Complex Interplay between FleQ, Cyclic Diguanylate and Multiple σ Factors Coordinately Regulates Flagellar Motility and Biofilm Development in Pseudomonas putida

Abstract

Most bacteria alternate between a free living planktonic lifestyle and the formation of structured surface-associated communities named biofilms. The transition between these two lifestyles requires a precise and timely regulation of the factors involved in each of the stages that has been likened to a developmental process. Here we characterize the involvement of the transcriptional regulator FleQ and the second messenger cyclic diguanylate in the coordinate regulation of multiple functions related to motility and surface colonization in Pseudomonas putida. Disruption of fleQ caused strong defects in flagellar motility, biofilm formation and surface attachment, and the ability of this mutation to suppress multiple biofilm-related phenotypes associated to cyclic diguanylate overproduction suggests that FleQ mediates cyclic diguanylate signaling critical to biofilm growth. We have constructed a library containing 94 promoters potentially involved in motility and biofilm development fused to gfp and lacZ, screened this library for FleQ and cyclic diguanylate regulation, and assessed the involvement of alternative σ factors σN and FliA in the transcription of FleQ-regulated promoters. Our results suggest a dual mode of action for FleQ. Low cyclic diguanylate levels favor FleQ interaction with σN-dependent promoters to activate the flagellar cascade, encompassing the flagellar cluster and additional genes involved in cyclic diguanylate metabolism, signal transduction and gene regulation. On the other hand, characterization of the FleQ-regulated σN- and FliA-independent PlapA and PbcsD promoters revealed two disparate regulatory mechanisms leading to a similar outcome: the synthesis of biofilm matrix components in response to increased cyclic diguanylate levels.

FleN and FleQ play a synergistic role in regulating lapA and bcs operons in Pseudomonas putida KT2440

“…A recent study by Jiménez‐Fernández and colleagues () has confirmed that FleQ positively regulates the expression of flagella‐associated genes in P. putida . Here, in P. putita KT2440, we found that deletion of fleN led to increased flagellar number (Fig.…”

“…Previous report from P. aeruginosa showed that FleN exerted an antagonistic effect on FleQ in flagella biosynthesis through inhibiting FleQ ATPase activity (Baraquet and Harwood, ), while FleQ ATPase activity and RpoN were not required for biofilm gene expression (Baraquet and Harwood, ), suggesting a different regulation mechanism between the flagella synthesis and biofilm formation in P. aeruginosa . Unlike FleQ in P. aeruginosa , FleQ of P. putida functioned as an activator for both flagella‐related genes and biofilm‐related gene lapA (Jiménez‐Fernández et al ., ; Xiao et al ., ). In addition, we discovered that ATPase of FleQ and RpoN were required for full expression of lapA and were strictly needed for the expression of fleSR and fliF (Xiao et al ., ), suggesting that FleQ probably relies on σ 54 to regulate lapA expression.…”

“…The former studies revealed that FleQ directly regulates the transcription of lapA and bcs operons in response to c‐di‐GMP in P. putida , and FleQ is an activator of lapA , but a repressor of bcs (Jiménez‐Fernández et al ., ; Molina‐Henares et al ., ; Xiao et al ., ). However, it is unclear whether and how FleN participates in the regulation of the biofilm matrix related genes.…”

The Wsp intermembrane complex mediates metabolic control of the swim‐attach decision of Pseudomonas putida

“…In general, cdGMP binding to FleQ antagonizes its repressor activity, thereby enabling transcription from the same promoter it was otherwise inhibiting. This TF is so important in lifestyle decisions that a P. putida strain lacking fleQ is both non-motile and unable to form biofilms (Jimenez-Fernandez et al, 2016;Molina-Henares et al, 2017). FleQ can also interact with another TF, FleN.…”

ThePseudomonas aeruginosaPilSR two-component system regulates both twitching and swimming motilities

“…Flagellum-dependent swimming motility, for example, is controlled through a regulatory cascade that includes the transcriptional regulator, FleQ (3) and the FleS-FleR TCS, which like many TCSs also requires the alternate sigma factor RpoN (σ 54 )(4). FleQ controls transcription of fleS-fleR in addition to multiple other flagellar, adhesion and biofilm-associated genes, in a c-di-GMP dependent manner (3,5). FleSR has been implicated in the expression of 20 or more flagellar biosynthetic genes in P. aeruginosa , as well as additional genes not previously known to be involved in flagellar assembly or function (6).…”