Role Of C-di-GMP And FleN In Regulation Of Swimming Motility And Biofilm Formatiom In Pseudomonas Putida KT2440

Pseudomonas putida KT2440 is a non-pathogenic Gram-negative bacterium with a wide range of metabolic diversity and strong environmental adaptability and plays an important role in production and application practice,such as bioremediation of environmental pollutants and production of special-purpose chemical products.Biofilm forming-ability and swimming ability are important abilities for bacteria to adapt to complex environments,and are also closely related to the application of KT2440 in production practice.Studying and understanding the regulation mechanism of KT2440 biofilm formation and swimming motility would help and guide the application of this strain in actual production practice.Cyclic-di-guanidine monophosphate（c-di-GMP）is a broadly conserved signaling molecule that regulates various physiological activities in bacteria,especially governs the transition between planktonic and biofilm states.In P.putida KT2440,42 genes encoding c-di-GMP synthesis/degradation domain proteins were identified.For the present,except for four proteins whose enzymatic activity and role in biofilm formation and swimming motility had been confirmed,the function of the remaining proteins remains unclear.In terms of the regulatory mechanisms,the c-di-GMP receptor Fle Q regulates the biofilm matrix-related genes expression in response to intracellular c-di-GMP level,thus affecting the biofilm formation.Fle N functions as a major cotroller of flagellar numbers and often works as an antagonist of Fle Q,participating in the physiological processes regulated by Fle Q,such as biofilm formation and swimming motility.However,whether Fle N regulates the biofilm formation of KT2440,and how Fle N interacts with Fle Q and c-di-GMP in regulating the biofilm formation remain unclear.Our study focused on the role of 42 c-di-GMP metabolic enzymes on biofilm formation and swimming motility of P.putida KT2440 and the regulation mechanism of Fle N on biofilm formation.The following conclusions were derived:1.Systematic analysis of the influences of 42 predicted diguanylate cyclases and phosphodiesterases on the biofilm formation and swimming motility in P.putida KT2440By analyzing the annotated genome of P.putida KT2440,we identified 42 potential c-di-GMP metabolic enzymes encoding genes.Of these,19 encode proteins with GGDEF domain,six encode proteins with EAL or HD-GYP domain,17 encodes proteins with dual GGDEF-EAL domain.Further analysis revealed that 37 of the 42 predicted c-di-GMP metabolic enzymes were coupled with signal-sensing and signal transduction domains,indicating that these c-di-GMP metabolic enzymes are closely related to the external environmental signal response.To investigate the role of 42 predicated c-di-GMP metabolic enzymes in regulating the biofilm formation and motility of P.putida KT2440,we systematically constructed 42 deletions and overexpression mutants targeting each gene involved in c-di-GMP metabolism in P.putida KT2440 and tested their biofilm formation and swimming motility.Our results showed that only four genes（PP₀165,PP₀218,PP₀914,and PP₁494）mutatated significantly affected biofilm formation.Of the four genes,three was reported previously,while PP₀218 was not.PP₀218 is a protein with a dual GGDEF-EAL domain,and deletion of PP₀218 significantly inhibited the biofilm formation.while 17 genes overexpressed obviously produced promotion on biofilm formation.As far as the 42 mutants and 42 overexpressed strains were concerned,the majority strains with mutation or overexpression in the c-di-GMP metabolic genes had an evident influence on swimming motility,indicating that swimming motility was more sensitive to cellular c-di-GMP concentrations relative to biofilm formation.In addition,we also examined the effect of the overexpression of these c-di-GMP metabolic genes on the transcription of the biofilm matrix（lap A and bcs）and flagella-related genes（fle SR and fli M）,and make a comparision between the phenotype variation and corresponding gene expression levels.Seen from the overall results,the strains with strong biofilm forming-ability produced impaired motility.Besides,the genes associated with biofilm formation had increased expression,while genes associated with flagellar biosynthesis had decreased expression.However,for some individual strains,there was insistence between phenotype and corresponding genes expression.For example,overexpression of PP₀369,PP₃581 and PP₃663 significantly affected biofilm formation and swimming motility,but had no influence on biofilm and flagella-related genes expression,indicating that the influence of the overexpression of these genes on biofilm formation may not be caused by altering gene transcription,but by affecting the protein translation,protein secretion or protein-protein interaction at the post-transcriptional level.These results provide us with a basic understanding of the 42 genes in regulating biofilm formation and swimming motility.Moreover,these results also offer us clues to predict the metabolic activity of those proteins,which lay the foundation for further study on function of c-di-GMP metabolic enzymes and regulation of intracellular c-di-GMP level in KT2440.2.Fle N and Fle Q/C-di-GMP play a synergistic role in regulating biofilm matrix encoding genes lapA and bcs expression in P.putida KT2440Previous study confirmed that Fle Q promoted P.putida KT2440 biofilm formation through directly regulating biofilm-related genes lap A and bcs expression.Here we found that in P.putida KT2440,Fle N and Fle Q play a synergistic role in regulating two biofilm matrix coding operons,lap A and bcs.Fle N deletion decreased transcription of lap A and increased transcription of bcs operon,the same trend was observed in fle Q deletion mutant before.In vitro EMSA experiments showed that Fle N promoted binding of Fle Q to the lap A/bcs promoter DNA especially in the presence of ATP.Besides,we constructed a mutated Fle N with a point mutation in the putative Fle N ATP binding motif（21K→Q）and observed influence of pointed Fle N on binding of Fle Q to lap A and bcs promoters.The results showed that the mutaed Fle N lost promotion effect on Fle Q binding to the DNA promoter,suggesting that the ATP binding motif of Fle N was of importance for Fle N interacting with Fle Q.In addition,we also observed influence of c-di-GMP on biofilm formation in the fle N mutant.Both phenotype observation and transcription analysis showed that,similar to fle Q deletion,fle N deletion significantly weaken the effect of high c-di-GMP level on biofilm formation,surface winkle phenotype and expression of lap A and bcs operons,indicating that c-di-GMP relied on both Fle Q and Fle N to regulate biofilm formation of KT2440.Our results initially characterize the role of 42 c-di-GMP metabolic enzymes in regulating biofilm and swimming motility,and confirmed the role and mechanism of Fle N in regulation of biofilm formation in P.putida KT2440.Our results offer us clues for further study on function of c-di-GMP and its metabolic enzymes.