转录因子SlyA调控腐败希瓦氏菌生物被膜形成的分子机制研究

Shewanella putrefaciens has been widely applied in environmental remediation and microbial fuel cell, and the biofilm formation can improve the efficiency of environmental remediation and power generation. Moreover, Shewanella putrefaciens is a common opportunistic pathogen and food spoilage bacteria, and biofilm make it hard to be cleaned up, which seriously threaten human health and food safety. However, the study of biofilm formation by Shewanella putrefaciens is still in its infancy. In our previous study, we found for the first time that core transcriptional factor SlyA significantly inhibits biofilm formation by regulating intracellular c-di-GMP level. However, the specific signal transduction pathways involved by SlyA remain unclear. This proposal aims to look for the downstream target genes regulated by SlyA through the methods of ChIP-seq and EMSA, to analyze regulatory patterns of SlyA on target genes by DNase I footprinting and 5′-RACE, to look for the upstream regulators that modulate the transcription of SlyA by DNA affinity capture assay and transposon mutagenesis technique, and to investigate the regulatory relationship between SlyA and c-di-GMP. This proposal can promote the studies on molecular mechanism of Shewanella putrefaciens biofilm formation and to establish theoretical basis for controlling biofilm formation.

腐败希瓦氏菌在环境修复和微生物燃料电池等领域具有广泛应用，生物被膜的形成能提高其环境修复效率和产电性能。同时腐败希瓦氏菌是常见的条件致病菌和食品腐败菌，生物被膜会使其难于被清除，严重威胁人类健康和食品安全。然而目前对腐败希瓦氏菌形成生物被膜的研究还处于起步阶段。在前期工作中，我们首次发现核心转录因子SlyA通过调控胞内c-di-GMP的含量显著抑制生物被膜的形成，但SlyA参与的具体的信号转导通路尚不清楚。本研究拟通过ChIP-seq，EMSA技术寻找SlyA调控的下游靶基因；利用5′-RACE和DNase I足迹法分析SlyA对靶基因的调控模式；通过DNA亲和捕获和启动子探测技术寻找调控slyA转录的上游调控因子；同时研究SlyA与c-di-GMP之间的相互调控关系，进而揭示SlyA调控腐败希瓦氏菌形成生物被膜的分子机制，为其生物被膜的充分利用和解决其生物被膜引起的危害奠定理论基础

腐败希瓦氏菌（S. putrefaciens CN32）是重要的食品腐败菌，同时在环境修复和微生物发电等多个领域具有广泛的应用。生物被膜的形成会显著增加其作为腐败菌和致病菌的耐药性，同时也可以增加其作为环境修复菌对有毒污染物质的耐受能力，提高其在微生物染料电池中的发电效率。MarR家族转录因子SlyA在调控S. putrefaciens CN32生物被膜形成过程中起着重要作用，然而其调控生物被膜形成的分子机制尚不清楚。.针对这一科学问题，本研究首先利用qPCR技术、EMSA实验和ChIP-seq技术发现转录因子SlyA调控其下游bpfA操纵子的转录，进而调控生物被膜的表型；通过DNA亲和捕获技术、SDS-PAGE和质谱检测鉴定到全局调控转录因子CRP蛋白调控slyA基因的转录；由于CRP是第二信使cAMP的受体蛋白，而且转录因子SlyA调控胞内第二信使c-di-GMP的含量，因此我们分析了第二信使cAMP与c-di-GMP之间的交叉调控作用，发现cAMP与c-di-GMP可以通过他们的受体蛋白CRP和BpfD的直接互作，调控BpfAGD系统调控S. putrefaciens CN32生物被膜的形成。本研究最终找到了SlyA调控的下游靶基因和调控slyA基因转录的上游调控因子，建立了一条信号传递途径，初步揭示了MarR家族转录因子SlyA调控S. putrefaciens CN32形成生物被膜的分子机制，为人为调控S. putrefaciens CN32生物被膜的形成奠定了理论基础。.在本项目的支持下，项目主持人以第一作者共发表SCI论文2篇，其中在自然指数期刊Nature Communications发表论文1篇，该文入选了亮点文章；另一成果发表在Frontiers in Microbiology上，本项目执行期间参加学术会议1次，较好地完成了本项目原定任务。