Dataset Information

AmrZ Regulates Swarming Motility Through Cyclic di-GMP-Dependent Motility Inhibition and Controlling Pel Polysaccharide Production in Pseudomonas aeruginosa PA14.

ABSTRACT: 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.

SUBMITTER: Hou L

PROVIDER: S-EPMC6707383 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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AmrZ Regulates Swarming Motility Through Cyclic di-GMP-Dependent Motility Inhibition and Controlling Pel Polysaccharide Production in <i>Pseudomonas aeruginosa</i> PA14.

Hou Lingli L Debru Alexander A Chen Qianqian Q Bao Qiyu Q Li Kewei K

Frontiers in microbiology 20190814

Swarming is a surface-associated motile behavior that plays an important role in the rapid spread, colonization, and subsequent establishment of bacterial communities. In <i>Pseudomonas aeruginosa</i>, 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 concep ...[more]

PMID: 31474950

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Project description:UnlabelledThe second messenger cyclic diguanylate (c-di-GMP) is an important regulator of motility in many bacterial species. In Pseudomonas aeruginosa, elevated levels of c-di-GMP promote biofilm formation and repress flagellum-driven swarming motility. The rotation of P. aeruginosa's polar flagellum is controlled by two distinct stator complexes, MotAB, which cannot support swarming motility, and MotCD, which promotes swarming motility. Here we show that when c-di-GMP levels are elevated, swarming motility is repressed by the PilZ domain-containing protein FlgZ and by Pel polysaccharide production. We demonstrate that FlgZ interacts specifically with the motility-promoting stator protein MotC in a c-di-GMP-dependent manner and that a functional green fluorescent protein (GFP)-FlgZ fusion protein shows significantly reduced polar localization in a strain lacking the MotCD stator. Our results establish FlgZ as a c-di-GMP receptor affecting swarming motility by P. aeruginosa and support a model wherein c-di-GMP-bound FlgZ impedes motility via its interaction with the MotCD stator.ImportanceThe regulation of surface-associated motility plays an important role in bacterial surface colonization and biofilm formation. c-di-GMP signaling is a widespread means of controlling bacterial motility, and yet the mechanism whereby this signal controls surface-associated motility in P. aeruginosa remains poorly understood. Here we identify a PilZ domain-containing c-di-GMP effector protein that contributes to c-di-GMP-mediated repression of swarming motility by P. aeruginosa We provide evidence that this effector, FlgZ, impacts swarming motility via its interactions with flagellar stator protein MotC. Thus, we propose a new mechanism for c-di-GMP-mediated regulation of motility for a bacterium with two flagellar stator sets, increasing our understanding of surface-associated behaviors, a key prerequisite to identifying ways to control the formation of biofilm communities.

Project description:With the opportunistic pathogen Pseudomonas aeruginosa, quorum sensing based on homoserine lactones was found to influence biofilm formation. Here we discern a mechanism by which quorum sensing controls biofilm formation by screening 5850 transposon mutants of P. aeruginosa PA14 for altered biofilm formation. This screen identified the PA3885 mutant, which had 147-fold more biofilm than the wild-type strain. Loss of PA3885 decreased swimming, abolished swarming, and increased attachment, although this did not affect production of rhamnolipids. The PA3885 mutant also had a wrinkly colony phenotype, formed pronounced pellicles, had substantially more aggregation, and had 28-fold more exopolysaccharide production. Expression of PA3885 in trans reduced biofilm formation and abolished aggregation. Whole transcriptome analysis showed that loss of PA3885 activated expression of the pel locus, an operon that encodes for the synthesis of extracellular matrix polysaccharide. Genetic screening identified that loss of PelABDEG and the PA1120 protein (which contains a GGDEF-motif) suppressed the phenotypes of the PA3885 mutant, suggesting that the function of the PA3885 protein is to regulate 3,5-cyclic diguanylic acid (c-di-GMP) concentrations as a phosphatase since c-di-GMP enhances biofilm formation by activating PelD, and c-di-GMP inhibits swarming. Loss of PA3885 protein increased cellular c-di-GMP concentrations; hence, PA3885 protein is a negative regulator of c-di-GMP production. Purified PA3885 protein has phosphatase activity against phosphotyrosine peptides and is translocated to the periplasm. Las-mediated quorum sensing positively regulates expression of the PA3885 gene. These results show that the PA3885 protein responds to AHL signals and likely dephosphorylates PA1120, which leads to reduced c-di-GMP production. This inhibits matrix exopolysaccharide formation, which leads to reduced biofilm formation; hence, we provide a mechanism for quorum sensing control of biofilm formation through the pel locus and suggest PA3885 should be named TpbA for tyrosine phosphatase related to biofilm formation and PA1120 should be TpbB.

Project description:Gene transfer agents (GTAs) are bacteriophage-like particles produced by several bacterial and archaeal lineages that contain small pieces of the producing cells' genomes that can be transferred to other cells in a process similar to transduction. One well-studied GTA is RcGTA, produced by the alphaproteobacterium Rhodobacter capsulatus RcGTA gene expression is regulated by several cellular regulatory systems, including the CckA-ChpT-CtrA phosphorelay. The transcription of multiple other regulator-encoding genes is affected by the response regulator CtrA, including genes encoding putative enzymes involved in the synthesis and hydrolysis of the second messenger bis-(3'-5')-cyclic dimeric GMP (c-di-GMP). To investigate whether c-di-GMP signaling plays a role in RcGTA production, we disrupted the CtrA-affected genes potentially involved in this process. We found that disruption of four of these genes affected RcGTA gene expression and production. We performed site-directed mutagenesis of key catalytic residues in the GGDEF and EAL domains responsible for diguanylate cyclase (DGC) and c-di-GMP phosphodiesterase (PDE) activities and analyzed the functions of the wild-type and mutant proteins. We also measured RcGTA production in R. capsulatus strains where intracellular levels of c-di-GMP were altered by the expression of either a heterologous DGC or a heterologous PDE. This adds c-di-GMP signaling to the collection of cellular regulatory systems controlling gene transfer in this bacterium. Furthermore, the heterologous gene expression and the four gene disruptions had similar effects on R. capsulatus flagellar motility as found for gene transfer, and we conclude that c-di-GMP inhibits both RcGTA production and flagellar motility in R. capsulatusIMPORTANCE Gene transfer agents (GTAs) are virus-like particles that move cellular DNA between cells. In the alphaproteobacterium Rhodobacter capsulatus, GTA production is affected by the activities of multiple cellular regulatory systems, to which we have now added signaling via the second messenger dinucleotide molecule bis-(3'-5')-cyclic dimeric GMP (c-di-GMP). Similar to the CtrA phosphorelay, c-di-GMP also affects R. capsulatus flagellar motility in addition to GTA production, with lower levels of intracellular c-di-GMP favoring increased flagellar motility and gene transfer. These findings further illustrate the interconnection of GTA production with global systems of regulation in R. capsulatus, providing additional support for the notion that the production of GTAs has been maintained in this and related bacteria because it provides a benefit to the producing organisms.

Project description:Various important cellular processes in bacteria are controlled by c-di-GMP, such as motility, biofilm formation and virulence factors production. C-di-GMP is synthesized from two molecules of GTP by diguanylate cyclases (DGCs) and its actions are terminated by EAL or HD-GYP domain phosphodiesterases (PDEs), which hydrolyze c-di-GMP to linear pGpG or GMP. Thus far the majority of efforts have been dedicated to the development of inhibitors of DGCs but not PDEs. This is probably because the old view was that inhibiting any c-di-GMP PDE would lead to biofilm formation, an undesirable phenotype. Recent data however suggest that some PDEs only change the localized (not global) concentration of c-di-GMP to increase bacterial virulence and do not affect biofilm formation. A challenge therefore is to be able to develop selective PDE inhibitors that inhibit virulence-associated PDEs but not inhibit PDEs that regulate bacterial biofilm formation. Using high throughput docking experiments to screen a library of 250 000 commercially available compounds against E. coli YahA (also called PdeL), a benzoisothiazolinone derivative was found to bind to the c-di-GMP binding site of YahA with favorable energetics. Paradoxically the in silico identified inhibitor (a benzoisothiazolinone derivative) did not inhibit the hydrolysis of c-di-GMP by YahA, the model PDE that was used in the docking, but instead inhibited RocR, which is a PDE from the opportunistic pathogen P. aeruginosa (PA). RocR promotes bacterial virulence but not biofilm dispersal, making it an ideal PDE to target for anti-virulence purposes. This newly identified RocR ligand displayed some selectivity and did not inhibit other P. aeruginosa PDEs, such as DipA, PvrR and PA4108. DipA, PvrR and PA4108 are key enzymes that reduce global c-di-GMP concentration and promote biofilm dispersal; therefore the identification of an inhibitor of a PA PDE, such as RocR, that does not inhibit major PDEs that modulate global c-di-GMP is an important step towards the development of selective c-di-GMP PDEs that could have interesting biomedical applications. The identified RocR ligand could also inhibit P. aeruginosa (PAO1) swarming but not swimming or biofilm formation. Rhamnolipid production was decreased, explaining the inhibition of swarming.

Dataset Information

AmrZ Regulates Swarming Motility Through Cyclic di-GMP-Dependent Motility Inhibition and Controlling Pel Polysaccharide Production in Pseudomonas aeruginosa PA14.

Publications

AmrZ Regulates Swarming Motility Through Cyclic di-GMP-Dependent Motility Inhibition and Controlling Pel Polysaccharide Production in <i>Pseudomonas aeruginosa</i> PA14.

Similar Datasets

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