草地贪夜蛾幼虫肠道细菌的分离鉴定及纤维素降解细菌的筛选

【目的】本研究旨在明确草地贪夜蛾Spodopterafrugiperda幼虫肠道可培养细菌组成,筛选纤维素降解细菌。【方法】采用传统细菌培养及16S rDNA分子标记相结合的方法分离鉴定草地贪夜蛾幼虫肠道可培养细菌;采用刚果红染色法筛选纤维素降解细菌,并通过3,5-二硝基水杨酸(DNS)法测定不同pH(5.0-9.0)条件下的纤维素酶活力。【结果】从草地贪夜蛾幼虫中筛选分离出14种肠道细菌菌株,分别隶属放线菌门(Actinobacteria)、厚壁菌门(Firmicutes)、变形菌门(Proteobacteria)等3门11属,即谷氨酸棒杆菌属(Glutamicibacter)、肠球菌属(Enterococcus)、芽胞杆菌属(Bacillus)、葡萄球菌属(Staphylococcus)、摩根菌属(Morganella)、肠杆菌属(Enterobacter)、志贺氏菌属(Shigella)、克洛诺杆菌属(Cronobacter)、克雷伯氏菌属(Klebsiella)、沙雷氏菌属(Serratia)、苍白杆菌属(Ochrobactrum)。2株产纤维素酶细菌隶属厚壁菌门芽胞杆菌属。酶活力测定结果显示:2株纤维素降解细菌的纤维素酶在pH 6.0-8.0均有相对较高的活性,在pH 8.0时,纤维素酶活最高。【结论】草地贪夜蛾幼虫肠道中细菌种类多样,其肠道内存在纤维素降解细菌。在偏碱性条件下,纤维素降解细菌的纤维素酶活力显著高于酸性条件。研究结果丰富了纤维素降解细菌资源,在饲料生产、食品加工、化学能源等方面具有应用前景。此外,草地贪夜蛾作为重大农业害虫,其肠道有益菌群,有望成为新的防治靶标。     

芽胞杆菌浸种对水稻内生细菌群落结构的影响

水稻内生细菌群落是反映植株内环境是否健康稳定的重要生物学指标,芽胞杆菌是防治水稻病害的重要生防微生物。为揭示芽胞杆菌浸种处理对水稻内生细菌群落结构的影响,采用Illumina MiSeq测序的方法对水稻内生细菌的16S rRNA基因进行测序,剖析了芽胞杆菌浸种处理对不同水稻组织内生细菌的微生态调控作用。结果表明,3种芽胞杆菌浸种处理可以提高水稻根和茎部内生细菌群落的丰富度和均匀度,降低叶部内生细菌群落的丰富度和均匀度,显著增加根部内生细菌群落多样性。变形菌门(Proteobacteria)、厚壁菌门(Firmicutes)和拟杆菌门(Bacteroidetes)是水稻根部和茎部共有优势菌门,厚壁菌门和芽胞杆菌属(Bacillus)是叶部共有优势菌门和属。芽胞杆菌浸种处理显著提高了叶部内生厚壁菌门和芽胞杆菌属的相对丰度,增加了根系和茎部组织内生细菌的分类单元OTU(Operational Taxonomic Units)数量,对叶部组织影响不明显;降低了茎部和叶部中参与各种代谢通路的内生细菌丰度,显著增加了根部参与代谢通路的内生细菌丰度。因此,3种芽胞杆菌浸种处理可以显著改变水稻根部、茎部和叶部内生细菌群落结构,改善水稻生长的微生态环境。     

小碎斑鱼蛉幼虫肠道细菌分离及鉴定研究

目的从微生态学角度研究小碎斑鱼蛉幼虫的营养生理活动。方法从小碎斑鱼蛉幼虫肠道环境中进行分离、纯化和培养,获得3个细菌菌株,对其菌体形态、染色反应、培养性状和生理生化反应进行系统研究。结果上述3个菌株均属于芽胞杆菌属(Bacillus),1号菌株为巨大芽胞杆菌(Bacillus megaterium),2号菌株为枯草芽胞杆菌(Bacillus subtilis),3号菌株为地衣芽胞杆菌(Bacillus licheniformis)。结论小碎斑鱼蛉幼虫肠道环境中的不同细菌,其数量存在明显差异。     

椰子织蛾幼虫肠道细菌的初步分离鉴定及功能分析

[目的] 研究椰子织蛾幼虫肠道微生物的种类和功能,以揭示其消化利用寄主老叶的机制。[方法] 采用传统微生物分离培养技术分离培养肠道细菌,用16S rRNA基因序列分析的方法鉴定菌株,采用透明圈染色法对所得菌株进行功能性验证。[结果] 基因序列检测对比鉴定得到9种可培养细菌菌株,主要属于变形菌门和厚壁菌门以及放线菌门;功能性验证结果表明,伯克霍尔德氏菌、解淀粉芽孢杆菌、贝莱斯芽孢杆菌、蜡样芽孢杆菌菌株具有纤维素降解酶,寒气玫瑰单胞菌、解淀粉芽孢杆菌含木聚糖降解酶。[结论] 椰子织蛾肠道中存在可培养的具有降解纤维素及木聚糖能力的细菌,这些细菌可能有助于椰子织蛾取食消化椰子等老叶,研究所获得的肠道微生物菌株也为后续研究该虫与环境的关系及相关菌株应用于农业、能源、环保价值的探索提供帮助。     

老年阿尔茨海默病患者肠道微生态改变及其与认知功能的关系探讨

目的探讨老年阿尔茨海默症患者肠道微生态结构与认知功能的关系。方法将2016年7月-2019年5月医院收治的86例老年阿尔茨海默病患者和73例健康志愿者,分别记为研究组和对照组。采用简易精神状态评价量表(MMSE)进行评分评价两组认知功能,采用高通量测序检测两组肠道微生物多样性。比较两组肠道微生物多样性与MMSE评分,并比较研究组中认知功能正常者与障碍者肠道微生物结构;采用Pearson相关性分析法探讨肠道微生物结构与认知功能障碍者的相关性。结果研究组肠道菌群Chao1指数、Shannon指数、放线菌门、拟杆菌门、放线菌科、乳杆菌科、拟杆菌科、双歧杆菌属、乳杆菌属、拟杆菌属、MMSE评分均低于对照组(均P0.05);研究组厚壁菌门、变形菌门、子囊菌门、梭菌科、假单胞菌科、葡萄球菌科、肠杆菌科、隐球酵母科、梭状芽孢杆菌属、假单胞菌属、葡萄球菌属、变形杆菌属、假丝酵母菌属相对丰度均高于对照组(均P0.05);研究组中认知功能障碍发生率为70.93%,且认知功能障碍者肠道菌群Chao1指数、Shannon指数,门、科、属水平相对丰度与认知功能正常者对比和上述结果一致;研究组与对照组肠道拟杆菌门、子囊菌门、拟杆菌科、梭状芽胞杆菌属、假丝酵母菌属、放线菌门、乳杆菌属、拟杆菌属相对丰度差异具有统计学意义(均P0.05);研究组认知功能障碍与认知功能正常者肠道未分类拟杆菌门、子囊菌门、假单胞菌属、拟杆菌属、变形杆菌属、梭状芽孢杆菌属、拟杆菌门、拟杆菌科、假丝酵母菌属相对丰度差异具有统计学意义(均P0.05);研究组中肠道菌群Chao1指数、Shannon指数,放线菌门、拟杆菌门、放线菌科、乳杆菌科、拟杆菌科、双歧杆菌属、乳杆菌属、拟杆菌属相对丰度与MMSE评分均呈正相关,厚壁菌门、变形菌门、子囊菌门、梭菌科、假单胞菌科、葡萄球菌科、肠杆菌科、隐球酵母科、梭状芽孢杆菌属、假单胞菌属、葡萄球菌属、变形杆菌属、假丝酵母菌属相对丰度与MMSE评分均呈负相关。结论老年阿尔茨海默病患者肠道微生物结构存在异常,且认知功能障碍者更加严重,认知功能与肠道微生物相关。     

家蝇幼虫肠道细菌对其取食选择性和生长发育的影响

目的探究家蝇幼虫肠道细菌对幼虫取食与发育的影响,进而明确肠道细菌的部分功能。方法取3日龄家蝇幼虫,采用细菌传统分离法分离纯化其肠道内可培养细菌并进行分子鉴定,利用这些肠道细菌进行家蝇幼虫爬行选择实验,测定幼虫对不同细菌的选择性;利用这些肠道细菌发酵液与麦麸混合饲养家蝇幼虫,记录家蝇幼虫发育过程中的生物学参数,分别以LB培养原液与麦麸混合饲养的幼虫为阴性对照,以无菌水与麦麸混合饲养的幼虫为空白对照。结果在家蝇幼虫肠道中共分离出9个属10种细菌,分别为霍氏肠杆菌(Enterobacter hormaechei)、肺炎克雷伯菌(Klebsiella pneumoniae)、铜绿假单胞菌(Pseudomonas aeruginosa)、别雷斯不动杆菌(Acinetobacter bereziniae)、斯氏普罗威登斯菌(Providencia stuartii)、阴沟肠杆菌(Enterobacter cloacae)、乳酸乳球菌(Lactococcus lactis)、纺锤形赖氨酸芽胞杆菌(Lysinibacillus fusiformis)、居幼虫普罗威登斯菌(Providencia vermicola)和沙福芽胞杆菌(Bacillus safensis)。其中,除铜绿假单胞菌和斯氏普罗威登斯菌外,其余8种细菌对幼虫有显著吸引作用;除铜绿假单胞菌对家蝇生长有明显抑制作用以外,其余9种细菌对家蝇幼虫生长均有一定的促进作用,其中霍氏肠杆菌和别雷斯不动杆菌对其生长有显著促进作用。结论明确了家蝇幼虫肠道细菌的组成,确定了8种家蝇幼虫肠道细菌对其取食有吸引作用,9种细菌对其生长发育有促进作用。霍氏肠杆菌和别雷斯不动杆菌可作为添加剂用于家蝇的规模化饲养,以提高养殖效率。铜绿假单胞菌可作为生长抑制剂用于家蝇幼虫的防治。     

小菌虫肠道可培养细菌的分离鉴定 及产消化酶活性分析

摘要：目的 分离小菌虫肠道可培养细菌,并研究其产消化酶活性,探讨肠道细菌对小菌虫消化食物的影响。方法 采用传统细菌分离培养方法分离小菌虫肠道细菌,利用16S rDNA序列进行细菌分子鉴定;利用筛选培养基鉴别各细菌的产蛋白酶、脂肪酶、淀粉酶和纤维素酶活性。结果 在小菌虫肠道中分离到4种可培养细菌,分别是枯草芽胞杆菌（Bacillus subtilis）、肺炎克雷伯菌（Klebsiella pneumoniae）、Pseudocitrobacter faecalis和芽胞杆菌（Bacillus sp.）。其中,2种芽胞杆菌属细菌有产消化酶活性。枯草芽胞杆菌有产纤维素酶、淀粉酶和蛋白酶活性;芽胞杆菌仅有产蛋白酶活性,但产酶能力低于枯草芽胞杆菌。结论 小菌虫肠道细菌中可培养细菌结构简单,但其中的芽胞杆菌属细菌有产纤维素酶、淀粉酶和蛋白酶能力,说明小菌虫肠道中的2种芽胞杆菌属细菌可能有协助小菌虫进行食物消化的功能。     

广东南岭森林土壤中可培养细菌多样性

广东南岭森林土壤中蕴藏着丰富的生物资源,但对其中的可培养细菌种类仍缺乏系统了解。本研究采用贫营养型的R2A培养基和富营养型的TSA培养基对南岭森林土壤中细菌进行了分离,获得细菌408株,分别从属于厚壁菌门、变形菌门、放线菌门和拟杆菌门的35属。其中的优势类群为厚壁菌门,占分离总数量的71%。在属水平,芽胞杆菌及其近缘属为优势类群。除芽胞杆菌外,假单胞菌、伯克霍尔德氏菌、草酸杆菌科Collimonas属和罗丹诺杆菌科Dyella属是分离获得的主要类群。R2A培养基在分离革兰氏阴性的变形菌门菌株方面表现出一定的偏好性,而TSA培养基分离得到的更多为快速生长的芽胞杆菌及其近缘的革兰氏阳性细菌。发现了15属的菌株具有一定的水解酶活性,大多表现出对淀粉和牛奶的水解活性,对有机磷的水解性能优于对无机磷的水解。降解纤维素的菌株则主要集中于芽胞杆菌及其近缘属中。发现了潜在新物种26株,分布于芽胞杆菌、Dyella、类芽孢杆菌等9属中。本研究仅使用了两种营养类型的培养基,进一步借助培养组学技术有望能更加全面反映南岭森林土壤中的可培养微生物多样性。     

红树林沉积物中天然多聚有机物厌氧降解菌多样性与细菌新类群分离

[目的]红树林沉积物中有机物丰富,通过研究认识参与难降解天然有机多聚物的微生物降解过程及其环境作用,并获得新颖的难培养厌氧微生物。[方法]对漳州九龙江河口红树林沉积物中降解纤维素、几丁质和木质素的厌氧细菌定向富集和平板分离纯化,并对其多样性进行分析。[结果]共筛选分离获得202株厌氧细菌(82株专性厌氧细菌,120株兼性厌氧细菌),包括4个疑似新属(Lachnotalea sp.MCCC 1A16036、Varunaivibrio sp.MCCC 1A15903、Clostridium sp.MCCC 1A15884、Caminicella sp.MCCC 1A17445)和4个疑似新种(Sunxiuqinia sp.MCCC 1A15904、Pseudodesulfovibrio sp.MCCC 1A16040、Pseudodesulfovibrio sp.MCCC 1A16038、Mangrovibacterium lignilyticum MCCC1A15882)。不同天然有机多聚物富集菌群分离到的优势可培养细菌主要属于变形菌门、拟杆菌门和厚壁菌门,但种群略有差异。在纤维素和几丁质富集菌群中,变形菌门和拟杆菌门菌株是纤维素和几丁质富集菌群的优势菌群,拟杆菌门的Prolixiacer bellariivorans、Mangrovibacterium lignilyticum和变形菌门的Desulfovibrio salexigenes、Vibrio alginolyticus分别在纤维素和几丁质富集菌群可培养细菌中占绝对优势。但降解实验表明,放线菌门细菌Demequina salsinemoris MCCC 1A15890和Brevibacterium celere MCCC 1A17451对纤维素降解活性最高。梭杆菌门细菌Propionigenium maris MCCC 1A15874和Ilyobacter polytropus MCCC 1A15889对几丁质降解活性最高。在木质素富集菌群中,拟杆菌门的Mangrovibacterium lignilyticum和厚壁菌门的Clostridium amygdalinum都具有较高的相对丰度。变形菌门细菌Desulfomicrobium apsheronum MCCC 1A15932和拟杆菌门细菌Mangrovibacterium lignilyticum MCCC 1A15882对木质素降解效果显著。[结论]红树林沉积物中存在丰富多样且新颖的厌氧难培养细菌,且多数具有纤维素、几丁质或木质素厌氧降解能力。该研究结果为探究红树林沉积物原位环境天然有机多聚物碳的生物地球化学循环机制提供了相关理论基础和纯培养微生物资源。     

不同品种酿酒葡萄表皮微生物群落多样性分析

葡萄酒酿造是多种微生物参与代谢的过程,其中的微生物在没有外源接种的情况下通常认为源自酿酒葡萄本身。应用高通量测序技术,分析沙城地区不同品种酿酒葡萄表皮的微生物群落,旨在从源头上了解葡萄酒酿造原料的品质,为研究酿酒葡萄微生物对酿酒品质的影响提供理论依据。结果显示,无论细菌还是真菌丰富度最大的均为雷司令,细菌主要分布在变形菌门(Proteobacteria)、放线菌门(Actinobacteria)、厚壁菌门(Firmicutes)等8个门,优势细菌主要是欧文氏菌属(Erwinia)、假单胞菌属(Pseudomonas)、芽孢杆菌属(Bacillus)等6个属。真菌仅分布在子囊菌门(Ascomycota)、担子菌门(Basidiomycota)和接合菌门(Zygomycota)3个门,链格孢菌属(Alternaria)、枝孢属(Cladosporium)、茎点霉属(Phoma)、镰孢属(Fusarium)等9个主要的属。细菌中心OTU最相似菌种分别为节杆菌(Arthrobacter sp.)、假单胞菌(Pseudomonas sp.)等,真菌为枝孢菌(Cladosporiumsp.)、茎点霉(Phoma sp.)、链格孢菌(Alternaria sp.)以及大量未知种属的OTU。研究表明,葡萄品种是影响微生物群落的最重要的因素,推测酿酒葡萄上的一些微生物会对葡萄植株的健康、葡萄果实的品质以及葡萄酒酿造产生有益或有害的影响。     

牛蒡根际可培养细菌多样性和镉耐性初步分析

从牛蒡根际土壤中分离可培养细菌,进行多样性分析,并对镉耐受性菌株进行筛选及其抗性和种群多样性进行了分析。限制性内切酶多态性分析显示,分离的菌株可分为9个操作分类单元(OUT),分别属于变形菌门、厚壁菌门和放线菌门,分属于6个科,9个属,其中隶属于肠杆菌属、芽胞杆菌属和假单胞菌属的是优势物种。分离到的耐镉菌株分别属于Bacillus subtilis、Enterobacter aerogenes、Enterobacter ludwigi、Klebsiellasp.、Pectobacterium carotovorum、Pseudomonassp.,而Pectobacterium carotovorumNP22、Enterobacter ludwigii NP23、Pseudomonassp.NP39三菌株可在Cd2+浓度为400 mg/L固体培养基上生长。     

Chilling and cultivar type affect the diversity of bacterial endophytes colonizing sweet pepper (Capsicum anuum L.)

A climate chamber experiment was conducted to assay the effect of low temperatures (chilling) on the diversity of bacteria colonizing the endospheres of two thermophilic sweet pepper (Capsicum anuum L.) cultivars, Milder Spiral and Ziegenhorn Bello. Structural diversity was analyzed by 16S rRNA-based terminal restriction fragment length polymorphism (T-RFLP) analysis and by the generation of 16S rRNA gene libraries to determine dominant community members in T-RFLP profiles. Cultivable community members colonizing lines Milder Spiral and Ziegenhorn Bello were identified by 16S rRNA gene analysis. T-RFLP profiles and 16S rRNA gene libraries revealed a high heterogeneity of community composition due to chilling and suggested further the existence of cultivar-specific communities. The majority of isolates obtained from the cultivar Milder Spiral were assigned as high-G+C Gram-positive bacteria (Microbacterium sp., Micrococcus sp., Rhodococcus sp.) and Firmicutes (Staphylococcus sp.). Of the isolated endophytes obtained from cultivar Zeigenhorn Bello, 93% were affiliated with Staphylococcus aureus and Bacillus sp. (Firmicutes). The experimental set-up was suited to demonstrate that chilling and cultivar type can influence the diversity of bacterial endophytes colonizing sweet pepper. We propose additional chilling experiments to investigate the effect of chilling on functional, plant-beneficial abilities of bacterial endophytes associated with low-temperature-sensitive crops, such as sweet pepper.     

The Gut Bacteria Associated with Camponotus japonicus Mayr with Culture-Dependent and DGGE Methods

The bacterial composition and distribution in the different gut regions of Camponotus japonicus were investigated using both culture-dependent method and culture-independent method of polymerase chain reaction and denaturing gradient gel electrophoresis (PCR–DGGE). Five different bacterial strains were isolated using culture-dependent method, and they all belong to the phylum Firmicutes, including three genera of bacteria Bacillus, Paenibacillus, and Enterococcus. Bacillus cereus and Enterococcus mundtii were found in the midgut; Paenibacillus sp. was isolated from the hindgut; and the other two Bacillus spp. were isolated from the crop. Twelve distinct DGGE bands were found using PCR–DGGE method, and their sequences blasting analysis shows that they are members of the Proteobacteria and the Firmicutes, respectively, including three genera (Pseudomonas, Candidatus Blochmannia, Fructobacillus) and one uncultured bacterium, in which Pseudomonas was the most dominant bacteria group in all the three gut regions. According to the DGGE profile, the three gut regions had very similar gut communities, and all the DGGE bands were presented in the midgut and hindgut, while just two bands representing Blochmannia were not present in the crop. The results of our study indicate that the gut of C. japonicus harbors several other bacteria besides the obligate endosymbionts Blochmannia, and more work should be carried on to verify if they are common in the guts of other Camponotus ants.     

Specific incorporation of L-glutamine into volicitin in the regurgitant of Spodoptera litura

Volicitin, [N-(17-hydroxylinolenoyl)-L-glutamine], was identified as an elicitor of plant volatiles from a Spodoptera exigua regurgitant. It has been proposed that gut microbes synthesize volicitin from glutamine, a predominant amino acid component in the insect gut. However, we found that glutamine was not a major component in the regurgitant of Spodoptera litura, although L-glutamine was exclusively incorporated into volicitin by S. litura fed on diets enriched with various amino acids. This selectivity of glutamine as a substrate was not due to a dominant occurrence in the insect gut.     

四川冬菜中细菌群落组成及多样性

【目的】了解腌制4年的四川南充冬菜中细菌群落组成及多样性。【方法】通过16S rDNA多样性分析样品细菌落组成;采用16S rDNA-RFLP方法分析从样品中分离出的纯培养细菌。【结果】16S rDNA多样性分析结果表明,样品中细菌主要属于变形杆菌门(Proteobacteria)和厚壁菌门(Firmicutes),分别占克隆文库的87.9%、7.1%,其中包括Virgibacillus kekensis,Marinococcus albus,Salinicoccus sp.,Lactobacillus halophilus和Halomonas等中度嗜盐菌,仅有5%属于放线菌门(Actinobacteria)。通过纯培养方法从冬菜中分离到35株菌,16S rDNA-RFLP分析结果表明,34株属于厚壁菌门(Firmicutes),包括Virgibacillus,Bacillus megaterium和Gracilibacillus saliphilus等中度嗜盐菌,1株属于放线菌门(Actinobacteria)。【结论】冬菜中细菌群落多样性较低,以中度嗜盐菌为主。     

Bacteria abundance and diversity of different life stages of Plutella xylostella (Lepidoptera: Plutellidae), revealed by bacteria culture‐dependent and PCR‐DGGE methods

Microbial abundance and diversity of different life stages (fourth instar larvae, pupae and adults) of the diamondback moth, Plutella xylostella L., collected from field and reared in laboratory, were investigated using bacteria culture‐dependent method and PCR‐DGGE analysis based on the sequence of bacteria 16S rRNA V3 region gene. A large quantity of bacteria was found in all life stages of P. xylostella. Field population had higher quantity of bacteria than laboratory population, and larval gut had higher quantity than pupae and adults. Culturable bacteria differed in different life stages of P. xylostella. Twenty‐five different bacterial strains were identified in total, among them 20 strains were presented in larval gut, only 8 strains in pupae and 14 strains in adults were detected. Firmicutes bacteria, Bacillus sp., were the most dominant species in every life stage. 15 distinct bands were obtained from DGGE electrophoresis gel. The sequences blasted in GenBank database showed these bacteria belonged to six different genera. Phylogenetic analysis showed the sequences of the bacteria belonged to the Actinobacteri, Proteobacteria and Firmicutes. Serratia sp. in Proteobacteria was the most abundant species in larval gut. In pupae, unculturable bacteria were the most dominant species, and unculturable bacteria and Serratia sp. were the most dominant species in adults. Our study suggested that a combination of molecular and traditional culturing methods can be effectively used to analyze and to determine the diversity of gut microflora. These known bacteria may play important roles in development of P. xylostella.     

转tcdA1B1杀虫基因的阴沟肠杆菌对4种农业害虫的杀虫活性与毒力

tcdA1B1为昆虫病原细菌一发光杆菌Photorhabdus luminescens的杀虫毒素基因。本文测定了转tcdA1B1基因的阴沟肠杆菌对甜菜夜蛾Spodoptera exigua、斜纹夜蛾Spodoptera litura、黄曲条跳甲Phyllotreta striolata和东亚飞蝗Locusta migratoria manilensis4种重要农业害虫的杀虫活性与毒力。在室内条件下,浓度为5．0×108cfu／mL的工程菌悬浮液,采用叶片浸渍法饲喂甜菜夜蛾2龄幼虫、斜纹夜蛾2龄幼虫、黄曲条跳甲成虫、东亚飞蝗2龄若虫,在11d、11d、15d、19d后的致死率分别为80．05％、81．83％、72．31％、39．74％;LT50分别为8．64d,8．38d、11．04d、28．22d。     

Gut bacteria may be involved in interactions between plants, herbivores and their predators: microbial biosynthesis of N-acylglutamine surfactants as elicitors of plant volatiles

N-Acylamino acids are dominant and widespread constituents of insect oral secretions (regurgitants), serving the insect as biosurfactants in the digestive process. During feeding the conjugates may be introduced into damaged leaves and contribute there to the elicitation of plant defenses such as the induction of volatile biosynthesis. From gut segments of Spodoptera exigua, Mamestra brassicae and Agrotis segetum 23 bacterial strains were isolated, ten of which were able to synthesise typical lepidopteran N-acylamino acids from externally added precursors. Four strains, Providencia rettgeri, Ochrobactrum spec., Myroides odoratus and Acinetobacter sp. genospecies 11 were identified on the basis of their 16 S rDNA. The organisms displayed a very broad substrate tolerance, since fatty acids of different chain length and different degree of saturation were converted into N-acylamino acids. Moreover, most of the proteinogenic amino acids, but not glutamic and aspartic acid, were used as substrates. The dominant occurrence of fatty acids conjugated with glutamine may result from a preferred transport of glutamine from the hemolymph into the gut of the insects. The involvement of bacteria in the biosynthesis of compounds which play a pivotal role in the interaction of plants, herbivores and their predators adds a new trophic level to this complex network of interactions. Due to their short generation cycle and the ease of adaptation endosymbiontic bacteria may have an outstanding importance for the coevolution of plant-insect interactions.     

Insecticidal activity and processing in larval gut juices of genetically engineered 130-kDa proteins of Bacillus thuringiensis subsp. aizawai.

The 130-kDa insecticidal protein (IP) of Bacillus thuringiensis subsp. aizawai is proteolytically processed in the gut juice of susceptible insect larvae to yield an insecticidally active 60-kDa fragment. Twenty-seven mutant IP genes with the replacement of codons for Arg and Lys with codons for Gln in the active fragment and its adjacent regions of the 130-kDa IP were constructed by site-directed mutagenesis and expressed in Escherichia coli cells. The produced mutant IPs at Arg87, Arg131, Arg198, Arg311, Arg368, Arg402, Arg458, Arg502, Arg512, Arg524, Arg526, Arg528, and Arg601 had reduced insecticidal activity against Spodoptera litura larvae. The mutant at Arg601 was sensitive to proteolytic digestion in the gut juice of S. litura larvae. Although the mutants at Arg619, Lys622, and Lys637 had nearly the same activity as that of the wild type, the mutant with the triple replacement at Arg619, Lys622, and Lys637 was 2.5 times more active against S. litura larvae than the wild type. This triple mutant showed a slightly different processing profile in the gut juice than that of the wild type.