利用ERIC-PCR对苏云金芽胞杆菌与蜡状芽胞杆菌进行鉴定的研究

为了探索ERIC-PCR技术在苏云金芽胞杆菌和蜡状芽胞杆菌的鉴定及分型中的应用价值,本研究采用PCR方法初步检测苏云金芽胞杆菌杀虫晶体蛋白基因的组成,并对苏云金芽胞杆菌和蜡状芽胞杆菌的总DNA进行ERIC-PCR扩增,分析ERIC-PCR指纹图谱的特点并采用NTSYS2.10软件对其进行聚类。结果显示,各菌株的ERIC指纹图谱表现出不同程度的多态性,但图谱与菌株所含cry基因的类型存在一定的相关性。聚类分析结果显示,含有相同或相近cry基因类型的Bt菌株在进化树上趋向聚为一类,而不含cry基因的蜡状芽胞杆菌趋向于与不含cry基因的Bt菌株聚为一类或单独聚类。若在多种模式菌株的参考下,该方法可用于苏云金芽胞杆菌的初步鉴定和分型。     

Conjugative transfer, stability and expression of a plasmid encoding a cry1Ac gene in Bacillus cereus group strains

The plasmid pHT73 containing cry1Ac and tagged with an erythromycin resistance gene was transferred from Bacillus thuringiensis subspecies kurstaki KT0 to several Bacillus cereus group strains by conjugation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and phase contrast microscopy showed that the transconjugants containing plasmid pHT73 could express Cry1Ac toxin and produce bipyramidal crystalline inclusion bodies during sporulation. The study demonstrated that pHT73 could be transferred to B. thuringiensis subsp. kurstaki, several B. cereus strains and Bacillus mycoides. Under non-selective conditions, the stability of the pHT73 plasmid in the transconjugants was found to be 58.2-100% after 100 generations and 4-96% after 200 generations. The variations are mainly caused by the choice of receptor strain.     

Psychrotolerant species from the Bacillus cereus group are not necessarily Bacillus weihenstephanensis

Twenty-six strains of Bacillus cereus from different sources were determined to be either mesophilic or psychrotrophic by growth at 6 and 42 degrees C. The strains were also screened by two polymerase chain reaction (PCR) methods designed to discriminate between mesophilic and psychrotrophic types. Seventeen of the 26 strains were able to grow at 6 degrees C, but only four conformed to the new psychrotolerant species Bacillus weihenstephanensis. Among the 26 strains were two which caused outbreaks of food poisoning in Norway, and three others that were isolated from food suspected of causing illness. The presence of the gene components encoding production of enterotoxins Nhe, Hbl, EntT and a recently described cytotoxin K was determined by PCR. All the strains possessed genes for at least one of these toxins, and 19 of the 26 strains were cytotoxic in a Vero cell assay. We conclude that there are psychrotrophic B. cereus strains which cannot be classified as B. weihenstephanensis, and that intermediate forms between the two species exist. No correlation between cytotoxicity and the growth temperature of the strains was found.     

Phenotypic and genotypic comparisons of 23 strains from the Bacillus cereus complex for a selection of known and putative B. thuringiensis virulence factors

Sixteen Bacillus thuringiensis, four Bacillus cereus and three Bacillus anthracis isolates were screened for a selection of known and putative B. thuringiensis virulence factors. PCR primers were designed to detect genes for phosphatidylcholine specific phospholipase C, phosphatidylinositol specific phospholipase C, immune inhibitor A, vegetative insecticidal protein 3A, a protein proposed to be involved in capsule synthesis, a newly identified Ser/Thr kinase homologue and enterotoxin entS. Motility, the presence of flagella, haemolysis, chitinase and lecithinase production were also evaluated. The widely varying profiles of the 23 strains from the complex provide a pool of different genotypes that can help to identify factors involved in pathogenicity.     

Natural occurrence of Bacillus thuringiensis and Bacillus cereus in eukaryotic organisms: a case for symbiosis

Bacillus thuringiensis and Bacillus cereus, two members of the Bacillus cereus sensu lato group, are most noted for their virulence in, respectively, arthropods and mammals including humans. Because of their pathogenicity to insects in particular, and their narrow host range, strains of B. thuringiensis have been utilised successfully as biocontrol agents of insect pests. Whereas B. cereus is not an established entomopathogen, certain strains of this species are well known to be etiological agents of gastrointestinal and emetic syndromes in humans. While much is known about the taxonomic properties and molecular basis for virulence of B. thuringiensis and B. cereus, comparatively less is known about their ecology in natural environments. Thus, there are limited data regarding their resilience, i.e. recycling of vegetative and sporulated phases of growth in soil, ecolgical niches including symbiotic interactions with other organisms, and the impact on ecosystems in which they proliferate. Nevertheless, based on recent data, a picture is beginning to emerge that B. thuringiensis and B. cereus are capable of establishing mutual and commensal relationships with both animals and plants. In this regard, these bacilli can proliferate in the digestive tracts of animals, where upon defecation they form dormant spores in the soil, and to a lesser extent on the phylloplane and rhizospheres of plants. Altogether, current evidence strongly suggests that B. thuringiensis and B. cereus are capable of completing their life cycles and recycling through various reservoirs, including animals, plants, and soil. This review focuses on the current knowledge pertaining to the ecologic interactions between B. thuringiensis and B. cereus and eukaryotic hosts, with special emphasis on symbiosis.     

The Bacillus thuringiensis linear double-stranded DNA phage Bam35, which is highly similar to the Bacillus cereus linear plasmid pBClin15, has a prophage state

Bam35, a 15-kbp double-stranded DNA phage, infects Bacillus thuringiensis. Recently, sequencing of the related Bacillus cereus revealed a 15.1-kbp linear plasmid, pBClin15. We show that pBClin15 closely resembles Bam35 and demonstrate conversion of Bam35 to a prophage. This state is common, as several B. thuringiensis strains release Bam35-related viruses.     

Multilocus sequence typing reveals that Bacillus cereus strains isolated from clinical infections have distinct phylogenetic origins

Eight strains of Bacillus cereus isolated from bacteremia and soft tissue infections were assigned to seven sequence types (STs) by multilocus sequence typing (MLST). Two strains from different locations had identical STs. The concatenated sequences of the seven STs were aligned with 65 concatenated sequences from reference STs and a neighbor-joining tree was constructed. Two strains were distantly related to all reference STs. Three strains were recovered in a clade that included Bacillus anthracis, B. cereus and rare Bacillus thuringiensis strains while the other three strains were assigned to two STs that were more closely affiliated to most of the B. thuringiensis STs. We conclude that invasive B. cereus strains do not form a single clone or clonal complex of highly virulent strains.     

The autolytic phenotype of the Bacillus cereus group

AIM: To determine the autolytic phenotype of five species in the Bacillus cereus group. METHODS AND RESULTS: The autolytic rate of 96 strains belonging to five species in the B. cereus group was examined under starvation conditions at pH 6, 6.5 and 8.5 in different buffers. The autolytic rate was strain-dependent with a wide variability at pH 6, but higher and more uniform at pH 6.5. At pH 8.5, and respect to the extent of autolysis at pH 6.5, it was relatively low for most of the strains with the lowest values between 13 and 52% in Bacillus mycoides and Bacillus pseudomycoides. Peptidoglycan hydrolase patterns evaluated by renaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis using cells of Bacillus thuringiensis ssp. tolworthi HD125 as an indicator, revealed complex profiles with lytic bands of about 90, 63, 46, 41, 38, 32, 28 and 25 kDa in B. cereus, B. thuringiensis and Bacillus weihenstephanensis. Bacillus mycoides and B. pseudomycoides had simpler profiles with lytic bands of 63, 46 and 38 kDa. Changes in the autolytic pattern were observed for cells harvested at the stationary phase of growth (72 h) showing an increase in the intensity of the 25 kDa band in the case of B. cereus, B. thuringiensis and B. weihenstephanensis, while no changes were observed for B. mycoides. Using Micrococcus lysodeicticus and Listeria monocytogenes as indicators lytic activity was retained by proteins of 63, 46, 38, 32 and 25 kDa and a new one of about 20 kDa in B. mycoides. Growth in the different media did not affect the autolytic pattern. NaCl abolished the activity of all the peptidoglycan hydrolases except for those of B. mycoides and B. weihenstephanensis. Lytic activity was retained in the presence of MgCl(2), MnCl(2) and EDTA and increased at basic pH. CONCLUSIONS: Bacillus cereus/B. thuringiensis/B. weihenstephanensis showed a high extent of autolysis around neutral pH, even though they presented relatively complex autolysin profiles at alkaline pH. Bacillus mycoides/B. pseudomycoides had a higher extent of autolysis at acidic pH and a simpler autolysin pattern. SIGNIFICANCE AND IMPACT OF THE STUDY: Information on the autolytic phenotype expand the phenotypic characterization of the different species in the B. cereus group.     

Presence of calmodulin-like calcium-binding protein in Bacillus cereus T spores

Abstract Bacillus cereus T spores were extensively washed, broken, and heated at 90°C for 2 min. Using calcium-dependent hydrophobic interaction chromatography, a single peak protein fraction was obtained which possessed calcium-binding capacity and some characteristics of calmodulin. This heat-stable protein fraction was retained by hydrophobic matrices (Phenyl-Sepharose) or a calmodulin antagonist (naphthalenesulfonamide) in a calcium-dependent manner. Calcium binding ability was verified by ⁴⁵Ca autoradiography and a competitive calcium binding assay using Chelex-100. The crude spore extract displaced bovine brain calmodulin from its antibody in a radioimmunoassay and the immunoreactive specific activity of the partially purified fraction was approx. 200-fold greater than the crude spore extract. Thus, B. cereus T spores have a calcium-binding protein with calmodulin-like properties.     

Glycosylation of flavonoids with a glycosyltransferase from Bacillus cereus

Microbial glycosyltransferases can convert many small lipophilic compounds such as phenolics, terpenoids, cyanohydrins and alkaloids into glycons using uridine-diphosphate-activated sugars. The main chemical functions of glycosylation processes are stabilization, detoxification and solubilization of the substrates. The gene encoding the UDP-glycosyltransferase from Bacillus cereus, BcGT-1, was cloned by PCR and sequenced. BcGT-1 was expressed in Escherichia coli BL21 (DE3) with a his-tag and purified using a His-tag affinity column. BcGT-1 could use apigenin, genistein, kaempferol, luteolin, naringenin and quercetin as substrates and gave two reaction products. The enzyme preferentially glycosylated at the 3-hydroxyl group, but it could transfer a glucose group onto the 7-hydroxyl group when the 3-hydroxyl group was not available. The reaction products made by biotransformation of flavonoids with E. coli expressing BcGT-1 are similar to those produced with the purified recombinant enzyme. Thus, this work provides a method that might be useful for the biosynthesis of flavonoid glucosides and for the glycosylation of related compounds.     

苏云金芽孢杆菌大型质粒DNA的小量提取

以苏云金芽孢杆菌库斯塔克亚种、猝倒亚种及以色列亚种为材料,介绍了低拷贝大型质粒DNA的小量提取方法,该法采用PEG 6000进行质粒纯化,省略了苯酚和氯仿抽提过程。实验证明,该法结果稳定,提取的质粒DNA产量和质量均符合大多数分子生物学实验的要求。     

Substrate binding to mononuclear metallo-beta-lactamase from Bacillus cereus

Structure and dynamics of substrate binding (cefotaxime) to the catalytic pocket of the mononuclear zinc-beta-lactamase from Bacillus cereus are investigated by molecular dynamics simulations. The calculations, which are based on the hydrogen-bond pattern recently proposed by Dal Peraro et al. (J Biol Inorg Chem 2002; 7:704-712), are carried out for both the free and the complexed enzyme. In the resting state, active site pattern and temperature B-factors are in agreement with crystallographic data. In the complexed form, cefotaxime is accommodated into a stable orientation in the catalytic pocket within the nanosecond timescale, interacting with the enzyme zinc-bound hydroxide and the surrounding loops. The beta-lactam ring remains stable and very close to the hydroxide nucleophile agent, giving a stable representation of the productive enzyme-substrate complex.     

Characterisation of a non-haemolytic enterotoxin complex from Bacillus cereus isolated after a foodborne outbreak

Abstract Three enterotoxic components have been isolated from a strain of Bacillus cereus which was involved in a large food poisoning outbreak in Norway in 1995. The components were purified by chromatography on three different columns. Three proteins of 39, 45 and 105 kDa, respectively, were found to be necessary for maximum cytotoxicity. The amino acid N-terminal sequences of the 39 and 45 kDa proteins were determined. The 45 kDa component was the same protein as the main antigen detected in the Bacillus Diarrhoeal Enterotoxin Visual Immunoassay (Tecra). The 39 kDa protein showed some similarity to the l₁ protein of haemolysin BL from B. cereus . Furthermore, the three toxic components were all recognised by a polyclonal antiserum reported to detect enterotoxin from B. cereus . The proteins were different from the B- and L₂-components of haemolysin BL, previously suggested to be a primary virulence factor, and had no detectable haemolytic activity.     

苏云金芽胞杆菌大质粒pBMB28的克隆

苏云金芽胞杆菌幕虫亚种YBT-020具有典型的晶胞粘连表型。在前期的研究中,通过质粒消除实验,推测晶胞粘连现象与YBT-020内生质粒pBMB28有关。为了定位质粒pBMB28上控制晶胞粘连表型的基因,首先对质粒pBMB28进行克隆。利用穿梭载体pEMB0557,成功构建了苏云金芽胞杆菌YBT-020的基因组人工染色体(BAC)文库。前期的研究表明晶体蛋白基因cry28Aa定位在质粒pBMB28上,根据cry28Aa基因序列设计引物,从文库中筛选到含有cry28Aa的重组质粒pBMB231。镜检和SDS-PAGE证明质粒pBMB231转化无晶体突变株BMB171形成的重组子BMB231可以产生Cry28Aa晶体蛋白,但不能恢复晶胞粘连表型。对重组质粒pBMB231的插入片段末端序列测定并设计引物筛选文库,通过染色体步移方式得到4个可以重叠覆盖质粒pBMB28不同区域的克隆子,从而克隆了该质粒。对这4个克隆子末端测序和酶切分析,测算出该质粒的大小约为140 kb。进一步确定应用基因组BAC文库以及重叠片段筛选的方法,可以快速有效的克隆苏云金芽胞杆菌大质粒。     

Radiation sensitivity of Bacillus cereus with and without a crystalline surface protein layer

The radiation sensitivity of four strains of Bacillus cereus was investigated with attention to bacterial surface structure. All four strains were sensitive to radiation with gamma rays (D(10)=0.4 kGy). No crystalline surface protein layer could be detected on the cell surface. When cultured on solid media, an S-layer covered the cells of the two strains, and they were 2.6 times as resistant to radiation as the two reference strains without an S-layer. In SDS-PAGE, a major 97-kDa band from the resistant strains from plate cultures was replaced by a ca. 85-kDa protein band in samples from broth cultures. Electron microscopy, SDS-PAGE, Western blot and fluorescent antibody staining indicated that the higher resistance to radiation of the clinical strains from plate cultures was associated with the presence of the S-layer on the cell surface.     

Detection of the single-stranded DNA of Streptomyces plasmid pSA1.1 and a binding histone-like protein

Abstract Streptomyces plasmid pSA1.1 accumulated single-stranded DNA as replication intermediates in S. lividans ; therefore, this plasmid was considered to replicate by a rolling-circle mechanism. A DNA-binding protein (pI > 9.7 and about 10 kDa) was purified on a denatured DNA-Cellulose column, then on a native DNA-Cellulose column. The N-terminal amino acid sequence of this protein has a high homology with bacterial histone-like proteins. In the gel retardation assay, this protein bound with the single-stranded DNA of pSA1.1. We propose that this protein may participate in the replication of pSA1.1.     

苏云金芽胞杆菌大质粒pBMB165的克隆与分析

以pBeloBAC11为载体,成功构建了苏云金芽胞杆菌YBT-1765的基因组人工染色体(BAC)文库和质粒BAC文库.根据已克隆的包含复制子ori165在内的3.6kb片段中编码复制蛋白Rep165的核苷酸序列设计探针,通过染色体步移方式,对质粒文库和基因组文库进行筛选,得到13个覆盖YBT-1765菌株中质粒pBMB165不同区域的克隆子.通过Hind Ⅲ和BamH Ⅰ酶切分析,建立了质粒pBMB165的物理图谱和线状重叠连锁图,并测算出该质粒的大小为82kb.根据部分核苷酸序列初步统计了pBMB165上转座因子的存在机率.YBT-1765菌株基因组文库的构建和物理图谱的绘制为克隆苏云金芽胞杆菌大质粒提供了一套可行的方案,成功解决了大质粒难克隆的问题.