Pyrrolnitrin and Hydrogen Cyanide Production by <i>Pseudomonas chlororaphis</i> Strain PA23 Exhibits Nematicidal and Repellent Activity against <i>Caenorhabditis elegans</i>

Pseudomonas chlororaphis strain PA23 is a biocontrol agent able to suppress growth of the fungal pathogen Sclerotinia sclerotiorum. This bacterium produces an arsenal of exometabolites including pyrrolnitrin (PRN), phenazine (PHZ), hydrogen cyanide (HCN), and degradative enzymes. Production of these compounds is controlled at both the transcriptional and posttranscriptional levels by the Gac-Rsm system, RpoS, PsrA, and the Phz quorum-sensing system. Beyond pathogen-suppression, the success of a biocontrol agent is dependent upon its ability to establish itself in the environment where predation by bacterivorous organisms, including nematodes, may threaten persistence. The focus of this study was to investigate whether PA23 is able to resist grazing by Caenorhabditis elegans and to define the role played by exoproducts in the bacterial-nematode interaction. We discovered that both PRN and HCN contribute to fast- and slow-killing of C. elegans. HCN is well-established as having lethal effects on C. elegans; however, PRN has not been reported to be nematicidal. Exposure of L4 stage nematodes to purified PRN reduced nematode viability in a dose-dependent fashion and led to reduced hatching of eggs laid by gravid adults. Because bacterial metabolites can act as chemoattractants or repellents, we analyzed whether PA23 exhibited attractant or repulsive properties towards C. elegans. Both PRN and HCN were found to be potent repellents. Next we investigated whether the presence of C. elegans would elicit changes in PA23 gene activity. Co-culturing the two organisms increased expression of a number of genes associated with biocontrol, including phzA, hcnA, phzR, phzI, rpoS and gacS. Exoproduct analysis showed that PHZ and autoinducer signals were upregulated, consistent with the gene expression profiles. Collectively, these findings indicate that PA23 is able to sense the presence of C. elegans and it is able to both repel and kill the nematodes, which should facilitate environmental persistence and ultimately biocontrol.绿针假单胞菌PA23菌株（Pseudomonas chlororaphis strain PA23）是一种生物防治剂，能够抑制真菌病原菌核盘菌（Sclerotinia sclerotiorum）的生长。该细菌可产生一系列胞外代谢物，包括吡咯尼群（pyrrolnitrin, PRN）、吩嗪（phenazine, PHZ）、氰化氢（hydrogen cyanide, HCN）以及各类降解酶。上述化合物的合成在转录与转录后水平上，均受到Gac-Rsm系统、RpoS、PsrA以及Phz群体感应系统的调控。除病原菌抑制功能外，生物防治剂的应用效果还取决于其在环境中的定殖能力，而食细菌生物（包括线虫）的捕食可能威胁其种群存续。本研究的核心目标为探究PA23是否能够抵御秀丽隐杆线虫（Caenorhabditis elegans）的捕食，并明确胞外产物在细菌-线虫互作中发挥的作用。本研究发现，吡咯尼群与氰化氢均可分别介导秀丽隐杆线虫的快速与慢速致死。氰化氢对秀丽隐杆线虫的致死效应已有定论，但此前尚未见吡咯尼群具有杀线虫活性的报道。将L4期线虫暴露于纯化的吡咯尼群后，线虫存活率呈剂量依赖性下降，同时孕成虫所产卵的孵化率也显著降低。由于细菌代谢物可充当化学引诱剂或驱避剂，本研究分析了PA23对秀丽隐杆线虫是否具有引诱或驱避活性。结果显示，吡咯尼群与氰化氢均为强效驱避剂。随后，本研究探究了秀丽隐杆线虫的存在是否会引发PA23的基因表达变化。将二者共培养后，多个与生物防治相关的基因表达量均出现上调，包括phzA、hcnA、phzR、phzI、rpoS与gacS。胞外产物分析结果表明，吩嗪与自诱导信号分子的产量显著提升，与基因表达谱的变化一致。综上，本研究结果表明PA23能够感知秀丽隐杆线虫的存在，并通过驱避与杀线虫双重策略提升自身在环境中的存续能力，最终助力其生物防治功能的实现。