Function Of C-di-GMP And Two C-di-GMP Metabolic Enzymes,BifA And GcbA,in Regulation Of Biofilm Formatiom And Swimming Motility In Pseudomonas Putida KT2440

Pseudomonas putida KT2440 is a biosafety gram negative model strain with wide range of metabolic diversity and strong adaptability to different environments,has been used as workhorse in discovering general basal metabolic pathway.Besides,KT2440 and its derived strains have been widely exploited in development of several biotechnological applications since isolation,such as bioremediation,production of specific chemicals,etc.Biofim formation and swimming motility are important abilities for bacteria survival in complex environments,and also shown closely related to its efficiency in production practice,thus study on the regulation of biofilm formation and swimming motility of KT2440 would help and guide the applications in the actual production of this strain.Cyclic di-GMP is an intracellular secondary messenger that is involved in a diverse range of cellular processes in bacteria.The most intensively studied process that c-di-GMP participated in is the transition between motile and sessile lifestyles.C-di-GMP promotes expression of biofilm matrix coding genes,and decreases expression of flagellar and motility related genes.However,types of extracellular matrix molecules are highly diverse in different bacteria,and whether and how c-di-GMP influence expression of biofilm matrix coding genes are still unclear.The cellular c-di-GMP level is inversely governed by diguanylate cyclase?DGC?enzymes and phosphodiesterase?PDE?enzymes,which synthesize and degrade c-di-GMP,respectively.Bif A is a conserved PDE in Pseudomonas,and plays an essential role in maintain the intracellular c-di-GMP concentration.Gcb A is a conserved DGC in Pseudomonas,former studies revealed that both Bif A and Gcb A involved in biofilm formation and swimming motility,but knowledge about regulation of the two emzymes are very poor.Our study focused on the regulation of c-di-GMP on biofilm formation,and regulation of the two c-di-GMP metabolic enzymes and their role in biofilm formation and swimming motility.The following conclusions were derived:1.C-di-GMP regulates the expression of two biofilm matrix coding operons,lapA and bcs,via Fle Q in Pseudomonas putida KT2440Former studies indicated that c-di-GMP positively modulated the production of biofilm matrix components from the transcriptional to the post-translational level in a variety of bacterial species.However,mechanisms by which it regulates these components in Pseudomonas putida KT2440 remain unclear.In this study,we confirmed the function of c-di-GMP in regulation of biofilm formation in KT2440 and found the same pattern as in other bacteria: Higher c-di-GMP level promoted biofilm formation and decreased swimming motility,while lower c-di-GMP level depressed biofilm formation and enhanced swimming motility.Then by comparing expression levels of biofilm matrix coding genes,we found that c-di-GMP regulated the adhesin Lap A,Lap F and exopolysaccharides Bcs,Pea at transcriptional level.Transcriptional regulator Fle Q is required for the modulation of lap A and bcs expression by c-di-GMP,but seemed not to be necessary for that of lap F and pea.We also found that fle Q mutant of P.putida was defective in biofilm formation and had smooth colony morphology.Transcription assay indicates that Fle Q acted as an activator of lap A,but a repressor of bcs.In vitro experiments showed that Fle Q binds to lap A and bcs promoter DNA.The binding to lap A promoter was slightly promoted by c-di-GMP,while binding to bcs promoter was inhibited by c-di-GMP.2.Expression of the phosphodiesterase Bif A facilitating swimming motility is partly controlled by Fli AFlagella-mediated motility is an important capability of many bacteria to survive in nutrient-depleted and harsh environments.Phosphodiesterase Bif A is involved in c-di-GMP concetration in Pseudomonas,bif A deletion caused an obvious increase in intracellular c-di-GMP level.Former study suggested that expression of Bif A was influenced by flagellar sigma factor Fli A(?²⁸)in KT2440.Here we confirmed that expression of Bif A was partly controlled by Fli A,Fli A deletion led to an approximately twofold decrease in transcription of bif A.5' race assay revealed two transcription start points in bif A promoter region,with the putative ?70 and ?28 promoter sequences upstream,respectively.Point mutations in-10 and spacer regions of ?28 promoter region significantly reduced the promoter activity,though the mutation in-35 region had no obvious impact on the promoter activity.Fli A overexpression decreased the intracellular c-di-GMP level in a Bif A dependent way,suggesting that Fli A was able to modulate intracellular c-di-GMP level and Bif A function was required for the modulation.Besides,Fli A overexpression enhanced swimming ability of wild type strain,while made no difference to the bif A mutant.Our results suggest that Fli A acts as a negative regulator to modulate c-di-GMP level via controlling transcription of bif A to facilitate swimming motility.3.Expression of the diguanylate cyclase Gcb A is regulated by FleQ in response to cyclic di-GMPGcb A is a well conserved DGC among Pseudomonas species,and has been reported to influence biofilm formation and flagellar motility in Pseudomonas fluorescens and Pseudomonas aeruginosa.Here by homology comparison,we found the gcb A homologous gene in KT2440,pp₀563,and we confirmed the function of pp₀563 in initial biofilm formation,biofilm maturation and swimming motility was similar to that in Pseudomonas fluorescens and Pseudomonas aeruginosa,thus we named PP₀563 Gcb A.Besides,our results revealed that expression of Gcb A was regulated by Fle Q in response to c-di-GMP.Deletion of the c-di-GMP effector Fle Q led to a significant decrease in transcription of gcb A.Moreover,reducing c-di-GMP levels promoted gcb A transcription in a Fle Q dependent way,while enhancing c-di-GMP levels abolished the promotion.In in vitro experiments we found that Fle Q bound to gcb A promoter DNA and the binding was inhibited by c-di-GMP.Besides,Fle N,an anti-activator of Fle Q,and the sigma factor Rpo N also participated in transcription of gcb A.ATPase of Fle Q and Rpo N were required for full expression of gcb A and lap A,and were strictly needed for expression of fle SR and fli F,but were totally not demanded for expression of bcs operon.This finding expands the complexity of Fle Q-dependent regulation and reveals a self-regulation function of c-di-GMP by regulating Gcb A expression via Fle Q.Our results confirmed the role and mechanism of c-di-GMP in regulation of biofilm formation in Pseudomonas putida KT2440,and revealed role and express regulation of two c-di-GMP metabolic enzymes.Our results lay the foundation for furture study on function of c-di-GMP and its metabolic enzymes,and regulation of intracellular c-di-GMP level in KT2440.