Vol. 56, No. 7APPLIED AND ENVIRONMENTAL MICROBIOLOGY, JUIY 1990, p. 2065-20720099-2240/90/072065-08$02.00/0Copyright ©D 1990, American Society for Microbiology

Starvation-Specific Formation of a Peripheral Exopolysaccharide bya Marine Pseudomonas sp., Strain S9

MICHAEL WRANGSTADH, ULRICH SZEWZYK, JORGEN OSTLING, AND STAFFAN KJELLEBERG*

Department of General and Marine Microbiology, University of Goteborg, Carl SkottsbergsGata 22, 5413 19 Goteborg, Sweden

Received 12 January 1990/Accepted 1 May 1990

The marine bacterium Pseudomonas sp. strain S9 produces exopolysaccharides (EPS) during both growthand total energy source and nutrient starvation. Transmission electron microscopy of immunogold-labeled cellsdemonstrated that the EPS is closely associated with the cell surface during growth (integral EPS), while boththe integral form and a loosely associated extracellular (peripheral) form were observed during starvation.Formation and release of the latter rendered the starvation medium viscous. In addition, after 3 h of starvationin static conditions, less than 5% of the cells were motile, compared with 100% at the onset of starvation andapproximately 80% subsequent to release of the peripheral EPS at 27 h of starvation. Inhibition of proteinsynthesis with chloramphenicol added before 3 h of starvation caused no increase in viscosity. However,addition of chloramphenicol at 3 h did not prevent the subsequent increase in viscosity displayed by S9 cells.The amount of integral EPS increased for both nontreated and chloramphenicol-treated S9 cells during the firsthour of starvation, with a subsequent equal decrease. The chloramphenicol-treated cells, as well as cells of a

transposon-generated mutant strain deficient in peripheral EPS formation, remained adhesive to a hydropho-bic inanimate surface during the initial 5 h of starvation, whereas nontreated wild-type cells had progressivelydecreased adhesion capacity. During the initial 5 h of starvation, most of the nontreated cells but only a smallfraction of the chloramphenicol-treated and mutant cells detached from the hydrophobic substratum. It wasconcluded that formation of peripheral EPS and the consequent phenotypical alterations observed are a specificresponse to starvation which involves de novo protein synthesis and is possibly responsible for the assembly ofshort-chain EPS into larger subunits.

The marine environment is characterized by heteroge-neous nutrient conditions in which large differences innutrient availability exist (3, 6, 12, 19, 21, 25). As a responseto nutrient depletion, copiotrophic (26) heterotrophic bacte-ria may undergo considerable morphological, physiological,and chemical changes (7, 14, 15, 17, 18, 20). In fact, tosurvive energy- and nutrient-deprived conditions, non-sporeforming heterotrophic bacteria are known to undergoan active adaptation program (20).

Proteins that are synthesized at the onset of the starvationphase have been demonstrated to be of importance forlong-term survival of a marine vibrio (24). Starvation-spe-cific proteins, other than those involved in conferring star-vation resistance, may contribute to the abilities of starvedVibrio and Pseudomonas cells to efficiently find and scav-enge substrates. These proteins include exoproteases (2) andproteins involved in high-affinity amino acid uptake (17).Further functions of the starvation-induced survival programmay include alteration of the ability of cells to adhere tosurfaces (7, 10, 15). Surfaces constitute a significant micro-biological habitat for reversibly and irreversibly bound bac-teria in aquatic systems (16, 19). These involve, for example,biofilm communities (8), particle-associated bacteria (4), andgas-liquid interfaces in the bulk-water and at air-waterboundaries (12). The organisms which initially adhere inmarine systems are gram-negative rods, including Pseudo-monas spp. and Vibrio spp. (34). Starvation-induced pheno-typic characteristics that may alter the ability of cells toadhere could be synthesis of outer membrane proteins (1, 23)and loss of 0 side chains (11). In addition, previous studiesof marine Pseudomonas sp. strain S9 showed that this strain

* Corresponding author.

transiently produces an extracellular polysaccharide (EPS)during the initial phase of starvation, thereby making thecells less adhesive to inanimate hydrophobic surfaces (32)and causing detachment from them (33).The aim of the work presented here was to study the EPS

of Pseudomonas sp. strain S9. It was revealed that twoforms of EPS were present on the cell surface, a closelyassociated, integral form and a loosely associated, peripheralform. The terminology proposed by Wicken was adopted(31). The study focused on the following three questions. (i)Is formation of peripheral EPS a specific response to star-vation? (ii) Is de novo synthesis of proteins necessary forformation of peripheral EPS during starvation? (iii) To whatextent does starvation-specific synthesis of peripheral EPSinfluence phenotypic characteristics, such as adhesion, de-tachment, and motility of Pseudomonas sp. strain S9 duringstarvation?

MATERIALS AND METHODS

Strains, media, and growth conditions. Pseudomonas sp.strain S9 was cultured in VNSS medium and starved innine-salt solution (NSS) as previously described (32, 33).Cells and test surfaces were washed with NSS. Escherichiacoli J100 containing pRK2013::mini-Mu (Tetr) (J. Ostling, A.Goodman, and S. Kjelleberg; submitted for publication) wasused to generate transposon insertion mutants of Pseudomo-nas sp. strain S9. Media, growth conditions, and the trans-poson mutation procedure, using pRK2013 as a vector inconjugation, are described by Ostling et al. (submitted).Streptomycin-resistant (Smr) Pseudomonas sp. strain S9,isolated by natural selection, was used as the recipient. Thisresistance did not influence production and release of theperipheral EPS compared with the wild type.

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Screening for a mutant strain deficient in the release ofperipheral EPS. To isolate a mutant deficient in the release ofperipheral EPS during starvation, VNSS agar plates contain-ing a total of more then 3,000 S9 Tcr Smr colonies werescreened by transferring the colonies from parallel masterplates onto nitrocellulose paper and incubating the sheetswith the colony side up for 24 h on NSS agar plates. Thisprocedure initiated starvation and thus polymer productionby the wild type. After incubation, the sheets were baked at80°C for 1 h. The filters were then blotted by using rabbitanti-polysaccharide antibodies diluted 1/10 in NSS (33) andgoat anti-rabbit horseradish peroxidase-conjugated antibod-ies (Bio-Rad Immuno-Blot Assay Kit) as recommended bythe manufacturer. The presence of released polysaccharideswas visualized as dark halos around the colonies. Thenitrocellulose sheets were screened for colonies that ap-peared to be release-deficient mutants (i.e., colonies lackingthe dark halo), and such colonies were picked from themaster plates, inoculated into VNSS containing 10 jig oftetracycline per ml, and incubated at 27°C until growth wasapparent. The viable cultures were reinoculated in themedium described above and grown to the mid-log phase(optical density at 610 nm, -1.0). The cells were subjected tocomplete energy and nutrient starvation in NSS, and releaseof polysaccharides was detected visually as an increase inviscosity of the starvation regimen. The growth, starvation,and EPS detection steps were repeated three times to ensurethat no polysaccharide was released and that stable muta-tions of the S9 genome had occurred. Finally, of three suchmutants, one (B29) was chosen on the basis of this selectionprocedure. As a control, the mutant was grown with orwithout addition of tetracycline and subjected to starvationto ensure no polysaccharide release and stable mutation ofthe S9 genome.

Viability determinations and chloramphenicol inhibitionexperiments. The viability of starving cell suspensions wasdetermined by spreading serial dilutions on VNSS agar

plates after 12 h, subsequent to addition of chloramphenicol(100 jig/ml; Kebo Laboratories, Goteborg, Sweden) at 0, 1.5,3, 4.5, and 6 h of starvation. No loss in viability wasobserved. The inhibitory effect of chloramphenicol (100jig/ml) was determined as described by Nystrom et al. (24).The concentrations used inhibited protein synthesis by 85%during the starvation period.

Viscosity measurements of chloramphenicol-treated andnontreated, starved S9 cell suspensions. Viscosity measure-ments of chloramphenicol-treated and non-chloramphenicol-treated S9 cells were carried out at 0, 1, 2, 3, 4, and 5 h ofstarvation. In addition, measurements of viscosity were

carried out at 5 h of starvation, subsequent to addition ofchloramphenicol to the starvation regimen at 0, 1, 2, and 3 h.The measurements were performed at ambient temperatureby using an Ubbelohde 501 microviscometer (Kebo Labora-tories) as recommended by the manufacturer. As a control,the viscosities of NSS- as well as log-phase-grown cells inVNSS growth medium were measured.

Motility of S9 cells during starvation in static and agitatedconditions. Washed log-phase cells were divided into twoportions and subjected to energy and nutrient starvation inNSS during static conditions or with agitation on a rotaryshaker. At 0, 1, 3, 5, and 27 h of starvation, samples weretaken from each starvation regimen and the percentage ofmotile cells was estimated microscopically.

Indirect estimation of integral EPS on bulk-phase cells

during starvation. Rabbit anti-polysaccharide serum pro-

duced against both integral and peripheral EPS (32), diluted

1:10 in TTBS (Tris-buffered saline containing 20 mM Tris,500 mM NaCl [pH 7.5], and 0.05% Tween 20) supplementedwith 1% gelatin, was used as the primary antibody. Peroxi-dase-linked goat anti-rabbit immunoglobulin G (0.2 ml at 1mg of protein per ml [Jackson Immunoresearch Laborato-ries, Inc.] diluted 1:3,000 in TTBS) was used as a secondaryreagent to detect and quantify the antigen-antibody reactionenzymatically. Washed log-phase cells were suspended intwo 100-ml volumes ofNSS in two Erlenmeyer flasks (250 mleach) to a final concentration of 2 x 108 to 5 x 108 cells perml. Chloramphenicol was added to one flask to a finalconcentration of 100 ,ug/ml, while the other flask served as acontrol. At 0, 1, 2, 3, 4, and 5 h of starvation, five 100-jIlsamples from each flask were transferred to 1 ml of NSS,mixed, and filtered through Nuclepore filters (pore size, 0.2,um) that had been pretreated for 1 h in 3% gelatin inTris-buffered saline. The filters were finally rinsed with 10 mlof TTBS to remove peripheral and released EPS. Primaryand secondary antibodies were sequentially added to eachfilter by 15-min room temperature incubations, one 10-mlTTBS rinse after each antibody, and a final rinse in 10 ml ofTris-buffered saline. The filters were placed in disposableplastic vials, and the amount of bound enzyme was mea-sured spectrophotometrically at 450 nm by using a TMBPeroxidase EIA Substrate Kit (Bio-Rad) as recommended bythe manufacturer. For the enzyme concentrations used inthe experiment, the reaction velocity was directly propor-tional to the enzyme concentration with a standard peroxi-dase solution. The reaction velocity was thus indirectlyproportional to the amount of integral EPS on the washedcells present on the filter (33). As a control, two filters eachtime were treated as described above but with 100 jil ofsterile NSS instead of the cell suspensions. To determine theenzyme concentration associated with the antibody com-plex, the activity on the control filter was subtracted fromthat of the sample. The results were expressed in A450 unitsand corrected for changes in cell numbers during the exper-iment. As a control, experiments were conducted to verifythat the secondary antibody did not bind nonspecifically tothe filters.Immunogold staining and transmission electron micros-

copy. Rabbit anti-polymer serum diluted 1/10 in NSS (seeabove) or absorbed against log-phase cells was used as theprimary reagent. Protein A-gold (10-nm-diameter particlesdiluted 1/10; Janssen Life Sciences Products, Beerse, Bel-gium) was used as a secondary reagent to visualize theantigen-antibody reaction. As a blocking buffer, 1% bovineserum albumin in NSS (pH 7.0) was included in all steps.Immunoelectron microscopy was used to study cells in thefollowing ways. (i) Starved wild-type (nontreated and chlor-amphenicol treated at the onset of starvation) and mutantcells were transferred to Formvar-coated copper grids at 0,1, 2, 3, 4, and 5 h of starvation and treated with rabbitanti-polymer antibodies. (ii) Starving wild-type cells (non-treated and chloramphenicol treated at the onset of starva-tion) were transferred to Formvar-coated copper grids at theabove-listed times and treated with antibodies that wereabsorbed with washed log-phase cells. The grids (from thetwo separate experiments) were transferred to 100-,I dropsof the different solutions on sterile Petri dishes as follows;buffer for 5 min, antibodies (absorbed or nonabsorbed; [seeabove]) for 30 min, buffer twice for 10 min each time, proteinA-gold for 30 min, buffer twice for 10 min each time, andrinsing with milli-Q water twice for 5 min each time. Thegrids were dried at room temperature and examined with aPhilips EM 301 transmission electron microscope. Controls

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were performed by incubating the grids with protein A-goldonly. Grids were qualitatively assessed, and photographswere randomly taken from each treatment.

Adhesion and detachment of chloramphenicol-treated andnontreated wild-type S9 cells and mutant B29 cells duringstarvation. Both strains were radioactively labeled duringgrowth in VNSS regimen (33), harvested by centrifugation(10 min at 13,000 x g), washed three times, and suspended instarvation medium (NSS) to a final concentration of 107/mI to108/ml. Plastic disposable 10-ml pipette tips were filled withequal amounts of hydrophobic glass beads (5 mm in diame-ter). The glass beads were siliconized as previously de-scribed (32) and stored in the pipette tips in sterile NSSbefore use. The adhesion experiment was performed as

follows: At 0, 1, 2, 3, and 5 h of starvation, 6-ml portions ofeach starvation regimen (chloramphenicol treated and non-

treated) were applied to the glass bead columns, incubatedfor 15 min, and eluted four times with 5 ml of NSS each time.The detachment experiment was performed by the followingprotocol. Immediately after resuspension, 6-ml portions ofeach starvation regimen (chloramphenicol treated and non-

treated) were applied to the glass bead columns. At 0, 2.5,and 5 h of starvation, the columns were eluted four timeswith S ml of NSS each time. Adhesion and detachmentexperiments with mutant B29 were conducted as describedabove, except that the sampling times were at 0, 2.5, and 5 hof starvation. The eluates containing labeled cells wereeither four separate samples containing 5 ml of eluate each or

one pooled sample containing 20 ml of eluate. Samples (0.5ml) from the eluate(s) were transferred to vials containing 4.5ml of scintillation cocktail (Aquassure; Dupont, NEN Re-search Products, Boston, Mass.). The glass beads weremixed four times with 5 ml of Econofluor 2 supplementedwith 7.5% Protosol (Dupont, New Research Products) eachtime to dissolve the attached cells. From this mixture, 0.5 mlof each sample was transferred to vials to which 4.5 ml ofAquassure was added. The total amount of radioactivity percell was measured by filtering subsamples (200 ,ul) of thebacterial suspensions at each sampling time from the starva-tion regimen, followed by a rinse in 5 ml of NSS. The filterswere dissolved in 5 ml of Econofluor 2 supplemented with7.5% Protosol, and the eluate (0.5 ml) was transferred tovials containing 4.5 ml of Aquassure. The total number ofcells at each sampling time was determined by direct lightmicroscopic counts of glutaraldehyde-fixed cells.The radioactivity on the glass beads and filters and in the

eluates was measured by a scintillation counter and con-verted to disintegrations per minute. The values obtainedwere recalculated to numbers of cells, and the degree ofadhesion or detachment was expressed as the percentage ofthe added population that remained on the glass bead columncompared with the number of cells found in the eluate. Threecolumns were used each time, and each experiment was

repeated three times to confirm reproducibility. Leakage ofradiolabel from the cells did not exceed 10%.

RESULTS

Detection of peripheral EPS by viscosity measurements ofthe starvation suspension of chloramphenicol-treated and non-treated Pseudomonas sp. strain S9. The viscosity of thenontreated S9 cell suspension increased gradually, while no

increase in viscosity was noted subsequent to addition ofchloramphenicol to the cell suspension (Fig. 1). Further-more, when the viscosity was measured 5 h after addition ofchloramphenicol at 0, 1, 2, or 3 h of starvation, an increase

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Time of starvation (h)FIG. 1. Detection of released peripheral EPS as increased vis-

cosity (c St = mm2 X s-') of starvation regimen containingchloramphenicol-treated or nontreated Pseudomonas sp. strain S9cells. Measurements of the starvation suspension were performed at0, 1, 2, 3, and 4 h for nontreated cells (-) and at 5 h of starvationsubsequent to addition of chloramphenicol to the starvation regimenat 0, 1, 2, and 3 h (L). The arrows denote addition of chloramphen-icol at different times of starvation. Data from a typical experimentare presented.

in viscosity was observed only when chloramphenicol wasadded at 3 h (Fig. 1). In this sample, the viscosity equalledthat of the nontreated cell suspension at 4 h.

Motility of cells starved in static and shaken conditions. Alarge decrease in the number of motile cells was observedmicroscopically subsequent to the onset of starvation understatic conditions but not during shaking (Table 1). Withcontinued starvation, however, an increase in motility tomore than 80% motile cells after 27 h was found. In contrast,a small decrease in motility was noted for S9 cells starvedduring shaking.

Transmission electron microscopy of immunogold-labeledchloramphenicol-treated and nontreated S9 cells and mutantB29 cells. Immunogold electron microscopy of starved cellstreated with nonabsorbed antibodies or antibodies afterabsorption of the original serum with washed log-phase cells(absorbed antibodies) allowed the EPS to be divided intointegral and peripheral EPS. With nonabsorbed antibodies,only integral (closely associated) EPS was observed onwild-type cells during growth (Fig. 2A) while both integraland peripheral (loosely associated or extracellular) EPSwere detected on free-living cells after 5 h of starvation (Fig.2B). However, no peripheral EPS was demonstrated on thechloramphenicol-treated wild-type cells (Fig. 2C) or mutantB29 cells during starvation (Fig. 2D). With absorbed anti-

TABLE 1. Motility of Pseudomonas sp. strain S9 during totalenergy and nutrient starvation

% of motile cells after starvation for":Starvation condition

3 h 5 h 27 h

Static 5 5 80Shaken 80 80 70

a The values were rounded off to the nearest multiple of five. At 0 and 1 h,100 and 90%o of the cells, respectively, were motile.

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bodies, small amounts of integral EPS were detected ongrowing cells (Fig. 3A). Peripheral EPS, however, visible asstrands on and extending away from the cell surface, in-creased gradually for nontreated wild-type cells (Fig. 3B),while no such structures were visible on chloramphenicol-treated cells after 5 h of starvation (Fig. 3C).

Detection of integral EPS on chloramphenicol-treated andnontreated S9 cells by immunoperoxidase measurements. Therelative amounts of integral EPS on rinsed chloramphenicol-treated and nontreated S9 cells are shown in Fig. 4. Bothchloramphenicol-treated and control cells showed an in-crease in the relative amount of integral polymer during the

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chloramphenicol-treated and nontreated Pseudomonas sp. strain S9cells. Antibodies absorbed to log-phase cells were used (see Materialsand Methods). Panels: A, log-phase cells; B, nontreated cells duringcomplete energy and nutrient starvation for 5 h; C, chloramphenicol-treated cells during complete energy and nutrient starvation for 5 h.

Time of starvation (h)FIG. 4. Detection of integral EPS by immunoperoxidase mea-

surements of washed chloramphenicol-treated (O) and nontreated(-) cells during starvation. Mean values ± standard deviations arethe results of five samples per analysis.

first hour of starvation and a subsequent decrease after thattime. No significant differences between treated and non-treated cells starved for up to 4 h were noted.

Adhesion of chloramphenicol-treated and nontreated Pseu-domonas sp. strain S9 and mutant B29 to hydrophobic sur-faces during starvation. The data presented in Fig. 5 illustratethe adhesion of nontreated and chloramphenicol-treated S9cells and mutant B29 cells during starvation. The previouslynoted (32) decrease in the adhesion of nontreated S9 cellsduring the first hours of starvation was confirmed. Theadhesion of chloramphenicol-treated S9 cells differed signif-icantly from that of nontreated cells. Adhesion of chloram-phenicol-treated cells, which remained between 90 and 110%during the entire starvation period, did not decrease. Adhe-

Time of starvation (h)FIG. 5. Adhesion of radioactively labeled chloramphenicol-

treated (C1) and nontreated (U) cells of Pseudomonas sp. strain S9and mutant B29 (A) to siliconized glass beads during completeenergy and nutrient starvation. Mean values + standard deviationsare the results of three samples per analysis.

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TABLE 2. Detachment of radioactively labeled chloramphenicol-treated and nontreated Pseudomonas sp. strain S9 andmutant B29 cells from siliconized glass beads during

complete energy and nutrient starvation

Mean ± SD % of attached cellsOrganism detached after starvation fora:(treatment)

2.5 h 5 h

S9 (none) 19 ± 0.8 70 ± 7.6S9 (chloramphenicol) 15 ± 2.8 25 t 3.7B29 (none) 18 ± 1.4 22 ± 2.15

a The data are based on three samples per analysis.

sion of mutant B29 cells was also relatively high during thisperiod. A small decrease in adhesion, 20%, was noted duringthe first 2.5 h of starvation. Subsequent starvation resulted inan increase in adhesion to 90% of the value obtained at theonset of starvation.Detachment of chloramphenicol-treated and nontreated

Pseudomonas sp. strain S9 and mutant B29 during starvation.The previously noted degree of detachment of the wild typewas confirmed (33). Between 60 and 70% of the initiallyattached cell population detached during the subsequent 5 h(Table 2). The degrees of detachment of chloramphenicol-treated and mutant cells differed significantly from that ofwild-type cells. The degrees of detachment of chlorampheni-col-treated and mutant cells were 25 and 22%, respectively.

DISCUSSION

It is known that certain nondifferentiating copiotrophicheterotrophic bacteria can actively respond to low-nutrientconditions to survive starvation (20). Sequential induction ofstarvation-specific proteins, essential for survival and recov-

ery of a marine Vibrio sp., has recently been demonstrated(24). Outer membrane starvation-specific proteins that mayalter cell surface characteristics and attachment capacityhave been shown to be synthesized during low-nutrientconditions (1, 23, 30). As outlined in the introduction,surfaces in marine waters constitute an important microbio-logical habitat.

It has previously been reported (32, 33) that an EPSproduced by the marine Pseudomonas sp. strain S9 duringstarvation promotes detachment of cells and reduces theability of cells to adhere to hydrophobic surfaces. Produc-tion of EPS during starvation was suggested to serve as a

dispersal strategy for attached cells subsequent to depletionof the surface-localized substrate, thereby allowing for col-onization of new surfaces by starved cells that displayadhesive properties (32). The existence of other polysaccha-rides that induce detachment of bacteria during growth hasbeen reported (9, 27, 29). However, few studies have fo-cused on polysaccharide production and adhesion of bacte-ria during low-nutrient conditions, although copiotrophicbacteria obviously experience intermittent periods of nutri-ent depletion in marine waters (13, 22). To address thesepossible starvation-specific processes, we used an inhibitorof protein synthesis added at different times of starvationand an EPS release-deficient mutant to study EPS expres-sion and its effects on the adhesion and detachment ofstarved S9 cells.As visualized by immunogold electron microscopy of

starved cells treated with nonabsorbed antibodies or anti-bodies after absorption of the original serum with washedlog-phase cells, the presence of two different forms of EPS

was demonstrated. With nonabsorbed antibodies, immu-nogold transmission electron microscopy showed that theEPS present on growing, non-chloramphenicol-treated (ex-ponential-phase) S9 cells was found to be integral only (Fig.2A), while both the integral and peripheral forms wereshown during starvation (Fig. 2B). When absorbed antibod-ies were used, however, only the peripheral EPS wasdetected, located as strands on or outside the cell (Fig. 3B).This is contrary to the results of previous studies (32, 33) inwhich no polysaccharides, integral or peripheral, were de-tected by use of fluorescence-labeled antibodies duringgrowth or during the first 2 h of starvation. The recent datacould be explained by the fact that the immunogold methodused in this study is more sensitive than the techniquepreviously used.

Inhibition of protein synthesis with chloramphenicol at theonset of starvation appears to prevent formation and releaseof peripheral EPS to the surrounding liquid phase. This wasdeduced by the transmission electron microscopy study, aswell as by viscosity measurements of the starvation regimen.It was shown that there was no increase in viscosity follow-ing addition of chloramphenicol at 0, 1, and 2 h of starvation,while addition of chloramphenicol at 3 h of starvation andlater (data not shown) did not inhibit the increase in viscosityof the starvation regimen (Fig. 1).While release of peripheral EPS that would increase the

relative viscosity of the starvation regimen was prevented inthe chloramphenicol-treated cell suspension, the possibilitythat release of unpolymerized material or short repeatingunits of sugars would occur cannot be ruled out. This is nowunder investigation by analysis of sugar components ofdialyzed and nondialyzed extracellular components.

In contrast to the formation and release of peripheral EPS,production of integral material was found to be an ongoingprocess during both growth and starvation. An immunoper-oxidase method was used to further quantitate the amount ofintegral EPS on washed chloramphenicol-treated and non-treated cells. The results obtained demonstrated a markedincrease in integral EPS during the first hour of starvation,despite inhibition of protein synthesis (Fig. 4). The enzymesresponsible for the formation of integral EPS seem to besynthesized during growth and appear to be stable (andactive) during the first hour of starvation. The decrease inthe amount of integral EPS subsequent to 1 h of starvationdisplayed by both chloramphenicol-treated and nontreatedcells may indicate either that synthesis is prevented or thatcontinuous active or passive release of integral EPS into thestarvation supernatant takes place. The decrease in integralEPS displayed by both chloramphenicol-treated and non-treated cells was followed by an increase in viscosity of thestarvation regimen for nontreated cells, while no such in-crease was noted for chloramphenicol-treated cells (Fig. 1).A model that explains the above-described results may be

proposed. Pseudomonas sp. strain S9 continually producesshort-chain integral polysaccharides during growth. Thiscontinues for at least 1 h subsequent to energy and nutrientdepletion as a result of active enzymes. Starvation-inducedsynthesis of proteins that either covalently connect thereleased integral EPS chains or intercalate between theintegral EPS chains may allow for the formation of largerpolysaccharide molecules, thereby rendering the starvationregimen viscous. Since addition of chloramphenicol inhibitssynthesis of starvation-induced proteins that presumablyconnect the short EPS strands to form larger molecules, noincrease in viscosity can occur. It should be noted thatpreliminary chemical data indicate that the release of sugars

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continues in the presence of chloramphenicol. The proteinsnecessary for assembly of extracellular material seem to besynthesized between 0 and 3 h of starvation, since additionof chloramphenicol at 3 h did not prevent a further increasein viscosity.The implications of these findings are reflected by both

adhesion and detachment of this strain during starvation. Byinhibition of protein synthesis and thereby assembly ofperipheral EPS, no reduction in adhesion occurred (Fig. 5).Instead, the cells maintained the same adhesive capacity,while nontreated cells demonstrated decreased adhesivecapacity during the experiment. The marked degree ofdetachment displayed by nontreated S9 cells was also af-fected. Between 60 and 70% of the initially attached non-treated cells detached up to 5 h, while only 25% of chloram-phenicol-treated cells detached during the same time (Table2). The adhesion data given for chloramphenicol-treated andnontreated wild-type S9 cells are in good agreement withresults obtained by the use of the mini-Mu transposon-generated mutant B29. The mutant, which displayed EPSphenotypic characteristics similar to those of nonstarvedwild-type cells and chloramphenicol-treated starved cells(Fig. 2D), behaved similarly to chloramphenicol-treated S9cells, demonstrating a very low decrease in adhesion (Fig. 5)and a small increase in detachment (Table 2) during starva-tion. Taken together, the adhesion and detachment dataobtained by the different strains demonstrate that the inte-gral form directly or indirectly mediates adhesion and pre-vents cells from detaching from surfaces, while the periph-eral EPS has the opposite effect.The decrease in motility displayed by S9 cells during static

but not shaken starvation (Table 1) was probably due toformation of peripheral EPS, which increases the viscosityaround the cells such that flagellar movement is obstructed.The subsequent increase in motility displayed by S9 cellsafter 27 h of starvation corresponds to release of most of thecell-associated peripheral EPS into the starvation medium.Starvation during shaking, however, previously shown notto cause EPS formation or increase the viscosity of thestarvation medium (32), resulted in only a small decrease inmotility. The effect of increased viscosity on flagellar move-ment has previously been reported for a marine vibrio (5).

This starvation-induced de novo synthesis of proteins,suggested to allow for the transient formation of peripheralEPS reported here, is proposed to regulate adhesion, detach-ment, and motility of Pseudomonas sp. strain S9. It is not,however, a unique phenomenon that microorganisms pro-duce polysaccharides (integral or peripheral) to interferewith adhesion (27, 29) or to detach from a surface (9, 28). Itmay be a response to nutrient deprivation and a result of thestarvation survival program.

ACKNOWLEDGMENTS

This work was supported by grants from the Swedish NaturalScience Research Council, BioInvent International AB, and the CarlTryggers Foundation. Ulrich Szewzyk was supported by a scholar-ship from the Deutsche Forschungsgemeinschaft.We thank Nan Albertson for performing the experiments on the

inhibitory effect of chloramphenicol on starved Pseudomonas sp.strain S9 cells.

LITERATURE CITED1. Albertson, N. H., G. W. Jones, and S. Kjelleberg. 1987. The

detection of starvation-specific antigens in two marine bacteria.J. Gen. Microbiol. 133:2225-2231.

2. Albertson, N., T. Nystrom, and S. Kjelleberg. 1990. Synthesis

and regulation of exoproteases during the starvation of twomarine bacteria. Appl. Environ. Microbiol. 56:218-223.

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