Bacillus subtilis自然感受态缺陷株蛋白质表达谱的初步分析

利用蛋白质双向电泳对枯草芽孢杆菌自然感受态缺陷突变株BR151pm感受态形成期的全细胞蛋白质进行比较分析,发现有28个蛋白质斑点出现变化。利用基质辅助激光解析/电离串联飞行时间质谱对其巾2个明显缺失的蛋白斑点进行分析鉴定,确定这2个蛋白质分别为直接参与自然感受态形成的Nin蛋白和RecA蛋白,进一步确证了BR151pm为自然感受态缺陷突变株。     

Bacillus subtilis自然感受态缺陷株蛋白质表达谱的初步分析

利用蛋白质双向电泳对枯草芽孢杆菌自然感受态缺陷突变株BR151pm感受态形成期的全细胞蛋白质进行比较分析,发现有28个蛋白质斑点出现变化。利用基质辅助激光解析/电离串联飞行时间质谱对其巾2个明显缺失的蛋白斑点进行分析鉴定,确定这2个蛋白质分别为直接参与自然感受态形成的Nin蛋白和RecA蛋白,进一步确证了BR151pm为自然感受态缺陷突变株。     

细菌感受态细胞摄取和分泌DNA的相关性研究① I. 含有质粒的枯草芽孢杆菌自然感受态缺陷突变株的诱变和分离

以携带pUB110质粒的枯草芽孢杆菌BR151菌株为出发菌株,利用亚硝基胍作诱变剂,直接在LB平板上进行诱变。从2949个菌落中筛选出一个转化频率低于出发菌株2～3个数量级的突变株,并对其营养缺陷型、UV的敏感性及Km抗性和质粒进行了检测,确证其转化能力降低为自然感受态缺陷而非营养缺陷型的改变或重组缺陷所致。 Abstract　Natural competence-deficient mutant of Bacillus subtilis　BR151, which carries plasmid pUB110, was obtained by nitrosoguanidine mutagenesis on solid medium. From 2949 clones, a mutant, the transformation frequency of which is 102～103　times lower than that of the control, was obtained and tested for the changes of trophic type, resistance to Km, plasmid and sensitivity to UV. The results indicate the reduction of transformation frequency is due to competence-deficient mutation.     

柔嫩艾美耳球虫地克株利抗药株与敏感株孢子化卵囊的蛋白质差异分析

利用双向电泳技术,对本实验室诱导保存的柔嫩艾美耳球虫地克株利抗药株与敏感株的蛋白质表达图谱进行差异比较和分析,发现两者之间差异有5个蛋白质斑点,利用MALDI_TOF_TOF质谱技术对其中4个差异明显的蛋白质斑点进行分析鉴定,获得4个明确的肽质量指纹图谱,通过在NCBInr数据库中检索分析,确定了其中2个蛋白质分别为球虫子孢子表面抗原TA4和热休克蛋白Hsp70 ,另外两种为真核细胞的功能蛋白。上述蛋白的鉴定将对球虫的抗药性产生机理和柔嫩艾美耳球虫地克株利抗药株的分子标志物提供了研究方向。     

利用双向电泳技术研究异烟肼耐药MTB感染U937引致的差异表达蛋白

利用双向电泳技术对结核分枝杆菌（MTB）异烟肼（INH）耐药株和敏感株感染人源巨 噬细胞（U937）后的全细胞蛋白表达图谱进行差异比较和分析,发现其中产生差异的有86个蛋白质斑点.利用基质辅助激光解吸/电离飞行时间质谱技术对其中8个差异表达蛋白质斑点进行分析鉴定,获得8个明确的肽质量指纹图谱.通过在蛋白质数据库中进行检索分析,确定这8个蛋白质中的2个为在INH耐药株感染的U937中差异表达的干细胞生长因子和主要组织相容性复合物Ⅰ类抗原,6个为在敏感株感染的U937中差异表达的微管蛋白β4、信号转导和转录激活子3、延长因子-2激酶、环指蛋白29、锌指蛋白193和SNARE Vti1a-β蛋白.实验结果显示,INH耐药株和敏感株感染后的U937表达蛋白有差异,这有助于分析解释临床中观察到的受INH耐药株感染的病例出现毒力下降、致病性下降、传染性降低以及潜伏期延长的现象.结果为针对INH耐药株进行的新疫苗的设计提供了探索方向.     

细菌感受态细胞摄取和分泌DNA的相关性研究I.含有质粒的枯草芽孢杆菌自然感受态缺陷突变株的诱变和分离

以携带ｐＵＢ１１０质粒的枯草芽孢杆菌ＢＲ１５１菌株为出发菌株,利用亚硝基胍作诱变剂,直接在ＬＢ平板上进行诱变。从２９４９个菌落中筛选出一个转化频率低于出发菌株２～３个数量级的突变株,并对其营养缺陷型、ＵＶ的敏感性及Ｋｍ抗性和质粒进行了检测,确证其转化能力降低为自然感受态缺陷而非营养缺陷型的改变或重组缺陷所致     

家蚕蛹期雌雄生殖腺蛋白质双向电泳比较分析

分析家蚕Bombyx mori雌雄生殖腺细胞蛋白质,有利于发现性别分化相关的功能蛋白质,探讨生殖腺发育相关基因的表达调控机理。本研究利用蛋白质双向凝胶电泳和图像分析技术,分析家蚕蛹期第2天的雌雄生殖腺细胞蛋白质。结果表明: 在雄蚕生殖腺蛋白质电泳图谱中共检测到435个蛋白斑点,其中特异性蛋白斑点73个,占总蛋白斑点数的16.8％;雌蚕生殖腺的电泳图谱中有417个蛋白斑点,其中特异性蛋白斑点55个,占总蛋白斑点数的13.2％。雌雄能匹配的蛋白斑点有362对,匹配率达85.0％。     

一种筛选自然感受态缺陷突变株方法的改进*

对平板筛选自然感受态缺陷突变株的方法进行了改进,采用改进后的方法可以节省转化ＤＮＡ用量８０％以上,而且筛选时转化ＤＮＡ相对浓度提高约１倍,筛选效果较好。     

解淀粉芽孢杆菌赖氨酸-3基因在枯草芽孢杆菌中的克隆

限制性核酸内切酶EcoRI酶切解淀粉芽孢杆菌ASl.1099染色体DNA和质粒pUB lloDNA,T4 DNA连接酶连接,转化枯草芽孢杆菌BRl51(trpC2痢“B5 ly.f3 Neo')感受态细胞。用选择性培养基两次筛选,得到Neo‘和与BR 151 lys3营养缺陷突变互补的菌落。用快速测定质粒的方法检查,其中有13株转化子带有大小相同的重组质粒。分离其中l株转化子BRl51-29的重组质粒pB--L29 DNA,再次转化BRl51感受态细胞和原生质体。测定第二次得到的转化子性状,均与转化子B1~151-29相同。琼脂糖曩胶电泳检查这些转化子的质粒,其分子大小也与t组质粒pB--L29相同。以上试验证明,重组质粒pB--L29是由pUBll0和解淀粉芽孢杆蕾的咖3基因片段构成的。根据琼脂糖凝胶电泳测定pB--L29的分子量约为5.0kb,推断出l~,J3基因片段约为O.5kb。     

炭疽芽孢杆菌A16R株eag基因缺失突变株构建

【目的】构建炭疽芽孢杆菌A16R株eag基因缺失突变株, 为研究eag基因的功能奠定了基础。【方法】本研究以我国人用炭疽杆菌活疫苗A16R株中eag基因为目的缺失基因,根据炭疽芽孢杆菌Ames株基因组序列,利用软件设计了扩增上下游同源臂以及抗性基因引物,构建了重组质粒,将该重组质粒电击转入炭疽杆菌A16R感受态细胞中,利用同源重组原理筛选到炭疽杆菌A16R株eag基因缺失突变株。在分子水平及蛋白质组学方面对基因缺失突变株进行验证。【结果】成功构建了重组质粒,经同源重组后获得eag基因缺失突变株。PCR鉴定表明目的基因已经丢失;SDS PAGE表明野生株与突变株在93 KDa处有差异蛋白条带,经质谱鉴定分析该条带为目的基因所表达的EA1蛋白;双向电泳结果显示突变株与野生株比较明显缺失3个蛋白点,经质谱分析后确定这3个点都是EA1蛋白。【结论】成功获得炭疽芽孢杆菌A16R株eag基因缺失突变株,为深入研究eag基因的功能奠定了基础,同时也为炭疽芽孢杆菌重要基因功能的研究建立了一个良好的技术平台。     

Bacillus subtilis mutants dependent on streptomycin

Summary Six streptomycin-dependent mutants of Bacillus subtilis, two of which were asporogenous, were isolated. All six mutants, SD1, SD2, SD6, SD7, SD9 and SD10, contained a single mutation causing streptomycin dependence and asporogeny, but four of these mutants (SD6, SD7, SD9, SD10) contained a second mutation which phenotypically suppressed the asporogenous character of the streptomycin dependence mutation. All six mutants grew more slowly than the wild type strain BR151, but those defective in sporulation grew the slowest. The streptomycin dependence mutations of SD9 and SD10B (a sporeplus transformant from SD10 carrying both the dependence mutation and the phenotypic suppressor) lie near or possibly within the strA locus. Ribosomes from SD9, SD10A (a spore-minus transformant from SD10 carrying only the dependence mutation), and SD10B were stimulated in vitro by concentrations of streptomycin that inhibit the activity of wild type strain BR151 ribosomes. The level of misreading as measured by poly(U)-directed isoleucine incorporation was greatly enhanced by streptomycin in wild type strain BR151 ribosomes, but misreading of mutant SD9, SD10A, and SD10B ribosomes, irrespective of the sporulation phenotype, was little affected by streptomycin. There were no apparent differences in the patterns obtained by two-dimensional polyacrylamide gel electrophoresis of the 70S ribosomal proteins of the mutants SD9, SD10A, SD10B, and wild type strain BS151.     

Functional substitution of the recE gene of Bacillus subtilis by the recA gene of Proteus mirabilis

Summary Rec mutants of Bacillus subtilis have been tested for complementation by the recA gene of Proteus mirabilis (recApm) which was introduced into B. subtilis via the plasmid pHP334. In the recE4 mutant of B. subtilis the plasmid pHP334 restored significantly the defects in RecE functions tested: UV-sensitivity, homologous recombination (transduction and transformation) and prophage induction.Although serological methods to detect the presence of RecApm protein in B. subtilis have been unsuccessful, our results strongly indicate that the recE function of B. subtilis is analogous to the recA function of P. mirabilis.Abbreviations Cm^r resistance to chloramphenicol - Em^r resistance to erythromycin - Tc^r resistance to tetracycline - SDS sodium dodecyl sulfate - UV ultraviolet - AS ammonium sulfate     

Transformation of Bacillus subtilis competent cells: Identification of a protein involved in recombination

Summary With the use of two-dimensional gel electrophoresis, the proteins present in a transformation-proficient B. subtilis strain were compared with those present in an isogenic, recombination-deficient strain carrying the recE4 mutation. One protein (molecular weight 45 kD, iso-electric point 5.4) was found to be virtually absent in the recE4 strain. This 45 kD protein is a prominent protein predominantly present in the competent fraction of a competent culture. The synthesis of the protein is substantially stimulated by irradiation with ultraviolet light or treatment with mitomycin C and, to a lesser extent, by treatment with nalidixic acid. Since the protein is also observed in a strain cured for SP and carrying non-inducible PBS X, it is unlikely that this protein is a gene product specified by one of these prophages usually present in B. subtilis strain 168. Based on these results we conclude that the 45 kD protein is involved in recombination in B. subtilis.     

Establishment of Monoclonal Antibodies Specific for Bacillus subtilis DB9011

Bacillus subtilis DB9011 is a strain with useful functions for agriculture. To establish a method for the discrimination of this strain from others, monoclonal antibodies (MAbs) were prepared. Although two established MAbs (MAb9B6 and MAb14D2) cross-react with some other Bacillus strains in ELISA, only B. subtilis DB9011 vegetative cells are recognized by both MAbs. MAb14D2 recognizes flagellin, a 34-kDa unit protein of flagella. The two MAbs established will provide powerful tools with which detailed analysis of this bacterial strain can be obtained under environmental conditions.     

Across Genus Plasmid Transformation Between <Emphasis Type="Italic">Bacillus subtilis</Emphasis> and <Emphasis Type="Italic">Escherichia coli</Emphasis> and the Effect of <Emphasis Type="Italic">Escherichia coli</Emphasis> on the Transforming Ability of Free Plasmid DNA

The results presented in this article show that direct plasmid transfer from Escherichia coli carrying shuttle plasmid to Bacillus subtilis occurred when close contact between the two species was established by mixing E. coli and B. subtilis onto selective agar plates. The data demonstrate that the production of resistant colonies by plasmid transformation through cell contact was DNase I sensitive and dependent on transformable B. subtilis strains. Furthermore, another observation indicated that the E. coli strain is able to affect the transformation capability of B. subtilis. It is assumed that the donor strain is a momentous factor for taking up plasmid DNA. This conclusion is significant in the assessment of both the possibility of intercellular DNA transfer in natural habitats of micro-organisms and the risk of the application of genetically engineered micro-organisms.     

Physiology of trans-translation deficiency in Bacillus subtilis – a comparative proteomics study

trans-Translation is the most effective ribosome rescue system known in bacteria. While it is essential in some bacteria, Bacillus subtilis possesses two additional alternative ribosome rescue mechanisms that require the proteins BrfA or RqcH. To investigate the physiology of trans-translation deficiency in the model organism B. subtilis, we compared the proteomes of B. subtilis 168 and a ΔssrA mutant in the mid-log phase using gel-free label-free quantitative proteomics. In chemically defined medium, the growth rate of the ssrA deletion mutant was 20% lower than that of B. subtilis 168. An ³⁵S-methionine incorporation assay demonstrated that protein synthesis rates were also lower in the ΔssrA strain. Alternative rescue factors were not detected. Among the 34 proteins overrepresented in the mutant strain were eight chemotaxis proteins. Indeed, both on LB agar and minimal medium the ΔssrA strain showed an altered motility and chemotaxis phenotype. Despite the lower growth rate, in the mutant proteome ribosomal proteins were more abundant while proteins related to amino acid biosynthesis were less abundant than in the parental strain. This overrepresentation of ribosomal proteins coupled with a lower protein synthesis rate and down-regulation of precursor supply reflects the slow ribosome recycling in the trans-translation-deficient mutant.     

Isolation and Identification of a Novel Antifungal Protein from a Rhizobacterium Bacillus subtilis Strain F3

Bacillus subtilis strain F3, isolated from peach rhizosphere soil, is an antifungal bacterium against many plant pathogens. In this study, the antifungal protein was isolated and purified by ammonium sulphate and chromatography, then identified by mass spectrum analysis. By sequential chromatography of Sephadex G‐50, DEAE‐Sephadex A‐25 anion exchange and Sephadex G‐100, a fraction designated as F3A was isolated to show a single protein band in SDS‐PAGE and be antagonistic towards Monilinia fructicola. The peptide mass fingerprinting of the protein band of F3A had high similarity with the amino acid sequences of several flagellin protein of B. subtilis. There were seven amino acid fragments matched with the protein having the highest score, and sequence coverage was 33%. F3A showed a strongly inhibitory effect to the growth and sporulation of M. fructicola. There were little aerial hyphae and conidia at the antifungal zone, and the hyphae were abnormal with some cell wall collapse and several vacuoles in cells.