Wsp system oppositely modulates antibacterial activity and biofilm formation via FleQ-FleN complex in Pseudomonas putida.

Type VI secretion systems (T6SS) are specific antibacterial weapons employed by diverse bacteria to protect themselves from competitors. Pseudomonas putida KT2440 possesses a functional T6SS (K1-T6SS) and exhibits antibacterial activity towards a broad range of bacteria. Here we found that the Wsp signal transduction system regulated K1-T6SS expression via synthesizing the second messenger cyclic di-GMP (c-di-GMP), thus mediating antibacterial activity in P. putida. High-level c-di-GMP produced by Wsp system repressed the transcription of K1-T6SS genes in structural operon and vgrG1 operon. Transcriptional regulator FleQ and ATPase FleN functioned as repressors in the Wsp system-modulated K1-T6SS transcription. However, FleQ and FleN functioned as activators in biofilm formation, and Wsp system promoted biofilm formation largely in a FleQ/FleN-dependent manner. Furthermore, FleQ-FleN complex bound directly to the promoter of K1-T6SS structural operon in vitro, and c-di-GMP promoted the binding. Besides, P. putida biofilm cells showed higher c-di-GMP levels and lower antibacterial activity than planktonic cells. Overall, our findings reveal a mechanism by which Wsp system oppositely modulates antibacterial activity and biofilm formation via FleQ-FleN, and demonstrate the relationship between plankton/biofilm lifestyles and antibacterial activity in P. putida.

Second Messenger c-di-GMP Modulates Exopolysaccharide Pea-Dependent Phenotypes via Regulation of eppA Expression in Pseudomonas putida.

The exopolysaccharide (EPS) Pea is essential for wrinkly colony morphology, pellicle formation, and robust biofilm production in Pseudomonas putida. The second messenger cyclic diguanylate monophosphate (c-di-GMP) induces wrinkly colony morphology in P. putida through an unknown mechanism(s). Herein, we found that c-di-GMP modulates wrinkly colony morphology via the regulation of expression of eppA (PP_5586), a small individually transcribed gene of 177 bp, and this gene was adjacent to the upstream region of the pea cluster. Phenotype observation revealed that eppA was essential for Pea-dependent phenotypes. The deletion of eppA led to a smooth colony morphology and impaired biofilm, which was analogous to the phenotypes with loss of the entire pea operon. eppA expression was positively regulated by c-di-GMP via the transcriptional effector FleQ, and eppA was essential for the c-di-GMP-induced wrinkly colony morphology. Structure prediction results implied that EppA had two transmembrane regions, and Western blotting revealed that EppA was located on the cell membrane. Transcriptomic analysis indicated that EppA had no significant effect on the transcriptomic profile of P. putida. A bacterial two-hybrid (BTH) assay suggested that there was no direct interaction between EppA and the proteins in the pea cluster and adjacent operons. Overall, these findings reveal that EppA is essential for Pea-dependent phenotypes and that c-di-GMP modulates Pea-dependent phenotypes via regulation of eppA expression in P. putida. IMPORTANCE Microbe-secreted EPSs are high-molecular-weight polysaccharides that have the potential to be used as industrially important biomaterials. The EPS Pea in P. putida is essential for wrinkly colony morphology and pellicle formation. Here, we identified a function-unknown protein, EppA, which was also essential for Pea-dependent wrinkly colony morphology and pellicle formation, and EppA was probably involved in Pea secretion. Meanwhile, our results indicated that the second messenger c-di-GMP positively regulated the expression of EppA, resulting in Pea-dependent wrinkly colony morphology. Our results reveal the relationship of c-di-GMP, EppA, and Pea-dependent phenotypes and provide a possible pathway to construct genetically engineered strains for high Pea production.

Mussel-Inspired Bisphosphonated Injectable Nanocomposite Hydrogels with Adhesive, Self-Healing, and Osteogenic Properties for Bone Regeneration.

Injectable hydrogels have received much attention because of the advantages of simulation of the natural extracellular matrix, microinvasive implantation, and filling and repairing of complex shape defects. Yet, for bone repair, the current injectable hydrogels have shown significant limitations such as the lack of tissue adhesion, deficiency of self-healing ability, and absence of osteogenic activity. Herein, a strategy to construct mussel-inspired bisphosphonated injectable nanocomposite hydrogels with adhesive, self-healing, and osteogenic properties is developed. The nano-hydroxyapatite/poly(l-glutamic acid)-dextran (nHA/PLGA-Dex) dually cross-linked (DC) injectable hydrogels are fabricated via Schiff base cross-linking and noncovalent nHA-BP chelation. The chelation between bisphosphonate ligands (alendronate sodium, BP) and nHA favors the uniform dispersion of the latter. Moreover, multiple adhesion ligands based on catechol motifs, BP, and aldehyde groups endow the hydrogels with good tissue adhesion. The hydrogels possess excellent biocompatibility and the introduction of BP and nHA both can effectively promote viability, proliferation, migration, and osteogenesis differentiation of MC3T3-E1 cells. The incorporation of BP groups and HA nanoparticles could also facilitate the angiogenic property of endothelial cells. The nHA/PLGA-Dex DC hydrogels exhibited considerable biocompatibility despite the presence of a certain degree of inflammatory response in the early stage. The successful healing of a rat cranial defect further proves the bone regeneration ability of nHA/PLGA-Dex DC injectable hydrogels. The developed tissue adhesive osteogenic injectable nHA/PLGA-Dex hydrogels show significant potential for bone regeneration application.