Attenuation of Quorum Sensing Regulated Virulence of <i>Pectobacterium carotovorum</i> subsp. <i>carotovorum</i> through an AHL Lactonase Produced by <i>Lysinibacillus</i> sp. Gs50

Quorum sensing (QS) is a mechanism in which Gram negative bacterial pathogens sense their population density through acyl homoserine lactones (AHLs) and regulate the expression of virulence factors. Enzymatic degradation of AHLs by lactonases, known as quorum quenching (QQ), is thus a potential strategy for attenuating QS regulated bacterial infections. We characterised the QQ activity of soil isolate Lysinibacillus sp. Gs50 and explored its potential for controlling bacterial soft rot of crop plants. Lysinibacillus sp. Gs50 inactivated AHL, which could be restored upon acidification, suggested that inactivation was due to the lactone ring hydrolysis of AHL. Heterologous expression of cloned gene for putative hydrolase (792 bp) designated adeH from Lysinibacillus sp. Gs50 produced a ~29 kDa protein which degraded AHLs of varying chain length. Mass spectrometry analysis of AdeH enzymatic reaction product revealed that AdeH hydrolyses the lactone ring of AHL and hence is an AHL lactonase. Multiple sequence alignment of the amino acid sequence of AdeH showed that it belongs to the metallo- β- lactamase superfamily, has a conserved “HXHXDH” motif typical of AHL lactonases. KM for AdeH for C6HSL was found to be 3.089 μM and the specific activity was 0.8 picomol min-1μg-1. AdeH has not so far been reported from any Lysinibacillus sp. and has less than 40% identity with known AHL lactonases. Finally we found that Lysinibacillus sp. Gs50 can degrade AHL produced by Pectobacterium carotovorum subsp. carotovorum (Pcc), a common cause of soft rot. This QQ activity causes a decrease in production of plant cell wall degrading enzymes of Pcc and attenuates symptoms of soft rot in experimental infection of potato, carrot and cucumber. Our results demonstrate the potential of Lysinibacillus sp. Gs50 as a preventive and curative biocontrol agent.群体感应（Quorum Sensing，QS）是革兰氏阴性病原菌通过酰基高丝氨酸内酯（acyl homoserine lactones，AHLs）感知种群密度，并调控毒力因子表达的信号机制。利用内酯酶对AHLs进行酶促降解的策略被称为群体淬灭（quorum quenching，QQ），因此这是一种减弱QS调控的细菌感染的潜在手段。本研究对土壤分离株赖氨酸芽孢杆菌属（Lysinibacillus sp.）Gs50的群体淬灭活性进行了表征，并探究了其用于防控农作物细菌性软腐病的潜力。赖氨酸芽孢杆菌Gs50可使AHL失活，该失活效应可通过酸化作用恢复，表明其失活机制源于AHL的内酯环水解。从赖氨酸芽孢杆菌Gs50中克隆得到的假定水解酶基因adeH（全长792 bp）经异源表达后，可产生分子量约29 kDa的蛋白质，该蛋白能够降解不同链长的AHLs。对AdeH酶促反应产物的质谱分析结果显示，AdeH可水解AHL的内酯环，因此属于AHL内酯酶。对AdeH氨基酸序列的多序列比对结果表明，其属于金属β-内酰胺酶超家族，且带有AHL内酯酶典型的保守基序‘HXHXDH’。经测定，AdeH对C6HSL的米氏常数（KM）为3.089 μM，比活性为0.8皮摩尔·分钟⁻¹·微克⁻¹。截至目前，尚未在任何赖氨酸芽孢杆菌属菌株中报道过AdeH，且其与已知AHL内酯酶的序列同源性低于40%。最终研究发现，赖氨酸芽孢杆菌Gs50能够降解胡萝卜软腐果胶杆菌胡萝卜亚种（Pectobacterium carotovorum subsp. carotovorum，Pcc，一种常见的软腐病致病菌）产生的AHL。该群体淬灭活性可降低Pcc的植物细胞壁降解酶产量，并在马铃薯、胡萝卜和黄瓜的实验感染模型中减弱软腐病症状。本研究结果证实，赖氨酸芽孢杆菌Gs50具备作为兼具预防与治疗效果的生防制剂的潜力。