INTRODUCTION

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Lectins are a group of diverse proteins which bind to specific configurations of sugar residues. The binding of lectins to sugar residues present in polysaccharides resembles the specific binding of antibodies to antigens (4). Lectins have been widely used to characterize surfaces of eucaryotic cells and polysaccharides. Recently, fluorescently labeled lectins have been applied in the study of biofilm formation and biofilm composition. Excretion of adhesive polymers during attachment of bacterial cells to surfaces has been described using a panel of fluorescent lectins (9, 14, 20). The formation of biofilms on living and nonliving surfaces has been investigated with lectins (17). Lectins in conjunction with confocal laser scanning microscopy (CLSM) have been valuable tools in the study of the three-dimensional structure of biofilms (12, 15) or of the composition of extracellular polymeric substances (EPS) involved in accumulation of chlorinated organic compounds (24). Strains of Sphingomonas spp. are known for their interesting catabolic capabilities to degrade a wide variety of environmentally hazardous compounds, including polycyclic aromatics (25), dioxine compounds (6), and chlorinated phenols (3). Theoretically, lectins may be used to study the interaction between Sphingomonas cells and environmental surfaces during biofilm formation or to investigate the interaction of EPS with organic compounds. The common approach has been to deduce the structure or composition of biofilm EPS on the basis of the specific binding of lectins to different sugar residues. In this study, we evaluate the use of lectins for the characterization of Sphingomonas biofilms by investigating the binding of five fluorescent lectins with known specificities to Sphingomonas biofilms and to industrially produced Sphingomonas exopolysaccharides (sphingans) with known molecular structures.

	MATERIALS AND METHODS

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Bacterial strains and growth conditions. Sphingomonas paucimobilis EPA505 was obtained from J. Mueller (19), and Sphingomonas sp. strain LH128 and Sphingomonas sp. strain LB126 were received from L. Bastiaens (1). All strains were stored in 43% glycerol at 80°C. The bacteria were grown at room temperature in phosphate minimal medium supplemented with glucose as the sole carbon source (PMMG) containing (in grams/liter) the following: glucose, 2; Na₂HPO₄ · 2H₂O, 0.875; KH₂PO₄, 0.1; (NH₄)₂SO₄, 0.25; MgCl₂ · 6H₂O, 0.05; CaCl₂ · 2H₂O, 0.015; NaNO₃, 0.018. The medium was amended with 5 ml of a trace element solution consisting of (in milligrams/liter) the following: Na-EDTA, 800; FeCl₂, 300; MnCl₂ · 4H₂O, 10; CoCl₂ · 6H₂O, 4; CuSO₄, 1; Na₂MoO₄ · 2H₂O, 3; ZnCl₂, 2; LiCl, 0.5; SnCl₂ · 2H₂O, 0.5; H₃BO₃, 1; KBr, 2; KI, 2; BaCl₂, 0.5. Phosphate and glucose were autoclaved separately.

Cultivation of biofilms on microscope slides. Single-species biofilms were grown on Cel-Line HTC printed microscope slides with six wells on each slide (Cel-Line Associates, Inc., Newfield, N.J.). The slides were sterilized overnight in an oven at 200°C and transferred to sterile petri dishes. One drop of a bacterial culture grown overnight in PMMG was added to each well, and the cells were allowed to attach to the surface for 1 h. PMMG was gently poured into the petri dishes to cover the slides with medium. The petri dishes with the slides were incubated on a slowly tilting table (Heidolph Duomax, Kelheim, Germany) for 3 days (LB126 and LH128) or 5 days (EPA505).

Binding of lectins to Sphingomonas biofilms. Each strain was tested for binding of the five fluorescently labeled lectins: Con A-TRITC type IV (Canavalia ensiformis), Pha-E-TRITC (Phaseolus vulgaris), PNA-TRITC (Arachis hypogaea), UEA 1-TRITC (Ulex europaeus), all obtained from Sigma (Deisenhofen, Germany), and WGA-Texas red (Triticum vulgaris) obtained from Molecular Probes (Eugene, Oreg.). The affinity, the molecular weight, and the fluorophore content of the lectins are listed in Table 1. The biofilms were checked for contamination by streaking 10 µl of the overlying growth medium on Luria-Bertani plates. Slides with biofilms were washed in phosphate-buffered saline (PBS) (pH 6.8) consisting of (in grams/liter) the following: Na₂HPO₄, 0.2; NaH₂PO₄, 1.44; NaCl, 8.0; KCl, 0.2; CaCl₂, 0.011; MnCl₂, 0.013. The areas around the wells were blotted dry. Lectin stock solutions of 1.0 or 2.5 mg/ml were prepared by dissolving lectins in PBS containing 0.05% NaN₃ to prevent microbial growth. Working solutions of 0.1 mg of lectin per ml were made by dilution of lectin stocks in PBS containing the nucleic acid stain PicoGreen (Molecular Probes) in a 1:500 dilution. Working solutions were centrifuged at 8,000 × g for 5 min before use. The supernatant (10 µl) was added to each well, and the slides were incubated for 15 min in the dark on a piece of wet paper in a petri dish. Biofilms incubated with PBS containing PicoGreen were used as controls. After incubation, each well was washed with 1 ml of PBS, the slides were immersed for 30 min in PBS in the dark, and slides were examined by epifluorescence microscopy.

Preincubation of lectins with target sugars. Binding sites of ConA were blocked with the target sugar D-glucose (4, 22), and the binding sites of WGA were blocked with the target sugar N-acetyl-D-glucosamine (23). Lectin stock solutions were diluted with sugar stock solutions to final concentrations of 1.0 or 0.1 mg/ml for lectins and 10 or 50 mg/ml for sugars (Table 2). The mixtures were incubated in the dark for 1 h, and the affinities of the lectins for EPA505 biofilms were assayed as described above, using the lectin-sugar solutions as working solutions. PicoGreen was included in the working solutions in a 1:500 dilution as a counterstain. Wells with no sugars, no lectins, or no sugars and no lectins were used as controls.

Binding of ConA and WGA to commercially available sphingans. Three of the Sphingomonas exopolysaccharides (sphingans), gellan, welan, and rhamsan, were produced by Kelco, Inc. (San Diego, Calif.). Clarified gellan was purchased from Sigma under the trade name Phytagel. Industrial grade welan and rhamsan were a gift from Mikael Grathwohl (Ringsted og Semler A/S, Copenhagen, Denmark). The sphingans (250 µg per ml) were hydrated in Tris buffer (20 µM Tris-HCl, 10 µM CaCl₂, pH 6.8) for 1 h and dissolved by autoclaving at 121°C for 5 min. The resulting solutions were centrifuged at 15,000 × g for 10 min. Printed microscope slides were coated with the sphingans by adding 50 µl of sphingan solution to each well and evaporating the water at 110°C for 2 h. The slides were washed in distilled water and stained with ConA and WGA as described above, omitting PicoGreen from the staining solutions.

Semiquantitative estimation of the binding of fluorescent lectins to biofilms or to commercially available polysaccharides. The degree of binding of lectins to biofilms was estimated simply by looking at the biofilms with the microscope in the fluorescence mode. The fluorescence intensities were divided into groups showing strong fluorescence (+++), medium fluorescence (++), low fluorescence (+), and no fluorescence (). In each experiment, a positive control slide (ConA, strong fluorescence) and a negative control slide (biofilm without lectin, no fluorescence) were compared to the biofilms under investigation. In the experiments with gellan, welan, and rhamsan, all three polysaccharides were compared to each other at the same time by switching back and forth between the slides. Biofilms were grown and screened at least twice. For replicates with divergent ratings, the experiments were done in triplicate.

Cultivation of biofilms in flow cells. The bacteria were cultivated as continuous cultures in flow cells, which could be fitted directly on a microscope table for noninvasive analysis of the biofilms. Four parallel flow cells, each 4 by 7 by 40 mm, were drilled in stainless steel and fitted with short inlet and outlet tubes also made of stainless steel. The flow cells were assembled by applying silicone rubber around each channel and adding glass coverslips on top of the silicone. Silicone tubing was connected to the inlets and outlets, and the flow system was autoclaved. Medium was pumped through the sterile flow system with a multichannel peristaltic pump at a rate of 5 ml/h. The flow cells were inoculated by stopping the flow, clamping off the inlet tubes, and injecting 0.3 ml of overnight cultures into the flow cells through the inlet tubes by using 1-ml syringes with thin hypodermic needles. The flow of the medium was restarted after 1 h, and the flow cells were incubated for 3 days at room temperature.

Lectin staining of biofilms in flow cells. ConA (Sigma) was tagged with the fluorophore Cy5 by using an Amersham Life Science FluoroLink Cy5 AB-Labelling Kit (Amersham Pharmacia Biotech UK, Buckinghamshire, England) as described by the manufacturer. The flow cells with the biofilms were washed with PBS for 30 min at a flow rate of 15 ml per h, and the liquid content of each flow cell was displaced by injection of 1.3 ml of lectin working solution through the inlet tubes. After 30 min of incubation, the flow cells were washed for 30 min at a flow rate of 15 ml per h. Stacks of digital images based on lectin and PicoGreen fluorescence were collected from selected areas of the biofilms with a CLSM 410 confocal laser scanning microscope coupled to an Axiovert 135M inverted microscope (both instruments from C. Zeiss, Jena, Germany). Images were generated using a 40×, 1.3 numerical aperture oil immersion lens, a pinhole value of 20 or 22, and a digital zoom factor of 2.5 or 5.0. WGA-Texas red images were collected using the 543-nm laser line and an LP590 emission filter. ConA-Cy5 was excited with the 633-nm laser line and collected using a LP 665 emission filter. PicoGreen images were obtained using the 488-nm laser line and a BP515-540 emission filter. The image dynamics were improved digitally with the computer program Adobe Photoshop 3.0.

	RESULTS AND DISCUSSION

Top Abstract Introduction Materials and Methods Results and Discussion References

Binding of lectins to Sphingomonas biofilms and to commercially available sphingans. Biofilms of the three Sphingomonas strains were analyzed for binding of the five lectins shown in Table 1. The reproducibility of results was generally high. The binding of WGA to strain LH128 (Fig. 1) and of ConA to strain EPA505 (Fig. 2) demonstrate that fluorescence from WGA and ConA is closely associated with the cells and that the strongest fluorescence emanated from groups of cells and microcolonies. Control experiments with PicoGreen alone gave no signals with the CLSM settings used for WGA-Texas red and ConA-Cy5.

View larger version (83K): [in this window] [in a new window]   FIG. 1.   (A) CLSM transmission image of a microcolony of Sphingomonas sp. strain LH128 grown as a biofilm in a flow cell. (B) CLSM-extended depth of focus image of the same microscopic field showing fluorescence from the Texas red-labeled WGA lectin. The image was constructed from 15 xy sections with a vertical distance of 2 µm between successive images. Scale bar, 10 µm.

View larger version (56K): [in this window] [in a new window]   FIG. 2.   (A) CLSM-extended depth of focus image of two microcolonies of S. paucimobilis strain EPA505 stained with the nucleic acid stain PicoGreen. (B) CLSM-extended depth of focus image of the same microscopic field showing ConA-TRITC fluorescence. Both images are projections constructed from 20 xy sections with a vertical distance of 2 µm between successive images. Scale bar, 20 µm.

Preincubation of lectins with target sugars. The lectins ConA and WGA were preincubated with target sugars to block the binding sites as a confirmation of the specificity of the lectin binding to the EPA505 biofilms (Table 2). There was no reduction in the binding of ConA and WGA when using 1 mg of lectin/ml and 50 mg of target sugars/ml compared to that of the controls without target sugars. This indicates that the target sugar-to-lectin ratio was not sufficiently high to block the binding sites of the lectins. Reducing the lectin concentration to 0.1 mg/ml yielded a reduction in the lectin signal intensity, compared to that of bound lectin at 1.0 mg/ml. Adding target sugars at concentrations of 10 and 50 mg/ml to the working solutions with 0.1 mg/ml lectin decreased the binding of the lectin, but surprisingly there was still some binding of the lectins even at a target sugar concentration of 50 mg/ml. This suggests that some lectin was bound nonspecifically to the biofilms.