基于转录组学分析Xenorhabdus bovienii肽基脯氨酰异构酶ppia-l20基因的表达调控

[目的]前期从广谱抗菌的Xenorhabdus bovienii NN6菌株的胞外蛋白中分离出一种新型杀菌蛋白PPIA-L20,为肽基脯氨酰异构酶A类似物,该蛋白能够抑制尖孢镰刀菌西瓜专化型的孢子的萌发,致使孢子死亡。经过多次试验验证ppia-l20基因的表达水平不稳定,与发酵时间没有明显的相关性。本文旨在探寻调控ppia-l20基因表达的调控因子,进而推测ppia-l20基因表达调控的分子机制,为进一步提高抗菌蛋白的产量提供参考。[方法]对ppia-l20的转录水平进行Real-time PCR分析并与菌株生长曲线进行相关性分析,选择ppia-l20基因表达量差异显著的2个发酵时间点进行转录组学比较,并对表达差异显著的DEG(differential expressed gene)进行GO功能注释、KEGG富集通路和蛋白网络互作分析。[结果]4~84 h发酵菌体中的ppia-l20基因表达趋势不稳定,其转录水平与时间不相关。与ppia-l20低表达量的8 h发酵菌体相比,ppia-l20高表达量的12 h发酵菌体中有466个DEG,包括193个上调基因和273个下调基因。Real-time PCR验证表明基因表达差异趋势与转录组测序结果一致,证明测序结果可信。对差异表达的基因进行GO功能注释、KEGG富集分析,发现DEG的功能与膜形成相关,富集通路与双组分调节系统、磷酸转移酶系统和阳离子抗菌肽抵制通路相关。其中,双组分调节系统中的qseC基因和opmR基因表达量均上调2倍,ppia-l20基因位于阳离子抗菌肽抵制通路,该通路上的nlpE基因(负责编码外膜脂蛋白)显著下调。蛋白互作网络中,PPIA-L20蛋白与位于外膜的AMP结合蛋白TycC、GrsB有互作关系,NlpE蛋白与双组分调节系统中cpxR基因、cpxA基因表达的蛋白有互作关系。[结论]Xenorhabdus bovienii NN6菌株可能通过双组分调节系统通路、阳离子抗菌肽抵制通路上的关键基因nlpE和磷酸转移酶系统共同调控ppia-l20的表达。

Xenorhabdus nematophilus BP品系杀虫毒素基因的克隆与鉴别

构建了昆虫病原线虫共生菌XenorhabdusnematophilusBP品系的粘粒文库并用生物测定的方法从中筛选到 2个对棉铃虫初孵幼虫有口服抑杀作用的克隆cos83和cos76。为初步确定这两个克隆的毒素基因与已报道基因的差异程度 ,根据已发表的共生菌毒素基因序列资料设计了 7对引物对这两个克隆进行PCR扩增并对扩增产物进行测序和分析。从毒力较强的cos83中 ,7对引物均扩增出与目标片段大小一致的产物 ,而从cos76中只有 5对扩增到目标片段。测序结果显示 ,cos76的 5个PCR扩增产物与cos83的对应扩增产物DAN序列同源性为 10 0 %。以cos83的 7个PCR扩增产物所编码氨基酸序列进行BLAST分析 ,它们与X .nematophilusPMF12 96和PhotorhabdusluminencencW14毒素相应区间的平均同源性分别为 94%和 5 8% ,说明cos83毒素是共生菌口服毒素家族的一员但与同类的其它毒素有一定的差异 ,值得对其杀虫谱 ,作用机理等进行进一步的研究。

嗜线虫致病杆菌Xenorhabdus ne matophilus HB310对3种十字花科蔬菜害虫杀虫活性的研究

嗜线虫致病杆菌XenorhabdusnematophilusHB310是与昆虫病原线虫SteinernemacarpocapsaeHB310共生的细菌。对X.nematophilusHB310杀虫活性的测定结果表明:该共生菌菌液对小菜蛾、菜粉蝶和云斑粉蝶等幼虫均有较高的胃毒杀虫活性,饲喂蘸有该菌液的甘蓝叶片72h后,小菜蛾2龄幼虫、云斑粉蝶1龄幼虫校正死亡率达100%,96h菜粉蝶1龄幼虫、云斑粉蝶4龄幼虫的校正死亡率也分别达到100%。云斑粉蝶1龄幼虫最敏感(LC50为6 4998×105cells/mL),其次为小菜蛾2龄幼虫(LC50为1 3614×106cells/mL)和菜粉蝶1龄幼虫(LC50为2 7418×106cells/mL)。深入研究发现胃毒杀虫活性物质在该菌对数生长初期就开始产生,对数生长中期已达到最大量。生测结果表明:胞内、胞外分泌物中都含有较强的胃毒杀虫活性物质,5 994×108cells/mL的原菌液、细胞及同样浓度的菌液上清和胞内上清120h对1龄菜粉蝶幼虫的胃毒杀虫活性均在90%以上,上清的胃毒活性最低,与原菌液、细胞及胞内上清的胃毒活性差异显著。X.nematophilusHB310的Ⅱ型菌菌液的胃毒杀虫活性较低,只有Ⅰ型菌的1/3左右。

响应面法优化Xenorhabdus nematophila发酵培养基的研究

【目的】提高Xenorhabdus nematophilaYL001的抗菌活性,为该菌株杀菌活性成分的提取分离及生物农药的开发应用奠定基础。【方法】以YSG培养基为基础,采用单因子试验方法,对最佳碳源和氮源进行筛选,并采用全因子中心组合试验设计和响应面法对其最佳配比进行优化。【结果】YL001菌株的最佳碳源为玉米粉,氮源为豆饼粉,培养基的最佳组成为:玉米粉9.69 g/L,豆饼粉76.50 g/L,MgSO41.07 g/L,(NH4)2SO41.79 g/L,KH2PO40.63 g/L,K2HPO40.80 g/L,Na2SO41.25 g/L,在此条件下,YL001菌株抗菌活性达268.9 U/mL。【结论】培养基优化后YL001菌株的抗菌活性与试验的预测值接近,表明响应面法在培养基优化中十分有效,相对简单,且节省时间和材料。

Xenorhabdus nematophila发酵动力学研究

在分批发酵中,研究了Xenorhabdus nematophila YL001的生长、基质消耗及抗菌物质产生的特性.基于Logistic方程和Luedeking-Piret方程,得到了描述分批发酵过程的动力学模型及模型参数,同时对实验数据与模型进行了验证比较.模型计算值与实验数据拟合良好,模型基本反映了Xenorhabdus nematophila YL001分批发酵过程的动力学特征.分批发酵中细胞生长与产物合成属于偶联型.

Xenorhabdus spp.菌株的菌落、菌体多样现象及抗菌特性

本文报道了二十多株Steinernema和Heterorhabditis线虫共生细菌在指示培养基上的菌落形态、菌体形态的多样性及菌株的色素与抗菌特性。试验发现菌株出现树枝状菌落,折光性内含物及粒状菌体(比正常菌体小1/4至1/8)的现象,在各菌株中有很大的不同。所有试验菌株都可出现树枝状菌落,但非所有菌株都可见折光性内含物和粒状菌体。实验还观察到H06及CB2B菌株在肉汤培养基中所产生的色素颜色随pH值而变化,但G12及ED/1的色素对酸碱度的变化不敏感,表明菌株产生的色素物质是不同的。直接比较Xenorhaadus菌株之间的抗菌活动显示各菌株间具拮抗作用。菌株产生的抗菌物质是不同的,抗菌物的产生可能与菌株的培养条件有关。

Xenorhabdus nematophila BP杀虫毒素基因的序列分析及其杀虫活性

XnBP83是从Xenorhabdus nematophila BP基因组粘粒文库中筛选出的一个对棉铃虫有较强口服杀虫活性的克隆.采用亚克隆结合primer-walkingDNA测序技术对粘粒XnBP83的插入片段进行序列测定.该插入片段全长38939bp,其中包括5个与杀虫活性相关的tc类基因xptA1、xptB1、xptC1、xptA2、xptD1.序列分析显示:a.插入片段中的xptD1不完整,与X.nematophila PMFI296 XptD1相应氨基酸序列有99%的相似性.b.BPxptA1读码框全长7569bp,编码2520个氨基酸,与PMF1296的XptA1氨基酸序列有98%的相似性,两者在第2200～2223氨基酸区域连续有23个氨基酸不同.c.BPxptB1读码框全长3051bp,编码1016个氨基酸,与PMF1296XptB1氨基酸序列有98%的相似性,在第620～650氨基酸之间有28个氨基酸差异.d.BPxptC1读码框全长4225bp,编码1408个氨基酸,与PMF1296的XptC1氨基酸序列有96%的相似性.在BP的第232氨基酸后插入了一个TAQRYLAK的氨基酸序列,在第627～646氨基酸区域内,有18个氨基酸不同.e.BPxptA2读码框全长7574bp,编码2524个氨基酸,与PMF1296的XptA2氨基酸序列有90%的相似性,在BP品系XptA2的第788～855氨基酸和第1630～1784氨基酸有两个明显变异区.将XnBP83培养物上清和沉淀饲喂棉铃虫、甜菜夜蛾、斜纹夜蛾和粉纹夜蛾,结果表明XnBP83对所测昆虫有广谱杀虫活性.

Xenorhabdus nematophila BP品系xpt B1基因的克隆和序列分析

采用PCR方法,从该室构建的Xenorhabdus nematophila BP品系粘粒文库的一个对棉铃虫有强口服杀虫活性的粘粒cos83中扩增出全长的xptB1基因,将扩增片段回收纯化后,连接到pMD-18T载体,转化大肠杆菌TG1后进行序列测定和分析。结果显示,该基因全长为3051bp,编码1016个氨基酸,其编码的毒素BP XptB1与X.nematophila PMF1296品系的XptB1的氨基酸序列同源性平均为95.8%,两侧高度一致,中间第607—738位差异明显。结构分析显示,两者在跨膜区,α螺旋等方面也有差异。BP XptB1与Photorhabdus luminescens的TccC毒素,Serratia entomophila的SepC,以及Yersinia pseudotubercusis和Y.pestis等的杀虫毒素均有58%以上的同源性。分析结果预示了XptB1可能是一个杀虫毒素。

培养方式对Xenorhabdus nematophila生长与抗菌活性的影响

为了明确不同培养方式对Xenorhabdus nematophilaYL001生长和抗菌活性的影响,提高YL001菌株的抗菌活性。采用分批发酵方式研究了摇瓶与发酵罐培养对X.nematophilaYL001生长和抗菌活性的影响。实验结果表明:在通气量2.5 L/min、搅拌转速300 r/min条件下,发酵罐的体积氧传递系数KLa明显高于摇瓶,通气及供氧状况好于摇瓶,细胞生长及代谢旺盛,细胞生长量较摇瓶发酵增加了23.9%;但由于pH变化幅度大,抗菌物质的活性单位仅达到摇瓶的发酵水平,为229 U/mL。发酵罐pH控制初步研究表明,发酵过程中控制pH为7.5时,细胞生长量和抗菌活性达到23.71 g/L和290 U/mL,较不控制pH分别增加了21%和25%。通风对X.nematophilaYL001生长和抗菌物质的产生有较大的影响,发酵罐培养时通气及供氧状况好于摇瓶,有利于YL001菌株的生长和抗菌物质的产生。

Histopathological Effects of the Protein Toxin from Xenorhabdus nematophila on the Midgut of Helicoverpa armigera

Xenorhabdus nematophila HB310, which is highly virulent for many insects, is symbiotic with Steinernema carpocapsae HB310. Toxin II was obtained using methods such as salting out and native-PAGE from the cells of X. nematophila HB310. The histopathology of toxin II on H. armigera larvae was studied by dissecting an olefin slice of the midgut. The symptoms showed that the histopathology of the H. armigera midgut was similar to that of other novel midgut-active toxins such as the δ-endotoxins from Bacillus thuringiensis, as well as Tca from Photorhabdus luminescens W14. The midgut tissues of H. armigera fourth-instar larvae began to transform after the oral intake of the toxin Ⅱ over 6 h. First, the anterior region of the peritrophic membrane （PM） began to degrade followed by the elongation of the columnar cells. The epithelium decomposed gradually, and the midgut tissues were either loose or disordered. The PM disappeared after 12 h but reappeared after 72 h following transient or sublethal exposure to the toxin Ⅱ. Toxin Ⅱ also directly destroyed in vitro PMs of H. armigera.

Microbial control of diamondback moth, Plutella xylostella L. （Lepidoptera： Yponomeutidae） using bacteria （Xenorhabdus nematophila） and its metabolites from the entomopathogenic nematode Steinernema carpocapsae

Cells and cell-free solutions of the culture filtrate of the bacterial symbiont, Xenorhabdus nematophila taken from the entomopathogenic nematode Steinernema carpocapsae in aqueous broth suspensions were lethal to larvae of the diamondback moth Plutella xylostella. Their application on leaves of Chinese cabbage indicated that the cells can penetrate into the insects in the absence of the nematode vector. Cell-free solutions containing metabolites were also proved as effective as bacterial cells suspension. The application of aqueous suspensions of cells of X.nematophila or solutions containing its toxic metabolites to the leaves represents a possible new strategy for controlling insect pests on foliage.

Pathogenicity of bacterium, Xenorhabdus nematophila isolated from entomopathogenic nematode (Steinernema carpocapsae) and its secretion against Galleria mellonella larvae

The entomopathogenic bacterium, Xenorhabdus nematophila was isolated from the hemolymph of Galleria mel- lonella infected with Steinernema carpocapsae. The bacterial cells and its metabolic secretions have been found lethal to the Galleria larvae. Toxic secretion in broth caused 95% mortality within 4 d of application whereas the bacterial cells caused 93% mortality after 6 d. When filter and sand substrates were compared, the later one was observed as appropriate. Similarly, bacterial cells and secretion in broth were more effective at 14% moisture and 25 °C temperature treatments. Maximum insect mortality (100%) was observed when bacterial concentration of 4×106 cells/ml was used. Similarly, maximum bacterial cells in broth (95%) were penetrated into the insect body within 2 h of their application. However, when stored bacterial toxic secretion was applied to the insects its efficacy declined. On the other hand, when the same toxic secretion was dried and then dissolved either in broth or water was proved to be effective. The present study showed that the bacterium, X. nematophila or its toxic secretion can be used as an important component of integrated pest management against Galleria.

Larvicidal activity of Xenorhabdus and Photorhabdus bacteria against Aedes aegypti and Aedes albopictus

Objective: To evaluate the efficacy of symbiotic bacteria, Xenorhabdus indica, Xenorhabdus stockiae, Photorhabdus luminescens subsp. akhurstii and Photorhabdus luminescens subsp. hainanensis as a larvicide against Aedes aegypti and Aedes albopictus. Methods: Larvae(L3-L4) of Aedes aegypti and Aedes albopictus were given 2 m L of a suspension 107-108 CFU/m L of each symbiotic bacterium. Distilled water and Escherichia coli ATCC襅25922 were used as the control. The mortality rate of the larval mosquitoes was observed at 24, 48, 72 and 96 h. The experiment was performed in triplicates. Results: The larvae of both Aedes species started to die at 24 h exposure. Aedes aegypti showed the highest mortality rate(87%-99%), 96 h after exposure to Xenorhabdus stockiae(b NBP22.2_TH). The mortality rate of Aedes albopictus was between 82% and 96% at 96 h after exposure to Xenorhabdus indica(b KK26.2_TH). Low effectiveness of distilled water and Escherichia coli ATCC襅25922 were observed in both Aedes larvae, with a mortality rate of 2% to 12%. Conclusions: The study confirms the oral toxicity of Xenorhabdus and Photorhabdus bacteria against Aedes spp. Xenorhabdus stockiae and Xenorhabdus indica may be an alternative agent for control Aedes spp. This is basic information for further study on the mechanism of action on Aedes larvae or application to control mosquito larvae in the community.

Toxic activity of a protein complex purified from Xenorhabdus nematophila HB310 to Plutella xylostella larvae

Xenorhabdus nematophila, a Gram-negative proteobacterium belonging to the family Enterobacteriaceae and associated symbiotically with soil entomopathogenic nematodes, Steinernema carpocapsae, is pathogenic to a wide range of insects. A protein complex with insecticidal activity was isolated from the cells ofX. nematophila HB310 strain using methods of salting out and native polyacrylamide gel electrophoresis （PAGE）. Seven polypeptides ranging 50~250 kDa were well separated from the protein complex （named Xnpt） by sodium dodecyl sulfate （SDS）-PAGE, five of which are identified as XptA2, xptC 1, XptB 1, GroEL and hypothetical protein by matrix-assisted laser desorption- time-of-flight mass spectrometry （MALDI-TOFMS）. Xnpt showed high oral virulence to larvae of diamondback moth （DBM）, Plutella xylostella L. （Lepidoptera, Plutellidae） as its median lethal concentration （LC50） against second and third instar larvae were 331.45 ng/mL and 553.59 ng/mL at 72 h, respectively. The histological analysis of Xnptfed DBM larvae showed extensive histopathological effects on the midgut. Biochemical analysis indicated that Xnpt markedly inhibited the activities of three important enzymes in the midgut. Overall, our data showed that the protein complex isolated from X. nematophila HB310 induced the antifeedant and death of insects by destroying midgut tissues and inhibiting midgut proteases activities.

Acaricidal activities of whole cell suspension,cell-free supernatant,and crude cell extract of Xenorhabdus stokiae against mushroom mite (Luciaphorus sp.)

Xenorhabdus bacterium has been used as a biological control agent against Luciaphorus sp., a mushroom mite endemic in Thailand. To develop an effective formulation ofXenorhabdus stokiae, treatments using different parts of X. stokiae isolate PB09 culture, including whole cell suspension, cell-free supernatant, and crude cell extract, were performed. The results show that different parts ofX. stokiae isolate PB09 culture could induce variable effects on mite mortality and fecundity. Application with cell-free supernatant of X. stokiae culture resulted in both the highest mite mortality rate [（89.00＋3.60）%] and the lowest mite fecundity [（41.33_＋23.69） eggs/gravid female]. Whole cell suspen- sion of X. stokiae isolate PB09 culture was found to be slightly less effective than its cell-free supernatant, suggesting that X. stokiae was more likely to release its metabolites with acaricidal activities to the surrounding culture media. Crude cell extract of X. stokiae was not effective against mites. Cell-free supematant of X. stokiae isolate PB09 was the most effective biological control agent and it could be conveniently used in future formulations instead of live bacteria.

Three metabolites from an entomopathogenic bacterium, Xenorhabdus nematophila, inhibit larval development of Spodoptera exigua （Lepidoptera： Noctuidae） by inhibiting a digestive enzyme, phospholipase A2

An entomopathogenic bacterium, Xenorhabdus nematophila, has been known to induce significant immunosuppression of target insects by inhibiting immune-associated phospholipase A2 （PLA2）, which subsequently shuts down biosynthesis of eicosanoids that are critical in immune mediation in insects. Some metabolites originated from the bacterial culture broth have been identified and include benzylideneacetone, proline-tyrosine and acetylated phenylalanine-glycine-valine, which are known to inhibit enzyme activity of PLA2 extracted from hemocyte and fat body. This study tested their effects on digestive PLA2 of the beet armyworm, Spodoptera exigua. Young larvae fed different concentrations of the three metabolites resulted in significant adverse effects on larval development even at doses below 100 #g/mL. In particular, they induced significant reduction in digestive efficiency of ingested food. All three metabolites significantly inhibited catalytic activity of digestive PLA2 extracted from midgut lumen of the fifth instar larvae at a low micromolar range. These results suggest that the inhibitory activities of the three bacterial metabolites on digestive PLA2 of S. exigua midgut may explain some of their oral toxic effects.

Oral JS-38,a metabolite from Xenorhabdus sp.,has both anti-tumor activity and the ability to elevate peripheral neutrophils

AIM: JS-38(mitothiolore), a synthetic version of a metabolite isolated from Xenorhabdus sp., was evaluated for its anti-tumor and white blood cell(WBC) elevating activities. METHOD: These anti-proliferative activities were assessed in vitro using a panel of ten cell lines. The anti-tumor activities were tested in vivo using B16 allograft mouse models and xenograft models of A549 human lung carcinoma and QGY human hepatoma in nude mice. The anti-tumor interactions of JS-38 and cyclophosphamide(CTX) or 5-fluorouracil(5-Fu) were studied in a S180 sarcoma model in ICR mice. Specific stimulatory effects were determined on peripheral neutrophils in normal and CTX- and 5-Fu-induced neutropenic mice. RESULTS: The IC50 values ranged from 0.1 to 2.0 μmol·L-1. JS-38(1 μmol·L-1) caused an increase in A549 tumor cell apoptosis. Multi-daily gavage of JS-38(15, 30, and 60 mg?kg-1?d-1) inhibited in vivo tumor progression without a significant effect on body weight. JS-38 additively enhanced the in vivo anti-tumor effects of CTX or 5-Fu. JS-38 increased peripheral neutrophil counts and neutrophil rates in normal BALB/c mice almost as effectively as granulocyte colony-stimulating factor(G-CSF). In mice with neutropenia induced by CTX or 5-Fu, JS-38 rapidly restored neutrophil counts. CONCLUSION: These results suggest that JS-38 has anti-tumor activity, and also has the ability to increase peripheral blood neutrophils.

昆虫病原菌Xenorhabdus cabanillasii JM26产生的一种新型抗真菌代谢产物

革兰阴性致病杆菌Xenorhabdus属菌株可以与斯氏科线虫形成共生体。由此发现了一种由Xenorhabdus cabanillasii产生的新型抗真菌代谢产物cabanillasin。使用离子交换色谱和反相色谱纯化该化合物。经过同核和异核NMR以及MS．MS分析确定该分子的化学结构。Cabanillasin对可致机会性感染的酵母和丝状真菌具有抑菌活性。

昆虫病原线虫共生细菌胞内晶体蛋白的研究进展

初生型发光杆菌属(Photorhabdus)及嗜线虫致病杆菌属(Xenorhabdus)细菌分别与异小杆线虫属(Heterorhabditis)和斯氏线虫属(Steinernema)昆虫病原线虫互惠共生。这类昆虫病原细菌在稳定生长期分别产生两种形态各异的胞内晶体蛋白。本文回顾了这类蛋白的研究历史和最新的研究进展,特别是胞内晶体蛋白的理化性质和生物学功能,同时讨论这种晶体蛋白的研究方法与技术。

具角斯氏线虫共生菌的分子鉴定

在我国的云南和贵州省进行昆虫病原线虫(Entomopathogenic nematodes)资源调查时从不同地区采集到4个品系的具角斯氏线虫Steinernema ceratophorum,以16S rDNA部分序列为分子标记对从这4个品系线虫中分离到的4株共生菌进行了初步的鉴定。测序结果显示,4株菌的16S rDNA部分序列完全一样。基于该段序列所构建的系统发育树表明,具角斯氏线虫共生菌属异杆菌属Xenorhabdus,很可能是该属的一个新种。