抗菌肽天蚕素B基因在纳豆芽胞杆菌中的表达

抗菌肽是生物体内经诱导产生的一类具有生物活性的小分子多肽。天蚕素B(Cecropin B)是最早从天蚕体内分离得到的一种热稳定的可溶性多肽,在已分离的众多抗菌肽中抗性较强。纳豆芽胞杆菌具有优良的益生特性,本研究选择枯草芽胞杆菌的一种表达载体p HT43,将抗菌肽天蚕素B基因导入纳豆芽胞杆菌中,验证其在目的菌中是否能够表达和稳定传代以及进行抗菌活性分析。结果表明,天蚕素B基因在纳豆芽胞杆菌中表达,并能稳定传代,能够提高纳豆芽胞杆菌的抑菌活性,抗金黄色葡萄球菌的活性优于干酪乳杆菌和枯草芽胞杆菌。本研究为该重组菌作为饲料添加剂的应用提供了技术基础。本文首次报道天蚕素B在纳豆枯草芽胞杆菌中表达。     

细菌群体感应抑制活性微生物Zou 03的筛选与鉴定

从近海区生态环境中分离纯化98株海洋菌株,以根癌农杆菌WCF47为敏感检测菌株,筛选出1株具细菌群体感应抑制活性的菌株Zou03,对其进行形态、生理生化特征鉴定和16S rDNA分子鉴定。结果显示,Zou03具枯草芽胞杆菌(Bacillus subtilis)的典型特征,其16S rDNA序列通过对比分析,与GenBank中枯草芽胞杆菌16SrDNA的部分序列同源性为100%。综合形态、生化特征及16S rDNA序列对比分分析,鉴定菌株Zou03为枯草芽胞杆菌。表明近海区生态环境中存在具有抑制细菌群体感应活性的微生物,有利于海洋微生物资源开发,为以致病菌群体感应系统为靶点的新型疗法提供新技术。     

1株枯草芽胞杆菌的鉴定及其弹性蛋白酶结构研究

鉴定弹性蛋白酶产生菌株EL32,确定该酶的基本结构。采用分子生物学、形态学及生理生化性质对菌株EL32进行鉴定;用硫酸铵沉淀、离子交换层析和分子筛层析纯化酶蛋白;借助肽指纹图谱及酶基因克隆技术研究该酶的一级结构;利用同源建模的方法研究该酶的空间结构。菌株EL32的16S rDNA与枯草芽胞杆菌16S rDNA的同源性达到99%,其菌落呈乳白色,其细胞革兰染色阳性,具有芽胞;发酵葡萄糖试验产酸不产气,明胶水解试验呈阳性,能够水解淀粉,V-P反应呈阳性,鉴定为枯草芽胞杆菌。从菌株BL32发酵液中纯化得到了弹性蛋白酶,SDS-PAGE分析显示其分子量为31 ku。用LTQ-MS测定肽指纹图谱表明该弹性蛋白酶是枯草芽胞杆菌蛋白酶subtilisin,该酶的基因和蛋白质序列与枯草芽胞杆菌蛋白酶subtilisin的同源性都高达99%。菌株EL32弹性蛋白酶的三维结构含有6个α-螺旋,7个扭曲的平行β-折叠以及2个反平行的β-折叠,His、Asp和Ser是其活性中心的关键基团。鉴定了1株产弹性蛋白酶的枯草芽胞杆菌,确定了其弹性蛋白酶是蛋白酶subtilisin,为该枯草芽胞杆菌蛋白酶的应用提供了基础。     

常见4种微生态制剂菌种产淀粉酶的比较

目的 探索常见4种微生态制剂菌种地衣芽胞杆菌、枯草芽胞杆菌、蜡样芽胞杆菌和纳豆芽胞杆菌产生淀粉酶的能力,以筛选和研制动物微生态制剂和饲料添加剂的使用菌种。方法 将各菌种接于淀粉酶试验培养基,培养后滴加稀碘溶液,观察透明圈,判定产酶能力。结果 地衣芽胞杆菌、枯草芽胞杆菌、蜡样芽胞杆菌和纳豆芽胞杆菌都能产生淀粉酶,以蜡样芽胞杆菌产生的淀粉酶较多。结论 4种常见芽胞杆菌产淀粉酶能力依次为：蜡样芽胞杆菌＞纳豆芽胞杆菌＞枯草芽胞杆菌＞地衣芽胞杆菌。     

佳西热带雨林土壤芽胞杆菌分离与多样性分析

采用热处理法从海南省佳西热带雨林土壤中分离到147株芽胞杆菌,并利用16S rDNA PCR-RFLP与序列分析技术对其遗传多样性进行了研究。16S rDNA PCR-RFLP酶切图谱UPGMA聚类分析结果表明,在100%的相似性水平上,这些芽胞杆菌分属13个遗传类群。不同遗传类型代表菌株的16S rRNA基因序列分析结果显示,它们分布在Bacillaceae、Planococcaceae和Paenibacillaceae科的Bacillus、Lysinibacillus、Paucisalibacillus、Bhargavaea和Paenibacillus五个属,其中Bacillus为优势属(占50%);有3株芽胞杆菌的16S rRNA基因序列与数据库中相应模式菌株的最大相似性在98.3%~98.9%之间。结果表明,佳西热带雨林土壤中芽胞杆菌有着较为丰富的遗传多样性。     

利用16S rRNA基因同源性分析鉴定两株明串珠菌

从酸马奶中分离出2株明串珠菌KLDS 5.0301和KLDS 5.0302,对2株菌的16S rRNA基因经PCR扩增测序,将测序结果同该属内菌株的16S rRNA序列作多序列比较,并建立明串珠菌属的系统发育树.结果表明,KLDS 5.0301的16S rRNA序列同L. garlicum的同源性百分比为100%.KLDS 5.0302的16S rRNA序列同L.mesenteroides LM2菌株的16S rRNA序列的同源性百分比为99.9%.根据系统发育树的结果,将KLDS5.0301鉴定为L.garlicum,KLDS 5.0302鉴定为L.mesenteroides.菌株KLDS 5.0301和KLDS 5.0302的16SrRNA序列已经在GeneBank申请国际序列注册号,分别为DQ239691和DQ297412.     

对胞必佳生产菌株的重新鉴定

主要是从形态学观察、菌株脂肪酸成分和16S rRNA基因全序列3个方面出发,重新对胞必佳生产菌株红色诺卡氏菌(Nocardia rubra)进行鉴定。结果表明,该菌株并非诺卡氏菌属中的红色诺卡氏菌,而属于红球菌属。16S rRNA序列相似性比较和系统进化树进一步说明,该菌株与Rhodococcus ruber(AY114117.1)的同源性最高,是1株红色红球菌。     

冬凌草内生细菌的分离鉴定及其对植物病害的生防作用

从冬凌草(Rabdosia rubescens (Hemsl.) H.Hara)中分离筛选出1株对多种作物真菌病害具有显著拮抗作用的细菌,命名为KD3.通过其形态特征和生理生化特性以及16S rRNA序列的同源性分析,鉴定该菌株为枯草芽胞杆菌(Bacillus subtilis).试验表明,KD3菌株能够显著抑制多种病原真菌的侵染,具有良好的应用开发潜力.     

基于脂肪酸生物标记与16S rRNA的芽胞杆菌系统发育分析比较

旨在探究脂肪酸作为一种有效的芽胞杆菌分类标记,以25种芽胞杆菌模式菌株为研究对象,对芽胞杆菌进行脂肪酸组分和16S rRNA基因系统进化分析比较。结果表明,脂肪酸系统发育分析能充分体现芽胞杆菌种类间的亲缘关系,并且按生物学特性进行聚类分群,而16S rRNA系统发育仅完美体现出种间的亲缘关系。利用脂肪酸分析可将25种芽胞杆菌完全准确分开,且将生物学特性相同的芽胞杆菌种类聚为一类,如碱性条件下生长良好的4种芽胞杆菌(B.agaradhaerens、B.alacalphilus、B.alkalitelluris和B.fastidiosus)聚为一类,芽胞杆菌为圆形的芽胞杆菌(B.fusiformis、B.odysseyi和B.sphaericus)聚为一类。结果表明,脂肪酸分析不仅根据亲缘关系进行聚类,还可以根据生物学特性对芽胞杆菌进行分类。     

1株抗菌植物内生菌EJH-2菌株的分离和鉴定

目的从中药植物金银花的组织中分离到1株具有抗菌活性的植物内生菌EJH-2菌株,并对其进行了分子生物学鉴定。方法通过总DNA提取,PCR扩增,得到1300bp的16S rRNA序列。PCR产物序列通过BLAST软件在NCBI网站中进行同源性比较。通过Bioedit7．0和Treedrawing软件绘制系统发育树。结果EJH-2的16SrRNA序列和数据库中的类多粘芽胞杆菌KCTC 1663菌株的序列的同源性为99．76％。在系统发育树中,EJH-2菌株和多粘类芽胞杆菌在同一分支。结论EJH-2菌株应归属于多粘类芽胞杆菌（Paenibacillus polymyxa）。     

Detection of molecular diversity in Bacillus atrophaeus by amplified fragment length polymorphism analysis

Phenotypically, Bacillus atrophaeus is indistinguishable from the type strain of Bacillus subtilis except by virtue of pigment production on certain media. Several pigmented variants of B. subtilis have been reclassified as B. atrophaeus, but several remain ambiguous in regard to their taxonomic placement. In this study, we examined strains within the American Type Culture Collection originally deposited as Bacillus globigii, B. subtilis var. niger, or Bacillus niger using 16S rRNA gene sequencing and amplified fragment length polymorphism (AFLP) analysis to determine the level of molecular diversity among these strains and their relationship with closely related taxa. The 16S rRNA gene sequences revealed little variation with one base substitution between the B. atrophaeus type strain ATCC 49337 and the other pigmented bacilli. AFLP analysis produced high-quality DNA fingerprints with sufficient polymorphism to reveal strain-level variation. Cluster analysis of Dice similarity coefficients revealed that three strains, ATCC 31028, ATCC 49760, and ATCC 49822, are much more closely related to B. atrophaeus than to B. subtilis and should be reclassified as B. atrophaeus. A very closely related cluster of B. atrophaeus strains was also observed; this cluster was genetically distinct from the type strain. The level of variation between the two groups was approximately the same as the level of variation observed between members of the two B. subtilis subspecies, subtilis and spizizenii. It is proposed that the cluster of strains typified by ATCC 9372 be designated a new subspecies, B. atrophaeus subsp. globigii.     

Identification and molecular characterization of a novel Bacillus strain capable of degrading Tween-80

A Tween-80-degrading novel marine Bacillus strain, N10, has recently been isolated in Alexandria University, Egypt. The taxonomic position of this endospore forming bacterium was investigated on the basis of fatty acid analysis and 16S rRNA gene sequencing. Comparative computer database analyses revealed that the bacterium is a Bacillus subtilis strain. The gene encoding the small acid-soluble protein gamma-type (SASP-B), sspE, was successfully utilized in this study as a tool for discrimination between the two B. subtilis subspecies W23 and 168. Based on the alignment of 16S rRNA sequences and analysis of SASP-B relatedness, it has been demonstrated that the novel marine B. subtilis strain N10 is more closely related to the B. subtilis reference strain W23 than to 168. The strain, N10, has been deposited in the Bacillus Genetic Stock Center (BGSC) and assigned the accession number 3A17.     

植物根际芽胞杆菌菌种资源采集及其具有过水解催化活性水解酶基因的克隆

[背景]芽胞杆菌源枯草杆菌蛋白酶(subtilisin carlsberg)、乙酰基木聚糖酯酶(acetyl xylan esterase)和头孢菌素乙酰水解酶(cephalosporin acetyl hydrolase)具有较高的过水解催化活性,有商业开发价值。[目的]挖掘芽胞杆菌菌株中具有过水解酶催化活性的水解酶蛋白基因,为后续制备过水解酶及酶法合成过氧乙酸奠定基础。[方法]利用定向筛选培养基,从植物根际及纳豆产品中筛选产蛋白酶芽胞杆菌候选菌株,并利用RFLP及16S rRNA基因对其进行鉴定。从蛋白酶高产芽胞杆菌菌株中克隆枯草杆菌蛋白酶、乙酰木聚糖醋酶和头孢菌素乙酰水解酶的全长基因。[结果]从植物根际土壤及纳豆产品中共分离到85个候选菌株,RFLP及16S rRNA基因鉴定结果表明候选菌株均为芽胞杆菌,分别属于Bacillus subtilis、Bacillus cereus、Bacillus pumilus和Bacillus megaterium四个类群。从B.subtilis NSYT-3克隆的枯草杆菌蛋白酶基因编码的多肽链全长381个氨基酸,从B.pumilus OSLJ-3克隆得到的乙酰基木聚糖酯酶基因编码的多肽链全长320个氨基酸,从B.subtilis NSYT-3克隆的头孢菌素乙酰水解酶基因编码的多肽链全长318个氨基酸,3D结构模拟表明这3个酶蛋白均具有α/β水解酶折叠家族蛋白结构特点。[结论]芽胞杆菌源具过水解催化活性水解酶基因的克隆,为后续开发酶法合成过氧乙酸工艺奠定了基础。     

Transformation of Bacillus subtilis in α-Amylase Productivity by Deoxyribonucleic Acid from B. subtilis var. amylosacchariticus

Deoxyribonucleic acid (DNA) of Bacillus subtilis var. amylosacchariticus showed almost the same ability as B. subtilis Marburg to induce transfer of several genetic markers in DNA-mediated transformation. DNA-DNA hybridization data also showed an intimate relationship between the two strains. Genetic elements involved in the production of extracellular alpha-amylase (EC 3.2.1.1.) in B. subtilis var. amylosacchariticus were studied by using DNA-mediated transformation. Two Marburg derivatives, NA20(amyR2) and NA20-22(amyR1), produced about 50 and 10 U of alpha-amylase per mg of cells, respectively, whereas B. subtilis var. amylosacchariticus produced as much as 150 U of the enzyme per mg of cells. When B. subtilis var. amylosacchariticus was crossed with strain NA20-22 as recipient, transformants that acquired high alpha-amylase productivity (about 50 U/mg of cells) were obtained. Genetic analysis revealed that a regulator gene (amyR) for alpha-amylase synthesis was found in B. subtilis var. amylosacchariticus, as in the case of B. natto 1212 (amyR2) and B. subtilis Marburg (amyR1). The allele was designated amyR3; it is phenotypically indistinguishable from amyR2, but is readily distinguishable from amyR1. The presence of amyR3 was not sufficient for an organism to render production of an exceptional amount of alpha-amylase. Extra-high alpha-amylase producers could be obtained by crossing B. subtilis var. amylosacchariticus as donor with strain NA20 as recipient. The transformants produced the same or even greater amounts of the enzyme than the donor strain. Results suggest the presence of another gene that is involved in the production of the exceptional amount of alpha-amylase.     

Detailed physical mapping of the ribosomal RNA genes of Bacillus subtilis

Characterization of patterns of ribosomal RNA (rRNA) homology with restriction digests of Bacillus subtilis 168 chromosomal DNA and with cloned DNA sequences has resulted in the construction of a physical map of the rRNA gene sets. There are two types of gene sets which differ in the size of "spacer" DNA sequences separating the 16S and 23S rRNA determinants. It was estimated that there are ten rRNA gene sets on the B. subtilis chromosome.     

Bacillus subtilis (natto) plasmid pLS20 mediates interspecies plasmid transfer.

The 55-kilobase plasmid, pLS20, of Bacillus subtilis (natto) 3335 promotes transfer of the tetracycline resistance plasmid pBC16 from B. subtilis (natto) to the Bacillus species B. anthracis, B. cereus, B. licheniformis, B. megaterium, B. pumilus, B. subtilis, and B. thuringiensis. Frequency of pBC16 transfer ranged from 2.3 x 10(-6) to 2.8 x 10(-3). Evidence for a plasmid-encoded conjugationlike mechanism of genetic exchange includes (i) pLS20+ strains, but not pLS20- strains, functioned as donors of pBC16; (ii) plasmid transfer was insensitive to the presence of DNase; and (iii) cell-free filtrates of donor cultures did not convert recipient cells to Tcr. Cotransfer of pLS20 and pBC16 in intraspecies matings and in matings with a restriction-deficient B. subtilis strain indicated that pLS20 was self-transmissible. In addition to mobilizing pBC16, pLS20 mediated transfer of the B. subtilis (natto) plasmid pLS19 and the Staphylococcus aureus plasmid pUB110. The fertility plasmid did not carry a selectable marker. To facilitate direct selection for pLS20 transfer, plasmid derivatives which carried the erythromycin resistance transposon Tn917 were generated. Development of this method of genetic exchange will facilitate the introduction of plasmid DNA into nontransformable species by use of transformable fertile B. subtilis or B. subtilis (natto) strains as intermediates.     

Restriction enzyme analysis of Bacillus subtilis ribosomal ribonucleic acid genes.

The organization of the ribosomal ribonucleic acid (rRNA) genes (rDNA) of Bacillus subtilis was examined by cleaving the genome with several restriction endonucleases. The rDNA sequences were assayed by hybridization with purified radioactive rRNA's. Our interpretation of the resulting electrophoretic patterns is strengthened by an analysis of a fragment of B. subtilis rDNA cloned in Escherichia coli. The results indicated that there are eight rRNA operons in B. subtilis. Each operon contains one copy of the sequences coding for 16S, 23S, and 5S rRNA. The sequences coding for 5S rRNA were shown to be more closely linked to the 23S rRNA genes than to the 16S rRNA genes.     

一株番茄青枯菌拮抗菌的鉴定及抗病效果初探

从形态、生理生化、16S rDNA3个方面确定了番茄青枯菌拮抗菌株3-1-16的分类地位。光学显微镜下观察到菌体为杆状细胞,革兰氏染色均匀,并可见菌体染成蓝紫色。透射电镜进一步观察到细胞内有许多颗粒状物质,无伴胞晶体。Biolog鉴定,3-1-16与巨大芽孢杆菌(Bacillus megaterium)具有最高相似率为98%。16S rRNA分析,3-1-16与巨大芽孢杆菌MO31同源性最高为99.4%。聚类分析显示3-1-16与3株巨大芽孢杆菌聚成一支,支持度为100%。生理生化特征及培养特征测定结果表明,菌株3-1-16鉴定为巨大芽孢杆菌(Bacillus megaterium)。盆栽试验表明该菌株对番茄青枯病防病效果达到81.3%。     

A cloned 23S rRNA gene fragment of Bacillus subtilis and its use as a hybridization probe of conserved character

Abstract A subclone of plasmid p14B8 containing the major part of a 23S rRNA gene of Bacillus subtilis was constructed and designated pJK1. Labeled plasmid pJK1 could be used as a DNA probe with conserved gene sequences. DNA-DNA hybridization experiments between filter-bound DNA from various bacteria and labeled pJK1 showed a good correlation between oligonucleotide sequence analysis of 16S rRNA and DNA homology values. Application of suboptimal or stringent hybridization conditions and an additional short incubation under the same conditions following hybridization yielded the best data for differentiating organisms related to B. subtilis from less or non-related bacteria.