AmrZ and FleQ Co-regulate Cellulose Production in Pseudomonas syringae pv. Tomato DC3000.
ABSTRACT: Pseudomonas syringae pv. tomato DC3000 carries the wssABCDEFGHI operon for the synthesis of acetylated cellulose, whose production is stimulated by increasing the intracellular levels of the second messenger c-di-GMP. This enhances air-liquid biofilm formation and generates a wrinkly colony morphotype in solid media. In the present study we show that cellulose production is a complex process regulated at multiple levels and involving different players in this bacterium. Using different in vitro approaches, including Electrophoretic Mobility Shift Assay (EMSA) and footprint analysis, we demonstrated the interrelated role of two transcriptional regulators, AmrZ and FleQ, over cellulose production in Pto DC3000 and the influence of c-di-GMP in this process. Under physiological c-di-GMP levels, both regulators bind directly to adjacent regions at the wss promoter inhibiting its expression. However, just FleQ responds to c-di-GMP releasing from its wss operator site and converting from a repressor to an activator of cellulose production. The additive effect of the double amrZ/fleQ mutation on the expression of wss, together with the fact that they are not cross-regulated at the transcriptional level, suggest that FleQ and AmrZ behave as independent regulators, unlike what has been described in other Pseudomonas species. Furthermore, this dual co-regulation exerted by AmrZ and FleQ is not limited to cellulose production, but also affects other important phenotypes in Pto DC3000, such as motility and virulence.
Project description:Cellulose, whose production is controlled by c-di-GMP, is a commonly found exopolysaccharide in bacterial biofilms. Pseudomonas syringae pv. tomato (Pto) DC3000, a model organism for molecular studies of plant-pathogen interactions, carries the wssABCDEFGHI operon for the synthesis of acetylated cellulose. The high intracellular levels of the second messenger c-di-GMP induced by the overexpression of the heterologous diguanylate cyclase PleD stimulate cellulose production and enhance air-liquid biofilm (pellicle) formation. To characterize the mechanisms involved in Pto DC3000 pellicle formation, we studied this process using mutants lacking flagella, biosurfactant or different extracellular matrix components, and compared the pellicles produced in the absence and in the presence of PleD. We have discovered that neither alginate nor the biosurfactant syringafactin are needed for their formation, whereas cellulose and flagella are important but not essential. We have also observed that the high c-di-GMP levels conferred more cohesion to Pto cells within the pellicle and induced the formation of intracellular inclusion bodies and extracellular fibres and vesicles. Since the pellicles were very labile and this greatly hindered their handling and processing for microscopy, we have also developed new methods to collect and process them for scanning and transmission electron microscopy. These techniques open up new perspectives for the analysis of fragile biofilms in other bacterial strains.
Project description:Flagellum mediated motility is an essential trait for rhizosphere colonization by pseudomonads. Flagella synthesis is a complex and energetically expensive process that is tightly regulated. In Pseudomonas fluorescens, the regulatory cascade starts with the master regulatory protein FleQ that is in turn regulated by environmental signals through the Gac/Rsm and SadB pathways, which converge in the sigma factor AlgU. AlgU is required for the expression of amrZ, encoding a FleQ repressor. AmrZ itself has been shown to modulate c-di-GMP levels through the control of many genes encoding enzymes implicated in c-di-GMP turnover. This cyclic nucleotide regulates flagellar function and besides, the master regulator of the flagellar synthesis signaling pathway, FleQ, has been shown to bind c-di-GMP. Here we show that AdrA, a diguanylate cyclase regulated by AmrZ participates in this signaling pathway. Epistasis analysis has shown that AdrA acts upstream of SadB, linking SadB with environmental signaling. We also show that SadB binds c-di-GMP with higher affinity than FleQ and propose that c-di-GMP produced by AdrA modulates flagella synthesis through SadB.
Project description:The AmrZ/FleQ hub has been identified as a central node in the regulation of environmental adaption in the plant growth-promoting rhizobacterium and model for rhizosphere colonization Pseudomonas ogarae F113. AmrZ is involved in the regulation of motility, biofilm formation, and bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) turnover, among others, in this bacterium. The mutants in amrZ have a pleiotropic phenotype with distinguishable colony morphology, reduced biofilm formation, increased motility, and are severely impaired in competitive rhizosphere colonization. Here, RNA-Seq and qRT-PCR gene expression analyses revealed that AmrZ regulates many genes related to the production of extracellular matrix (ECM) components at the transcriptional level. Furthermore, overproduction of c-di-GMP in an amrZ mutant, by ectopic production of the Caulobacter crescentus constitutive diguanylate cyclase PleD*, resulted in increased expression of many genes implicated in the synthesis of ECM components. The overproduction of c-di-GMP in the amrZ mutant also suppressed the biofilm formation and motility phenotypes, but not the defect in competitive rhizosphere colonization. These results indicate that although biofilm formation and motility are mainly regulated indirectly by AmrZ, through the modulation of c-di-GMP levels, the implication of AmrZ in rhizosphere competitive colonization occurs in a c-di-GMP-independent manner.
Project description:High levels of the intracellular signalling molecule cyclic diguanylate (c-di-GMP) supress motility and activate exopolysaccharide (EPS) production in a variety of bacterial species. In many bacteria part of the effect of c-di-GMP is on gene expression, but the mechanism involved is not known for any species. We have identified the protein FleQ as a c-di-GMP-responsive transcriptional regulator in Pseudomonas aeruginosa. FleQ is known to activate expression of flagella biosynthesis genes. Here we show that it also represses transcription of genes including the pel operon involved in EPS biosynthesis, and that this repression is relieved by c-di-GMP. Our in vivo data indicate that FleQ represses pel transcription and that pel transcription is not repressed when intracellular c-di-GMP levels are high. FleN, a known antiactivator of FleQ also participates in control of pel expression. In in vitro experiments we found that FleQ binds to pel promoter DNA and that this binding is inhibited by c-di-GMP. FleQ binds radiolabelled c-di-GMP in vitro. FleQ does not have amino acid motifs that resemble previously defined c-di-GMP binding domains. Our results show that FleQ is a new type of c-di-GMP binding protein that controls the transcriptional regulation of EPS biosynthesis genes in P. aeruginosa.
Project description:The transcriptional regulator AmrZ is a global regulatory protein conserved within the pseudomonads. AmrZ can act both as a positive and a negative regulator of gene expression, controlling many genes implicated in environmental adaption. Regulated traits include motility, iron homeostasis, exopolysaccharides production and the ability to form biofilms. In Pseudomonas fluorescens F113, an amrZ mutant presents a pleiotropic phenotype, showing increased swimming motility, decreased biofilm formation and very limited ability for competitive colonization of rhizosphere, its natural habitat. It also shows different colony morphology and binding of the dye Congo Red. The amrZ mutant presents severely reduced levels of the messenger molecule cyclic-di-GMP (c-di-GMP), which is consistent with the motility and biofilm formation phenotypes. Most of the genes encoding proteins with diguanylate cyclase (DGCs) or phosphodiesterase (PDEs) domains, implicated in c-di-GMP turnover in this bacterium, appear to be regulated by AmrZ. Phenotypic analysis of eight mutants in genes shown to be directly regulated by AmrZ and encoding c-di-GMP related enzymes, showed that seven of them were altered in motility and/or biofilm formation. The results presented here show that in P. fluorescens, AmrZ determines c-di-GMP levels through the regulation of a complex network of genes encoding DGCs and PDEs.
Project description: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.
Project description:The plant innate immune system employs plasma membrane-localized receptors that specifically perceive pathogen/microbe-associated molecular patterns (PAMPs/MAMPs). This induces a defence response called pattern-triggered immunity (PTI) to fend off pathogen attack. Commensal bacteria are also exposed to potential immune recognition and must employ strategies to evade and/or suppress PTI to successfully colonize the plant. During plant infection, the flagellum has an ambiguous role, acting as both a virulence factor and also as a potent immunogen as a result of the recognition of its main building block, flagellin, by the plant pattern recognition receptors (PRRs), including FLAGELLIN SENSING2 (FLS2). Therefore, strict control of flagella synthesis is especially important for plant-associated bacteria. Here, we show that cyclic-di-GMP [bis-(3'-5')-cyclic di-guanosine monophosphate], a central regulator of bacterial lifestyle, is involved in the evasion of PTI. Elevated cyclic-di-GMP levels in the pathogen Pseudomonas syringae pv. tomato (Pto) DC3000, the opportunist P.?aeruginosa?PAO1 and the commensal P.?protegens?Pf-5 inhibit flagellin synthesis and help the bacteria to evade FLS2-mediated signalling in Nicotiana benthamiana and Arabidopsis thaliana. Despite this, high cellular cyclic-di-GMP concentrations were shown to drastically reduce the virulence of Pto?DC3000 during plant infection. We propose that this is a result of reduced flagellar motility and/or additional pleiotropic effects of cyclic-di-GMP signalling on bacterial behaviour.
Project description:Swarming is a surface-associated motile behavior that plays an important role in the rapid spread, colonization, and subsequent establishment of bacterial communities. In Pseudomonas aeruginosa, swarming is dependent upon a functional flagella and aided by the production of biosurfactants. AmrZ, a conserved transcription factor across pseudomonads, has been shown to be a global regulator of multiple genes important for virulence and ecological fitness. In this study, we expand this concept of global control to swarming motility by showing that deletion of amrZ results in a severe defect in swarming, while multicopy expression of this gene stimulates swarming of P. aeruginosa. Mechanistic studies showed that the swarming defect of an amrZ mutant does not involve changes of biosurfactant production but is associated with flagellar malfunction. The ?amrZ mutant exhibits increased levels of the second messenger cyclic di-GMP (c-di-GMP) compared to the wild-type strain, under swarming conditions. We found that the diguanylate cyclase GcbA was the main contributor to the increased accumulation of c-di-GMP observed in the ?amrZ mutant and was a strong inhibitor of flagellar-dependent motility. Our results revealed that the GcbA-dependent inhibition of motility required the presence of two c-di-GMP receptors containing a PilZ domain: FlgZ and PA14_56180. Furthermore, the ?amrZ mutant exhibits enhanced production of Pel polysaccharide. Epistasis analysis revealed that GcbA and the Pel polysaccharide act independently to limit swarming in ?amrZ. Our results support a role for AmrZ in controlling swarming motility, yet another social behavior besides biofilm formation that is crucial for the ability of P. aeruginosa to colonize a variety of surfaces. The central role of AmrZ in controlling these behaviors makes it a good target for the development of treatments directed to combat P. aeruginosa infections.
Project description:The transcription factor AmrZ regulates genes important for P. aeruginosa virulence, including type IV pili, extracellular polysaccharides, and the flagellum; however, the global effect of AmrZ on gene expression remains unknown, and therefore, AmrZ may directly regulate many additional genes that are crucial for infection. Compared to the wild type strain, a ?amrZ mutant exhibits a rugose colony phenotype, which is commonly observed in variants that accumulate the intracellular second messenger cyclic diguanylate (c-di-GMP). Cyclic di-GMP is produced by diguanylate cyclases (DGC) and degraded by phosphodiesterases (PDE). We hypothesized that AmrZ limits the intracellular accumulation of c-di-GMP through transcriptional repression of gene(s) encoding a DGC. In support of this, we observed elevated c-di-GMP in the ?amrZ mutant compared to the wild type strain. Consistent with other strains that accumulate c-di-GMP, when grown as a biofilm, the ?amrZ mutant formed larger microcolonies than the wild-type strain. This enhanced biofilm formation was abrogated by expression of a PDE. To identify potential target DGCs, a ChIP-Seq was performed and identified regions of the genome that are bound by AmrZ. RNA-Seq experiments revealed the entire AmrZ regulon, and characterized AmrZ as an activator or repressor at each binding site. We identified an AmrZ-repressed DGC-encoding gene (PA4843) from this cohort, which we named AmrZ dependent cyclase A (adcA). PAO1 overexpressing adcA accumulates 29-fold more c-di-GMP than the wild type strain, confirming the cyclase activity of AdcA. In biofilm reactors, a ?amrZ ?adcA double mutant formed smaller microcolonies than the single ?amrZ mutant, indicating adcA is responsible for the hyper biofilm phenotype of the ?amrZ mutant. This study combined the techniques of ChIP-Seq and RNA-Seq to define the comprehensive regulon of a bifunctional transcriptional regulator. Moreover, we identified a c-di-GMP mediated mechanism for AmrZ regulation of biofilm formation and chronicity.
Project description:The bacterial second messenger cyclic diguanylate (c-di-GMP) modulates plankton-to-biofilm lifestyle transition of <i>Pseudomonas</i> species through its transcriptional regulatory effector FleQ. FleQ regulates transcription of biofilm- and flagellum-related genes in response to c-di-GMP. Through transcriptomic analysis and FleQ-DNA binding assay, this study identified five new target genes of c-di-GMP/FleQ in <i>P. putida</i>, including <i>PP_0681</i>, <i>PP_0788</i>, <i>PP_4519</i> (<i>lapE</i>), <i>PP_5222</i> (<i>cyaA</i>), and <i>PP_5586</i> Except <i>lapE</i> encoding an outer membrane pore protein and <i>cyaA</i> encoding an adenylate cyclase, the functions of the other three genes encoding hypothetical proteins remain unknown. FleQ and c-di-GMP coordinately inhibit transcription of <i>PP_0788</i> and <i>cyaA</i> and promote transcription of <i>PP_0681</i>, <i>lapE</i>, and <i>PP_5586</i> Both <i>in vitro</i> and <i>in vivo</i> assays show that FleQ binds directly to promoters of the five genes. Further analyses confirm that LapE plays a central role of in the secretion of adhesin LapA and that c-di-GMP/FleQ increases <i>lapE</i> transcription, thereby promoting adhesin secretion and biofilm formation. The adenylate cyclase CyaA is responsible for synthesis of another second messenger, cyclic AMP (cAMP). FleQ and c-di-GMP coordinate to decrease the content of cAMP, suggesting that c-di-GMP and FleQ coregulate cAMP by modulating <i>cyaA</i> expression. Overall, this study adds five new members to the c-di-GMP/FleQ-regulated gene family and reveals the role of c-di-GMP/FleQ in LapA secretion and cAMP synthesis regulation in <i>P. putida</i> <b>IMPORTANCE</b> c-di-GMP/FleQ promotes the plankton-to-biofilm lifestyle transition at the transcriptional level via FleQ in <i>Pseudomonas</i> species. Identification of new target genes directly regulated by c-di-GMP/FleQ helps to broaden the knowledge of c-di-GMP/FleQ-mediated transcriptional regulation. Regulation of <i>lapE</i> by c-di-GMP/FleQ guarantees highly efficient LapA secretion and biofilm formation. The mechanism of negative correlation between c-di-GMP and cAMP in both <i>P. putida</i> and <i>P. aeruginosa</i> remains unknown. Our result concerning transcriptional inhibition of <i>cyaA</i> by c-di-GMP/FleQ reveals the mechanism underlying the decrease of cAMP content by c-di-GMP in <i>P. putida</i>.