Identification and characterization of Bacillus anthracis by multiplex PCR analysis of sequences on plasmids pXO1 and pXO2 and chromosomal DNA

Abstract Bacillus anthracis can be identified on the basis of the detection of virulence factor genes located on two plasmids, pXO1 and pXO2. Thus isolates lacking both pXO1 and pXO2 are indistinguishable from closely related B. cereus group bacteria. We developed a multiplex PCR assay for characterization of B. anthracis isolates, and simultaneous confirmation of the species identity independent of plasmid content. The assay amplifies lef, cya, pag (pXO1) and cap (pXO2) genes, and a B. anthracis specific chromosomal marker, giving an easy-to-read profile. This system unambiguously identified virulent (pXO1⁺/2⁺) and avirulent (pXO1⁺/2⁻, pXO1⁻/2⁺ and pXO1⁻/2⁻) strains of B. anthracis and distinguished 'anthrax-like' strains from other B. cereus group bacteria.     

Degradation of mistargeted OEE33 in the chloroplast stroma

OEE33, a component of the oxygen-evolving enzyme in chloroplasts, normally resides in the thylakoid lumen. In an attempt to study the fate of mistargeted proteins in chloroplasts, we substituted the bipartite transit peptide of OEE33 with that of CAB7, an integral thylakoid-membrane protein. As a result, when imported into isolated chloroplasts, the chimeric protein was targeted to the stroma instead of the thylakoid lumen. Whereas the wild-type OEE33 was totally stable for at least 2 h, the chimeric protein was rapidly degraded, with a half-life of 60 min. Degradation of the chimeric protein was stimulated by ATP supplementation. Degradation could also be observed in lysed chloroplasts, in an ATP-stimulated manner. When lysates were fractionated, the proteolytic activity was found to be associated mainly with the stromal fraction. This activity was very effectively inhibited by all tested inhibitors of serine proteases. Western blot analysis demonstrated that the stromal fraction active in degrading the chimeric OEE33 contains ClpC and ClpP, homologues of the regulatory and proteolytic subunits, respectively, of the bacterial, ATP-dependent, serine-type Clp protease.     

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.     

炭疽芽孢杆菌保护性抗原在大肠杆菌中的表达及纯化

按照炭疽芽孢杆菌保护性抗原（PA）基因成熟肽编码序列设计引物,从炭疽杆菌pOX1质粒中扩增出PA基因片段,将该片段定向插入到原核表达载体pET-28a中,获得了pET-PA原核表达重组质粒,限制性酶切分析和DNA序列测定均证实该克隆插入片段为PA基因的成熟呔编码序列。将该重组质粒转化大肠杆菌BL21（DE3）,经IPTG诱导,重组蛋白在大肠杆菌表达系统中获得了高效表达;Western印迹分析表明表达产物具有良好的免疫学活性。     

炭疽杆菌致病性研究进展

炭疽杆菌是人类历史上第一个被发现的病原菌。炭疽杆菌的研究在近几年取得了较大进展 ,特别是本年度其基因组序列测定已完成并向全世界公布 ,进一步深化了对炭疽杆菌的研究。炭疽杆菌致病性的研究一直是炭疽杆菌研究的重点 ,近年来此方面的研究取得了很多新进展 ,从基因组、致病物质及致病机制 3个方面对此作一个简单的介绍。     

Expression and secretion of the protective antigen of Bacillus anthracis in Bacillus brevis

We used the Bacillus brevis-pNU212 system to develop a mass production system for the protective antigen (PA) of Bacillus anthracis. A moderately efficient expression-secretion system for PA was constructed by fusing the PA gene from B. anthracis with the B. brevis cell-wall protein signal-peptide encoding region of pNU212, and by introducing the recombinant plasmid, pNU212-mPA, into B. brevis 47-5Q. The clone producing PA secreted about 300 microg of recombinant PA (rPA) per ml of 5PY-erythromycin medium after 4 days incubation at 30 degrees C. The rPA was fractionated from the culture supernatant of B. brevis 47-5Q carrying pNU212-mPA using ammonium sulfate at 70% saturation followed by anion exchange chromatography on a Hitrap Q, a Hiload 16/60 Superdex 200 gel filtration column and a phenyl sepharose hydrophobic interaction column, yielding 70 mg rPA per liter of culture. The N-terminal sequence of the purified rPA was identical to that of native PA from B. anthracis. The purified rPA exhibited cytotoxicity towards J774A.1 cells when combined with lethal factor. The rPA formulated in either Rehydragel HPA or MPL-TDM-CWS adjuvant (Ribi-Trimix) elicited the expression of a large amount of anti-PA and neutralizing antibodies in guinea pigs and completely protected them against a 100 LD50 challenge with fully virulent B. anthracis spores.     

炭疽芽胞杆菌疫苗研究进展

炭疽芽胞杆菌引起的炭疽病死亡率非常高 ,当前的疫苗具有效力不稳定、对吸入性炭疽的保护率低、免疫程序繁琐、存在副作用等缺点。近年来人们在改造传统疫苗的同时又有一些新的发现 ,如保护性抗原 (PA)的抗体在体内可杀死芽胞 ;通过粘膜免疫能够诱导机体分泌IgA抗体 ;抗多聚谷氨酸 (γ D PGA)抗体可以同炭疽杆菌的繁殖体作用 ,从而杀死繁殖体 ;寻找到新的免疫原。DNA疫苗、活载体疫苗的出现为新一代安全、免疫程序简单、具更高保护率的疫苗奠定了基础     

Extreme variation in rates of evolution in the plastid Clp protease complex

Eukaryotic cells represent an intricate collaboration between multiple genomes, even down to the level of multi‐subunit complexes in mitochondria and plastids. One such complex in plants is the caseinolytic protease (Clp), which plays an essential role in plastid protein turnover. The proteolytic core of Clp comprises subunits from one plastid‐encoded gene ( clpP1 ) and multiple nuclear genes. The clpP1 gene is highly conserved across most green plants, but it is by far the fastest evolving plastid‐encoded gene in some angiosperms. To better understand these extreme and mysterious patterns of divergence, we investigated the history of clpP1 molecular evolution across green plants by extracting sequences from 988 published plastid genomes. We find that clpP1 has undergone remarkably frequent bouts of accelerated sequence evolution and architectural changes (e.g. a loss of introns and RNA ‐editing sites) within seed plants. Although clpP1 is often assumed to be a pseudogene in such cases, multiple lines of evidence suggest that this is rarely true. We applied comparative native gel electrophoresis of chloroplast protein complexes followed by protein mass spectrometry in two species within the angiosperm genus Silene , which has highly elevated and heterogeneous rates of clpP1 evolution. We confirmed that clpP1 is expressed as a stable protein and forms oligomeric complexes with the nuclear‐encoded Clp subunits, even in one of the most divergent Silene species. Additionally, there is a tight correlation between amino acid substitution rates in clpP1 and the nuclear‐encoded Clp subunits across a broad sampling of angiosperms, suggesting continuing selection on interactions within this complex.     

环介导等温扩增技术在快速检测炭疽芽胞杆菌中的应用

本文旨在建立适合国境口岸现场应用的生物恐怖防控快速检测方法,从而保障口岸安全.针对生物恐怖炭疽芽胞杆菌,选择目标菌种特异性基因片段,设计引物,运用环介导等温扩增(LAMP)技术建立一套简便、高效的检测方法,并模拟生物恐怖炭疽芽胞杆菌可能存在的基质条件,评价LAMP技术在快速筛查中的适用性.结果显示,LAMP技术排查生物恐怖炭疽芽胞杆菌简便、快速、特异,检测灵敏度为102～103 CFU/ml;且能有效检出在偏酸、偏碱及黏稠基质中的炭疽芽胞杆菌.而高盐环境对该反应影响较大,有必要采用能有效去除盐分的核酸抽提方法.     

Evaluation of two selective media for the isolation of Bacillus anthracis

Aims:  To evaluate two selective media, polymyxin, lysozyme, ethylenediaminetetraacetic acid, thallium acetate (PLET) agar and R&F Anthracis chromogenic agar (ChrA), for the isolation and selection of Bacillus anthracis .
Methods and Results:  Sixteen genotypically diverse B. anthracis strains were sub-cultured onto PLET and ChrA to test the sensitivity (ability of B. anthracis to grow and produce expected colony morphology) of both media. Fourteen of the 16 B. anthracis strains produced the expected morphology on PLET (88% sensitive) while 13/16 produced the expected morphology on the ChrA medium (81% sensitive). Seventeen other Bacillus strains and 18 non Bacillus spp. strains were used to evaluate the media's selectivity (ability to inhibit non- B. anthracis growth). PLET inhibited growth of 14/35 strains (40% selective), including six Bacillus strains, while ChrA inhibited 3/35 (9% selective). In addition, we did not observe any differences between the recovered CFU on PLET or ChrA when plating extractions of spiked soil.
Conclusions:  Polymyxin, lysozyme, ethylenediaminetetraacetic acid, thallium acetate agar was more selective and sensitive than ChrA.
Significance and Impact of the Study:  Although both media are more expensive than sheep blood agar, for samples with high numbers of bacteria, they can be used to isolate B. anthracis with proper training and experience and with the knowledge that there are limitations to each media.     

Proteomics reveals that proteins expressed during the early stage of Bacillus anthracis infection are potential targets for the development of vaccines and drugs

In this review, we advance a new concept in developing vaccines and/or drugs to target specific proteins expressed during the early stage of Bacillus anthracis (anthrax) infection and address existing challenges to this concept. Three proteins (immune inhibitor A, GPR-like spore protease, and alanine racemase) initially identified by proteomics in our laboratory were found to have differential expressions during anthrax spore germination and early outgrowth. Other studies of different bacillus strains indicate that these three proteins are involved in either germination or cytotoxicity of spores, suggesting that they may serve as potential targets for the design of anti-anthrax vaccines and drugs.     

Disruption and analysis of the clpB, clpC, and clpE genes in Lactococcus lactis: ClpE, a new Clp family in gram-positive bacteria

In the genome of the gram-positive bacterium Lactococcus lactis MG1363, we have identified three genes (clpC, clpE, and clpB) which encode Clp proteins containing two conserved ATP binding domains. The proteins encoded by two of the genes belong to the previously described ClpB and ClpC families. The clpE gene, however, encodes a member of a new Clp protein family that is characterized by a short N-terminal domain including a putative zinc binding domain (-CX2CX22CX2C-). Expression of the 83-kDa ClpE protein as well as of the two proteins encoded by clpB was strongly induced by heat shock and, while clpC mRNA synthesis was moderately induced by heat, we were unable to identify the ClpC protein. When we analyzed mutants with disruptions in clpB, clpC, or clpE, we found that although the genes are part of the L. lactis heat shock stimulon, the mutants responded like wild-type cells to heat and salt treatments. However, when exposed to puromycin, a tRNA analogue that results in the synthesis of truncated, randomly folded proteins, clpE mutant cells formed smaller colonies than wild-type cells and clpB and clpC mutant cells. Thus, our data suggest that ClpE, along with ClpP, which recently was shown to participate in the degradation of randomly folded proteins in L. lactis, could be necessary for degrading proteins generated by certain types of stress.     

炭疽杆菌噬菌体裂解酶基因在大肠杆菌中的表达及鉴定

利用PCR方法扩增炭疽杆菌噬菌体裂解酶 (γlysin)基因 ,克隆至大肠杆菌表达载体pET2 2b中 ,经菌落PCR筛选、序列测定和酶切鉴定证实表达载体pET22b-γlysin构建成功 ,并在EscherichiacoliBL21(DE3)中获得了高表达。目的蛋白约占菌体总蛋白的40% ,5L发酵罐中的产酶水平高达 15g L。菌体经超声破碎 ,制备无细胞抽提液 ,StreamlineSP和SPHP柱层析以及SephacrylS-100凝胶过滤三步纯化 ,得到分子量为 2 7kD单一条带的目的蛋白 ,薄层扫描分析显示其纯度大于 95 %。目的蛋白的收率为19.1% ,纯化倍数为350。生物活性鉴定重组的γ噬菌体裂解酶具有特异性 :可快速裂解炭疽杆菌 ,比活为 1400u mg左右 ;而对大肠杆菌、枯草杆菌及蜡样芽孢杆菌没有裂解活性。     

The extracellular and cytoplasmic proteomes of the non-virulent Bacillus anthracis strain UM23C1-2

The recently published genome sequence of Bacillus anthracis Ames has facilitated the prediction of proteins associated with the virulence of this bacterium. The aim of this study was to define reference maps for the extracellular and cytoplasmic proteomes of the avirulent B. anthracis strain UM23C1-2 that are useful for physiological studies and the development of improved vaccines. Using 2-DE and subsequent MALDI-TOF-TOF MS, 64 proteins were identified in the extracellular proteome, only 29 of which were predicted to be exported into the culture medium. The latter included chitinases, proteases, nucleotidases, sulfatases, phosphatases and proteins of unknown function. Of the remaining proteins in the culture medium, 18 were predicted to be associated with the cell wall or anchored on the trans side of the cytoplasmic membrane while 17 other proteins lacked identifiable export signals and were predicted to be cytoplasmic proteins. Among the S-layer proteins, Sap and Eag account for 10% of the total extracellular proteome. Many of the proteins are predicted to contribute to the virulence and antigenic signature of B. anthracis. We have also studied the composition of the cytoplasmic proteome, identifying 300 distinct proteins. The most abundant cytoplasmic proteins are primarily those involved in glycolysis, amino acid metabolism, protein translation, protein folding and stress adaptation. The presence of a variety of proteases, peptidases, peptide binding proteins, as well as enzymes required for the metabolism of amino acids, suggests that B. anthracis is adapted to life in a protein-rich environment rather than the soil. We therefore speculate that proteases and peptidases could be useful targets for the development of improved vaccines. In addition, both of these B. anthracis compartment-specific proteomes can be used as reference maps to monitor changes in the production of secreted and cytosolic proteins that occur, for example, during growth in macrophages.     

炭疽芽胞杆菌基因芯片探针文库的构建

为制备炭疽芽胞杆菌的基因芯片探针文库,以炭疽芽胞杆菌毒素质粒pX01和荚膜质粒pX02为原材料,用Sau3A I酶切pX01和pX02质粒DNA,Taq DNA聚合酶72℃补平加A,经AT克隆,PCR初步鉴定筛选出炭疽质粒片段的阳性克隆．DNA自动分析仪对克隆片段进行序列测定;用生物信息学方法对其片段进行同源性分析;并将克隆的探针打印于玻片上,制备成炭疽芽胞杆茵基因芯片,与炭疽杆茵质粒DNA样品进行初步芯片杂交的实验,杂交实验的阳性率达到了90％以上,证明大部分克隆探针属于炭疽芽胞杆菌．炭疽芽胞杆菌基因芯片探针文库的构建为基因芯片探针的制备摸索出一条简便、高效、可行的方法．     

炭疽杆菌水肿因子基因的克隆与序列测定

采用聚合酶链反应（ＰＣＲ）从炭疽芽孢杆菌减毒株ＹＢ１中扩增其水肿因子（ＥＦ）的编码区基因,将其克隆至ｐＧＥＭ－Ｔ载体中,并分步测定其序列。序列测定表明,该基因长２ ３０１ ｂｐ,编码７６７个氨基酸,与已报道的Ｓｔｅｒｎｅ标准株的ＥＦ基因完全一致。     

炭疽杆菌保护性抗原基因的克隆与序列测定

采用聚合酶链反应从炭疽芽孢杆菌减毒株ＹＢ１中扩增其保护性抗原（ＰＡ）的编码区基因,将其克隆至ｐＧＥＭ－Ｔ载体中,并分步测定其序列。序列测定表明,该基因长２２０５ｂｐ,编码７３５个氨基酸残基,与献报道的标准菌株Ｓｔｅｒｎｅ株的ＰＡ序列只有４个碱基的差异。