mBio. 2017 Mar 21;8(2). pii: e00294-17. doi: 10.1128/mBio.00294-17.
Cohesive Properties of the Caulobacter crescentus Holdfast Adhesin Are Regulated by a Novel c-di-GMP Effector Protein.
Sprecher KS1, Hug I1, Nesper J1, Potthoff E2, Mahi MA3, Sangermani M1, Kaever V4, Schwede T3, Vorholt J2, Jenal U5.
Focal Area of Infection Biology, Biozentrum, University of Basel, Basel, Switzerland.
Institute of Microbiology, ETH Zurich, Zurich, Switzerland.
Focal Area of Computational & Systems Biology, Biozentrum, University of Basel, Basel, Switzerland.
Research Core Unit Metabolomics and Institute of Pharmacology, Hannover Medical School, Hannover, Germany.
Focal Area of Infection Biology, Biozentrum, University of Basel, Basel, Switzerland urs.jenal@unibas.ch.
When encountering surfaces, many bacteria produce adhesins to facilitate their initial attachment and to irreversibly glue themselves to the solid substrate. A central molecule regulating the processes of this motile-sessile transition is the second messenger c-di-GMP, which stimulates the production of a variety of exopolysaccharide adhesins in different bacterial model organisms. In Caulobacter crescentus, c-di-GMP regulates the synthesis of the polar holdfast adhesin during the cell cycle, yet the molecular and cellular details of this control are currently unknown. Here we identify HfsK, a member of a versatile N-acetyltransferase family, as a novel c-di-GMP effector involved in holdfast biogenesis. Cells lacking HfsK form highly malleable holdfast structures with reduced adhesive strength that cannot support surface colonization. We present indirect evidence that HfsK modifies the polysaccharide component of holdfast to buttress its cohesive properties. HfsK is a soluble protein but associates with the cell membrane during most of the cell cycle. Coincident with peak c-di-GMP levels during the C. crescentus cell cycle, HfsK relocalizes to the cytosol in a c-di-GMP-dependent manner. Our results indicate that this c-di-GMP-mediated dynamic positioning controls HfsK activity, leading to its inactivation at high c-di-GMP levels. A short C-terminal extension is essential for the membrane association, c-di-GMP binding, and activity of HfsK. We propose a model in which c-di-GMP binding leads to the dispersal and inactivation of HfsK as part of holdfast biogenesis progression.IMPORTANCE Exopolysaccharide (EPS) adhesins are important determinants of bacterial surface colonization and biofilm formation. Biofilms are a major cause of chronic infections and are responsible for biofouling on water-exposed surfaces. To tackle these problems, it is essential to dissect the processes leading to surface colonization at the molecular and cellular levels. Here we describe a novel c-di-GMP effector, HfsK, that contributes to the cohesive properties and stability of the holdfast adhesin in C. crescentus We demonstrate for the first time that c-di-GMP, in addition to its role in the regulation of the rate of EPS production, also modulates the physicochemical properties of bacterial adhesins. By demonstrating how c-di-GMP coordinates the activity and subcellular localization of HfsK, we provide a novel understanding of the cellular processes involved in adhesin biogenesis control. Homologs of HfsK are found in representatives of different bacterial phyla, suggesting that they play important roles in various EPS synthesis systems.
Copyright © 2017 Sprecher et al.
10.1128/mBio.00294-17
HfsK specifically binds c-di-GMP. (A) ITC measuring the interaction of His-HfsK with c-di-GMP. Heat release upon ligand injection (top), integrated heat plotted against the molar ratio of ligand and protein (bottom), and the resulting disassociation constant (Kd) are shown. (B) Quantified autoradiographs of 1 μM purified His-HfsK that has been UV cross-linked to 10 μM [33P]c-di-GMP in the presence or absence of a 10-fold excess of nonlabeled nucleotides to test competition. norm., normalized.
Cohesive Properties of the Caulobacter crescentus Holdfast Adhesin Are Regulated by a Novel c-di-GMP Effector Protein
mBio. 2017 Mar-Apr;8(2):e00294-17.
HfsK deletion leads to an incoherent holdfast that does not support surface colonization. (A) Surface colonization by the wild type (wt) and mutants lacking the hfsK paralog family determined by crystal violet staining of adherent cells after 30 min (dark red bars) and 24 h (light red bars) of growth in microtiter plates. norm, normalized per condition. Error bars show standard deviations (SD) of three independent experiments. *, **, and *** represent P values of <0.1, <0.01, and <0.001, respectively. (B) Analysis of holdfast shedding in hfsK and cc2278 mutants, as well as in strains that shed (ΔhfaB) or form incoherent (ΔhfsH) holdfast. Shown are overlays of phase-contrast (Ph) and fluorescence images of adhered WGA-stained holdfast on glass coverslips after 2 h of cell adsorption. (C) Holdfasts analyzed under shear stress after 15 h of growth in a microfluidic channel with a constant flow of fresh medium containing OG-WGA. Shown is an overlay of fluorescence and inverted differential interference contrast (DIC) images. Arrowheads point to holdfast filaments. Scale bars in panels B and C, 5 μm.
c-di-GMP controls HfsK localization. (A, B) HfsK localization in wild-type (wt), rcdG0 (c-di-GMP low), and rcdG0::dgcZ (c-di-GMP high) cells analyzed by cell fractionation, followed by immunoblotting. CtrA (cytosolic) and CC0164 (inner membrane) were used as controls. (A) Lanes: cell lysates, L; soluble fraction, S; membrane fraction, M. (B) Quantification of three independent cell fractionations showing the fractions of soluble (light red) and membrane-associated (dark red) HfsK. Error bars show the SD, * and ** represent P values of <0.1 and <0.01, respectively. (C) 3D-SIM images of HfsK-eGFP in cells with different c-di-GMP levels. Staining was done with the membrane dye FM4-64. Arrowheads indicate cells with cytosolic HfsK-eGFP. Scale bar, 2 µm. (D) Quantification of HfsK-eGFP localization in standard fluorescence microscopy images. The ratio of the average signal intensities in the membrane and cytosolic compartments is correlated with cell length. The pie chart inset shows the fraction of cells with a membrane-associated (ratiomembrane-cytosol, > 0.7; dark red) or cytosolic (ratio, ≤0.7; light red) GFP signal. n = 250 cells per strain in two independent experiments. (E) Time-lapse video of ΔhfsK Pvan-hfsK-egfp mutant cells grown on PYE agarose pads supplemented with rhodamine-WGA and vanillic acid at 30°C. Cells with dispersed HfsK-eGFP (arrowheads) and the first appearance of holdfast in each cell (asterisks) are indicated. Scale bar, 1 µm. In all of the images, expression of egfp constructs and dgcZ was induced with 0.55 mM vanillic acid and 0.5 mM IPTG, respectively.
The C terminus of HfsK is an important determinant of its localization and function. (A) Schematic representation of the HfsK-eGFP mutants used in this experiment. Amino acid positions are indicated at the top. The drawing is not to scale. TM, transmembrane domain of SecE. (B) Localization quantification of different HfsK-eGFP mutants expressed by the addition of 0.55 mM vanillic acid as described in the legend to . n = 260 cells per strain from two independent experiments. Representative 3D-SIM images for visualization are shown at the top. Arrowheads indicate cells with dispersed HfsK-eGFP. Note that because of the weak signal intensity of HfsKtrnc-TM, the 3D-SIM image recreation resulted in images with a high background signal level. Scale bar, 3 µm. (C) Functionality of HfsK-eGFP mutants compared to that of a wild-type strain harboring the control empty vector (e.v.) in surface colonization after 24 h of growth in microtiter plates in the presence of 0.1 mM vanillic acid. Error bars show the SD of three independent experiments. ** and *** represent P values of <0.01 and <0.001, respectively.
c-di-GMP binding is required for HfsK delocalization but not for HfsK-mediated surface colonization. (A) Quantified autoradiographs of 1 μM purified StrepII-HfsK mutants that were UV cross-linked to increasing concentrations of [33P]c-di-GMP. (B) Localization analysis of different arginine mutants fused to GFP expressed by the addition of 0.55 mM vanillic acid. Representative 3D-SIM images for visualization (left) and localization quantification, as described in the legend to , are shown. n = 575 cells per strain from two independent experiments. Expression of dgcZ was induced with 0.5 mM IPTG. Scale bar, 3 µm. (C) Functionality of HfsK arginine mutants compared to that of a wild-type (wt) strain harboring the control empty vector (e.v.) in surface colonization after 24 h of growth in microtiter plates in the presence of 0.1 mM vanillic acid. (D) Analysis of adhered OG-WGA-stained holdfast on coverslips after 2 h of cell adsorption. Representative overlays of fluorescence and phase-contrast (Ph) images are shown. Scale bar, 5 μm. (E) Holdfasts of a strain with high c-di-GMP levels analyzed under shear stress after 15 h of growth in a microfluidic channel with a constant flow of fresh medium containing OG-WGA. Shown is an overlay of fluorescence and inverted differential interference contrast (DIC) images. Scale bar, 5 μm. (F) Model of HfsK regulation. General protein localization is represented by red shades. Active HfsK binds to membrane component X in the absence of c-di-GMP via its C terminus. Upon c-di-GMP binding, the C terminus rearranges to accommodate the nucleotide and the protein is inactivated and disperses in the cytosol. M, membrane. The error bars in panels A and B show the SD of three independent experiments. **, P < 0.01.
Adhesins, Bacterial/metabolism*
Arylamine N-Acetyltransferase/genetics
Arylamine N-Acetyltransferase/metabolism*
Bacterial Adhesion*
Caulobacter crescentus/genetics
Caulobacter crescentus/metabolism*
Caulobacter crescentus/physiology*
Cyclic GMP/analogs & derivatives*
Cyclic GMP/metabolism
Gene Deletion
Gene Expression Regulation, Bacterial*
Adhesins, Bacterial
bis(3',5')-cyclic diguanylic acid
Arylamine N-Acetyltransferase
Cyclic GMP
HighWire