Oriented Adhesion of Escherichia coli to Polystyrene Particles

The adhesion of nonflagellated Escherichia coli strain K-12to polystyrene (PS) latex spheres or glass capillaries has beenobserved by using several techniques. Attention was focusedon the orientation of the rod-shaped bacteria as they adheredto the surfaces in 100 mM phosphate-buffered saline. Data showthat PS particles adhered to the ends of the bacteria more than90% of the time. Moreover, the PS particles adhered to one endonly, never to both. Similarly, for experiments with bacteriaadhering to glass, the bacteria adhered on their ends. In orderto determine whether the end of a bacterium had a differentcharge density from that of the middle, rotational electrophoresisexperiments were used. These experiments indicated no measurablecharge nonuniformity. In order to examine how strongly adheredthe bacteria were to the PS particles, differential electrophoresiswas used. Almost always, bacteria were found to be irreversiblyadhered to the PS spheres. The cause of the oriented adhesionis not likely due to surface lipopolysaccharides (LPS), sincethe three strains of K-12 that were used, each having a differentlength of LPS, showed similar behavior. The results are discussedin terms of bacterial cell polarity. The data indicate thatnanodomains on the bacterial ends are important for adhesionand that the time scale for irreversible adhesion is short.

INTRODUCTION

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Introduction

Bacterial adhesion and biofilms are a critical problem for insitu bioremediation (25, 12), heat exchanger fouling (18), biomaterialinfections (9), and ship hull drag increase (8, 18). Prior tothe anchoring of a bacterium onto a surface (28), a number ofinitial adhesion events must occur as a bacterium approachesa surface. First, the bacterium must overcome the repulsiveelectrostatic forces (40, 36). This is at least partly doneby the attractive van der Waals forces (27, 35), although thehydrophobic forces, which are less well understood, are oftenimportant in causing attractive forces between bacteria andsurfaces (47). These initial adhesion events occur over lengthscales of the order of 100 nm [i.e., O(100 nm)] and time scalesof O(seconds), rather than over a few nanometers and hours,like long-term adhesion processes (39). Not until these initialevents have occurred can long-term events such as surface conformationalchanges (11), extracellular polymeric substance production (44,53), DNA production (52), and cell signaling (14) dominate theadhesion process.

In this work we show that bacterial surface nonuniformitiesplay an important role in adhesion. Such nonuniformities couldresult from bacterial polarity, which means that specializedstructures or dynamic molecular localization occurs at or nearthe ends of the cell (42). While bacterial polarity has beenshown to be important in cell division (24) and cell life cycle(2), bacterial polarity has been studied with regard to adhesionprimarily for specific adhesion events (29), not for physicalforces only. The mechanism of how bacterial polarity affectsadhesion is not known. One possibility is surface charge nonuniformity,which is the localization of charged regions on a length scaleof O(10 nm) or O(100 nm). Charge nonuniformity has been shownto affect adhesion in colloidal systems (17), and it could ariseon bacterial surfaces if molecules at the bacterial surfacelocalized on a larger-than-molecular scale.

Techniques to measure and observe initial bacterial adhesionevents are usually macroscopic in nature, and they are bettersuited to examining populations than probing effects of individualbacterial cell surface polarity. Incubation and rinse (19, 46),column flow (4, 38), and flow chamber experiments (21, 45) cannotbe used to study bacteria on a submicrometer level. The techniqueof atomic force microscopy (AFM) is a powerful tool that hasbeen used to study individual bacteria and bacterial adhesion,and researchers have recently examined polymer and extracellularpolymeric substance attachment strength related to bacterialadhesion (7, 41). However, the method has not been used to characterizethe adhesion of the ends of rod-shaped bacteria (6, 37). Thereare several challenges to using AFM to study bacterial adhesionin more depth. (i) The adhered portion of the bacterium facesaway from the AFM tip and is therefore very difficult to probe.(ii) Only the central region of the bacterium can be studied,since the tip slides off the edges. (iii) Bacterial measurementson soft surfaces are difficult to conduct and to interpret (50,51).

The literature contains several references concerning orientedbacterial adhesion to particles or surfaces. Several researchershave examined the role of flagella, which can cause orientedbacterial adhesion. Marshall et al. observed the preferentialorientation, dependent upon motility, of a flagellated Pseudomonasstrain attaching to a surface (28). A Pseudomonas strain wasobserved by Fletcher to attach to hydrophilic surfaces via flagella,while assuming random orientations on hydrophobic surfaces (20).McClaine and Ford have found that flagellar rotation increasesattachment rates of Escherichia coli bacteria to glass (30,31), although it could not be ascertained whether the bacteriawere adhering by their cell bodies or by their flagella. Otherresearchers have observed polar (oriented) adhesion due to specificmechanisms (5), such as adhesin-mediated adherence to fibronectin(10), lectin-mediated adherence to Sepharose beads covalentlyderivatized with lactose (26), or pilus adherence to trachealcells (54). Using a synthesized wettability gradient surfacefor nonflagellated bacteria, Ellen et al. have shown that Treponemadenticola adheres flat onto hydrophobic surfaces but adhereson its ends to hydrophilic surfaces (15). More recently, Haruffet al. caused rod-shaped Klebsiella pneumoniae organisms toadhere in specific orientations by positioning them with a lasertrap (22). In the present study, we examined the oriented adhesionof nonflagellated E. coli to PS latex spheres that have onlysulfate charge groups and that were not treated to promote adhesion.

We combined four methods for examining bacterial adhesion. (i)Video microscopy orientation experiments reveal not only whethera bacterium is adhered to a particle or a surface, but theyalso reveal the precise location on a bacterium where the adherenceoccurs. (ii) Differential electrophoresis allows us to measurebacterium-particle attractive forces with subpiconewton resolution,up to a force of about 50 pN. This technique can thus discernirreversible adhesion. (iii) Rotational electrophoresis enablesus to determine whether the bacteria are uniformly charged,which might be important since we are considering bacterialpolarity. (iv) Shear swaying experiments enable us to visualizewhether bacteria adhere to a flat surface by their ends or bytheir entire surface.

MATERIALS AND METHODS

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Materials and Methods

Bacteria.
Three strains of Escherichia coli K-12—D21f2 and D21 (obtainedfrom the E. coli Genetic Stock Center [Department of Biology,Yale University, New Haven, Conn.]) and JM109 (donated by ShahriarMobashery [Wayne State University, Detroit, Mich.])—werestudied. The wild type is D21. The D21f2 strain has a shorterlipopolysaccharide (LPS) chain on the surface, while the JM109strain has a longer LPS chain. All bacteria were grown in ashaker incubator at 150 rpm and 37°C in Miller's Luria broth,and they were harvested in mid-exponential growth. All threestrains were nonmotile and nonflagellated. While E. coli K-12organisms have peritrichous flagella under many conditions (1),we saw no motility in our experiments other than Brownian motion,and neither transmission electron micrographs nor scanning electronmicrographs revealed flagella on the bacteria under our growthconditions (M. Elimelech, personal communication).

Doubling times were determined from growth curves conductedin our laboratory. These were 39 min for JM109, 35 min for D21,and 28 min for D21f2. Cells were prepared for experimentationby washing them in the suspending media three times with centrifugation(Sorvall Biofuge Primo) at 5,000 rpm (3,466 x g) for 10 min.The bacterial solution was then prepared for experimentation.All three strains were found to be >90% viable after 30 min(the approximate time required for our experiments) in solutionby using a Live/Dead BacLight (kit L-7007, lot 02A1-3; MolecularProbes, Eugene, Oreg.). The temperature during the experimentswas typically 20 to 25°C, with the temperature holding constantto within 0.5°C during each experiment.

Experimental techniques.
A Nikon TE 300 Eclipse inverted optical microscope coupled withvideo equipment (a Cohu 4010 charge-coupled device VHS camera)was used for all experiments with a x100 magnification oil objectiveand differential interference contrast for visualization ofthe bacteria. Experiments were conducted in 100 mM phosphate-bufferedsaline (PBS) solutions consisting of potassium hydrogen phosphateand potassium dihydrogen phosphate dissolved in Milli-Q water.Glassware for the experiments was cleaned by sonication, soakedin 16 N nitric acid for 24 h, and rinsed with Milli-Q water.

Bacteria were mixed by hand-shaking them with 1.5-µm sulfatedPS particles (batch no. 6951; Interfacial Dynamics Corporation,Portland, Oreg.). The bacteria and PS particles were then ableto undergo Brownian aggregation in a 0.2-by-2.0-mm (inside dimensions)capillary tube (Vitrocom, Mountain Lakes, N.J.). The capillarywas mounted on a standard 25-by-75-mm microscope slide, andwe observed the resulting couplets by using video microscopy,recording whether the PS particles adhered to the middle orends of the bacteria (Fig. 1). A motorized stage (Prior) alloweda linear progression across the length of the capillary tubeso that couplets were not recorded twice.

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FIG. 1. Bacterium-particle couplets. (a) One of the rare couplets for which the PS latex particles adhere to the center region of the bacterium. (b) One sphere adhered to the end of a bacterium. (c) Two spheres adhered to one end of a bacterium. (d) Three spheres adhered to the end of a bacterium. Note that particles never adhere to both ends.

For observations of bacteria adhering to glass surfaces, solutionscontaining only bacteria in PBS were placed in the glass microelectrophoresiscell (48) and allowed to contact the glass surface. A low-intensityelectric field ( $~$ 1 V/cm) was applied and then reversed; the electro-osmoticflow within the electrophoresis cell produced a gentle shearfield that caused many bacteria to sway back and forth. Thus,we could determine whether the bacteria were adhered, and moreover,whether they were adhered on one end.

For the D21 strain, adhesion forces were measured between thebacterial cells and the PS sphere in many couplets by usingdifferential electrophoresis. Differential electrophoresis isa technique that exploits differences in zeta ( ${zeta}$ ) potential ofcolloidal species to measure the attractive forces between thetwo moieties (3, 43, 48). Since the bacterium and the PS spherecomposing the couplet have different ${zeta}$ potentials, they tendto move at different velocities in an applied electric field(E₀). As the magnitude of E₀ increases, a stronger tension forceis applied to the couplet. In our experiments, we located aparticle-bacterium couplet, applied an electric field (E₀),and incrementally increased its strength. The couplet was thenmonitored visually. This process was continued until eitherthe couplet broke or the couplet escaped the viewing plane.Figure 2 shows the only particle-bacterium couplet that brokein all our experiments with PS particles. From the value ofE₀ and the ${zeta}$ potentials of the two parts of the couplet, a valuefor the attractive force (F) holding together the bacteriumand particle can be estimated by using the equation (describedpreviously in reference 48) F = 8.76 ${pi}$ a ${varepsilon}$ | ${zeta}$ ₂ - ${zeta}$ ₁|E₀, where a is anaverage particle radius; ${varepsilon}$ is the fluid permittivity for water,and the subscript 1 and 2 represent the bacterium and the particle,respectively. This equation was derived for two spherical particlesand can therefore give only estimates for the bacterium-PS spherecouplets. Importantly, this relationship does not depend uponany particular model for interparticle forces. In order to interpretour experiments, we chose an a value of 0.75 µm. The ${zeta}$ potentials on the bacteria and the PS particles were measuredwith a Brookhaven Zeta PALS analyzer.

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FIG. 2. Differential electrophoresis image of PS particle-bacterium couplet breaking. The images are taken approximately 0.5 s apart. Of the 25 couplets that we have observed, this is the only couplet that broke.

The fact that bacteria adhere by one end indicated that thebacteria were perhaps nonuniformly charged, and so we used thetechnique of rotational electrophoresis to measure the chargenonuniformity on the bacterial surfaces (16, 17, 49). The essenceof this technique is that particles that are uniformly chargeddo not rotate by electrophoresis, regardless of shape (34).However, nonuniformly charged particles will rotate by electrophoresis,and we have recently developed the experimental and theoreticaltools with which to measure and interpret charge nonuniformity(16, 17, 49). Thus, if we see a bacterium rotating in an appliedelectric field, we know that this bacterium is nonuniformlycharged, and we can interpret the angular velocity in termsof a standard deviation of ${zeta}$ potential over the bacterial surface.

RESULTS

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Results

Orientation observations.
Initially on the basis of observations with differential electrophoresis,the bacteria (E. coli D21) were found to adhere to the colloidalPS particles most often in an end-on fashion (Fig. 1b through d).Only rarely did the PS particles adhere to the central sectionof a bacterium (Fig. 1a). Because of the size comparison ofthe particles to that of the bacteria, adhesion to the endsof the bacteria versus the middle was visually unambiguous.In the systematic studies, we found that an overwhelming majority(>90%) of all three strains of bacteria adhered by theirends to the PS particles (Table 1). Furthermore, only one ofthe ends of the bacteria adhered to the PS particles; out of246 couplets observed, no bacterium was found with particlesadhered to both ends. In fact, for many couplets, several particleswere adhered to one end of a bacterium (Fig. 1c and d) but neverto both.

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TABLE 1. Adhesion statistics for E. coli K-12 to PS latex spheres^a

Attachment of the bacteria to the glass capillary was also observedto occur in an end-on geometry. This geometry was observed byapplying a small electric field and observing the bacteria swayingin the electroosmotic flow. Figure 3a shows two bacteria attachedto a glass surface in the absence of flow, while Fig. 3b and cshow the same bacteria in the presence of a flow field. Thebacteria are anchored by one end but otherwise sway in the flow.

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FIG. 3. Swaying experiment images. (a) Bacteria attached to glass surface under static conditions. (b) Bacteria beginning to sway in a flow field. (c) Bacteria aligned with the flow field (to the right). The image is cropped and enlarged to make the bacterial orientations clear.

Force measurements.
Differential electrophoresis was done on 25 additional bacterium(D21)-PS couplets in order to quantify adhesion forces. Thetechnique can apply roughly 50 pN or less, which is more thansufficient for measuring the weak, reversible (often subpiconewton)forces associated with adhesion in a Derjaguin-Landau-Verwey-Overbeek(DLVO) secondary energy minimum (23). Although the literaturedoes not define irreversible bacterial adhesion in quantitativeterms, we define irreversible here as an adhesion force greaterthan 10 pN, based on calculations from classical colloid theory.However, during experiments on 25 couplets, only one coupletbroke. This strong adhesion is not typical of DLVO forces butrather of much stronger forces (e.g., hydrophobic forces andspecific interactions). Zeta potential values in 100 mM PBS,used in calculating the applied force of differential electrophoresis,were -26 ± 1 mV for D21 and -73 ± 2 mV for PS.

Charge nonuniformity.
All three E. coli strains were tested for surface charge nonuniformitywith rotational electrophoresis. No rotation was found for anystrain, indicating that charge nonuniformity was too small tobe measurable with this technique. That is, the angular velocitiescould not be distinguished from the Brownian motion of the bacteria.Figure 4 shows the rotation angles of a strain D21f2 bacteriumin an electric field compared to a Bacillus subtilis bacteriumwith a polar flagellum. While B. subtilis rotated with the electricfield to a steady angle and then rotated back when the fieldwas reversed at 20 s, E. coli exhibited only small, random changesin angle associated with Brownian motion. B. subtilis had aquantifiable surface charge nonuniformity based on its angularvelocity, while E. coli did not. The experiments representedin Fig. 4 were conducted in 1 mM Tris buffer. However, for allthree E. coli strains, these experiments were repeated in 100mM PBS, and again no measurable charge nonuniformity was observed.

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FIG. 4. Measuring charge nonuniformity on bacteria using rotational electrophoresis. Each open circle ( ${circ}$ ) represents an E. coli bacterium. ${theta}$ is the angle in degrees between the applied electric field and the long axis of the bacterium. Since this bacterium is undergoing only random Brownian rotation, its charge nonuniformity is too small to be measured. On the other hand, the filled circles (•) represent a B. subtilis bacterium, which rotated in the electric field. When the field direction was switched at about 20 s, the rotation reversed since this bacterium was nonuniformly charged.

DISCUSSION

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Discussion

Table 1 clearly shows that E. coli adheres end-on to PS latexspheres. This result is complemented by the observations ofbacteria adhered on end to bulk glass. One end of the bacteriumclearly showed adhesion properties different from those of therest of the bacterium. One explanation of the data is bacterialcell polarity (42). In E. coli, this bacterial polarity is causedby the localization of peptidoglycan (13), proteins (33), andphospholipids (32) on the surface of the bacterium. Differencesin the surface composition and chemistry at the bacterium endcould result in preferential adhesion at the end.

Nevertheless, the rotational electrophoresis experiments revealedno charge nonuniformity on the bacteria, and this finding remainspuzzling. Although no charge nonuniformity could be discerned,localization of particular surface molecules must still be occurring.The small nanodomain where the PS latex particles adhere isa small fraction of the total bacterial surface area, and sobased on geometry, it is highly improbable that 90% of the PSparticles would adhere to the end of the bacteria. More importantly,the PS particles adhere to one end but never to both. Apparently,this bacterial polarity is not exhibited through charged functionalities,or at least the differences in charge across the surface arenot detectable by rotational electrophoresis. To maintain theintegrity of the membrane structure, it is unlikely that a largelocalization of molecules other than LPS occurs. However, thecombination of several patches of molecules at a polar end couldresult in significant differences in adhesion properties.

Although the three strains of E. coli had differing lengthsof LPS surface molecules, the adhesion orientation of thesebacteria did not change, remaining at 90% end-on adhesion. Thisresult indicates that LPS does not seem to be the importantmolecule involved in adhesion. Our differential electrophoresisexperiments showed that the initial adhesion process was mostoften irreversible, consistent with the findings of Rijnaartset al. (39). The one couplet (out of 25) that did break in ourexperiments had an attractive force that was roughly <10pN.

Polar adhesion of E. coli bacteria was shown to occur to bothPS latex particles and to standard glass microscope slides.Greater than 90% of the adhering bacteria adhered on their ends,which cannot be accounted for by geometric considerations. Theadhesion of individual bacteria was found to occur at only asingle end, indicating different functionalities in those regions.And yet rotational electrophoresis measurements revealed nocharge nonuniformity on the E. coli organisms. Since the threestrains of K-12, each with a different length of LPS, behavedsimilarly in the experiments, it is possible that the bacterialpolarity due to protein localization causes the polar adhesion.This adhesion is essentially irreversible, since the classicalcolloidal force models cannot explain the large attractive forcesmeasured using differential electrophoresis.

We are currently working to apply the techniques we used inthis research—in particular, the electrophoresis and videomicroscopy techniques—to other bacteria. Bacteria adhereto surfaces by a variety of mechanisms, and by using these techniques,we will examine the generality or specificity of end-on adhesionfor various strains of bacteria. As of yet we do not know, forinstance, whether the polar adhesion results from the divisionprocess. But our aim is to isolate and identify the molecularcomposition that causes the adhesion to the sticky nanodomains,with the long-term goal of controlling the expression of thesemolecules.

ACKNOWLEDGMENTS

This work was funded by the National Science Foundation throughNSF CRAEMS grant no. CH3-0089156 and NSF CAREER grant no. CTS-9984443.

FOOTNOTES

* Corresponding author. Mailing address: Department of Chemical Engineering, The Pennsylvania State University, University Park, PA 16802. Phone: (814) 865-8739. Fax: (814) 865-7846. E-mail: velegol{at}psu.edu.

REFERENCES

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