林业病害之一.本研究测评了在农业上广泛使用的、我国微生物肥料行业主要菌种资源之一——贝莱斯芽孢杆菌,对松材线虫的潜在抑杀性能.[方法]选用贝莱斯芽孢杆菌的代表性菌株FZB42为材料,测定对不同条件下的菌液上清、不同菌株的菌液上清、细菌素plantaz...