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Feedback regulation of Caulobacter crescentus holdfast synthesis by flagellum assembly via the holdfast inhibitor HfiA.

ABSTRACT: To permanently attach to surfaces, Caulobacter crescentusproduces a strong adhesive, the holdfast. The timing of holdfast synthesis is developmentally regulated by cell cycle cues. When C. crescentusis grown in a complex medium, holdfast synthesis can also be stimulated by surface sensing, in which swarmer cells rapidly synthesize holdfast in direct response to surface contact. In contrast to growth in complex medium, here we show that when cells are grown in a defined medium, surface contact does not trigger holdfast synthesis. Moreover, we show that in a defined medium, flagellum synthesis and regulation of holdfast production are linked. In these conditions, mutants lacking a flagellum attach to surfaces over time more efficiently than either wild-type strains or strains harboring a paralyzed flagellum. Enhanced adhesion in mutants lacking flagellar components is due to premature holdfast synthesis during the cell cycle and is regulated by the holdfast synthesis inhibitor HfiA. hfiA transcription is reduced in flagellar mutants and this reduction is modulated by the diguanylate cyclase developmental regulator PleD. We also show that, in contrast to previous predictions, flagella are not necessarily required for C. crescentus surface sensing in the absence of flow, and that arrest of flagellar rotation does not stimulate holdfast synthesis. Rather, our data support a model in which flagellum assembly feeds back to control holdfast synthesis via HfiA expression in a c-di-GMP-dependent manner under defined nutrient conditions.

SUBMITTER: Berne C

PROVIDER: S-EPMC6195837 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Feedback regulation of Caulobacter crescentus holdfast synthesis by flagellum assembly via the holdfast inhibitor HfiA.

Berne Cécile C Ellison Courtney K CK Agarwal Radhika R Severin Geoffrey B GB Fiebig Aretha A Morton Robert I RI Waters Christopher M CM Brun Yves V YV

Molecular microbiology 20181005 2

To permanently attach to surfaces, Caulobacter crescentusproduces a strong adhesive, the holdfast. The timing of holdfast synthesis is developmentally regulated by cell cycle cues. When C. crescentusis grown in a complex medium, holdfast synthesis can also be stimulated by surface sensing, in which swarmer cells rapidly synthesize holdfast in direct response to surface contact. In contrast to growth in complex medium, here we show that when cells are grown in a defined medium, surface contact do ...[more]

PMID: 30079982

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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:Due to their intimate physical interactions with the environment, surface polysaccharides are critical determinants of fitness for bacteria. Caulobacter crescentus produces a specialized structure at one of its cell poles called the holdfast that enables attachment to surfaces. Previous studies have shown that the holdfast is composed of carbohydrate-based material and identified a number of genes required for holdfast development. However, incomplete information about its chemical structure, biosynthetic genes, and regulatory principles has limited progress in understanding the mechanism of holdfast synthesis. We leveraged the adhesive properties of the holdfast to perform a saturating screen for genes affecting attachment to cheesecloth over a multiday time course. Using similarities in the temporal profiles of mutants in a transposon library, we defined discrete clusters of genes with related effects on cheesecloth colonization. Holdfast synthesis, flagellar motility, type IV pilus assembly, and smooth lipopolysaccharide (SLPS) production represented key classes of adhesion determinants. Examining these clusters in detail allowed us to predict and experimentally define the functions of multiple uncharacterized genes in both the holdfast and SLPS pathways. In addition, we showed that the pilus and the flagellum control holdfast synthesis separately by modulating the holdfast inhibitor hfiA. This report defines a set of genes contributing to adhesion that includes newly discovered genes required for holdfast biosynthesis and attachment. Our data provide evidence that the holdfast contains a complex polysaccharide with at least four monosaccharides in the repeating unit and underscore the central role of cell polarity in mediating attachment of C. crescentus to surfaces.IMPORTANCE Bacteria routinely encounter biotic and abiotic materials in their surrounding environments, and they often enlist specific behavioral programs to colonize these materials. Adhesion is an early step in colonizing a surface. Caulobacter crescentus produces a structure called the holdfast which allows this organism to attach to and colonize surfaces. To understand how the holdfast is produced, we performed a genome-wide search for genes that contribute to adhesion by selecting for mutants that could not attach to cheesecloth. We discovered complex interactions between genes that mediate surface contact and genes that contribute to holdfast development. Our genetic selection identified what likely represents a comprehensive set of genes required to generate a holdfast, laying the groundwork for a detailed characterization of the enzymes that build this specialized adhesin.

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

Feedback regulation of Caulobacter crescentus holdfast synthesis by flagellum assembly via the holdfast inhibitor HfiA.

Publications

Feedback regulation of Caulobacter crescentus holdfast synthesis by flagellum assembly via the holdfast inhibitor HfiA.

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