Ubiquity of parasporin-1 producers in <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> natural populations of Japan

Parasporin, a Bacillus thuringiensis parasporal protein, is unique in having a strong cytocidal activity preferential for human cancer cells. In this study, we characterized parasporin activities associated with three novel geographical isolates of B. thuringiensis. Parasporal inclusion proteins of the three isolates were highly toxic to human uterus cervix cancer cells (HeLa), but not to non-cancer uterine smooth muscle cells (UtSMC). Inclusions of the isolates lacked insect toxicity and hemolytic activity against sheep erythrocytes. Ouchterlony immunodiffusion tests revealed that the proteins of the three isolates are immunologically closely related to parasporin-1 (Cry31A), but dissimilar to the three other existing parasporin groups. Our results provide evidence that the parasporin-1-producing organism is a common member in B. thuringiensis populations occurring in natural environments of Japan.     

磺胺类耐药菌中抗性基因sul的表达规律

抗生素环境污染是影响抗生素抗性基因传播和扩散的主要因素,然而关于抗生素耐药菌在抗生素暴露下抗性基因的表达机制研究甚少。本研究利用RT-PCR方法检测了土壤中优势耐药菌菌株炭疽芽孢杆菌(Bacillus anthracis SYN201,G+)和弗氏志贺菌菌株(Shigella flexneri NJJN802,G-)在含有不同浓度磺胺类药物的培养基中生长不同时间后抗性基因(sul 1、sul 2、sul 3)的表达变化。结果发现:无论培养基中是否存在磺胺类药物,菌株SYN201和NJJN802中的3种磺胺类抗性基因均分别在培养的第72小时或36小时出现一个明显的表达峰,而在其他培养时间下不表达或表达量处于相对极低的水平;磺胺嘧啶的存在有助于提高菌株在此特征表达时间下抗性基因的表达水平,与未加磺胺嘧啶组相比,暴露于磺胺嘧啶(60μg·m L~(-1))组的sul 1、sul 2和sul 3在菌株SYN201中的相对表达量分别提高了2.5、4.8和3.2倍,而在菌株NJJN802中的相对表达量分别提高了3.7、6.0和5.0倍。通过将耐药菌暴露于不同磺胺嘧啶浓度下考察sul基因相对表达情况,研究发现随着培养基中磺胺嘧啶浓度的升高(0~1 024μg·m L~(-1)),菌株SYN201和NJJN802中sul基因的表达量整体上呈现出明显的上升趋势。本研究对揭示磺胺耐药菌的抗性表达规律及抗生素暴露对其抗性表达发挥的作用具有重要意义。     

Etude sur l'activite initiale et la persistance d'un larvicide experimental a base de bacillus thuringiensis var. israelensis et d'une preparation commerciale contenant l'insecticide organophosphore temephos

Abstract

The larvicide activity of the experimental preparation SAN 402 I WDC containing B. Thuringiensis var. Israelensis and that of Abate 500 E (44 % temephos) were tested on the larvae of Aedes aegypti. The studies were performed on the following media : distilled water, pure and buffered at pH 9, 7 and 4 and pond water whether or not free from materials in suspension. The activity of both preparations is not influenced by the composition of the media, exception made for the presence of materials in suspension. The persistance varies according to the kind of the media and the concentration level. It is in general rather low. Both preparations behave rather similar, the microbial insecticide however is more influenced by the presence of materials in. suspension.     

Alternative low-cost process for large-scale production of Bacillus thuringiensis in a simple and novel culture system

Abstract

An industrial-scale, profitable method for production of the most widely used bioinsecticide, Bacillus thuringiensis (Bt), is challenging because of its widespread application. The aim of this study is to present a strategy to develop a low-cost, large-scale bioprocess to produce Bt H14.

This study was first focused on the design of a culture medium composed of economical and available components, such as glycerol and lysed Saccharomyces cerevisiae. The production goal of 1200 ITU was achieved using a medium composed of 20:20 g L^?1of glycerol:lysed yeast in batch cultures. Efforts were subsequently focused on the design of an appropriate culture system, and an original two-stage culture system was proposed. First, yeast (the primary component of the culture medium) are cultivated using a minimal mineral medium and lysed, and in the second stage, Bt is cultivated in the same bioreactor using the lysed yeasts as culture medium (supplemented with a feeding pulse of 10 g L^?1 glycerol). This system was called fed batch one pot (FOP). A new inoculation strategy is also presented in this study, since these Bt cultures were inoculated directly with heat pre-treated spores instead of vegetative bacteria to facilitate the bioprocess. This study was developed from the laboratory to production-scale bioreactors (measuring from 500 mL to 2500 L), and the efficiency of the proposed strategy was evident in LD₅₀ tests results, achieving 1796 ITU in large-scale processes. Both the use of non-conventional sources and the process development for biomass production are important for cost-effective production of Bt-based insecticides in mosquito control projects.     

Behavior of fenhexamid in soil and water

A study was conducted to investigate fenhexamid (FEX) behavior in soil and in water. FEX proved to be rather stable at acid pH but showed slight degradation at neutral and alkaline pH. After 101 days of FEX spiking of a soil sample, 94% at pH 4, 12% at pH 7 and 23% at pH 9 of the active ingredient was still present. In natural water the rate of FEX disappearance appeared to be slow which may be due to abiotic rather than biotic processes. The soil degradation tests showed low persistence of the active ingredient if a good microflora activity is guaranteed (DT₅₀ about 1 day). Moreover, in absence of microorganisms, FEX proved to be stable. Humidities of 25 and 50% of Water Holding Capacity (WHC) influenced in equal measure the rate of degradation. From the same soil, a bacterium was isolated and identified as Bacillus megaterium, which was able to metabolize FEX with the hydroxylation of the cyclohexane ring. Moreover, FEX showed an elevated affinity for humic acid (73%), smectite (31%), and ferrihydrite(20%) and low affinity for vermiculite (11%) and kaolinite (7%).     

地衣芽孢杆门菌（Bacillus licheniformis）对Cr^6＋的吸附动力学研究

从污染土壤中分离出地衣芽孢杆菌（Bacillus licheniformis），利用其死菌体对Cr^6＋溶液进行吸附动力学研究．在Ci=300mg／L、pH=2．5和日=50℃条件下，吸附120min获得最大吸附量60．5mg／g．应用Langmuir和Freundlich吸附等温线研究，结果表明，Langmuir吸附等温线更为适合．动力学研究显示，地衣芽孢杆菌对Cr^6＋的吸附动力学可以用拟二级速度方程进行描叙．图3表4参14     

地衣芽孢杆菌2709和 6816碱性蛋白酶基因在大肠杆菌中的克隆、表达及序列分析

用PCR方法从地衣芽孢杆菌 2 70 9和 6 816中扩增了碱性蛋白酶基因 (apr1和apr2 ) ,扩增的DNA片段插入到大肠杆菌载体 pET -2 8a中 ,构建成重组分泌型表达载体pAPR1、pAPR2 .pAPR1、pAPR2中碱性蛋白酶基因在大肠杆菌宿主JM 10 9(DE3)中得到表达 .SDS -PAGE分析显示融合表达产物的分子量均为 30 .5× 10 3 ,同核酸序列测定所推导的值相符 .表达产物分别占细胞总蛋白的 8.0 %和 7.5 % .2 70 9重组菌所得酶活为 12 10u/mL ,6 816重组菌所得酶活为 1175u/mL .研究发现 ,重组的碱性蛋白酶在进入大肠杆菌周质空间时可能存在前肽自动脱落的现象 .同时 ,对地衣芽孢杆菌 2 70 9碱性蛋白酶基因序列进行了测定和比较分析 ,发现与地衣芽孢杆菌 6 816碱性蛋白酶基因的同源性为 98% .图 5参 11     

克劳氏芽孢杆菌(Bacillus clausii S-4)吸附Zn2+的研究

为了验证生物吸附剂去除废水中重金属离子Zn²⁺的可行性，筛选了一株高效吸附Zn²⁺的微生物菌株克劳氏芽孢杆菌Bacillus clausii S-4。采用火焰原子吸收、红外光谱和扫描电镜能谱分析，对这种新型生物吸附剂在水相中吸附Zn²⁺的性能和机理进行了研究。结果表明，B. clausii S-4菌体在20 ℃、30 ℃、40 ℃条件下，吸附Zn²⁺的最佳pH为4.5，吸附容量为57.5 mg/g，吸附容量随温度的升高而增加，吸附过程是吸热反应，吸附平衡时间约30 min。吸附前后，B. clausii S-4菌体表面上的化学官能团和金属离子发生了明显的变化，—OH、—NH⁺₄、—COOH、（—CO—NH—）、—C₆H₅等官能团可能参与了吸附过程。0.1 mol/L HNO₃、HCl和EDTA对锌的解吸附效果较好。在矿山废水中，B. clausii S-4菌体对Zn²⁺的吸附效果也比较理想，显示出其潜在的应用价值。     

紫外线杀菌效能的研究

自来水的消毒目前普遍采用氯及含氯化合物药剂，消毒过程中产生三卤甲烷等致癌或致突变物质，从而造成水体的二次污染，许多国家已经禁止在消毒中使用含氯药剂。因此，具有安全可靠高效杀菌作用的紫外线消毒技术逐渐发展起来，并越来越受到人们的关注。但是紫外线的使用也有一定的限制，通过紫外线照射时间及进水浊度的变化，研究了具有一定照射强度的紫外灯的杀菌效能变化，并得出了紫外线具有最佳杀菌效果的进水极限浊度和照射时间。同时还研究了紫外线对具有不同抗性的细菌的系灭效率，为饮用水和污水的消毒处理提供了实验基础。     

豆豉纤溶酶在枯草杆菌WB800中的高水平表达

一个来源于豆豉产生菌DC12的新的纤溶酶基因序列被克隆测序.为了实现在枯草杆菌中高水平表达豆豉纤溶酶,将来源于枯草杆菌168的重叠启动子P43序列以及转录终止信号序列通过重叠延伸融合,将融合序列克隆到大肠杆菌-枯草杆菌穿梭载体pBE3中,构建了pBEP43载体.将豆豉纤溶酶基因插入到载体pBEP43后转化枯草杆菌WB800.结果表明,在P43启动子驱动下,豆豉纤溶酶基因在重组菌中的对数生长期和平衡期均获得了表达且分泌到培养基中.重组菌经培养后上清液中的纤溶酶活性最高达1270U/mL,是豆豉纤溶酶基因在自身启动子驱动下表达量的1.87倍和野生菌株枯草杆菌DC12表达量的4.1倍.图5参16