一株芽孢杆菌的分离和鉴定

从中国农业科学院北京畜牧兽医研究所鸡舍附近土壤中分离到一株芽孢杆菌P-25,并进行了分子鉴定。通过形态鉴定、革兰氏染色、生理生化测定、16SrRNA序列分析和系统发育树构建,确定该菌株为蜡状芽孢杆菌(Bacillus cereus),其16SrRNAGenBank登录号为GU271135。     

球形赖氨酸芽孢杆菌(Lysinibacillus sphaericus)和嗜冷芽孢八叠球菌(Sporosarcina psychrophila)介导形成白云石晶体

【目的】白云石(Dolomite)是一种含有钙镁的碳酸盐矿物[CaMg(CO3)2],广泛存在于陆地和海洋等环境并常与油气埋藏共存。尽管白云石(或岩)的发现已经有三百多年的历史,但是对白云石的成因仍然没有定论,地质学上称之为"白云石之谜"。20世纪90年代Vasconcelos C.提出了"微生物白云石模型",为白云石成因的研究带来了新的思路。但是这一模型并不完善,白云石的形成与所介导的微生物生理状态以及环境参数之间的关系不明确。另外,所有报道的实验都是在地表压强条件下进行,无法表征自然界中白云石所处的高压环境。本研究中引入压力这一环境参数,结合菌株本身生理特性参数,综合考察多重因子对微生物介导形成白云岩的影响。【方法】利用球形赖氨酸芽孢杆菌(Lysinibacillus sphaericus)和嗜冷芽孢八叠球菌(Sporosarcina psychrophila)两株具有尿素水解活性的细菌作为生物材料,在不同的温度(15°C和30°C)压强(常压和20 MPa)氧气浓度(常压好氧条件和常压微氧条件)不同的尿素水解活性下进行生物矿化实验。通过SEM(扫描电子显微镜)和EDS(X射线能谱分析)相结合的方法观察沉淀物形貌和矿物成分构成。通过XRD(X射线衍射分析)定性测定碳酸盐矿物沉淀物的种类。【结果】球形赖氨酸芽孢杆菌和嗜冷芽孢八叠球菌在实验中所设计的所有矿化条件下都能够介导形成碳酸盐矿物沉淀。XRD和SEM检测均证实球形赖氨酸芽孢杆菌在30°C的20 MPa微氧条件下能够介导形成不规则菱面型和椭球型白云石。高压条件更有助于白云石的形成。除了白云石晶体,实验中还观察到有其他矿物(如方解石,碳氢镁石,钙镁碳酸石等)。【结论】实验证实球形赖氨酸芽孢杆菌和嗜冷芽孢八叠球菌具有矿化能力,特别是球形赖氨酸芽孢杆菌具有介导形成白云石的能力。微生物介导形成的碳酸盐矿物组分受到微生物的代谢活性以及温度、压力等生物矿化实验条件控制。这一研究结果帮助完善"微生物白云石模型",为解释白云石的深部成因提供数据支持。     

一株抗真菌内生多粘芽孢杆菌的分离鉴定及对水稻恶苗病菌的抑制作用

目的：从番茄叶片中筛选具广谱抑真菌活性的拮抗内生细菌,研究其对水稻恶苗病菌的抑制作用。方法：采用对峙培养法筛选拮抗内生细菌,根据菌株形态、生理生化特性结合16S rRNA基因序列分析鉴定菌株;采用硫酸铵沉淀法提取抗菌粗蛋白,研究其对水稻恶苗病菌菌丝生长和孢子萌发的影响。结果：从番茄叶片中筛选到一株抗真菌内生多粘芽孢杆菌（Paenibacillus polymyxa）SD-6,该菌株具有广谱抑菌活性,对供试的13种植物病原真菌均具较强的抑制作用;该菌株产生的抗菌粗蛋白能够显著抑制水稻恶苗病菌菌丝生长和孢子萌发,并能导致萌发孢子畸形和破裂。结论：从番茄叶片中分离到一株能产生抗真菌蛋白并具有广谱高效抑真菌作用的内生多粘芽孢杆菌,该菌株及其抗菌蛋白具有防治水稻恶苗病的潜力。     

赖氨酸芽孢杆菌GW-2菌株作用下碳钙镁石的形成

摘要:【目的】微生物诱导碳酸盐矿物沉淀机理的研究有可能为CO2的矿物捕获提供科学依据。【方法】本研究在Mg/Ca 为2的B4培养基中对赖氨酸芽孢杆菌GW-2菌株进行了为期50天的培养实验,同时完成了一组无菌对照实验。在实验过程中,对沉淀物重量、培养液的pH值、电导率及其中细菌数量、Ca2+ 和Mg2+浓度等进行了动态监测。利用扫描电子显微镜对矿物形态进行了跟踪观察,利用X-射线衍射仪对矿物成分进行了测定。【结果】(1)在对GW-2菌株进行培养的过程中发现,沉淀物的质量随着培养时间的延长而逐 渐增加,而在无菌对照实验中未收集到沉淀物;(2)各时间段平均沉淀速率与细菌数量之间(r=0.67,P＜0.05)、沉淀物质量与培养液pH值之间(r=0.79,P＜0.05)均具有显著的正相关关系;(3)沉淀物质量与电导率、Ca2+和Mg2+浓度均呈极显著的负相关关系(r分别为0.89、0.93和0.98,P＜0.001);(4)在赖氨酸芽孢杆菌作用下,3种碳酸盐矿物按如下顺序先后形成:非晶态碳酸钙→碳钙镁石→高镁方解石。【结论】(1)赖氨酸芽孢杆菌GW-2菌株具有诱导碳酸盐矿物形成的能力;(2)细菌数量是控制碳酸盐矿物沉淀的直接因素,而较高的pH值是碳酸盐沉淀的必要条件;(3)电导率以及Ca2+和Mg2+浓度的明显降低可以间接地指示碳酸盐矿物沉淀的发生;(4)碳钙镁石可能主要是非晶态碳酸钙经过老化作用而形成,碳钙镁石可能经脱镁作用转变为高镁方解石。     

一株种内拮抗的海洋枯草芽孢杆菌的分离与鉴定

从中国东海海域筛选到好氧耐盐菌株A01,此菌株显示出抗枯草芽孢杆菌和白色念珠菌的活性采取对该菌株形态特征、培养特征、生理生化特征和遗传特性进行研究的方法,结果表明此菌株与枯草芽孢仟菌（Bacillus subitilis）的特征一致;将此菌株的16S rDNA序列在GenBank中进行序列比对,结果亦显示其与Bacillus subitilis的16SrDNA的序列片段的相似性为100％;以相似性为基础构建系统发育树,分析表明该菌株与Bacillus subitilis同源关系最近最终得出结论为菌株A01是一株来源于海洋的枯草芽孢杆菌,且具有种内拮抗的特性。     

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。     

一株水稻根内生拮抗细菌SM13的分离及鉴定

【背景】作物根内生细菌具有固氮、分泌激素、产生病原真菌抗性物质等特性,根系内生菌的分离及应用成为环境友好型防控技术研究的热点之一。近年来盐碱地水稻种植面积逐年增加,而关于盐碱地水稻根内生菌的分离与应用鲜有报道。【目的】从大庆盐碱地水稻根中分离内生细菌,筛选对植物真菌病害有拮抗作用的促生菌株,初步探讨其抑菌和促生功效,为进一步研究其抑菌和促生机理提供菌种资源。【方法】对水稻根表面灭菌后研磨涂布分离内生细菌,采用对峙培养法和改良Salkowski比色法筛选具有广谱抑菌效果并有分泌吲哚-3-乙酸(Indole-3-acetic acid,IAA)能力的菌株,通过形态鉴定、革兰氏染色、生理生化测定、16S rRNA结构基因以及srfA、ituA、fenB功能基因序列系统进化分析,确定细菌的分类地位。【结果】从水稻根部分离到一株内生细菌SM13,该菌株具有广谱性抑菌作用,对玉米新月弯孢菌、大豆菌核病菌、稻瘟病菌、禾谷镰刀菌的抑菌率分别为59.38%、78.13%、53.12%、37.50%,分泌IAA的能力为5.56±0.41μg/mL (n=6)。经形态学、生理生化试验结合系统进化分析初步鉴定SM13菌株属于枯草芽孢杆菌(Bacillus subtilis),该菌株在pH 11.0、盐浓度10%的NA培养基中生长良好,具有较高的耐盐碱性。【结论】水稻根内生菌SM13菌株具有耐盐碱性、促生和生防性能,可作为微生物农药及菌肥的材料。     

水稻内生枯草芽孢杆菌G87抗菌蛋白的分离纯化及理化特性

【目的】为得到枯草芽孢杆菌(Bacillus subtilis)G87的抗菌蛋白,明确其蛋白理化特性。【方法】采用硫酸铵沉淀和柱层析法进行分离纯化。【结果】获得单一抗菌活性蛋白(峰6-2-1),此抗菌蛋白分子量为50.8 kDa,等电点为5.90。经初步分析,抗菌蛋白不含脂,而含有少量(0.62%)糖;其蛋白部分具有脯氨酸或羟脯氨酸,但不含芳香族氨基酸。抗菌蛋白在高温(≥60℃)和较碱(pH8)环境下活性明显下降,但较抗紫外线、氯仿和胰蛋白酶、蛋白酶K、胃蛋白酶。【结论】枯草芽孢杆菌G87的抗菌蛋白为不含芳烃的糖蛋白,对高温和碱性条件敏感,而对蛋白酶类和紫外线等不敏感。     

两株茶树内生草螺菌的微生物学特性

【目的】从健康茶树叶片内分离两株内生乳白色短杆菌(编号WT00C和WT00F)并进行微生物学特性调查。【方法】通过细菌培养和染色的方法进行了形态观察;通过微生物生理生化分析的方法进行了生物活性测定,还进行了16S rDNA序列分析以及生理生化特性调查;通过系统发育学分析及各项指标的比较,确定两个菌株的分类归属。【结果】两株细菌菌落形态为圆形、不透明、乳白色、中央隆起、边缘整齐。菌体呈杆状,大小为(0.5-0.7)μm×(1.4-1.8)μm,有鞭毛,无芽孢,革兰氏染色阴性,产生IAA、NH4+和嗜铁载体但无固氮酶活性。WT00C和WT00F菌株产生IAA量分别为18.7±1.2 mg/L和24.9±1.5 mg/L。除不能利用丙酸盐外,它们的生理特征与伯杰氏手册中草螺菌属生化指标中的可利用碳源情况基本一致,并且与已鉴定的13种草螺菌的16S rDNA高度同源,相似度达99%。基于16S rDNA序列的系统发育学分析结果显示,两株细菌形成一个独立的分支,与已报道的13种草螺菌聚类并保持着一定的距离,两个菌株的生理生化特征与其它草螺菌有许多共性但存在明显的差别。【结论】分离获得的两株茶树内生细菌WT00C和WT00F为草螺菌属的新菌株。     

武夷山地衣表生和内生芽孢杆菌种群的多样性

摘要:【目的】分析武夷山地衣表生和内生芽孢杆菌种群的多样性。【方法】从武夷山自然保护区采集扁枝衣属(Evernia)、珊瑚枝属(Stereocaulon)、孔叶衣属(Menegazzia)等分属于7科9属的地衣样品9份,分离地衣表面附生(表生)和内生芽孢杆菌,并根据16S rRNA基因序列同源性对分离得到的芽孢杆菌进行种的鉴定。【结果】未从扁枝衣属、树花属(Ramalina)和茶渍属(Lecanora)的3个样品中分离出表生或内生芽孢杆菌,从另外6个样品中分离出芽孢杆菌34株,分别鉴定为芽孢杆菌属(Bacillus)、类芽孢杆菌属(Paenibacillus)、短芽孢杆菌属(Brevibacillus)、赖氨酸芽孢杆菌属(Lysinibacillus)和绿芽孢杆菌属(Viridiibacillus)的24个种。类芽孢杆菌属、芽孢杆菌属是这些地衣表生、内生芽孢杆菌的优势种群,分别占分离得到菌株的41.2%(14株)和35.3%(12株);短芽孢杆菌属、赖氨酸芽孢杆菌属和绿芽孢杆菌属为首次报道从地衣中分离获得。从不同的地衣样品分离的表生和内生芽孢杆菌的种类、数量存在差异。蜈蚣衣属(Physcia)表生的芽孢杆菌数量最多、可达3.85×106 cfu/g,而珊瑚枝属表面附生和内生芽孢杆菌的种类最多、共有12个种。分离到的芽孢杆菌大部分来自单独的一种地衣;台中类芽孢杆菌(Paenibacillus taichungensis)、土壤短芽孢杆菌(Brevibacillus agri)等4个种存在于2－3种地衣中;蕈状芽孢杆菌(Bacillus mycoides)分布最广,在蜈蚣衣属、珊瑚枝属等4种地衣中都有存在。【结论】武夷山地衣表生和内生芽孢杆菌存在种类和数量的多样性。     

Lysinibacillus composti sp. nov., isolated from compost

A Gram-negative, motile, rod-shaped, endospore-forming bacterial strain, designated as NCCP-36^T, was isolated from the compost of fruit and vegetable wastes. The strain NCCP-36^T grew within a temperature range of 10–45?^○C (optimum 28?^○C) and a pH range of 6.5–8.5 (optimum 7.0), and its cells tolerated <50 mM boron (optimum growth without boron) and 0–5 % NaCl (w/v) in tryptic soya broth medium. Based on comparative analysis of 16S rRNA gene sequence, strain NCCP-36^T showed the highest similarity to Lysinibacillus sinduriensis BLB-1^T (97.52 %) and L. xylanilyticus XDB9^T (96.96 %), and <97 % similarity with other closely related taxa. However, DNA–DNA relatedness between strain NCCP-36^T and the closely related type strains of genus Lysinibacillus was ≤37 %. Phylogenetic and chemotaxonomic analyses [major polar lipids: diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, and phospholipids; predominant menaquinone: MK-7; major cellular fatty acids: iso-C_15:0, antieso-C_15:0, and iso-C_16:0; DNA G+C contents: 37 mol %; Lys-Asp (type A4α) in cell-wall peptidoglycans as diagnostic amino acids] also support the affiliation of strain NCCP-36^T to genus Lysinibacillus. Based upon DNA–DNA relatedness as well as distinctive chemotaxonomic, phylogenetic, and genotypic data, we conclude that strain NCCP-36^T belongs to a novel species of genus Lysinibacillus, for which the name Lysinibacillus composti sp. nov. is proposed. The type strain is NCCP-36^T (JCM 18777^T?=?KCTC 13796^T?=?DSMZ 24785^T).     

Transformation of malathion by Lysinibacillus sp. isolated from soil

An axenic bacterial strain, Lysinibacillus sp. KB1, was isolated from malathion-contaminated soil. It tolerated malathion up to 0.15?% and, under aerobic conditions, utilized it as sole carbon source. 20?% malathion and 47?% malaoxon were degraded out of the initially provided malathion. Two metabolites, mal-monocarboxylic acid and mal-dicarboxylic acid, were detected within 7?days at 30?°C. Esterase activity of the strain was 240?±?2.5?U/ml after 7?days of growth. Sterilized soil mixed with malathion showed rapid degradation of malathion when inoculated with strain KB1 as compared to the uninoculated soil.     

Lysinibacillus jejuensis sp. nov., isolated from swinery waste

A Gram-positive, endospore-forming, rod-shaped bacterium, designated strain N2-5^T, was isolated from swinery waste collected in Jeju, Republic of Korea. A phylogenetic analysis based on 16S rRNA gene sequences demonstrated that strain N2-5^T formed a phyletic group within the phylum Firmicutes with less than 97.0% similarities to members of the genus Lysinibacillus, its nearest phylogenetic neighbors. The highest levels of sequence similarity to the isolate were observed against Lysinibacillus xylanilyticus XDB9^T (96.8%), Lysinibacillus macroides LMG 18474^T (95.6%), and Lysinibacillus parviboronicapiens BAM-582^T (95.6%). The organism grew optimally at 30°C and pH 7 and in the presence of 1–3% (w/v) NaCl. Strain N2-5^T was chemotaxonomically characterized by possessing menaquinone-7 (MK-7) as the major menaquinone, and iso-C_15:0 (54.9%), iso-C_17:1 ω10c (12.0%), and C_16:1 ω7c alcohol (11.8%) as the predominant fatty acids. The genomic DNA G+C content of the novel strain was 43.3 mol% and the cell-wall peptidoglycan was type A4α. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylethanolamine. Based on its phenotypic properties and phylogenetic data, strain N2-5^T (=DSM 28310^T =KCTC13837^T) represents a novel species in the genus Lysinibacillus, for which the name Lysinibacillus jejuensis sp. nov. is proposed.     

Lysinibacillus tabacifolii sp. nov., a novel endophytic bacterium isolated from Nicotiana tabacum leaves

A Gram-positive, catalase- and oxidase-positive, strictly aerobic, endospore-forming rod bacterium, designated K3514^T, was isolated from the leaves of Nicotiana tabacum. The strain was able to grow at temperatures of 8–40°C, pH 5.0–10.0 and NaCl concentrations of 0–7%. The predominant quinones (>30%) of this strain were MK-7(H2) and MK-7. Phylogenetic analysis of 16S rRNA gene sequence showed that strain K3514^T was affiliated to the genus Lysinibacillus, with its closest relatives being Lysinibacillus mangiferihumi (98.3% sequence similarity), Lysinibacillus sphaericus (97.9% sequence similarity), Lysinibacillus fusiformis (97.4% sequence similarity), and Lysinibacillus xylanilyticus (97.3% sequence similarity). However, low levels of DNA-DNA relatedness values suggested that the isolate was distinct from the other closest Lysinibacillus species. Additionally, based on analysis of morphological, physiological, and biochemical characteristics, the isolate could be differentiated from the closest known relatives. Therefore, based on polyphasic taxonomic data, the novel isolate likely represents a novel species, for which the name Lysinibacillus tabacifolii sp. nov. and the type strain K3514^T (=KCTC 33042^T =CCTCC AB 2012050^T) are proposed.     

Biosequestration, transformation, and volatilization of mercury by Lysinibacillus fusiformis isolated from industrial effluent

In the present study, an efficient mercury-tolerant bacterial strain (RS-5) was isolated from heavy-metalcontaminated industrial effluent. Under shake flask conditions, 97% of the supplemented mercuric chloride was sequestered by the biomass of RS-5 grown in a tryptone soy broth. The sequestered mercuric ions were transformed inside the bacterial cells, as an XRD analysis of the biomass confirmed the formation of mercurous chloride, which is only feasible following the reaction of the elemental mercury and the residual mercuric chloride present within the cells. Besides the sequestration and intracellular transformation, a significant fraction of the mercury (63%) was also volatilized. The 16S rRNA gene sequence of RS-5 revealed its phylogenetic relationship with the family Bacillaceae, and a 98% homology with Lysinibacillus fusiformis, a Gram-positive bacterium with swollen sporangia. This is the first observation of the sequestration and volatilization of mercuric ions by Lysinibacillus sp.     

Populous: a tool for building OWL ontologies from templates

BACKGROUND: Ontologies are being developed for the life sciences to standardise the way we describe and interpret the wealth of data currently being generated. As more ontology based applications begin to emerge, tools are required that enable domain experts to contribute their knowledge to the growing pool of ontologies. There are many barriers that prevent domain experts engaging in the ontology development process and novel tools are needed to break down these barriers to engage a wider community of scientists. RESULTS: We present Populous, a tool for gathering content with which to construct an ontology. Domain experts need to add content, that is often repetitive in its form, but without having to tackle the underlying ontological representation. Populous presents users with a table based form in which columns are constrained to take values from particular ontologies. Populated tables are mapped to patterns that can then be used to automatically generate the ontology's content. These forms can be exported as spreadsheets, providing an interface that is much more familiar to many biologists. CONCLUSIONS: Populous's contribution is in the knowledge gathering stage of ontology development; it separates knowledge gathering from the conceptualisation and axiomatisation, as well as separating the user from the standard ontology authoring environments. Populous is by no means a replacement for standard ontology editing tools, but instead provides a useful platform for engaging a wider community of scientists in the mass production of ontology content.