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Two-component systems (TCSs) are predominant means by which bacteria sense and respond to environment signals. Genome of Pseudomonas putida contains dozens of putative TCS-encoding genes, but phenotypical-genotypical correlation and transcriptional regulation of these genes are largely unknown. Herein, we characterized function and transcriptional regulation of a conserved P. putida TCS, named TarR-TarS. TarS (PP_0769) encodes a potential histidine kinase, and tarR (PP_0768) encodes a potential response regulator. Protein-protein interaction assay and phosphorylation assay confirmed that TarR-TarS was a functional TCS. Growth assay under antibiotics revealed that TarR-TarS positively regulated bacterial resistance to multiple antibiotics. Pull-down assay revealed that TarR directly interacted with PP_0800 (a hypothetical protein) and GroEL (the chaperonin). GroEL played a positive role in antibiotic resistance, while PP_0800 seemed to have no effect on antibiotic resistance. The regulator FleQ indirectly activated tarR-tarS transcription. However, the second messenger c-di-GMP antagonized FleQ activation to inhibit tarR-tarS transcription. The sigma factor FliA directly activated tarR-tarS transcription via a consensus motif. These findings reveal function and transcriptional regulation of TarR-TarS, and enrich knowledge regarding the relationship between c-di-GMP and antibiotic susceptibility in P. putida.

The bacterial second messenger cyclic diguanylate (c-di-GMP) modulates plankton-to-biofilm lifestyle transition of Pseudomonas species through its transcriptional regulatory effector FleQ. FleQ regulates transcription of biofilm- and flagellum-related genes in response to c-di-GMP. Through transcriptomic analysis and FleQ-DNA binding assay, this study identified five new target genes of c-di-GMP/FleQ in P. putida, including PP_0681, PP_0788, PP_4519 (lapE), PP_5222 (cyaA), and PP_5586. Except lapE encoding an outer membrane pore protein and cyaA encoding an adenylate cyclase, the functions of the other three genes encoding hypothetical proteins remain unknown. FleQ and c-di-GMP coordinately inhibit transcription of PP_0788 and cyaA and promote transcription of PP_0681, lapE, and PP_5586. Both in vitro and in vivo assays show that FleQ binds directly to promoters of the five genes. Further analyses confirm that LapE plays a central role of in the secretion of adhesin LapA and that c-di-GMP/FleQ increases lapE transcription, thereby promoting adhesin secretion and biofilm formation. The adenylate cyclase CyaA is responsible for synthesis of another second messenger, cyclic AMP (cAMP). FleQ and c-di-GMP coordinate to decrease the content of cAMP, suggesting that c-di-GMP and FleQ coregulate cAMP by modulating cyaA expression. Overall, this study adds five new members to the c-di-GMP/FleQ-regulated gene family and reveals the role of c-di-GMP/FleQ in LapA secretion and cAMP synthesis regulation in P. putida. IMPORTANCE c-di-GMP/FleQ promotes the plankton-to-biofilm lifestyle transition at the transcriptional level via FleQ in Pseudomonas species. Identification of new target genes directly regulated by c-di-GMP/FleQ helps to broaden the knowledge of c-di-GMP/FleQ-mediated transcriptional regulation. Regulation of lapE by c-di-GMP/FleQ guarantees highly efficient LapA secretion and biofilm formation. The mechanism of negative correlation between c-di-GMP and cAMP in both P. putida and P. aeruginosa remains unknown. Our result concerning transcriptional inhibition of cyaA by c-di-GMP/FleQ reveals the mechanism underlying the decrease of cAMP content by c-di-GMP in P. putida.

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.

The ubiquitous bacterial second messenger c-di-GMP is synthesized by diguanylate cyclase (DGC) and degraded by phosphodiesterase (PDE). Pseudomonas putida has dozens of DGC/PDE-encoding genes in its genome, but the phenotypical-genotypical correlation and transcriptional regulation of these genes are largely unknown. Herein, we characterize function and transcriptional regulation of a P. putida c-di-GMP-metabolizing enzyme, GcsA. GcsA consists of two per-ARNT-sim (PAS) domains, followed by a canonical conserved central sequence pattern (GGDEF) domain and a truncated EAL domain. In vitro analysis confirmed the DGC activity of GcsA. The phenotypic observation revealed that GcsA inhibited swimming motility in an FlgZ-dependent manner. In terms of transcriptional regulation, gcsA was found to be cooperatively regulated by c-di-GMP and cAMP via their effectors, FleQ and Crp respectively. The transcription of gcsA was promoted by c-di-GMP and inhibited by cAMP. In vitro binding analysis revealed that FleQ indirectly regulated the transcription of gcsA, while Crp directly regulated the transcription of gcsA by binding to its promoter. Besides, an inverse relationship between the cellular c-di-GMP and cAMP levels in P. putida was confirmed. These findings provide basic knowledge regarding the function and transcriptional regulation of GcsA and demonstrate a crosstalk between c-di-GMP and cAMP in the regulation of the expression of GcsA in P. putida.

Oxidative stress is an unavoidable consequence of interactions with various reactive oxygen species (ROS)-inducing agents that would damage cells or even cause cell death. Bacteria have developed defensive systems, including induction of stress-sensing proteins and detoxification enzymes, to handle oxidative stress. Cyclic diguanylate (c-di-GMP) is a ubiquitous intracellular bacterial second messenger that coordinates diverse aspects of bacterial growth and behavior. In this study, we revealed a mechanism by which c-di-GMP regulated bacterial oxidative stress resistance in Pseudomonas putida KT2440. High c-di-GMP level was found to enhance bacterial resistance towards hydrogen peroxide. Transcription assay showed that expression of two oxidative stress resistance genes, fpr-1 and katE, was promoted under high c-di-GMP level. Deletion of fpr-1 and katE both decreased bacterial tolerance to hydrogen peroxide and weakened the effect of c-di-GMP on oxidative stress resistance. The promoted expression of fpr-1 under high c-di-GMP level was caused by increased cellular ROS via a transcriptional regulator FinR. We further demonstrated that the influence of high c-di-GMP on cellular ROS depend on the existence of FleQ, a transcriptional regulatory c-di-GMP effector. Besides, the regulation of katE by c-di-GMP was also FleQ dependent in an indirect way. Our results proved a connection between c-di-GMP and oxidative stress resistance and revealed a mechanism by which c-di-GMP regulated expression of fpr-1 and katE in P. putida KT2440.

In Pseudomonas putida KT2440, the exopolysaccharide Pea is associated with biofilm stability and pellicle formation; however, little is known about its regulatory pathway. In this study, we identified that the gene cluster pea was transcribed from 25 bp upstream of the operon and the stationary phase alternative sigma factor RpoS regulated the transcription of pea. When RpoS was absent, another sigma factor, likely the housekeeping sigma factor RpoD, could also mediate pea transcription but at a low level. The function of Pea polysaccharide was further confirmed to be necessary for full production of biofilm, formation of pellicle and c-di-GMP-dependent wrinkly colony morphology. Additionally, evidences were provided to demonstrate that the transcriptional regulator AmrZ was a negative regulator for pea expression. DNase I footprinting studies verified that AmrZ bound directly to the site overlapping the pea promoter, which might interfere with the binding of RNA polymerase to the promoter and resulted in inhibition of transcription initiation.

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.

More than 90% of potassium (K) in soil exists in forms of insoluble silicate minerals and cannot be directly utilized by plants. K-solubilizing bacteria (KSB) can improve soil fertility and plant growth as biofertilizers by decomposing silicate minerals and releasing insoluble K into soluble forms. The objectives of this study were to isolate and characterize KSB from rape rhizospheric soil and to evaluate their effects on ryegrass growth. In this study, 16 cultivable potential KSB were isolated from rape rhizosphere at first. Then, quantitative analysis revealed that three KSB strains, named S-15, S-17 and S-18, showed the best K mineral solubilizing ability and they were identified as Mesorhizobium sp., Paenibacillus sp. and Arthrobacter sp. Inoculating the three strains into available K limit soil increased available K content significantly. The result of the pot experiment revealed that the three strains increased ryegrass growth vigor, biomass yield and K uptake to different degrees in availa...

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.

Many proteobacteria harbor FinR homologues in their genomes as putative LysR-type proteins; however, the function of FinR is poorly studied except in the induction of fpr-1 under superoxide stress conditions in Pseudomonas putida and Pseudomonas aeruginosa. Here, by analyzing the influence of finR deletion on the transcriptomic profile of P. putida KT2440 through RNA sequencing and real-time quantitative PCR (RT-qPCR), we found 11 operons that are potentially regulated by FinR. Among them, the expression of nicC and nicX operons, which were reported to be responsible for the aerobic degradation of nicotinic acid (NA), was significantly decreased in the finR mutant, and complementation with intact finR restored the expression of the two operons. The results of bacterial NA utilization demonstrated that the deletion of finR impaired bacterial growth in minimal medium supplemented with NA/6HNA (6-hydroxynicotinic acid) as the sole carbon source and that complementation with intact finR restored the growth of the mutant strain. The expression of nicC and nicX operons was previously revealed to be repressed by the NicR repressor and induced by NA/6HNA. Our transcriptional assay revealed that the deletion of finR weakened the induction of nicC and nicX by NA/6HNA. Meanwhile, the deletion of finR largely decreased the effect of nicR deletion on the expression of nicC and nicX operons. These results suggest that finR plays a positive role and cooperates with NicR in the regulation of nicC and nicX operons. In vitro experiments showed that both FinR and NicR bound to nicX and nicC promoter regions directly. The results of this study deepened our knowledge of FinR function and nicotinic acid degradation in P. putida. IMPORTANCE This study analyzed the influence of finR deletion on the transcriptomic profile of Pseudomonas putida KT2440. The FinR regulator is widely distributed but poorly studied in diverse proteobacteria. Here, we found 11 operons that potentially are regulated by FinR in KT2440. We further demonstrated that FinR played a positive role and cooperated with the NicR repressor in bacterial nicotinic acid (NA) degradation via regulating the expression of nicC and nicX operons. Furthermore, a transcriptomic analysis also indicated a potentially negative role of FinR in the expression of the hut cluster involved in bacterial histidine utilization. The work deepened our knowledge of FinR function and nicotinic acid degradation in P. putida.

The global regulatory molecule (p)ppGpp is synthesized under limited nutrition conditions and involves in many cellular processes in bacteria. (p)ppGpp has been reported to affect biofilm formation in several bacterial species. Here, we found that deletion of (p)ppGpp synthase genes of Pseudomonas putida KT2440 led to enhanced biofilm formation in polystyrene microtitre plates. Besides, the pellicle of this mutant formed at the air-liquid interface lost the robust structure and became frail. The biofilm formation and its structure are mainly determined by exopolysaccharides (EPSs) and adhesins. Transcriptional analysis of four EPS operons designated as pea, peb, alg and bcs and two adhesin genes nominated as lapA and lapF showed that the deletion of (p)ppGpp synthase genes increased the expression of peb, bcs and lapA but repressed the expression of pea and lapF. Furthermore, expression of the regulation factor FleQ was significantly augmented in (p)ppGpp-synthase mutants while the expression of sigma factor RpoS was reduced. Since FleQ and RpoS play important roles in regulating expression of EPS and adhesin genes, (p)ppGpp may mediate the synthesis of biofilm matrix via influencing these regulators to control the biofilm formation and pellicle structure.

Flagella‐mediated motility is an important capability of many bacteria to survive in nutrient‐depleted and harsh environments. Decreasing the intracellular cyclic di‐GMP (c‐di‐GMP) level by overexpression of phosphodiesterase BifA promotes flagellar‐mediated motility and induces planktonic lifestyle in Pseudomonas. The mechanism that regulates expression of bifA gene was poorly studied. Here we showed that expression of BifA was partly controlled by flagellar sigma factor FliA (σ28) in Pseudomonas putida KT2440. FliA deletion led to an approximately twofold decrease in transcription of bifA. 5′ race assay revealed two transcription start points in bifA promoter region, with the putative σ70 and σ28 promoter sequences upstream, respectively. Point mutation in σ28 promoter region reduced transcriptional activity of the promoter in wild‐type KT2440, but showed no influence on that in fliA deletion mutant. FliA overexpression decreased the intracellular c‐di‐GMP level in a BifA‐dependent way, suggesting that FliA was able to modulate the intracellular c‐di‐GMP level and BifA function was required for the modulation. Besides, FliA overexpression enhanced swimming ability of wild‐type strain, while made no difference to the bifA mutant. Our results suggest that FliA acts as a negative regulator to modulate the c‐di‐GMP level via controlling transcription of bifA to facilitate swimming motility.