枯草芽孢杆菌在抑制植物病原菌中的研究进展

枯草芽孢杆菌是芽孢杆菌中比较具应用潜力的菌种之一。近年来国内外对于芽孢杆菌各方面应用的研究日益增多,枯草芽孢杆菌作为一种生防细菌越来越引起人们的关注。主要综述了枯草芽孢杆菌在抑制植物病原菌生物防治领域的研究进展,阐述了枯草芽孢杆菌的控病作用机制,包括竞争作用、拮抗作用、溶茵作用、诱导植物产生抗性及促进植物生长5个方面。简要介绍了枯草芽孢杆菌及其制剂在国内外的应用情况及在植物病害防治应用中存在的问题、解决措施及发展前景。     

枯草芽孢杆菌微生态制剂制备及在仿刺参养殖中的应用

采用2%海藻酸钠为壁材,4%氯化钙作为固定化剂,将发酵后收集的枯草芽孢杆菌菌体制备成微生态制剂。将该枯草芽孢杆菌微生态制剂添加到仿刺参基础饲料中,对仿刺参进行喂养,观察其对仿刺参消化酶活性以及生长和仿刺参养殖水质的影响。试验组在基础饲料上添加5 mL的枯草芽孢杆菌微生态制剂,其投喂量为仿刺参体重的5%,并且换水周期为3 d。试验结果表明,添加枯草芽孢杆菌微生态制剂的试验组水质中亚硝酸盐、氨氮、化学需氧量均低于对照组。通过2个生长阶段的比较,试验组内仿刺参肠道内淀粉酶、蛋白酶和纤维素酶活性均高于对照组,尤其淀粉酶活力提高明显。通过对试验组和对照组中仿刺参的质量增加率和质量日增重量进行比较,在生长第1个阶段里两组数值差距不明显,但在生长第2个阶段里,两组数值差距逐渐增大,这说明微生态制荆对仿刺参生长的影响,随着时间逐渐增大。     

动物用芽孢杆菌微生态制剂中蜡样芽孢杆菌的分离及安全性

【背景】芽孢杆菌是仅次于乳酸菌常用于微生态制剂中的菌种,然而部分芽孢杆菌微生态制剂规范不严,应用存在安全隐患。【目的】调查我国在售动物用芽孢杆菌微生态制剂中蜡样芽孢杆菌携带情况,揭示蜡样芽孢杆菌应用的潜在风险。【方法】对微生态制剂预处理,选择性筛选分离蜡样芽孢杆菌,通过全基因组测序测绘细菌毒素基因谱与耐药基因谱,细胞计数试剂盒-8法测定菌株对细胞的毒性,利用微量肉汤稀释法确定菌株耐药值。【结果】从50份微生态制剂产品中筛选分离得到23株蜡样芽孢杆菌群细菌,它们对氨苄西林、林可霉素和泰妙菌素3种抗生素均耐药,主要毒力基因nhe、hbl、cytK、ces的检出率分别为100%、30%、39%和4%,分离株均有溶血性且39%菌株产生热稳定毒素,不同菌株对非洲绿猴肾细胞呈现出不同程度的毒性。【结论】微生态制剂来源的蜡样芽孢杆菌毒性与耐药性严重,携带毒素基因与耐药基因广泛,多株菌株呈高细胞毒性且产生热稳定毒素。芽孢杆菌微生态制剂存在安全性问题,应加强对蜡样芽孢杆菌的质量安全监管力度,规范微生态制剂的市场秩序,杜绝安全隐患。     

枯草芽孢杆菌二联活菌的生物学特性及其基因组分析

摘要 目的：枯草芽孢杆菌二联活菌是我国临床上应用比较广泛的微生态制剂。为了评估该种微生态制剂对肠道健康的影响,对其含有的两种细菌枯草芽孢杆菌R-179和屎肠球菌R-026的生物学特性和耐药性展开研究。方法：通过细菌生长曲线的测定,分析了两种细菌在好氧和厌氧条件下生长状况和氧化还原电位,并对两种菌的抗生素耐药特性进行了检测;同时采用三代测序技术对两株细菌进行了基因组测序和生物信息学分析。结果：枯草芽孢杆菌R-179在好氧条件下生长,厌氧情况下并不生长,其在肠道环境中可以有效的消耗氧气;两种菌混合培养可以有效降低氧化还原电位,耐药性分析表明该两种菌对青霉素、万古霉素敏感,基因组分析表明该两种菌并不含有耐药转移元件,而且枯草芽孢杆菌还分泌多种抗菌活性物质。结论：利用现代研究手段,结合最新测序技术,挖掘和探讨了枯草芽孢杆菌二联活菌在肠道中的作用机制,为枯草杆菌二联活菌用于临床消化道疾病治疗的机理研究和安全应用评价提供了重要的临床参考。     

益生芽孢杆菌对动物免疫功能影响研究进展

益生芽孢杆菌是一种新型的微生态制剂,其抗性好,具有调节动物肠道微生态平衡、促进机体消化与吸收、增强动物免疫功能、提高动物生产性能等益生效应,在动物养殖业中得到了广泛的应用。本文主要综述了益生芽孢杆菌对动物免疫器官、特异性免疫、非特异性免疫和红细胞免疫等免疫功能的影响,免疫调节和产生抗菌物质两方面的作用机理,以及影响免疫效果的因素,提出有待研究的方向和解决的方法,为益生芽孢杆菌的应用提供理论参考。     

蜡状芽孢杆菌LW9809芽孢生成条件及发酵代谢产物研究

对影响蜡状芽孢杆菌 (Bacilluscereus)LW 980 9的芽孢生成条件进行了研究 ,确定了影响芽孢生成的主要因素。对其代谢产物进行了分析测定 ,为合理应用微生态制剂提供了理论依据。     

海水养殖系统中37株枯草芽孢杆菌生理代谢、遗传特性及抑菌效果差异分析

【背景】由水产致病菌导致的病害不断暴发,寻找安全有效的抗生素替代品是目前生产的迫切需求。人们通常过于关注益生菌效应,而对其安全性评价重视度不够。【目的】分析我国海水养殖系统中不同来源枯草芽孢杆菌菌株的表型及遗传特征,并寻找绿色安全且具有多重抑菌作用的菌株。【方法】以2009-2021年从我国海水养殖系统中分离的37株枯草芽孢杆菌为对象,利用纸片扩散法(K-B法)检测其对不同抗生素的抗性;利用培养基平板法测定淀粉酶、蛋白酶和溶血能力;通过PCR方法检测枯草芽孢杆菌溶血相关基因携带风险;采用牛津杯法测定其对副溶血弧菌、溶藻弧菌、爱德华氏菌、哈维氏弧菌、美人鱼发光杆菌和假交替单胞菌等6种病原菌的抑菌作用;并对候选益生性枯草芽孢杆菌的安全性进行评估。【结果】药敏检测结果显示,37株枯草芽孢杆菌对甲氧苄啶、吡哌酸、链霉素表现出强耐药性,对磺胺嘧啶表现出中等耐药,对头孢噻肟、环丙沙星、舒巴坦的耐药率低,对克拉霉素、诺氟沙星、氟苯尼考、氟甲喹、复方新诺明、四环素表现为完全敏感。蛋白酶、淀粉酶活性测试结果显示,37株枯草芽孢杆菌能不同程度地水解酪蛋白和淀粉。溶血性测试结果显示,37株枯草芽孢杆菌中有4株出现溶血现象,而8个溶血相关基因在37株枯草芽孢杆菌中均有检出,溶血表型与检测基因关联分析表明,产生溶血现象的菌株与其溶血基因携带间无直接相关性。抑菌试验分析表明,37株枯草芽孢杆菌均对2种及以上病原菌有抑制作用,对6种病原菌均具有良好抑菌作用的有2株(菌株Bs4和Bs7)。对凡纳滨对虾的安全试验表明,菌株Bs4对凡纳滨对虾具有高安全性,7 d对虾存活率为100%。【结论】通过对37株枯草芽孢杆菌生理代谢表型、遗传特性及病原拮抗特性进行比较分析,揭示了我国海水养殖系统中枯草芽孢杆菌具有多元化的表型及遗传特征,并筛选出一株生态安全且具有多重抑菌活性的益生性枯草芽孢杆菌,为水产养殖病害防控、开发抑菌类微生态制剂及水产养殖行业健康绿色发展提供了理论基础和技术支撑。     

微生物制剂对奥尼罗非鱼生长及消化酶活性的影响

选用192尾初始体重(34.500.25) g的健康奥尼罗非鱼(Oreochromis niloticusO. aureu), 研究在基础饲料中分别添加相同剂量(活菌含量为3.01011 cfu/kg饲料)的汉逊德巴利酵母、枯草芽孢杆菌和凝结芽孢杆菌对奥尼罗非鱼生长及消化酶活性的影响, 试验期56d。试验结果表明, 与对照组相比, 添加枯草芽孢杆菌和凝结芽孢杆菌, 奥尼罗非鱼的增重率分别提高12.27%和8.56%(P0.05), 饵料系数分别降低10.92%和8.18%(P0.05)。饲料干物质表观消化率分别提高10.54%和10.07%(P0.05), 蛋白质表观消化率分别提高4.18%和3.63%(P0.05)。添加枯草芽孢杆菌和凝结芽孢杆菌组, 奥尼罗非鱼肠道、肝胰脏和胃蛋白酶活性显著高于对照组和汉逊德巴利酵母组(P 0.05), 而添加三种微生物制剂对奥尼罗非鱼肠道、肝胰脏和胃的淀粉酶和脂肪酶没有显著影响(P0.05)。结果显示, 三种微生物制剂相互比较, 饲料中添加剂量为3.01011 cfu/kg饲料的枯草芽孢杆菌和凝结芽孢杆菌能显著提高肠道、肝胰脏和胃的蛋白酶活性, 满足最佳生长。       

微生态叶面肥促进小麦生长的效应

研究了稀释100倍的枯草芽孢杆菌、沼泽红假单胞菌、乳酸菌、假丝酵母菌单一菌种微生态叶面肥对小麦生长的效应。结果表明微生态叶面肥对小麦生长有促进作用,其活菌制剂对小麦的生长促进效应更为显著。其中,沼泽红假单胞菌有效的提高成穗率,较对照组增幅为10.2%;枯草芽孢杆菌对小麦的穗粒数和百粒重增加效果明显,较对照组的增幅分别为14.19%和17.01%;乳酸菌则有效的促进了小麦的分蘖及穗长,较对照组的增幅分别为16.24%和6.37%。而酵母菌的促进效果不明显。     

生防用枯草芽孢杆菌固态发酵工艺的研究

目的:提高一株生防用枯草芽孢杆菌固体发酵生产过程中的芽孢产量。方法:研究通过优化固体发酵培养基及发酵生产工艺条件等方法提高了固体发酵枯草芽孢杆菌的芽孢产量。结果:固体发酵过程中,豆饼粉作用显著,能显著提高固体发酵枯草芽孢杆菌的芽孢数,可达到7.1×1010CFU/g。结论:该枯草芽孢杆菌的最优培养基为:麸皮84.4%、稻壳粉10%、豆饼粉5%、硫酸铵0.5%、硫酸镁0.1%、硫酸锰0.05%。生产工艺为料水比为1:1.2,发酵温度为37℃,发酵培养时间为52 h。     

Characterization of sporulation histidine kinases of Bacillus anthracis

The initiation of sporulation in Bacillus species is regulated by the phosphorelay signal transduction pathway, which is activated by several histidine sensor kinases in response to cellular and metabolic signals. Comparison of the protein components of the phosphorelay between Bacillus subtilis and Bacillus anthracis revealed high homology in the phosphorelay orthologs of Spo0F, Spo0B, and Spo0A. The sensor domains of sensor histidine kinases are poorly conserved between species, making ortholog recognition tenuous. Putative sporulation sensor histidine kinases of B. anthracis were identified by homology to the HisKA domain of B. subtilis sporulation sensor histidine kinases, which interacts with Spo0F. Nine possible kinases were uncovered, and their genes were assayed for complementation of kinase mutants of B. subtilis, for ability to drive lacZ expression in B. subtilis and B. anthracis, and for the effect of deletion of each on the sporulation of B. anthracis. Five of the nine sensor histidine kinases were inferred to be capable of inducing sporulation in B. anthracis. Four of the sensor kinases could not be shown to induce sporulation; however, the genes for two of these were frameshifted in all B. anthracis strains and one of these was also frameshifted in the pathogenic pXO1-bearing Bacillus cereus strain G9241. It is proposed that acquisition of plasmid pXO1 and pathogenicity may require a dampening of sporulation regulation by mutational selection of sporulation sensor histidine kinase defects. The sporulation of B. anthracis ex vivo appears to result from any one or a combination of the sporulation sensor histidine kinases remaining.     

Construction and detection of bioluminescent strains of Bacillus subtilis

Bioluminescence ( lux ) genes from Vibrio fischeri and V. harveyi were introduced into Bacillus subtilis on a plasmid vector and by chromosomal integration. The plasmid-bearing strain was highly luminescent and stable under antibiotic selection, but luminescence was lost in the absence of selection and following sporulation and germination. The chromosomally marked strains emitted less light but were found to be stable without the requirement for antibiotic selection and following sporulation and germination. Individual luminescing colonies of both B. subtilis strains could be detected against a high background of non-bioluminescent indigenous soil microbial colonies on agar plates using a charge-coupled device camera. These bioluminescent Gram-positive strains could be of value in studies concerning the survival and spread of genetically-modified micro-organisms in soil environments.     

Characterization of a new sporulation factor in Bacillus subtilis.

We report the existence and partial purification of sporulation factor, which stimulates sporulation of Bacillus subtilis at low cell density. Proline or arginine is required for stimulation under the conditions of our assay. Sporulation factor is a small heat-stable substance produced by the cells during exponential growth phase. It is required in small amounts and is resistant to various proteolytic agents. Several spo mutants were tested for the ability to produce functional sporulation factor. All of these mutants produce factor and do not sporulate in the presence of factor from wild-type cells. Sporulation factor is not involved in the induction of alpha-amylase synthesis at the initiation of sporulation.     

Mapping of Asporogenous Mutations of Bacillus subtilis: a Minimum Estimate of the Number of Sporulation Operons

Sporulation-specific mutations in Bacillus subtilis have been mapped by transduction and transformation. The mutations caused blocks at stages 0, II, III, and IV of sporulation; more than one phenotype was found for each of these stages. On the basis of the criteria used to define a sporulation operon, a minimum estimate could be made of the number of operons activated during sporulation. Nine operons were identified for stage 0, eight for stage II, five for stage III, and six for stage IV. It is probable that several of these 28 operons are activated in groups so that the number of steps in the dependent sequence of sporulation events should turn out to be less than the number of operons.     

Growth and sporulation of entrapped Bacillus subtilis cells

Growing Bacillus subtilis cells were immobilized in k-carrageenan gel beads. Growth and sporulation of entrapped cells were studied by two different methods: cell enumeration and transmission electron microscopy. Immobilized growing cells had a shorter generation time than free cells did, whereas sporulation timing was unchanged. When steady state was reached, cell density was very high in gel beads.     

Growth and sporulation of auxotrophs of Bacillus subtilis in a medium with limited nutrients.

Growth and sporulation were examined for 30 auxotrophs of Bacillus subtilis in a chemically defined medium with suboptimal amounts of nutrients. All strains except for some adenine-requiring mutants could not overtake sporulation stage II when amino acids, vitamins, or bases were limited, whereas they sporulated fairly well without limitation. Abnormal structures, a cell with thickened cell wall and a cell with several refractile bodies, were found in some strains after the vegetative growth stopped.     

A mathematical model for examining growth and sporulation processes of Bacillus subtilis

A mathematical model for the growth process of the bacterium Bacillus subtilis is described. The model is a highly structured one. The driving motivation for development of the model and explicit accounting of major interactions of metabolic networks in the model is related to our eventual goal that the model will be used in the analysis of complex biological patterns. Bacillus subtilis was chosen in our study due to the interesting sporulation process that these cells undergo in response to adverse environmental conditions including nutrient limitation. Sporulation process in B. subtilis represents a primordial prototype of cellular differentiation in higher cellular systems. Thus a model for the B. subtilis growth process should prove extremely useful for understanding questions of developmental biology. The model is capable of simulating the transition between the exponential and stationary phase of growth in a batch culture. Since during the transition period the growth process and the metabolism become decoupled and many transient processes are taking place, such predictions are a severe test for the validity of any model. A strategy to examine the leading hypothesis on B. subtills sporulation implementing GTP as a component which signals sporulation initiation is described.     

The Evolution of Cell-to-Cell Communication in a Sporulating Bacterium

Traditionally microorganisms were considered to be autonomous organisms that could be studied in isolation. However, over the last decades cell-to-cell communication has been found to be ubiquitous. By secreting molecular signals in the extracellular environment microorganisms can indirectly assess the cell density and respond in accordance. In one of the best-studied microorganisms, Bacillus subtilis, the differentiation processes into a number of distinct cell types have been shown to depend on cell-to-cell communication. One of these cell types is the spore. Spores are metabolically inactive cells that are highly resistant against environmental stress. The onset of sporulation is dependent on cell-to-cell communication, as well as on a number of other environmental cues. By using individual-based simulations we examine when cell-to-cell communication that is involved in the onset of sporulation can evolve. We show that it evolves when three basic premises are satisfied. First, the population of cells has to affect the nutrient conditions. Second, there should be a time-lag between the moment that a cell decides to sporulate and the moment that it turns into a mature spore. Third, there has to be environmental variation. Cell-to-cell communication is a strategy to cope with environmental variation, by allowing cells to predict future environmental conditions. As a consequence, cells can anticipate environmental stress by initiating sporulation. Furthermore, signal production could be considered a cooperative trait and therefore evolves when it is not too costly to produce signal and when there are recurrent colony bottlenecks, which facilitate assortment. Finally, we also show that cell-to-cell communication can drive ecological diversification. Different ecotypes can evolve and be maintained due to frequency-dependent selection.     

Chromosome segregation in<Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Bacillus subtilis, a Gram-positive bacterium commonly found in soil, is an excellent model organism for the study of basic cell processes, such as cell division and cell differentiation, called sporulation. In B. subtilis the essential genetic information is carried on a single circular chromosome, the correct segregation of which is crucial for both vegetative growth and sporulation. The proper completion of life cycle requires each daughter cell to obtain identical genetic information. The consequences of inaccurate chromosome segregation can lead to formation of anucleate cells, cells with two chromosomes, or cells with incomplete chromosomes. Although bacteria miss the classical eukaryotic mitotic apparatus, the chromosome segregation is undeniably an active process tightly connected to other cell processes as DNA replication and compaction. To fully understand the chromosome segregation, it is necessary to study this process in a wider context and to examine the role of different proteins at various cell life cycle stages. The life cycle of B. subtilis is characteristic by its specific cell differentiation process where, two slightly different segregation mechanisms exist, specialized in vegetative growth and in sporulation.     

Genome engineering reveals large dispensable regions in Bacillus subtilis

Bacterial genomes contain 250 to 500 essential genes, as suggested by single gene disruptions and theoretical considerations. If this view is correct, the remaining nonessential genes of an organism, such as Bacillus subtilis, have been acquired during evolution in its perpetually changing ecological niches. Notably, approximately 47% of the approximately 4,100 genes of B. subtilis belong to paralogous gene families in which several members have overlapping functions. Thus, essential gene functions will outnumber essential genes. To answer the question to what extent the most recently acquired DNA contributes to the life of B. subtilis under standard laboratory growth conditions, we initiated a "reconstruction" of the B. subtilis genome by removing prophages and AT-rich islands. Stepwise deletion of two prophages (SPbeta, PBSX), three prophage-like regions, and the largest operon of B. subtilis (pks) resulted in a genome reduction of 7.7% and elimination of 332 genes. The resulting strain was phenotypically characterized by metabolic flux analysis, proteomics, and specific assays for protein secretion, competence development, sporulation, and cell motility. We show that genome engineering is a feasible strategy for functional analysis of large gene clusters, and that removal of dispensable genomic regions may pave the way toward an optimized Bacillus cell factory.