Spatial Periodicity of Escherichia coli K-12 Biofilm Microstructure Initiates during a Reversible, Polar Attachment Phase of Development and Requires the Polysaccharide Adhesin PGA

Using fast Fourier transform (FFT) analysis, we previously observedthat cells within Escherichia coli biofilm are organized innonrandom or periodic spatial patterns (K. Agladze et al., J.Bacteriol. 185:5632-5638, 2003). Here, we developed a gravitydisplacement assay for examining cell adherence and used itto quantitatively monitor the formation of two distinct formsof cell attachment, temporary and permanent, during early biofilmdevelopment. Temporarily attached cells were mainly surfaceassociated by a cell pole; permanent attachments were via thelateral cell surface. While temporary attachment precedes permanentattachment, both forms can coexist in a population. Exposureof attached cells to gravity liberated an unattached populationcapable of rapidly reassembling a new monolayer, composed oftemporarily attached cells, and possessing periodicity. A csrAmutant, which forms biofilm more vigorously than its wild-typeparent, exhibited an increased proportion of permanently attachedcells and a form of attachment that was not apparent in theparent strain, permanent polar attachment. Nevertheless, itformed periodic attachment patterns. In contrast, biofilm mutantswith altered lipopolysaccharide synthesis (waaG) exhibited increasedcell-cell interactions, bypassed the polar attachment step,and produced FFT spectra characteristic of aperiodic cell distribution.Mutants lacking the polysaccharide adhesin ß-1,6-N-acetyl-D-glucosamine( ${Delta}$ pgaC) also exhibited aperiodic cell distribution, but withoutapparent cell-cell interactions, and were defective in formingpermanent attachments. Thus, spatial periodicity of biofilmmicrostructure is genetically determined and evident duringthe formation of temporary cell surface attachments.

INTRODUCTION

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Introduction

Surface-associated bacterial microbial communities known asbiofilms have a profound impact on processes throughout thebiosphere (5, 6), suggesting that mechanisms responsible forthe design of their structure are likely to be of wide importance.Biofilm development includes stages of cell attachment, growth,differentiation, and dispersal (15, 21). In a previous study,we used fast Fourier transform (FFT) analysis to examine thecellular architecture or microstructure of Escherichia colibiofilm (1). These analyses revealed that a two-dimensionallayer of attached cells exhibits distinct spatial periodicity,which persists within the body of the developing three-dimensionalbiofilm (1). This periodicity is formed independently of surfacechemistry and is similar in wild-type strains and in a csrAmutant, which forms elevated amounts of biofilm (10, 11, 26,27). Possible models for the origination of the spatial patternswere considered, including an activator-inhibitor or Turingmodel (1). Computer simulations have also suggested that anautoinhibitory process could guide the development of biofilmstructure (4).

In E. coli K-12, attachment to abiotic surfaces and cell-celladhesion depends on surface organelles and the adhesin poly-ß-1,6-N-acetyl-D-glucosamine(PGA) (9, 17, 26, 27). Attachment is also affected by physicalfactors, including shear force and gravity. Although in somecases shear may enhance cell adhesion (25), we observed thatflow tended to remove adhering E. coli cells and created anisotropyin the observed attachment patterns (1). Gravitational settlingof bacterial cells facilitates attachment and is widely usedfor the primary inoculation of biofilm support surfaces (2).However, under static conditions or at low flow rates, whenthe bacterial cells are allowed to settle freely, it can bedifficult to distinguish between attached versus settled cells.

Two major classes of bacterial cells are immediately apparentwithin a biofilm-forming population: unattached planktonic cellsand attached cells. Studies dating back to the 1940s revealthat bacterial interaction with a surface often involves aninitial reversible or temporary attachment, followed by irreversibleor permanent attachment (reviewed in reference 24). Temporaryattachments are reversible within a short period of time, withinseconds or a few minutes, and are thought to afford the bacteriuman opportunity to sample the environment before forming permanentattachments or releasing from the surface (discussed in reference3). Permanently attached cells remain surface associated forat least tens of minutes and provide the foundation for furtherbiofilm development. Although the molecular mechanisms responsiblefor these two forms of attachment remain to be defined, theywere recently shown to be genetically distinguishable in Pseudomonasspecies. Mutants lacking lapA or sadB in Pseudomonas fluorescensand P. aeruginosa, respectively, were unable to undergo thetransition from reversible to irreversible attachment (3, 8).

The attachment force should be different for temporary versuspermanently attached cells. In principle, sheer force can beused to distinguish strength of attachment, e.g., in a flowcell. Although bulk flow rate is easily measured, the actualspeed of the flow and thus the shear force may vary significantlywithin the chamber. Unlike shear force, gravitational fieldis homogeneous and affects all bacterial cells equally. Withthis idea, we developed a quantitative assay to distinguishcells according to their strength of attachment by monitoringtheir displacement by gravitational field.

The gravity displacement assay revealed two classes of surfaceattachment: temporary and permanent. As shown for Pseudomonasspecies (3, 8), temporary attachment was mainly by a cell pole,whereas permanent attachment was mainly by the lateral cellsurface. A csrA mutant of E. coli (reviewed in reference 18)formed an increased proportion of permanently attached cellsand a new subclass of cells that were permanently attached viaa cell pole. The major role of CsrA in biofilm formation isposttranscriptional repression of pgaABCD expression, whichis needed for PGA synthesis (28). Mutants lacking the glycosyltransferasethat synthesizes PGA ( ${Delta}$ pgaC) still formed polar attachments butdid not undergo the transition to permanent attachment and formedaperiodic or quasirandom attachment patterns. In contrast, polarattachments were not observed for lipopolysaccharide mutants(waaG), which formed permanently attached cells and cell clustersthat exhibited quasirandom FFT patterns. Thus, waaG mutantsappear to bypass the temporary attachment step in development.

Gravity displacement was reversible; a displaced populationwas able to rapidly reestablish temporary cell attachments possessingspatial periodicity. These findings suggest that the decisionto remain temporarily attached or return to the planktonic phaseis a pivotal step in E. coli K-12 biofilm formation, designedto promote the formation of spatial periodicity within the developingmicrostructure, and that PGA synthesis is integral to this process.

MATERIALS AND METHODS

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Materials and Methods

Growth and media. Bacteria (Table 1) were routinely grown in Luria-Bertani (LB)medium (13) overnight at 37°C with shaking. Such cultureswere used to inoculate (1:100) biofilm cultures, which wereincubated at ambient temperature ( $~$ 24°C) in colonizationfactor antigen (CFA) medium (per liter 10 g of Casamino Acids[Difco, Detroit, MI], 1.5 g of yeast extract [Difco], 50 mgof MgSO₄, and 5 mg of MnCl₂ [pH 7.4]) (12). For microscopy studies,sterile borosilicate coverslips were placed into a petri dishcontaining 25 ml of a freshly inoculated medium, and colonizedslides were withdrawn at defined time intervals. Antibioticswere used as required at the following concentrations: ampicillin,150 µg/ml; chloramphenicol, 10 µg/ml; kanamycin,100 µg/ml; and spectinomycin, 100 µg/ml.

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TABLE 1. E. coli K-12 strains used in this study

Slide preparation and handling for microscopy. After a defined period of time, a coverslip containing attachedbacteria was removed from the incubation medium, immersed intoa petri dish with sterile CFA medium, and gently rinsed for30 s to remove unbound cells. A microscopic coverslip-slidewas then assembled by placing the coverslip onto a sterile slide,separated by a Parafilm gasket ( $~$ 0.18 mm). All borosilicate coverslipsand slides that were used to assess cell binding were pretreatedwith poly-L-lysine, which stabilizes cell-surface interactionsbut does not alter the cell attachment patterns or biofilm microstructure(1). The space between coverslip with the attached bacteriaand the opposite slide contained a drop of sterile CFA medium.This arrangement allows the observation of cells attached toeither glass surface, as well as unattached cells within thecentral space of the slide. Immediately after preparation, theslide was maintained so that the surface of the coverslip withthe attached bacteria was kept facing up, i.e., the cells wereon the bottom surface of the enclosed space. Within 1 to 2 minafter the preparation of the slide, the cell attachment patternwas visually examined and photographed. Afterward, the slidewas inverted, so that the glass surface with the attached bacteriawas facing down, exposing cells to gravitational detachment.The pictures of cell distribution were taken immediately afterinverting the slide and at measured time intervals thereafter.The number of attached cells was electronically counted andplotted versus time. As a rule, the counting was conducted twoto four times in different fields of the slide, and the averagevalue was used. This precaution was found to be more than adequate;variability was low for cell densities between 0.5 x 10⁴ and8 x 10⁴/mm², which was no more than 3 to 5%. At lower cell densities,statistical disorder increased variability. Error was also increasedat higher cell densities because of the small distances betweencells and cell overlap, causing the counting of individual cellsto be less reliable, e.g., influenced by the settings of thesoftware. A final consideration of this method is that the presenceof the cell clusters will lead to underestimates of cell numbers.

Imaging. The cells were observed under phase contrast with an OlympusBX60 microscope (Thornwood, NY). Images were recorded with aCOHU-4910 charge-coupled device camera (COHU, Inc., Florence,KY) connected to a frame-grabber board (GMS500; Scion Corp.,Frederick, MD), installed in a personal computer (PC). All imageprocessing used Image Pro-Plus 4.5 software (MediaCybernetics,Silver Spring, MD). The PRIOR ProScan motorized microscopicstage was controlled by a PC and was used for aligning the sampleas well as for focusing. The periodicity of the cell attachmentpattern in a biofilm monolayer was analyzed by two-dimensionalFFT as described previously (1).

Quantitative biofilm assay. Biofilms were assayed by crystal violet staining, as describedpreviously (11). Overnight cultures were diluted 1:100 intofresh medium and grown at 26°C in 96-well polystyrene microtiterplates. Bacterial growth was determined by measuring the absorbanceat 630 nm. At least six replicas were conducted for each sample,and each experiment was performed at least twice. The resultswere calculated as averages and standard errors of two or moreexperiments.

Biofilm mutants. The procedures for the isolation and genetic and molecular characterizationof biofilm transposon mutants of strain DJ25, which containrandom mini-Tn10cam insertions, have been described in detail(26). Briefly, strain DJ25 (MG1655 ${Delta}$ fimB-H ${Delta}$ motB csrA) was infectedwith ${lambda}$ NK1324, containing a mini-Tn10cam transposon, at a multiplicityof infection of 0.2 (12), and chloramphenicol-resistant colonieswere isolated and evaluated for growth in planktonic shakingcultures and for biofilm formation by crystal violet stainingof adherent cells. The genetic linkage between the biofilm phenotypeand the transposon insertion was confirmed by P1vir transduction(13) back into the parent strain, DJ25; the wild-type E. coliK-12 strain MG1655; and/or its csrA mutant (TR1-5). Insertionsites were determined by arbitrarily primed PCR and DNA sequencingas described previously (26). P1vir transduction was also usedto move the csrA::kan marker among strains.

RESULTS

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Results

Kinetics of cell detachment from a glass surface. Previous publications reveal that, besides planktonic and sessilebacterial cells, a cell surface interaction referred to as "adsorption"or "reversible attachment" (discussed in reference 2) precedescell incorporation into a biofilm structure. Our observationsfurther indicated that in static cultures surface-associatedcells consist of truly attached cells and loosely sedimentedcells (data not shown). The latter cells were removed by gentlerinsing for a few seconds before slides were assembled for microscopicexamination (see Materials and Methods).

Exposure of surface-attached cells to the gravitational fieldby inverting the slide caused a portion of the cells to detach.Images taken immediately after inverting the slide and 30 minthereafter are shown in Fig. 1. The kinetics of detachment weremonitored by collecting images over time and counting the numberof cells remaining on the original attachment surface, now atthe top of the assembly, during exposure to gravity. The accumulationof cells on the bottom surface of this slide assembly was simultaneouslymonitored. The wild-type strain MG1655 (Fig. 2A) and an isogenichigh biofilm-producing csrA mutant TR1-5 (Fig. 2B) were bothexamined in this assay. In addition, cell detachment was examinedafter the biofilms were grown for 2 or 4 h in petri dishes priorto slide assembly. Within 10 min after the slide was inverted,ca. 90 and 82% of the population of wild-type cells detachedfrom the 2- and 4-h slides, respectively. During that time,the interior space of the slide contained numerous, slowly precipitatingcells (data not shown). After 20 min, the number of cells remainingat the upper (original attachment) surface was essentially constant,indicating that these cells were permanently attached. At thebottom surface of the slide assembly, the accumulation of bacteriacontinued for up to 40 to 50 min. The csrA mutant showed similarbehavior, although the proportion of permanently attached cellswas much higher, i.e., ca. 50 and 60% of the total for the 2-and 4-h slides, respectively (Fig. 2B). Comparison of totalcell numbers for a given strain indicated that only a modestincrease in cell number ( $≤$ 25%) occurred during these experiments.

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FIG. 1. Appearance of bacterial attachments before and after inversion of the microscopic slide assembly. A culture (MC4100) was incubated for 4 h, and images of attached cells were captured before (A), immediately after (B), and 30 min after (C) inversion of the slide and exposure to gravitational displacement. The frame width of each image is 0.3 mm.

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FIG. 2. Bacterial cell density versus time on the top and bottom surfaces of microscopic slide assemblies. Wild-type E. coli strain MG1655 (A) and its isogenic csrA mutant, TR1-5 (B), were allowed to form biofilm for 2 or 4 h on coverslips in CFA medium, as described in Materials and Methods. A slide was assembled and inverted so that the coverslip with the attached bacteria was now facing down. Cell detachment from the coverslip and cell accumulation on the bottom surface of the slide assembly were monitored over time. Density values were calculated as the number of cells per square millimeter divided by 100.

Kinetics of permanent attachment. The experiment described above examined the proportion of cellsthat were temporarily or permanently attached at 2 or 4 h afterinoculation of batch cultures. In order to examine systematicallythe time required for cells to attach permanently to a surface,the attachment process was monitored over time, within the slideassembly. For this experiment, a slide containing the rinsedcells was assembled and then inverted to expose attached cellsto gravity, as before. The cells were allowed to detach andprecipitate toward the bottom surface for 20, 30, 40, or 60min, at which times cells on the bottom of the assembly werephotographed. Thereafter, the slide was overturned a secondtime, and the cells that were now at the top of the assemblyand exposed to gravity were monitored for detachment up to 1h. The dynamics of the latter displacement process allowed usto determine the time required for the wild-type and csrA mutantstrains to form temporary and permanent attachments (Fig. 3).For both strains, the majority of cells that reached the bottomsurface of the slide within the first 20 min were either temporarilyattached or had settled but not yet attached. More than 90%of these cells dissociated from the surface within 5 to 10 min.Thereafter, the number of bound cells remained relatively constant,a finding indicative of permanent attachment (Fig. 3A and B).Thus, $~$ 10% of the cells were permanently attached after 20 minof incubation. Permanent attachment in the wild-type strainincreased only modestly beyond 20 min, accounting for $~$ 12% ofthe population at 60 min (Fig. 3A and C). In contrast, boththe number and the proportion of permanently attached csrA mutantcells continued to increase throughout the experiment, reaching $~$ 60% of the population by 60 min (Fig. 3B and C).

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FIG. 3. Kinetics of permanent attachment. A series of coverslips were incubated for 4 h with MG1655 (A) or its isogenic csrA mutant, TR1-5 (B), and slides were assembled and inverted, as described in Fig. 2. Individual slides were inverted a second time at intervals of 20 to 60 min thereafter, as indicated, and the densities of bacterial cells attached to the upper surfaces of the slides were monitored over time. This allowed both the number of cells and the proportion of permanently attached cells to be determined at each time interval. (C) The proportion of permanently attached cells from panels A and B were plotted against the time that was permitted for the gravity-displaced cells to reattach. Cell density values were determined as in Fig. 2.

Gravity displacement of cells with polar versus lateral attachments. Visual examination of colonized coverslips revealed the presenceof polar and laterally attached cells, which were readily distinguishedbecause the former cells were seen to rotate about the attachedpole whereas the latter were immobile (data not shown). Furthermore,it was possible to capture still images that could be used toassess cell orientation relative to the substrate and therebydistinguish these two cell types quantitatively. This was accomplishedby focusing 1.5 µm below or above the attachment surface,depending upon whether the cells were attached to the top orbottom of the slide assembly, respectively. Cells attached tothe upper surface of a slide assembly in which the focal planecoincides with the surface of the glass are shown in Fig. 4A.A micrograph of the same field, taken with the focal plane 1.5µm below the glass, is shown in Fig. 4B. Polar attachedcells are clearly seen in the latter image as the dark roundspots (the focal plane cuts through them). Laterally attachedcells are no longer in focus and become pale in the latter image.By selecting an appropriate gray level cutoff, it was possibleto electronically distinguish images of laterally attached cellsfrom the darker images of polar attached bacteria and thus toautomatically count the two cell types. This approach gave satisfactoryreproducibility (variability, $≤$ 5%) for up to 3 x 10⁴/mm² celldensity and somewhat lower reproducibility (variability, $≤$ 10%)for up to 6 x 10⁴/mm². Microscopic observations of cells incubatedfor 4 h with coverslips in CFA medium showed that a substantialfraction of the attached cells were adhering by a pole: ca.80% for MG1655 and ca. 40% of the population for its isogeniccsrA mutant (Fig. 5). The number of polar and laterally attachedcells versus time at the upper surface of the slide is shownfor MG1655 (Fig. 5A) and its isogenic csrA mutant TR1-5 (Fig.5B). From these data, it is apparent that under gravitationalfield, the number of polar attached cells of both strains decreasesrapidly, whereas the number of laterally attached cells remainsrelatively constant. This indicates that temporarily attachedcells are associated with the surface by a cell pole, whilepermanently attached cells are bound laterally. Interestingly,the csrA mutant exhibited a substantial subpopulation of cellsthat were permanently attached by a cell pole (Fig. 5B). Thisbehavior appeared to be negligible in the wild-type strain.

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FIG. 4. Images of polar and laterally attached bacteria. Cells of TR1-5 (csrA) were grown and allowed to attach to a coverslip for 4 h. A slide was assembled containing the cells at the upper surface (see Materials and Methods). Images (x640 magnification) of these cells were taken at the surface of the coverslip (A) or at 1.5 µm below the surface (B). The frame width for each image is 0.2 mm.

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FIG. 5. Bacterial cells density versus time for polar and laterally attached bacteria. Analysis of the gravitational detachment of polar and laterally attached cells of MG1655 or its csrA mutant, TR1-5, grown for 2 or 4 h, was performed as described in Fig. 4 legend. Cell density values were determined as in Fig. 2.

These relationships were also supported by direct microscopicobservations of single cell interactions with the surface, whichrevealed that detachment generally involved cells that had beenattached by a pole (data not shown). Conversely, when cellsof either strain were seen to reattach, they predominantly didso by a cell pole, as observed by their movement around an anchoringpoint (data not shown).

Correlation of temporary attachment with periodic cell density patterns. The periodicity of the spatial patterns produced by attachedbacteria was examined by FFT analysis of images. The spatialdistribution of cells that attach to the bottom surface of theslide after detachment and sedimentation was nonrandom and,after 15 to 20 min of accumulation, the FFT spectrum showeddistinct periodicity (Fig. 6), indicated by the symmetricalring with a central bright spot (discussed in reference 1).Taking into account that 5 min after overturning the slide,almost no cells were observed on the bottom surface, the estimatedtime for the periodic pattern to form was ca. 10 to 15 min.The images of cells that were collected at the latter timesappeared uniformly shorter than those observed in longer incubationsbecause the majority of the cells were in a relatively uprightposition on the surface. In conjunction with observations onthe kinetics of permanent attachment (Fig. 3), this confirmsthat these cells were associated with the surface by reversiblepolar attachments. These findings reveal that cell attachmentsexhibit spatial periodicity during the temporary attachmentphase of biofilm formation.

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FIG. 6. Density pattern of cells detached from a coverslip by gravity and allowed to settle at the bottom of the slide assembly. Strain MG1655 was inoculated and allowed to attach to a coverslip for 4 h. A slide was prepared and inverted, and cells were permitted to detach, settle, and reattach for 20 min before they were photographed (x400 magnification). The corresponding two-dimensional FFT spectrum of the image is shown on the right.

Altered attachment behavior of biofilm mutants. While the csrA mutant forms periodic cell density patterns (1),examination of additional biofilm mutants revealed two classesthat exhibited distinct behavior in FFT analysis. In a randomtransposon mutagenesis screen, we isolated E. coli biofilm mutantsthat were altered in genes affecting LPS biosynthesis (X. Wangand T. Romeo, unpublished observations), as was previously reportedfor Salmonella enterica LPS mutants (14). The FFT spectrum ofone of these mutants, 12E12-6, its isogenic parent DJ24 ( ${Delta}$ motB ${Delta}$ fimB-H csrA), and TR1-5 (csrA) are compared in Fig. 7. Strain12E12-6 contains a transposon insertion in waaG, which encodesthe glucosyltransferase that attaches glucose I to heptose IIin the LPS core (28). Although strains TR1-5 and DJ24 both boundto the slide as single cells and exhibited spectra indicativeof periodic cell distribution (Fig. 7A and B), 12E12-6 tendedto form clusters of cells adhering to the microscope slide (Fig.7C). Its FFT spectrum showed a uniform circular cloud, indicativeof a quasirandom cell distribution (1). A well-studied nonpolarwaaG mutant (28) behaved similarly in both csrA mutant and wild-typegenetic backgrounds and formed cell clusters and an aperiodiccell distribution pattern as assessed by FFT (data not shown).All of the waaG mutants examined formed few or no polar attachments.A very small proportion of the population of waaG mutants wasobserved to detach during these experiments, although, becauseof cluster formation, accurate cell counting was not possible.The latter finding suggests that an increased proportion ofthe population was permanently attached in these strains.

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FIG. 7. Images of attached cells (x400 magnification) after 4 h of biofilm growth and corresponding FFT spectra. (A) TR1-5 (csrA); (B) DJ24 (csrA ${Delta}$ fimB-H ${Delta}$ motB); (C) 12E12-6 (DJ24 waaG).

A distinct attachment behavior was observed in mutants alteredin the production of the adhesin PGA. These mutants formed singlecell attachments to poly-L-lysine-treated glass surfaces (Fig.8 and data not shown) and to polystyrene (data not shown), althoughthey were defective in adhering to untreated borosilicate glass(26). Analysis of the cell attachment patterns of a nonpolarpgaC mutant, TRXWMG ${Delta}$ C ( ${Delta}$ pgaC csrA), by FFT revealed no detectableperiodicity, either initially or upon subjecting the strainto gravitational detachment for various times (Fig. 8). ThePgaC gene product is a family-2 glycosyltransferase needed forPGA synthesis (26, 27). The pgaC mutant cells continuously detachedfrom the surface throughout a period of an hour exposure togravity, until very few cells remained (Fig. 8D). From the experimentdepicted in Fig. 8, the initial density of cells was 380 x 10²/mm²,while the initial density of polar attached cells was 360 x10²/mm² (data not shown). Thus, almost 95% of the initial attachmentswere by a cell pole, a higher proportion than for its isogenicparent, TR1-5 (csrA), or MG1655 (Fig. 5). Therefore, these cellsdid not establish a permanently attached, adherent population.Upon continued incubation, pga mutants formed closely packedcells with little intervening spacing and yet failed to formthe cell-cell interactions required to construct three dimensionalbiofilm (26; data not shown).

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FIG. 8. Cell attachment patterns and displacement kinetics of a ${Delta}$ pgaC mutant of TR1-5. The culture was grown for 4 h in CFA medium at ambient ( $~$ 24°C) temperature, slides were assembled, and photographs were taken immediately (A), 20 min (B), and 60 min (C) later. FFT spectra are shown to the right of the corresponding images. Cell density versus time under gravitational field was calculated as in Fig. 2 and is shown for the top and bottom surfaces of the slide assembly in panel D.

cpxR and nlpE mutants. The Cpx two-component signal transduction system and a Cpx-regulatedlipoprotein, NlpE, play a role in surface sensing and regulationof gene expression in response to adhesion and have been proposedto affect mechanisms leading to stable surface adhesion (16,20). Thus, we tested mutations in the genes for the responseregulator, cpxR, and the lipoprotein, nlpE, on the attachmentprocess in four strain backgrounds. When present in the originalMC4100 strain, MG1655, or in csrA mutants of these two strains,neither mutation had an effect on biofilm formation in the crystalviolet microtiter plate assay or spatial periodicity in FFTanalysis (data not shown). Thus, under our experimental conditions,this surface sensing system did not appear to influence biofilmdevelopment.

DISCUSSION

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Discussion

The use of gravitational field allowed subpopulations of attachedbacteria to be quantitatively monitored in a simple format,to our knowledge for the first time. Our primary reason fordevising this gravitational displacement assay was to have atool for studying processes involved in the generation of spatialperiodicity of cells within the microstructure of E. coli biofilm.In addition, this approach should be useful for studying otherattachment and/or detachment processes on surfaces amenableto microscopic imaging. This assay revealed that surface-attachedE. coli cells can be physically separated into reversibly ortemporarily attached versus permanently attached forms, whichreact differently with respect to persistent exposure to gravity(Fig. 2 and 3).

An important feature of temporary attachment is that it is mediatedprimarily by the cell pole (Fig. 5). Preferential localizationof structures at the bacterial cell pole, i.e., polarity, iswell known (22, 23). Thus, polar attachment might reflect polarlocalization of an adhesin, although direct evidence for thisis lacking. The different strengths of temporary versus permanentattachments may result from the smaller area of contact of polarversus lateral attachments with the substrate. The finding thata csrA mutant is able to permanently attach by a cell pole ismost likely due to its overproduction of the cell-bound adhesin,PGA (26). Although temporary attachment is reversible, it isnevertheless able to resist modest shear force, as evidencedby the polar attached cells that persist on coverslips aftergentle rinsing (Fig. 4).

Although the present study provides a quantitative evaluationof cell populations undergoing reversible attachment and detachment,it cannot determine whether (i) the temporary attachment forceis insufficient to resist gravity over time or (ii) temporarilyattached cells occasionally detach and are removed by gravityor (iii) both of these processes contribute to the removal ofcells. Because cells of early biofilms that are not exposedto gravity occasionally detach and reattach, at least some contributionmust be from possibility ii. Detachment in the absence of gravitationalforce may involve Brownian motion, as well as forces contributedby the bacterial cell, e.g., motility or charge repulsion. Nevertheless,we found that flagellum-dependent motility, type I fimbriae,and cpx signal transduction were not needed for reversible attachmentor generation of spatial periodicity (Fig. 7 and data not shown).It is possible that the experimental design of the gravitationaldisplacement studies, i.e., examination of cells within a microscopeslide assembly, might have had an influence on the attachmentor detachment behavior during the relatively brief ( $≤$ 1-h) timecourse of our experiments. However, cells that were allowedto detach under gravity and reform attachments within the slideassembly exhibited the characteristic periodic distributionpatterns observed in batch culture (compare Fig. 6 and 7). Furthermore,E. coli attachment or detachment behavior appeared to be similarto that described for Pseudomonas species (3, 8).

The pgaC mutant of E. coli behaves similarly to lapA and sadBmutants of P. fluorescens and P. aeruginosa, respectively (3,8), in that it fails to make the transition from temporary topermanent attachment. This finding strongly suggests that transitionto permanent attachment is mediated by synthesis of the adhesinPGA in E. coli. The lapA and sadB genes encode a large proteinthat is loosely associated with the outer membrane protein anda cytoplasmic protein of unknown function, respectively. Theunderlying mechanism of this transition in the two Pseudomonasspecies and the precise roles played by LapA and SadB are stilluncertain. Whether the biofilm matrices of the latter speciesexhibit spatial periodicity also remains to be determined.

We suggested previously that spatial periodicity might requirethe sensing of a chemical gradient produced by attached cells,which causes altered detachment frequency (1). Here, we foundthat a subpopulation of cells, detached by gravity, is ableto reconstruct temporary attachments (Fig. 3) that exhibit spatialperiodicity within <15 min of reaching the surface (Fig.6). Thus, the process that generates periodicity in the attachmentpattern must be ongoing within this time frame. Other researchershave concluded that temporary attachment affords cells an opportunityto sample the environment before choosing to take up residence(discussed in reference 3). We propose that environmental samplingmay serve to determine proximity to other attached cells. Thus,permanent attachment might result from association with a surfacelong enough for the cell to sense that it is positioned appropriatelywithin the developing biofilm matrix and produce an adhesin.We caution that neither the putative signals that might be samplednor the relevant attachment and detachment mechanisms have beendefined for any developing biofilm, let alone the interveningsignal transduction pathway. These three kinds of informationare needed for the sampling hypothesis to be validated.

Examination of biofilm mutants by FFT analysis revealed celldistribution patterns distinct from those of parental E. colistrains, providing the first direct evidence that these patternsare genetically based. Mutations in waaG, which result in ashortened LPS molecule (28), increased cell-cell interaction,decreased the formation of polar attachments, caused the lossof spatial periodicity, and stabilized the attachment of cellsto the abiotic surface. Although it is possible that LPS mighteither physically or through a regulatory mechanism have anintegral influence on the pattern-generating mechanism, a trivialexplanation for this attachment behavior may be that increasedcell-cell adhesion by this mutant and the presence of cell clustersoverwhelms more-subtle interactions of the pattern-forming process.

Perhaps the more interesting attachment phenotype was exhibitedby pga mutants, including a nonpolar deletion of pgaC, whichlacks PGA (26, 27). Although PGA is not essential for polarattachment to a poly-L-lysine-treated glass surface, the ${Delta}$ pgaCmutant nevertheless failed to form periodic cell density patternsand was defective in permanent attachment (Fig. 8). Furthermore,a csrA mutation, which derepresses PGA synthesis (27), causesincreased lateral attachment, as well as permanent attachmentvia the cell pole (Fig. 2, 3, and 5). These findings confirmand extend our understanding of the crucial role played by PGAin E. coli attachment behavior.

Taken together, our observations support the idea that PGA promotesstabilization of interactions necessary for the cell to undergothe transition from temporary to permanent attachment. However,the transition to permanent attachment alone does not appearto explain the origination of periodicity, since cells thatare temporarily attached (Fig. 6) still exhibit periodicity.We suggest that regulated secretion of PGA and/or asymmetricdistribution of this polymer on the abiotic surface during thetemporary attachment step might account for its role in patternformation. The later hypothesis is depicted within a workingmodel for the origination of biofilm microstructure in Fig.9. In this model, periodicity is generated by the interplayof the attachment activator, PGA, and a putative soluble inhibitorof attachment. PGA production permits the conversion from temporaryto permanent attachment and is suggested to lead to recruitmentof additional cells to the surface at close proximity to existingattachments. A critical density of closely attached cells permitsthe putative inhibitory signal to accumulate and generates aninhibitory field, which is sensed by newly attached cells. Withinthis inhibitory field, the cell attachment does not become permanent,perhaps due to inhibition of PGA synthesis in newly attachedcell, unless the cell has encountered surface-associated PGA,which is only present very close to cells that have alreadyattached. The inability to synthesize PGA eventually resultsin a monolayer of closely packed cells with an aperiodic distributionpattern. Because three-dimensional biofilms exhibit the characteristicperiodic cell distribution patterns, the process that generatesspatial periodicity appears to persist as the biofilm grows(1). We hope that the findings and hypotheses of the presentstudy will foster an increased appreciation of the sophisticationand complexity of the biofilm development process.

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FIG. 9. Working model for the initiation of E. coli K-12 biofilm microstructure. This model is described in the Discussion. Hypothetical steps of the model are indicated by "(?)".

ACKNOWLEDGMENTS

We thank T. J. Silhavy, B. Clarke, and C. Whitfield for strains;Rebecca Desplas for arbitrarily primed PCR analyses; and CarlosGoller and June Scott for reviewing the manuscript.

These studies were funded in part by the National Institutesof Health (GM066794). Kane Biotech, Inc., may develop applicationsrelated to the findings herein. T.R. serves as Chief ScientificAdvisor for, owns equity in, and may receive royalties fromthis company. The terms of this arrangement have been reviewedand approved by Emory University in accordance with its conflict-of-interestpolicies.

FOOTNOTES

* Corresponding author. Mailing address: Department of Microbiology and Immunology, Emory University School of Medicine, 3105 Rollins Research Center, 1510 Clifton Rd. N.E., Atlanta, GA 30322. Phone: (404) 727-3734. Fax: (404) 727-3659. E-mail: romeo{at}microbio.emory.edu.

REFERENCES

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