C‐di‐GMP and biofilm are regulated in Pseudomonas putida by the CfcA/CfcR two‐component system in response to salts

• Published in: Environmental microbiology 2022-01, Vol.24 (1), p.158-178
• Main Authors: Tagua, Víctor G., Molina‐Henares, María Antonia, Travieso, María L., Nisa‐Martínez, Rafael, Quesada, José Miguel, Espinosa‐Urgel, Manuel, Ramos‐González, María Isabel
• Format: Article
• Language: English
• Subjects: Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Biofilms; Cyclic GMP - analogs & derivatives; Cyclic GMP - metabolism; Domains; Escherichia coli Proteins - genetics; Gene Expression Regulation, Bacterial; Histidine; Histidine kinase; Kinases; Membrane proteins; Membranes; Phenotypes; Phosphorus-Oxygen Lyases - genetics; Phosphorus-Oxygen Lyases - metabolism; Proteins; Pseudomonas putida; Pseudomonas putida - genetics; Pseudomonas putida - metabolism; Salts; Sessile species
• ISSN: 1462-2912; 1462-2920
• DOI: 10.1111/1462-2920.15891

Summary In Pseudomonas putida KT2440, cfcR encodes an orphan multidomain response regulator with diguanylate cyclase activity, which is responsible for the synthesis of c‐di‐GMP, a second messenger key in the transition from planktonic to sessile bacterial lifestyles. When overexpressed, cfcR enhances biofilm formation and causes other phenotype alterations. The cfcA gene, encoding a membrane‐anchored multisensory CHASE3/GAF hybrid histidine kinase (HK), is required to develop this pleiotropic phenotype. Here we show autophosphorylation of CfcA through HisKA/HATPase_c domains and then transfer of the phosphoryl group to an internal receiver (REC) domain. CfcA REC domains are nonessential for phosphotransfer from CfcA~P to the REC domain of CfcR. CfcA~P also phosphorylates the REC domain of CfcD, a second HK encoded in the same gene cluster as CfcA, which negatively regulates the CfcA/CfcR pathway. To evaluate the impact of CfcA domains on CfcR activity, a battery of mutants with in‐frame domain deletions was generated, whose CfcA protein locations were also examined. CfcA membrane anchorage contributes to protein stability and CfcR activation. Salt enhances c‐di‐GMP levels through CfcR, a response which is hampered by alteration of a presumed ligand‐binding motif in the CHASE3 sensor domain. Thus, in P. putida, c‐di‐GMP is salt‐regulated through the CfcA/CfcR/CfcD system.