丁香假单胞菌环式脂肽毒素的生理和分子生物学研究

综合评述了近１０年来在丁香假单胞菌脂肽毒素生理和分子生物学研究上的发现。这些毒素依肽部ＡＡ数目可分两组。丁香假单胞霉素组（ｓｙｒｉｎｇｏｍｙｃｕｎｓ）已报告４个成员,肽部有９个ＡＡ;丁香假单胞肽毒素组有２个成员,肽部分别有２２个和２５个ＡＡ。肽部Ｃ－端羧基与分子内羟基氨基酸残基（ＡＡ）的羟基酯化闭合成环,再由羟基脂肪酸酰化。两组毒素都诱导植物电解质渗漏、人和动物红血球溶解,其机制在于在细胞膜上形成     

假单胞菌合成环脂肽转录调控的研究进展

假单胞菌所合成的环脂肽是一类由环状的寡肽连接一个脂肪酸链组成的两亲性分子,利用巯基化模块由非核糖体肽合成酶合成。环脂肽的生物合成受到严格、复杂的调控,GacS/GacA双组分系统和群体感应系统是其中两类重要的调控系统。本文总结假单胞菌合成环脂肽的调控机制及相关调控因子;对基于PCR的高通量分子筛选方法获取特定环脂肽进行分析,同时对基于调控机制的遗传改造提高假单胞菌产环脂肽的能力和获取更多新型环脂肽等方面的应用进行 展望。     

丁香假单胞菌中的III型分泌系统及其调控机制研究进展

丁香假单胞菌(Pseudomonas syringae)是引起许多作物病害的一种革兰氏阴性病原细菌。该细菌入侵寄主植物细胞主要通过其III型分泌系统(type III secretion system,T3SS)将效应蛋白转入到寄主真核细胞内,抑制寄主免疫功能,以达到成功侵染和定殖的目的。III型分泌系统的主调控因子RhpR/S通过感受环境信号的变化直接调控hrpR/S及其他毒力相关通路。同时III型分泌系统基因的表达也受到其他调控因子的影响,包括σ因子HrpL、双组分系统GacA/S、Lon蛋白酶、第二信使分子和环境信号等。本文在简要介绍丁香假单胞菌III型分泌系统组成和功能的基础上,综述丁香假单胞菌III型分泌系统调控机制的最新研究进展,以期为深入探究病原菌的致病机制提供参考和思路。     

丁香假单胞菌冰核基因inaQ变速箱启动子结构与活性分析

序列比对与结构预测显示丁香假单胞菌(Pseudomonas syringae)野生型菌株MB03的冰核基因inaQ启动子为一种在细菌中罕见的变速箱型启动子。通过克隆长度为522bp的inaQ基因启动子区(P522)并与绿色荧光蛋白基因gfp构建融合基因P522gfp后,在恶臭假单胞菌AB92019菌株中进行表达分析。结果表明,包含结构模块A-Box和B-Box的P522在该菌株中具有启动子活性,且在寡营养条件和较低温度下具有更高的活性,是一种可调控启动子。     

沼泽红假单胞菌对微囊藻毒素的降解

以铜绿微囊藻为材料,通过固相萃取-高效液相色谱方法(SPE-HPLC),研究了沼泽红假单胞菌对微囊藻毒素MC-LR的降解作用。结果表明:沼泽红假单胞菌在厌氧、光照强度2000lx、35℃、pH7.0、乙酸钠为碳源、菌液初始浓度OD680为0.325和初始MC-LR为3mg.L-1时,6d降解率为36.5%,12d达到最高,降解率为78.7%。此降解条件和蓝藻水华爆发的环境条件基本一致,因此该菌株在水华爆发季节消除水中的微囊藻毒素方面具有应用潜力。     

生防绿针假单胞菌HT66中脂肽的鉴定与功能

【目的】脂肽(Lipopeptide,LP)是微生物合成的一类重要的生物表面活性剂,不仅影响细菌的生物学功能,还对多种植物和人类病原菌具有广谱的拮抗作用。然而至今未见绿针假单胞菌(Pseudomonas chlororaphis)中脂肽产物的报道。【方法】通过生物信息学手段预测绿针假单胞菌HT66中脂肽的氨基酸组成及顺序,构建脂肽合成基因缺失突变株HT66Δclp,根据突变株缺失代谢产物的UPLC/QTOF-MS信息验证预测结果,并研究了脂肽对该菌株的生长、吩嗪-1-甲酰胺(PCN)合成、生物膜形成和群集运动性的影响。【结果】预测菌株HT66的脂肽氨基酸顺序为L-Leu–D-Glu–D-allo-Thr–D-Val–L-Leu–D-Ser–L-Leu–D-Ser–L-Ile,通过比对野生型和突变株代谢产物的质谱信息确定该产物为黏液菌素(Viscosin);脂肽合成基因缺失后,菌株HT66的生长无明显变化,但其PCN合成、生物膜形成和群集运动性均有不同程度地下降。【结论】菌株HT66的脂肽产物为黏液菌素,对菌株的代谢、生物膜形成和运动性等生物学功能具有重要的调控作用。研究报道了绿针假单胞菌中一种脂肽分子的结构与功能,为研究其合成和调控机制及开发和应用奠定了基础。     

一株假单胞菌(Pseudomonas sp．)产生的胞外脂酶

假单胞菌(Psendomonas sp．)生长在一定的培养条件中能产生胞外脂酶。 最适碳源为1．0％淀粉,氮源为1．0％蛋白胨。一些植物油,如橄榄油、糠油、菜油等能诱导脂酶的大量产生,诱导脂酶产生的橄榄油最适浓度为0．5％。无机离子在菌培养过程中对脂酶产率影响很大,K+、Na+、Mg2+、Ca2+等对脂酶产生有促进作用,而Mn2+、Ba2+、Zn2+、Fe3+、Co2+、Cu2+件等则抑制脂酶产生。非离子表面活性剂(tween、span及糖脂)能刺激胞外脂酶的产生。     

松材线虫携带一株荧光假单胞菌分泌毒素的初步研究

本文研究了离体情况下松材线虫携带的致病菌一株荧光假单胞菌（Pseudomonas fluorescens GcM5-1A）在LB、NB和PD三种培养基中的毒性,以及产生的毒素对黑松（Pinus thunbergii）切根苗和悬浮细胞的效应。结果显示,菌体在LB和NB培养液的毒性较高,其中LB培养液的毒性最高,且培养液的pH值为7时比pH值为5时毒性高,而该菌EPD培养基中几乎不产毒。细菌培养液经硫酸铵分级沉淀,得到了主要含有50kDa蛋白的蛋白组分,该蛋白组分对黑松悬浮细胞和切根苗均有较高的毒性,并能改变黑松悬浮细胞细胞膜的透性,导致胞内可溶性糖和游离氨基酸外渗。     

松材线虫携带一株荧光假单胞菌分泌毒素的初步研究

本文研究了离体情况下松材线虫携带的致病菌一株荧光假单胞菌(Pseudomonas fluorescens GcM5-1A)在LB、NB和PD三种培养基中的毒性, 以及产生的毒素对黑松(Pinus thunbergii)切根苗和悬浮细胞的效应。结果显示, 菌体在LB和NB 培养液的毒性较高, 其中 LB培养液的毒性最高, 且培养液的pH值为7时比pH值为5时毒性高, 而该菌在PD培养基中几乎不产毒。细菌培养液经硫酸铵分级沉淀, 得到了主要含有50 kDa蛋白的蛋白组分, 该蛋白组分对黑松悬浮细胞和切根苗均有较高的毒性, 并能改变黑松悬浮细胞细胞膜的透性, 导致胞内可溶性糖和游离氨基酸外渗。     

Syringomicin production is stimulated by cysteine in Pseudomonas syringae pv. syringae

Abstract The influence of cysteine and serine in the production of syringomycin by Pseudomonas syringae pv. syringae has been studied. Both amino acids increased toxin synthesis in wild-type strains, although cysteine has a higher stimulatory effect than serine. To corroborate the role of cysteine in the production of syringomycin, a Cys⁻ mutant of P. syringae pv. syringae was isolated by transpositional mutagenesis with Tn5; this Cys⁻ mutant did not produce syringomycin. Nevertheless, and after the addition of high concentrations of cysteine, the cys ∷Tn5 mutant recovered its ability to produce syringomycin. On the other hand, the addition of serine did not return the production of syringomycin to the sys ∷ Tn5 strain: all these data indicated that cysteine modulates the synthesis of syringomycin in P. syringae pv. syringae positively.     

First Report of Bacterial Leaf Spot of Parsley Caused by Pseudomonas syringae pv. apii in Turkey

Since March, 2011, typical leaf spot symptoms were observed on parsley in several fields inspected in Hatay and Adana provinces of Turkey. Incidence of the disease was 5–15% in the regions. Symptoms were characterized as angular to irregular, initially water soaked later brown to dark black spots. Spots often limited by veins which were visible from both adaxial and abaxial sides of leaves but were not present on stems. Fluorescent bacterial colonies were consistently isolated from typical leaf spots. Biochemical tests, fatty acid methyl ester (FAME) analysis, molecular, pathogenicity tests and sequence of 16S ribosomal DNA of bacterial isolates were performed to identify possible causal disease agent. The causal disease agent was identified as Pseudomonas syringae pv. apii based on symptoms, biochemical, molecular, pathogenicity tests and sequencing. To our knowledge, this is the first report of bacterial leaf spot on parsley caused by Pseudomonas syringae pv. apii in Turkey.     

Yeast genes involved in growth inhibition by Pseudomonas syringae pv. syringae syringomycin family lipodepsipeptides

Abstract Saccharomyces cerevisiae genes encoding functions necessary for inhibition by the Pseudomonas syringae pv. syringae cyclic lipodepsipeptide, syringomycin-E, were identified by mutant analyses. Syringomycin-E-resistant mutants were isolated, shown to contain single recessive mutations, and divided into eight gene complementation groups. Representative strains from five groups were resistant to nystatin, and deficient in the plasma membrane lipid, ergosterol. All of the mutant strains were resistant to the related cyclic lipodepsipeptides, syringotoxin and syringostatin. The findings show that: 1) at least eight gene-encoded functions participate in the inhibitory response to syringomycin; 2) ergosterol is important for this response; 3) the three related lipodepsipeptides have similar modes of action.     

植物病原细菌Pseudomonas syringae pv. tomato基因组中的信号肽分析

应用SignalP 3.0 对植物病原细菌Pseudomonas syringae pv. tomato DC3000菌株基因组中的全部5 615个ORFs进行了分析,确定其中679个ORFs所编码蛋白质的N-端有信号肽序列,其中已经命名并有注释的有107个ORFs。信号肽的长度以19 ~31 个氨基酸居多,其中最多的是23 个氨基酸的信号肽。具有信号肽的ORFs编码蛋白的长度大多为101~400 个氨基酸之间。同时,对组成信号肽的氨基酸种类作了系统的分析,发现组成信号肽的氨基酸中非极性氨基酸占48.54%,极性氨基酸占18.67%,带负电荷氨基酸占24.54%,带正电荷氨基酸仅占8.00%,出现最多的3种氨基酸依次为亮氨酸、丙氨酸和丝氨酸,最少的氨基酸是异亮氨酸,在切割位点-1端的氨基酸中83.211%均为丙氨酸,在切割位点后3位的氨基酸中最多的氨基酸也是丙氨酸。通过分析确定628个分泌类信号肽,36个信号肽具有RR-motif的保守区段,15个脂蛋白类信号肽,未发现Prepilin-like 信号肽和Bacteriocin and Pheromone信号肽。     

Host specificity,pathogenicity and the presence of virulence genes in Iranian strains of Pseudomonas syringae pv. syringae from different hosts

Pseudomonas syringae pv. syringae (Pss) strains were isolated from almond, apricot, peach, pear, sweet cheery and wheat in Kohgiluye and Boyer-Ahmad, Kordestan, Fras and Chaharmahal and Bakhtiari provinces of Iran. The strains were examined for host specificity, the presence of virulence genes and pathogenicity on different hosts. After inoculation of isolates, in compatible reactions bacterial populations increased within six days of inoculation and final cell numbers increased several-fold over initial inoculum levels, but in incompatible reactions, bacterial populations declined within four days of inoculation. Almond, sweet cherry and wheat isolates induced progressive necrotic symptoms on almond leaves and stems. Apricot, peach and sweet cherry isolates induced necrotic lesions when inoculated on apricot leaves. On pear leaves and stems, only the pear isolate incited pathogenic reaction and isolates from other hosts did not. The syrB gene was detected in all of the tested isolates. Almond and pear isolates did not have the syrD gene. The sypA gene was detected in the almond, peach, pear and sweet cherry isolates while the sypB gene was detected in the apricot, peach, sweet cherry and wheat isolates. Almond, apricot, pear and wheat isolates gave negative results for the detection of nit gene. The gene Ach, was detected only in the peach isolate and gene hrmA, was detected only in the wheat isolate. This study indicates that host specificity exists among different Pss strains, and genes responsible for syringomycin and syringopeptin production contribute to the virulence of Pss strains.     

大豆细菌性斑点病菌harpin编码基因的克隆与表达

摘要：【方法、目的】利用PCR方法从丁香假单胞菌大豆致病变种（Pseudomonas syringae pv. glycinea）Psg12菌株中克隆到1026bp的hrp基因。将其定向插入到表达载体pGEX-4T-1上,并转化宿主菌BL21,IPTG诱导表达后,SDS-PAGE显示其表达产物为分子量为61 kDa的融合蛋白质。【结果】该蛋白质在性质与功能上类似于已发现的harpins,即富含甘氨酸、不含半胱氨酸,热稳定以及对蛋白酶K敏感,能够在烟草上引起典型的过敏性反应,过敏性反应还可被真核生物代谢抑制     

Efficiency of procedures for induction and cultivation of Pseudomonas syringae pv. pisi L-form

The L-form of Pseudomonas syringae pv. phaseolicola has been proved to induce resistance to bean halo blight.Various procedures were tested to induce the L-form of Pseudomonas syringae pv. pisi for its potential use as biocontrol agent of pea bacterial blight. Cell-wall deficient cells were induced in a liquid medium with penicillin following a protocol described for P. s. pv. phaseolicola. Cell growth on solid induction medium developed as typical granular and vacuolated structures, and characteristic colonies were observed in the first transfer. However, there was poor growth in subsequent transfers and some reversion to the parental type. To improve the induction procedure, the following new procedures were applied: (1) viability of cells was monitored during induction. The optimum induction time in liquid medium with penicillin was lower for pv. pisi than for pv. phaseolicola. Viability of L-forms in solid induction medium with penicillin was low and decreased in time. (2) the inducer ticarcillin was combined with clavulanic acid, which prevented the reversion to the parental type and (3) a range of concentrations of penicillin and ticarcillin/clavulanic acid was applied by the spiral gradient endpoint method for calculation of minimum inhibitory concentrations (MIC). Based on the results from these tests an induction method for P. s. pv. pisi L-form is proposed and the relevance of L-form is discussed for practice.     

Transgenic tobacco cultivars resistant to Pseudomonas syringae pv. tabaci

 Six oriental cultivars of tobacco (Nicotiana tabacum L.) were evaluated for transformation and foreign gene expression. Leaf-disc explant tissue was transformed with Agrobacterium tumefaciens strain LBA4404 carrying the plasmid pARK21, which contains NPTII gene and ttr (tabtoxin resistance) gene conferring the resistance to Pseudomonas syringae pv. tabaci. The disease resistance of regenerated plants and segregation of this trait up to R₇ progeny were investigated in a greenhouse and under field conditions. Our results indicated that the resistance to Pseudomonas syringae pv. tabaci introduced by transformation is heritable. Received: 10 June 1997 / Accepted: 31 March 1998     

丁香假单胞大豆致病变种两个III型效应因子的克隆及功能分析

为了研究Ⅲ型泌出效应因子在丁香假单胞大豆致病变种中的作用,利用反向PCR技术,首次从丁香假单胞大豆致病变种全基因组中克隆得到两个效应因子HopAB1和HopAF1基因的同源物,分别命名为HopAB1s和HopAF1s。生物信息学分析表明,HopAB1s基因全长是1 572 bp,编码523个氨基酸;HopAF1s基因全长是855 bp,编码284个氨基酸。即基因的登录号分别为JF826562和JF826563。保守功能区预测显示HopAB1s在N末端包含一个E3泛素连接酶功能区。将这2个基因克隆到PVX二元表达载体并转化农杆菌,利用农杆菌介导的瞬时侵染技术在本生烟中表达,发现2个效应因子均能抑制由鼠凋亡因子激发的细胞程序性死亡;将烟草疫霉接种在表达效应基因的区域,发现效应因子能促进烟草疫霉侵染烟草,因此本研究得到的两个效应因子是免疫抑制因子,为进一步研究该菌的致病机理奠定基础。     

Comparative genomics-guided loop-mediated isothermal amplification for characterization of Pseudomonas syringae pv. phaseolicola

Aims:  To design and evaluate a loop-mediated isothermal amplification (LAMP) protocol by combining comparative genomics and bioinformatics for characterization of Pseudomonas syringae pv. phaseolicola (PSP), the causal agent of halo blight disease of bean ( Phaseolus vulgaris L.).
Methods and Results:  Genomic sequences of Pseudomonas syringae pathovars, P. fluorescens and P. aeruginosa were analysed using multiple sequence alignment. A pathovar-specific region encoding pathogenicity-related secondary metabolites in the PSP genome was targeted for developing a LAMP assay. The final assay targeted a polyketide synthase gene, and readily differentiated PSP strains from other Pseudomonas syringae pathovars and other Pseudomonas species, as well as other plant pathogenic bacteria, e.g. species of Pectobacterium , Erwinia and Pantoea .
Conclusion:  A LAMP assay has been developed for rapid and specific characterization and identification of PSP from other pathovars of P. syringae and other plant-associated bacteria .
Significance and Impact of the Study:  This paper describes an approach combining a bioinformatic data mining strategy and comparative genomics with the LAMP technology for characterization and identification of a plant pathogenic bacterium. The LAMP assay could serve as a rapid protocol for microbial identification and detection with significant applications in agriculture and environmental sciences.