绿针假单胞菌HT66中ompR基因的功能

【背景】植物根部存在大量对植物生长有促进或对病原菌有拮抗作用的有益细菌,是当前农业微生物研究的热点之一。其中,绿针假单胞菌HT66是一株可高效合成吩嗪-1-甲酰胺(PCN)的环境友好型生防菌株。【目的】探究在绿针假单胞菌HT66中ompR基因的生理功能,以及其对菌株生防作用的影响。【方法】通过基因无痕敲除的方法构建HT66菌株的ompR基因缺失突变株,对比研究突变株与野生株在生长速率、渗透压感应、生物膜的合成、pH耐受性、群集运动和PCN产量的变化。【结果】与野生株相比,ompR基因缺失突变株的细胞生物量微量减少,生物膜的合成减少31.5%,群集运动以及对渗透压和pH的耐受性明显下降,但是其PCN产量较野生株提高了57.8%。【结论】在HT66菌株中,ompR基因对其运动性、环境耐受性和生理生防功能均有一定程度的调控作用。本研究丰富了绿针假单胞菌的代谢通路,此报道将对后续PCN合成机制的研究和应用提供一定的理论依据。     

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。     

绿针假单胞菌HT66中luxR_19对吩嗪-1-甲酰胺合成的影响

旨在研究调控基因lux R_19对绿针假单胞菌HT66中的抑菌物质吩嗪-1-甲酰胺(phenazine-1-carboxamide,PCN)合成及菌体生长的影响,通过分子操作构建了lux R_19基因敲除株、回补菌株、过表达菌株以及lux R_19(G85A)点突变菌株,检测了目标菌株生长曲线、PCN发酵结果、细菌泳动性与从动性等。结果显示,将lux R_19敲除后PCN产量明显降低,对lux R_19基因进行过表达,发现能够将PCN产量提高2倍;lux R_19对于吩嗪合成的多个调控基因以及合成基因表达均产生影响;lux R_19的敲除对HT66的生长及泳动性不产生影响,而对细菌的从动性有一定的促进作用;lux R_19(G85A)点突变菌株吩嗪产量较野生型相差很小。结果表明,lux R_19对PCN的合成为正调控基因,一定程度上影响HT66的从动性,第254位碱基突变对PCN合成产量不产生影响。     

铜绿假单胞菌M18 rsmY突变株的构建及其对PCA和Plt的区别性调控

【目的】在假单胞菌中,小RNA(sRNA)参与初级和次级代谢产物、多种毒素因子以及菌群传感系统的调控,通过在植物根际促生铜绿假单胞菌M18中研究RsmY对吩嗪-1-羧酸(PCA)和藤黄绿菌素(Plt)两种抗生素的调控作用,深入了解假单胞菌中次级代谢的途径并为构建高产抗生素工程菌株提供了一定的理论基础。【方法】运用同源重组技术,构建了铜绿假单胞菌M18株的rsmY突变菌株M18RY,通过基因过表达、lacZ报告基因融合分析实验,进一步验证了RsmY对抗生素合成基因的调控作用。【结果】比较野生型M18和突变株M18RY中PCA和Plt在同一培养条件下的生物合成量,突变菌株M18RY中PCA的产量显著增加,为野生型菌株的5倍左右,而Plt的产量降为野生型的1/8。LacZ报告基因融合分析进一步证明了RsmY对PCA的负调控作用主要是通过phz2基因簇来实现的。【结论】结果表明,rsmY基因区别性调控PCA和Plt的生物合成。     

铜绿假单胞菌PAO1中c-di-GMP代谢相关基因PA2072的生物学分析

【背景】铜绿假单胞菌为革兰氏阴性杆菌,是医院感染的常见条件致病菌之一。广泛存在于细菌中的第二信使分子环鸟苷二磷酸(cyclic-di-guanosine monophosphate,c-di-GMP)对细菌生理生化功能具有重要的调节作用。铜绿假单胞菌PAO1中存在参与c-di-GMP代谢的基因PA2072。【目的】探讨铜绿假单胞菌PAO1中c-di-GMP代谢相关基因PA2072的生物学功能。【方法】运用PCR及分子克隆技术构建PA2072基因及各结构域的自杀载体,运用基因敲除方法获取PA2072基因的3个突变株;利用泳动性(swimming)、蜂群运动(swarming)、蹭行运动(twitching)和生物膜定量实验对细菌进行初步的表型分析,进一步通过刚果红染色法对菌株进行分析。【结果】成功构建PA2072基因敲除突变菌株及回补菌株;生物膜定量结果发现基因PA2072的敲除会影响细菌生物膜的形成,PA2072蛋白的不同结构域对生物膜的合成也起到了重要作用;细菌运动能力检测中发现PA2072相关基因的敲除对细菌运动能力也有一定影响。刚果红平板检测结果显示,与野生型PAO1菌株相比,P...     

异油酸含量对铜绿假单胞菌群集运动能力的影响

摘要:【目的】研究铜绿假单胞菌的脂肪酸组成对群集运动(swarming)的影响及可能的原因。【方法】通过双交换原理敲除铜绿假单胞菌PAO1脂肪酸合成的一个重要基因fabF,构建fabF缺失突变体;构建pUCP18Gm-fabF质粒大量表达FabF,对缺失体进行回补。比较野生型、缺失体和回补后菌株的swarming能力。利用气相色谱分析野生型、缺失体和回补后菌株的脂肪酸组成变化,以揭示FabF表达影响swarming能力的可能原因。【结果】获得了假单胞菌的fabF缺失突变体YFF-1,证实了fabF敲除后假单胞菌的 swarming能力消失,fabF回补后细菌的swarming能力得以恢复。气相色谱结果显示,突变后脂肪酸组成发生明显变化,异油酸(C18:1 Δ11)含量由33.6%急剧下降至8.9%,不饱和脂肪酸:饱和脂肪酸(USA:SFA)也由0.96下降至0.74;fabF回补后,异油酸含量恢复至20.9%,USA:SFA也上升至1.09。【结论】铜绿假单胞菌fabF基因的表达水平对细菌的swarming能力具有重要影响,突变体中异油酸含量的下降及脂肪酸不饱和程度的降低(尤其是异油酸含量的急剧下降),可能是YFF-1 swarming能力消失的重要原因。     

细菌群体感应参与铜绿假单胞菌胞内聚羟基脂肪酸酯合成的调控

群体感应是细菌根据细胞密度变化调控基因表达的一种调节机制。铜绿假单胞菌中QS系统由lasI和rhlI合成的信号分子3OC12-HSL和C4-HSL以及各自的受体蛋白LasR、RhlR组成,它们以级联方式调控多个基因表达。【目的】研究细菌群体感应(QS)对聚羟基脂肪酸酯合成的调控。【方法】利用铜绿假单胞菌PAO1及其QS突变株为材料通过气相色谱、荧光定量PCR在生理和分子水平上研究QS对聚羟基脂肪酸酯合成的调控。【结果】QS信号分子合成抑制剂阿奇霉素处理铜绿假单胞菌PAO1和QS突变株导致胞内PHA积累量显著减少;铜绿假单胞菌PAO1中C4-HSL合成酶基因rhlI缺失突变株PAO210胞内PHA积累量与野生型无差别;而3OC12-HSL合成酶基因lasI缺失突变株PAO55、3OC12-HSL受体合成酶基因lasR缺失突变株PAO56以及lasI/lasR双缺失突变株PAO57胞内PHA含量与野生型相比明显减少;lasI和lasR的突变株体内PHA合成酶基因phaC1的表达量显著降低,信号分子3OC12-HSL回补实验使phaC1的表达量可恢复到野生株水平,但只可部分恢复lasI缺失导致的胞内PHA合成。【结论】由此推测,铜绿假单胞菌群体感应系统中lasI/lasR系统参与胞内聚羟基脂肪酸酯合成的调控。     

植物发酵液提取物对铜绿假单胞菌生物膜的作用

【目的】探讨植物发酵液提取物(plant fermentation extract,PFE)对铜绿假单胞菌生物膜的抑制作用,为临床上铜绿假单胞菌感染相关疾病的治疗提供参考。【方法】通过划线法分离临床标本中的铜绿假单胞菌并进行鉴定,通过报告菌株测定铜绿假单胞菌的毒力因子,采用试管法和激光共聚焦扫描显微镜测定生物膜的形成。【结果】在分离出的16株铜绿假单胞菌中,PFE对PA007菌株的作用效果最好,1%PFE显著降低PA007菌株生物膜、绿脓菌素和N-(3-oxododecanoyl)-HSL(3-oxo-C12-HSL)的产量(P0.05)。同时,也显著降低Las A蛋白酶的活性以及持留菌存活率(P0.05)。荧光定量PCR实验结果表明PFE能显著抑制las I和pqs A基因的表达(P0.05)。【结论】PFE具有抗铜绿假单胞菌感染能力,在临床上铜绿假单胞菌感染疾病的治疗中具有巨大的潜在价值。     

ARTP技术选育吩嗪-1-甲酰胺高产菌株及发酵优化

从一株高产吩嗪-1-甲酰胺(PCN)的绿针假单胞菌P3株出发,利用常压室温等离子体诱变技术进行诱变育种,从初筛的20株突变株中获得了一株PCN产量达到2 093 mg/L的突变株P3-9,为出发菌株的125%。随后通过单因素实验考察了各种营养因子对该高产菌株合成PCN的影响,结果表明发酵培养基的最佳碳源、氮源分别为甘油和蛋白胨,外源添加Fe3+或Fe2+对于积累PCN有显著促进作用,而添加苯丙氨酸、色氨酸和酪氨酸对PCN产量无明显影响。优化后,该突变株的PCN产量高达2 810 mg/L,是目前国际上通过诱变育种获得的较高PCN产量。     

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。     

Geminiviruses: a tale of a plasmid becoming a virus

Background  

Geminiviruses (family Geminiviridae) are small single-stranded (ss) DNA viruses infecting plants. Their virion morphology is unique in the known viral world – two incomplete T = 1 icosahedra are joined together to form twinned particles. Geminiviruses utilize a rolling-circle mode to replicate their genomes. A limited sequence similarity between the three conserved motifs of the rolling-circle replication initiation proteins (RCR Reps) of geminiviruses and plasmids of Gram-positive bacteria allowed Koonin and Ilyina to propose that geminiviruses descend from bacterial replicons.     

Testing a Primary and a Secondary Endpoint in a Group Sequential Design

Summary We consider a clinical trial with a primary and a secondary endpoint where the secondary endpoint is tested only if the primary endpoint is significant. The trial uses a group sequential procedure with two stages. The familywise error rate (FWER) of falsely concluding significance on either endpoint is to be controlled at a nominal level α. The type I error rate for the primary endpoint is controlled by choosing any α‐level stopping boundary, e.g., the standard O'Brien–Fleming or the Pocock boundary. Given any particular α‐level boundary for the primary endpoint, we study the problem of determining the boundary for the secondary endpoint to control the FWER. We study this FWER analytically and numerically and find that it is maximized when the correlation coefficient ρ between the two endpoints equals 1. For the four combinations consisting of O'Brien–Fleming and Pocock boundaries for the primary and secondary endpoints, the critical constants required to control the FWER are computed for different values of ρ. An ad hoc boundary is proposed for the secondary endpoint to address a practical concern that may be at issue in some applications. Numerical studies indicate that the O'Brien–Fleming boundary for the primary endpoint and the Pocock boundary for the secondary endpoint generally gives the best primary as well as secondary power performance. The Pocock boundary may be replaced by the ad hoc boundary for the secondary endpoint with a very little loss of secondary power if the practical concern is at issue. A clinical trial example is given to illustrate the methods.     

Vegetation as a component of a non-nested hierarchy: a conceptual model

Abstract. A general conceptual model of vegetation based on hierarchy theory is presented. The model emphasizes that prediction of vegetation requires consideration of both mechanisms of vegetation change and the constraints within which it occurs. The mechanisms of vegetation change are the responses to and effects upon their surroundings of individual plants. The most general constraints upon vegetation are aspects of the environment not affected by vegetation over successional time, and the pool of species within dispersal range. Examples of such environmental factors include macroclimate and soil parent material. In some cases, vegetation may alter important labile environmental factors such as soil nutrient and water availability. Some vegetation compositions appear to be resistant to changes in the general constraints. Due to both sources, there are multiple possible vegetation compositions given the same general constraints. Disturbance is defined as an abrupt change in the constraints on the vegetation resulting in a change in the vegetation's state or dynamics. Both the recognition of disturbance and the distinction between independent and labile environmental factors depend on the spatial and temporal scale of observation. For example, a particular wildfire at a given stand may be a disturbance, whereas at a larger scale of observation the same event may contribute to the wildfire regime, part of the constraints at that scale. Similarly, levels of soil organic matter may constrain vegetation over short time scales, due to influencing availability of water and nutrients. Over long time scales, the vegetation itself is a primary determinant of soil organic matter content. This model contains elements of both the initial, holistic theory of vegetation and recent, reductionistic approaches. It reiterates the need to considerboth mechanisms and constraints, stressed by contemporary and earlier workers. Hierarchy theory provides new insights concerning sufficient conditions for prediction, possible limits on predictability, and appropriate research strategy.     

Transformation of a psychrotrophic bacterium with a plasmid from a mesophile

The psychrotrophic bacteriumBacillus psychrophilus was transformed with the broadhost-range plasmid pC194. The ability of the transformant to express chloramphenicol (CAM) resistance and the possible effects of such expression on the physiology of the psychrotroph were examined. The transformant exhibited growth rates, filament formation at elevated temperatures, synthesis of cold shock proteins and cold acclimation proteins, similar to the parentalB. psychrophilus.