A Symphony of Cyclases: Specificity in Diguanylate Cyclase Signaling

Dahlstrom, Kurt M.; O’Toole, George A.

doi:10.1146/annurev-micro-090816-093325

Cited by 87 publications

(93 citation statements)

References 69 publications

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“…These observations confirmed that biofilm formation is not directly promoted by a higher concentration of c-di-GMP in the cell but is regulated by discrete and interconnected pathways that respond to local concentrations of c-di-GMP 26 . The DipA protein plays a crucial role in biofilm formation and dispersion in P. aeruginosa as a ∆dipA mutant exhibits reduced swarming motility, increased EPS production and reduced cell dispersal 35 .…”

Section: Discussionsupporting

confidence: 67%

“…Cyclic diguanylate (c-di-GMP), a near universal intracellular signaling messenger, regulates many aspects of bacterial growth and behavior 26,27 , including the transition from motile-to-sessile lifestyle leading to biofilm formation and virulence 28,29 . Intracellular levels of c-di-GMP can be modulated through the balanced activities of two classes of enzymes: (i) the diguanylate cyclases (DGCs) that synthetize c-di-GMP from two GTPs and (ii) the diguanylate phosphodiesterases (PDEs) that degrade c-di-GMP in pGpG and GMP 28,29 .…”

Section: Introductionmentioning

confidence: 99%

“…In addition to DCGs (which contain GGDEF domains) and PDEs (which contain EAL or HY-GYP domains), bacterial genomes generally encode multiple c-di-GMP-binding proteins, including PilZ-domain effectors and proteins with 'degenerate' GGDEF/EAL domains that are enzymatically inactive but still bind c-di-GMP 30,31 . Although the propensity of bacteria to form biofilm correlates with higher intracellular concentrations of c-di-GMP 32 , it is not currently understood how a subset of the multiple DGCs and PDEs can act to produce a specific phenotypic outcome 26 . Extensive studies in P. aeruginosa uncovered the roles of many DGCs, PDEs and c-di-GMP-binding effectors during biofilm development, from the initial stage of cell adhesion on a surface to colony formation and biofilm maturation and dispersion 28 .…”

Section: Introductionmentioning

confidence: 99%

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Interrogation of genes controlling biofilm formation using CRISPR interference in Pseudomonas fluorescens

Noirot‐Gros

Forrester

et al. 2018

Preprint

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Bacterial biofilm formation involves multigenic signaling and regulatory pathways that control the transition from motile to sessile lifestyle, production of extracellular polymeric matrix, and maturation of the biofilm complex 3D structure. Biofilms are extensively studied because of their importance in biomedical, ecological and industrial settings. Genetic approaches based on gene inactivation are powerful for mechanistic studies but often are labor intensive, limiting systematic gene surveys to the most tractable bacterial hosts. Here, we adapted the CRISPR interference (CRISPRi) system for use in P. fluorescens. We found that CRISPRi is applicable to three genetically and physiologically diverse species, SBW25, WH6 and Pf0-1 and affords extended periods of time to study complex phenotypes such as cell morphology, motility and biofilm formation. In SBW25, CRISPRi-mediated silencing of the GacA/S two-component system and genes regulated by cylic-di-GMP produced phenotypes similar to those previously described after gene inactivation in various Pseudomonas. Combined with detailed confocal microscopy of biofilms, our study also revealed novel phenotypes associated with biofilm architecture and extracellular matrix biosynthesis as well as the potent inhibition of SBW25 biofilm formation mediated by the PFLU1114 protein. Thus, CRISPRi is a reliable and scalable approach to interrogate gene networks in the diverse P. fluorescens group.

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Section: Discussionsupporting

confidence: 67%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Interrogation of genes controlling biofilm formation using CRISPR interference in Pseudomonas fluorescens

Noirot‐Gros

Forrester

et al. 2018

Preprint

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“…We also demonstrated previously that the l-site of GcbC contributes to the interaction of this enzyme with LapD (13). This direct interaction model was proposed as one means to confer signaling specificity (12, 14).…”

Section: Introductionmentioning

confidence: 99%

Ligand-Mediated Biofilm Formation via Enhanced Physical Interaction Between a Diguanylate Cyclase and Its Receptor

Giacalone

Smith

Collins

et al. 2017

Preprint

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The second messenger, cyclic dimeric GMP (c-di-GMP) regulates biofilm formation for many bacteria. The binding of c-d¡-GMP by the inner-membrane protein LapD controls biofilm formation, and the LapD receptor is central to a complex network of c-di-GMP-mediated biofilm formation. In this study, we examine how c-di-GMP signaling specificity by a diguanylate cyclase (DGC), GcbC, is achieved via interactions with the LapD receptor and by small ligand sensing via GcbC’s calcium channel chemotaxis (CACHE) domain. We provide evidence that biofilm formation is stimulated by the environmentally relevant organic acid citrate (and a related compound, isocitrate) in a GcbC-dependent manner through enhanced GcbC-LapD interaction, which results in increased LapA localization to the cell surface. Furthermore, GcbC shows little ability to synthesize c-di-GMP in isolation. However, when LapD is present GcbC activity is significantly enhanced ~8-fold, indicating that engaging the LapD receptor stimulates the activity of this DGC; citrate-enhanced GcbC-LapD interaction further stimulates c-di-GMP synthesis. We propose that the l-site of GcbC serves two roles beyond allosteric control of this enzyme: promoting GcbC-LapD interaction and stabilizing the active conformation of GcbC in the GcbC-LapD complex. Finally, given that LapD can interact with a dozen different DGCs of P. fluorescens, many of which have ligand-binding domains, the ligand-mediated enhanced signaling via LapD-GcbC interaction described here is likely a conserved mechanism of signaling in this network. Consistent with this idea, we identify a second example of ligand-mediated enhancement of DGC-LapD interaction that promotes biofilm formation.ImportanceIn many bacteria, dozens of enzymes produce the dinucleotide signal c-di-GMP, however it is unclear how undesired crosstalk is mitigated in the context of this soluble signal, and how c-di-GMP signaling is regulated by environmental inputs. We demonstrate that GcbC, a DGC, shows little ability to synthesize c-d¡-GMP in the absence of its cognate receptor LapD; GcbC-LapD interaction enhances c-di-GMP synthesis by GcbC, likely mediated by the l-site of GcbC. We further show evidence for a ligand-mediated mechanism of signaling specificity via increased physical interaction of a DGC with its cognate receptor. We envision a scenario wherein a “cloud” of weakly active DGCs can increase their activity by specific interaction with their receptor in response to appropriate environmental signals, concomitantly boosting c-di-GMP production, ligand-specific signaling and biofilm formation.

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“…cyclic-diguanylate monophosphate (c-diGMP) (21). c-diGMP is produced by DGCs, typically having a GG(D/E)EF domain and is degrade by phosphodiesterases (PDE), often having an EAL domain (22). The ZM4 genome has five genes annotated to have GG(D/E)EF and/or EAL domains ( Fig.…”

Section: Clustering Of Cells Into Flocs or Biofilms Is Commonly Coordmentioning

confidence: 99%

Cell aggregation and aerobic respiration facilitate survival of Zymomonas mobilis ZM4 in an aerobic minimal medium

Jones-Burrage

Kremer

McKinlay

2019

Preprint

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Zymomonas mobilis produces ethanol from glucose near the theoretical maximum yield, making it a potential alternative to yeast for industrial ethanol production. A potentially useful industrial feature is the ability to form multicellular aggregates called flocs, which can settle 15 quickly and exhibit higher resistance to harmful chemicals. While spontaneous floc-forming Z. mobilis mutants have been described, little is known about the natural conditions that induce Z. mobilis floc formation and the genetic factors involved. Here we found that wild-type Z. mobilis forms flocs in response to aerobic growth conditions but only in a minimal medium. We identified a cellulose synthase gene cluster and a single diguanylate cyclase that are essential 20 for both floc formation and survival in an aerobic minimal medium. We also found that NADH dehydrogenase 2, a key component of the aerobic respiratory chain, is important for survival in an aerobic minimal medium, providing a physiological role for this enzyme which has previously been found to be disadvantageous in aerobic rich media. Supplementation of the minimal medium with vitamins also promoted survival but did not inhibit floc formation. 25

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A Symphony of Cyclases: Specificity in Diguanylate Cyclase Signaling

Cited by 87 publications

References 69 publications

Interrogation of genes controlling biofilm formation using CRISPR interference in Pseudomonas fluorescens

Interrogation of genes controlling biofilm formation using CRISPR interference in Pseudomonas fluorescens

Ligand-Mediated Biofilm Formation via Enhanced Physical Interaction Between a Diguanylate Cyclase and Its Receptor

Cell aggregation and aerobic respiration facilitate survival of Zymomonas mobilis ZM4 in an aerobic minimal medium

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