铜绿假单胞菌antABC操纵子的生物学效应分析  

Biological effects of antABC operon on Pseudomonas aeruginosa

作　　者：李治盈 李盼欣 宁婉清 李兰 韩嘉仪 成娟丽 林金水 LI Zhiying;LI Panxin;NING Wanqing;LI Lan;HAN Jiayi;CHENG Juanli;LIN Jinshui(Shaanxi Key Laboratory of Research and Utilization of Resource Plants on the Loess Plateau,College of Life Sciences,Yan’an University,Yan’an 716000,Shaanxi,China)

机构地区：[1]延安大学生命科学学院,陕西省黄土高原资源植物研究与利用省市共建重点实验室,陕西延安716000

出　　处：《微生物学报》2024年第11期4403-4424,共22页Acta Microbiologica Sinica

基　　金：国家自然科学基金(32070103,31860012,32360015);陕西省秦创原“科学家+工程师”队伍建设项目(2023KXJ-019);陕西省“特支计划”区域发展人才项目(2020-44);陕西省普通高等学校青年杰出人才支持计划(2018-111);陕西高校青年创新团队项目(2022-943);陕西省大学生创新创业训练项目(S202210719127,S202310719142);延安大学科研计划(2023HBZ-001,2023CGZH-007)。

摘　　要：在铜绿假单胞菌中,色氨酸可通过KynABU犬尿氨酸途径被转化邻氨基苯甲酸,邻氨基苯甲酸进一步作为底物在烷基喹诺酮类化合物(alkylquinolones,AQs)合成基因簇pqsABCDE的作用下被转化为AQs信号分子,包括2-庚基-3-羟基-4-喹诺酮(Pseudomonas quinolone signal, PQS)和2-庚基-4-喹诺酮(2-heptyl-4-hydroxyquinoline, HHQ)。同时,邻氨基苯甲酸还能在邻氨基苯甲酸双加氧酶复合物AntABC的催化下被降解进入三羧酸循环,但AntABC在铜绿假单胞菌中所介导的生物学效应依然不清楚。【目的】构建铜绿假单胞菌antABC的缺失突变株,并对该突变株的表型进行分析。【方法】以铜绿假单胞菌PAO1为亲本菌株,通过同源重组的方法构建antABC缺失突变株,研究该操纵子对铜绿假单胞菌色氨酸降解、生物被膜形成、绿脓菌素合成、运动性和毒力等的影响。【结果】缺失突变antABC或kynU均完全抑制了铜绿假单胞菌以色氨酸为唯一碳源的生长,而ΔpqsA则无该表型,说明antABC是铜绿假单胞菌降解色氨酸所必需的,并且在本研究指定的培养条件下KynABU-AntABC途径是该菌降解色氨酸的唯一途径。除了影响铜绿假单胞菌的色氨酸降解,缺失突变antABC通过诱导胞外多糖合成操纵子pel的表达进而促进铜绿假单胞菌生物被膜的形成,还通过诱导绿脓菌素合成操纵子phz1和phz2的表达进而促进铜绿假单胞菌绿脓菌素的合成。缺失突变antABC还促进了铜绿假单胞菌的集群运动和蹭行运动。另外,进一步缺失突变pqsA则完全逆转ΔantABC的这些生理表型。因此,antABC对这些生理表型的调控依赖于AQs。然而缺失突变antABC确实使铜绿假单胞菌积累了更多的HHQ,但却抑制了PQS的合成。说明antABC对这些生理表型的调控主要依赖于HHQ。此外,缺失突变antABC还增强了铜绿假单胞菌对白菜和大蜡螟幼虫的毒力,但是进一步缺失突变pqsA只能部分逆转ΔantABC的这一In Pseudomonas aeruginosa,tryptophan can be converted into anthranilate via the KynABU pathway,and anthranilate as a substrate is further converted into alkyl quinolones(AQs),including 2-heptyl-3-hydroxy-4-quinolone(PQS)and 2-heptyl-4-quinolone(HHQ),under the action of pqsABCDE,a synthetic gene cluster of AQs.At the same time,anthranilate can be degraded into the tricarboxylic acid cycle under the catalysis of the anthranilate dioxygenase complex AntABC,while the biological effect of AntABC on P.aeruginosa remains unclear.[Objective]To construct and characterize the phenotype of the antABC-deleted mutant of P.aeruginosa.[Methods]With P.aeruginosa PAO1 as the starting strain,we constructed the antABC-deleted mutant by homologous recombination to study the effects of the operon on tryptophan degradation,biofilm formation,pyocyanin synthesis,motility,and virulence of P.aeruginosa.[Results]The deletion of antABC or kynU completely inhibited the growth of P.aeruginosa with tryptophan as the sole carbon source,whileΔpqsA did not present this phenotype,indicating that antABC was essential for the degradation of tryptophan by P.aeruginosa,and KynABU-AntABC pathway was the only way for the degradation of tryptophan by the bacterium under the culture conditions in this study.In addition to affecting tryptophan degradation in P.aeruginosa,the deletion of antABC promoted the biofilm formation of P.aeruginosa by inducing the expression of the extracellular polysaccharide synthesis operon pel,and it promoted the synthesis of pyocyanin by inducing the expression of the pyocyanin synthesis operons phz1 and phz2.In addition,the deletion of antABC enhanced the swarming motility and twitching motility of P.aeruginosa.Interestingly,further deletion of pqsA completely reversed the physiological phenotypes ofΔantABC.Therefore,the regulation of antABC on these physiological phenotypes depended on AQs.The deletion of antABC increased the HHQ accumulation while inhibiting the synthesis of PQS in P.aeruginosa.These results indicated tha

关 键 词：铜绿假单胞菌 AntABC 邻氨基苯甲酸 烷基喹诺酮类化合物 毒力 

分 类 号：R378[医药卫生—病原生物学]