Physiochemical properties of Caulobacter crescentus holdfast: a localized bacterial adhesive.
ABSTRACT: To colonize surfaces, the bacterium Caulobacter crescentus employs a polar polysaccharide, the holdfast, located at the end of a thin, long stalk protruding from the cell body. Unlike many other bacteria which adhere through an extended extracellular polymeric network, the holdfast footprint area is tens of thousands times smaller than that of the total bacterium cross-sectional surface, making for some very demanding adhesion requirements. At present, the mechanism of holdfast adhesion remains poorly understood. We explore it here along three lines of investigation: (a) the impact of environmental conditions on holdfast binding affinity, (b) adhesion kinetics by dynamic force spectroscopy, and (c) kinetic modeling of the attachment process to interpret the observed time-dependence of the adhesion force at short and long time scales. A picture emerged in which discrete molecular units called adhesins are responsible for initial holdfast adhesion, by acting in a cooperative manner.
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:Attachment to surfaces by the prosthecate bacterium Caulobacter crescentus is mediated by an adhesive organelle, the holdfast, found at the tip of the stalk. Indirect evidence suggested that the holdfast first appears at the swarmer pole of the predivisional cell. We used fluorescently labeled lectin and transmission electron microscopy to detect the holdfast in different cell types. While the holdfast was readily detectable in stalked cells and at the stalked poles of predivisional cells, we were unable to detect the holdfast in swarmer cells or at the flagellated poles of predivisional cells. This suggests that exposure of the holdfast to the outside of the cell occurs during the differentiation of swarmer to stalked cells. To investigate the timing of holdfast synthesis and exposure to the outside of the cell, we have examined the regulation of a holdfast attachment gene, hfaA. The hfaA gene is part of a cluster of four genes (hfaABDC), identified in strain CB2A and involved in attachment of the holdfast to the polar region of the cell. We have identified the hfaA gene in the synchronizable C. crescentus strain CB15. The sequence of the CB2A hfaA promoter suggested that it was regulated by sigma54. We show that the transcription of hfaA from either strain is not dependent on sigma54. Using a hfaA-lacZ fusion, we show that the transcription of hfaA is temporally regulated during the cell cycle, with maximal expression in late-predivisional cells. This increase in expression is largely due to the preferential transcription of hfaA in the swarmer pole of the predivisional cell.
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: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.
Project description:Caulobacter crescentus cells adhere to surfaces by using an extremely strong polar adhesin called the holdfast. The polysaccharide component of the holdfast is comprised in part of oligomers of N-acetylglucosamine. The genes involved in the export of the holdfast polysaccharide and the anchoring of the holdfast to the cell were previously discovered. In this study, we identified a cluster of polysaccharide biosynthesis genes (hfsEFGH) directly adjacent to the holdfast polysaccharide export genes. Sequence analysis indicated that these genes are involved in the biosynthesis of the minimum repeat unit of the holdfast polysaccharide. HfsE is predicted to be a UDP-sugar lipid-carrier transferase, the glycosyltransferase that catalyzes the first step in polysaccharide biosynthesis. HfsF is predicted to be a flippase, HfsG is a glycosyltransferase, and HfsH is similar to a polysaccharide (chitin) deacetylase. In-frame hfsG and hfsH deletion mutants resulted in severe deficiencies both in surface adhesion and in binding to the holdfast-specific lectin wheat germ agglutinin. In contrast, hfsE and hfsF mutants exhibited nearly wild-type levels of adhesion and holdfast synthesis. We identified three paralogs to hfsE, two of which are redundant to hfsE for holdfast synthesis. We also identified a redundant paralog to the hfsC gene, encoding the putative polysaccharide polymerase, and present evidence that the hfsE and hfsC paralogs, together with the hfs genes, are absolutely required for proper holdfast synthesis.
Project description:Growth in a surface-attached bacterial community, or biofilm, confers a number of advantages. However, as a biofilm matures, high-density growth imposes stresses on individual cells, and it can become less advantageous for progeny to remain in the community. Thus, bacteria employ a variety of mechanisms to control attachment to and dispersal from surfaces in response to the state of the environment. The freshwater oligotroph Caulobacter crescentus can elaborate a polysaccharide-rich polar organelle, known as the holdfast, which enables permanent surface attachment. Holdfast development is strongly inhibited by the small protein HfiA; mechanisms that control HfiA levels in the cell are not well understood. We have discovered a connection between the essential general protein chaperone, DnaK, and control of C. crescentus holdfast development. C. crescentus mutants partially or completely lacking the C-terminal substrate binding "lid" domain of DnaK exhibit enhanced bulk surface attachment. Partial or complete truncation of the DnaK lid domain increases the probability that any single cell will develop a holdfast by 3- to 10-fold. These results are consistent with the observation that steady-state levels of an HfiA fusion protein are significantly diminished in strains that lack the entire lid domain of DnaK. While dispensable for growth, the lid domain of C. crescentus DnaK is required for proper chaperone function, as evidenced by observed dysregulation of HfiA and holdfast development in strains expressing lidless DnaK mutants. We conclude that DnaK is an important molecular determinant of HfiA stability and surface adhesion control.Regulatory control of cell adhesion ensures that bacterial cells can transition between free-living and surface-attached states. We define a role for the essential protein chaperone, DnaK, in the control of Caulobacter crescentus cell adhesion. C. crescentus surface adhesion is mediated by an envelope-attached organelle known as the holdfast. Holdfast development is tightly controlled by HfiA, a small protein inhibitor that directly interacts with a WecG/TagA-family glycosyltransferase required for holdfast biosynthesis. We demonstrate that the C-terminal lid domain of DnaK is not essential for growth but is necessary for proper control of HfiA levels in the cell and for control of holdfast adhesin development.
Project description:Caulobacter crescentus firmly adheres to surfaces with a structure known as the holdfast, which is located at the flagellar pole of swarmer cells and at the stalk tip in stalked cells. A three-gene cluster (hfaAB and hfaC) is involved in attachment of the holdfast to the cell. Deletion and complementation analysis of the hfaAB locus revealed two genes in a single operon; both were required for holdfast attachment to the cell. Sequence analysis of the hfaAB locus showed two open reading frames with the potential to encode proteins of 15,000 and 26,000 Da, respectively. A protein migrating with an apparent size of 21 kDa in gel electrophoresis was encoded by the hfaA region when expressed in Escherichia coli under the control of the lac promoter, but no protein synthesis could be detected from the hfaB region. S1 nuclease analysis indicated that transcription of the hfaAB locus was initiated from a region containing a sequence nearly identical to the consensus for C. crescentus sigma 54-dependent promoters. In addition, a sequence with some similarity to ftr sequences (a consensus sequence associated with other Caulobacter sigma 54-dependent genes) was identified upstream of the hypothesized sigma 54 promoter. At least one of the hfaAB gene products was required for maximal transcription of hfaC. The sequence of hfaB showed some similarity to that of transcriptional activators of other bacteria. The C-terminal region of the putative gene product HfaA was found to be homologous to PapG and SmfG, which are adhesin molecules of enteropathogenic E. coli and Serratia marcescens, respectively. This information suggests that the protein encoded by the hfaA locus may have a direct role in the attachment of the holdfast to the cell, whereas hfaB may be involved in the positive regulation of hfaC.
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.
Project description:Alphaproteobacteria commonly produce a polar adhesin that is anchored to the exterior of the cell envelope. In Caulobacter crescentus, this adhesin enables permanent attachment to solid surfaces and is known as the holdfast. An ensemble of two-component signal transduction (TCS) proteins control C. crescentus holdfast biogenesis by indirectly regulating expression of HfiA, a potent holdfast inhibitor. We designed a genetic screen to identify regulators of hfiA that function downstream of the TCS adhesion regulatory system. This screen identified a hypothetical protein that we have named RtrC. Though the primary structure of RtrC does not match any defined sequence family, we demonstrate that RtrC directly binds and regulates dozens of sites on the C. crescentus chromosome via a pseudo-palindromic motif. Among these binding sites is the hfiA promoter where RtrC functions to repress transcription and thereby activate holdfast biogenesis. RtrC forms an OR-gated type I coherent feedforward loop with the DNA-binding response regulator SpdR and the adhesion regulator RtrB. This type of network motif is known to buffer gene expression against transient loss of regulating signals. We further demonstrate that, in addition to promoting adhesion, RtrC expression influences cell motility in soft agar. We conclude that the formerly hypothetical gene, rtrC, encodes a transcription factor that functions downstream of the C. crescentus TCS adhesion control system to regulate holdfast biogenesis and motility. Overall design: RNA-sequencng from cells overexpressing rtrC
Project description:Alphaproteobacteria commonly produce a polar adhesin that is anchored to the exterior of the cell envelope. In Caulobacter crescentus, this adhesin enables permanent attachment to solid surfaces and is known as the holdfast. An ensemble of two-component signal transduction (TCS) proteins control C. crescentus holdfast biogenesis by indirectly regulating expression of HfiA, a potent holdfast inhibitor. We designed a genetic screen to identify regulators of hfiA that function downstream of the TCS adhesion regulatory system. This screen identified a hypothetical protein that we have named RtrC. Though the primary structure of RtrC does not match any defined sequence family, we demonstrate that RtrC directly binds and regulates dozens of sites on the C. crescentus chromosome via a pseudo-palindromic motif. Among these binding sites is the hfiA promoter where RtrC functions to repress transcription and thereby activate holdfast biogenesis. RtrC forms an OR-gated type I coherent feedforward loop with the DNA-binding response regulator SpdR and the adhesion regulator RtrB. This type of network motif is known to buffer gene expression against transient loss of regulating signals. We further demonstrate that, in addition to promoting adhesion, RtrC expression influences cell motility in soft agar. We conclude that the formerly hypothetical gene, rtrC, encodes a transcription factor that functions downstream of the C. crescentus TCS adhesion control system to regulate holdfast biogenesis and motility. Overall design: Chromatin Immunoprecipitation sequencing (ChIP-seq) for 3xFLAG-tagged rtrC