摘要
【目的】探究铜绿假单胞菌(Pseudomonas aeruginosa)鸟苷酸环化酶(diguanylate cyclase,DGC)SadC合成的环二鸟苷酸(cyclic di-GMP,c-di-GMP)信号与PilZ结构域受体间的信号传递关系,分析鉴定出特定PilZ结构域受体的调控功能和机制。【方法】SadC突变株和过表达菌株的构建及泳动能力分析;SadC过表达背景下,PilZ结构域受体突变各菌株的泳动表型分析和筛选;基因敲除和过表达解析筛选出的PilZ结构域受体功能;定点突变和遗传互补检测筛选出的PilZ结构域受体是否参与SadC合成c-di-GMP对泳动能力的调控。【结果】SadC通过影响鞭毛功能而非鞭毛形成抑制铜绿假单胞菌的泳动能力;PilZ结构域受体突变菌株筛选发现PilZ、FlgZ这2个受体参与了SadC介导的泳动能力抑制;功能分析发现ΔpilZ或ΔflgZ的泳动能力相比野生型PA14显著增强,而过表达PilZ或FlgZ则抑制了泳动能力;定点突变和回补实验发现PilZ第10位和FlgZ第140位氨基酸R对其介导SadC负调控泳动能力至关重要,多序列比对分析表明这些位点是其保守的c-di-GMP结合位点。【结论】SadC合成的c-di-GMP信号通过PilZ和FlgZ调控铜绿假单胞菌的泳动能力。
[Objective]To identify the PilZ domain-containing receptor(s)that sense the second messenger cyclic di-GMP(c-di-GMP)produced by the diguanylate cyclase SadC in Pseudomonas aeruginosa and investigate the functions and regulatory mechanisms of the identified receptor(s).[Methods]We constructed the strains in which sadC gene was deleted or overexpressed and tested their ability to swim by using a plate-based approach.We then added sadC in multicopy in each deletion mutant of the eight PilZ domain-containing receptors and screened for the mutants with alleviated swimming repression compared to the wild-type PA14 overexpressing SadC.For the mutations screened out,single gene knockout and overexpression strategies were used to explore the function of the identified receptor(s).Furthermore,site-directed mutagenesis and genetic complementation were employed to test whether the identified receptor’s role in SadC-mediated swimming repression requires its c-di-GMP-binding motif.[Results]The SadC-mediated repression of swimming motility was associated with flagellar malfunction rather than flagellum formation.Two PilZ domain-containing receptors,PilZ and FlgZ,were identified to be involved in SadC-mediated swimming repression.The deletion of gene pilZ or flgZ increased the swimming motility,while overexpression of them significantly impaired swimming.A R10A substitution in the conserved c-di-GMP-binding motif of PilZ,or a R140A substitution in FlgZ,resulted in a variant that was no longer able to repress swimming inΔpilZ orΔflgZ overexpressing SadC,indicating that the conserved residue required for c-di-GMP binding is critical for PilZ or FlgZ to repress swimming in response to SadC-derived c-di-GMP.[Conclusion]PilZ and FlgZ are the effector relay proteins that respond to SadC c-di-GMP signaling to mediate swimming repression in P.aeruginosa.
作者
董友明
林心铭
付春情
王倩琪
吴玲娟
卢冰倩
林羲
包其郁
李科伟
DONG Youming;LIN Xinming;FU Chunqing;WANG Qianqi;WU Lingjuan;LU Bingqian;LIN Xi;BAO Qiyu;LI Kewei(Key Laboratory of Laboratory Medicine,Ministry of Education,School of Laboratory Medicine and Life Sciences,Wenzhou Medical University,Wenzhou 325035,Zhejiang,China)
出处
《微生物学报》
CAS
CSCD
北大核心
2023年第3期1115-1127,共13页
Acta Microbiologica Sinica
基金
浙江省自然科学基金(LY21C010004)
浙江省大学生科技创新活动计划(新苗人才计划)(2020R413040)
温州市基础性科研项目(Y20210082)。
