Study On The Role Of Fap Gene Cluster In Biofilm Formation And Its Expression Regulation Mechanism Of Pseudomonas Fluorescens

Research background:Biofilm is an important cause of food spoilage and pathogen contamination.As a representative food spoilage bacterium in high-protein fresh food with aerobic refrigeration,Pseudomonas fluorescens has a strong biofilm forming ability,causing huge hidden dangers to food quality and safety.The extracellular matrix is essential for the biofilm formation of food spoilers.Pseudomonas fluorescens PF07 was previously isolated from spoiled marine fish.PF07 has a strong biofilm forming ability,and can form mature macrocolony biofilms,solid surface-associated（SSA,solid surface-associated）biofilms,and pellicles.However,the genes involved in the extracellular matrix formation of PF07 biofilm and the molecular mechanism of its synthesis and regulation remain poorly defined,which is not conducive to the effective control of biofilm in food.Research objective:Selecting Pseudomonas fluorescens PF07 as the research object to explore the mechanism for the formation of biofilm.This study elucidates the role of fap gene clusters of Pseudomonas fluorescens in biofilm formation and the transcriptional regulation mechanism of the new transcription factor Brf A on fap gene clusters,providing new ideas and new targets for the effective control of biofilm in food.Research methods:The differentially expressed genes in PF07＿5d compared with PF07＿1d were screened and identified by RNA-seq-dependent transcriptomic analysis.The5’-RACE method was performed to identify the transcriptional start site of fap A.Three deletion mutants of fap C,rpo N and brf A（a novel gene coding for an Rpo N-dependent transcriptional regulator,which we have named brf A for“biofilm regulator of fap”）were constructed by homologous recombination method.Macrocolony biofilms were observed by Congo red assay and transmission electron microscopy（TEM）.Pellicles were assayed by visual inspection of the air-liquid interface of the standing culture.The ability of the bacteria to form SSA biofilms in plastic microplates was analyzed by crystal violet staining.To further confirm the regulatory effect of Brf A on fap gene clusters,the full-length brf A gene including its own promoter and the N-terminal REC domain deletion brf A gene were added intoΔbrf A via the expression vector p BBR1MCS-5.The full-length complement strainΔbrf A+MCS5-brf A and partial complement strainΔbrf A+MCS5-brf A^ΔRECwere constructed.In addition,Brf A,Brf A^ΔREC and Rpo N proteins were induced the expression and purification by E.coli prokaryotic expression system,and detected the binding activity of Brf A,Brf A^ΔREC and Rpo N proteins to the transcriptional regulatory sequence of fap A gene and the identifiable target sequence by EMSA assay.Further,we constructed the p MCS5-lac Z promoter activity reporter vector,inserted the full-length fap A regulatory sequence and the fap A regulatory sequence with Site1,Site2 and Site3 deletion respectively into the lac Z reporter gene,and introduced the reporter vector into PF07.Promoter activity was detected byβ-galactosidase activity.All data were analyzed by SPSS software,P<0.01was considered significant difference.Research results:A total of 1,403 DEGs were identified,including 641 significantly upregulated genes and 762 significantly downregulated genes in PF07＿5d,compared with PF07＿1d.In addition,159 DEGs were identified by comparing the macrocolony biofilms of rpo N mutant and PF07 on day 5,including 29 significantly upregulated genes and 130significantly downregulated genes.The gene cluster fap A-E,encoding functional amyloids,was significantly upregulated in PF07＿5d compared with PF07＿1d,and significantly downregulated inΔrpo N compared with PF07.The operon fap A-E had a-24/-12 promoter dependent on the sigma factor Rpo N.The gene cluster fap A-E is likely to be involved in the formation of biofilm matrix and is positively regulated by Rpo N.In addition,the deletion mutants of fap C,rpo N,and brf A were defective in forming mature macrocolony biofilms,solid surface-associated biofilms,and pellicles,and they showed significantly reduced biofilm matrices.The fap genes were significantly downregulated inΔbrf A,as inΔrpo N.Full-length complemented strainΔbrf A+MCS5-brf A could basically restore the phenotype of wild type PF07 on Congo red plate,and the partial complemented strainΔbrf A+MCS5-brf A^ΔREC appeared to form a wrinkled macrocolony biofilm in Congo red plate earlier than wild type and full-length complemented strain,and the colony was larger.EMSA assay showed that Brf A,Brf A^ΔREC and Rpo N can bind to the transcriptional regulatory region of fap A,and Brf A can specifically bind to three 20-bp conserved sequences of Site1,Site2 and Site3 in the regulatory region.We found that the activities of the promoters without Site1,Site2,or Site3 were significantly reduced.Research conclusion:In summary,we found that the functional amyloid Fap is the main component of PF07 biofilm matrices.In this study,the newly identified transcription factors Brf A and Rpo N directly regulate the transcription of the fap gene clusters and the biofilm formation.The N-terminal REC domain of Brf A does not affect its binding activity to regulatory sequence of fap A.Brf A can specifically recognize 3 enhancer sequences of fap A,and all 3 sequences are essential for promoter activity.