信号分子AI-2合成关键基因luxS对嗜盐喜盐芽孢杆菌盐适应性的影响  

Effects of the critical gene luxS related to synthesis of AI-2 on salt adaptability of Halobacillus halophilus

作　　者：王卓 王传旭 杨静 李云杰 牛亚洁 李新[1] WANG Zhuo;WANG Chuanxu;YANG Jing;LI Yunjie;NIU Yajie;LI Xin(Shanxi Key Laboratory of Yuncheng Salt Lake Ecological Protection and Resource Utilization,Yuncheng University,Yuncheng 044000,Shanxi,China)

机构地区：[1]运城学院运城盐湖生态保护与资源利用厅市共建山西省重点实验室,山西运城044000

出　　处：《微生物学通报》2024年第11期4517-4534,共18页Microbiology China

基　　金：国家自然科学基金(32300111);山西省科技创新人才团队项目(202204051001035);山西省基础研究计划(202303021212258);运城市基础研究计划(YCKJ-2023030);博士来晋科研专项(QZX-2023006);运城学院博士启动基金(YQ-2023005);运城学院应用研究项目(YY-202302)。

摘　　要：【背景】群体感应信号分子AI-2是由S-核糖基高半胱氨酸裂解酶(S-ribosylhomocysteinase,LuxS)和S-腺苷高半胱氨酸核苷酶(S-adenosylhomocysteine nucleosidase,Pfs,也被称为MtnN)催化S-腺苷高半胱氨酸(S-adenosylhomocysteine,SAH)生成的4,5-二羟基-2,3-戊二酮(4,5-dihydroxy-2,3-pentanedione,DPD)自发环化形成,在细菌中广泛参与调节多种重要的生理过程,如趋化、生物发光、生物膜形成等;然而该信号分子对嗜盐喜盐芽孢杆菌盐适应性的影响尚未报道。【目的】对嗜盐喜盐芽孢杆菌(Halobacillus halophilus)群体感应信号分子AI-2进行体外合成并揭示AI-2对嗜盐喜盐芽孢杆菌盐适应性的影响。【方法】以嗜盐喜盐芽孢杆菌为研究对象,首先通过实时定量PCR分析luxS转录水平与盐浓度之间的联系;之后对LuxS、MtnN-1、MtnN-2、MtnN-3进行多序列比对分析寻找其关键氨基酸位点并通过异源表达纯化4种蛋白进行AI-2的体外合成;最后使用同源重组方法敲除luxS并通过盐胁迫、胞内相容性溶质含量检测、生物膜形成检测等试验研究luxS对嗜盐喜盐芽孢杆菌盐适应性的影响。【结果】实时定量分析表明嗜盐喜盐芽孢杆菌中luxS的转录水平随盐浓度上升而上调,而且受到Cl离子浓度的正向调控。生长曲线结合生物发光试验表明随着嗜盐喜盐芽孢杆菌的生长,培养液上清中AI-2活性在15 h达到最大,此外随着盐浓度的提高,其AI-2活性也会增强。体外酶促反应试验表明LuxS能够与MtnN-1或MtnN-2共同催化合成AI-2。利用同源重组技术成功构建了嗜盐喜盐芽孢杆菌luxS缺失突变体,在低盐条件下(0.5 mol/L NaCl)野生型、luxS缺失突变体和互补菌株的生长曲线无明显差异,而在高盐条件下(3.5 mol/L NaCl)luxS突变体的生长较为缓慢,互补菌株则能够恢复到与野生型相似的生长状况。此外,luxS的缺失会导致嗜盐喜盐芽孢杆菌在高盐条件下的盐胁迫存活率、�[Background]The quorum sensing signal molecule AI-2 is spontaneously cyclized from 4,5-dihydroxy-2,3-pentanedione(DPD),which is derived from S-adenosylhomocysteine(SAH)via catalysis by S-ribosylhomocysteinase(LuxS)and S-adenosylhomocysteine nucleosidase(Pfs,also known as MtnN).AI-2 regulates a variety of physiological processes including chemotaxis,bioluminescence,and biofilm formation of bacteria.However,the effect of AI-2 on the salt adaptability of Halobacillus halophilus has not been reported.[Objective]To synthesize AI-2 in vitro and reveal its effect on the salt adaptability of H.halophilus.[Methods]The relationship between luxS transcript level in H.halophilus and salt concentration was assessed by real-time quantitative PCR.Then,multiple sequence alignments of LuxS,MtnN-1,MtnN-2,and MtnN-3 were performed to identify the key amino acid sites,and the four proteins were heterologously expressed and purified for the synthesis of AI-2 in vitro.Finally,luxS was knocked out by homologous recombination,and the effect of luxS on the salt adaptability of H.halophilus was studied through salt stress assay,intracellular compatible solute content determination,and biofilm formation assay.[Results]The transcript level of luxS in H.halophilus increased with the elevation in salt concentration and was positively regulated by the Cl–concentration.The growth curve and bioluminescence assay showed that as H.halophilus grew,the AI-2 activity in the supernatant of the culture medium reached the maximum at 15 h,and it enhanced with the increase in salt concentration.The in vitro enzymatic reaction assay showed that LuxS collaborated with MtnN-1 or MtnN-2 to catalyze the synthesis of AI-2.The luxS-deleted mutant of H.halophilus was successfully constructed by homologous recombination.Under a low salt concentration(0.5 mol/L NaCl),there were no significant differences in the growth curves between the wild type,luxS-deleted mutant,and complementary strain.However,under high salt(3.5 mol/L NaCl),the luxS-deleted mutant grew slow

关 键 词：嗜盐喜盐芽孢杆菌 AI-2 LUXS 盐适应性 相容性溶质 

分 类 号：Q933[生物学—微生物学]