Dataset Information

A localized multimeric anchor attaches the Caulobacter holdfast to the cell pole.

ABSTRACT: Caulobacter crescentus attachment is mediated by the holdfast, a complex of polysaccharide anchored to the cell by HfaA, HfaB and HfaD. We show that all three proteins are surface exposed outer membrane (OM) proteins. HfaA is similar to fimbrial proteins and assembles into a high molecular weight (HMW) form requiring HfaD, but not holdfast polysaccharide. The HfaD HMW form is dependent on HfaA but not on holdfast polysaccharide. We show that HfaA and HfaD form homomultimers and that they require HfaB for stability and OM translocation. All three proteins localize to the late pre-divisional flagellar pole, remain at this pole in swarmer cells, and localize at the stalk tip after the stalk is synthesized at the same pole. Hfa protein localization requires the holdfast polysaccharide secretion proteins and the polar localization factor PodJ. An hfaB mutant is much more severely deficient in adherence and holdfast attachment than hfaA and hfaD mutants. An hfaA, hfaD double mutant phenocopies either single mutant, suggesting that HfaB is involved in holdfast attachment beyond secretion of HfaA and HfaD. We hypothesize that HfaB secretes HfaA and HfaD across the outer membrane, and the three proteins form a complex anchoring the holdfast to the stalk.

SUBMITTER: Hardy GG

PROVIDER: S-EPMC2908716 | biostudies-literature | 2010 Apr

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

A localized multimeric anchor attaches the Caulobacter holdfast to the cell pole.

Hardy Gail G GG Allen Rebecca C RC Toh Evelyn E Long Maria M Brown Pamela J B PJ Cole-Tobian Jennifer L JL Brun Yves V YV

Molecular microbiology 20100310 2

Caulobacter crescentus attachment is mediated by the holdfast, a complex of polysaccharide anchored to the cell by HfaA, HfaB and HfaD. We show that all three proteins are surface exposed outer membrane (OM) proteins. HfaA is similar to fimbrial proteins and assembles into a high molecular weight (HMW) form requiring HfaD, but not holdfast polysaccharide. The HfaD HMW form is dependent on HfaA but not on holdfast polysaccharide. We show that HfaA and HfaD form homomultimers and that they require ...[more]

PMID: 20233308

Similar Datasets

Project description:Attachment is essential for microorganisms to establish interactions with both biotic and abiotic surfaces. Stable attachment of Caulobacter crescentus to surfaces requires an adhesive polysaccharide holdfast, but the exact composition of the holdfast is unknown. The holdfast is anchored to the cell envelope by outer membrane proteins HfaA, HfaB, and HfaD. Holdfast anchor gene mutations result in holdfast shedding and reduced cell adherence. Translocation of HfaA and HfaD to the cell surface requires HfaB. The Wzx homolog HfsF is predicted to be a bacterial polysaccharide flippase. An hfsF deletion significantly reduced the amount of holdfast produced per cell and slightly reduced adherence. A ?hfsF ?hfaD double mutant was completely deficient in adherence. A suppressor screen that restored adhesion in the ?hfsF ?hfaD mutant identified mutations in three genes: wbqV, rfbB, and rmlA Both WbqV and RfbB belong to a family of nucleoside-diphosphate epimerases, and RmlA has similarity to nucleotidyltransferases. The loss of wbqV or rfbB in the ?hfsF ?hfaD mutant reduced holdfast shedding but did not restore holdfast synthesis to parental levels. Loss of wbqV or rfbB did not restore adherence to a ?hfsF mutant but did restore adherence and holdfast anchoring to a ?hfaD mutant, confirming that suppression occurs through restoration of holdfast anchoring. The adherence and holdfast anchoring of a ?hfaA ?hfaD mutant could be restored by wbqV or rfbB mutation, but such mutations could not suppress these phenotypes in the ?hfaB mutant. We hypothesize that HfaB plays an additional role in holdfast anchoring or helps to translocate an unknown factor that is important for holdfast anchoring.IMPORTANCE Biofilm formation results in increased resistance to both environmental stresses and antibiotics. Caulobacter crescentus requires an adhesive holdfast for permanent attachment and biofilm formation, but the exact mechanism of polysaccharide anchoring to the cell and the holdfast composition are unknown. Here we identify novel polysaccharide genes that affect holdfast anchoring to the cell. We identify a new role for the holdfast anchor protein HfaB. This work increases our specific knowledge of the polysaccharide adhesin involved in Caulobacter attachment and the general knowledge regarding production and anchoring of polysaccharide adhesins by bacteria. This work also explores the interactions between different polysaccharide biosynthesis and secretion systems in bacteria.

Project description:Adhesion allows microbes to colonize surfaces and is the first stage in biofilm formation. Stable attachment of the freshwater alphaproteobacterium Caulobacter crescentus to surfaces requires an adhesive polysaccharide called holdfast, which is synthesized at a specific cell pole and ultimately found at the tip of cylindrical extensions of the cell envelope called stalks. Secretion and anchoring of holdfast to the cell surface are governed by proteins HfsDAB and HfaABD, respectively. The arrangement and organization of these proteins with respect to each other and the cell envelope, and the mechanism by which the holdfast is anchored on cells, are unknown. In this study, we have imaged a series of C. crescentus mutants using electron cryotomography, revealing the architecture and arrangement of the molecular machinery involved in holdfast anchoring in cells. We found that the holdfast is anchored to cells by a defined complex made up of the HfaABD proteins and that the HfsDAB secretion proteins are essential for proper assembly and localization of the HfaABD anchor. Subtomogram averaging of cell stalk tips showed that the HfaABD complex spans the outer membrane. The anchor protein HfaB is the major component of the anchor complex located on the periplasmic side of the outer membrane, while HfaA and HfaD are located on the cell surface. HfaB is the critical component of the complex, without which no HfaABD complex was observed in cells. These results allow us to propose a working model of holdfast anchoring, laying the groundwork for further structural and cell biological investigations.IMPORTANCE Adhesion and biofilm formation are fundamental processes that accompany bacterial colonization of surfaces, which are of critical importance in many infections. Caulobacter crescentus biofilm formation proceeds via irreversible adhesion mediated by a polar polysaccharide called holdfast. Mechanistic and structural details of how the holdfast is secreted and anchored on cells are still lacking. Here, we have assigned the location and described the arrangement of the holdfast anchor complex. This work increases our knowledge of the relatively underexplored field of polysaccharide-mediated adhesion by identifying structural elements that anchor polysaccharides to the cell envelope, which is important in a variety of bacterial species.

Project description: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.

Dataset Information

A localized multimeric anchor attaches the Caulobacter holdfast to the cell pole.

Publications

A localized multimeric anchor attaches the Caulobacter holdfast to the cell pole.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets