FleQ, FleN and c-di-GMP coordinately regulate cellulose production in Pseudomonas syringae pv. tomato DC3000.

The second messenger cyclic di-GMP (c-di-GMP) controls the transition between motility and sessility in many bacterial species by a variety of mechanisms, including the production of multiple exopolysaccharides. Pseudomonas syringae pv. tomato (Pto) DC3000 is a plant pathogenic bacteria able to synthesize acetylated cellulose under high c-di-GMP levels thanks to the expression of the wssABCDEFGHI operon. Increased cellulose production enhances air-liquid biofilm formation and generates a wrinkled colony phenotype on solid media. We previously showed that under low levels of c-di-GMP, the regulators FleQ and AmrZ bound to adjacent sequences at the wss promoter inhibiting its expression, but only FleQ responded to the presence of c-di-GMP by activating cellulose production. In the present work, we advance in the knowledge of this complex regulation in Pto DC3000 by shedding light over the role of FleN in this process. The distinctive features of this system are that FleN and FleQ are both required for repression and activation of the wss operon under low and high c-di-GMP levels, respectively. We have also identified three putative FleQ binding sites at the wss promoter and show that FleQ/FleN-ATP binds at those sites under low c-di-GMP levels, inducing a distortion of DNA, impairing RNA polymerase binding, and repressing wss transcription. However, binding of c-di-GMP induces a conformational change in the FleQ/FleN-ATP complex, which relieves the DNA distortion, allows promoter access to the RNA polymerase, and leads to activation of wss transcription. On the other hand, AmrZ is always bound at the wss promoter limiting its expression independently of FleQ, FleN and c-di-GMP levels.

Transcriptional organization and regulation of the Pseudomonas putida flagellar system.

A single region of the Pseudomonas putida genome, designated the flagellar cluster, includes 59 genes potentially involved in the biogenesis and function of the flagellar system. Here, we combine bioinformatics and in vivo gene expression analyses to clarify the transcriptional organization and regulation of the flagellar genes in the cluster. We have identified 11 flagellar operons and characterized 22 primary and internal promoter regions. Our results indicate that synthesis of the flagellar apparatus and core chemotaxis machinery is regulated by a three-tier cascade in which fleQ is a Class I gene, standing at the top of the transcriptional hierarchy. FleQ- and σ54 -dependent Class II genes encode most components of the flagellar structure, part of the chemotaxis machinery and multiple regulatory elements, including the flagellar σ factor FliA. FliA activation of Class III genes enables synthesis of the filament, one stator complex and completion of the chemotaxis apparatus. Accessory regulatory proteins and an intricate operon architecture add complexity to the regulation by providing feedback and feed-forward loops to the main circuit. Because of the high conservation of the gene arrangement and promoter motifs, we believe that the regulatory circuit presented here may also apply to other environmental pseudomonads.

Transcriptional organization, regulation and functional analysis of flhF and fleN in Pseudomonas putida.

The Pseudomonas putida flhA-flhF-fleN-fliA cluster encodes a component of the flagellar export gate and three regulatory elements potentially involved in flagellar biogenesis and other functions. Here we show that these four genes form an operon, whose transcription is driven from the upstream PflhA promoter. A second promoter, PflhF, provides additional transcription of the three distal genes. PflhA and PflhF are σN-dependent, activated by the flagellar regulator FleQ, and negatively regulated by FleN. Motility, surface adhesion and colonization defects of a transposon insertion mutant in flhF revealed transcriptional polarity on fleN and fliA, as the former was required for strong surface adhesion and biofilm formation, and the latter was required for flagellar synthesis. On the other hand, FlhF and FleN were necessary to attain proper flagellar location and number for a fully functional flagellar complement. FleN, along with FleQ and the second messenger c-di-GMP differentially regulated transcription of lapA and the bcs operon, encoding a large adhesion protein and cellulose synthase. FleQ positively regulated the PlapA promoter and activation was antagonized by FleN and c-di-GMP. PbcsD was negatively regulated by FleQ and FleN, and repression was antagonized by c-di-GMP. FleN promoted FleQ binding to both PlapA and PbcsD in vitro, while c-di-GMP antagonized interaction with PbcsD and stimulated interaction with PlapA. A single FleQ binding site in PlapA was critical to activation in vivo. Our results suggest that FleQ, FleN and c-di-GMP cooperate to coordinate the regulation of flagellar motility and biofilm development.

PP4397/FlgZ provides the link between PP2258 c-di-GMP signalling and altered motility in Pseudomonas putida.

Bacteria swim and swarm using rotating flagella that are driven by a membrane-spanning motor complex. Performance of the flagella motility apparatus is modulated by the chemosensory signal transduction system to allow navigation through physico-chemical gradients - a process that can be fine-tuned by the bacterial second messenger c-di-GMP. We have previously analysed the Pseudomonas putida signalling protein PP2258 that has the capacity to both synthesize and degrade c-di-GMP. A PP2258 null mutant displays reduced motility, implicating the c-di-GMP signal originating from this protein in control of P. putida motility. In Escherichia coli and Salmonella, the PilZ-domain protein YcgR mediates c-di-GMP responsive control of motility through interaction with the flagellar motors. Here we provide genetic evidence that the P. putida protein PP4397 (also known as FlgZ), despite low sequence homology and a different genomic context to YcgR, functions as a c-di-GMP responsive link between the signal arising from PP2258 and alterations in swimming and swarming motility in P. putida.

The stringent response promotes biofilm dispersal in Pseudomonas putida.

Biofilm dispersal is a genetically programmed response enabling bacterial cells to exit the biofilm in response to particular physiological or environmental conditions. In Pseudomonas putida biofilms, nutrient starvation triggers c-di-GMP hydrolysis by phosphodiesterase BifA, releasing inhibition of protease LapG by the c-di-GMP effector protein LapD, and resulting in proteolysis of the adhesin LapA and the subsequent release of biofilm cells. Here we demonstrate that the stringent response, a ubiquitous bacterial stress response, is accountable for relaying the nutrient stress signal to the biofilm dispersal machinery. Mutants lacking elements of the stringent response - (p)ppGpp sythetases [RelA and SpoT] and/or DksA - were defective in biofilm dispersal. Ectopic (p)ppGpp synthesis restored biofilm dispersal in a ∆relA ∆spoT mutant. In vivo gene expression analysis showed that (p)ppGpp positively regulates transcription of bifA, and negatively regulates transcription of lapA and the lapBC, and lapE operons, encoding a LapA-specific secretion system. Further in vivo and in vitro characterization revealed that the PbifA promoter is dependent on the flagellar σ factor FliA, and positively regulated by ppGpp and DksA. Our results indicate that the stringent response stimulates biofilm dispersal under nutrient limitation by coordinately promoting LapA proteolysis and preventing de novo LapA synthesis and secretion.

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

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.

Mechanism of Antiactivation at the Pseudomonas sp. Strain ADP σN-Dependent PatzT Promoter.

UNLABELLED: PatzT is an internal promoter of the atzRSTUVW operon that directs the synthesis of AtzT, AtzU, AtzV, and AtzW, components of an ABC-type cyanuric acid transport system. PatzT is σ(N) dependent, activated by the general nitrogen control regulator NtrC with the assistance of protein integration host factor (IHF), and repressed by the LysR-type transcriptional regulator (LTTR) AtzR. We have used a variety of in vivo and in vitro gene expression and protein-DNA interaction assays to assess the mechanisms underlying AtzR-dependent repression of PatzT Here, we show that repression only occurs when AtzR and NtrC interact simultaneously with the PatzT promoter region, indicating that AtzR acts as an antiactivator to antagonize activation by NtrC. Furthermore, repression requires precise rotational orientation of the AtzR and NtrC binding sites, strongly suggesting protein-protein interaction between the two proteins on the promoter region. Further exploration of the antiactivation mechanism showed that although AtzR-dependent repression occurs prior to open complex formation, AtzR does not alter the oligomerization state of NtrC or inhibit NtrC ATPase activity when bound to the PatzT promoter region. Taken together, these results strongly suggest that PatzT-bound AtzR interacts with NtrC to prevent the coupling of NtrC-mediated ATP hydrolysis with the remodeling of the interactions between E-σ(N) and PatzT that lead to open complex formation. IMPORTANCE: Here, we describe a unique mechanism by which the regulatory protein AtzR prevents the activation of the σ(N)-dependent promoter PatzT Promoters of this family are always positively regulated, but there are a few examples of overlapping negative regulation. The mechanism described here is highly unconventional and involves an interaction between the repressor and activator proteins to prevent the action of the repressor protein on the RNA polymerase-promoter complex.

Biofilm formation-defective mutants in Pseudomonas putida.

Out of 8000 candidates from a genetic screening for Pseudomonas putida KT2442 mutants showing defects in biofilm formation, 40 independent mutants with diminished levels of biofilm were analyzed. Most of these mutants carried insertions in genes of the lap cluster, whose products are responsible for synthesis, export and degradation of the adhesin LapA. All mutants in this class were strongly defective in biofilm formation. Mutants in the flagellar regulatory genes fleQ and flhF showed similar defects to that of the lap mutants. On the contrary, transposon insertions in the flagellar structural genes fliP and flgG, that also impair flagellar motility, had a modest defect in biofilm formation. A mutation in gacS, encoding the sensor element of the GacS/GacA two-component system, also had a moderate effect on biofilm formation. Additional insertions targeted genes involved in cell envelope function: PP3222, encoding the permease element of an ABC-type transporter and tolB, encoding the periplasmic component of the Tol-OprL system required for outer membrane stability. Our results underscore the central role of LapA, suggest cross-regulation between motility and adhesion functions and provide insights on the role of cell envelope trafficking and maintenance for biofilm development in P. putida.

A Pseudomonas putida cbrB transposon insertion mutant displays a biofilm hyperproducing phenotype that is resistant to dispersal.

The CbrAB two-component system in the Pseudomonads controls a variety of metabolic and behavioural traits required for its adaptation to changing environmental conditions, including the uptake or assimilation of certain carbon sources, and processes such as chemotaxis or stress tolerance. In this work we characterize a miniTn5-luxAB-Km transposon insertion mutant in cbrB (MPO406) in Pseudomonas putida leading to a biofilm overproducing phenotype that is not dispersed when nutrients are depleted. Comparison with a cbrB deletion mutant revealed that all phenotypes previously attributed to CbrB in P. putida correlated in both strains, with the exception of biofilm overproduction and absence of dispersal. We show that in the insertion mutant, the expression of the downstream regulatory RNA CrcZ is upregulated, and also show the presence of a truncated form of CbrB. Also, two additional point mutations in lapG and lapD have been detected in MPO406 by whole genome sequencing. Combination of these effects provides a robust biofilm overproducing phenotype. We present the mutant strain MPO406 as a good candidate to perform bio-production of substances of biotechnological interest or other processes such as bioremediation, which take advantage of immobilized cells on solid surfaces.

The c-di-GMP phosphodiesterase BifA regulates biofilm development in Pseudomonas putida.

We previously showed the isolation of biofilmpersistent Pseudomonas putida mutants that fail to undergo biofilm dispersal upon entry in stationary phase. Two such mutants were found to bear insertions in PP0914, encoding a GGDEF/EAL domain protein with high similarity to Pseudomon asaeruginosa BifA. Here we show the phenotypic characterization of a ΔbifA mutant in P. putida KT2442.This mutant displayed increased biofilm and pellicle formation, cell aggregation in liquid medium and decreased starvation-induced biofilm dispersal relative to the wild type. Unlike its P. aeruginosa counterpart, P. putida BifA did not affect swarming motility. The hyperadherent phenotype of the ΔbifA mutant correlates with a general increase in cyclic diguanylate (c-di-GMP) levels, Congo Red-binding exopolyaccharide production and transcription of the adhesin-encoding lapA gene. Integrity of the EAL motif and a modified GGDEF motif (altered to GGDQF)were crucial for BifA activity, and c-di-GMP depletion by overexpression of a heterologous c-di-GMP phosphodiesterase in the ΔbifA mutant restored wild-type biofilm dispersal and lapA expression.Our results indicate that BifA is a phosphodiesterase involved in the regulation of the c-di-GMP pool and required for the generation of the low c-di-GMP signal that triggers starvation-induced biofilm dispersal.

New methods for the isolation and characterization of biofilm-persistent mutants in Pseudomonas putida.

Here we describe two new methods for the genetic characterization of bacterial biofilm development. First, we have designed a microtitre dish-based approach for high-throughput screening of Pseudomonas putida mutants showing increased biofilm under dispersal conditions. Using this method, nine such biofilm-persistent mutants, bearing transposon insertions in four loci: lapG, bifA, mvaB and dksA, were isolated. Second, we have developed a serial dilution-based scheme to monitor biofilm development and dispersal in microtitre dish wells in a simple, time-efficient and reproducible manner. Using this method, we showed that (i) mutants in bifA and dksA do not undergo starvation-induced biofilm dispersal in LB or minimal medium, (ii) a mvaB mutant does not disperse the biofilm in LB, but shows a normal dispersal response in minimal medium, and (iii) unlike the lapG mutant, the bifA, mvaB and dksA mutants do not show an increase in biofilm production. The procedures shown here are useful tools for the identification of previously uncharacterized biofilm-related genes and considerably simplify the characterization of biofilm growth phenotypes.

An A-tract at the AtzR binding site assists DNA binding, inducer-dependent repositioning and transcriptional activation of the PatzDEF promoter.

The LysR-type regulator AtzR activates the Pseudomonas sp. ADP atzDEF operon in response to nitrogen limitation and cyanuric acid. Activation involves repositioning of the AtzR tetramer on the PatzDEF promoter and relaxation of an AtzR-induced DNA bend. Here we examine the in vivo and in vitro contribution of an A5 -tract present at the PatzDEF promoter region to AtzR binding and transcriptional activation. Substitution of the A-tract for the sequence ACTCA prevented PatzDEF activation and high-affinity AtzR binding, impaired AtzR contacts with the activator binding site and shifted the position of the AtzR-induced DNA bend. Analysis of a collection of mutants bearing different alterations in the A-tract sequence showed that the extent of AtzR-dependent activation does not correlate with the magnitude or orientation of the spontaneous DNA bend generated at this site. Our results support the notion that indirect readout of the A-tract-associated narrow minor groove is essential for the AtzR-DNA complex to achieve a conformation competent for activation of the PatzDEF promoter. Conservation of this motif in several binding sites of LysR-type regulators suggests that this mechanism may be shared by other proteins in this family.

Tetralin-induced and ThnR-regulated aldehyde dehydrogenase and beta-oxidation genes in Sphingomonas macrogolitabida strain TFA.

A new cluster of genes has been found downstream of the previously identified thnA2 gene. The gene products are similar to nonacylating aldehyde dehydrogenases (ThnG) and to proteins representing a complete beta-oxidation pathway (ThnH to ThnP). ThnG has a nonacylating NAD-dependent pimelic semialdehyde dehydrogenase activity that renders pimelic acid a seven-carbon dicarboxylic acid. For further metabolism via beta-oxidation, pimelic acid could be acylated by a constitutive acyl coenzyme A (acyl-CoA) ligase found in Sphingomonas macrogolitabida strain TFA or by ThnH, which would transfer CoA from a previously acylated molecule. The first round of beta-oxidation is expected to render glutaryl-CoA and acetyl-CoA. Glutaryl-CoA dehydrogenase (ThnN) would catalyze the oxidation and decarboxylation of glutaryl-CoA and yield crotonyl-CoA, which enters the central metabolism via acetyl-CoA. Mutagenesis studies have shown that these genes are not essential for growth on tetralin or fatty acids, although a thnG disruption mutant showed threefold less pimelic semialdehyde dehydrogenase activity. Transcriptional analysis indicated that these genes are induced by tetralin, subjected to catabolite repression, and regulated by the same regulatory factors previously identified to regulate other thn structural genes. In the present study, transcription initiation upstream of thnH and thnM has been detected by primer extension analysis, and putative promoters were identified by sequence analysis. In addition, binding of the activator ThnR to its putative binding sites at the PH and PM promoter regions has been characterized. These results provide a complete characterization of the biodegradation pathway of tetralin to central metabolites and describe the transcriptional organization of the thn operons in S. macrogolitabida strain TFA.

Co-ordinated regulation of two divergent promoters through higher-order complex formation by the LysR-type regulator ThnR.

The genes required for tetralin biodegradation by Sphingomonas macrogolitabida strain TFA are clustered in two divergent and closely linked operons. ThnR, a LysR-type regulator, activates transcription from each operon in response to tetralin. The regulatory thnR gene is co-transcribed with the catabolic genes thnC, thnA3 and thnA4, resulting in positive autoregulation. ThnR binds with different affinity to two primary binding sites, designated B and C, in the intervening region between the two operons and makes additional contact with secondary sites that extend towards the promoters. In addition, ThnR may interact with itself when bound to each site via the formation of a DNA loop, as evidenced by the distortion of the DNA between the primary binding sites and the elimination of the higher-order complexes following the introduction of a half-turn of the DNA helix between the primary binding sites. Transcription from each promoter is not fully independent since mutations in each binding site affected transcription from both promoters. Based on these results, we propose a model of transcription activation that involves the formation of a complex structure by interactions between ThnR molecules bound to distant binding sites and favours transcription from one promoter to the detriment of the other.

Integrated response to inducers by communication between a catabolic pathway and its regulatory system.

Efficient gene regulation of metabolic pathways implies that the profile of molecules inducing the pathway matches that of the molecules that are metabolized. Gratuitous induction, a well-known phenomenon in catabolic pathways, is the consequence of differences in the substrate and inducer profiles. This phenomenon is particularly evident in pathways for biodegradation of organic contaminants that can be induced by a variety of molecules similar to the real substrates. Analysis of the regulation of tetralin biodegradation genes in mutant strains with mutations that affect each component of the initial dioxygenase enzymatic complex indicated that the response of the regulatory system to potential inducers is altered differently depending on the mutated component. Based on the expression phenotypes of a number of single or double mutants, we propose a model that represents an unprecedented way of communication between a catabolic pathway and its regulatory system to prevent efficient induction by a molecule that is not a real substrate. This communication allows a better fit of the substrate and inducer profiles, thus minimizing gratuitous induction, without a requirement for optimal coevolution to match the specificity of catabolic enzymes and their regulatory systems. Modulation of the regulatory system in this way not only provides a more appropriate response to potential inducers recognized by the regulatory system but also may properly adjust the levels of gene expression to the substrate availability.