Monitoring growth phase-related changes in phosphatidylcholine-specific phospholipase C production,adhesion properties and physiology of <Emphasis Type="Italic">Bacillus cereus</Emphasis> vegetative cells

The physiological status and metabolic heterogeneity of Bacillus cereus cells within a culture during an 8-h batch fermentation process was measured using flow cytometry (FCM). Concurrently, production of the toxin, PC-PLC, and the extent of cell adhesion of live and dead cells were monitored using novel fluorescent assays. Flow cytometry analysis detected growth phase-related changes in the physiological profiles of cells over the course of the fermentation, with variation in the percentage of cells displaying membrane damage and intracellular esterase and redox activities. As the exponential phase proceeded, populations became more uniform in terms of protein content as measured using FCM in tandem with a cell tracking dye, with the majority of cells becoming membrane intact, esterase positive and redox active. PC-PLC activity appeared strongly related to cell density. Permeabilisation of cells was accompanied by a loss in adherent properties, while 25–100% of cells with intracellular esterase activity possessed adhesion properties. Cells in late exponential phase appeared to have reduced adherence properties compared to cells in early exponential or lag phase. As well as demonstrating the utility of FCM for measuring heterogeneity in terms of cell physiological status throughout the course of batch cultures, the methods utilised in this study could be used to relate processes such as toxin production or cell adhesion to cell physiological state.     

Functional characterization of truncated fragments of Bacillus sphaericus binary toxin BinB

Bacillus sphaericus produces a mosquitocidal binary toxin composed of two subunits, BinA (42 kDa) and BinB (51 kDa). Both components are required for maximum toxicity against mosquito larvae. BinB has been proposed to provide specificity by binding to the epithelial gut cell membrane, while BinA may be responsible for toxicity. To identify regions in BinB responsible for receptor binding and for interaction to BinA, we used six BinB shorter constructs derived from both the N-terminal and the C-terminal halves of the protein. All constructs expressed as inclusion bodies in Escherichia coli, similarly to the wild-type protein. A marked decrease in larvicidal activity was observed when BinA was used in combination with these BinB constructs, used either individually or in pairs from both N and C-halves of BinB. Nevertheless, immunohistochemistry analyses demonstrate that these constructs are able to bind to the epithelium gut cell membrane, and in vitro protein-protein interaction assays revealed that these constructs can bind to BinA. These results show that fragments corresponding to both halves of BinB are able to bind the receptor and to interact with BinA, but both halves are required by the toxin to exhibit full larvicidal activity.     

苏云金芽孢杆菌gerA操纵子在芽孢萌发中的作用

芽孢萌发的营养诱导剂通过与特异的萌发受体结合激活下游的萌发过程,从而使芽孢经过一系列的遗传变化及生化反应恢复营养生长.从苏云金芽孢杆菌(Bacillus thuringiensis)中克隆到一个与枯草芽孢杆菌(Bacillus subtilis)gerA操纵子和蜡状芽孢杆菌(Bacillus cereus)gerR操纵子同源的gerA操纵子.苏云金芽孢杆菌gerA操纵子含有3个开放读码框:gerAA、gerAC和gerAB,该操纵子在产孢起始3个小时后开始转录.gerA的破坏阻断了L-丙氨酸诱导的芽孢萌发并且延迟了肌苷诱导的萌发.在L-丙氨酸诱导芽孢萌发的过程中D-环丝氨酸能够提高芽孢的萌发率.     

枯草芽孢杆菌渗透压调节基因proB的克隆和表达

用PCR扩增的方法从耐盐的枯草杆菌中克隆出一个13kb长的DNA片段,经功能检测,证明正向插入片段与大肠杆菌的脯氨酸营养缺陷特性(proB-)能够营养互补。含有该重组质粒的大肠杆菌DH5α在基本培养基上的耐盐能力从2%提高至4%。通过引物步行法测定了该插入片段的核苷酸序列。利用DNAsis软件进行序列分析发现,该片段第122～1235bp核苷酸编码一个由370个氨基酸组成的蛋白质分子,其上游存在非典型的-10区,典型的-35区和核糖体结合位点,起始密码子处有最佳翻译起始效率的侧翼核苷酸序列。将其与Genebank中的已知基因的序列和编码的氨基酸序列进行同源性比较,结果表明该片段与枯草杆菌168的核苷酸序列、氨基酸序列的同源性分别为81%和90%。证明该基因确实是一个proB基因。通过与三十个不同种属微芽生物proB基因的氨基酸序列比较,发现该蛋白存在有可能与形成酶的活性中心和三维结构有密切关系的几个绝对保守的区域。     

枯草芽孢杆菌CAS15嗜铁素基因dhbC的克隆、表达及功能鉴定

通过PCR技术扩增得到dhbC基因,对其进行序列分析发现,dhbC基因片段长为1197bp,预期编码398个氨基酸,蛋白分子量大小为43.8kD。将目的片段连接到表达载体pET-30a(+),转化大肠杆菌Escherichia coli BL21(DE3)获得重组菌株BL21(DE3)/pET-30a-dhbC,以IPTG在30oC诱导4h实现高效表达,获得一个分子量为48.8kD的融合蛋白。重组蛋白可溶性分析结果表明:融合蛋白主要为可溶性蛋白。Western blotting分析结果表明:重组蛋白可与兔抗His-tag多克隆抗体发生特异性反应,在48.8kD处有特异条带,与预期结果一致,证明重组质粒中含有dhbC基因。通过同源重组的策略将dhbC基因敲除后重新导入,验证了dhbC基因与嗜铁素的生物合成密切相关。     

苏云金杆菌δ-内毒素、芽孢及苏云金素A对棉铃虫毒性及拒食性的比较

苏云金杆菌库斯塔克变种HD-1的晶体蛋白与芽孢、HD-1无晶体突变株(Cry-)的芽孢以及苏云金素A对棉铃虫Helicoverpa armigera毒性和拒食性的比较研究显示,HD-1晶体蛋白对棉铃虫的杀虫毒力高, 拒食作用强; HD-1芽孢对棉铃虫具有一定的杀虫活性和生长抑制作用, 并有很强的拒食作用; HD-1无晶体突变株(Cry-)芽孢对棉铃虫无毒也无拒食作用;苏云金素A对棉铃虫的生长发育有极显著的抑制作用, 但对棉铃虫无拒食作用,由此证明晶体蛋白是苏云金杆菌杀虫活性和拒食作用的主要来源。苏云金素A与苏云金杆菌芽孢晶体混合物一起使用, 可使棉铃虫的死亡率显著提高。     

新型数值分类软件X-Cluster的开发及应用

针对目前常用数值分类软件的不足,采用多种设计模式开发了新型数值分类软件X-C luster。该软件具有界面友好、操作方便、体积小、速度快、功能强大、应用范围广等特点,能够满足大多数情况下数值分类研究工作的需求,并在芽孢杆菌分类研究中得到了验证。     

Energetics of alkalophilic representatives of the genus <Emphasis Type="Italic">Bacillus</Emphasis>

Cytochrome and lipid composition of membranes is considered as the attributes required for adaptation of the alkalophiles to alkaline conditions. Respiratory chains of alkalophilic representatives of the genus Bacillus are discussed. Special attention is paid to the features of the Na⁺-cycle of these bacteria and to the features determining halo- and alkalotolerant phenotype, which have been reported due to recent achievements in genomics.Translated from Biokhimiya, Vol. 70, No. 2, 2005, pp. 171–176.Original Russian Text Copyright © 2005 by Muntyan, Popova, Bloch, Skripnikova, Ustiyan.This revised version was published online in April 2005 with corrections to the post codes.     

Mosquito control actions affect chironomid diversity in temporary wetlands of the Upper Rhine Valley

The Upper Rhine Valley, a “hotspot of biodiversity” in Germany, has been treated with the biocide Bacillus thuringiensis var. israelensis (Bti) for mosquito control for decades. Previous studies discovered Bti nontarget effects in terms of severe chironomid abundance reductions. In this study, we investigated the impact of Bti on species level and addressed the community composition of the nontarget family Chironomidae by use of community metabarcoding. Chironomid emergence data were collected in three mosquito‐control relevant wetland types in the Upper Rhine Valley. For all three sites the chironomid species composition, based on operational taxonomic units (OTUs), was different to varying degrees in the Bti‐treated samples versus control samples, ranging from a significant 63% OTU reduction to an OTU replacement. We assumed that predatory chironomids are less prone to Bti than filter feeders, as the latter feed on floating particles leading to direct ingestion of Bti. However, a comparable percentage of predators and filter feeders (63% and 65%, respectively) was reduced in the Bti samples, suggesting that the feeding strategy is not the main driver for Bti sensitivity in chironomids. Finally, our data was compared to a three‐year‐old data set, indicating possible chironomid community recovery due to species recolonization a few years after the last Bti application. Considering the currently discussed worldwide insect decline we recommend a rethinking of the usage of the biocide Bti, and to prevent its ongoing application especially in nature protection reserves to enhance ecological resilience and to prevent boosting the current biodiversity loss.