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.
Highlights
The alternation between a free-swimming planktonic lifestyle and the formation of structured highly cooperative polymer-encased sessile communities, named biofilms, is a common feature of bacterial life in the environment [1]
In order to characterize the contribution of FleQ to the biofilm developmental cycle of P. putida, we examined the biofilm development kinetics of strains KT2442 and MRB35 in LB by means of a serial dilution-based growth curve method we described recently, in which a dilution series is used to recapitulate the time course of planktonic and biofilm growth in microtiter plate wells [47] (Fig 1A)
The set of FleQ-activated motility-related promoters could be further divided into two subsets, three promoters directly activated by FleQ and strongly dependent on σN, and six promoters indirectly activated by FleQ, and co-dependent on σ factors σN and FliA
Summary
The alternation between a free-swimming planktonic lifestyle and the formation of structured highly cooperative polymer-encased sessile communities, named biofilms, is a common feature of bacterial life in the environment [1]. Transition between the planktonic and biofilm lifestyles and through the different stages of biofilm development requires the timely production of different factors in response to environmental and physiological signals, involving a variety of signal transduction and regulatory pathways to connect such cues to the adequate physiological responses [3]. The nucleotide cyclic diguanylate (c-di-GMP) is a second messenger that is ubiquitously used in bacteria for signaling the transition between the planktonic and sessile lifestyles. Changes in c-di-GMP concentration are sensed by effectors, which in turn regulate a variety of processes, generally related to motility, biofilm development or virulence, acting at the transcriptional, translational or posttranslational levels. The biology of cdi-GMP signaling has been extensively reviewed in recent years [4,5,6,7,8]
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