Complete genome sequence of Bacillus thuringiensis serovar finitimus strain YBT-020

Bacillus thuringiensis is a gram-positive, spore-forming bacterium that forms parasporal crystals at the onset of the sporulation phase of its growth. Here, we report the complete genome sequence of B. thuringiensis serovar finitimus strain YBT-020, whose parasporal crystals consist of Cry26Aa and Cry28Aa crystal proteins and are located between the exosporium and the spore coat and remain adhering to the spore after sporulation.     

Promoters of crystal protein genes do not control crystal formation inside exosporium of Bacillus thuringiensis ssp. finitimus strain YBT-020

Oenococcus oeni strains from traditional Italian red wines of the Basilicata region were investigated on the basis of their physiological and molecular response to different temperatures and ethanol concentrations. All strains were highly resistant to different ethanol concentrations and it has been observed that 7% ethanol was able to stimulate the growth of strains in wine, and 12–13% of ethanol allowed their proliferation. Moreover, strain tolerances to 18 and 42 °C were observed. Fingerprinting analysis with fluorescent differential display-PCR and investigation of changes in gene expression during the tolerance process were carried out. The expression gene pattern reflects mechanisms involved in tolerance to environmental conditions. This study establishes and validates a method that enables, with a high reproducibility, different gene expression identification under stress conditions in lactic acid bacteria.     

晶胞粘连苏云金芽胞杆菌C15杀线虫毒力因子发掘与功能鉴定

苏云金芽胞杆菌（Bacillus thuringiensis,Bt）制剂作为一种高效的微生物杀虫剂,在植物病虫害防控领域有着广泛的应用。Bt制剂的主效成分为杀虫晶体和芽胞,其中,杀虫晶体的环境低持久性是Bt农药应用的重要限制因素之一。自然界中存在着一些Bt菌株,其产生的杀虫晶体位于芽胞外壁和芽胞衣之间,这种特殊的表型被称为晶胞粘连（spore-crystal association, SCA）表型。由于芽胞外壁对晶体的保护作用,SCA表型可以提升晶体抵抗不良环境因素的能力,是开发新型Bt生物囊杀虫剂的有效育种策略。本文选取对线虫具有强毒杀能力的 SCA菌株C15作为研究对象。获得了C15菌株的完整基因组序列,包括一个5 637 049 bp的环状染色体和8个不同大小的环形质粒（240 314 bp到3 188 bp）。C15基因编码了5个杀虫蛋白（Cry蛋白）基因：cry21-99、cry21-67、cry21-66、cry21-46和cry-N。在Bt无晶体突变株BMB171中异源表达cry21-99基因,发现其表达产物形成菱形晶体,且对秀丽隐杆线虫（Caenorhabditis elegans）和南方根结线虫（Meloidogyne incognita）均有毒杀活性。同时,还在全基因组范围内预测了Cry毒素以外的杀线虫毒力因子和次生代谢产物。此外,在C15基因组中预测了本团队已报道的苏云金芽胞杆菌幕虫亚种（B. thuringiensis serovar finitimus）菌株YBT-020 SCA表型决定因子的同源基因,缺失后突变体仍然保留稳定的SCA表型,说明C15菌株的SCA表型形成机制与YBT-020不同,该菌株代表了一种新的SCA表型形成机制。本研究为转基因作物防控线虫提供了新的遗传资源,也为研究SCA表型形成机制,开发新型高效Bt制剂提供了新线索。     

Genetic characterization of two putative toxin-antitoxin systems on cryptic plasmids from Bacillus thuringiensis strain YBT-1520

A novel putative toxin-antitoxin segregational stability system named KyAB system was identified in a novel native plasmid pBMB8240 from Bacillus thuringiensis strain YBT-1520, based on sequences homology with other toxin-antitoxin systems, the lethal activity of the KyB putative toxin in Escherichia coli and the stabilizing effect of the kyAB system in Bacillus thuringiensis. Secondarily, the native plasmid pBMB9741 from the same strain was resequenced and the corrected plasmid was named as pBMB7635. Based on sequence homology with the tasAB system and the lethal activity of toxin protein in Escherichia coli, a tasAB-like putative toxin-antitoxin system was identified on pBMB7635.     

Molecular characterization of two novel crystal protein genes from Bacillus thuringiensis subsp. thompsoni.

Two genes encoding the predominant polypeptides of Bacillus thuringiensis subsp. thompsoni cuboidal crystals were cloned in Escherichia coli and sequenced. The polypeptides have electrophoretic mobilities of 40 and 34 kDa, with the deduced amino acid sequences predicting molecular masses of 35,384 and 37,505 Da, respectively. No statistically significant similarities were detected between the 40- or 34-kDa crystal protein and any other characterized B. thuringiensis crystal protein, nor were they detected between the 40- and 34-kDa crystal proteins. A 100-MDa plasmid carries both crystal protein genes, which appear to be part of an operon, with the 40-kDa gene 64 nucleotides upstream of the 34-kDa gene. Both crystal proteins are synthesized in approximately the same amounts. Even though small compared with other crystal proteins, the 34-kDa crystal protein has insecticidal activity against lepidopteran larvae (Manduca sexta). The 40-kDa polypeptide appears to have no insecticidal activity, but it could have a role in crystal structure.     

苏云金芽胞杆菌cry26Aa和cry28Aa基因共表达可在芽胞外壁内侧形成伴胞晶体

苏云金芽胞杆菌幕虫亚种的伴胞晶体在预芽胞外壁内侧形成,呈现晶体芽胞粘连的现象。根据已发表的cry26Aa1和cry28Aa1基因序列设计引物,从苏云金芽胞杆菌幕虫亚种T02中扩增得到cry26Aa和cry28Aa基因,通过穿梭载体将这两个基因分别和同时转化到苏云金芽胞杆菌无晶体突变株BMB171后,透射电镜下可在芽胞外壁内侧和外侧同时观察到伴胞晶体,而单独表达时可在芽胞外壁外侧观察到伴胞晶体。结果表明,伴胞晶体在芽胞外壁内侧表达不单独依赖于启动子的时空调控,可能还受到晶体蛋白相互作用的影响。     

Evidence for plasmid-associated crystal toxin production in Bacillus thuringiensis subsp. israelensis

Three crystalliferous (Cry⁺) strains of Bacillus thuringiensis subsp. israelensis (serotype 14) that produce parasporal protein crystals toxic to dipteran larvae and several acrystalliferous (Cry^?) mutants, either induced or spontaneously derived from a single Cry⁺ parent, were examined for the presence of covalently closed circular (CCC) DNA in attempts to correlate toxin production with the presence of a specific plasmid. The plasmid profiles of both Cry⁺ and Cry^? variants were analyzed by both a cleared lysate- and a modified Eckhardt lysateelectrophoresis technique. All of the Cry^? mutants derived from the Cry⁺ parental strain had lost a 4.0- to 4.4-megadalton (Mdal) plasmid. Bioassay data confirmed loss of toxin production by the Cry^? variants. All three Cry⁺ strains, including the parent of the Cry^? strains, contained CCC plasmids DNAs of the following approximate molecular weights: 4.0 to 4.4, 5.2 to 6.0, and 11.4 to 13.0 Mdal. One Cry⁺ strain contained an additional CCC plasmid of 6.7 to 7.2 Mdal. The plasmid patterns for several Cry^? derivatives differed in other respects from the pattern for their parent strain. The various Cry⁺ and Cry^? strains could be distinguished either by phenotypical differences in antibiotic sensitivity, crystal production, and toxicity, or by differences in their plasmid profiles.     

Bacterial (Bacillus thuringiensis subsp. thuringiensis and B. thuringiensis subsp. kurstaki) Entomocidal Preparations Decrease Chlorophyll Accumulation in Larch Needles

Chlorophyll a plus b content and absorption spectra of the homogenates from the cotyledonary leaves of 30-day-old seedlings of two larch species, Larix gmelinii (Rupr.) Rupr. and L. sibirica Ldb. were studied. The seedlings were grown on Perlite containing aqueous solutions of entomocidal biopreparations isolated from Bacillus thuringiensis subsp. thuringiensis (bitoxybacillin) and B. thuringiensis subsp. kurstaki (lepidocide) at various final concentrations (2, 6, and 12 g/l). Changes in the form of chlorophyll absorption spectra induced by biopreparations were established. A marked inhibition of pigment accumulation in the needles dependent on the biopreparation concentration was noted. At a low concentration (2 g/l), the biopreparations virtually did not affect the chlorophyll content; an increase in their concentrations resulted in a decrease in chlorophyll content in leaves by 20% (at 6 g/l) and 40% (at 12 g/l). It is concluded that bitoxybacillin and lepidocide inhibited the chlorophyll accumulation in larch needles to a similar extent.     

L-2,3-diaminopropionate: one of the building blocks for the biosynthesis of Zwittermicin A in Bacillus thuringiensis subsp. kurstaki strain YBT-1520

Zwittermicin A (ZwA) is a hybrid polyketide-non-ribosomal peptide that is thought to be biosynthesized from five proposed building blocks, including the 2,3-diaminopropionate. Candidate genes for de novo biosynthesis of 2,3-diaminopropionate, zwa5A and zwa5B, have been identified in a previous study. In this research, zwa5A was interrupted and chemically synthesized 2,3-diaminopropionate was used to feed the zwa5A(-) mutant. Results showed that feeding with 2,3-diaminopropionate restored the ability of the zwa5A(-) mutant to produce ZwA. Another non-ribosomal peptide synthase gene, designated orf3, was identified. Amino acid dependent PPi release assay showed that the adenylation domain ZWAA2 of ORF3 acyl-adenylated l-2,3-diaminopropionate effectively. Taken together, it can be concluded that l-2,3-diaminopropionate is indeed one of the building blocks for the biosynthesis of Zwittermicin A.     

Structural disulfide bonds in the Bacillus thuringiensis subsp. israelensis protein crystal.

We examined disulfide bonds in mosquito larvicidal crystals produced by Bacillus thuringiensis subsp. israelensis. Intact crystals contained 2.01 X 10(-8) mol of free sulfhydryls and 3.24 X 10(-8) mol of disulfides per mg of protein. Reduced samples of alkali-solubilized crystals resolved into several proteins, the most prominent having apparent molecular sizes of 28, 70, 135, and 140 kilodaltons (kDa). Nonreduced samples contained two new proteins of 52 and 26 kDa. When reduced, both the 52- and 26-kDa proteins were converted to 28-kDa proteins. Furthermore, both bands reacted with antiserum prepared against reduced 28-kDa protein. Approximately 50% of the crystal proteins could be solubilized without disulfide cleavage. These proteins were 70 kDa or smaller. Solubilization of the 135- and 140-kDa proteins required disulfide cleavage. Incubation of crystals at pH 12.0 for 2 h cleaved 40% of the disulfide bonds and solubilized 83% of the crystal protein. Alkali-stable disulfides were present in both the soluble and insoluble portions. The insoluble pellet contained 12 to 14 disulfides per 100 kDa of protein and was devoid of sulfhydryl groups. Alkali-solubilized proteins contained both intrachain and interchain disulfide bonds. Despite their structural significance, it is unlikely that disulfide bonds are involved in the formation or release of the larvicidal toxin.     

Mobilization of small plasmids in Bacillus thuringiensis subsp. israelensis is accompanied by specific aggregation.

Mobilizations of pBC16 and pAND006, containing the replicon of the Bacillus thuringiensis subsp. israelensis plasmid pTX14-3, between strains of B. thuringiensis subsp. israelensis were examined. Transconjugants appeared after a few minutes and reached a maximum frequency after approximately 2 h. Plasmid pBC16 was mobilized at a frequency approximately 200 times that of pAND006. However, pAND006 was consistently transferred, suggesting that the replicon of pTX14-3 is sufficient to sustain mobilization in B. thuringiensis subsp. israelensis. A specific protease-sensitive coaggregation between strains of B. thuringiensis subsp. israelensis was found to be unambiguously correlated with plasmid transfer. Two aggregation phenotypes, Agr+ and Agr-, were identified in this subspecies. Aggregation disappeared when the optical density of the mating mixture at 600 nm exceeded approximately 1, and it did not reappear upon dilution. Aggregation was shown to involve interactions of cells with opposite aggregation phenotypes, and evidence of a proteinaceous molecule on the surface of the Agr- that is cells involved in aggregation formation is presented. Matings and selection for the presence of two antibiotic resistance plasmids followed by identification of the host cell revealed that mobilization was unidirectional, from the Agr+ cell to the Agr- cell. The aggregation phenotype was found to be transferred with high frequency (approximately 100%) in broth matings, and the appearance of Agr- isolates from Agr+ strains suggested that the loci involved in aggregation formation are located on a plasmid. No excreted aggregation-inducing signals were detected in the supernatant or culture filtrate of either the donor, the recipient, or the mating mixture.     

苏云金芽胞杆菌拟步行甲亚种YBT-1765中稳定质粒pBMB175的克隆和功能分析

从苏云金芽胞杆菌拟步行甲亚种YBT_1765中克隆得到一个大小约15.2kb的质粒pBMB175,构建了该质粒的限制性图谱,通过功能验证,将其最小的复制区定位在一个1151bp的片段上。分析了包含有这个复制区的一个大小为4152bp的核苷酸序列,该片段包含有3个编码框(ORF1、OFR2和ORF3)。氨基酸序列同源性比较发现,ORF1(767AA)与UvrD_旋促酶、重组酶RecD和RecB家族具有20%～30%的相似性;ORF2(149AA)没有发现与任何已知序列具有同源性;ORF3(83AA)与pGI3中一个未知功能的蛋白(ORF7)具有34%的相似性。通过缺失及序列比较分析推测ORF2可能编码一种新的复制蛋白。因此pBMB175的复制类型可能属于一类新的复制家族。利用最小复制区构建的重组质粒在无抗生素选择压力下可稳定遗传40多代,具备构建稳定遗传质粒载体的潜力。     

The genetic basis of the aggregation system in Bacillus thuringiensis subsp. israelensis is located on the large conjugative plasmid pXO16.

The aggregation phenotypes Agr+ and Agr- of Bacillus thuringiensis subsp. israelensis are correlated with a conjugation-like plasmid transfer and characterized by the formation of aggregates when the bacteria are socialized during exponential growth. We present evidence for the association of the Agr+ phenotype with the presence of the large (135-MDa) self-transmissible plasmid pXO16.     

Expression of mosquitocidal crystal protein genes in non-insecticidal Bacillus thuringiensis subsp. israelensis

Bacillus thuringiensis NTB-1 isolated from soil samples in Korea produces ovoidal parasporal inclusions with proteins of approximately 24–40 kDa in size. Although serological study indicated that the isolate has a flagella (H) antigen identical with subsp. israelensis , it seemed to be non-insecticidal against Lepidoptera and Coleoptera as well as Diptera. To investigate the activity of non-insecticidal B. thuringiensis transformed with insecticidal crystal protein genes, cryIVD and cytA genes of B. thuringiensis subsp. morrisoni PG-14, highly toxic to mosquito larvae, were introduced into the isolate NTB-1. The expression of mosquitocidal crystal protein genes in NTB-1 was characterized by SDS–PAGE analysis and electron microscopy. The results showed that crystalline inclusions of host, CryIVD and CytA were stably expressed in the transformant. However, the mosquitocidal activity of transformant was similar to that of B. thuringiensis subsp. kurstaki Cry⁻ B harbouring cryIVD and cytA genes, demonstrating that a synergistic effect by an interaction of both introduced insecticidal and resident non-insecticidal crystal proteins was not observed.     

Accumulation of the insecticidal crystal protein of Bacillus thuringiensis subsp. kurstaki in post-exponential-phase Bacillus subtilis.

A gene from Bacillus thuringiensis subsp. kurstaki that codes for a Lepidoptera-specific insecticidal toxin (delta-endotoxin) was engineered for expression in Bacillus subtilis. A low-copy-number plasmid vector that replicates in Escherichia coli and B. subtilis was constructed to transform B. subtilis with gene fusions first isolated and characterized in E. coli. Naturally occurring promoter sequences from B. subtilis (43, veg, ctc, and spoVG) were inserted upstream from the plasmid-borne structural gene. In the most prolific case, when the sporulation-specific spoVG promoter was fused to the heterologous toxin gene, the toxin product accumulated during postexponential growth to greater than 25% of the total cell protein. However, the resulting specific activity of the insecticidal toxin product was not commensurate with the abundance of the protein.     

Parasporal bodies of Bacillus thuringiensis subsp. morrisoni (PG-14) and Bacillus thuringiensis subsp. israelensis are similar in protein composition and toxicity

Abstract The mosquitocidal parasporal bodies of the PG-14 isolate of Bacillus thuringiensis ssp. morrisoni and B. thuringiensis ssp. israelensis were purified on sodium bromide gradients and compared using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) electron microscopy and bioassays against mosquito larvae. The parasporal bodies of both subspecies were spherical/ovoidal, approx. 0.7–1.2 μm in diameter, and contained major proteins of 28, 65, 126 and 135 kDa. In addition to these, the parasporal body of B. thuringiensis ssp. morrisoni contained at least one other major protein, of 144 kDa, which correlated with the presence of a quasi-bi-pyramidal inclusion not present in the B. thuringiensis ssp. israelensis parasporal body. The LC₅₀ for parasporal bodies of each subspecies was in the range of 3 ng/ml for fourth-instars of Aedes aegypti . These results indicate that B. thuringiensis Serotype 8a:8b, which is generally considered to produce proteins toxic to lepidopterous insects, is capable of producing a protein toxin complement similar to B. thuringiensis Serotype 14.     

苏云金芽胞杆菌幕虫亚种晶胞粘连现象与晶体蛋白基因cry26Aa所在质粒有关

苏云金芽胞杆菌幕虫亚种T02菌株的伴胞晶体在芽胞外壁内侧形成,呈现晶胞粘连的现象。在此菌株中克隆了cry26 Aa和cry28 Aa两个基因,并对晶胞粘连现象与质粒的相关性做了系统研究。通过消除幕虫亚种T02菌株的质粒,得到了仅消除cry26 Aa所在质粒的菌株BMB1151和无质粒的菌株BMB1152。通过穿梭载体将cry26 Aa和cry28 Aa两个基因分别和同时转化无质粒突变株BMB1152并表达,形成的晶体与芽胞独立存在不能粘连,表明在幕虫亚种染色体背景下仅仅cry的表达不能形成晶胞粘连现象,从而推断晶胞粘连现象可能与幕虫亚种两个基因所在的质粒有关;进一步的研究发现将cry26 Aa在仅消除cry26 Aa所在质粒的突变株BMB1151中表达,形成的晶体与芽胞也分别独立存在不能粘连,从而进一步推断幕虫亚种晶胞粘连现象与cry26 Aa所在质粒有关。     

Accumulation of the insecticidal crystal protein of Bacillus thuringiensis subsp. kurstaki in post-exponential-phase Bacillus subtilis

A gene from Bacillus thuringiensis subsp. kurstaki that codes for a Lepidoptera-specific insecticidal toxin (delta-endotoxin) was engineered for expression in Bacillus subtilis. A low-copy-number plasmid vector that replicates in Escherichia coli and B. subtilis was constructed to transform B. subtilis with gene fusions first isolated and characterized in E. coli. Naturally occurring promoter sequences from B. subtilis (43, veg, ctc, and spoVG) were inserted upstream from the plasmid-borne structural gene. In the most prolific case, when the sporulation-specific spoVG promoter was fused to the heterologous toxin gene, the toxin product accumulated during postexponential growth to greater than 25% of the total cell protein. However, the resulting specific activity of the insecticidal toxin product was not commensurate with the abundance of the protein.     

Bacillus thuringiensis subsp. sichuansis strain MC28 produces a novel crystal protein with activity against Culex quinquefasciatus larvae

The Bacillus thuringiensis subsp. sichuansis MC28 strain produces spherical parasporal crystals during sporulation and exhibits remarkable insecticidal activity against dipteran and lepidopteran pests. We characterized a novel cry gene (cry69Aa1), which was found in the pMC95 plasmid of the MC28 strain. The cry69Aa1 gene was inserted into a shuttle vector (pSTK) and expressed in an acrystalliferous mutant B. thuringiensis HD73^?. In this transformant, a large number of spherical parasporal crystals, which were toxic to Culex quinquefasciatus (Diptera), were formed.