申氏杆菌37-1中群体感应淬灭内酯酶AiiS的功能分析

许多植物病原细菌通过群体感应（quorum sensing,QS）系统调控相关毒性因子的表达,而群体感应淬灭（quorum quenching,QQ）是通过干扰QS系统,达到防治植物细菌性病害的重要策略之一。本研究利用原位培养法分离得到2000多株不同菌株形态的植物根围细菌,结合QS系统信号分子检测平板筛选到7株具有QQ活性的候选细菌,其中菌株37-1可完全降解信号分子。16S rDNA序列分析表明,菌株37-1属于Shinella sp.。全基因组序列分析发现菌株37-1中存在一个可能的QQ降解酶编码基因aiiS（autoinducer inactivation gene from Shinella sp.）。系统发育分析表明AiiS属于α/β水解酶家族蛋白。液相色谱-串联质谱分析进一步表明AiiS可水解N-乙酰高丝氨酸内酯（N-acylhomoserine lactone,AHL）类QS信号分子中的内酯健,生成酰基高丝氨酸,因此AiiS属于AHL内酯酶。将aiiS基因导入胡萝卜软腐果胶杆菌Pectobacterium carotovorum subsp.carotovorum Z3-3中,可显著降低该菌AHL信号分子和果胶酸盐裂解酶的产生及其在白菜、马铃薯和胡萝卜上的致病性。以上结果表明菌株37-1中AiiS蛋白是一种AHL内酯酶;病原细菌中异源表达aiiS基因可有效干扰相关病原细菌中QS系统的调控功能,表明AiiS蛋白具备开发为潜在新型生防制剂的价值。     

淬灭酶AiiO-AIO6酶学性质及对嗜水气单胞菌毒力因子的表达调控

将N-乙酰高丝氨酸内酯酶基因(aiiO-AIO6)插入表达载体pET28a构建了原核表达载体pET28a/ aiiO-AIO6.转化大肠杆菌后筛选具有活性的重组子并对纯化的目的蛋白AiiO-AIO6的酶学性质进行了研究,同时研究了纯化的酶液对嗜水气单胞菌ATCC7966溶血素( Hemolysin,Hem)、细胞肠毒素(Cytotonic enterotoxin,Ast)、胞外蛋白酶(Extracellular protease,Ep)、丝氨酸蛋白酶(Serine protease,Sp)及磷脂酶Pho( Phospholipase Al,Pho)等5个毒力因子表达的影响.结果表明,pET28a/aiiO-AIO6在大肠杆菌中大量表达N-乙酰高丝氨酸内酯酶,纯化后的N-乙酰高丝氨酸内酯酶的最适作用条件为pH8.0,30℃,在pH 6.0 ～11.0的范围内能够稳定的存在,在80℃条件下保温30 min后酶活力能够维持在65％以上；且该酶对多种金属离子、化合物和中性蛋白酶都具有很好的抗性；该酶对多种底物都具有一定的降解作用；以3-oxo-C8-HSL为底物时的km值为0.015 mmol/L；比活力为583.33 U/mg.N-乙酰高丝氨酸内酯酶在培养8h及12 h时对嗜水气单胞菌5个毒力因子的表达量都具有明显的下调趋势(P＜0.05).本实验通过测定AiiO-AIO6的酶学性质及证明AiiO-AIO6可以通过降解嗜水气单胞菌产生的信号分子来抑制其毒力因子的表达,从而为将其应用于水产环境及致病性嗜水气单胞菌的生物防治奠定理论基础.     

酰基高丝氨酸内酯酶在蜡状芽胞杆菌中可增强双效菌素抗软腐病

双效菌素(zwittermicin A,ZwA)是一种广谱、新型的抗生素,对很多植物病原菌具有抑制作用;酰基高丝氨酸内酯酶(AHL)通过降解植物病原菌群体感应(quorum-sensing)系统的信号分子,减弱致病基因的表达从而达到抗病的效果。结合这两种物质的不同抗病机理,将来自苏云金芽胞杆菌(Bacillus thuringiensis)中的AHL基因导入到蜡状芽胞杆菌(B.cereus)中,得到相应的重组菌株。实验表明,AHL基因在重组菌中可正常表达,并且不影响受体菌ZwA的表达和产量;感病实验证明,同时表达ZwA和AHL的重组菌对于胡萝卜软腐欧文氏菌(Erwinia carotovora)感染马铃薯(Solanum tuberosum)所引起的病害的抗病效果比单独含产ZwA或AHL的蜡状芽胞杆菌的抗病效果有明显增强。说明结合ZwA和AHL的不同抗病机理来提高抗病效果是可行的。     

苏云金芽胞杆菌N-酰基高丝氨酸内酯酶基因(aiiA)在毕赤酵母中的优化表达

苏云金芽胞杆菌(Bacillus thuringiensis)N-酰基高丝氨酸内酯酶基因(auto inducer inactivation A,aiiA)编码的N-酰基高丝氨酸内酯酶(N-acylhomoserine lactonase,AiiA)能够水解植物病原菌群体效应的信号分子N-酰基高丝氨酸内酯(N-acylhomoserine lactone,AHL),进而使革兰氏阴性细菌群体感应受到抑制,减弱病原菌的致病性。本研究将aiiA基因连接至分泌型穿梭表达载体pPICZαB,获得重组表达质粒p PICZαB-aiiA,线性化后电击转化毕赤酵母(Pichia pastoris)GS115,获得重组工程菌GS115-p PICZαBaiiA。利用定点突变技术,对aiiA基因进行密码子优化,获得重组工程菌GS115-p PICZαB-MaiiA。以终浓度为1%的甲醇、28℃条件下诱导表达,重组工程菌GS115-p PICZαB-aiiA和GS115-p PICZαB-MaiiA均成功表达并分泌出AiiA蛋白。抗病性分析表明,分泌表达的AiiA蛋白能有效抑制胡萝卜软腐欧文氏杆菌(Erwinia carotovora)的致病性。AiiA蛋白在毕赤酵母中的分泌表达,拓宽了AiiA蛋白的获取途径,为AiiA蛋白的产业化提供理论依据。密码子优化为今后改造AiiA蛋白,提高AiiA蛋白的表达效率提供了新的思路。     

Silencing of Erwinia amylovora sy69 AHL‐quorum sensing by a Bacillus simplex AHL‐inducible aiiA gene encoding a zinc‐dependent N‐acyl‐homoserine lactonase

Quorum sensing in Gram‐negative bacteria is regulated by diffusible signal molecules called N‐acyl‐l ‐homoserine lactones (AHLs). These molecules are degraded by lactonases. In this study, six Bacillus simplex isolates were characterized and identified as a new quorum‐quenching species of Bacillus. An aiiA gene encoding an AHL‐lactonase was identified based on evidence that: (i) it showed high homology with other aiiA genes of Bacillus sp.; (ii) the deduced amino acid sequence contained two conserved regions, ¹⁰⁴SHLHFDH¹¹¹ and ¹⁶⁵TPGHTPGH¹⁷³, characteristic of the metallo‐β‐lactamase superfamily; and (iii) the protein had zinc‐dependent AHL‐degrading activity. Additionally, the expression of the aiiA gene was significantly up‐regulated by 3‐oxo‐AHL. The AHL‐lactonase inhibited multiplication of the 3‐oxo‐C6‐AHL‐producing plant pathogen Erwinia amylovora sy69 both in vitro and in planta. The results provide support for the use of the quorum‐quenching functionality of B. simplex in the integrated control of the devastating fire blight pathogen.     

Use of Quorum Sensing Inhibition Strategies to Control Microfouling

Interfering with the quorum sensing bacterial communication systems has been proposed as a promising strategy to control bacterial biofilm formation, a key process in biofouling development. Appropriate in vitro biofilm-forming bacteria models are needed to establish screening methods for innovative anti-biofilm and anti-microfouling compounds. Four marine strains, two Pseudoalteromonas spp. and two Vibrio spp., were selected and studied with regard to their biofilm-forming capacity and sensitivity to quorum sensing (QS) inhibitors. Biofilm experiments were performed using two biofilm cultivation and quantification methods: the xCELLigence^® system, which allows online monitoring of biofilm formation, and the active attachment model, which allows refreshment of the culture medium to obtain a strong biofilm that can be quantified with standard staining methods. Although all selected strains produced acyl-homoserine-lactone (AHL) QS signals, only the P. flavipulchra biofilm, measured with both quantification systems, was significantly reduced with the addition of the AHL-lactonase Aii20J without a significant effect on planktonic growth. Two-species biofilms containing P. flavipulchra were also affected by the addition of Aii20J, indicating an influence on the target bacterial strain as well as an indirect effect on the co-cultured bacterium. The use of xCELLigence^® is proposed as a time-saving method to quantify biofilm formation and search for eco-friendly anti-microfouling compounds based on quorum sensing inhibition (QSI) strategies. The results obtained from these two in vitro biofilm formation methods revealed important differences in the response of biosensor bacteria to culture medium and conditions, indicating that several strains should be used simultaneously for screening purposes and the cultivation conditions should be carefully optimized for each specific purpose.     

Marine Microbiological Enzymes: Studies with Multiple Strategies and Prospects

Marine microorganisms produce a series of promising enzymes that have been widely used or are potentially valuable for our daily life. Both classic and newly developed biochemistry technologies have been broadly used to study marine and terrestrial microbiological enzymes. In this brief review, we provide a research update and prospects regarding regulatory mechanisms and related strategies of acyl-homoserine lactones (AHL) lactonase, which is an important but largely unexplored enzyme. We also detail the status and catalytic mechanism of the main types of polysaccharide-degrading enzymes that broadly exist among marine microorganisms but have been poorly explored. In order to facilitate understanding, the regulatory and synthetic biology strategies of terrestrial microorganisms are also mentioned in comparison. We anticipate that this review will provide an outline of multiple strategies for promising marine microbial enzymes and open new avenues for the exploration, engineering and application of various enzymes.     

高丝氨酸内酯酶AI-96对嗜水气单胞菌NJ-1浸浴攻毒斑马鱼保护效应的研究

为评价斑马鱼口服高丝氨酸内脂酶AI-96对嗜水气单胞菌NJ-1浸浴攻毒的保护效应,实验设置基础料与实验料两组饲料,实验料是在基础料中按3 U/g饲料添加N酰基高丝氨酸内酯酶AI-96(以下简称AI-96),通过高浓度(2.5×108 cfu/mL)及低浓度(0.7×108 cfu/mL)两组剂量的嗜水气单胞菌NJ-1(以下简称NJ-1)分别浸浴攻毒斑马鱼,在12h、24 h、3d、7d和14d取鳃丝、肠道壁、肝和肾样,采用实时荧光定量PCR法检测取样器官中NJ-1量,并统计攻毒周期内的死亡率,来评价高丝氨酸内酯酶AI-96的保护力.结果显示:在攻毒周期内所取组织内均检测到NJ-1,按菌数肠＞鳃＞肝＞肾,其中高NJ-1剂量未加酶组各组织NJ-1数量均分别明显高于低剂量处理组.在高剂量攻毒条件下,加酶组各组织NJ-1数量均显著低于未加酶处理组(P＜0.05),鳃除外；在低剂量攻毒条件下,未加酶组的NJ-1数量在鳃(3 d)、肠(0.5、1、3、7及14 d)、肝(3 d)和肾(7 d)显著高于加酶组(P＜0.05),其余差异不显著(P＞0.05).此外无论在高、低剂量攻毒条件下,加酶组的死亡率均低于未加酶组,其中低剂量攻毒7d及以后其死亡率显著低于对照(P＜0.05).结果表明,口服AI-96可以有效预防NJ-1≤0.7×108 cfu/mL范围内的侵袭.