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Applied and Environmental Microbiology, January 2005, p. 428-435, Vol. 71, No. 1
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.1.428-435.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Use of Fluorophore-Conjugated Lectins To Study Cell-Cell Interactions in Model Marine Biofilms

Barbara Wigglesworth-Cooksey and Keith E. Cooksey^*

Department of Microbiology, Montana State University, Bozeman, Montana

Received 14 April 2004/ Accepted 13 July 2004

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Biofilms dominated by pennate diatoms are important in fields as diverse as ship biofouling and marine littoral sediment stabilization. The architecture of a biofilm depends on the fact that much of its mass consists of extracellular polymers. Although most illuminated biofilms in nature are dominated by phototrophs, they also contain heterotrophic bacteria. Given the close spatial association of the two types of organisms, cell-cell interaction is likely. Fluorophore-conjugated lectins were used to demonstrate the sites of the various extracellular polymers in three species of diatoms. Based on their lectin staining properties, the polymers in different species appeared to be similar, but their involvement in the process of attachment to a surface differed. In a coculture Pseudoalteromonas sp. strain 4 or its sterilized spent medium reduced the ability of Amphora coffeaeformis and Navicula sp. strains 1 and D to adhere, inhibited motility, and caused agglutination and eventually diatom cell lysis. Diatoms could be protected from the negative effects of the bacterial spent medium if D-galactose or mannan was included in the incubation medium. The active principle of the spent medium is probably a lectin/agglutinin that is able to bind to the extracellular polymers of the diatoms that are involved in adhesion and motility. Awareness of interactions of this type is important in the study of natural biofilms.

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Marine biofilms are studied for both ecological and technological reasons. For instance, in the near-shore environment it is now well accepted that microorganisms, notably raphid diatoms, through their production of extracellular polymers, are at least partially responsible for the stabilization of the sediment (12). These polymers are secreted through pores in the silicon dioxide cell wall of the diatom and through the raphe structure, which is a longitudinal slit in the cell wall. Single raphes are found on the ventral and dorsal cell walls of some diatoms (e.g., Navicula sp.), whereas in other species (e.g., Amphora sp.) two raphes are on the ventral surface of the cell. Raphes have been shown to be a major connection between the cell membrane and the environment (39). It is conceivable that the loss of the diatom component of the sediment microflora could be responsible for the sediment erosion seen in salt marshes over the last 40 years (26) and may contribute to hypoxia in the near-shore environment (Cooksey and Wigglesworth-Cooksey, Abstr. 6th USEPA Int. Symp. Fish Physiol., Toxicol., Water Quality: Effects of Hypoxia, abstr. 15, 2001). There is currently no universally accepted view concerning which of the several exopolymers (37, 41) (Table 1) made by diatoms are responsible for the stabilization activity. However, in the case of Amphora coffeaeformis in laboratory simulations, only the matrix polymer appears to be involved in this activity (41).

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TABLE 1. Lectin staining properties of two biofilm-forming diatoms, A. coffeaeformis and Navicula sp. strain 1a

There are many reasons for this lack of consensus. In the first place, it is very difficult to compare work on extracellular polymers (extracellular polymeric substances [EPS]) extracted from field samples (3, 15, 26, 30, 36, 38, 43, 46) with work on EPS extracted from axenic cultures in the laboratory (14, 17, 33, 34, 43, 44). Nutritional status is important in regulating EPS (34), and the nutritional status of field samples is usually unknown at the spatial resolution influencing the cell. Similarly, the light regimen under which the diatom cells are grown influences radically both the synthesis and the accumulation of EPS (17, 30). Thus, the time of day at which field samples are taken is also important. It is recognized that diatoms synthesize more than one type of EPS (3, 16, 33, 37, 41, 44) and that the different types can have different roles (16, 17, 32). Many workers regard the EPS as being wholly carbohydrate. We have shown that tunicamycin, an inhibitor of glycoprotein synthesis, specifically inhibits adhesion and motility in the marine pennate epipelic diatom A. coffeaeformis (11). Both of these processes are mediated by extracellular polymers (18, 39), so it is very likely that some of the diatom EPS is in the form of glycoprotein. The results of Lind et al. (25) support this suggestion. It is not known whether all diatom EPS molecules are similar in different species and thus have similar physical properties. We have shown that the polymers of A. coffeaeformis and a Navicula sp. do not behave similarly in extraction protocols (41), but de Brouwer et al. (16) observed that the results of sugar analyses of two operationally defined extracellular polymers of Cylindrotheca and Nitzschia were similar. Although the effects of indigenous bacteria are usually purposely excluded in laboratory experiments, the effects of these organisms in natural samples are only sometimes considered (41, 43, 46). Two factors that are more general but very important are the ways in which field samples are processed and the subsequent EPS extraction method that is used. It is now recognized that lyophilization of diatom cells compromises their cell membranes (15, 17, 33, 41); thus, extracts made from such cells may contain intracellular, as well as extracellular, components of the cells. A comprehensive inventory of diatom polymers was prepared for Pinnularia viridis by Chiovitti et al. (8). These workers demonstrated that after removal of the outer layers of extracellular polymers of Pinnularia with warm water alone, the material remaining had a reduced viscoelasticity as measured by atomic force microscopy; i.e., it was more rigid. In summary, investigation of the extracellular polymers of diatom-containing biofilms by extractive methods is, in our opinion and in the opinion of Underwood et al. (37), likely to lead to information that is difficult to interpret.

The activities of marine biofilms are also studied from the point of view of biofouling (i.e., the loss of efficiency of man-made devices and structures in the aquatic environment because of the accumulation of organisms and their products at interfaces) (7). For example, a biofilm is responsible for a decrease in the performance of a ship, even when the biofilm is only a few tens of micrometers thick (4). In contrast to the work on sediments, a much smaller proportion of the work in this area has concerned diatoms. Studies of fouling bacteria dominate the literature (1, 22), but algal involvement in the fouling process has been reviewed recently (6, 12). Both reviews indicate that diatoms of the genus Amphora are able to dominate marine biofilms, but there is even less information on the algal extracellular polymers of marine fouling films than there is on biofilms involved in sediment stabilization.

Control of algal fouling has not been completely successful, even with tributyl tin coatings (6), but these highly toxic materials will be phased out entirely by 2008 and new applications of such material were not allowed after 2003 (35). It is proposed that new nontoxic, fouling release coatings should replace the toxic coatings currently in use. These new coatings depend on the effects of hydrodynamic shear over a ship's hull while it is underway to remove organisms from the low-surface-energy polymers in the coating systems. Successful assessment of candidate coating polymers requires laboratory-based assays that mimic conditions found in the field.

We report here that diatoms synthesize several extracellular polymers which can be differentiated after lectin staining and that there are significant interactions between diatoms and bacteria in mixed-species biofilms. Both of these results are significant for understanding sediment stabilization processes and also for designing meaningful bioassays for antifouling coatings.

(Preliminary reports of this work have been made previously [Cooksey and Wigglesworth-Cooksey, Abstr. 102nd Gen. Meet. Am. Soc. Microbiol., abstr. Q-257, 2002; Cooksey and Wigglesworth-Cooksey, Abstr. 103rd Gen. Meet. Am. Soc. Microbiol., abstr. Q-149, 2003].)

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Isolation of organisms. (i) Diatoms.
The isolation and growth characteristics of A. coffeaeformis have been described previously (9, 10). Navicula sp. strain 1 and Navicula sp. strain D were isolated from surface diatom blooms on sandy intertidal sediment obtained in False Bay, San Juan Island, Wash. The medium used was ASP₂ medium modified to contain 5 mM Ca²⁺ (9, 10). All cultures were axenic.

(ii) Bacteria.
Bacteria were isolated from the same sediment as the Navicula species. The sediment was washed aseptically with sterile medium to remove unattached organisms. It was then treated with ultrasound by using a low-power cleaning bath to detach adherent bacteria. Experiments in which bacteria attached to glass culture vessel walls were used confirmed the efficiency of removal of attached organisms by this method without lysis. Standard techniques with marine broth 2216 (Difco) were used to isolate the bacteria from the supernatant medium. The identity of the bacterial strain used here, Pseudoalteromonas sp. strain 4, was determined by MIDI Laboratories Inc. (Newark, Del.) by 16S rRNA methodology. This bacterium was identified only to the genus level and appears to be a new isolate. Aseptic precautions were used in all experiments

Growth of organisms. (i) Diatoms.
Diatoms were grown in modified ASP₂ medium, but when they were eventually going to be used in conjunction with bacteria or their products in short-term experiments, they were grown in ASP₂ medium containing 0.1% Casamino Acids (Difco) and 0.05% yeast extract (ASP₂-C). Long-term (i.e., stationary-phase) cultures were grown in ASP₂ medium. A daily cycle consisting of 16 h of light (100 µmol m^–2 s^–1) and 8 h of darkness and incubation at 25°C were used. Under these conditions, the generation time for both diatoms was about 24 h.

(ii) Bacteria.
Bacteria were grown in ASP₂-C at 25°C in baffled flasks shaken at 200 rpm. Under these conditions the generation time for Pseudoaltermonas sp. strain 4 was 51 min.

For epifluorescence and confocal microscopy, both bacteria and diatoms were grown in 0.8 ml of the appropriate medium together or individually in four-place culture slides (BD-Falcon, Franklin Lakes, N.J.). The walls of the slides were removable for microscopy, and the growth surface was hydrophilic glass.

Lectins.
The following lectins were investigated to determine their abilities to bind to diatom extracellular polymers: concanavalin A-fluorescein isothiocyanate (FITC) (lectin L1; specific for glucose and mannose; Sigma Chemical Co., St. Louis, Mo.), Bandeiraea simplicifolia lectin-FITC (lectin L2; specific for galactose and N-acetylgalactose; Sigma), Tetraconolobus purpurea lectin-FITC (lectin L3; specific for fucose; Sigma), Arachis hypogaea lectin-FITC (lectin L4; specific galactosyl-N-galactose; Sigma), succinyl concanavalin A-FITC (lectin L5; specific for glucose and mannose; Sigma), concanavalin A-Oregon Green (lectin L6; specific for glucose and mannose; Molecular Probes Inc., Eugene, Oreg.), and Phaseolus vulgaris lectin (lectin L7; specific for general oligosaccharides; Sigma). For use, the lectins were diluted with sterile ASP₂ medium to a concentration of 10 to 20 µg ml^–1.

Staining procedures.
The diatom cells were grown in culture slides for 2 days (logarithmic phase) or 4 days (stationary phase), after which the walls of the growth chambers were removed, which left a monolayer of cells attached to each glass growth surface. Each slide was rinsed twice with 2 ml of medium to remove unattached cells. The areas where the cells had grown were surrounded by a hydrophobic barrier which effectively isolated one area from another and thus allowed individual treatments to be performed. Fifty microliters of staining solution was applied to each set of cells. After 1 h in the dark at room temperature, the staining solution was removed by washing the slide in a stream of medium. The cells were washed three more times to remove unbound dye. The washing medium was allowed to remain in contact with the sample for 5 min between treatments. This was found to be the minimal washing procedure necessary to remove all unbound lectin and to obtain an unstained background. Glass coverslips were used for microscopy. Samples prepared in this manner were stable at 2°C for up to 24 h.

Microscopy.
Stained diatoms and bacteria were examined with a Nikon Photomic microscope by using epifluorescent illumination (excitation filter, 450 to 490 nm; emission filter, 20 nm; dichroic mirror, 510 nm). For confocal microscopy we utilized a Leica model TCS-SP microscope equipped with a x40 water-immersible lens. Optical slice images (thickness, 0.3 to 0.5 µm) were collected at 500 to 560 nm (FITC) by using excitation at a wavelength of 488 nm from an argon laser. Chlorophyll fluorescence (red) was collected at 580 to 680 nm by using excitation from a krypton laser (568 nm). Images were processed by using Adobe Photoshop and Imaris-3D software.

Diatom-bacterium interactions.
Diatoms were grown until the late logarithmic phase. When grown in tubes or flasks, they were harvested by centrifugation and resuspended at a concentration of 1.5 x 10⁶ cells ml^–1 in growth medium, test medium, or test medium containing a putative hapten. After gentle mixing, samples were removed aseptically with a pipette and examined microscopically after incubation at 25°C in the presence of 100 µmol of light m^–2 s^–1 for 15 min to 24 h. The assay volume was 0.5 ml in 96-well plates, test tubes, or culture slides. When culture slides were used, the diatom cells were not harvested prior to treatment with test materials. The growth medium was removed aseptically and replaced by an equal volume of spent bacterial medium. Image analysis of moving diatom cells was performed by using a Cell Trak system (Motion Analysis Corp., Santa Rosa, Calif.) as described in detail previously (40). Briefly, five or more microscopic fields (optical magnification, x10; electronic magnification, x50; field size, 390 by 280 µm) were examined. The Cell Trak software counted all cells and calculated the speed of movement of the cells moving at a rate of more than 0.25 µm s^–1 (38). Typically, each field contained about 100 cells, and the motility was recorded for 2 min, during which each cell moved about 20 to 25 cell lengths. When cells were treated with spent bacterial medium, many of the cells detached or aggregated, so there were fewer motile cells per field; nevertheless, it was still possible to measure the motility of the remaining cells. Data in Table 2 were obtained by using BD-Falcon slide chambers. When attached cells were rinsed before counting, the technique described above for staining procedures was used. Bacteria were also grown in ASP₂-C until the stationary phase. Cells were removed from the medium by centrifugation, and the spent medium was sterilized by filtration (pore size, 0.2 µm). Some spent medium was frozen and thawed to verify the stability, and some was concentrated by using a 5-kDa-cutoff ultrafiltration device (Millipore Ultrafree-15). All experiments were carried out in duplicate unless indicated otherwise.

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TABLE 2. Influence of Pseudoalteromonas sp. strain 4 on the short-term adhesion of A. coffeaeformis

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Table 1 shows which diatom extracellular polymers were stained by lectins conjugated to a fluorescent probe and indicates where, when traditional extraction methods were used, these fractions were found (37, 41, 45). It is important to realize that all extraction procedures are only operationally defined (37, 41). As far as we are aware, no one has purified to a single entity any extracellular polymer from a diatom and provided evidence for its physiological role. Underwood et al. (37) made the point that EPS fractions produced by current methods may contain a range of polymeric materials which may be present in various degrees. Since most methods currently used to characterize exopolymers are time-consuming and require large samples, we performed an imaging time of flight secondary ion mass spectrometry study to compare diatom exopolymers extracted in standard ways with the results of analyses of materials imaged in situ.

Some staining was seen with all samples of polymers, especially at lectin protein concentrations greater than 50 µg ml^–1, but only staining that could be inhibited by the appropriate lectin-specific sugar or its derivative is reported here (Table 1). Optimal staining and its inhibition by haptens were observed at a lectin protein concentration of 10 µg ml^–1. We found that the mannan and maltotriose competitors more completely inhibited the binding of succinyl concanavalin A to the polymers than the binding of the underivatized lectin. Thus, in most experiments reported here we used this derivative. It is evident that the Amphora polymers associated with the cell walls, motility polymer (Fig. 1a), footpads (Fig. 1b), and matrix (Fig. 1c) were stained with lectins specific for D-glucose, D-mannose, and D-galactose. The motility polymer in Amphora was found only in log-phase cells because stationary-phase cells were not motile. We found that the lectin specific for D-fucose (lectin L3) did not stain polymers of any diatom tested. In similar work, Wustman et al. (45) fractionated the extracellular polymers of A. coffeaeformis and found D-fucose in one fraction; however, fucosyl residues were not detected by fucose-specific lectins from Ulex europaeus or Lotus tetragonolobus (identical to lectin L3). We demonstrated that the ventral raphe imprint of Amphora is obvious either as two parallel lines inside the raphe in the case of a whole cell (Fig. 2) or where the cell has detached from the surface, leaving behind the footpad (Fig. 1b and c). However, while the raphe of Navicula sp. strain 1 was also in contact with or close to the substratum when it adhered, no raphe-shaped staining was seen (Fig. 1d); furthermore, the raphe itself contained no entity that were stained with the lectins which we used. This suggests that the extracellular materials secreted by these two diatoms reach the outside of the cell by different means. It is obvious from this study and that of Webster et al. (39) that in the case of Amphora, this occurs via the raphe canal; however, in Navicula sp. strain 1, it is likely that the valvar pores close to the pole of the cell are involved in the secretory process. In support of this, whole cells of Navicula sp. strain 1 washed with medium and treated with lectin L1 exhibited staining only in a terminal pore (results not shown). Note that in log-phase cultures of Amphora, no matrix was evident (Fig. 1b) and that the extracellular material stained by lectin L1 was dendritic. This was not an artifact of dehydration as the preparations were always fully hydrated. The lack of the sheet-like polymer (matrix [Fig. 1c]) in logarithmic-phase cultures supports results reported previously (41) which showed that only diatoms which were phosphate limited and formed extracellular matrices of polymeric material were able to stabilize columns of glass beads used as sediment core analogues. The general increase in extracellular polymer synthesis when diatoms are phosphate limited is well known (20, 28, 34, 37). However, Underwood et al. (37) showed that, at least in Cylindrotheca fusiformis, this increase involved synthesis of an exopolymer not found in nutrient-replete cells.

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FIG. 1. EPS of diatoms (A. coffeaeformis and Navicula sp. strain 1) stained green with concanavalin A-FITC. Chloroplasts autofluoresce red. (a) The tracks of polymer were used to determine motility. There are two parallel lines of polymer (arrows) because Amphora has two raphes on the ventral surface of the cell. Bar = 50 µm. (b) EPS from Amphora in the logarithmic phase of growth. The arrows indicate the footpads remaining when cells are detached. Note the absence of a well-developed matrix polymer and the fact that there are string-like polymers between cells. These strings are not artifacts of fixation or drying. Bar = 50 µm. (c) Stationary-phase culture of Amphora with cells embedded in a polymer matrix. The preparation was deliberately torn with a needle to show the cohesive nature of the polymer. Bar = 50 µm. (d) Cells and footpads of Navicula sp. strain 1. The arrows indicate footpads whose shape is distinct from that of the footpads shown in panel b. Navicula has one raphe each on the ventral and dorsal surfaces. Cell walls are not visible, but the red autofluorescence of the H-shaped chloroplast is visible. Bar = 15 µm. Large versions of Fig. 1 to 4 have been placed on an open-access website (http://www.montana.edu/wwmb/homepages/kcooksey.htm).

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FIG. 2. Black and white confocal microscope images of a raphe imprint or footpad polymer of Amphora stained with concanavalin A-FITC. (a) There is less polymer near the middle of the footpads than that at either end because the raphes (18 µm) do not run the length of the cell (25 µm) but are in fact divided into two portions near the middle of the cell by the central node. (b) Image in panel a turned 90° by using the confocal microscope software. The height of the polymer pads is about 7 µm. Bar = 60 µm.

Figure 2a shows a confocal microscope image of a footpad that remained attached to the substrate when the cell (Amphora) was removed as a result of shear during the slide-washing process. Structures such as this in bacterial preparations have been referred to as footprints by Neu and Lawrence (29). As hypothesized previously (11), the formation of footpads or footprints involves cohesive rupture of the adhesive polymer rather than its detachment from the substratum. However, in our previous work we observed footpads that were desiccated and provided no information on their thickness in the Z direction. In Fig. 2b the image in Fig. 2a is rotated 90° so that the height of the footpad from the surface is shown. We estimated that this height was about 5 to 7 µm. This indicated that the frustule of Amphora is not very closely associated with the substratum, as was generally assumed based on previous transmission electron micrographs in which dehydration artifacts were almost certainly present (39). It is likely, therefore, that the almost universal presence of Amphora in marine fouling films undergoing shear forces (5) and the film's resistance to removal by a shear force are more a result of the properties of the adhesive polymers than a result of the geometry of the adherent cell.

Our results obtained with A. coffeaeformis and lectin L1 indicate that there is a more universal interaction of this lectin with the extracellular polymers than that reported by Wustman et al. (45). Wustman et al. showed that only the tightly attached polymeric organic sheath reacts with concanavalin A. An explanation for this may be that the operational definitions of the extracellular polymeric fractions are different in different laboratories or that the staining differences were due to dissimilar culture conditions. It is also important to realize that diatom taxonomy and thus the names of species depend entirely on the physical shape of the cell wall. It is known that physiologically different organisms may have the same name (24), so that even comparing organisms with the same name can be misleading. In any event, results such as these make interspecies or interlaboratory comparisons difficult.

The manner in which Navicula sp. strain D grows on a surface is distinct from the pattern shown by the other diatoms mentioned here. Whereas the other diatoms grow as a uniform biofilm that usually is only one cell deep, Navicula sp. strain D grows in what appears to be small colonies that are up to 100 µm high surrounded by a film of cells. Examination of optical slices through these colonies by confocal laser scanning microscopy showed no chlorophyll a autofluorescence in the interior, indicating the absence of cells (Fig. 3). A comparison of a series of horizontal and vertical optical slices demonstrated that below the veneer of diatom cells there were only exopolymers. The fact that this material did not stain uniformly throughout the colony was likely due to a lack of penetration of the lectin.

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FIG. 3. Confocal micrograph of a colony of Navicula sp. strain D stained with concanavalin A-FITC. (a) Colony seen from above. The red areas are the chloroplasts in cells, and the green-yellow areas are the polymer matrix. (b) Image identical to panel a but with the autofluorescence of the chloroplast removed by using the image analysis software of the microscope (Imaris), leaving only the matrix EPS visible. (c) Side view of the same colony (height, 90 µm). Bar = 50 µm.

In general, the lectin binding properties of the three sets of diatom polymers were rather similar, but they were not identical. For instance, the matrix polymer of Amphora was stained by lectin L2 (galactose specific), but the matrix polymer of Navicula sp. strain 1 was not stained by this lectin. Similarly, the polymers associated with the frustule (cell wall) of Amphora were stained by several lectins, but those of Navicula sp. strain 1 were not stained. The footpad polymers of Amphora and Navicula could not be distinguished by their abilities to bind the lectins studied. These polymers are likely to be closely related to the polymers involved in cellular motility (41), and thus we concluded that, at least in these two diatoms, they are similar. Using an immunological approach, Lind et al. (25) showed that these types of polymers were also similar in Stauroneis decipiens.

Using columns of glass beads as sediment analogues, we showed previously that diatoms are able to stabilize the columns against mechanical disturbance (41). We considered it likely that a defined mixed culture of diatoms and bacteria may act synergistically in the stabilization process. We did not find that this was so; in fact, the opposite effect was seen (i.e., the bacteria and diatoms were less effective in these stabilizing sediments than the diatoms alone were). To investigate this phenomenon further, we grew Pseudoalteromonas sp. strain 4 in a mixed culture with diatoms. We found that in these circumstances, the matrix component of the extracellular polymers was not detectable with a fluorescently conjugated lectin. At the moment we cannot say whether the matrix was not synthesized or was consumed in some way by the bacteria (19). To discover whether the process required the presence of bacterial cells or merely a bacterial product, we also prepared spent medium from the bacterial cultures and tested its effect on diatom cells. Figure 4 shows that the spent medium had an immediate effect on Navicula sp. strain 1. Whereas control cells in ASP₂-C showed dispersal behavior, as explained in detail previously (42), cells in the presence of medium in which Pseudoalteromonas had been grown agglutinated. Similar information for Amphora and Navicula is shown in Tables 2 and 3. In these experiments, the number of cells that were enumerated for motility or adhesion in the treatments was less than the number in the controls, as most cells had either detached from the substratum, had aggregated, or had lysed. Table 2 shows that there was no statistical difference (t test, 95% confidence) between controls 1 and 2 or between controls 3 and 4; however, is it evident that the presence of bacteria in the system reduced the attached diatom component considerably. In some experiments, no attached diatoms could be detected. The results in Table 3 obtained by using Navicula sp. strain 1 showed that there is no need for the presence of the bacteria, since some entity produced by them and secreted into the medium had the same effect as the bacteria alone. Navicula sp. strain D biofilms were affected like Amphora and Navicula sp. strain biofilms. The mounds of EPS mentioned above (Fig. 3) became undetectable by fluorophore-conjugated lectins, and the cells detached from the substratum. The areas that had previously been the sites of EPS mounds were devoid of diatoms and were covered by a bacterial biofilm. The order of events in the action of the spent medium component(s) on the diatom cells is as follows: diatom cells move more slowly and detach, all motility stops, the cells become agglutinated (in minutes to hours), and finally the cells begin to lyse (6 to 24 h). It is possible that there is more than one active component in the spent medium. Similar results were obtained with Bacillus simplex, which was isolated from the same sediment sample as Pseudoalteromonas sp. strain 4 (Cooksey and Wigglesworth-Cooksey, Abstr. 103rd Gen. Meet. Am. Soc. Microbiol.)

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FIG. 4. Experiment carried out with a 96-well plate which was sampled for microscopy. (a) Dispersing cells of Navicula sp. strain 1 with directed movement from a clump (42). (b) Agglutinated cells as a result of resuspension in spent medium from Pseudoalteromonas sp. strain 4. No motile cells were present in this preparation. Bar = 40 µm.

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TABLE 3. Bacterial cells are not necessary to influence motility in Navicula sp. strain 1

The experiments described here can be explained if a binding event takes place and the active component(s) in the spent medium binds initially to one of the extracellular polymers. To investigate this, we measured the effect of the haptens which were successful in preventing fluorophore-conjugated lectin binding (i.e., D-galactose and mannan) (Table 4). Glucose was not used as it is also a heterotrophic growth substrate for both Amphora and Navicula sp. strain 1. Both galactose- and mannan-containing spent medium protected the diatoms from the lethal effects of the spent medium (i.e., lysis). However, the effects of these compounds were distinct. Whereas galactose prevented formation of aggregates and allowed full motility of the culture, mannan initially allowed motility but some aggregation took place. After 24 h, cells in the presence of galactose and spent medium were indistinguishable from controls, but with mannan all cells had aggregated. It is well accepted that some lectins bind to surface receptors, which in turn are connected to the actin microfilament network of the cell (2). Actin microfilaments have been implicated in diatom motility and thus adhesion (18, 39). If, as it appears, the putative lectins from Pseudoalteromonas sp. strain 4 bind to diatom surface receptors, inhibition of motility and adhesion should take place, but specific sugars should have a protective action. This idea also complements a hypothesis for surface sensing by diatoms that use their secreted polymers as surface probes (40). Lind et al. (25) showed that antibodies to S. decipiens cell surface proteoglycans inhibited motility.

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TABLE 4. D-Galactose and mannan protect Navicula sp. strain 1 from the effects of Pseudoalteromonas sp. strain 4 spent medium

Other workers have found similar effects of pseudomonad or pseudoalteromonad cultures on algae. For instance, Sharabi and Gilboa-Garber (31) described the agglutination effects of Pseudomonas aeruginosa PA-I and PA-II lectins on a variety of cells, including planktonic freshwater algae. The PA-I and -II lectins bind, among other sugars, D-galactose and mannan, respectively. Thus, there appears to be a similarity between our results and those of Sharabi and Gilboa-Garber. On the other hand, our results and those of Dakhama et al. (13) are not comparable. The active materials which we investigated had molecular masses of at least 5 kDa since they were found in the retentate after ultrafiltration experiments. The materials investigated by Dakhama et al. had much lower molecular masses. Several groups (21, 23, 27) have also found antialgal compounds, including antidiatom compounds, in pseudoalteromonad species. The active substances were proteases with algicidal activity. Since D-galactose and mannan protected the diatoms studied here against the active materials in spent medium, it is not likely that a protease is involved. As we were unable to demonstrate the presence of the diatom matrix polymers after spent medium treatment, it is possible that a matrix lytic agent was present. An alternative hypothesis to EPS removal by bacterial enzymes is that binding of a bacterial lectin to the diatom extracellular polymers prevents the attachment of the analytical fluorophore-conjugated lectin to the EPS. The net result should be that the diatom polymer does not stain, but it still is present. However, this explanation does not take into account the fact that whatever happens to the EPS, the diatom cell physiology is compromised.

In summary, this work supports a potential role for indigenous bacteria in biofilms that are dominated by diatoms. In view of the importance of such biofilms in sediment stabilization and the fouling of man-made structures, we believe that this phenomenon warrants further investigation.

 
This work was supported in part by the U.S. Office of Naval Research (grant NOOO14-00-1-0754).

We thank Eric Donaldson for alignment of the 16S rRNA sequence of Pseudoalteromonas sp. strain 4 and Deborah Berglund for help with the confocal microscopy.

 
* Corresponding author. Mailing address: Department of Microbiology, Montana State University, Bozeman, MT 59717. Phone: (406) 994-6136. Fax: (406) 994-4926. E-mail: umbkc{at}montana.edu.

Top Abstract Introduction Materials and Methods Results and Discussion References

 

1
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Applied and Environmental Microbiology, January 2005, p. 428-435, Vol. 71, No. 1
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.1.428-435.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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