生防假单胞菌2P24中mvaT和mvaV基因对PcoI/PcoR群体感应系统的调控作用

【目的】自小麦全蚀病自然衰退土壤分离得到的荧光假单胞菌(Pseudomonas fluorescens)2P24,可防治多种由植物病原菌引起的土传病害。菌株2P24具有群体感应(quorum-sensing,QS)系统PcoI/PcoR,该系统影响生防菌2P24生物膜的形成以及其在小麦根围的定殖能力,从而影响2P24的生防能力。本文利用遗传学方法进一步研究了2P24中QS系统的调控途径。【方法】将QS系统信号合成基因pcoI的转录报告质粒p970Gm-pcoIp转入gacA基因突变菌株PM201中,再利用Tn5转座子对该菌株进行随机突变,筛选影响pcoI基因表达的调控因子。【结果】根据菌落颜色的变化筛选到2株突变菌株。Tn5插入位点和基因序列分析表明这2个突变体中Tn5破坏了同一个基因mvaT;设计引物利用PCR方法从2P24基因组中获得mvaT基因及其同源基因mvaV。转录融合报告实验表明:与野生菌株2P24相比,mvaT及mvaV突变体中pcoI基因的表达和N-乙酰高丝氨酸内酯的产量显著提高;HPLC试验表明mvaT和mvaV基因影响抗生素2,4-二乙酰基间苯三酚的合成。细菌双杂交试验证实,MvaT蛋白和MvaV蛋白在体内发生自身互作,这两个蛋白也可相互作用。【结论】以上结果表明mvaT和mvaV参与调控生防假单胞菌2P24的PcoI/PcoR群体感应系统,并可能影响其生防功能基因的表达。     

荧光假单胞菌2P24中PcoI-PcoR群体感应系统的自体反馈调控

荧光假单胞菌2P24的PcoI-PcoR 群体感应(QS)系统信号合成基因pcoI的表达受多种因子的调控, 其中GacS-GacA双因子调控系统在转录水平正调控信号合成基因pcoI的表达。为进一步研究QS系统调控因子, 将2P24基因组文库转入gacA缺失的pcoI基因转录报告菌株PM203 (pcoI-lacZ, gacA-), 筛选可提高pcoI表达的基因。结果表明粘粒pP32-24可显著提高pcoI转录水平, 亚克隆实验证明其中的功能基因为pcoI; 外源添加标准信号分子3-氧-己酰高丝氨酸内酯(3-oxo-C8-HSL)同样可显著提高pcoI基因的表达, 表明pcoI基因的表达对自身有正调控作用。同时构建了QS系统的另外一个组分pcoR基因的缺失突变体, pcoR基因缺失后pcoI的表达和N-乙酰高丝氨酸内酯信号分子(AHL)的产量明显低于野生菌株及其互补菌株, 并显著降低该菌株的生物膜(Biofilm)形成能力。这些结果表明菌株2P24的PcoI-PcoR QS系统中, 信号合成基因pcoI的表达受自体反馈调控, pcoR基因参与pcoI基因表达的调控以及生物膜的形成。     

荧光假单胞菌2P24中opgGH基因影响抗生素2,4-二乙酰基间苯三酚的产生

【目的】抗生素2,4-二乙酰基间苯三酚(2,4-diacetylphloroglucinol,2,4-DAPG)是生防菌株荧光假单胞菌(Pseudomonas fluorescens) 2P24防治植物病害的关键因子,然而对2,4-DAPG生物合成的调控通路并未完全解析。【方法】前期利用Tn5随机突变的方法获得一株对棉花立枯丝核菌(Rhizoctonia solani)拮抗能力完全丧失的突变菌株W3,本研究利用基因互补等方法研究该突变体中被破坏的基因对菌株2P24分泌2,4-DAPG和其他生防相关性状的影响。【结果】Tn5插入位点及其序列分析表明突变菌株W3中Tn5破坏了opgG基因。鉴于opgG和opgH基因组成操纵子,利用同源重组技术构建了opgGH内缺失突变菌株。与野生菌株2P24相比,opgGH突变菌株中2,4-DAPG的产量显著降低。对其他生防相关性状的检测发现,突变opgGH基因并不影响群体感应系统(quorum sensing,QS)信号分子的产生、氢氰酸的产生以及生物膜的形成,但可抑制菌株2P24的游动性。转录融合实验进一步表明opgGH基因并不调控gacA基因及其调控...     

酰基高丝氨酸内脂合成酶LasI对禽致病性大肠杆菌生物学特性的影响

【背景】禽致病性大肠杆菌(Avian pathogenic Escherichia coli, APEC)是禽类主要病原菌之一,群体感应(Quorumsensing,QS)系统可通过信号分子调控其生物学特性。在APEC中信号分子AHL对其生物学特性的影响目前尚不清楚。【目的】研究信号分子AHL对APEC生物学特性的影响。【方法】将含铜绿假单胞菌酰基高丝氨酸内脂合成酶(Acyl-homoserine-lactone synthase,lasI)基因的表达质粒转化至APEC菌株DE17中,构建重组菌株DE17-lasI,利用LasI在DE17中合成AHL。比较野生株和重组菌株产生AHL信号分子、生长特性、生物被膜形成能力、运动性以及耐药性等生物学特性的差异;运用Real-timePCR技术,比较野生株和重组菌株中与生物被膜形成、运动性以及毒力因子相关基因的转录水平。【结果】对重组菌株AHL信号分子检测表明,DE17-lasI能够产生AHL信号分子,与野生株DE17相比,DE17-lasI生物被膜形成能力和运动性显著降低(P0.01),但其生长特性和耐药性无显著变化(P0.05);Real-time PCR检测结果表明,重组菌株的毒力因子fimH转录水平上调了58.8倍,而ompA、iss分别下调了95.4%、77.3%。与生物被膜形成相关基因agn43下调了75%,鞭毛合成基因flhA下调了80.8%。此外,AHL受体sdiA的转录水平上调了19.8倍。【结论】转化lasI至APEC中,能促进其在APEC中合成信号分子AHL,并显著影响APEC的部分生物学特性,为进一步探讨AHL型群体感应系统对APEC的调控作用提供参考。     

不同碳源对生防荧光假单胞菌2P24产抗生素2,4-二乙酰基间苯三酚的影响

【目的】包括碳源代谢等不同环境因子可调控生防菌株生防相关因子表达,进而影响其防病效果。荧光假单胞菌2P24可防治多种植物病原真菌、细菌引起的土传病害,抗生素2,4-二乙酰基间苯三酚(2,4-diacetylphoroglucinol,2,4-DAPG)是其主要生防因子之一。本文利用平板对峙法及遗传学方法研究不同碳源对菌株2P24产生2,4-DAPG的影响及相关的调控途径。【方法】利用平板对峙法检测了菌株2P24在添加葡萄糖、果糖和蔗糖等碳源的土豆浸液培养基中对棉花立枯丝核菌(Rhizoctonia solani)的拮抗能力及菌株2P24中影响2,4-DAPG产生的相关基因的表达。另外,利用Tn5转座子对含有2,4-DAPG合成基因phl A报告质粒p970Gm-phl Ap的野生型菌株2P24进行随机突变,在果糖土豆浸液培养基中筛选提高phl A基因表达的突变菌株。【结果】平板对峙实验表明,菌株2P24以葡萄糖为碳源时其抑菌活性最强,蔗糖次之,而以果糖等为碳源时菌株2P24无抑菌活性;转录融合实验进一步表明葡萄糖可促进phl A基因的表达,果糖则不影响phl A基因的表达。在果糖土豆浸液培养基中,转座子随机突变实验获得了5株可明显提高phl A基因表达的突变菌株。Tn5插入位点和序列分析显示其中一个突变体是Tn5破坏了che B基因。转录检测表明与野生菌株相比,che B突变体中phl A基因的表达和2,4-DAPG的前体物质间苯三酚(phloroglucinol,PG)产量都显著提高。游动性实验发现突变che B基因可显著降低该菌株的游动性。【结论】上述结果表明菌株2P24中不同碳源在转录水平上可影响phl A基因的表达,进而影响2,4-DAPG产生。遗传学结果也显示,che B基因参与调控2,4-DAPG生物合成过程。     

假单胞菌株M18 psrA突变株的构建及其对吩嗪-1-羧酸和藤黄绿菌素合成的调控

【目的】假单胞菌M18是一株能同时合成吩嗪-1-羧酸(PCA)和藤黄绿菌素(Plt)两种抗生素的植物根际促生细菌。PsrA为细菌TetR家族转录调控因子。为了研究PsrA对PCA与Plt生物合成的影响,从M18菌株基因组中扩增psrA基因。【方法】通过同源重组技术,构建庆大霉素抗性片段置换psrA的突变菌株M18psrA。利用基因互补、lacZ报告基因融合分析实验,验证PsrA对抗生素合成基因的调控作用。【结果】在PPM和KMB培养基中,分别比较野生型菌株M18和突变菌株M18psrA的PCA与Plt产量,突变菌株M18psrA的PCA产量显著下降;Plt产量显著升高,为野生型菌株的10-15倍。基因互补、lacZ报告基因融合分析,进一步证明了psrA正调控PCA的phz2合成基因簇,负调控Plt的合成基因簇。【结论】PsrA区别性调控抗生素PCA与Plt的生物合成。     

假单胞菌株M18中pqsA突变株的构建及其对plt的调控

【目的】根际铜绿假单胞菌M18能产生藤黄绿菌素(Plt)和吩嗪-1-羧酸(PCA)两种主要的抗生素。其PqsR/PQS群体感应系统由应答调控蛋白PqsR与信号分子PQS组成。前期研究已经表明pqsR负调控Plt生物合成及基因簇表达。本论文旨在研究PQS分子对Plt合成及基因表达的调控作用。【方法】从M18基因组中扩增PQS合成基因pqsA,通过同源重组技术构建假单胞菌M18的pqsA突变菌株M18pqsA。利用lacZ报告基因分析、信号分子添加实验等,研究PQS对Plt合成及基因表达的调控作用。【结果】在KMB培养基中,分别比较野生型菌株M18和突变菌株M18pqsA的Plt产量,突变菌株的Plt产量存在较小幅度的升高,约为野生型菌株的1.53倍。添加PQS对plt表达存在一定程度但不是很显著的负调控作用。【结论】PQS分子对Plt生物合成及基因表达存在部分负调控作用。     

铜绿假单胞菌PAO1中c-di-GMP代谢相关基因PA0575对表型的影响

【背景】铜绿假单胞菌PAO1中存在与环鸟苷二磷酸(cyclic-di-guanosine monophosphate,c-di-GMP)代谢相关基因PA0575。【目的】探讨铜绿假单胞菌PAO1中环鸟苷二磷酸代谢相关基因PA0575对运动能力及生物膜的影响。【方法】通过PCR对菌株遗传背景进行确认;利用刚果红结合实验及电转PcdrA-gfp质粒间接测量胞内c-di-GMP水平;利用泳动性(swimming)、蜂群泳动(swarming)、蹭行运动(twiching)和生物膜定量实验对细菌进行表型分析,并在运动培养基中添加抗生素研究其对运动能力的影响;针对PA0575基因进行融合蛋白表达载体的构建,并对蛋白进行原核诱导表达。【结果】3株突变体菌株的转座子插入突变位点不一致,胞内c-di-GMP水平检测结果显示,PA0575-1菌株的c-di-GMP含量高于野生型PAO1菌株(P0.05),PA0575-2、PA0575-3菌株胞内c-di-GMP水平与野生型PAO1菌株无差异(P0.05)。运动能力检测实验中,与野生型PAO1菌株相比,PA0575-1菌株泳动性增强(P0.05);PA0575-2、PA0575-3菌株的泳动性、蜂群运动均增强(P0.05);该基因不同位点的突变均导致氯霉素对菌株的运动能力产生抑制作用。生物膜定量结果显示,与野生型PAO1菌株相比,细菌培养18 h后PA0575-1的生物膜含量降低(P0.05),PA0575-2、PA0575-3菌株的生物膜含量升高。最后成功构建了PA0575基因不同结构域的8个表达载体,并获得了异源表达蛋白。【结论】PA0575基因降低铜绿假单胞菌胞内c-di-GMP的水平,影响表型的同时也抑制了氯霉素抗性基因的表达。以上研究为PA0575基因对表型的影响奠定了基础。     

铜绿假单胞菌中III型分泌系统受Rhl和PQS群体感应系统调节

摘要：【目的】研究铜绿假单胞菌中群体感应系统（Quorum sensing, QS）与III型分泌系统（Type III secretion system, T3SS）的关系。【方法】通过基因敲除的方法破坏铜绿假单胞菌QS系统相关基因,将T3SS相关基因exoS、exoY、exoT、exsD-pscA-L启动子-报道子luxCDABE融合体整合到野生型菌株及QS系统突变菌株的染色体组上,通过检测启动子活性,比较这些基因在不同菌株中的表达情况。【结果】研究结果表明,T3SS中的exoS与exoT在pqsR基因突变体中的表达有明显的增强,Rhl系统对这四种基因的表达具有抑制作用,而Las系统存在与否对T3SS基本没有影响。【结论】铜绿假单胞菌中的Rhl系统和奎诺酮信号（Pseudomonas Quinolone Signal, PQS）系统对T3SS相关基因的表达具有重要的调节作用。     

suhB基因对绿针假单胞菌HT66生防能力的影响

【背景】绿针假单胞菌(Pseudomonas chlororaphis) HT66是一株兼具生防安全性和吩嗪-1-甲酰胺(Phenazine-1-Carboxamide,PCN)高产的植物根际促生菌,在生物防治、生态农业及可持续发展农业领域具有广阔的应用前景。非编码RNA (ncRNA) SuhB参与了细胞中多个过程的代谢调控。【目的】探究suhB基因对绿针假单胞菌HT66生防能力的影响。【方法】以同源重组的方法无痕敲除suhB基因构建突变菌株HT66ΔsuhB,利用质粒回补suhB基因构建突变菌株HT66ΔsuhB-pBBR-suhB,研究suhB基因对菌株生长状态、生物膜形成、群集运动及PCN合成的影响。【结果】缺失suhB基因后,菌株HT66生长缓慢,平台期滞后12 h,而且生物量减少为野生型的61.6%;在KMB培养基中单位细胞PCN产量最高达109.5mg/g,为野生株的2.1倍;生物膜形成量明显增加,为野生型的1.8倍;在运动性检测平板上,野生株的运动半径为21 mm,而suhB突变株的运动半径缩减至9.7 mm,群集运动能力明显下降。suhB基因回补突变株上述生物学功能同野生株相似。在突变株HT66ΔsuhB中,pME6015-phzI-lacZ融合质粒表达的LacZ酶活与野生型差异不显著;pME6015-phzR-lacZ融合质粒表达的LacZ酶活显著上升,为野生型的3.1倍;pME6522-phzAp-lacZ融合质粒表达的LacZ酶活为野生型的1.8倍。【结论】绿针假单胞菌HT66中suhB基因参与了菌株生长、生物膜形成、群集运动及PCN合成等多个过程的调控。本研究为该菌株的代谢改造与生防应用提供了理论基础。     

Quorum-sensing system influences root colonization and biological control ability in Pseudomonas fluorescens 2P24

Pseudomonas fluorescens 2P24 is a biocontrol agent isolated from a wheat take-all decline soil in China. This strain produces several antifungal compounds, such as 2,4-diacetylphloroglucinol (2,4-DAPG), hydrogen cyanide and siderophore(s). Our recent work revealed that strain 2P24 employs a quorum-sensing system to regulate its biocontrol activity. In this study, we identified a quorum-sensing system consisting of PcoR and PcoI of the LuxR–LuxI family from strain 2P24. Deletion of pcoI from 2P24 abolishes the production of the quorum-sensing signals, but does not detectably affect the production of antifungal metabolites. However, the mutant is significantly defective in biofilm formation, colonization on wheat rhizosphere and biocontrol ability against wheat take-all, whilst complementation of pcoI restores the biocontrol activity to the wild-type level. Our data indicate that quorum sensing is involved in regulation of biocontrol activity in P. fluorescens 2P24.     

Contribution of quorum‐sensing system to hexadecane degradation and biofilm formation in Acinetobacter sp. strain DR1

Aims: To investigate roles of quorum‐sensing (QS) system in Acinetobacter sp. strain DR1 and rifampicin‐resistant variant (hereinafter DR1R). Methods and Results: The DR1 strain generated three putative acyl homoserine lactones (AHLs), while the DR1R produced only one signal and QS signal production was abrogated in the aqsI (LuxI homolog) mutant. The hexadecane‐degradation and biofilm‐formation capabilities of DR1, DR1R, and aqsI mutants were compared, along with their proteomic data. Proteomics analysis revealed that the AHL lactonase responsible for degrading QS signal was highly upregulated in both DR1R and aqsI mutant, also showed that several proteins, including ppGpp synthase, histidine kinase sensors, might be under the control of QS signalling. Interestingly, biofilm‐formation and hexadecane‐biodegradation abilities were reduced more profoundly in the aqsI mutant. These altered phenotypes of the aqsI mutant were restored via the addition of free wild‐type cell supernatant and exogenous C₁₂‐AHL. Conclusions: The QS system in strain DR1 contributes to hexadecane degradation and biofilm formation. Significance and Impact of the Study: This is the first report to demonstrate that a specific QS signal appears to be a critical factor for hexadecane degradation and biofilm formation in Acinetobacter sp. strain DR1.     

Pseudomonas corrugata contains a conserved N-acyl homoserine lactone quorum sensing system; its role in tomato pathogenicity and tobacco hypersensitivity response

Pseudomonas corrugata is a phytopathogenic bacterium, causal agent of tomato pith necrosis, yet it is an ubiquitous bacterium that is part of the microbial community in the soil and in the rhizosphere of different plant species. Although it is a very heterogeneous species, all the strains tested were able to produce short chain acyl homoserine lactone (AHL) quorum sensing signal molecules. The main AHL produced was N-hexanoyl-L-homoserine lactone (C(6)-AHL). An AHL quorum sensing system, designated PcoI/PcoR, was identified and characterized. The role of the quorum sensing system in the expression of a variety of traits was evaluated. Inactivation of pcoI abolished the production of AHLs. The pcoR mutant, but not the pcoI mutant, was impaired in swarming, unable to cause a hypersensitivity response on tobacco and resulted in a reduced tomato pith necrosis phenotype. The pcoI mutant showed a reduced antimicrobial activity against various fungi and bacteria when assayed on King's B medium. These results demonstrate that the AHL quorum sensing in Ps. corrugata regulates traits that contribute to virulence, antimicrobial activity and fitness. This is the first report of genes of Ps. corrugata involved in the disease development and biological control activity.     

The acyl-homoserine lactone (AHL)-type quorum sensing system affects growth rate,swimming motility and virulence in <Emphasis Type="Italic">Acidovorax avenae</Emphasis> subsp. <Emphasis Type="Italic">citrulli</Emphasis>

Acidovorax avenae subsp. citrulli is a Gram-negative bacterium and is the causal agent of bacterial fruit blotch (BFB) in cucurbits. In this study, the role played by the acyl-homoserine lactone (AHL)-type quorum sensing (QS) system in growth, swimming motility and virulence was characterized in A. avenae subsp. citrulli strain XJL12. The AHL synthase gene of the QS system from strain XJL12, defined as aacI, was cloned and characterized, and an aacI disruption mutant was generated. The aacI mutant XJL13 abolished the ability to produce AHL molecules, whereas the corresponding complemented strain CPXJL13 produced wild-type levels of AHL. The aacI mutant exhibited a significant decrease in growth rate relative to the wild type in minimal medium, and was partially impaired in swimming motility. In plants, the aacI mutant showed a significant reduction of virulence in watermelon fruits and melon seedlings when compared to the wild-type strain. However, the aacI mutation in strain XJL12 had no effects on biofilm formation, exopolysaccharide production, or induction of hypersentitive response in Nicotiana tabacum. Our data suggest that the AHL-type QS may play a key role in pathogen virulence and this may provide an opportunity to explore novel approaches for managing BFB in cucurbits by QS interference.