The HfaB and HfaD adhesion proteins of Caulobacter crescentus are localized in the stalk

The differentiating bacterium Caulobacter crescentus produces two different cell types at each cell division, a motile swarmer cell and an adhesive stalked cell. The stalked cell harbours a stalk, a thin cylindrical extension of the cell surface. The tip of the stalk is decorated with a holdfast, an adhesive organelle composed at least in part of polysaccharides. The synthesis of the stalk and holdfast occur at the same pole during swarmer cell differentiation. Mutations in the hfaABDC gene cluster had been shown to disrupt the attachment of the holdfast to the tip of the stalk, but the role of individual genes was unknown. We used lacZ fusions of various DNA fragments from the hfaABDC region to show that these genes form an operon. In order to analyse the relative contribution of the different genes to holdfast attachment, mutations were constructed for each gene. hfaC was not required for holdfast attachment or binding to surfaces. The hfaA and hfaD mutants shed some holdfast material into the surrounding medium and were partially deficient in binding to surfaces. Unlike hfaA and hfaB mutants, hfaD mutants were still able to form rosettes efficiently. Cells with insertions in hfaB were unable to bind to surfaces, and lectin binding studies indicated that the hfaB mutants had the strongest holdfast shedding phenotype. We determined that HfaB and HfaD are membrane-associated proteins and that HfaB is a lipoprotein. Purification of stalks and cell bodies indicated that both HfaB and HfaD are enriched in the stalk as compared to the cell body. These results suggest that HfaB and HfaD, and probably HfaA, serve to anchor the holdfast to the tip of the stalk.     

Dynamics and control of biofilms of the oligotrophic bacterium Caulobacter crescentus

Caulobacter crescentus is an oligotrophic alpha-proteobacterium with a complex cell cycle involving sessile-stalked and piliated, flagellated swarmer cells. Because the natural lifestyle of C. crescentus intrinsically involves a surface-associated, sessile state, we investigated the dynamics and control of C. crescentus biofilms developing on glass surfaces in a hydrodynamic system. In contrast to biofilms of the well-studied Pseudomonas aeruginosa, Escherichia coli, and Vibrio cholerae, C. crescentus CB15 cells form biphasic biofilms, consisting predominantly of a cell monolayer biofilm and a biofilm containing densely packed, mushroom-shaped structures. Based on comparisons between the C. crescentus strain CB15 wild type and its holdfast (hfsA; DeltaCC0095), pili (DeltapilA-cpaF::Omegaaac3), motility (motA), flagellum (flgH) mutants, and a double mutant lacking holdfast and flagellum (hfsA; flgH), a model for biofilm formation in C. crescentus is proposed. For both biofilm forms, the holdfast structure at the tip of a stalked cell is crucial for mediating the initial attachment. Swimming motility by means of the single polar flagellum enhances initial attachment and enables progeny swarmer cells to escape from the monolayer biofilm. The flagellum structure also contributes to maintaining the mushroom structure. Type IV pili enhance but are not absolutely required for the initial adhesion phase. However, pili are essential for forming and maintaining the well-defined three-dimensional mushroom-shaped biofilm. The involvement of pili in mushroom architecture is a novel function for type IV pili in C. crescentus. These unique biofilm features demonstrate a spatial diversification of the C. crescentus population into a sessile, "stem cell"-like subpopulation (monolayer biofilm), which generates progeny cells capable of exploring the aqueous, oligotrophic environment by swimming motility and a subpopulation accumulating in large mushroom structures.     

Characterization of the Adhesive Holdfast of Marine and Freshwater Caulobacters

Caulobacters are prosthecate (stalked) bacteria that elaborate an attachment organelle called a holdfast at the tip of the cellular stalk. We examined the binding of lectins to the holdfasts of 16 marine Caulobacter strains and 10 freshwater species or strains by using a panel of fluorescein-conjugated lectins and fluorescence microscopy. The holdfasts of all the marine isolates bound to only wheat germ agglutinin (WGA) and other lectins that bind N-acetylglucosamine (GlcNac) residues. The freshwater caulobacters showed more variability in holdfast composition. Some bound only to WGA and comparable lectins as the marine strains did. Others bound additional or other lectins, and some did not bind to the lectins tested. The binding of WGA appeared to involve the regions of the holdfast involved with adhesion; a holdfast bound to WGA was significantly less adhesive to glass. Competition experiments with WGA-binding holdfasts and oligomers of GlcNac demonstrated that trimers of GlcNac (the preferred substrate for WGA binding) were more effective than dimers or monomers in preventing WGA binding to holdfasts, suggesting that stretches of contiguous GlcNac residues occur in the WGA-binding holdfasts. In addition, differences between freshwater and marine holdfasts in the strength of WGA binding were noted. The effect of a number of proteolytic and glycolytic enzymes on holdfast integrity was examined; the proteases had no effect for all caulobacters. None of the glycolytic enzymes had an effect on marine caulobacter holdfasts, but chitinase and lysozyme (both attack oligomers of GlcNac) disrupted the holdfasts of those freshwater caulobacters that bound WGA. Despite some similarity to chitin, holdfasts did not bind Calcofluor and no measurable effects on holdfast production were detectable after cell growth in the presence of diflubenzuron or polyoxin D, inhibitors of chitin synthesis in other systems. Finally, the holdfasts of all caulobacters bound to colloidal gold particles, without regard to the coating used to stabilize the gold particles. This binding was stronger or more specific than WGA binding; treatment with colloidal gold particles prevented WGA binding, but the reverse was not the case.     

The Caulobacter crescentus polar organelle development protein PodJ is differentially localized and is required for polar targeting of the PleC development regulator

Regulation of polar development and cell division in Caulobacter crescentus relies on the dynamic localization of several proteins to cell poles at specific stages of the cell cycle. The polar organelle development protein, PodJ, is required for the synthesis of the adhesive holdfast and pili. Here we show the cell cycle localization of PodJ and describe a novel role for this protein in controlling the dynamic localization of the developmental regulator PleC. In swarmer cells, a short form of PodJ is localized at the flagellated pole. Upon differentiation of the swarmer cell into a stalked cell, full length PodJ is synthesized and localizes to the pole opposite the stalk. In late predivisional cells, full length PodJ is processed into a short form which remains localized at the flagellar pole after cell division and is degraded during swarmer to stalked cell differentiation. Polar localization of the developmental regulator PleC requires the presence of PodJ. In contrast, the polar localization of PodJ is not dependent on the presence of PleC. These results indicate that PodJ is an important determinant for the localization of a major regulator of cell differentiation. Thus, PodJ acts directly or indirectly to target PleC to the incipient swarmer pole, to establish the cellular asymmetry that leads to the synthesis of holdfasts and pili at their proper subcellular location.     

Development of surface adhesion in Caulobacter crescentus

Caulobacter crescentus has a dimorphic life cycle composed of a motile stage and a sessile stage. In the sessile stage, C. crescentus is often found tightly attached to a surface through its adhesive holdfast. In this study, we examined the contribution of growth and external structures to the attachment of C. crescentus to abiotic surfaces. We show that the holdfast is essential but not sufficient for optimal attachment. Rather, adhesion in C. crescentus is a complex developmental process. We found that the attachment of C. crescentus to surfaces is cell cycle regulated and that growth or energy or both are essential for this process. The initial stage of attachment occurs in swarmer cells and is facilitated by flagellar motility and pili. Our results suggest that strong attachment is mediated by the synthesis of a holdfast as the swarmer cell differentiates into a stalked cell.     

Turning off flagellum rotation requires the pleiotropic gene pleD: pleA, pleC, and pleD define two morphogenic pathways in Caulobacter crescentus.

We have identified mutations in three pleiotropic genes, pleA, pleC, and pleD, that are required for differentiation in Caulobacter crescentus. pleA and pleC mutants were isolated in an extensive screen for strains defective in both motility and adsorption of polar bacteriophage phi CbK; using temperature-sensitive alleles, we determined the time at which the two genes act. pleA was required for a short period at 0.7 of the swarmer cell cycle for flagellum biosynthesis, whereas pleC was required during an overlapping period from 0.6 to 0.95 of the cell cycle to activate flagellum rotation as well as to enable loss of the flagellum and stalk formation by swarmer cells after division. The third pleiotropic gene, pleD, is described here for the first time. A pleD mutation was identified as a bypass suppressor of a temperature-sensitive pleC allele. Strains containing this mutation were highly motile, did not shed the flagellum or form stalks, and retained motility throughout the cell cycle. Since pleD was required to turn off motility and was a bypass suppressor of pleC, we conclude that it acts after the pleA and pleC gene functions in the cell cycle. No mutants defective in both flagellum biosynthesis and stalk formation were identified. Consequently, we propose that the steps required for formation of swarmer cells and subsequent development into stalked cells are organized into at least two developmental pathways: a pleA-dependent sequence of events, responsible for flagellum biosynthesis in predivisional cells, and a pleC-pleD-dependent sequence, responsible for flagellum activation in predivisional cells and loss of motility and stalk formation in progeny swarmer cells.     

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

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.     

Identification of genes affecting production of the adhesion organelle of Caulobacter crescentus CB2

Transposon (Tn5) mutagenesis was used to identify regions in the genome involved with production, regulation, or attachment to the cell surface of the adhesive holdfast of the freshwater bacterium Caulobacter crescentus CB2. A total of 12,000 independently selected transposon insertion mutants were screened for defects in adhesion to cellulose acetate; 77 mutants were detected and examined by Southern blot hybridization mapping methods and pulsed-field gel electrophoresis. Ten unique sites of Tn5 insertion affecting holdfast function were identified that were clustered in four regions of the genome. Representative mutants of the 10 Tn5 insertion sites were examined by a variety of methods for differences in their phenotype leading to the loss of adhesiveness. Four phenotypes were identified: no holdfast production, production of a smaller or an altered holdfast, production of a holdfast that was unable to remain attached to the cell, and a fourth category in which a possible alteration of the stalk was related to impaired adhesion of the cell. With the possible exception of the last class, no pleiotropic mutants (those with multiple defects in the polar region of the cell) were detected among the adhesion-defective mutants. This was unexpected, since holdfast deficiency is often a characteristic of pleiotropic mutants obtained when selecting for loss of other polar structures. Overall, the evidence suggests that we have identified regions containing structural genes for the holdfast, genes involved with proper attachment or positioning on the caulobacter surface, and possibly regions that regulate the levels of holdfast production.     

Holdfast formation in motile swarmer cells optimizes surface attachment during Caulobacter crescentus development

The adhesive holdfast is required for irreversible surface anchoring of Caulobacter crescentus cells. The holdfast is synthesized early during swarmer cell development and, together with pili and a functional flagellum, contributes to optimal attachment during cell differentiation. We present evidence that the timing of holdfast formation in swarmer cells is regulated posttranslationally and is dependent on the diguanylate cyclase PleD.     

Identification of genes affecting production of the adhesive holdfast of a marine caulobacter.

Caulobacters are stalked bacteria that produce a structure termed a holdfast which enables firm attachment to surfaces. Tn5 insertion mutagenesis was used to identify genes affecting holdfast production or function in the marine strain MCS6. Twelve thousand Tn5 insertion mutants were screened for adhesion defects by an assay involving the attachment of cells to polystyrene microtiter dish wells. Among adhesion-defective mutants, those with multiple polar (pleiotropic) defects were excluded and the remainder were examined for the presence of holdfast. Forty-one mutants that produced no detectable holdfast or a significantly reduced amount were found. Southern blot and pulsed-field gel electrophoresis analyses indicated that 11 unique Tn5 insertions were clustered in three regions of the genome. In addition, 71 mutants that adhered poorly or not at all to polystyrene, yet still produced a holdfast, were found. Southern blot and pulsed-field gel electrophoresis analyses of 15 of these mutants showed eight unique Tn5 insertion sites clustered in two additional regions of the genome. An assay involving attachment to glass treated with siloxane chemicals (producing surfaces with varying degrees of hydrophobicity or hydrophilicity) was used to attempt characterization of this phenotype. Unexpectedly, no simple pattern of differences in binding between the mutants and wild-type caulobacters was found. In particular, no reduction in the ability of the mutants to bind to hydrophobic surfaces was noted. Complementation with cosmid clones was successful in nearly all cases and confirmed the designation of five genomic regions of holdfast-related genes. No detectable cross-hybridization was observed with several holdfast-related gene regions from a freshwater caulobacter, providing further evidence that the marine and freshwater caulobacters are genetically distinct.     

Synthesis and Structure of Caulobacter crescentus Flagella

During the normal cell cycle of Caulobacter crescentus, flagella are released into the culture fluid as swarmer cells differentiate into stalked cells. The released flagellum is composed of a filament, hook, and rod. The molecular weight of purified flagellin (subunit of flagella filament) is 25,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The formation of a flagellum opposite the stalk has been observed by microscope during the differentiation of a stalked cell in preparation for cell division. By pulsing synchronized cultures with (14)C-amino acids it has been demonstrated that the synthesis of flagellin occurs approximately 30 to 40 min before cell division. Flagellin, therefore, is synthesized at a discrete time in the cell cycle and is assembled into flagella at a specific site on the cell. A mutant of C. crescentus that fails to synthesize flagellin has been isolated.     

Fluctuation analysis of Caulobacter crescentus adhesion

The aquatic bacterium Caulobacter crescentus divides asymmetrically to a flagellated swarmer cell and a cell with a stalk. At the end of the stalk is an adhesive organelle known as the holdfast, which the stalked cell uses to attach to a solid surface. Often there are two or more cells with their stalks attached to the same holdfast. By analyzing the fluctuations in the stalk angle for a pair of cells attached to a single holdfast, we determine the elastic stiffness of the holdfast. We model the holdfast as three torsional springs in series and find that the effective torsional spring constant for the holdfast is of the order of (10(-17)-10(-18)) Nm, with unequal spring constants. The asymmetry suggests the sequence in which the cells attach to each other, and in some cases suggests that strong crosslinks form between the stalks as they make a shared holdfast.     

Localization of Surface Structures During Procaryotic Differentiation: Role of Cell Division in Caulobacter crescentus

Asymmetric cell division in Caulobacter crescentus produces two cell types, a stalked cell and a new swarmer cell, with characteristics surface structures. We have examined the role of the cell cycle in the differentiation of these two cells using the adsorption of bacteriophage øLC72, the assembly of the polar flagellum, and stalk formation as assays for changes in surface morphology. Previous studies of this aquatic bacterium [17, 25] have suggested that the replicating chromosome acts as a 'clock' in timing the formation of the flagellar filament at one pole of the new swarmer cell. The analysis of conditional cell cycle mutants presented here extends these results by showing that DNA synthesis is also required for adsorption of phage øLC72 and, more importantly, they also suggest that a late cell division step is involved in determining the spatial pattern in which the phage receptors and flagella are assembled. We propose that this cell division step is required for formation of 'organizational' centers which direct the assembly of surface structures at the new cell poles, and for the polarity reversal in assembly that accompanies swarmer cell to stalked cell development.     

Cell cycle arrest of a Caulobacter crescentus secA mutant.

Cell differentiation is an inherent component of the Caulobacter crescentus cell cycle. The transition of a swarmer cell, with a single polar flagellum, into a sessile stalked cell includes several morphogenetic events. These include the release of the flagellum and pili, the proteolysis of chemotaxis proteins, the biogenesis of the polar stalk, and the initiation of DNA replication. We have isolated a group of temperature-sensitive mutants that are unable to complete this process at the restrictive temperature. We show here that one of these strains has a mutation in a homolog of the Escherichia coli secA gene, whose product is involved in protein translocation at the cell membrane. This C. crescentus secA mutant has allowed the identification of morphogenetic events in the swarmer-to-stalked cell transition that require SecA-dependent protein translocation. Upon shift to the nonpermissive temperature, the mutant secA swarmer cell is able to release the polar flagellum, degrade chemoreceptors, and initiate DNA replication, but it is unable to form a stalk, complete DNA replication, or carry out cell division. At the nonpermissive temperature, the cell cycle blocks prior to the de novo synthesis of flagella and chemotaxis proteins that normally occurs in the predivisional cell. Although interactions between the chromosome and the cytoplasmic membrane are believed to be a functional component of the temporal regulation of DNA replication, the ability of this secA mutant to initiate replication at the nonpermissive temperature suggests that SecA-dependent events are not involved in this process. However, both cell division and stalk formation, which is analogous to a polar division event, require SecA function.     

Dissection of functional domains of the polar localization factor PodJ in Caulobacter crescentus

The polar organelle development protein, PodJ, is important for proper establishment of polarity in Caulobacter crescentus. podJ null mutants are unable to form holdfast or pili, have reduced swarming motility, and have difficulty ejecting the flagellum during the swarmer to stalked cell transition. In this study, we create a series of truncation mutants to investigate functional domains of PodJ. We show that PodJ has a transmembrane domain between amino acids 600 and 670. We identify a periplasmic region important for pili production and a cytoplasmic region required for holdfast formation and swarming motility, and establish that PleC localization is not required for holdfast formation and motility in soft agar. Analysis of the mutants reveals that the last 54 amino acids of the protein negatively regulate processing of the full-length form of the protein, PodJ(L), to a shorter form, PodJ(S). Finally, we identify a cytoplasmic region of PodJ involved in targeting it to the flagellar pole, and a periplasmic region required for localization of PleC.     

Bypassing the need for subcellular localization of a polysaccharide export‐anchor complex by overexpressing its protein subunits

Subcellular protein localization is thought to promote protein–protein interaction by increasing the effective concentration and enabling spatial co‐ordination and proper segregation of proteins. We found that protein overexpression allowed the assembly of a productive polysaccharide biosynthesis‐export‐anchoring complex in the absence of polar localization in Caulobacter crescentus. Polar localization of the holdfast export protein, HfsD, depends on the presence of the other export proteins, HfsA and HfsB, and on the polar scaffold protein PodJ. The holdfast deficiency of hfsB and podJ mutants is suppressed by the overexpression of export proteins. Restored holdfasts are randomly positioned and colocalize with a holdfast anchor protein in these strains, indicating that functional complexes can form at non‐polar sites. Therefore, overexpression of export proteins surpasses a concentration threshold necessary for holdfast synthesis. Restoration of holdfast synthesis at non‐polar sites reduces surface adhesion, consistent with the need to spatially co‐ordinate the holdfast synthesis machinery with the flagellum and pili. These strains lack the cell‐specific segregation of the holdfast, resulting in the presence of holdfasts in motile daughter cells. Our results highlight the fact that multiple facets of subcellular localization can be coupled to improve the phenotypic outcome of a protein assembly.     

Characterization of the holdfast region of wild-type cells and holdfast mutants of Asticcacaulis biprosthecum

Mutants of Asticcacaulis biprosthecum lacking the ability to attach to various surfaces were selected by serial transfer in liquid media containing cheesecloth, to which wild-type cells attach but holdfast mutants do not. Congo red, incorporated into solid media, distinguishes between colonies of wild-type cells and those of holdfast mutants. Holdfast mutants were characterized and compared to wild-type cells according to their ability to swim, to attach to each other or to wild-type cells, for the presence on the cells of polar surface structures (holdfast, flagella, pili), and for sensitivity to phages. All holdfast mutants produced flagella, even though some mutants were nonmotile. Eighteen holdfast mutants fell into two groups: those apparently defective only in holdfast function and those defective in additional structures localized at the holdfast pole of the cell. None of these holdfast mutants was defective in prosthecal development. All holdfast mutants are capable of forming rosettes with wild-type cells, even though they are incapable of initiating attachment on their own, suggesting polymeric bridging as a likely mechanism for attachment.Abbreviation PYE peptone-yeast extract     

Cell cycle-dependent degradation of a flagellar motor component requires a novel-type response regulator

The poles of each Caulobacter crescentus cell undergo morphological development as a function of the cell cycle. A single flagellum assembled at one pole during the asymmetric cell division is later ejected and replaced by a newly synthesized stalk when the motile swarmer progeny differentiates into a sessile stalked cell. The removal of the flagellum during the swarmer-to-stalked cell transition coincides with the degradation of the FliF flagellar anchor protein. We report here that the cell cycle-dependent turnover of FliF does not require the structural components of the flagellum itself, arguing that it is the initial event leading to the ejection of the flagellum. Analysis of a polar development mutant, pleD, revealed that the pleD gene was required for efficient removal of FliF and for ejection of the flagellar structure during the swarmer-to-stalked cell transition. The PleD requirement for FliF degradation was also not dependent on the presence of any part of the flagellar structure. In addition, only 25% of the cells were able to synthesize a stalk during cell differentiation when PleD was absent. The pleD gene codes for a member of the response regulator family with a novel C-terminal regulatory domain. Mutational analysis confirmed that a highly conserved motif in the PleD C-terminal domain is essential to promote both FliF degradation and stalk biogenesis during cell differentiation. Signalling through the C-terminal domain of PleD is thus required for C. crescentus polar development. A second gene, fliL, was shown to be required for efficient turnover of FliF, but not for stalk biogenesis. The possible roles of PleD and FliL in C. crescentus polar development are discussed.     

Reflections on a sticky situation: how surface contact pulls the trigger for bacterial adhesion

Adhesion of bacterial cells to surfaces can be mediated by a wide variety of extracellular structures, which can either recognize specific molecular motifs or adhere in non-specific ways to multiple types of surfaces. The attachment is thought to be highly regulated, but the underlying sensory mechanism(s) are poorly understood. In the α-proteobacterium Caulobacter crescentus, the formation of adhesive organelles is 'hardwired' into the cell cycle regulatory circuitry. In this issue of Molecular Microbiology, Li et al. (2011) employed this model organism to examine the adhesion process and the transition from temporary to permanent attachment using total internal reflection fluorescence (TIRF) microscopy. Surprisingly, they observed that adhesin production was not only under developmental control, but was also stimulated by surface contact. Initial reversible contact of the pili with the surface was followed by flagellum rotation arrest and subsequent induction of the holdfast to allow irreversible surface adhesion. These findings demonstrate that Caulobacter produces its holdfast only at the appropriate time for surface attachment, preventing premature export of the adhesin, which could then be inactivated by 'curing' or be masked by occluding particles. Importantly, their results support the notion that the flagellum serves as a mechanosensor for adhesion.