The Pseudomonas putida CsrA/RsmA homologues negatively affect c‐di‐GMP pools and biofilm formation through the GGDEF/EAL response regulator CfcR

Abstract

Expression of cfcR, encoding the only GGDEF/EAL response regulator in Pseudomonas putida, is transcriptionally regulated by RpoS, ANR and FleQ, and the functionality of CfcR as a diguanylate cyclase requires the multisensor CHASE3/GAF hybrid histidine kinase named CfcA. Here an additional level of cfcR control, operating post-transcriptionally via the RNA-binding proteins RsmA, RsmE and RsmI, is unraveled. Specific binding of the three proteins to an Rsm-binding motif (5'CANGGANG3') encompassing the translational start codon of cfcR was confirmed. Although RsmA exhibited the highest binding affinity to the cfcR transcript, single deletions of rsmA, rsmE or rsmI caused minor derepression in CfcR translation compared to a ΔrsmIEA triple mutant. RsmA also showed a negative impact on c-di-GMP levels in a double mutant ΔrsmIE through the control of cfcR, which is responsible for most of the free c-di-GMP during stationary phase in static conditions. In addition, a CfcR-dependent c-di-GMP boost was observed during this stage in ΔrsmIEA confirming the negative effect of Rsm proteins on CfcR translation and explaining the increased biofilm formation in this mutant compared to the wild type. Overall, these results suggest that CfcR is a key player in biofilm formation regulation by the Rsm proteins in P. putida.

Arginine as an environmental and metabolic cue for cyclic diguanylate signalling and biofilm formation in Pseudomonas putida

“…There is still limited information about the mechanisms of action of amino acids that lead to changes in the turnover of the second messenger. Our results indicate that in P. putida KT2440, the response regulator with DGC activity CfcR, the chief contributor to c-di-GMP levels in stationary phase 6 , is the main element in the increase in c-di-GMP levels caused by exogenous l -arginine, despite its lack of amino acid-binding or protein–protein interaction domains. Still, a Δ cfcR mutant retains some response to l -arginine, suggesting additional protein(s) with DGC activity yet to be identified also participate in the process.…”

“…In the wild type, significantly increased rpoS expression was only observed with 5 mM l -arginine. Activity of a transcriptional cfcR::lacZ fusion, harboured in pMIR200 6 , was also tested in the Δ argG and Δ argH mutants. As shown in Fig.…”

“…This phenotype includes increased biofilm formation, cell flocculation in liquid cultures, pellicle formation in the air–liquid interface, and altered (crinkly) colony morphology in solid medium. Expression of cfcR is subject to a complex, multi-level control that involves global regulators, including the stationary phase sigma factor RpoS 5 and the three post-transcriptional regulators of the CsrA/RsmA family identified in KT2440, RsmA, RsmE and RsmI 6 .…”

Homologues of the RNA binding protein RsmA in Pseudomonas syringae pv. tomato DC3000 exhibit distinct binding affinities with non‐coding small RNAs and have distinct roles in virulence

“…In Pseudomonas protegens , RsmA and RsmE controlled metabolism and antibiotic biosynthesis (Reimmann et al , ; Wang et al , ). In Pseudomonas putida , RsmA, RsmE and RsmI negatively affected c‐di‐GMP pools and biofilm formation (Huertas‐Rosales et al , , ).…”

A feed‐forward signalling circuit controls bacterial virulence through linking cyclic di‐GMP and two mechanistically distinct sRNAs, ArcZ and RsmB

“…An Hfq‐dependent sRNA has been shown to bind RsmA to regulate biofilm formation in E. coli (Jørgensen et al ., ). RsmA has been shown to down‐regulate c‐di‐GMP signalling by repressing DGCs in Pseudomonas aeruginosa and Pseudomonas putida (Moscoso et al ., ; Huertas‐Rosales et al ., ). Our previous data showed that c‐di‐GMP post‐transcriptionally inhibits RsmB in D. dadantii (Yuan et al ., ).…”