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.     

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.     

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.     

Attachment of the adhesive holdfast organelle to the cellular stalk of Caulobacter crescentus

Caulobacters attach to surfaces in the environment via their holdfasts, attachment organelles located at the base of the flagellum in swarmer cells and later at the end of the cellular stalk in the stalked cells which develop from the swarmer cells. There seems to be little specificity with respect to the types of surfaces to which holdfasts adhere. A notable exception is that the holdfast of one cell does not adhere to the cell surface of another caulobacter, except by joining holdfasts, typically forming "rosettes" of stalked cells. Thus, the localized adhesion of the holdfasts to the cells is in some way a specialized attachment. We investigated this holdfast-cell attachment by developing an adhesion screening assay and analyzing several mutants of Caulobacter crescentus CB2A selected to be defective in adhesion. One class of mutants made a normal holdfast by all available criteria, yet the attachment to the cell was very weak, such that the holdfast was readily shed. Another class of mutants made no holdfast at all, but when mixed with a wild-type strain, a mutant of this class participated in rosette formation. The mutant could also attach to the discarded holdfast produced by a shedding mutant. In addition, when rosettes composed of holdfast-defective and wild-type cells were examined, an increase in the number of holdfast-defective cells was correlated with a decrease in the ability of the holdfast material at the center of the rosette to bind colloidal gold particles. Gold particles are one type of surface to which holdfasts adhere well, suggesting that the stalk end and the colloidal gold particles occupy the same sites on the holdfast substance. Taken together, the data support the interpretation that there is a specialized attachment site for the holdfast at the base of the flagellum which later becomes the end of the stalk, but not a specialized region of the holdfast for attachment to this site. Also, attachment to the cell is accomplished by bond formations that occur not only at the time of holdfast production. Thus, we propose that the attachment of the holdfast to the cell is a true adhesion process and that the stalk tip and base of the flagellum must have compositions distinctly different from that of the remainder of the caulobacter cell surface.     

ppGpp and polyphosphate modulate cell cycle progression in Caulobacter crescentus

Caulobacter crescentus differentiates from a motile, foraging swarmer cell into a sessile, replication-competent stalked cell during its cell cycle. This developmental transition is inhibited by nutrient deprivation to favor the motile swarmer state. We identify two cell cycle regulatory signals, ppGpp and polyphosphate (polyP), that inhibit the swarmer-to-stalked transition in both complex and glucose-exhausted media, thereby increasing the proportion of swarmer cells in mixed culture. Upon depletion of available carbon, swarmer cells lacking the ability to synthesize ppGpp or polyP improperly initiate chromosome replication, proteolyze the replication inhibitor CtrA, localize the cell fate determinant DivJ, and develop polar stalks. Furthermore, we show that swarmer cells produce more ppGpp than stalked cells upon starvation. These results provide evidence that ppGpp and polyP are cell-type-specific developmental regulators.     

Plasmid and chromosomal DNA replication and partitioning during the Caulobacter crescentus cell cycle

Cell division in Caulobacter crescentus yields a swarmer and a stalked cell. Only the stalked cell progeny is able to replicate its chromosome, and the swarmer cell progeny must differentiate into a stalked cell before it too can replicate its chromosome. In an effort to understand the mechanisms that limit chromosomal replication to the stalked cell, plasmid DNA synthesis was analyzed during the developmental cell cycle of C. crescentus, and the partitioning of both the plasmids and the chromosomes to the progeny cells was examined. Unlike the chromosome, plasmids from the incompatibility groups Q and P replicated in all C. crescentus cell types. However, all plasmids tested showed a ten- to 20-fold higher replication rate in the stalked cells than the swarmer cells. We observed that all plasmids replicated during the C. crescentus cell cycle with comparable kinetics of DNA synthesis, even though we tested plasmids that encode very different known (and putative) replication proteins. We determined the plasmid copy number in both progeny cell types, and determined that plasmids partitioned equally to the stalked and swarmer cells. We also reexamined chromosome partitioning in a recombination-deficient strain of C. crescentus, and confirmed an earlier report that chromosomes partition to the progeny stalked and swarmer cells in a random manner that does not discriminate between old and new DNA strands.     

SpoT regulates DnaA stability and initiation of DNA replication in carbon-starved Caulobacter crescentus

Cell cycle progression and polar differentiation are temporally coordinated in Caulobacter crescentus. This oligotrophic bacterium divides asymmetrically to produce a motile swarmer cell that represses DNA replication and a sessile stalked cell that replicates its DNA. The initiation of DNA replication coincides with the proteolysis of the CtrA replication inhibitor and the accumulation of DnaA, the replication initiator, upon differentiation of the swarmer cell into a stalked cell. We analyzed the adaptive response of C. crescentus swarmer cells to carbon starvation and found that there was a block in both the swarmer-to-stalked cell polar differentiation program and the initiation of DNA replication. SpoT is a bifunctional synthase/hydrolase that controls the steady-state level of the stress-signaling nucleotide (p)ppGpp, and carbon starvation caused a SpoT-dependent increase in (p)ppGpp concentration. Carbon starvation activates DnaA proteolysis (B. Gorbatyuk and G. T. Marczynski, Mol. Microbiol. 55:1233-1245, 2005). We observed that SpoT is required for this phenomenon in swarmer cells, and in the absence of SpoT, carbon-starved swarmer cells inappropriately initiated DNA replication. Since SpoT controls (p)ppGpp abundance, we propose that this nucleotide relays carbon starvation signals to the cellular factors responsible for activating DnaA proteolysis, thereby inhibiting the initiation of DNA replication. SpoT, however, was not required for the carbon starvation block of the swarmer-to-stalked cell polar differentiation program. Thus, swarmer cells utilize at least two independent signaling pathways to relay carbon starvation signals: a SpoT-dependent pathway mediating the inhibition of DNA replication initiation, and a SpoT-independent pathway(s) that blocks morphological differentiation.     

Proteolysis of the Caulobacter McpA chemoreceptor is cell cycle regulated by a ClpX-dependent pathway

Proteolysis is involved in cell differentiation and the progression through the cell cycle in Caulobacter crescentus. We have constitutively expressed the transmembrane chemoreceptor McpA from a multicopy plasmid to demonstrate that McpA degradation is modulated during the cell cycle. The level of McpA protein starts to decrease only when the swarmer cells differentiate into stalked cells. The reduction in McpA protein levels is maintained until the stalked cells develop into predivisional cells, at which point the level returns to that observed in swarmer cells. The cell-cycle-regulated degradation of McpA does not require the last 12 C-terminal amino acids, but it does require three amino acids (AAL) located 15 residues away from the C terminus. The ClpXP protease is essential in C. crescentus for viability, and thus, we tested McpA degradation in xylose conditional mutants. The effect on McpA degradation occurred within two generations from the start of ClpX depletion. The conditional mutants' growth rate was only slightly affected, suggesting that ClpX is directly involved in McpA proteolysis.     

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.     

Caulobacter crescentus pili: structure and stage-specific expression.

Pili are functionally expressed during the predivisional and swarmer stages of the Caulobacter crescentus differentiation cycle. They appear on the developing swarmer pole and at the same cellular location as flagella and the phiCbK receptor sites. Pili disappear when the swarmer cell differentiates into a stalked cell; this occurs with the loss of flagella and the disappearance of phage receptor sites. C. crescentus CB13B1a pili have been purified and characterized. Monomeric pilin is a protein with an apparent molecular weight of 8,500 that stains weakly with periodic acid-Schiff reagent. The amino acid composition of purified pilin reveals very low quantities of basic amino acids and a complete absence of methionine. Pilin is synthesized throughout the C. crescentus differentiation cycle. Neither free pili nor pilin monomers are detectable in the growth media, suggesting that loss of piliation in the swarmer- to stalked-cell transition occurs via pilus retraction.     

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.     

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.     

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.     

Differential membrane phospholipid synthesis during the cell cycle of Caulobacter crescentus.

The pattern of phospholipid synthesis during the cell cycle of Caulobacter crescentus has been determined. Although the phospholipid composition of swarmer and stalked cells was indistinguishable in continuously labeled cultures if the two cell types were pulse-labeled for a short time period, marked differences in the pattern of phospholipid synthesis were detected. Pulse-labeled swarmer cells exhibited a higher proportion of phosphatidic acid and a lower proportion of phosphatidylglycerol. In addition, minor phospholipids were detected in the swarmer cells that were not detected in stalked cells. Stalked cells that developed directly from swarmer cells showed that same phospholipid profile as the swarmer cells. The switch to the second phospholipid profile was observed to occur at the predivisional cell stage. Because cell division then yielded a swarmer cell with a different phospholipid profile than its sibling stalked cell, the cell division process may trigger a mechanism which alters the pattern of phospholipid synthesis.     

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.