十字花科黑腐病菌分泌蛋白质双向电泳条件的构建及优化

本研究旨在建立一套适合于十字花科黑腐病菌(Xanthomonas campestris pv.campestris 8004,Xcc8004)分泌蛋白质的双向电泳技术(two-dimensional electrophoresis,2-DE),以便更好的利用蛋白质组学技术鉴定Xcc 8004的分泌蛋白质。我们就MME和XCM2两种培养基对分泌蛋白质的诱导能力、蛋白质提取方法以及样品上样量等方面对2-DE的影响进行了比较,结果表明,XCM2培养基对Xcc 8004分泌蛋白质的诱导能力比MME强。用TCA/丙酮法沉淀蛋白质得到的双向电泳图谱背景清晰,分辨率高。分别采用400μg、300μg、250μg和150μg四个不同的上样量进行双向电泳,结果显示在上样量为250~300μg时(IPG p H 3-10,24 cm),电泳图谱分辨率最好。综上可知,XCM2培养基比较适合用于Xcc 8004分泌蛋白质的诱导,TCA/丙酮法提取分泌蛋白质为较优方法,250~300μg是较为合适的上样量。     

野油菜黄单胞菌野油菜致病变种8004菌株wxcA基因与EPS的产量有关

在以前的工作中,采用转座子Tn5 gusA5对野油菜黄单胞菌野油菜致病变种(Xcc)8004菌株进行诱变,获得一批胞外多糖(EPS)合成减少的突变体,对这些突变体的Tn5 gusA5的插入位点进行分析后,发现有两株突变体是wxcA基因不同插入位点的突变体。以前认为wxcA基因与脂多糖(LPS)的O-抗原合成有关而与EPS的合成无关。为明确wxc4基因的功能,对8004菌株的wxcA基因进行缺失,获得的△wxcA突变体的EPS产量与野生型菌株相比,减少了50％,并且一段PCR合成的包含wxcA基因的DNA片段能反式互补△wxcA突变体,恢复突变体的EPS产量。这证实了8004菌株的wxcA基因与EPS的合成产量有关。     

十字花科黑腐病菌dsbA基因控制的亚细胞蛋白质组分析

本研究以十字花科黑腐病菌(Xarcthomorcas campestris pv.campestris,Xcc)8004菌株为研究对象,采用双向电泳—质谱技术分析比较了dsbA1A2突变体和野生型菌株的分泌蛋白、周质蛋白表达谱。与野生型菌株相比,选取的31个差异蛋白中,14个蛋白点显著上调,17个蛋白点显著下调。对这些蛋白质点进行质谱鉴定和分析,结果显示,dsbA1A2基因的缺失导致了周质蛋白中与β桶结构膜蛋白形成有关的伴侣蛋白SurA的含量降低,几种未知功能的分泌蛋白的分泌量减少以及外膜蛋白OmpW、OmpW1在周质空间的积累。推测这些蛋白质对Xcc 8004的致病性至关重要,为进一步研究Xcc 8004中DSB系统的作用机理提供了依据。     

野油菜黄单胞菌6-磷酸果糖激酶基因的初步分析

6-磷酸果糖激酶是糖酵解途径中的关键酶,它催化糖酵解途径中第一个不可逆反应。本研究利用pK18mobsacB自杀质粒采用同源双交换的方法对野油菜黄单胞菌Xcc8004中的6-磷酸果糖激酶基因(XC_0872)进行缺失突变,获得无标记的缺失突变体DM0872。表型检测结果显示DM0872突变体不影响野油菜黄单胞菌对葡萄糖和果糖的利用,不影响胞外多糖的合成,也不影响其致病性。该结果显示糖酵解途径在野油菜黄单胞菌的地位并不重要。另外,我们利用RT-PCR方法检测了XC_0872的转录情况,结果显示XC_0872在Xcc8004中是转录的。而之前曾有报道称黄单胞菌中无法检测出6-磷酸果糖激酶活性,这表明XC_0872进行了转录后调控从而使6-磷酸果糖激酶活性受到限制。本研究为野油菜黄单胞菌中糖酵解途径的调控提供了理论依据,对揭示野油菜黄单胞菌中该途径的调控机制具有一定的意义。     

十字花科黑腐病菌中一个与趋化性相关基因的突变分析

十字花科作物黑腐病,又称为野油菜黄单胞菌野油菜变种(Xanthomonas campestris pv.campestris,简称Xcc),该细菌是引起十字花科作物等植物发生黑腐病的病原菌,同时该细菌也是人们研究寄主与病原微生物相互作用的具体分子机理的模式菌之一。在Xcc 8004菌株基因组中,XC_2304编码的产物为一个趋化性蛋白。由于细菌的趋化性在病原学方面的意义是非常重要的,为评估XC_2304的功能,本研究利用p K18mob Sac B对XC_2304进行缺失突变,获得缺失突变体DM2304。植株试验发现,突变体DM2304对寄主植物的致病力下降约30%,其互补菌株CDM2304的致病力基本恢复至野生型水平,这表明XC_2304与Xcc致病力有关。利用毛细管法检测DM2304对18种物质的趋化性,结果表明突变体对木糖、苯丙氨酸、精氨酸、蔗糖以及核糖的趋化性比野生型弱。运动性分析发现,DM2304在含有0.3%、0.6%琼脂的NYGA板的游动性稍微降低,表明XC_2304与游动性有关。而DM2304的胞外纤维素酶、胞外蛋白酶、胞外淀粉酶、EPS产量、HR与野生型菌株相比均没有明显差异。本研究为十字花科黑腐病菌中其它与趋化性相关基因提供实验思路,对病原菌如何趋利避害的机制的研究具有一定的意义。     

野油菜黄单胞菌野油菜致病变种8004菌株wxcA基因与EPS的产量有关

在以前的工作中,采用转座子Tn5gusA5对野油菜黄单胞菌野油菜致病变种(Xcc) 8004菌株进行诱变,获得一批胞外多糖(EPS)合成减少的突变体,对这些突变体的Tn5gusA5的插入位点进行分析后,发现有两株突变体是wxcA基因不同插入位点的突变体。以前认为wxcA基因与脂多糖(LPS)的O抗原合成有关而与EPS的合成无关。为明确wxcA基因的功能,对8004菌株的wxcA基因进行缺失,获得的ΔwxcA突变体的EPS产量与野生型菌株相比,减少了50%,并且一段PCR合成的包含wxcA基因的DNA片段能反式互补ΔwxcA突变体,恢复突变体的EPS产量。这证实了8004菌株的wxcA基因与EPS的合成产量有关。     

十字花科黑腐病菌中GGDEF结构域蛋白差异的in silico分析

近年来,含有GGDEF结构域(含有甘氨酸(G) (2个),天冬氨酸(D),谷氨酸(E),苯丙氨酸(F)保守氨基酸)的蛋白受到重视,已证实含GGDEF结构域蛋白在细胞信号转导、生长和致病性等方面发挥了重要作用.十字花科黑腐菌8004菌株(Xanthomonas campestris pv.campestris str.8004,Xcc 8004)有32个基因编码含GGDEF结构域蛋白,实验证明其中部分蛋白与Xcc致病性、胞外酶产生、生物膜形成和泳动等生命活动相关.本文利用互联网提供的生物信息学资源,对Xcc 8004不同功能含GGDEF结构域蛋白进行生物信息学分析,着重分析其结构域架构.对蛋白结构域架构整体比较显示,这些蛋白的整体结构域架构具有多样性,共有结构域架构仅有PAS_4-GGDEF-EAL (分布于参与致病的蛋白中);对结构域架构局部比较显示,在参与致病性的含GGDEF结构域蛋白中,PAS_4-GGDEF和GGDEF-EAL为共有结构域架构;在参与内切葡聚糖酶产生的蛋白中,PAS_4-PAS_4、PAS_4-GGDEF和GGDEF-EAL为共有结构域架构.本研究结果将为蛋白质功能预测提供线索.     

十字花科黑腐病菌一个新的III型效应物XC3176的鉴定

摘要:【目的】在十字花科黑腐病菌(Xanthomonas campestris pv． campestris,Xcc)的致病因子中,III型分泌系统(Type III Secretion System,T3SS)是至关重要的致病系统,III型效应物通过III 型分泌系统直接转运到寄主植物细胞内。本研究通过效应物水平转移的特征获得候选基因,旨在鉴定一个新的依赖于III型分泌的效 应物。【方法】以缺失了N-端58个氨基酸的AvrBs1作为报告系统构建效应物鉴定报告质粒pLJB3176,导入ΔavrBs1和ΔhrcV,通过检测报告菌株在辣椒ECW-10R上的过敏反应来鉴定XC3176是否为III型效应物。构建XC3176融合GUS报告质粒pLGUS3176,导入野生菌株Xcc 8004、ΔhrpG和ΔhrpX,通过测定菌株GUS活性检测hrpG、hrpX对XC3176 的调控作用。构建XC3176缺失突变体和互补菌株,通过剪叶法接种检测XC3176对Xcc 8004致病性的影响。【结果】XC3176融合AvrBs1报告菌株在非寄主辣椒ECW-10R上能 引发过敏反应,GUS活性检测显示ΔhrpG/pLGUS3176、ΔhrpX/pLGUS3176比8004/pLGUS3176的GUS酶活显著降低,致病性检测显示突变体Δ3176与野生型Xcc 8004相比在寄主满身红萝卜上的病斑长度有显著减少,互补菌株C3176 的病斑长度能补回到野生型水平。【结论】XC3176是依赖于hrcV分泌的III型效应物,hrpG、hrpX正调控XC3176,XC3176与Xcc致病相关。     

胆管癌胆汁蛋白质样品制备和双向电泳条件的优化

采用合适的裂解液和沉淀方法,提取胆管癌胆汁中的蛋白质,获得分辨率高、重复性好的蛋白质双向电泳图谱.通过不同裂解液和蛋白质沉淀方法提取胆汁蛋白的效果比较,设计了不同的样品制备方法,并且对双向电泳(2-DE)的条件进行优化.结果显示,试验确定了适合胆管癌胆汁的裂解液配方(LSⅣ),丙酮沉淀的蛋白分布完整,沉淀效率相对较高.高伏时、长时间的等电聚焦可以获得高分辨率、重复性好的蛋白质双向电泳图谱.因此,本方法可以应用到胆管癌胆汁蛋白的提取,也可以对其他体液蛋白质样品的制备和双向电泳提供借鉴.     

野油莱黄单胞菌两个γPON基因的突变和功能分析

广西大学生命科学与技术学院李恒聪、蒋瑞萍、姜伯乐与唐纪良(广西亚热带生物资源保护利用重点实验室)等5位科研工作者看到细菌的σ54(γPON)是一类可选择性识别启动子序列的σ因子,参与环境适应、细胞生理过程等,如氮代谢、鞭毛和菌毛的生物合成,在一些病原菌中,σ54还与致病性密切有关。为了明确σ54在野油菜黄单胞菌野油菜病变种(Xcc)中是否参与致病过程,用同源单交换定点突变方法,将Xcc 8004中的两个σ54编码基因γPON1(XC1256)和γPON2(XC2168)做单突变及双突变。其突变体表型分析结果表明:单个和同时突     

DsbB is required for the pathogenesis process of Xanthomonas campestris pv. campestris

The DsbA/DsbB oxidation pathway is one of the two pathways that catalyze disulfide bond formation of proteins in the periplasm of gram-negative bacteria. It has been demonstrated that DsbA is essential for multiple virulence factors of several animal bacterial pathogens. In this article, we present genetic evidence to show that the open reading frame XC_3314 encodes a DsbB protein that is involved in disulfide bond formation in periplasm of Xanthomonas campestris pv. campestris, the causative agent of crucifer black rot disease. The dsbB mutant of X. campestris pv. campestris exhibited attenuation in virulence, hypersensitive response, cell motility, and bacterial growth in planta. Furthermore, mutation in the dsbB gene resulted in ineffective type II and type III secretion systems as well as flagellar assembly. These findings reveal that DsbB is required for the pathogenesis process of X. campestris pv. campestris.     

Evaluation of rhizospheric Pseudomonas and Bacillus as biocontrol tool for Xanthomonas campestris pv campestris

Xanthomonas campestris pv campestris (Xcc), causing black rot, is one of the most yield-limiting and destructive pathogens of cruciferous crops. The intention of this study was to evaluate the potential of rhizobacteria in black rot management. Fifty-four isolates from rhizosphere soil of Brassica campestris were screened against Xcc. Two isolates namely, KA19 and SE, with inhibition radius >11 mm were selected. The combined use of them produced an average inhibition zone of 18.1 ± 1.4 mm radius (P < 0.05). 16S rRNA gene sequencing and phylogenetic analysis identified KA19 and SE as the nearest homologs (>99.4%) of Pseudomonas aeruginosa and Bacillus thuringiensis, respectively. In greenhouse study, both isolates were effective (P < 0.05) in reducing black rot lesions compared to untreated control involving either a foliar spray or the combined seed soak and soil drench. However, the combined strains (KA19 + SE) were significantly more effective (P < 0.05) when the mode of application was combined seed and soil drench. The lipid content of seeds increased significantly with the application of these strains, especially with SE alone and in combination. After 9 weeks, the Xcc population was significantly lower in soil treated with combined strains (P < 0.05). KA19 produced extracellular siderophores, influenced by various carbon sources and identified as 4-hydroxy-2-nonyl-quinoline by NMR. In Bacillus SE, two antibacterial factors corresponding to autolysins (β-N-acetylglucosaminidase) and AHL-lactonases were established. This study would strengthen our understanding for application of different rhizobacteria with various active principles like Pseudomonas and Bacillus as ingredients of a biocontrol mixture.     

Isolation and characterization of a generalized transducing phage for Xanthomonas campestris pv. campestris.

We have isolated and characterized a lytic double-stranded DNA Xanthomonas campestris pv. campestris bacteriophage (XTP1) capable of mediating generalized transduction. The phage transduces chromosomal markers at frequencies of 10(-5) to 10(-6) transductants per PFU. We demonstrated its genetic utility by the isolation and cotransduction of linked transposon insertions to a nonselectable locus, xgl, required for the cleavage of 5-bromo-3-chloro-indoyl-beta-D-galactoside and showed that rif and str alleles in X. campestris are 75% linked. One-step growth experiments showed that the latent and rise periods were each 2 h and the average burst size was 35. The DNA genome is approximately 180 kb, presumably modified in a sequence-specific manner, and may be covalently attached to protein(s). Electron micrographs show the phage particle to have an icosahedral head and contractile tail with tail fibers uniquely attached to a location 40 nm proximal from the end of the tail.     

Transformation of Xanthomonas campestris pathovar campestris with plasmid DNA

Procedures for the introduction of plasmid DNA into Gram-negative bacteria have been adapted and optimized to permit transformation of the plant pathogen Xanthomonas campestris pathovar campestris with the cloning vector pKT230 and other broad-host-range plasmids. The technique involves CaCl2-induced competence and heat shock and is similar to that routinely used for Escherichia coli. Wild-type X. c. campestris strains appear to restrict incoming unmodified DNA, so that plasmid DNA for transformation must be prepared from X. c. campestris (into which it has previously been introduced by conjugation). To overcome this disadvantage a restriction-deficient mutant has been isolated.     

Efficacy of four semi-selective media for recovery of Xanthomonas campestris pv. campestris from tropical soils

R. FUKUI, R. ARIAS AND R. ALVAREZ. 1994. Four semi-selective media, CS20 ABN, aesculin—trehalose (ET), Fieldhouse—Sasser (FS), and starch—methionine (SM), were compared for efficacy in recovering Xanthomonas campestris pv. campestris from artificially and naturally infested soils. Recoveries of X. c. campestris from soils infested with relatively large populations were similar on the four media. The FS and ET media exhibited higher selectivity against background saprophytes, whereas enumeration of X. c. campestris on CS20 ABN or SM was often hampered by the overgrowth of background saprophytes. Among three starch-containing media (CS20 ABN, ET and FS media), the zones of starch hydrolysis, characteristic of colonies of X. c. campestris, were most distinctive for FS medium. This allowed easier identification of the target colonies among numerous non-target colonies in tests with soil containing smaller numbers of X. c. campestris. Although the starch zone was also distinctive on CS20 ABN, this medium was not as effective as FS because the starch zones were so large that neighbouring zones fused with each other and many saprophytes formed colonies within the zones. Overall, FS was most suitable for soil studies in terms of the consistent recovery of the pathogen, the selectivity against saprophytes, and the differentiation from non-target organisms.     

The bifunctional effector AvrXccC of Xanthomonas campestris pv. campestris requires plasma membrane-anchoring for host recognition

Bacterial pathogens use type III secretion systems (TTSS) to deliver effector proteins into eukaryotic cells for pathogenesis. In bacterial–plant interactions, one effector may function as an avirulence factor to betray the pathogen to the plant surveillance system and induce the hypersensitive response (HR) in the resistant host carrying a corresponding resistance ( R ) gene. However, the same effector can also sustain the growth of the pathogen by acting as a virulence factor to modulate plant physiology in the susceptible host lacking the corresponding R gene. Here, we identified and characterized a bifunctional TTSS effector AvrXccC belonging to the AvrB effector family in Xanthomonas campestris pv. campestris 8004. This effector is required for full bacterial virulence in the susceptible host cabbage ( Brassica oleracea ) and avirulence in the resistant host mustard ( Brassica napiformis L.H. Baily). Expressing avrXccC in mustard-virulent strain Xcc HRI 3849A converts its virulence to avirulence. The effector AvrXccC is anchored to the plant plasma membrane, and the N-terminal myristoylation site (amino acids 2–7: GLcaSK) is essential for its localization. In addition, the avirulence function of AvrXccC for host recognition depends on its plasma membrane localization. Promoter activity assays showed that the expression of avrXccC is hrpG/hrpX -dependent. Moreover, the secretion of AvrXccC displayed hrp -dependency and the core sequence for AvrXccC translocation was defined to the N-terminal 40 amino acids.     

Requirement of a mip-like gene for virulence in the phytopathogenic bacterium Xanthomonas campestris pv. campestris

Macrophage infectivity potentiators (Mips) are FKBP domain-containing proteins reported as virulence factors in several human pathogens, such as members of genera Legionella, Salmonella and Chlamydia. The putative peptidylprolyl cis-trans isomerase (PPIase) encoded by XC2699 of the plant bacterial pathogen Xanthomonas campestris pv. campestris 8004 exhibits a 49% similarity at the amino-acid level to the Mip protein of Legionella pneumophila. This mip-like gene, XC2699, was overexpressed in Escherichia coli and the purified (His)6-tagged Mip-like protein encoded by XC2699 exhibited a PPIase activity specifically inhibited by FK-506. A mutation in the mip-like gene XC2699 led to significant reductions in virulence and replication capacity in the host plant Chinese radish (Raphanus sativus L. var. radiculus Pers.). Furthermore, the production of exopolysaccharide and the activity of extracellular proteases, virulence factors of X. campestris pv. campestris, were significantly decreased in the mip-like mutant. These results reveal that the mip-like gene is involved in the pathogenesis of X. campestris pv. campestris through an effect on the production of these virulence factors.     

Xanthomonas campestris pv. campestris possesses a single gluconeogenic pathway that is required for virulence

Disruption of ppsA, a key gene in gluconeogenesis, of Xanthomonas campestris pv. campestris resulted in the failure of the pathogen to grow in medium with pyruvate or C4-dicarboxylates as the sole carbon source and a significant reduction in virulence, indicating that X. campestris pv. campestris possesses only the malic enzyme-PpsA route in gluconeogenesis, which is required for virulence.     

The zinc uptake regulator Zur is essential for the full virulence of Xanthomonas campestris pv. campestris

Zur is a regulator of the high-affinity zinc uptake system in many bacteria. In Xanthomonas campestris pv. campestris 8004, a putative protein encoded by the open reading frame designated as XC1430 shows 42% amino acid similarity with the Zur of Escherichia coli. An XC1430-disrupted mutant 1430nk was constructed by homologous suicide plasmid integration. 1430nk failed to grow in rich medium supplemented with Zn2+ at a concentration of 400 microM and in nonrich medium supplemented with Zn2+ at a concentration of 110 microM, whereas the wild-type strain grew well in the same conditions. In rich medium with 400 microM Zn2+, 1430nk accumulated significantly more Zn2+ than the wild-type strain. 1430nk showed a reduction in virulence on the host plant Chinese radish (Raphanus sativus L. var. radiculus Pers.) and produced less extracellular polysaccharide (EPS) than did the wild-type strain in the absence of added zinc. These results revealed that XC1430 is a functional member of the Zur regulator family that controls zinc homeostasis, EPS production, and virulence in X. campestris pv. campestris.