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1 Molecular Microbial Ecology Group, BioCentrum-DTU, Technical University of Denmark, DK-2800 Lyngby, Denmark
2 School of Engineering and Sciences, International University Bremen, Campusring 1, D-28759 Bremen, Germany
3 Department of River Ecology, UFZ Centre for Environmental Research, D-39114 Magdeburg, Germany
Correspondence
Matthias S. Ullrich
m.ullrich{at}iu-bremen.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Present address: Arpida Ltd, CH-4142 Muenchenstein, Switzerland.
Present address: Statens Serum Institut, DK-2100 Copenhagen, Denmark.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), but DNA (Whitchurch et al., 2002), proteins and lipids (Wingender et al., 1999) can make up a significant proportion of the matrix in biofilms. This matrix is considered to be the key component that determines the physico-chemical and biological properties of biofilms (Wolfaardt et al., 1999). EPSs have been shown to be required for the initial attachment to a surface of Vibrio cholerae (Watnick & Kolter, 1999) and Staphylococcus epidermidis (McKenney et al., 1998) or for the structural development of mature biofilms like colanic acid in Escherichia coli K-12 biofilms (Danese et al., 2000).
Pseudomonas syringae pv. glycinea is the causal agent of bacterial blight on soybean plants. The bacterium is known to produce two different EPSs, alginate and levan (Osman et al., 1986). Levan is a high-molecular-mass 
-(2,6)-polyfructan with extensive branching through -(2,1)-linkages. Its synthesis from sucrose is catalysed by one single, extracellular enzyme, levansucrase (EC 2.4.1.10). Many Gram-negative (e.g. Acetobacter diazotrophicus, P. syringae, Erwinia amylovora; Arrieta et al., 1996; Bogs & Geider, 2000; Hettwer et al., 1995) and Gram-positive bacteria (e.g. Bacillus subtilis, Streptococcus mutans; Dedonder, 1966; Sato et al., 1984) produce levansucrases. Three lsc genes encoding levansucrase (of which only two are expressed) have been identified in P. syringae pv. glycinea PG4180 and mutants defective in levan formation were constructed (Li & Ullrich, 2001).
With the exception of dental biofilms, the role of levan in biofilm formation has not been studied so far. Fructans produced by S. mutans are believed to function primarily as extracellular storage compounds that are metabolized during periods of nutrient deprivation (Burne et al., 1996). P. syringae mainly produces levan when sucrose is present. In contrast, alginate has been shown to be produced by this bacterium when it infects plant leaves (Fett & Dunn, 1989; Osman et al., 1986). Alginate is a co-polymer of -1,4-linked D-mannuronic and L-guluronic acid, which is normally O-acetylated at the 2 and/or 3 position(s) of the D-mannuronate residues (Osman et al., 1986). The arrangement of the alginate structural gene cluster of P. syringae is virtually identical to that described for Pseudomonas aeruginosa (Penaloza-Vazquez et al., 1997). Alginate production by P. syringae has been associated with increased epiphytic fitness, resistance to desiccation and toxic molecules (Yu et al., 1999), and the induction of water-soaked lesions on infected leaves (Fett & Dunn, 1989). The algA gene is one of the genes induced during P. syringae pv. tomato infection of Arabidopsis thaliana as shown by IVET technology (Boch et al., 2002). Likewise, the algD gene is expressed when P. syringae attempts to colonize both susceptible and resistant plant hosts (Keith et al., 2003).
Due to their specific binding to carbohydrates, fluorescently labelled lectins combined with confocal laser scanning microscopy (CLSM) (Lawrence et al., 1998; Mathee et al., 1999; Neu & Lawrence, 1999; Strathmann et al., 2002) or with enzyme-linked lectin-sorbent assays (ELLAs) have been applied for analysis of EPSs in biofilms (Leriche et al., 2000; Strathmann et al., 2002; Thomas et al., 1997).
The goal of this study was to determine the spatial distribution and function of EPSs during biofilm formation by P. syringae using defined alginate- and/or levan-deficient mutants in combination with specific EPS staining. We hypothesized that alginate and levan might have either structure-determining or nutrient storage function(s).
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. Strain PG4180 produces levan but not alginate due to a nonsense mutation in the key regulatory gene, algT, as confirmed by nucleotide sequence determination of this gene (A. Schenk, L. M. Keith, C. L. Bender & M. Ullrich, unpublished data). The mucoid derivative of PG4180, PG4180.muc, emerged spontaneously from a PG4180 culture and is characterized by a reversion of this mutation (L. M. Keith & C. L. Bender, unpublished data). PG4180.muc produces both alginate and levan. The levan- and alginate-deficient mutant PG4180.M6 had previously been generated from PG4180 as an lscB lscC double mutant by marker exchange mutagenesis (Li & Ullrich, 2001). This mutant could be complemented to a levan-positive phenotype by either lscB or lscC. The alginate-producing transconjugant PG4180.M6 (pBBR3-AXSalgT) carries a plasmid containing the intact algT gene and its upstream promoter region cloned from strain PG4180.muc (A. Schenk & M. S. Ullrich, unpublished data). PG4180.M6 (pRLB7.2) represents a transconjugant restored in levan formation by providing a functional lscB gene in trans. PG4180.muc-GFP was fluorescently tagged with a gfp gene from a mini-Tn7 construct (Klausen et al., 2003; Koch et al., 2001) at an intergenic neutral chromosomal locus. The integration of the gfp-containing insert was confirmed by nucleotide sequencing.
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) supplemented with 10 mM glucose and 10 mM sucrose was used for batch overnight cultures grown at 20 °C. Mannitol-glutamate (MG) medium (Keane et al., 1970) agar plates supplemented with 5 % sucrose and incubated at 20 °C were used to determine levan production of P. syringae. The following antibiotics (mg ml–1) were added to the media when needed: gentamicin, 2; streptomycin, 25; tetracycline, 25; chloramphenicol, 25.
Microtitre plate biofilm formation assay.
To compare the impact of levan and alginate production on initial biofilm formation, biofilms of P. syringae PG4180, PG4180.muc, PG4180.M6, PG4180.M6(pBBR3-AXSalgT) and PG4180.M6 (pRLB7.2) were grown in wells of polystyrene 96-well microtitre plates at 20 °C for 48 h as follows. Overnight cultures of P. syringae were diluted to an OD600 of 0.02 in FAB minimal medium supplemented with 60 mM glucose or 10 mM glucose plus 50 mM sucrose. Subsequently, aliquots of 100 µl were placed into the wells of microtitre plates. The amount of surface-attached biofilm was determined by using a modified crystal violet method (O'Toole & Kolter, 1998) as described elsewhere (Klausen et al., 2003). The biofilm cell-associated dye was measured as its absorption at 590 nm.
ELLA of microtitre plate biofilms.
An ELLA was performed according to the procedure described by Leriche et al. (2000). Microtitre plate biofilms of P. syringae were grown for 72 h as described above. Following the removal of planktonic cells by washing, 100 µl of a 10 µg peroxidase-linked ConA ml–1 solution (Sigma) was applied to each well and incubated for 15 min. Unbound ConA was removed by three washing steps and the bound enzyme conjugate was visualized after addition of 100 µl 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) substrate solution (Sigma). Following an incubation of 15 min, the absorbance was measured at 405 nm. To determine unspecific binding of the lectin, medium without cells was used as control. Wells containing samples without added lectin were treated by the same procedure to detect intrinsic bacterial peroxidase activity. To elucidate the role of levan for its interaction with peroxidase-linked ConA, wells containing P. syringae biofilms grown in presence and absence of sucrose were incubated with 50 µg levanase for 1 h prior to the addition of enzyme linked-lectin. At least four parallel samples were run in each experiment, and each experiment was repeated at least twice.
Cultivation of biofilms in flow cells.
Biofilms were grown in flow cells with individual channel dimensions of 1x4x40 mm at 20 °C. The flow system was assembled and prepared as described previously (Møller et al., 1998). The flow-chambers were inoculated by injecting 300 µl of a bacterial overnight culture diluted to an OD600 of 0.4 into each flow channel with a 0.5 ml syringe. Upon inoculation, the flow channels were left upside down without flow for 2 h, after which medium was pumped (0.2 mm s–1) through the channels using a Watson Marlow 205S peristaltic pump. Channels were irrigated with FAB minimal medium supplemented with 1 mM glucose or citrate plus 1 mM sucrose, or with 50-fold diluted Luria–Bertani broth (LB) or King's B medium (KB) (King et al., 1954).
Microscopy and image acquisition.
Biofilm structures were thoroughly monitored to compare the phenotypes of the tested strains and mutants of P. syringae. CLSM was used to visualize biofilms of PG4180.muc-GFP directly. All other derivatives were monitored by CLSM upon cell staining by injection of 300 µl SYTO 62 (Molecular Probes) into the flow chambers. A Zeiss LSM 510 confocal laser scanning microscope equipped with detectors and filter sets for monitoring GFP, SYTO 62, FITC and TRITC fluorescence was used for all microscopic observations and image acquisitions. Images were obtained using a x63/1.4 or a x40/1.3 objective. Simulated three-dimensional images and sections were generated by using the IMARIS software package (Bitplane AG).
Lectin staining of biofilms in flow cells.
Previously, a screening of all commercially available lectins revealed suitable probes for P. syringae glycoconjugate staining (Staudt et al., 2003). To allow for a simultaneous screening of several of these lectins in the same flow cell, we applied a protocol of fixing and embedding the biofilm using polyacrylamide as has been applied for FISH in flow-chamber biofilms (Christensen et al., 2002). However, a lower polyacrylamide concentration (8 %) was used in the present study. After embedding with polymerizing acrylamide, the biofilm was carefully removed from the flow cell and cut into small pieces, which were each stained with an individual lectin and subsequently washed with distilled water to remove unbound lectin.
Application of fluorescently labelled lectins to flow-chamber biofilms was performed as described by Johnsen and co-workers (Mathee et al., 1999). Briefly, the flow-chamber biofilm was fixed using 4 % p-formaldehyde for 1 h, washed and incubated for 30 min in the presence of the fluorescently labelled lectins (final concn 0.1 mg ml–1). Finally, the biofilms were washed to remove unbound lectin and cells were stained using 300 µl SYTO 62. FAB medium without carbon source was used for all washing steps. Lectins investigated in more detail were FITC-labelled Concanavalin A (Sigma) and the lectin from Naja mossambica (Sigma), which was labelled with Alexa Fluor 488 using the Alexa Fluor Protein labelling kit (Molecular Probes).
Transcriptional analysis of lscB expression.
Plasmid pRLB7.2egfp contains a transcriptional fusion of the lscB gene from PG4180 with promoterless egfp encoding enhanced green fluorescence protein. To construct this fusion, egfp was excised from pRcCMV3.EGFP (kindly provided by I. B. Leibiger, Karolinska Institute, Sweden) using ApaI and HindIII. The resulting fragment was blunt-ended using Klenow fragment and ligated into XhoI-digested and Klenow-treated plasmid pRLB7.2, which contains a unique XhoI site inside the lscB gene. The resulting plasmid was designated pRLB7.2egfp and introduced into PG4180 cells by triparental mating. Transconjugant PG4180 (pRLB7.2egfp) was subsequently grown in liquid FAB medium supplemented with 10 mM glucose and 50 mM sucrose at 20 °C for 48 h. Likewise, the transconjugant was incubated in wells of polyvinylchloride 96-well microtitre plates at 20 °C for 96 h. To assess fluorescence during growth of bacteria in liquid culture, aliquots were harvested at distinct optical densities, centrifuged, suspended in PBS (pH 7.4) and adjusted to an OD value of 0.5. To assess fluorescence of attached bacterial cells in microtitre plate wells, the wells were rinsed carefully and filled with PBS (pH 7.4) prior to fluorescence determination. Parallel wells were stained as described for the microtitre plate biofilm formation assay with crystal violet in order to estimate the amount of attached cells. Fluorescence was quantified using a Dynatech Laboratories Fluorolite-100 microplate reader set to an excitation wavelength of 470 nm with emission detection at 560 nm.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). With the exception of biofilms of PG4180.muc which produced slightly more biomass, no substantial differences in the amount of attached biomass could be observed by crystal violet staining. When sucrose was added, PG4180 (levan+ alginate–), PG4180.M6 (levan– alginate–) and PG4180.M6(pBBR3-AXSalgT) (levan– alginate+) showed the same phenotype as observed above. However, a considerable decrease in biofilm formation was observed when PG4180.muc (levan+ alginate+) was incubated in the presence of sucrose. Our results suggest that neither levan nor alginate is required for biofilm formation of P. syringae in a static system. Interestingly, the presence of sucrose seemed to inhibit biofilm formation when both alginate and levan were produced.
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). Cells initially formed flat, irregularly shaped microcolonies, which later became ball-shaped microcolonies and towers. In the beginning, microcolonies were predominantly formed at the edges of the flow chamber, i.e. in regions with lower laminar flow. Subsequently, the flow channel was completely covered with microcolonies after about 7 d. Mature biofilms of P. syringae showed a heterogeneous appearance and consisted of towers (up to 120 µm thick), ball-shaped microcolonies, irregularly shaped network-forming microcolonies and substratum-proximal areas with only a few attached cells (Fig. 2).
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). Numerous motile cells were observed in mature biofilms. Experiments were repeated with untagged PG4180.muc either not stained or stained with the nucleic acid-binding dye SYTO 62, yielding very similar results. Biofilms of PG4180.muc-GFP grown in rich medium (LB or KB medium) supplemented with or without sucrose exhibited similarly heterogeneous structures as biofilms grown in minimal medium. However, biofilms developed faster in rich as opposed to minimal medium. After 8 d, the number of cells dispersing from biofilms grown under rich medium conditions was higher than that of the minimal medium control. Consequently, rich-medium biofilms were generally thinner than those grown in minimal medium and consisted of attached cells and small microcolonies at some areas of the substratum. As expected, there were no effects of sugar supplementation in biofilm development in rich medium.
Interestingly, when biofilms of levan-producing PG4180 derivatives were supplemented with sucrose, characteristic blebs of different sizes (about 2–15 µm diam.) developed in mature biofilms (5–8 days). These blebs did not develop under sucrose-deficient conditions. They were clearly distinguishable from air bubbles and thus were probably filled with liquid.
Flow-chamber biofilms of EPS-deficient P. syringae
To investigate the individual impact of the two EPSs on P. syringae biofilm structure, PG4180 derivatives deficient in levan or alginate synthesis, or both, were compared with PG4180.muc (alginate+ levan+). Experimental conditions were made appropriate for levan production by supplementing the cultures with sucrose. Biofilms were stained after 6 and 9 days with SYTO 62 prior to CSLM analysis. All four derivatives formed heterogeneous, dynamic biofilms with similar structures, i.e. dense towers and ball-shaped microcolonies (Fig. 3). PG4180.muc and PG4180 biofilms were thicker than those of the levan-deficient derivatives, PG4180.M6 and PG4180.M6(pBBR3-AXSalgT), with about 100 vs 63 µm maximum height. Dissolution of the biofilms by cell motility or physical sloughing proceeded regardless of whether or not levan or alginate was formed.
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Specific binding of fluorescently labelled lectins to P. syringae flow-chamber biofilms
To determine the specificity of diverse fluorescently labelled lectins for levan or alginate produced in P. syringae biofilms, we tested 64 commercially available lectins for binding to PG4180.muc biofilms. The lectins were applied to flow-chamber biofilms, which had been fixed and embedded in polyacrylamide. This procedure allowed individual staining of several samples from the same biofilm using each individual lectin. A total of 11 out of the 64 tested fluorescently labelled lectins bound to the PG4180.muc biofilm.
Furthermore, the specificity of the 11 positive lectins was screened in flow-chamber biofilms from all four PG4180 derivatives using CSLM analysis. Surprisingly, none of the 11 lectins was found to bind specifically to the levan-deficient and alginate-producing transconjugant PG4180.M6(pBBR3-AXSalgT). In contrast, all but one of the lectins showed no significant difference in dependence of the genotype of the other three tested PG4180 derivatives. Interestingly, FITC-labelled ConA did not bind to biofilms of the levan-deficient mutant PG4180.M6, nor its alginate-producing derivative, PG4180.M6(pBBR-AXSalgT), suggesting that ConA might specifically interact with levan, but not with alginate. Binding of this lectin to biofilms of P. syringae was studied in more detail (see below).
Spatial distribution of levan in P. syringae biofilms
Biofilms of levan-producing PG4180.muc and PG4180 were grown in minimal or rich medium supplemented with sucrose as described above, incubated with FITC-labelled ConA and analysed by CLSM. The fluorescently labelled ConA specifically bound to the centre of microcolonies in a condensed form (Fig. 4a, b, c). Interestingly, ConA mainly accumulated in the internal voids of mature microcolonies and in the blebs characteristic of levan production (Fig. 4a–d). However, neither all voids nor all microcolonies were stained evenly by ConA (Fig. 4a). Moreover, ConA did not significantly bind to biofilm regions consisting of a network of flat microcolonies. Control experiments with SYTO 62 alone gave no signals under the CLSM settings used for FITC-linked ConA. ConA also bound to microcolonies of non-fixed biofilms, suggesting that the fixation step had no influence on the specificity of the binding.
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). Additionally, no ConA binding could be observed for the levan-deficient mutant PG4180.M6 grown either with or without sucrose (data not shown). In summary, these findings suggested that ConA binds specifically to levan which mainly accumulates in the internal voids of mature microcolonies of P. syringae.
To investigate the dynamics of levan production in biofilms, we stained channels of PG4180.muc biofilms at different time points (3–10 d). No binding of ConA to early-stage biofilms was observed. In contrast, the lectin bound to mature biofilms after about 7 d growth. As described above, the preferred sites of binding were internal voids of microcolonies and differently sized blebs (Fig. 4a–d). Consequently, the blebs might either consist of or partially contain levan.
Peroxidase-linked ConA binds to levan in microtitre plate biofilms of P. syringae
To further investigate whether ConA binds specifically to levan, we applied an ELLA using peroxidase-linked ConA on 7-day-old biofilms of all four PG4180 derivatives grown in the presence or absence of sucrose in microtitre plates (Fig. 5). Additionally, a transconjugant of mutant PG4180.M6, PG4180.M6(pRLB7.2), complemented for levan production by the introduction of a functional lscB gene in trans, was used. This transconjugant had initially been tested positive for levan formation on agar plates containing sucrose (data not shown). ConA bound to PG4180.muc, PG4180 and PG4180.M6(pRLB7.2) biofilms grown in the presence of sucrose. A significantly lower level of binding of ConA was observed when these strains were grown in the absence of sucrose. As expected, biofilms of the levan-deficient controls, PG4180.M6 and PG4180.M6(pBBR-AXSalgT), could not be stained with ConA. Controls involving incubation of P. syringae biofilms in the presence of ABTS substrate without prior treatment with peroxidase-linked ConA revealed no interfering endogenous peroxidase activity (data not shown).
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Temporal analysis of levansucrase gene expression
To assess the function of void-associated levan accumulation in either structure formation or nutrient storage, respectively, a time study on lscB gene expression was conducted (Fig. 6). First, this experiment was done in FAB liquid cultures. Transconjugant PG4180(pRLB7.2egfp) containing a transcriptional lscB : egfp fusion expressed the highest normalized fluorescence levels in the early exponential growth phase with moderately declining levels of reporter gene expression during late exponential and stationary growth phases (Fig. 6a). The plateau of fluorescence in the developing late exponential phase is possibly due to the stability of green fluorescent protein (personal observation, data not shown). This result suggested that levansucrase is mainly produced during the onset of bacterial growth. Next, the experiment was repeated in 96-well microtitre plates under conditions identical to those used for the ELLA. Fluorescence values normalized towards bacterial growth and attachment via crystal violet staining peaked in the early phase of this experiment with slightly declining levels in later stages, thus confirming the results from the liquid culture incubations (Fig. 6b). In summary, lscB is likely to be mainly expressed in the early exponential phase, making a potential quorum-sensing-associated or stationary-phase regulation of this gene rather questionable.
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). In contrast to ConA, binding of Naja lectin was not detected in the voids of large microcolonies or blebs but was found specifically surrounding dense microcolonies of non-fixed biofilms. Additionally, fibres penetrating the dense microcolonies were stained by Naja lectin (Fig. 4j–l). Naja lectin bound to biofilms of levan-deficient PG4180 derivatives and to those of levan-positive PG4180 strains in the absence of sucrose, suggesting that this lectin was not specific to levan (Fig. 4k–l). Consequently, Naja lectin might stain a yet-to-be-characterized novel EPS or other polymeric structure(s). |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Watnick & Kolter, 1999). Herein, the impact and distribution of levan and alginate in P. syringae biofilms was investigated in either static microtitre plate assays or in continuous flow chamber experiments. Surprisingly, neither EPS had a major impact on biofilm formation, suggesting that as-yet-uncharacterized additional polymeric substance(s) mainly contribute to the formation and structure of biofilms. Fluorescently labelled lectins were applied to study the spatial localization of EPSs, demonstrating for the first time that the lectin ConA specifically binds to levan of P. syringae. ConA binding in mature biofilms suggested that levan might accumulate within cell-depleted voids in the centre of microcolonies and in blebs. We ruled out that ConA labelling was due to better accessibility of this lectin to these spaces, since wild-type biofilms grown without sucrose formed the same structures but did not stain with ConA. In support of this hypothesis, ConA only bound to biofilms of cells which produced levansucrase and were supplemented with sucrose. The results were confirmed by applying ELLA to P. syringae biofilms. Moreover, pre-treatment of biofilms of levansucrase-expressing cells grown in the presence of sucrose with levanase reduced the binding of peroxidase-linked ConA to background levels.
The association of ConA binding to levan inside the cell-free voids and blebs of microcolonies allows us to speculate that the voids and blebs may function as reservoirs of carbohydrates. Breakdown of levan chains to fructosyl residues during starvation periods may provide nutrients to the biofilm cells. Thus, levan might act as an energy storage complex for P. syringae. This hypothesis is supported by two findings from this study: (i) levan does not contribute to the overall biofilm structure, as shown by the phenotype of levan-deficient strains; and (ii) levansucrase gene expression occurs mainly in the early stages of bacterial growth and biofilm development. Since levansucrase is an extremely stable extracellular protein (Hettwer et al., 1995; Li & Ullrich, 2001) it might be synthesized during the onset of biofilm formation and then might contribute to the successive accumulation of nutrient storage macromolecules inside cell-free areas of the biofilm when sucrose is available. Burne et al. (1996) had suggested a similar function of levan produced by S. mutants in the oral cavity.
ConA did not bind to biofilms of the levan-deficient, alginate-producing transconjugant PG4180.M6(pBBR3-AXSalgT). This finding clearly ruled out P. syringae alginate being bound by ConA, and this is contradictory to results for alginate-containing P. aeruginosa biofilms in which ConA was demonstrated to bind to alginate (Strathmann et al., 2002). This discrepancy may be due to the different chemical properties of the alginate(s) produced by either pseudomonad as reflected in different proportions of mannuronic and guluronic acid monomers and different degrees of O-acetylation. Our data give no conclusive information on any particular structure-determining role for alginate in P. syringae. It has long been assumed that alginate is the primary secreted EPS in P. aeruginosa biofilms (Evans & Linker, 1973), but recent data have indicated that alginate is not a significant component of the primary structural matrix of P. aeruginosa biofilms (Friedman & Kolter, 2004a; Hentzer et al., 2001; Jackson et al., 2004; Matsukawa & Greenberg, 2004; Nivens et al., 2001; Wozniak et al., 2003). Besides structural differences between the EPSs involved in ConA binding for P. syringae and P. aeruginosa biofilms, respectively, the topological binding pattern was distinct for each organism. In contrast to mature P. syringae biofilms, ConA yielded cloud-like cell-containing regions that were heterogeneously distributed throughout P. aeruginosa biofilms (Strathmann et al., 2002). When comparing biofilms of both species, it should be noted that the experimental set-up of Wingender et al. (1999) and our study differ significantly.
When analysing the growth-stage dependency of levan production during development of P. syringae biofilms, significant ConA binding was only observed in mature 7- to 10-day-old biofilms. In contrast to our findings, time-resolved studies on EPS production in Sphingomonas sp. flow-chamber biofilms using ConA revealed a particular role of EPSs in the initial establishment of the biofilm (Kuehn et al., 2001). Similarly, we used a ConA-specific ELLA with static microtitre plates and found an earlier appearance of levan build-up under those conditions. The latter result was supported by the transcriptional analysis of lscB for which an association with quorum sensing or stationary-phase dependence remained highly unlikely.
Binding of a Naja mossambica lectin indicated the presence of at least one additional EPS in biofilms of P. syringae, regardless of the investigated genotypes. It might stabilize the biofilm structure by forming a dense web of fibres. Consequently, our future experiments will aim to identify the missing EPS(s), which might be fundamental to biofilm formation, and to further dissect the potential role of levan as a long-term nutrient storage source for P. syringae.
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ACKNOWLEDGEMENTS |
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Received 28 January 2006;
revised 21 June 2006;
accepted 27 June 2006.
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