Miao Guo , Siqi Tan , Yinying Wu , Chongni Zheng , Peng Du , Junli Zhu , Aihua Sun , Xiaoxiang Liu
{"title":"BrfA 作为一种细菌增强子结合蛋白,通过感知环状单磷酸二鸟苷来调节荧光假单胞菌中功能性淀粉样蛋白 Fap 依赖性生物膜的形成。","authors":"Miao Guo , Siqi Tan , Yinying Wu , Chongni Zheng , Peng Du , Junli Zhu , Aihua Sun , Xiaoxiang Liu","doi":"10.1016/j.micres.2024.127864","DOIUrl":null,"url":null,"abstract":"<div><p>The functional amyloid of <em>Pseudomonas</em> (Fap) is essential for the formation of macrocolony biofilms, pellicles, and solid surface-associated (SSA) biofilms of <em>Pseudomonas fluorescens</em> PF07, an isolate from refrigerated marine fish. However, limited information on the expression regulation of <em>fap</em> genes is available. Herein, we found that a novel bacterial enhancer-binding protein (bEBP), BrfA, regulated Fap-dependent biofilm formation by directly sensing cyclic diguanosine monophosphate (c-di-GMP). Our <em>in vivo</em> data showed that the REC domain deletion of BrfA promoted <em>fap</em> gene expression and biofilm formation, and c-di-GMP positively regulated the transcription of <em>fapA</em> in a BrfA-dependent manner. In <em>in vitro</em> experiments, we found that the ATPase activity of BrfA was inhibited by the REC domain and was activated by c-di-GMP. BrfA and the sigma factor RpoN bound to the upstream region of <em>fapA</em>, and the binding ability of BrfA was not affected by either deletion of the REC domain or c-di-GMP. BrfA specifically bound to the three enhancer sites upstream of the <em>fapA</em> promoter, which contain the consensus sequence CA-(N4)-TGA(A/T)ACACC. <em>In vivo</em> experiments using a <em>lacZ</em> fusion reporter indicated that all three BrfA enhancer sites were essential for the activation of <em>fapA</em> transcription. Overall, these findings reveal that BrfA is a new type of c-di-GMP-responsive transcription factor that directly controls the transcription of Fap biosynthesis genes in <em>P</em>. <em>fluorescens</em>. Fap functional amyloids and BrfA-type transcription factors are widespread in <em>Pseudomonas</em> species. The novel insights into the c-di-GMP- and BrfA-dependent expression regulation of <em>fap</em> provided by this work will contribute to the development of antibiofilm strategies.</p></div>","PeriodicalId":18564,"journal":{"name":"Microbiological research","volume":null,"pages":null},"PeriodicalIF":6.1000,"publicationDate":"2024-07-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"BrfA functions as a bacterial enhancer-binding protein to regulate functional amyloid Fap-dependent biofilm formation in Pseudomonas fluorescens by sensing cyclic diguanosine monophosphate\",\"authors\":\"Miao Guo , Siqi Tan , Yinying Wu , Chongni Zheng , Peng Du , Junli Zhu , Aihua Sun , Xiaoxiang Liu\",\"doi\":\"10.1016/j.micres.2024.127864\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><p>The functional amyloid of <em>Pseudomonas</em> (Fap) is essential for the formation of macrocolony biofilms, pellicles, and solid surface-associated (SSA) biofilms of <em>Pseudomonas fluorescens</em> PF07, an isolate from refrigerated marine fish. However, limited information on the expression regulation of <em>fap</em> genes is available. Herein, we found that a novel bacterial enhancer-binding protein (bEBP), BrfA, regulated Fap-dependent biofilm formation by directly sensing cyclic diguanosine monophosphate (c-di-GMP). Our <em>in vivo</em> data showed that the REC domain deletion of BrfA promoted <em>fap</em> gene expression and biofilm formation, and c-di-GMP positively regulated the transcription of <em>fapA</em> in a BrfA-dependent manner. In <em>in vitro</em> experiments, we found that the ATPase activity of BrfA was inhibited by the REC domain and was activated by c-di-GMP. BrfA and the sigma factor RpoN bound to the upstream region of <em>fapA</em>, and the binding ability of BrfA was not affected by either deletion of the REC domain or c-di-GMP. BrfA specifically bound to the three enhancer sites upstream of the <em>fapA</em> promoter, which contain the consensus sequence CA-(N4)-TGA(A/T)ACACC. <em>In vivo</em> experiments using a <em>lacZ</em> fusion reporter indicated that all three BrfA enhancer sites were essential for the activation of <em>fapA</em> transcription. Overall, these findings reveal that BrfA is a new type of c-di-GMP-responsive transcription factor that directly controls the transcription of Fap biosynthesis genes in <em>P</em>. <em>fluorescens</em>. Fap functional amyloids and BrfA-type transcription factors are widespread in <em>Pseudomonas</em> species. The novel insights into the c-di-GMP- and BrfA-dependent expression regulation of <em>fap</em> provided by this work will contribute to the development of antibiofilm strategies.</p></div>\",\"PeriodicalId\":18564,\"journal\":{\"name\":\"Microbiological research\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":6.1000,\"publicationDate\":\"2024-07-31\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Microbiological research\",\"FirstCategoryId\":\"99\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/S0944501324002659\",\"RegionNum\":1,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"MICROBIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Microbiological research","FirstCategoryId":"99","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S0944501324002659","RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"MICROBIOLOGY","Score":null,"Total":0}
BrfA functions as a bacterial enhancer-binding protein to regulate functional amyloid Fap-dependent biofilm formation in Pseudomonas fluorescens by sensing cyclic diguanosine monophosphate
The functional amyloid of Pseudomonas (Fap) is essential for the formation of macrocolony biofilms, pellicles, and solid surface-associated (SSA) biofilms of Pseudomonas fluorescens PF07, an isolate from refrigerated marine fish. However, limited information on the expression regulation of fap genes is available. Herein, we found that a novel bacterial enhancer-binding protein (bEBP), BrfA, regulated Fap-dependent biofilm formation by directly sensing cyclic diguanosine monophosphate (c-di-GMP). Our in vivo data showed that the REC domain deletion of BrfA promoted fap gene expression and biofilm formation, and c-di-GMP positively regulated the transcription of fapA in a BrfA-dependent manner. In in vitro experiments, we found that the ATPase activity of BrfA was inhibited by the REC domain and was activated by c-di-GMP. BrfA and the sigma factor RpoN bound to the upstream region of fapA, and the binding ability of BrfA was not affected by either deletion of the REC domain or c-di-GMP. BrfA specifically bound to the three enhancer sites upstream of the fapA promoter, which contain the consensus sequence CA-(N4)-TGA(A/T)ACACC. In vivo experiments using a lacZ fusion reporter indicated that all three BrfA enhancer sites were essential for the activation of fapA transcription. Overall, these findings reveal that BrfA is a new type of c-di-GMP-responsive transcription factor that directly controls the transcription of Fap biosynthesis genes in P. fluorescens. Fap functional amyloids and BrfA-type transcription factors are widespread in Pseudomonas species. The novel insights into the c-di-GMP- and BrfA-dependent expression regulation of fap provided by this work will contribute to the development of antibiofilm strategies.
期刊介绍:
Microbiological Research is devoted to publishing reports on prokaryotic and eukaryotic microorganisms such as yeasts, fungi, bacteria, archaea, and protozoa. Research on interactions between pathogenic microorganisms and their environment or hosts are also covered.