JOURNAL OF BACTERIOLOGY, Jan. 2007, p. 119–130 Vol. 189, No. 10021-9193/07/$08.00�0 doi:10.1128/JB.00930-06Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Phenotypic Diversification and Adaptation of Serratia marcescens MG1Biofilm-Derived Morphotypes�

Kai Shyang Koh,1,2 Kin Wai Lam,1,2 Morten Alhede,3 Shu Yeong Queck,1,2† Maurizio Labbate,1,2‡Staffan Kjelleberg,1,2* and Scott A. Rice1,2

The Centre for Marine Biofouling and Bio-Innovation, The University of New South Wales, Sydney, NSW 2052, Australia1;The School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, NSW 2052,

Australia2; and Center for Biomedical Microbiology, BioCentrum-DTU, Technical University ofDenmark, DK-2800 Kgs. Lyngby, Denmark3

Received 28 June 2006/Accepted 2 October 2006

We report here the characterization of dispersal variants from microcolony-type biofilms of Serratia marc-escens MG1. Biofilm formation proceeds through a reproducible process of attachment, aggregation, micro-colony development, hollow colony formation, and dispersal. From the time when hollow colonies were observedin flow cell biofilms after 3 to 4 days, at least six different morphological colony variants were consistentlyisolated from the biofilm effluent. The timing and pattern of variant formation were found to follow apredictable sequence, where some variants, such as a smooth variant with a sticky colony texture (SSV), couldbe consistently isolated at the time when mature hollow colonies were observed, whereas a variant thatproduced copious amounts of capsular polysaccharide (SUMV) was always isolated at late stages of biofilmdevelopment and coincided with cell death and biofilm dispersal or sloughing. The morphological variantsdiffered extensively from the wild type in attachment, biofilm formation, and cell ultrastructure properties. Forexample, SSV formed two- to threefold more biofilm biomass than the wild type in batch biofilm assays, despitehaving a similar growth rate and attachment capacity. Interestingly, the SUMV, and no other variants, wasreadily isolated from an established SSV biofilm, indicating that the SUMV is a second-generation geneticvariant derived from SSV. Planktonic cultures showed significantly lower frequencies of variant formation thanthe biofilms (5.05 � 10�8 versus 4.83 � 10�6, respectively), suggesting that there is strong, diversifyingselection occurring within biofilms and that biofilm dispersal involves phenotypic radiation with divergentphenotypes.

It is now recognized that bacteria in most habitats predom-inantly live as sessile multicellular consortia, termed biofilms(12), and biofilms form in many environments, including waterdistribution pipes, river rocks, contact lenses and virtually allindwelling biomedical devices (13). Although biofilm commu-nities are key to many ecologically important processes, such asthe biogeochemical cycles, they can also be detrimental, suchas in the corrosion of industrial steel pipes, and 60 to 80% ofall infections are biofilm based (36). As a result of these bio-film-mediated processes, much emphasis is now given to animproved understanding of the mechanisms of biofilm forma-tion and dispersal.

Recently, phenotypic diversification has emerged as a com-mon theme in biofilms. Significant phenotypic variation hasbeen found to occur within many different biofilm communities(25, 32, 41, 45). Although the biofilm is proposed to provideprotection from external stresses, high cell densities inside a

biofilm may result in stressful environments characterized by ascarcity of nutrients and oxygen, nonoptimal pH, and accumu-lation of metabolic by-products (8, 16, 51). Therefore, theability to evolve and adapt to the different microniches withinbiofilms is an important survival strategy to thrive in highlyvariable and adverse environmental conditions (2, 7). In theopportunistic pathogen Pseudomonas aeruginosa, numerousbiofilm-derived morphological variants, such as the small-col-ony variants (SCVs), sticky variants, wrinkly variants, and ru-gose variants, have been identified (7, 19, 21, 32, 52). Webb etal. (52) demonstrated that the emergence of SCVs was linkedto the activity of the Pf4 filamentous phage, which was ob-served during the dispersal process in P. aeruginosa PAO1biofilms (53). Furthermore, a dense network of the Pf4 phagewas found to be associated with the SCVs (52). Interestingly,these SCV cells exhibited enhanced attachment and biofilmdevelopment, and it was proposed that the emergence of theSCVs allows for recolonization of new surfaces during biofilmdispersal. Similarly, Kirisits et al. (32) isolated SCVs of P.aeruginosa, which they termed “sticky” variants, from bothlaboratory biofilms and cystic fibrosis sputum. It was found thatthe sticky variants displayed autoaggregative behavior in liquidcultures and exhibited a hyper-biofilm-forming phenotype, po-tentially facilitating the infection process. The formation ofsticky or mucoid variants have been associated with poor out-comes for cystic fibrosis patients (30). In other habitats, phe-notypic diversification has been shown to be important for the

* Corresponding author. Mailing address: The School of Biotech-nology and Biomolecular Sciences, The University of New SouthWales, Sydney, NSW 2052, Australia. Phone: 61-2-9385-2102. Fax:61-2-9385-1779. E-mail: S.Kjelleberg@unsw.edu.au.

† Present address: Laboratory of Human Bacterial Pathogenesis,Rocky Mountain Laboratories, National Institute of Allergy and In-fectious Diseases, National Institutes of Health, Hamilton, MT 59840.

‡ Present address: Department of Chemistry and Biomolecular Sci-ences, Macquarie University, Sydney, Australia.

� Published ahead of print on 27 October 2006.

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success of competitive rhizosphere colonization by Pseudomo-nas fluorescens strains (17, 39).

Serratia marcescens is an emerging pathogen that is respon-sible for endemic and epidemic nosocomial infections as aresult of resistance to many antimicrobial agents (27, 28) andcan lead to severe complications, such as Serratia bacteremia(24). This gram-negative bacterium is also implicated in arange of ocular infections such as keratitis, keratoconjunctivi-tis, conjunctivitis, and endophthalmitis (3, 6, 11, 35). Environ-mentally, S. marcescens can also cause Cucurbit yellow vinedisease in cucurbit crops such as squash (Cucurbita maxima)and pumpkin (Cucurbita pepo) (9), resulting in heavy economiclosses. Indeed, S. marcescens MG1 was initially isolated from arotting cucumber (23). Because biofilms play an important rolein diseases of humans and plants, it is important to understandthis process in S. marcescens. We have previously characterizedthe development of the filamentous biofilm of S. marcescensMG1 (34), and it was subsequently shown that under nutrient-limiting conditions S. marcescens MG1 can also form a micro-colony-type biofilm (46). In the present study, we have char-acterized the development of the microcolony-type biofilm ofS. marcescens MG1. It was found that the timing and pattern ofemergence of six different stable morphological variantsemerge in a predictable progression throughout the develop-ment of the microcolony biofilm-type of S. marcescens MG1. Inaddition, phenotypic diversification occurs only in the micro-colony biofilm-type and has not to date been identified in thehighly porous filamentous biofilm-type, indicating that the in-ternal conditions differ greatly between the two biofilm mor-photypes. Comparison of various recolonization traits of themorphological variants, such as motility, attachment, and bio-film formation, showed that these dispersal variants exhibitspecialized traits that appear to correlate to the different stagesof biofilm development at which they were isolated, suggestinga relationship between biofilm development and functionalphenotypic diversification in S. marcescens MG1 biofilms.

MATERIALS AND METHODS

Organisms and culture media. The wild-type S. marcescens strain MG1 (23)was used in the present study. This strain was originally classified as Serratialiquefaciens MG1 but, upon 16S rRNA gene sequencing and phylogenetic anal-ysis, was shown to be S. marcescens and has been reclassified (34a). All S.marcescens strains were routinely grown in M9 minimal medium (47) supple-mented with 0.5% (wt vol�1) glucose or on Luria-Bertani (LB) medium (5)supplemented with 1.5% (wt vol�1) agar, unless otherwise indicated. The platecultures were incubated at 30°C for 3 days, while liquid cultures were incubatedwith constant agitation at 180 rpm overnight.

Biofilm flow cell experiments. Biofilms were cultivated in glass flow cells,constructed as described previously (46), which were incorporated into the bio-film flow cell system as described by Christensen et al. (10) with some modifi-cations. Overnight cultures of S. marcescens strains were concentrated fourfoldwith fresh M9 minimal medium supplemented with 0.05% (wt vol�1) glucose andused as inocula for the biofilm experiments. After inoculation of the bacterialconcentrate (500 �l) into the glass flow chamber, cells were allowed to incubatefor 1 to 1.5 h, after which M9 minimal medium supplemented with 0.05% (wtvol�1) glucose was pumped into the flow cell at a constant rate of 0.15 ml min�1.Three independent experiments were conducted in duplicate for 10 days at roomtemperature, unless otherwise indicated.

Isolation of phenotypic variants from biofilm effluent. Aliquots of biofilmeffluent samples were collected from the 10-day biofilm flow cell experiment ona daily basis. The biofilm effluent samples were vortexed at high speed for 1 minand were serially diluted 10�2 to 10�6 in sterile saline solution. Portions (100 �l)of the serially diluted biofilm effluent samples were plated on LB agar in tripli-cate and incubated at 30°C for 3 days. Phenotypic dispersal variant colonies were

confirmed through visual examination using a Leica ZOOM 2000 dissectingmicroscope. Frequencies of phenotypic variation of a particular colony pheno-type were recorded as the percentage of the total colony counts, i.e., 100 � (thenumber of variant colonies/the total number of colonies).

Staining and microscopic analysis of biofilms. Biofilms were prepared forconfocal scanning laser microscopy by staining with the LIVE/DEAD BacLightviability probe (Molecular Probes, Inc., Eugene, OR), prepared according to themanufacturer’s specifications. Two milliliters of working concentration of theLIVE/DEAD BacLight viability probe was pumped into the glass flow cell at aconstant rate of 0.15 ml min�1 to minimize the disruption to the biofilms.

Microscopic observation and image acquisition of biofilms was performed byusing an Olympus LSMGB20 confocal microscope (Olympus Optical Co., Ltd.,Tokyo, Japan) equipped with an argon ion laser at 488 nm for excitation and a522- to 533-nm band-pass filter or a 605- to 632-nm band-pass for emission.Images were captured by using Olympus LSMGB200 confocal scanning lasermicroscopy bundled programs and were further processed by using PHOTO-SHOP software (version 7.0.1; Adobe Systems, Inc.).

Swarming and swimming assays. Swarming and swimming motility were ex-amined on minimal broth Davis without dextrose (Difco Laboratories, Detroit,MI) supplemented with 0.5% (wt vol�1) Casamino Acids, 0.2% (wt vol�1)glucose, and 0.7% (wt vol�1) or 0.4% (wt vol�1) Bacto agar (Difco Laboratories,Detroit, MI), respectively. Overnight cultures of S. marcescens strains were pointinoculated into the center of the agar in 50-mm petri dishes (Sarstedt, Inc.),unless otherwise indicated, and incubated at 30°C for 24 h. Swarming andswimming motility were assessed quantitatively by measuring the diameter of thecircular zone formed by the swarming or swimming colony of each strain.

Quantification of bacterial cell attachment and biofilm formation. Biofilmbiomass of S. marcescens strains was quantified by a crystal violet staining assayas described previously (42, 43), with some modification. Briefly, seed cultures ofS. marcescens strains were prepared by incubation at 30°C for 12 to 16 h, withconstant agitation at 50 rpm. For attachment assays, 100 �l of seeding culturesin M9 minimal medium were inoculated into 96-well polystyrene microtiterdishes, while for the biofilm formation assay 1 ml of 1:100 diluted seeding culturein M9 minimal medium supplemented with 0.5% (wt vol�1) glucose was inocu-lated into 24-well polystyrene microtiter dishes. The microtiter dishes wereincubated at 30°C for 2 h without agitation and at 30°C for 24 h with constantagitation at 150 rpm, respectively. The aqueous phase was removed from eachwell, rinsed three times with phosphate-buffered saline (PBS; pH 7.2), andstained with a 1% (wt vol�1) solution of filtered crystal violet for 20 min at roomtemperature. The wells were subsequently washed thrice with PBS before thecrystal violet-stained biofilms were solubilized in 95% ethanol. The absorbancewas measured at 490 nm with a Wallac Victor2 1420 Multilabel counter (Perkin-Elmer, Inc.).

TEM. Bacterial strains used for transmission electron microscopy (TEM)analysis were grown in M9 minimal medium supplemented with 0.5% (wt vol�1)glucose at 30°C overnight, under constant agitation at 50 rpm. Formvar-coatedcopper 200 mesh grids were floated on drops of the overnight bacterial cellsuspension for 1 min, rinsed once with PBS (pH 7.4), and stained with a 2% (wtvol�1) aqueous solution of phosphotungstic acid (pH 7.0) for 20 s. Samples wereexamined by using a Hitachi H7000 transmission electron microscope at anaccelerating voltage of 80 kV. Formvar-coated copper grids were prepared asdescribed by Davison and Colquhoun (15).

Statistical analysis. Data were analyzed by one-way analysis of variance usingthe SPSS 12.0.2 package for Windows (SPSS, Inc.). Tukey tests provided posthoccomparisons of means.

RESULTS

Characterization of microcolony-type biofilm developmentin S. marcescens MG1. When grown in M9 minimal mediumsupplemented with 0.05% (wt vol�1) glucose in a glass flow cellsystem, S. marcescens MG1 formed a microcolony-type biofilmthat followed a consistent pattern of development. Monitoringof the biofilm by confocal microscopy (Fig. 1) indicated thatthe biofilm developed a limited number of microcolonies byday 2, followed by microcolony maturation and the formationof hollow colonies by days 3 to 4. In contrast to numerousreports in which dead cells could be observed within micro-colonies (4, 31, 37, 53), staining using BacLight LIVE/DEADviability probes (Molecular Probes) clearly revealed the ab-

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sence of red propidium iodide-stained material in the micro-colonies (Fig. 1B). Thus, the microcolonies of S. marcescensappear as empty shells, where the shells are comprised of a thinlayer of viable cells. After the formation of large hollow colony

structures (�100 �m in diameter), an obvious radiation ofbiofilm microcolony types was observed (days 5 to 6). Thisexpansion occurred over a very short period, approximately24 h rather than resulting from a gradual increase in biomass.

FIG. 1. Biofilm development of S. marcescens MG1 in M9 minimal medium supplemented with 0.05% (wt vol�1) glucose over a 10-day period. (Ato F) Confocal microscopic images showing the formation of differentiated microcolonies at day 2 (A); large hollow colony structures at days 3 to 4 (B);biofilm expansion at days 5 to 6 (C); and cell death, biofilm detachment, and biofilm regeneration at days 7 to 10 (D to F). In each panel the top imageis the x-y plane, and the arrowhead indicates the position corresponding to the x-z cross-section in the lower image. Magnifications: A, B, and F, �400(bar, 50 �m); C, �100 (bar, 100 �m); D and E, �200 (bar, 100 �m).

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Subsequently, cell death and biofilm sloughing was observedover the next few days (days 7 to 9), after which biofilm regen-eration was noted (day 10). This pattern of biofilm develop-ment was consistently observed in at least five independentexperiments, each of which were performed in duplicate.

We have previously shown that SwrI, a quorum-sensing (QS)signal synthase that synthesizes N-butanoyl-L-homoserine lac-tone (20), is required for normal biofilm development into ahighly differentiated filamentous biofilm in minimal Davisbroth without dextrose (Difco Laboratories) supplementedwith 0.05% (wt vol�1) glucose and Casamino Acids (34). Todetermine whether cell-to-cell signaling (quorum sensing) wasalso required for the normal development of the microcolony-type biofilm, the signal synthase mutant, S. marcescens MG44,was allowed to form biofilms in M9 minimal medium supple-mented with 0.05% (wt vol�1) glucose. When grown in M9minimal medium (data not shown), the swrI mutant formedmicrocolonies and proceeded through the normal biofilm de-velopmental pathway. This suggests that quorum sensing,which is essential for filamentous biofilm development, is notrequired for the differentiation and maturation of the micro-colony biofilm-type in S. marcescens MG1.

Occurrence of phenotypic variation in the dispersal cellpopulation of S. marcescens MG1 microcolony-type biofilm.Biofilm effluent was sampled daily from the S. marcescensMG1 biofilm over a period of 10 days and plated onto LB agarplates. Six distinct phenotypic variants were identified (Fig. 2).These morphological colony variants have been termed: sticky-smooth variant (SSV); sticky-rough variant (SRV), sticky-rough-umbonate variant (SRUV), smooth-ultramucoid variant(SUMV), non-sticky-smooth variant (NSV), and non-sticky-small-colony-variant (NSCV). For comparison purposes, after3 days of incubation at 30°C, the S. marcescens MG1 parentalwild-type colonies were �4 mm in diameter, had a “non-sticky-to-touch” colony texture, and had a rough or grainy appear-ance (Fig. 2A).

The morphological variants were never isolated from thebiofilm on days 1 to 2 but first began to appear on days 3 to 4at the time when the hollow colonies formed (Fig. 1B). Sub-sequently, the degree of variation in colony morphology in-creased with the duration of biofilm growth (Fig. 3). Variationin the cell population of the biofilm effluent (for convenience,this will be referred to as the dispersed or dispersal population

or the dispersal cells and defined as the cells that are in thebiofilm effluent), reached a maximum percentage of total mor-photypes between at days 4 to 9, when biofilm expansion wastaking place. During the time period of biofilm development,ca. 50% of the dispersing cell population consisted of pheno-typic variants. Interestingly, the timing and pattern of variantformation were consistent. For example, SSV always appearedfirst at low frequencies (3.85% of the population, Fig. 3) at day3, and SUMV always appeared at the latter stages (days 8 to10) of biofilm dispersal. The colony morphotypes were stablewhen passaged daily in liquid culture for 12 days (data notshown), with the exception of NSCV. Some of the NSCVvariants were stable, one of which was selected for subsequentphenotypic characterization. This suggests that a geneticchange is involved in variant formation for the stable morpho-types as opposed to transient gene expression. Sequencing of

FIG. 2. S. marcescens MG1 phenotypic variants isolated from biofilm effluent. All colonies were grown on LB agar plates for 3 days at 30°C.(A to G) S. marcescens MG1 wild-type (A), SSV (B), SRV (C), SRUV (D), SUMV (E), NSV (F), and NSCV (G) colonies. Bar, 2.5 mm.

FIG. 3. Frequency of phenotypic variation (expressed as the per-centage of total colony morphotypes) occurring in the effluent of S.marcescens MG1 biofilm grown in M9 minimal medium supplementedwith 0.05% (wt vol�1) glucose over a 10-day period. Colonies of dis-persal cells bearing typical wild-type colony morphology and the vari-ant phenotypes SSV (�), SRV (s), SRUV (o), SUMV (2), NSV (`),and NSCV (p) were identified after biofilm effluent were plated on LBagar plates and incubated for 3 days. The wild-type morphotype (notshown) makes up the remaining percentages on each day. The datapresented in this graph were obtained from duplicate flow cells and arerepresentative of five independent experiments.

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the 16S rRNA gene, between positions 27 and 1492 (Esche-richia coli 16S rRNA gene sequence numbering), of the phe-notypic variants was performed to ensure that the variantswere not contaminants. Based on 16S rRNA gene sequenceanalysis (data not shown), the phenotypic variants exhibited�99.9% identity to the S. marcescens MG1 16S rRNA genesequence (GenBank accession number AY498856). Also, thevariants only appeared during microcolony biofilm formationin S. marcescens and to date have not been observed duringfilamentous biofilm growth (data not shown).

Phenotypic variation results in extensive variation in motil-ity, attachment, and biofilm formation traits. Based on theobservations that the timing of the emergence of the pheno-typic variants coincided with the development of hollow colo-nies, biofilm expansion, and biofilm dispersal (days 3 to 10),five of the variants were investigated for changes in traitsinvolved in surface colonization, such as motility, attachment,and biofilm formation. A colony with the typical wild-typecolony morphology, termed “BMG1001,” isolated at day 10 ofbiofilm formation was included for comparative purposes. Thedata obtained showed that the swimming and swarming motil-ity, attachment to different surfaces, and biofilm formation ofBMG1001 were similar to the parental wild type (Table 1).There was no significant difference between the growth rates ofthe morphological variants and the wild-type (data not shown).

Motility. Assessment of swimming and swarming motilityshowed that the NSCV exhibited delayed swarming andslightly reduced swimming motility, whereas the rest of thevariants (SSV, SRV, SRUV, and SUMV) were only reduced inswimming motility compared to the wild-type (Table 1). Im-paired motility for the small colony variants found in P. aerugi-nosa biofilms has been linked to abnormal pili or flagella (18,26). TEM of the variants revealed that all of the variants, withthe exception of SUMV, were hyperpiliated (Fig. 4), whereasno pili were observed for MG1 wild-type and BMG1001 cells.SUMV cells displayed reduced numbers of pilus structures thatwere spaced at regular intervals along the bacterial cell andalso expressed fewer flagella (one to two flagella per cell) incontrast to abundant peritrichous flagella observed on wild-

type cells. The significantly impaired swimming motility (P �0.001) observed for SUMV could be explained by the reducednumber of flagella. In striking contrast to the rod-shaped MG1wild-type cells (Fig. 4A), NSCV cells were small coccoids thatwere approximately 0.5 �m in diameter (Fig. 4F). In compar-ison, the SCVs from P. aeruginosa are microscopically identicalto the parent despite forming strikingly different colonies (52).To our knowledge, this is the first report of a biofilm-isolatedmorphological colony variant that has a cell morphology dif-ferent from that of wild-type cells.

Attachment and biofilm formation. To determine the effectsof phenotypic variation on the capacity to colonize and formbiofilms on surfaces, the attachment to different surfaces andbiofilm formation by the five variants in microtiter plates wasmeasured. Crystal violet staining assays showed that NSCVand SUMV did not attach significantly to either 96-well poly-styrene plates (hydrophobic) or tissue culture-treated 96-wellpolystyrene plates (hydrophilic) and failed to form significantbiofilms in 24-well polystyrene plates after 24 h of incubation(Table 1). In contrast, SSV and SRV attached at wild-typelevels but formed two- to threefold more biofilm (Table 1),despite showing no differences in growth rates (data notshown).

Phenotypic variation affects differential biofilm formation inflow cells systems. The extensive variation in biofilm formationin a batch system (Table 1) and the unusual biofilm expansionobserved for the parental wild-type biofilm within the flow cell(Fig. 1) suggested that the variants were likely to exhibitunique biofilm phenotypes. Using confocal microscopy, wecompared and characterized the biofilms formed individuallyby the five morphological variants in glass flow cell systems,including BMG1001, which displayed the typical wild-type col-ony morphology. Figure 5 shows the different biofilms formedby BMG1001 and the five variants when cultivated in M9minimal medium supplemented with 0.05% (wt vol�1) glucose.At day 1, all variants formed either small cell clusters or mi-crocolonies, a finding consistent with microcolony biofilm typedevelopment (data not shown). However, extensive differencesin biofilm formation by the variants were observed by days 2 to

TABLE 1. Comparison of motility, colonization, and biofilm formation traits between parental wild-type and biofilm phenotypic variants

Wild type orvariant

Mean � SDa

Motility AttachmentcBatch biofilm

formation(A490)dSwarming

(mm)bSwimming

(mm)Hydrophilic(A490/A600)

Hydrophobic(A490/A600)

MG1 wild-type 65.50 � 23.88 50.0 � 0.00 0.070 � 0.026 1.033 � 0.168 0.323 � 0.036SSV 71.17 � 17.65 42.0 � 3.31* 0.073 � 0.037 0.953 � 0.106 1.069 � 0.036*SRV 66.33 � 12.66 41.6 � 2.07* 0.069 � 0.012 1.124 � 0.100 0.666 � 0.024*SRUV 47.50 � 12.86 39.0 � 2.83* 0.060 � 0.019 0.967 � 0.171 0.200 � 0.017*SUMV 47.50 � 12.21 29.8 � 1.48* 0.013 � 0.019* 0.072 � 0.017* 0.140 � 0.013*NSCV 3.67 � 1.37*e 45.4 � 1.52* 0.009 � 0.013* 0.190 � 0.037* 0.078 � 0.004*BMG1001 59.50 � 22.50 50.0 � 0.00 0.084 � 0.010 1.177 � 0.088 0.388 � 0.020

a Data are means of five replicates from three independent experiments. *, Significant differences from MG1 wild-type (P � 0.001 analysis of variance).b Swarming experiments were performed in 90.0-mm-diameter petri dishes.c A490 of ethanol-solubilized crystal violet (CV) of CV-stained cells, which is normalized against the cell density (prior washing) of individual wells measured at A600.

Sarstedt 96-well polystyrene plates (Sarstedt, Inc.) and Costar tissue culture-treated 96-well polystyrene plates (Corning, Inc.) were used for attachment studies onhydrophobic and hydrophilic surfaces, respectively.

d Measurements of ethanol-solubilized CV of CV-stained biofilms at A490. Biofilm formation assays were performed on Sarstedt 24-well polystyrene plates (Sarstedt,Inc.).

e Swarming is delayed, and NSCV eventually swarmed to 90.00 � 0.00 after 4 days of incubation at 30°C.

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FIG. 4. Transmission electron micrographs of phosphotungstic acid-stained S. marcescens MG1 wild-type (A and i), S. marcescens MG1 biofilmdispersal variants SSV (B and ii), SRUV (C and iii), SRV (D and iv), SUMV (E and v), and NSCV (F and vi) and biofilm dispersal morphotypeBMG1001 (G and vii). Panels i to vii represent higher-resolution micrographs of panels A to G, respectively. Black arrows indicate flagellarstructures, while the white arrows indicate pili-like structures. Magnifications: A to G, �40,000 (bar, 500 nm); i to vii, �100,000 (bar, 200 nm).

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3. Phenotypic variants SSV, SRV, SRUV, and SUMV formedcopious amounts of biofilm which were �100 �m thick,whereas NSCV biofilms were initially composed of differenti-ated cell chains (Fig. 5E) that coaggregated to form compactirregular microcolony-like structures by days 4 to 5 in M9minimal medium supplemented with 0.05% (wt vol�1) glucose(data not shown). The SSV biofilms were comprised of largemicrocolonies (�100 �m in diameter), most of which hadhollow interiors (Fig. 5A). The SRV, SRUV, and SUMV bio-films, on the other hand, were generally undifferentiated, form-ing thick flat biofilms (Fig. 5B to D). Interestingly, the copiousamounts of biofilm observed for SRUV and SUMV in flow cellsystems contradicts the biofilm formation profile observed inbatch biofilm studies, since crystal violet staining of microtiterplates indicated that SRUV and SUMV formed significantlyless biofilm than the parental wild-type (Table 1). Similar to

the parental MG1 wild-type, BMG1001 formed a limited num-ber of small microcolonies (Fig. 5F) and developed throughthe typical MG1 microcolony biofilm-type pathway (data notshown).

SUMV is a phenotypic variant derived from SSV biofilms.When the SSV biofilm was monitored for 10 days under flowconditions, it was noted that the biofilm structures shifted fromlarge compact microcolonies (day 4 biofilm), which is typical ofthe SSV, to biofilms comprised of cells that were sparselydistributed within the microcolonies (day 6 biofilm), which istypical of a young SUMV biofilm and an indication of theoverexpression of extracellular polymeric substances (Fig. 6Ato C). ImageJ (National Institutes of Health) analysis of theSSV biofilms at days 2 and 6 revealed significant differences(P � 0.0001 [Student t test]) in microcolony architecture (90.73 �10.00 �m versus 48.54 � 3.74 �m in diameter, respectively).

FIG. 4—Continued.

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Analysis of the cells dispersing from the SSV biofilms revealedthat the SUMV morphotype could be consistently isolatedfrom the biofilm effluent from day 5 onward (Fig. 6D). More-over, the timing of appearance of SUMV in the SSV biofilm

was consistent with its emergence in the parental wild-typebiofilm (5 days after the appearance of SSV), relative to theemergence of SSV (Fig. 3). Furthermore, the frequency ofSUMV in the SSV biofilm effluent increased gradually, from

FIG. 5. Differential biofilm formation of S. marcescens MG1 biofilm dispersal variants in M9 minimal medium supplemented with 0.05% (wtvol�1) glucose. (A to C) Confocal microscopic images of 2-day-old biofilms of SSV (A), SRV (B), and SRUV (C). (D to F) Confocal microscopicimages of 3-day-old biofilms of SUMV (D), NSCV (E) and BMG1001 (F). In each panel the top image is the x-y plane, and the arrowhead indicatesthe position corresponding to the x-z cross-section in the lower image. Magnifications: A to C, �200 (bar, 100 �m); D to F, �400 (bar, 50 �m).

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2% (at day 5) to 55% of the dispersing population at day 8.Conversion to the SUMV phenotype appeared to be stable atca. 50% of the dispersing cell population over the remaining 2days. No other variants with different colony morphotypeswere observed, and the SSV morphotype was never observedto convert into the SUMV when passaged as a planktonicculture or on agar plates.

DISCUSSIONQuorum sensing is not required for microcolony develop-

ment in Serratia marcescens MG1. This study showed that mi-crocolony biofilm development in S. marcescens MG1 usingM9 minimal medium supplemented with 0.05% (wt vol�1)glucose was quorum sensing independent. In contrast, it waspreviously demonstrated that the swrI mutant grown in mini-

mal broth Davis supplemented with 0.05% (wt vol�1) glucoseand 0.05% (wt vol�1) Casamino Acids remained as a flat un-differentiated biofilm and was unable to progress to formhighly differentiated cell chain structures observed for the wildtype (34). In a recent report, Rice et al. (46) demonstrated thatnumerous quorum-sensing-dependent traits, such as biofilmformation and swarming motility, can be circumvented bygrowing the swrI mutant in 0.1X LB medium. Similarly, Daviset al. (14) demonstrated that quorum sensing is important forbiofilm development when P. aeruginosa PAO1 was grown inminimal glucose medium, whereas several groups (29, 44)found that there were no marked differences in biofilm struc-tures between the PAO1 wild type and the lasI quorum-sensingmutant. The present study further supports the concept thatthe role for quorum sensing in biofilm formation is not abso-

FIG. 6. Extended flow cell biofilm experiments (10-day-old biofilm) of SSV biofilms revealed a morphological switch to the SUMV biofilm. (Aand B) The formation of microcolonies in a 1- to 2-day-old SSV biofilm (A) is followed by the presence of large hollow colonies structures (�100�m thick) in a 2- to 4-day-old biofilm (B). (C) Sloughing of the biofilm takes place between days 3 to 5, and this was followed by biofilmregeneration at day 5 to 6, resulting in biofilm structural characteristics that resemble a SUMV biofilm. (D) The frequency of phenotypic variation(expressed as the percentage of total colony morphotypes) was determined by plating the biofilm effluent sampled from the SSV biofilm onto LBagar plates daily, and differentiation of SSV (data not shown) and SUMV (2) morphotypes were identified after 3 days of incubation at 30°C. Inpanels A to C the top image is the x-y plane, and the arrowhead indicates the position corresponding to the x-z cross-section in the lower image.Magnifications: A and C, �400 (bar, 50 �m); B, �100 (bar, 200 �m).

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lute but is dependent on the culture conditions used and thatbiofilm structures are similarly medium dependent.

Phenotypic variation of biofilm derived morphotypical vari-ants of S. marcescens MG1. Under the conditions reportedhere, S. marcescens MG1 biofilm development consistentlyprogressed through a series of stages: formation of microcolo-nies, emergence of hollow microcolonies, rapid biofilm expan-sion, cell death, and biofilm detachment. The rapid expansionin biofilm development over a 24-h period in S. marcescens,which has not been described for other organisms, consistentlytakes place after the appearance of hollow microcolonies (Fig.1). Analysis of the biofilm effluent resulted in the identificationof six colony morphological variants that were stable whenpassaged on agar plates or in liquid medium. Interestingly, therapid biofilm expansion at day 4 (Fig. 1C) coincided with thesudden increase in the percentage of morphological variantsdetected in the biofilm effluent (Fig. 3). The frequencies ofvariation peaked at ca. 50 to 55% from days 4 to 9, and thebiofilm morphotypes observed during biofilm expansion ap-pear to match individual biofilms formed by the morphologicalvariants (Fig. 1C and Fig. 5). Hence, we postulate that rapidbiofilm expansion and variant formation are linked. Since wehave only selected morphological variants that were easily dis-tinguished from the wild type, the natural frequency of varia-tion in S. marcescens biofilm is likely to be higher. This issupported by the pronounced phenotypic variation observedfor Pseudalteromonas tunicata D2 cells derived from coloniesof wild-type appearance collected from biofilm effluent (38).

S. marcescens MG1 biofilm variant morphotypes exhibit spe-cialized traits. We investigated several traits that are impor-tant for recolonization by dispersal cells: cell surface struc-tures, motility, attachment, and biofilm formation. UsingTEM, it was observed that all of the S. marcescens biofilmphenotypic variants, with the exception of the SUMV, dis-played hyperpiliated phenotypes (Fig. 4). We found, in con-trast to previous studies (18, 26, 33), no clear correlationbetween hyperpiliation and enhanced attachment or hyper-biofilm-forming ability in the S. marcescens biofilm variants.Either the extensive piliation does not translate into increasedattachment or the pili interact with specific receptors and mayshow preferential adhesion to specific surfaces. For example,piliation in S. marcescens was demonstrated to be importantfor adherence to biotic surfaces such as epithelial cells but notto abiotic surfaces (34a). Interestingly, all variant morphotypesthat had sticky-to-touch colony texture exhibited enhanced bio-film formation in flow cell conditions (Fig. 5), while biofilm-forming ability varied extensively under batch conditions (Ta-ble 1). It has been widely reported that the “sticky” variantsthat were isolated from numerous bacterial species were asso-ciated with the hyper-biofilm-forming phenotype (32, 54, 55).It may be that the physiological conditions differ between thebatch biofilm and flow cell systems, and we hypothesize thatphenotypic variants such as SUMV and SRUV are not welladapted to cope under batch environments in which nutrientand oxygen depletion and the accumulation of undesirablemetabolic by-products take place rapidly. It is also possible thatbiofilm formation as determined by the crystal violet-baseddetection may be an underestimate due to the excessive extra-cellular polymeric substances not stained by crystal violet, pro-duced by the variants. In the context of the present study,

characterization of these phenotypic variants indicated thatthey were different from the parental strain in a number ofproperties, such as swimming, swarming, attachment, and bio-film formation.

Variant formation appears to involve genetic radiation andis increased in frequency within the biofilm. In some organ-isms, phenotypic variation is an unstable trait, such as thepersister cells of P. aeruginosa, where the phenotypic change islikely due to gene regulation. However, the morphologicalvariants described here are stable, suggesting a permanentgenetic change. This is supported by the observation that thetiming and diversification of phenotypic variants followed apredictable sequence and was strongly correlated with the spe-cific stages of biofilm development. For example, the emer-gence of the SSV consistently occurred during hollow micro-colony formation, and the SUMV consistently emerged whencell death and biofilm detachment occurred (Fig. 3). Further-more, we demonstrated that SUMV could be derived fromSSV biofilms (Fig. 6D), and the timing of the appearance ofSUMV is consistent with its appearance relative to SSV inbiofilms made by the parental wild-type strain (Fig. 3), sug-gesting that SUMV is a genetic variant derived from SSV andmay share common mutations. Given the specific pattern andtiming of variant formation and that the same variants arealways observed, we hypothesize that either the genes involvedcontain hotspots for mutation or the local conditions andstresses within the biofilm change over time, leading to muta-tions in different genes by separate mechanisms.

Is the formation of variants a strategy for creating dispersalcells or simply a response to stress? The formation of variantsfrom the biofilm has several implications. One possibility,raised above, is that the biofilm represents a stressful environ-ment and the formation of variants is a consequence of anincreased mutation rate as a response to the stress. Indeed, acomparison of the frequency of genetic variation between bio-film and planktonic cultures revealed that the diversity andfrequency of phenotypic variation were significantly differentbetween the two culture conditions. In planktonic cultures, alow percentage of phenotypic variation was observed (reachinga frequency of 5.05 � 10�8 at day 9 and comprising only twomorphological variants) compared to the variation frequenciesof 4.83 � 10�6 from biofilms (Fig. 3). Variant formation inplanktonic cultures was tested in the same medium as for thebiofilms, was followed over a 10-day period, and evaluatedboth shaken and nonshaken cultures incubated at room tem-perature. The differences in the frequencies of variation in thevariant population suggests that biofilm-specific conditions or aselective pressure that is inherent to biofilms may be requiredto trigger and maintain phenotypic diversification. This bio-film-specific physiology is supported by a comparison betweenP. aeruginosa biofilms and planktonic cells, which revealed anoverall change in more than 50% of the proteome which wasspecific to biofilm development (49). Furthermore, the pro-teomic studies by Sauer et al. (49) showed that there weredistinct structural and metabolic changes that could be corre-lated to different stages of biofilm development, indicating thatthere are physiological characteristics that are unique to bio-films. Our findings revealed that the generation of phenotypicvariants is correlated to microcolony biofilm-type develop-ment, since phenotypic variation could not be detected in the

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highly porous filamentous biofilm-type in S. marcescens. Thesefindings suggest that the internal conditions for the two biofilmtypes, microcolony and filamentous, may differ dramaticallywhere the microcolony biofilm-type may provide the appropri-ate conditions (e.g., stress or starvation) required for the in-duction of variant morphotypes. It should be noted that star-vation or stress may not be the only factor required for theformation of variants, since the 10-day-old planktonic cultureswould also experience starvation and stress conditions. Per-haps the combination of stress and a biofilm-specific regulonare required for variant formation. One interesting possibilitythat remains to be resolved, is whether the increased frequencyof mutations observed for the biofilm grown cells occur ingenes that would alter the cells’ ability to colonize differentsites or to compete in particular niches. Our data clearly indi-cate that the variants do differ in several characteristics, such asattachment, swarming motility, and biofilm formation, that areimportant for surface colonization. In this way, the biofilm lifecycle may involve the formation of dispersal cells that differfrom the parental strain, and these dispersal cells may beconsidered as an “insurance policy” (7) for the subsequentoccupation of new niches. Clearly, there are several conditionsthat would need to be met for this to accurately reflect theeffects observed. First, the mutations would need to specificallyoccur in genes that alter surface colonization phenotypes. Thisis possible if such genes contain hot spots for mutations andhence would be over-represented against the general muta-tional background. Our data suggest that these variants mayoccur through such a process because of their high frequencyand consistent timing of appearance. Moreover, the formationof the SUMV from the SSV would seem to favor this hypoth-esis. Second, such mutations in a dispersal population shouldtranslate into a selective advantage. This remains to be dem-onstrated for these specific variants, but it has been demon-strated for other microorganisms that variant formation is im-portant for competitive fitness. For example, the wss gene hasbeen shown to be involved in formation of the “wrinklyspreader” variant of Pseudomonas fluorescens (50), and thisvariant was less fit when grown in competition on roots (22).Sanchez-Contrares et al. (48) noted for P. fluorescens that thewild-type cells attached to the entire root system, whereas themorphological variants attached in a more specialized fashionand were mostly recovered from either the central part of theroot or the last centimeter. Finally, Matz et al. (40) showedthat for Vibrio cholerae, the biofilm morphotypes were predom-inantly rugose, whereas the planktonic cells were smooth, sug-gesting niche specificity for the two morphotypes. Thus, iden-tification of the mutations involved in variant formation in S.marcescens, their frequency, and fitness under different condi-tions represent very exciting possibilities for understanding theecological implications of this process.

Microbial persistence in a stressful environment or in animalhosts has been described as survival in the evolutionary fasttrack (1, 7). In the present study, we have demonstrated thatwithin a highly controlled environment, microorganisms re-spond through phenotypic diversification to enhance the adap-tive potential of the microbial population. Moreover, special-ized traits displayed by different phenotypic variants correlatedwith specific stages of biofilm development. Studies of suchfunctional diversification and the mechanisms by which it oc-

curs will contribute to a better understanding of microbialevolutionary processes, the adaptability of microbial popula-tions, and how to design biofilm control systems.

ACKNOWLEDGMENTS

We thank Michael Givskov for providing the S. marcescens strainsMG1 and MG44 used in this study.

This study was supported by the Australian Research Council andthe Centre for Marine Biofouling and Bio-Innovation at The Univer-sity of New South Wales.

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