Table_1_The global role of Lrp in Vibrio alginolyticus and its response to diverse physicochemical factors.docx

Leucine-responsive regulatory protein (Lrp) is an essential transcriptional regulator in prokaryotes. However, the regulatory role of lrp in Vibrio alginolyticus has still not been studied in detail. In this study, an lrp mutant strain was constructed to gain insight into the role of lrp in Vibrio alginolyticus. The absence of lrp significantly enhances swarming motility, biofilm formation, extracellular protease secretion activity, and tolerance to copper ions. The cumulative mortality of zebrafish (Danio rerio) challenged by intraperitoneal injection against the lrp mutant strain reached 68.89%, significantly higher than the 40.00% suffered by fish injected with the wild-type strain. The expression levels of lrp decreased gradually with increasing culture time under the influence of various physicochemical factors. The expression level of lrp was significantly increased after two hours of culture at pH 5, 22°C, 5% NaCl, the presence of 1 mM Cu2+, 1/4/7 mM ferric citrate, 0.1 mg/L NaNO3, and 0.1 mg/L KH2PO4. The mRNA level of lrp decreased significantly after six hours of culture at 37°C, 1% and 5% NaCl, and the presence of 1/7 mM ferric citrate, 0.1/5 mg/L NaNO3, and 0.1/0.5/2.0 mg/L KH2PO4. The expression of lrp increased after ten hours of culture at pH 5/9, 22°C, 1% NaCl, and the presence of 1 mM Cu2+, 7 mM ferric citrate, 5 mg/L NaNO3, and 0.1 mg/L KH2PO4. Overall, this study indicates that lrp negatively controls the virulence of V. alginolyticus, probably by reducing its swarming motility, biofilm formation, extracellular protease secretion activity, and tolerance to copper ions, and that the expression of lrp is affected by numerous physicochemical factors, and is especially up-regulated after 2 hours of culture in bacterial growth.

亮氨酸响应调控蛋白（Leucine-responsive regulatory protein, Lrp）是原核生物中一类必需的转录调控因子。然而，lrp基因在溶藻弧菌（Vibrio alginolyticus）中的调控作用尚未得到详尽研究。本研究构建了lrp缺失突变菌株，以深入解析其在溶藻弧菌中的功能。缺失lrp可显著增强菌株的群体迁动能力、生物被膜形成能力、胞外蛋白酶分泌活性以及铜离子耐受能力。通过腹腔注射lrp突变菌株攻毒斑马鱼（Danio rerio），实验组累计死亡率达68.89%，显著高于注射野生型菌株组的40.00%。在多种理化因子作用下，lrp的表达水平随培养时长呈现动态变化：当在pH 5、22℃、5% NaCl、添加1 mM Cu²+、1/4/7 mM柠檬酸铁、0.1 mg/L硝酸钠及0.1 mg/L磷酸二氢钾的培养基中培养2小时后，lrp的表达水平显著上调；当在37℃、1%及5% NaCl、添加1/7 mM柠檬酸铁、0.1/5 mg/L硝酸钠及0.1/0.5/2.0 mg/L磷酸二氢钾的培养基中培养6小时后，lrp的mRNA水平显著下调；当在pH 5/9、22℃、1% NaCl、添加1 mM Cu²+、7 mM柠檬酸铁、5 mg/L硝酸钠及0.1 mg/L磷酸二氢钾的培养基中培养10小时后，lrp的表达水平有所上调。综上，本研究表明lrp负向调控溶藻弧菌的毒力，其机制可能是通过降低菌株的群体迁动能力、生物被膜形成能力、胞外蛋白酶分泌活性与铜离子耐受能力实现；同时lrp的表达受多种理化因子调控，尤其在细菌生长初期（培养2小时）会显著上调。