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Cyclic diguanylate (c-di-GMP) is a broadly conserved bacterial signalling molecule that modulates diverse cellular processes, such as biofilm formation, colony morphology and swimming motility. The intracellular level of c-di-GMP is controlled by diguanylate cyclases (DGCs) with GGDEF domain and phosphodiesterases (PDEs) with either EAL or HD-GYP domain. Pseudomonas putida KT2440 has a large group of genes on its genome encoding proteins with GGDEF/EAL/HD-GYP domains. However, phenotypic-genotypic correlation and c-di-GMP metabolism of these genes were largely unknown. Herein, by systematically constructing deletion mutants/overexpression strains of the 42 predicted c-di-GMP metabolism-related genes and analysing the phenotypes, we preliminarily revealed the role of each gene in biofilm formation, colony morphology and swimming motility. Subsequent results from protein sequence alignments and cellular c-di-GMP assessment indicated that 25 out of the 42 genes were likely to encode DGCs, nine genes were predicted to encode PDEs, four genes encoded bifunctional enzymes and the other four genes encoded enzymatically inactive proteins. This study offers a basic understanding of the roles of these 42 genes and can serve as a toolkit for investigators to further elucidate the functions of these GGDEF and EAL/HD-GYP domain-containing proteins.

FleN generally functions as an antagonist of FleQ in regulating flagellar genes and biofilm matrix related genes in Pseudomonas aeruginosa. Here, we found that in Pseudomonas putida KT2440, FleN and FleQ play a synergistic role in regulating two biofilm matrix coding operons, lapA and bcs. FleN deletion decreased the transcription of lapA and increased the transcription of bcs operon, and the same trend was observed in fleQ deletion mutant before. In vitro experiments showed that FleN promoted the binding of FleQ to the lapA/bcs promoter DNA especially in the presence of ATP. Both phenotype observation and transcription analysis showed that, similar to fleQ deletion, fleN deletion significantly weaken the effect of high c-di-GMP level on biofilm formation, surface winkle phenotype and expression of lapA and bcs operons. Mutagenesis of the putative ATP binding motif in FleNK21Q revealed that FleN ATPase activity played an essential role in the regulation of flagellar number and swimming motility but was not critical for biofilm formation. Our results revealed that FleN was not an antagonist of FleQ but a synergistic factor of FleQ in regulating the two biofilm matrix coding operons in P. putida KT2440.

Cyclic di-GMP (c-di-GMP), a ubiquitous bacterial second messenger that regulates diverse cellular processes, is synthesized by diguanylate cyclase (DGC) and degraded by phosphodiesterase (PDE). GcbA 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 we confirm the function of GcbA in Pseudomonas putida and reveal that expression of GcbA is regulated by FleQ in response to c-di-GMP. GcbA deletion impaired initial biofilm formation and enhanced swimming motility, but showed no influence on biofilm maturation in Pseudomonas putida. Deletion of the c-di-GMP effector FleQ led to a significant decrease in transcription of gcbA. Moreover, reducing c-di-GMP levels promoted gcbA transcription in a FleQ dependent way, while enhancing c-di-GMP levels abolished the promotion. In in vitro experiments we found that FleQ bound to gcbA promoter DNA and the binding was inhibited by c-di-GMP. Besides, FleN, an anti-activator of FleQ, and the sigma factor RpoN also participated in transcription of gcbA. Our finding expands the complexity of FleQ-dependent regulation and reveals a self-regulation function of c-di-GMP by regulating GcbA expression via FleQ.

Cyclic diguanylate (c-di-GMP) positively modulates 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 opponents in Pseudomonas putida KT2440 remain unclear. Here we show that c-di-GMP regulates the adhesin LapA, LapF and exopolysaccharides Bcs, Pea at transcriptional level. Transcriptional regulator FleQ is required for the modulation of lapA and bcs expression by c-di-GMP, but seems not to be necessary for that of lapF and pea. We also found that fleQ mutant of P. putida was defective in biofilm formation and had smooth colony morphology. Transcription assay indicates that FleQ acts as an activator of lapA, but a repressor of bcs. In vitro experiments show that FleQ binds to lapA and bcs promoter DNA. The binding to lapA promoter was slightly promoted by c-di-GMP, while binding to bcs promoter was inhibited by c-di-GMP. Our results show that c-di-GMP regulates the expression of lapA and bcs operons via FleQ in P. putida.

FleN generally functions as an antagonist of FleQ in regulating flagellar genes and biofilm matrix related genes in Pseudomonas aeruginosa. Here, we found that in Pseudomonas putida KT2440, FleN and FleQ play a synergistic role in regulating two biofilm matrix coding operons, lapA and bcs. FleN deletion decreased the transcription of lapA and increased the transcription of bcs operon, and the same trend was observed in fleQ deletion mutant before. In vitro experiments showed that FleN promoted the binding of FleQ to the lapA/bcs promoter DNA especially in the presence of ATP. Both phenotype observation and transcription analysis showed that, similar to fleQ deletion, fleN deletion significantly weaken the effect of high c-di-GMP level on biofilm formation, surface winkle phenotype and expression of lapA and bcs operons. Mutagenesis of the putative ATP binding motif in FleN

Cyclic di-GMP (c-di-GMP), a ubiquitous bacterial second messenger that regulates diverse cellular processes, is synthesized by diguanylate cyclase (DGC) and degraded by phosphodiesterase (PDE). GcbA 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 we confirm the function of GcbA in Pseudomonas putida and reveal that expression of GcbA is regulated by FleQ in response to c-di-GMP. GcbA deletion impaired initial biofilm formation and enhanced swimming motility, but showed no influence on biofilm maturation in Pseudomonas putida. Deletion of the c-di-GMP effector FleQ led to a significant decrease in transcription of gcbA. Moreover, reducing c-di-GMP levels promoted gcbA transcription in a FleQ dependent way, while enhancing c-di-GMP levels abolished the promotion. In in vitro experiments we found that FleQ bound to gcbA promoter DNA and the binding was inhibited by c-di-GMP. Besides, FleN, an anti-activator of FleQ, and the sigma factor RpoN also participated in transcription of gcbA. Our finding expands the complexity of FleQ-dependent regulation and reveals a self-regulation function of c-di-GMP by regulating GcbA expression via FleQ.

Cyclic diguanylate (c-di-GMP) positively modulates 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 opponents in Pseudomonas putida KT2440 remain unclear. Here we show that c-di-GMP regulates the adhesin LapA, LapF and exopolysaccharides Bcs, Pea at transcriptional level. Transcriptional regulator FleQ is required for the modulation of lapA and bcs expression by c-di-GMP, but seems not to be necessary for that of lapF and pea. We also found that fleQ mutant of P. putida was defective in biofilm formation and had smooth colony morphology. Transcription assay indicates that FleQ acts as an activator of lapA, but a repressor of bcs. In vitro experiments show that FleQ binds to lapA and bcs promoter DNA. The binding to lapA promoter was slightly promoted by c-di-GMP, while binding to bcs promoter was inhibited by c-di-GMP. Our results show that c-di-GMP regulates the expression of lapA and bcs operons via FleQ in P. putida.