Journal of Bacteriology, March 2004, p . 1438-1447, Vol . 186, No . 5

Development of Surface Adhesion in Caulobacter crescentus

Diane Bodenmiller, Evelyn Toh, and Yves V . Brun^*

Department of Biology, Indiana University, Bloomington, Indiana 47405

Received 5 June 2003/ Accepted 12 November 2003

 
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 contributionof growth and external structures to the attachment of C . crescentus to abiotic surfaces . We show that the holdfast is essentialbut not sufficient for optimal attachment . Rather, adhesionin C . crescentus is a complex developmental process . We foundthat the attachment of C . crescentus to surfaces is cell cycle regulated and that growth or energy or both are essential forthis process . The initial stage of attachment occurs in swarmercells and is facilitated by flagellar motility and pili . Ourresults suggest that strong attachment is mediated by the synthesisof a holdfast as the swarmer cell differentiates into a stalkedcell.

 
Aquatic bacteria can live in both planktonic and sessile states.In the planktonic state, a bacterium is free to move throughoutthe environment to find nutrients . However, in nature, bacteriaare predominantly found in the sessile state attached to surfaceswhere they form communities called biofilms [14, 25] . Biofilmsare defined as matrix-enclosed bacterial populations adherentto each other and/or to surfaces or interfaces [7] . Bacteriain a biofilm are more resistant to antibiotics and are able to form symbiotic relationships with other members of the biofilm community [4, 6, 22, 46].

The transition from a planktonic state to a sessile state isa developmental process involving different environmental cuesand the coordination of various molecular pathways and extra-cellular structures [9, 25, 33] . There are three stages of biofilm formation,early attachment, maturation, and detachment . Initially, bacteriaare found swimming close to a surface until they are able toovercome surface tension and bind, forming a monolayer biofilm.This monolayer biofilm eventually becomes tightly packed withadditional cells, and microcolonies begin to form . During thematuration stage, a three-dimensional structure emerges thatis made up of a matrix of exopolysaccharide and cells . The laststage is that of detachment, where planktonic cells are releasedfrom the biofilm [25, 43].

Limited studies on a small number of -proteobacteria have beenconducted to determine the genetic pathways and structures thatplay a role in biofilm development . Three structures show interspeciesimportance: pili, flagella, and exopolysaccharides . Flagellaand type IV pili facilitate early attachment events in Pseudomonasaeruginosa [26] and Vibrio cholerae El Tor [44] . Exopolysaccharidesplay a role in the initial stages of biofilm development inV . cholerae El Tor [44] as well as V . cholerae O139 [45], andcontribute to biofilm maturation in Escherichia coli [8] . Althoughthere are overlaps in the requirements for adhesion to surfaces,some requirements are not common to all bacteria . For example,unlike the bacteria discussed above, V . cholerae O139 does notrequire type IV pili for initial attachment [45].

Caulobacter crescentus is a gram-negative -purple bacteriumcommonly found in aquatic environments; it participates in formingbiofilms that have a biofouling effect on a variety of surfaces[47] . C . crescentus has a dimorphic life cycle, spending partof its life as a nonreplicating motile swarmer cell and partas a replicating sessile stalked cell [2] . Each cell type hasspecific polar structures that predispose the cell to its distinctlifestyle . The swarmer cell is characterized by the presenceof a flagellum and multiple pili at the swarmer pole . Eventually,the swarmer cell differentiates into a stalked cell, and thepili and flagellum are replaced with a stalk that is tipped by an adhesive organelle called a holdfast.

Little is known about the physical mechanism that results in stable attachment of C . crescentus to surfaces . The holdfast, composed in part of a polysaccharide containing N-acetylglucosamine, is essential for adhesion [20] . All of the mutants identifiedso far that are completely deficient in surface adhesion lackdetectable N-acetylglucosamine at the tip of the stalk [5, 21,40, 41]; this indicates that the holdfast N-acetylglucosamineplays a critical role in adhesion . Even though the holdfastN-acetylglucosamine cannot be detected in swarmer cells [15],swarmer cells can attach to surfaces [15, 23, 30] . Therefore,other structures are likely to facilitate the attachment ofswarmer cells . It has been suggested that the pilus may be onesuch structure [15].

In order to determine whether other polar structures besidesthe holdfast are necessary for attachment, we examined the attachmentof mutant strains lacking pili or flagella . In this paper, weshow that the presence of a holdfast is essential but not sufficientfor the attachment of C . crescentus cells to surfaces . Flagellaand pili facilitate attachment of cells to surfaces, attachmentis cell cycle regulated, and it requires growth or energy orboth . These results suggest that optimal attachment to surfacesinvolves an ordered series of developmental events during theC . crescentus cell cycle.

 
Medium and strains. Strains of C . crescentus were grown in peptone yeast extract[PYE] or 0.2 mM phosphate Hutner imidazole glucose glutamatemedium [HIGG] at 30°C, unless otherwise specified [31]. When required, antibiotics were used at the following concentrations: kanamycin, 5 or 20 µg/ml; and nalidixic acid, 20 µg/ml.E . coli was grown in Luria-Bertani [LB] medium at 37°C and supplemented with kanamycin [50 µg/ml] when necessary.

The mutant YB3756 [motB] was generated by amplifying a region of motB with the primers MotBXbaI and MotBPstI [oligonucleotide sequences are available from the authors upon request], and ligating the product into the nonreplicating plasmid pBGST18[M . R . K . Alley, unpublished] at the XbaI and PstI sites . The plasmid was transformed into E . coli S17-1 [38] and introducedinto the C . crescentus strain CB15 by conjugation . Homologousrecombination occurred between the plasmid and genomic DNA,generating an insertional mutation in motB . The phenotype ofthe mutation was confirmed by flagellum staining and a swarm assay . motB does not appear to be part of an operon, and therefore the motB mutation should not be polar.

The mutant YB3373 [cpaA] was identified among a collection of CBK-resistant [pilus minus] Mariner [37] transposon mutants[D . Klein and Y . V . Brun, unpublished] . Mutations in cpaA maybe polar on the cpaBCDE genes also involved in pilus synthesis[39] . Transposon locations in the cpa region were shown by Southern hybridization and sequencing to be in the cpa region . Genomic DNA from the mutants was digested with BamHI, releasing a 5.5-kb fragment in the pilA-cpa region . The probe was a 614-bp fragmentincluding pilA and was generated with the primers pilAup andpilAdn . The probe was labeled with digoxigenin according tothe DIG HIGH prime kit [Boehringer Mannheim, Indianapolis, Ind.]. Hybridization was done with QuikHyb [Stratagene, La Jolla, Calif.]. An antidigoxigen-alkaline phosphatase conjugate [Boehringer Mannheim] diluted 1:5,000, along with a solution of nitroblue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate [NBT/BCIP],was used to develop the blot.

PCR products of the regions adjacent to the estimated transposon locations were generated for sequencing purposes with the following primers: pilAHindIII and MarRseq, pilAXbaone and MarRseq, or pilAXbaone and MarLseq . PCR products were gel purified withthe Qiagen kit QIAquick . Sequencing reactions of the PCR productswere done in an MJ Research PTC-150 minicycler with MarRseqor MarLseq and the ABI Big Dye kit version 2 . Sequencing wasperformed at the Institute for Molecular and Cellular Biologylocated at Indiana University in Bloomington, Ind., with theABI 3700 capillary DNA sequencer . Sequence analysis was donewith the NCBI Blast program [http://www.ncbi.nlm.nih.gov/]. A transducing phage lysate was made of the cpaA mutant and was used to transduce the cpaA mutation into CB15 and SC305 [flaR] [11, 16].

Coverslip assay. A coverslip cell binding assay was adapted from reference 24.Overnight cultures were diluted with fresh PYE to an opticaldensity at 600 nm [OD₆₀₀] of 0.15, and 1.5 ml was added to eachwell of a 6-well polystyrene cell culture dish [Corning Costar3506] containing ethanol-flamed glass coverslips or ethanol-washedplastic coverslips [Corning Incorporated, catalog no . 3512].Samples were placed on an orbiter rotating between 90 and 100rpm and incubated at 30°C for 1.5 to 4.5 h or overnight.After incubation, coverslips were removed from the wells andrinsed with a steady stream of deionized distilled water for10 to 20 s . Coverslips were then placed on a slide containing 2 µl of SlowFade antifade reagent in 1x phosphate-bufferedsaline and glycerol [catalog S-2828, Molecular Probes, Eugene,Oreg.] . At this time, the culture density of each sample wasmeasured at 600 nm.

If the experiment required holdfast labeling, instead of being placed on a slide, coverslips were put in a darkened humid chamber cell side up and incubated with 50 µl of fluorescein isothiocyanate-labeled wheat germ agglutinin [FITC-wheat germ agglutinin] [Molecular Probes, Eugene, Oreg.] at a concentration of 0.05 µg/µlfor 30 min . Coverslips were again rinsed with a steady streamof distilled H₂O, and placed onto a slide.

Growth requirements for surface adhesion. Overnight cell cultures were diluted to an OD₆₀₀ of 0.40 inPYE with a final concentration of 2 µg of chloramphenicol/ml,0.05% [wt/vol] sodium azide, 1.7% [wt/vol] formaldehyde, orno additives . The diluted cultures were incubated for 30 minat 30°C or placed in an ice slurry . The tissue culture dishwas chilled at 4°C for 30 min for the change in temperatureexperiment . Coverslips and cell culture were added to each welland incubated for 4 h as described above.

To determine the effect of chemicals on cells already attachedto glass coverslips, cells were grown in PYE with a coverslipfor 5.5 h with a starting OD₆₀₀ of 0.15 . Chloramphenicol, sodium azide, or formaldehyde was added at the same concentrationsas above and incubation at 30°C was continued overnight.

Microscopy. Slides were examined with a Nikon Eclipse E800 microscope witha fluorescein isothiocyanate-HYQ filter [Ex 480/40 DM 505 BA535/50] with a 40x objective or a 100x oil immersion objective. Images were captured with a Princeton Instruments cooled charge-coupled device camera and the software Metamorph Imaging versions 4.0 and 4.5.

Plastic binding assay. Plastic binding assays were done as described [28] with modifications[C . Fuqua, personal communication] . Briefly, overnight cultureswere diluted with fresh PYE to an OD₆₀₀ of 0.15 and incubatedat 30°C until cultures reached an OD₆₀₀ between 0.35 and0.60 . Cultures were diluted to an OD₆₀₀ of 0.30 and added tothe wells of a 12-well tissue culture dish [750 µl perwell, four wells per strain] . Dishes were incubated with shakingat room temperature for 15 min . The cell culture was removed,and the wells were washed twice with fresh PYE to remove anyunattached cells . Each well was stained with 400 µl of1% [wt/vol] crystal violet in H₂O for 15 min . Wells were washedfour times with H₂O to remove excess crystal violet . After washing,the crystal violet was sequentially eluted with 200 µland 400 µl of methanol . The samples were diluted withthe addition of 400 µl of H₂O . Color intensity was measuredat 589 nm.

Synchrony. Cells were synchronized by taking advantage of the fact thatswarmer cells pellet more tightly than stalked cells, so thatrelatively pure cultures of swarmer cells can be obtained by centrifugation followed by the removal of loosely pelleted cells. A culture of C . crescentus ATCC 19089 [American Type Culture Collection, Manassas, Va.] [lab strain YB1360] was grown in HIGG at 30°C . The saturated culture was aliquoted into Corextubes [20-ml volume] and centrifuged in a Beckman JA-20 rotorfor 10 min at 9,000 rpm . The supernatant of each tube was thenpoured off until a 5- to 10-ml volume remained . The tubes wereswirled gently by hand to resuspend the loose stalk pellet butnot the tight swarmer pellet . The supernatant was poured off,and 5 ml of 1x M2 salts [29] was added to each tube . Any remaining stalk pellet was removed by gentle swirling.

The supernatant was poured off, and the swarmer cell pelletfrom each tube was resuspended in 1 ml of 1x M2 salts . The swarmercells from five tubes were combined into one tube and centrifugedas described above . The loose stalk pellet was removed as describedabove . Resuspended swarmer cells were then examined for thepresence of stalked cells . If the population of stalked cellsexceeded 5%, the washing and centrifugation steps were repeateduntil the population of stalked cells diminished to below 5%.Once the population of swarmer cells was 95% pure, swarmer cells were resuspended in fresh medium to an OD₆₀₀ of 0.30 . Swarmer cells were allowed to recover at 30°C for 1 min with shaking and samples were taken for the assays described below.

The plastic binding assay was carried out as described abovewith two modifications . Cells were incubated for 20 min in a12-well tissue culture dish instead of 15 min . Following thewashes with fresh medium, cold 1x M2 salts were added to eachwell, and the tissue culture dish was stored at 4°C until the synchrony was completed.

Samples were taken at 0, 34, 60, 146, 180, 210, and 240 minfor phage absorption assays, which were carried out as described[13] . For immunoblots, 1-ml samples were taken at each timepoint, and centrifuged at 6,000 rpm in a microcentrifuge for5 min . Cell pellets were then resuspended in 100 µl ofsodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE]loading dye, boiled for 5 min, and stored at -20°C . An equivalentvolume of cell lysate from each synchrony time point was loadedonto a 10% acrylamide gel and transferred onto nitrocellulose.The nitrocellulose was incubated for 2 h with an antiflagellinantibody at 1:40,000 or overnight with an anti-FtsZ antibodyat 1:20,000 . Blots were incubated for 1 h with the horseradishperoxidase-labeled secondary antibody, goat anti-rabbit immunoglobulin[InVitrogen Life Technologies, Carlsbad, Calif.] diluted 1:20,000and preabsorbed with CB15 acetone powder [18] . Blots were developed with the chemiluminescent substrate SuperSignal West Pico [Pierce Biotechnology, Inc., Rockford, Ill.] for 5 min.

Swarm and lectin binding assays. We stabbed 0.5 µl of log-phase cells into 0.3% semisolidPYE medium and grew them at room temperature in a humid chamberfor 3 to 5 days . Fluorescent lectin binding assays were performedas described [15] with fluorescein isothiocyanate-wheat germagglutinin, except that cells were washed twice.

 
Presence of a holdfast is not sufficient for efficient attachment of C . crescentus to surfaces. Although it has been shown that the holdfast is essential forattachment of C . crescentus to surfaces [21], it may not bethe only requirement . In other bacteria, attachment has beenshown to be a multistep process, requiring multiple structuressuch as pili, flagella, and exopolysaccharides [25] . To determineif the presence of a holdfast is sufficient for C . crescentus attachment, we tested the ability of nongrowing C . crescentus cells to attach to glass . Cells were incubated at 30°C inPYE with chloramphenicol, sodium azide, or formaldehyde, orin PYE at 4°C to inhibit growth or to kill cells . We comparedthe ability of treated cells to attach to glass coverslips tothat of untreated cells.

As shown in Fig . 1A, attachment to glass was severely affectedby all four conditions . Under these conditions, approximately75% of cells are stalked cells [42] and have a holdfast [15].These holdfast-bearing cells would be expected to randomly comeinto contact with the glass during the 5 h of incubation andshould bind to glass if the holdfast is sufficient for attachment.In order to ensure that the lack of attachment was not due toa treatment-induced detachment of cells, cells already attachedto glass were incubated with formaldehyde and sodium azide.The results in Fig . 1B show that the majority of cells remainedbound after incubation with formaldehyde and sodium azide, suggestingthat growth inhibition or cell death does not cause cells todetach . These results indicate that the presence of a holdfastis not sufficient for the efficient attachment of C . crescentusto a surface . One possibility to explain these results is thatenergy and/or growth is required for attachment.

 
Pili and flagellar motility are required for optimal attachment. Since the holdfast is necessary but not sufficient for bindingto a surface, it is likely that events preceding holdfast synthesisare required for the primary binding event . The fact that swarmercells can bind to glass [23, 30] suggested the possibility thatthe flagellum and/or the pili could contribute to the primarybinding stages . To analyze the contribution of various structuresto attachment, we used a plastic binding assay [28] as a semiquantitativemeasure of the attachment of various mutants.

To validate this assay, we compared mutants whose relative binding efficiencies had already been evaluated with cellulose acetateand glass binding assays [5] . We used six holdfast mutants with different degrees of attachment defects . NA1000 [12] and theYB3372 holdfast synthesis mutant [hfsA] [41] cannot synthesizea holdfast and are incapable of attachment according to celluloseacetate and glass binding assays [41] . The results of the plasticbinding assay confirmed that both strains were essentially unableto attach to surfaces; binding efficiencies were 4% for YB3372[hfsA] and 5% for NA1000 compared to wild-type strain CB15 [100%][Fig . 2].

 
Four of the mutants have insertional mutations located in the holdfast attachment hfa operon, YB2533 [hfaA], YB2536 [hfaB], YB2539 [hfaC], and YB2542 [hfaD] [5] . The hfa genes play a rolein securing the holdfast to the tip of the stalk [5, 17], andwith the exception of YB2539 [hfaC], these mutants display aholdfast shedding phenotype [5] . Of the three mutants, YB2536 [hfaB] has the most severe attachment defect, showing negligible binding to glass and cellulose acetate [5] . The plastic bindingassay indicated that YB2536 [hfaB] had a 5% binding efficiencycompared to CB15, which is similar to YB3372 [hfsA] and NA1000[Fig . 2] . The mutant YB2533 [hfaA] bound more efficiently thanYB2536 [hfaB] to glass and cellulose acetate [5] . The plasticbinding assay indicated that YB2533 [hfaA] had a 16% bindingefficiency compared to wild-type CB15 [Fig . 2] . Both the cellulose acetate and glass binding assays showed that although YB2542[hfaD] was not capable of binding as well as CB15, it clearlybound more efficiently than YB2533 [hfaA] and YB2536 [hfaB][5] . Cultures of the hfaD mutant have the highest percentageof cells with holdfasts of the hfa mutants [5] . The plasticbinding assay showed that the YB2542 [hfaD] mutant had a bindingefficiency of 47% compared to CB15 [Fig . 2] . The mutant YB2539[hfaC] consistently bound as well as CB15 in all three assays[5] . Thus, the binding patterns of the holdfast mutants determinedby the plastic binding assay mirror the results of the glassand cellulose acetate binding assays [5] . This indicates thatthe plastic binding assay is at least an equivalent means ofmeasuring attachment in C . crescentus compared to binding toglass or cellulose acetate.

As previously indicated, pili may play a role in attachmentof C . crescentus to surfaces [15, 23, 30] . We obtained nonpiliatedmutants by selecting transposon mutants for resistance to thephage CbK, which requires pili for infection, and used Southernhybridization to identify mutations in the pil gene cluster.We examined the binding efficiency of a mutant with a transposoninsertion in the cpaA gene . CpaA is thought to be a prepilinpeptidase [39] . The plastic binding assay showed that YB3373[cpaA] has a binding efficiency of 54% compared to the wildtype [Fig. 2], indicating that the presence of pili contributes to the ability of C . crescentus to attach to surfaces but that the pili are not crucial binding elements.

Since the pilus mutant could still attach substantially to the surface in the plastic binding assay, it seemed likely thatflagella might contribute to attachment . Flagella are key componentsin the initial stage of biofilm development in E . coli [32] and Pseudomonas aeruginosa [26] and facilitate attachment inV . cholerae El Tor [44] . We used SC305, which has a point mutationlocated in the flaR gene required for flagellar filament assembly.According to the plastic binding assay, SC305 [flaR] had a bindingefficiency of 20% compared to wild-type CB15 [Fig . 2] . This indicates that the flagellum, while not as important as theholdfast for binding to surfaces, is more important than pili.

The flagellum could be playing a role through direct adherenceto surfaces by acting as an adhesin, by breaking the electrostatic repulsion barrier at the surface, or perhaps by positioningthe cell so that it is in the correct orientation for binding.Another possibility to explain the strong binding defect ofthe mutant SC305 [flaR] is that the flagellar mutation couldreduce the efficiency of pilus biosynthesis . Indeed, flagellarmutants have a decreased rate of CbK absorption, suggestingthat normal flagellum synthesis is required for optimal pilussynthesis or function [1] . SC305 [flaR] appears to have fewerpili than wild-type cells because the plaque-forming efficiencyof CbK was 2.3-fold less in SC305 than in wild-type CB15 . Therefore,we examined the attachment ability of a double mutant, YB3757[flaR cpaA] . This double mutant had a binding efficiency of28%, which is comparable to the binding efficiency of the SC305[flaR] mutant [Fig . 2] . The lack of an additive effect between the flagellar and pilus mutations suggests that flagella and pili act in the same attachment pathway and that part of the attachment defect of SC305 [flaR] is due to a deficiency in pilus synthesis.

The effect of a flagellar mutation on pilus synthesis is not sufficient to explain the strong adhesion defect of a flagellar mutant . The motility imparted by the flagellum may also be important for attachment . In order to test this possibility, a motB mutant, YB3756, was generated by insertional inactivation . As detected by flagellar staining and a swarm assay, YB3756 [motB] has a flagellum but lacks the ability to swim [data not shown] . Theresults of the plastic binding assay showed that YB3756 [motB]has a binding efficiency of 32% compared to wild-type CB15 [Fig. 2] . This result is comparable to the flaR mutant SC305, whichsuggests that it is not the presence of a flagellum per se that is necessary for attachment but rather it is the motility imparted by the flagellum . It is also possible that attachment relieson chemotactic ability.

A fluorescent lectin binding assay was done to ensure that the decrease in surface attachment observed in the flagellar andpilus mutants was not due to the absence of a holdfast . Fluorescentlectin binding observed for the mutant was equivalent to thatseen in wild-type cells; this eliminates the possibility thatany of these mutations have an indirect effect on holdfast synthesis[data not shown].

Time course attachment assays. The plastic binding assay involves an attachment period of 15min and thus provides a measure of primary adhesion . To observethe effect of mutations on the later stages of biofilm formation,we analyzed the attachment of cells to plastic coverslips duringextended incubations . Coverslips were removed at each time pointand washed extensively with water to remove loosely bound cells.Assays were repeated three times, and the different strainswere consistently found to bind as described below.

Figure 3 shows the binding patterns observed during 4.5 h ofincubation . Wild-type cells initially bound to the surface as a dispersed monolayer of cells, as seen at 1.5-h . A few small aggregates of cells were also seen at that time . These small aggregates are probably not due to rosettes [groups of cellsattached to each other holdfast-to-holdfast that form in liquidculture] since flagellar mutants, which make rosettes in liquidculture, do not form these aggregates [see below] . The aggregatesare most likely due to the binding of new cells from the culture,since the growth rate of attached cells [greater than or equalto 1.5-h doubling time] is not sufficient to explain their formation.By 2.5 to 3.5 -h, wild-type cells began to form larger aggregates,which continued to increase in size until the coverslip wascovered by a high density of cells, as previously described[5, 40, 41].

 
Although we established that flagellar motility contributes significantly to surface attachment in C . crescentus, the temporal contribution of flagella remained unclear . Microscopic analysis of SC305 [flaR], YB3757 [flaR cpaA], and YB3756 [motB] throughoutthe attachment time course showed that very few cells were ableto bind compared to wild-type CB15 and that they never formed aggregates . YB3756 [motB] and SC305 [flaR] had very similar binding patterns and after 4.5 h exhibited less binding than wild-type CB15 after 1.5 h . YB3757 [flaR cpaA] exhibited the lowest binding of all the mutants tested . The pilus mutant YB3373[cpaA] had a binding pattern similar to wild-type cells, startingwith a dispersed monolayer of cells followed by some aggregation,but lagged slightly behind for the first 4.5 h . Note that thesudden increase in attached cells between 2.5 and 3.5 h forthe cpaA mutant was not reproducibly observed in other experiments.When cells were allowed to bind to coverslips overnight, thecpaA mutant was unable to form the dense monolayer biofilmsformed by wild-type cells [data not shown] . These results suggestthat flagellar motility and pili play a role in the primaryattachment of C . crescentus to surfaces and that motility isimportant for the formation of aggregates in the subsequentstage of adhesion . In addition, pili seem to contribute to thebinding process at a late stage.

Attachment occurs at a specific stage of the cell cycle. The developmental cycle of C . crescentus involves the ordered appearance and disappearance of various polar structures . The flagellum is synthesized in predivisional cells, and this isfollowed by the production of pili at the same pole in swarmercells . The differentiation of a swarmer cell into a stalkedcell is marked by the ejection of the flagellum and the retractionof the pili; this is coordinated with the synthesis of a stalkand holdfast [2] . Since primary adhesion is facilitated by theflagellum and pili and stable attachment is cemented by theholdfast, perhaps optimal attachment is dependent upon the completionof multiple ordered steps.

To determine the ability of various cell types to attach to surfaces, attachment was measured throughout the cell cycleof a synchronized population of C . crescentus . At each timepoint indicated, cells were allowed to bind to a cell culturedish for 20 min and attachment was measured with the plasticbinding assay . Results are shown as a percentage of the maximumattachment and represent the average of two experiments [Fig.4A] . Attachment was relatively constant during the first 80min of the cell cycle, with approximately 30% of the maximalattachment . This time period included the initial swarmer phaseand the differentiation of swarmer cells into stalked cells.The time period between 80 and 140 min showed a marked decreasein attachment, with the lowest relative attachment of approximately10% at 140 min . During this portion of the cell cycle, cellselongated and began to divide . The lowest point of attachmentat 140 min correlated with the presence of late predivisionalcells . Swarmer cells began to reappear at 160 min and steadilyincreased in number between 180 and 200 min . The reappearanceof swarmer cells paralleled an increase in attachment, withthe highest attachment occurring at 200 min.

 
An FtsZ immunoblot was used to establish the time of swarmerto stalked cell differentiation and of cell division [data notshown] . FtsZ controls the initiation of cell division, is absentfrom swarmer cells, appears during swarmer cell differentiation,is expressed at its highest level as cell division begins, andis degraded when cells are at the end of the division process[34, 35] . FtsZ appeared at 60 min, just prior to the decrease in the surface binding of cells, confirming that surface binding decreased after swarmer cell differentiation . The FtsZ level was highest between 140 and 160 min, which corresponded to the appearance of deeply constricted predivisional cells . An obvious decrease in the FtsZ level occurred at 180 min, which was marked microscopically by the appearance of numerous swarmer cells[data not shown] . These results confirm that cell division tookplace between 160 and 180 min, coincident with the increasein surface adhesion.

Since the number of swarmer cells at time zero should be equivalent to the number of swarmer cells after cell division, we expected to see equivalent attachment at both stages . However, swarmercells at the beginning of the cell cycle exhibited only one-thirdof the attachment observed after cell division . A possible explanationfor this discrepancy is that the process of synchronizationmay have caused damage to flagella and/or pili, both of whichplay a role in attachment . In order to determine if the synchronizationprocess affected the presence of pili, we performed a phagebinding assay . The amount of CbK absorbed by cells at differentstages of the cell cycle is indicative of the level of piliation.According to the phage assay, pili were present in large amountsduring the initial swarmer phase and during the reappearanceof swarmer cells and the increase in surface adhesion between160 and 200 min [Fig . 4A] . Therefore, it appears that the lackof attachment seen at the beginning of the cell cycle is notdue to a lack of pili.

Another possible explanation for the lack of binding by synchronized swarmer cells is that flagella might have been damaged and motility reduced during the synchrony process . Indeed, it is common to observe a loss of motility after the synchronization process.An antiflagellin antibody was used to assay for the presenceof the flagellum throughout the cell cycle [Fig . 4B] . The immunoblot revealed that the level of flagellin was lower at time zero compared to the end of the cell cycle . This correlated with microscopic observations that indicated that the cells wereless motile at 0 to 40 min compared to 160 to 200 min [datanot shown] . This decrease in flagellin level and motility isprobably due to a side effect of the synchronization process,which may cause flagella to shear off of the swarmer cells.We hypothesize that the reduced ability of swarmer cells tobind to surfaces at the beginning of the synchrony comparedto after cell division is due to flagellar defects . Therefore,it appears that attachment is cell cycle regulated and is coordinatedwith appearance of swarmer cells and the differentiation ofswarmer cells into stalked cells.

The possibility remained that cells attached at the predivisional stage . In order to test this possibility, exponentially growingcells from a mixed culture were allowed to attach to a coverslipfor 10 min, and the attached cell types were quantitated . Swarmercells accounted for 77% of the attached cells, 20% were stalkedcells, and only 3% were predivisional cells [Table 1] . The attached stalked cells had short stalks, suggesting that they were newborn stalked cells [data not shown] . Attached cells were also quantitated after 4 h of attachment . In this case, predivisional cells predominated with 36%, and the percentage of swarmer cells dropped to 34% [Table 1] . Furthermore, the stalks of attached cells were slightlylonger than after 10 min [data not shown] . This is expected because swarmer cells that attached early in the experiment would have differentiated into stalked and predivisional cells.These results clearly indicate that the strong increase in attachment around the time of cell division in the synchrony experimentwas not due to the binding of predivisional cells or stalkedcells and thus support the model that attachment is initiatedby swarmer cells.

 
The life cycle of C . crescentus involves the ordered synthesis of various polar structures, including the flagellum, the pili,and the holdfast . The holdfast is critical for attachment ofC . crescentus cells to surfaces [3] . Pili and flagella have been shown to be involved in the attachment of other types of bacteria [26, 27, 32, 44, 45] . In this paper, we determined the relative contribution of these polar structures to surface adhesion in C . crescentus . We show that the presence of a holdfast is essential but not sufficient for attachment of C . crescentus to surfaces; optimal attachment also requires both pili anda motile flagellum . We show that growth and/or energy is essentialfor attachment and that attachment is cell cycle regulated.Specifically, we show that stalked cells are unable to attachefficiently and that attachment is highest following the productionof swarmer cells . These results indicate that adhesion in C.crescentus is a developmental process whose initial stage occursin swarmer cells, where it is mediated by motility, pili, andperhaps other properties, followed by cementing of the attachmentby the holdfast during swarmer to stalked cell differentiation.

Attachment of C . crescentus to surfaces was severely affected by sodium azide, formaldehyde, chloramphenicol, and growth at 4°C, conditions that hinder the cell's ability to produceand use energy and to grow . Cells that were treated after theirattachment to a surface still remained bound, indicating thatthe holdfast was able to maintain its adhesive properties . Sinceholdfast-bearing cells were still present during the variousgrowth-inhibiting treatments and should undergo random collisionswith surfaces, these results suggest that random collisionsof holdfast-bearing cells with surfaces are not sufficient forattachment . These results correlate with the finding that showedthat the initial cell attachment of Pseudomonas fluorescenswas decreased by the inhibition of protein synthesis [27].

Since the presence of a holdfast is not sufficient for the efficient attachment of C . crescentus to surfaces, other structures may facilitate the initial binding events that lead to stable holdfast-to-surfaceinteractions . The observation that swarmer cells, which lacka holdfast [15], are able to attach to glass [23, 30] suggestedthat flagella and pili may play a role in primary binding events.Plastic binding assays showed that a mutant lacking pili hada 54% binding efficiency compared to wild-type cells when exposedto a surface for 20 min, indicating that pili are contributingto the primary attachment process but are not essential . Microscopicexamination over the course of hours revealed that the pilusmutant was deficient in early and late stages of attachment.Both the pilus mutant and wild-type cells first attached asindividual cells seemingly randomly, but the accumulation ofbound cells was slower in the pilus mutant . This was followedby the formation of tightly packed aggregates of cells.

The pilus mutant deviated from wild-type cells late in the attachment process; it appeared to lack the ability to go beyond the cell aggregation stage and to form the dense monolayers formed by wild-type cells . This attachment defect is similar to the typeIV pilus mutants of P . aeruginosa [26] . A time course attachmentassay revealed that a mutant of P . aeruginosa defective in typeIV pilus biosynthesis was able to form an initial monolayersimilar to wild-type cells but was unable to form microcoloniesor the dense cell populations seen with wild-type cells . Thetype IV pilus of P . aeruginosa may play a role in stabilizinginitial interactions with a surface or between cells . The twitchingmotility imparted by type IV pili may be necessary for cellsto migrate along the surface to form the microcolony cell aggregates[26] . To our knowledge, twitching motility has not been observedin C . crescentus.

Bacteria usually have to overcome electrostatic repulsion witha surface in order to attach, since most bacteria and inertsurfaces are negatively charged [10] . During the initial stages of attachment, the C . crescentus pili may help attachment by reducing the radius of interaction between the cell and the surface . As the swarmer cell differentiates into a stalked cell,the pili retract, and a stalk and holdfast are synthesized atthe pole that bore the pili [2] . An initial attachment via pili may help position the cell so that a newly synthesized holdfast would be in close enough proximity to contact the surface, establishing a stronger, more permanent attachment than the one mediatedby the pili [15] . Pili have also been implicated in promoting the primary adhesion event in Hyphomonas spp., another prosthecate bacterium [36] . Therefore, in cells lacking pili, holdfast contactwith a surface would still be possible, but its efficiency wouldbe reduced . In addition, it is possible that the pili of C.crescentus are capable of mediating twitching motility and actlike the pili of P . aeruginosa to establish cell-to-cell interactions.

We found that the flagellum makes a substantial contributionto attachment, since a flagellar mutant had a binding efficiencyof 20% compared to wild-type cells . This was four times higherthan a holdfast mutant and 2.5 times less than a pilus mutant.While the flagellum is similar to a pilus in that it is a thinstructure that can help break the electrostatic repulsion ata surface, it clearly has additional roles . Indeed, a motB mutant,which synthesizes a paralyzed flagellum, had a 32% binding efficiencycompared to wild-type cells, slightly more than the 20% of theflagellar mutant . Therefore, we conclude that the force of motility,the ability to chemotax, or both is more important for efficientattachment than the role of the flagellum in breaking the electrostaticrepulsion with the surface.

Motility plays an important role in binding of V . cholerae O139 [45], E . coli [32], and P . aeruginosa [26] . Motility may alsoplay a role in biofilm expansion [32] . The direction of flagellumrotation can affect attachment and detachment of E . coli cells[19] . It has been hypothesized that counterclockwise rotationmay play an important role in cell detachment and that a clockwiserotation may play a role in anchoring cells to a surface . Sinceflagellar rotation is required for efficient binding of C . crescentusto surfaces and the proton motive force is needed for flagellarrotation, the lack of binding that we observed with nongrowingcells may in part be due to the lack of flagellar rotation.

We hypothesize that the lack of adhesion in nongrowing cells reflects the requirement for an ordered succession of structuresat the adhesive pole of the cell . The lack of an additive effectbetween flagellum and pilus mutations suggests that flagellaand pili are acting in the same attachment pathway . Furthermore,analysis of a synchronized population indicated that attachmentcorrelated with the production of pilus-bearing swarmer cells.The elongating stalked cell and predivisional cell phases ofthe cell cycle exhibited the lowest attachment, confirming thatholdfasts are not sufficient for attachment to a surface . Theimportance of flagellar motility for the attachment of swarmercells was also indicated by the results of the cell synchronyexperiment . Immunoblot analysis indicated that the synchronizationprocedure resulted in a reduction of flagellins, suggestingthat flagella were broken off . When cells were examined by microscopy,they appeared to be less motile at the beginning of the synchronythan after cell division, correlating with the respective levelsof adhesion at each step [data not shown].

Our results suggest a model for the succession of events that allow C . crescentus cells to bind efficiently to surfaces . Since binding correlates with the appearance of swarmer cells and since the pili and flagellar motility are required for efficient attachment, we hypothesize that the primary binding event occursin swarmer cells . Indeed, when cells from a mixed culture wereexposed to a surface for only 10 min, the vast majority of attachedcells [77%] were swarmer cells . Motility is likely to increasethe occurrence of contacts between the cell and a surface . Inaddition, motility probably provides the force that is necessaryto overcome the repulsive barrier between the negatively chargedsurface and the negatively charged cell surface . The presenceof thin polar structures such as the pili and flagellum mayalso help break this repulsive barrier.

After the initial association with the surface, the retractionof bound pili during swarmer to stalked cell differentiationwould bring the pole at which the holdfast is about to be synthesizedin close proximity to the surface . This would help ensure thatcell orientation is optimal for the subsequent cementing ofthe adhesion by the holdfast . Export of the holdfast directlyfollowing flagellum ejection and pili retraction would thencement the permanent attachment to the surface . This successionof events explains why adhesion depends on growth; growth isrequired for the transition from the primary adhesion swarmerstage to the permanent adhesion stalked stage . Dissection ofthe mechanisms that coordinate motility, pilus retraction, flagellumejection, and holdfast synthesis should provide major insightinto the developmental process of biofilm formation.

 
We thank members of our laboratory and Clay Fuqua for critical reading of the manuscript and for helpful discussions.

This work was supported by National Institutes of Health grant GM51986 to Y.V.B.

 
* Corresponding author . Mailing address: Indiana University, Department of Biology, Jordan Hall 142, 1001 E . 3rd St., Bloomington IN 47405-3700 . Phone: [812] 855-8860 . Fax: [812] 855-6705 . E-mail: ybrun@bio.indiana.edu.

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