野油菜黄单胞菌中3-羟基脂酰ACP脱水酶Fab Z的生理功能

【背景】野油菜黄单胞菌(Xanthomonas campestris pv. campestris, Xcc)引起十字花科植物黑腐病,在全球范围内造成经济损失,亟须深入研究其致病机理,开发新的黑腐病防控措施。细菌脂肪酸合成系统不仅为细胞膜合成提供原料,其中间代谢产物还是许多生物活性分子合成的底物,具有重要的生理功能,也是抗菌药物筛选的重要靶标。【目的】研究XccfabZ对扩散信号分子(diffusible signal factor, DSF)类信号产量、致病力、胞外酶、胞外多糖和运动性等方面的影响。【方法】利用报告菌株检测法分析了不同替换突变株的DSF类群体感应信号产量。利用同源重组原理,在DSF类信号高产菌株中获得替换突变株,利用高效液相色谱(highperformanceliquid chromatography, HPLC)法测定DSF类信号产量。利用剪叶法检测替换突变株对寄主植物甘蓝的致病力,并分析了不同菌株的胞外多糖、胞外酶和运动性差异。【结果】报告菌株检测法和HPLC法都证明大肠杆菌fabZ替换突变株(XccΔfabZ/pSRK-EcfabZ)中DSF类信号产量显著下降。...     

Pan Proteome of Xanthomonas campestris pv. campestris Isolates Contrasting in Virulence

Fully sequenced genomes of Xanthomonas campestris pv. campestris (Xcc) strains are reported. However, intra‐pathovar differences are still intriguing and far from clear. In this work, the contrasting virulence between two isolates of Xcc ‐ Xcc51 (more virulent) and XccY21 (less virulent) is evaluated by determining their pan proteome profiles. The bacteria are grown in NYG and XVM1 (optimal for induction of hrp regulon) broths and collected at the max‐exponential growth phase. Shotgun proteomics reveals a total of 329 proteins when Xcc isolates are grown in XVM1. A comparison of both profiles reveals 47 proteins with significant abundance fluctuations, out of which, 39 show an increased abundance in Xcc51 and are mainly involved in virulence/adaptation mechanisms, genetic information processing, and membrane receptor/iron transport systems, such as BfeA, BtuB, Cap, Clp, Dcp, FyuA, GroEs, HpaG, Tig, and OmpP6. Several differential proteins are further analyzed by qRT‐PCR, which reveals a similar expression pattern to the protein abundance. The data shed light on the complex Xcc pathogenicity mechanisms and point out a set of proteins related to the higher virulence of Xcc51. This information is essential for the development of more efficient strategies aiming at the control of black rot disease.     

The role of PilZ domain proteins in the virulence of Xanthomonas campestris pv. campestris

Cyclic di‐GMP [(bis‐(3′–5′)‐cyclic di‐guanosine monophosphate)] is an almost ubiquitous second messenger in bacteria that is implicated in the regulation of a range of functions that include developmental transitions, aggregative behaviour, adhesion, biofilm formation and virulence. Comparatively little is known about the mechanism(s) by which cyclic di‐GMP exerts these various regulatory effects. PilZ has been identified as a cyclic di‐GMP binding protein domain; proteins with this domain are involved in regulation of specific cellular processes, including the virulence of animal pathogens. Here we have examined the role of PilZ domain proteins in virulence and the regulation of virulence factor synthesis in Xanthomonas campestris pv. campestris (Xcc), the causal agent of black rot of crucifers. The Xcc genome encodes four proteins (XC0965, XC2249, XC2317 and XC3221) that have a PilZ domain. Mutation of XC0965, XC2249 and XC3221 led to a significant reduction of virulence in Chinese radish. Mutation of XC2249 and XC3221 led to a reduction in motility whereas mutation of XC2249 and XC0965 affected extracellular enzyme production. All mutant strains were unaffected in biofilm formation in vitro. The reduction of virulence following mutation of XC3221 could not be wholly attributed to an effect on motility as mutation of pilA, which abolishes motility, has a lesser effect on virulence.     

大肠杆菌3-酮基脂酰ACP还原酶110位天冬酰胺突变后的结构与功能

3-酮基脂酰ACP还原酶催化3-酮基脂酰ACP还原为3-羟基脂酰ACP,是细菌脂肪酸合成反应的关键酶之一.为了明确该酶中110位的保守天冬酰胺残基在酶催化活性和酶结构中的作用,本研究采用基因定点突变和蛋白质表达纯化技术,获得了大肠杆菌3-酮基脂酰ACP还原酶FabG的两个突变蛋白：FabG N110Q和FabG N110L.圆二色谱结果显示,天冬酰胺残基的突变改变了FabG的空间结构,使突变蛋白的α螺旋结构明显增加.以3-酮脂酰ACP为底物的酶活性测定表明,突变蛋白的酶活性均有下降,但残存的酶活性达到了FabG的75%以上.突变蛋白FabG N110Q和FabG N110L具有3-酮基脂酰ACP还原酶的活性,能在体外重建细菌脂肪酸合成反应.对fabG温度敏感突变株的遗传互补分析表明,FabG蛋白110位天冬酰胺突变为谷氨酰胺或亮氨酸后,在一定的条件下仍能互补大肠杆菌的生长.本研究结果提示,FabG 110位的天冬酰胺残基不是参与3-酮基脂酰ACP还原酶催化反应的必需氨基酸,它只是作为结构氨基酸,在维持FabG的空间结构的稳定性方面起作用.     

Virulence analysis and genetic diversity of Xanthomonas campestris pv. campestris causing black rot of crucifers

Xanthomonas campestris pv. campestris (Xcc) is a devastating bacterium to cause black rot disease in crucifers. To study the genetic diversity and virulence analysis, 24 isolates of Xcc were collected from cole crops including cauliflower, cabbage, broccoli and knol khol from different agro-climatic regions of India ranging from temperate to subtropical climates. For virulence analysis, 24 isolates of Xcc were tested on 27 cultivars of crucifers including seven species of Brassica spp. (B. campestris, B. carinata, B. juncea, B. napus, B. nigra, B. oleracea and B. rapa), Sinapsis alba, Eruca sativa and Raphanus sativus under field conditions at IARI, New Delhi, during November 2010–March 2011. Maximum disease incidence 85.15% was found in the cultivars of crucifers caused by strains Xcc-C124, Xcc-C6, Xcc-C125, Xcc-C111 and Xcc-C131 after 15 days of inoculation and significantly increased after 30 days. Black rot severity in cultivars of crucifers varied from 0 to 6.9 and 0 to 7.9 out of 9 scale after 15 and 30 days of inoculation, respectively. But, no disease incidence was recorded on all the tested cultivars of B. juncea (Pusa Bold, Varuna, Pusa Mustard-21 and Pusa Vijay) against all the strains of Xcc after 15 days. Genetic diversity of 24 strains of Xcc was studied using REP- and BOX-PCR, indicating the existence of wide range of genetic diversity among the strains. The strains were clustered into two groups at 50% similarity coefficient and designated as Group 1 and Group 2. The majority of the strains (23 strains) were clustered under Group 1 except Xcc-C120, which formed separate group (Group 2). In the present study, genetic diversity and virulence pattern in Indian strains of Xcc were established, which will be helpful in the development of resistant genotypes against this bacterial pathogen.     

The type III effector AvrXccB in Xanthomonas campestris pv. campestris targets putative methyltransferases and suppresses innate immunity in Arabidopsis

Xanthomonas campestris pv. campestris (Xcc) causes black rot, one of the most important diseases of brassica crops worldwide. The type III effector inventory plays important roles in the virulence and pathogenicity of the pathogen. However, little is known about the virulence function(s) of the putative type III effector AvrXccB in Xcc. Here, we investigated the immune suppression ability of AvrXccB and the possible underlying mechanisms. AvrXccB was demonstrated to be secreted in a type III secretion system‐dependent manner. AvrXccB tagged with green fluorescent protein is localized to the plasma membrane in Arabidopsis, and the putative N‐myristoylation motif is essential for its localization. Chemical‐induced expression of AvrXccB suppresses flg22‐triggered callose deposition and the oxidative burst, and promotes the in planta growth of Xcc and Pseudomonas syringae pv. tomato in transgenic Arabidopsis plants. The putative catalytic triad and plasma membrane localization of AvrXccB are required for its immunosuppressive activity. Furthermore, it was demonstrated that AvrXccB interacts with the Arabidopsis S‐adenosyl‐l ‐methionine‐dependent methyltransferases SAM‐MT1 and SAM‐MT2. Interestingly, SAM‐MT1 is not only self‐associated, but also associated with SAM‐MT2 in vivo. SAM‐MT1 and SAM‐MT2 expression is significantly induced upon stimulation of microbe‐associated molecular patterns and bacterial infection. Collectively, these findings indicate that AvrXccB targets a putative methyltransferase complex and suppresses plant immunity.     

Genetic and Proteomic Analyses of a Xanthomonas campestris pv. campestris purC Mutant Deficient in Purine Biosynthesis and Virulence

Bacterial proliferation in hosts requires activation of a number of housekeeping pathways, including purine de novo biosynthesis. Although inactivation of purine biosynthesis genes can attenuate virulence, it is unclear which biochemical or virulence factors are associated with the purine biosynthesis pathway in vivo. We report that inactivation of purC, a gene encoding phosphoribosylaminoimidazole-succinocarboxamide synthase, caused complete loss of virulence in Xanthomonas campestris pv. cam- pestris, the causal agent of black rot disease of cruciferous plants. The purC mutant was a purine auxotroph; it could not grow on minimal medium, whereas addition of purine derivatives, such as hypoxanthine or adenine plus guanine, restored growth of the mutant. The purC mutation also significantly enhanced the production of an unknown purine synthesis associated pigment and extracellular polysaccharides by the bacterium. In addition, comparative proteomic analyses of bacteria grown on rich and minimal media revealed that the purC mutation affected the expression levels of diverse proteins involved in purine and pyrimidine synthesis, carbon and energy metabolisms, iron uptake, proteolysis, protein secretion, and signal transduction. These results provided clues to understanding the contributions of purine synthesis to bacterial virulence and interactions with host immune systems.     

The essential mycobacterial genes, <Emphasis Type="Italic">fabG1</Emphasis> and <Emphasis Type="Italic">fabG4</Emphasis>, encode 3-oxoacyl-thioester reductases that are functional in yeast mitochondrial fatty acid synthase type 2

Mycobacterium tuberculosis represents a severe threat to human health worldwide. Therefore, it is important to expand our knowledge of vital mycobacterial processes, such as that effected by fatty acid synthase type 2 (FASII), as well as to uncover novel ones. Mycobacterial FASII undertakes mycolic acid biosynthesis, which relies on a set of essential enzymes, including 3-oxoacyl-AcpM reductase FabG1/Rv1483. However, the M. tuberculosis genome encodes four additional FabG homologs, designated FabG2–FabG5, whose functions have hitherto not been characterized in detail. Of the four candidates, FabG4/Rv0242c was recently shown to be essential for the survival of M. bovis BCG. The present work was initiated by assessing the suitability of yeast oar1Δ mutant cells lacking mitochondrial 3-oxoacyl-ACP reductase activity to act as a surrogate system for expressing FabG1/MabA directed to the mitochondria. Mutant yeast cells producing this targeted FabG1 variant were essentially wild type for all of the chronicled phenotype characteristics, including respiratory growth on glycerol medium, cytochrome assembly and lipoid acid production. This indicated that within the framework of de novo fatty acid biosynthesis in yeast mitochondria, FabG1 was able to act on shorter (C₄) acyl substrates than was previously proposed (C_8–20) during mycolic acid biosynthesis in M. tuberculosis. Thereafter, FabG2–FabG5 were expressed as mitochondrial proteins in the oar1Δ strain, and FabG4 was found to complement the mutant phenotype and contain high levels of 3-oxoacyl-thioester reductase activity. Hence, like FabG1, FabG4 is also an essential, physiologically functional 3-oxoacyl-thioester reductase, albeit the latter’s involvement in mycobacterial FASII remains to be explored.     

Plant responses underlying nonhost resistance of Citrus limon against Xanthomonas campestris pv. campestris

Citrus is an economically important fruit crop that is severely afflicted by citrus canker, a disease caused by Xanthomonas citri ssp. citri (X. citri); thus, new sustainable strategies to manage this disease are needed. Although all Citrus spp. are susceptible to this pathogen, they are resistant to other Xanthomonas species, exhibiting non-host resistance (NHR), for example, to the brassica pathogen X. campestris pv. campestris (Xcc) and a gene-for-gene host defence response (HDR) to the canker-causing X. fuscans ssp. aurantifolii (Xfa) strain C. Here, we examine the plant factors associated with the NHR of C. limon to Xcc. We show that Xcc induced asymptomatic type I NHR, allowing the bacterium to survive in a stationary phase in the non-host tissue. In C. limon, this NHR shared some similarities with HDR; both defence responses interfered with biofilm formation, and were associated with callose deposition, induction of the salicylic acid (SA) signalling pathway and the repression of abscisic acid (ABA) signalling. However, greater stomatal closure was seen during NHR than during HDR, together with different patterns of accumulation of reactive oxygen species and phenolic compounds and the expression of secondary metabolites. Overall, these differences, independent of Xcc type III effector proteins, could contribute to the higher protection elicited against canker development. We propose that Xcc may have the potential to steadily activate inducible defence responses. An understanding of these plant responses (and their triggers) may allow the development of a sustained and sustainable resistance to citrus canker.     

细菌3-酮脂酰ACP还原酶研究进展

3-酮脂酰ACP还原酶(FabG)在细菌中广泛存在并且十分保守,已经发现的所有FabG及其同系物都具有类似的催化活性中心序列,隶属于短链醇脱氢酶/还原酶(SDRs)超家族成员。它是Ⅱ型脂肪酸合成反应中的关键酶,将3-酮脂酰ACP还原为3-羟脂酰ACP多以NADPH作为辅酶。从搜集的文献来看,国内外针对不同细菌中3-酮脂酰ACP还原酶同系物的研究报道体现了其多样性的特点。但是,近年来,该方面的专题综述十分少见。本文主要对3-酮脂酰ACP还原酶的结构特征、在脂肪酸合成和其他方面的生物学功能,以及以该酶为作用靶点的抑菌剂等方面进行概述,以期为将来3-酮脂酰ACP还原酶的深入研究提供理论参考。     

野油菜黄单胞菌中烯脂酰ACP还原酶的功能鉴定

烯脂酰ACP还原酶是细菌脂肪酸合成的关键酶之一.本研究通过生物信息学分析发现,野油菜黄单胞菌Xanthomonas campestris(Xcc)8004基因组中XC_0119(Xccfab V)注释为反-2-烯脂酰Co A还原酶基因.但其编码产物与铜绿假单胞菌的烯脂酰ACP还原酶Fab V具有较高的同源性,并含有相同的催化活性中心Tyr-(Xaa)8-Lys序列.用携带Xccfab V的质粒载体互补大肠杆菌fab I温度敏感突变株JP1111,转化子能在42℃生长,表明Xccfab V能遗传互补大肠杆菌fab I突变.体外重建脂肪酸合成反应表明,Xcc Fab V能催化不同链长的烯脂酰ACP还原为脂酰ACP,且催化活性不受三氯森抑制.遗传学研究表明,Xccfab V是必需基因,不能获得Xccfab V基因敲除突变株.将携带大肠杆菌fab I的外源质粒导入野生菌后,可敲除染色体上的fab V基因,获得的替换突变株生长特性和脂肪酸组成未发生显著变化,但替换突变株对三氯森敏感.上述结果证实,野油菜黄单胞菌fab V是必需基因,编码烯脂酰ACP还原酶,参与脂肪酸从头合成反应,且Fab V是Xcc对三氯森耐受的根本原因.     

Biological Role of Xanthomonadin Pigments in Xanthomonas campestris pv. Campestris

Previous studies have indicated that the yellow pigments (xanthomonadins) produced by phytopathogenic Xanthomonas bacteria are unimportant during pathogenesis but may be important for protection against photobiological damage. We used a Xanthomonas campestris pv. campestris parent strain, single-site transposon insertion mutant strains, and chromosomally restored mutant strains to define the biological role of xanthomonadins. Although xanthomonadin mutant strains were comparable to the parent strain for survival when exposed to UV light; after their exposure to the photosensitizer toluidine blue and visible light, survival was greatly reduced. Chromosomally restored mutant strains were completely restored for survival in these conditions. Likewise, epiphytic survival of a xanthomonadin mutant strain was greatly reduced in conditions of high light intensity, whereas a chromosomally restored mutant strain was comparable to the parent strain for epiphytic survival. These results are discussed with respect to previous results, and a model for epiphytic survival of X. campestris pv. campestris is presented.     

Isolation and characterization of beta-ketoacyl-acyl carrier protein reductase (fabG) mutants of Escherichia coli and Salmonella enterica serovar Typhimurium

FabG, beta-ketoacyl-acyl carrier protein (ACP) reductase, performs the NADPH-dependent reduction of beta-ketoacyl-ACP substrates to beta-hydroxyacyl-ACP products, the first reductive step in the elongation cycle of fatty acid biosynthesis. We report the first documented fabG mutants and their characterization. By chemical mutagenesis followed by a tritium suicide procedure, we obtained three conditionally lethal temperature-sensitive fabG mutants. The Escherichia coli [fabG (Ts)] mutant contains two point mutations: A154T and E233K. The beta-ketoacyl-ACP reductase activity of this mutant was extremely thermolabile, and the rate of fatty acid synthesis measured in vivo was inhibited upon shift to the nonpermissive temperature. Moreover, synthesis of the acyl-ACP intermediates of the pathway was inhibited upon shift of mutant cultures to the nonpermissive temperature, indicating blockage of the synthetic cycle. Similar results were observed for in vitro fatty acid synthesis. Complementation analysis revealed that only the E233K mutation was required to give the temperature-sensitive growth phenotype. In the two Salmonella enterica serovar Typhimurium fabG(Ts) mutants one strain had a single point mutation, S224F, whereas the second strain contained two mutations (M125I and A223T). All of the altered residues of the FabG mutant proteins are located on or near the twofold axes of symmetry at the dimer interfaces in this homotetrameric protein, suggesting that the quaternary structures of the mutant FabG proteins may be disrupted at the nonpermissive temperature.     

野油菜黄单胞菌中LipB-LipA是唯一的硫辛酰化途径

【目的】硫辛酸是细胞内重要的辅因子,参与多种基础代谢过程。野油菜黄单胞菌(Xcc)是十字花科植物黑腐病的病原菌,在全球范围内引起植物病害,引起重大经济损失。为此研究Xcc中硫辛酸的合成途径,为防治黑腐病提供新思路。【方法】利用大肠杆菌硫辛酸合成关键酶LipA和LipB序列,同源比对发现Xcc基因组中XC_0713 (XccLipA)和XC_0712 (XccLipB)具有较高的同源性。采用PCR方法分别扩增XccLipA和XccLipB基因,并连入表达载体pBAD24M后分别互补大肠杆菌突变株,并检测转化子生长表型。利用同源重组方法,获得替换突变株,分析其生长性状,并利用剪叶法检测替换突变株对寄主植物甘蓝的致病力。【结果】XcclipA和XcclipB能分别恢复大肠杆菌lipA和lipB突变株在基础培养上生长。XcclipA和XcclipB都是菌体生长的必需基因,不能直接被敲除。但导入pSRK-EclplA后,成功分别获得XcclipA和XcclipB敲除突变株。两种EclplA替换后的敲除突变株在基础培养上都不能生长,添加硫辛酸后能恢复生长表型。在丰富培养基上,XcclipB敲除突变株能正常生长,而XcclipA敲除突变株不能生长,添加硫辛酸后生长也能恢复。分别测定不同培养条件下生长曲线,也得到同样的结果。寄主植物侵染结果显示,与野生菌相比,XcclipA敲除突变株致病性几乎丧失,而XcclipB敲除突变株的致病性与野生菌无显著性差异。【结论】Xcc中lipA编码硫辛酸合成酶,lipB编码辛酰转移酶,两者都是必需基因。Xcc中LipB-LipA途径是唯一的硫辛酰化途径,而没有外源性的硫辛酸途径。lipA敲除后显著影响Xcc的致病性,可作为抗菌药物筛选的靶点。