Bacterial biofilms and biofouling

Bacterial biofilms and biofouling i

Madilyn Fletcher University of Maryland Biotechnology Institute, Baltimore, USA Advances in the development and application of investigative tools, such as scanning confocal laser microscopy and genetic techniques, are leading to new insights concerning the composition, structure, and function of bacterial biofilms. Molecular biology has also demonstrated that bacteria are able to 'sense' surface environments, altering their pattern of gene expression. Recent studies on mechanisms of bacterial attachment provide further evidence for the diversity of attachment mechanisms, as do investigations of attachment interactions between bacteria and invertebrates. Current Opinion in Biotechnology 1994, 5:302-306

Introduction Solid surfaces in aqueous environments are invariably colonized by resident microorganisms and macroorganisms, which are e m b e d d e d in a polymeric matrix. Frequently, biofilms have damaging effects (e.g. corrosion) or result in loss of performance of the object to which they are attached (e.g. heat exchangers). In these cases, the colonization process is termed biofouling. Biofilm formation may, however, be desirable in some situations, such as trickling filters. Bacteria are generally the first colonizers of surfaces, and it is likely that they have a significant influence on the attachment of subsequent colonizers and the ultimate nature of the biofilm. Consequently, most studies on biofilm formation have concentrated o n the adhesion of bacteria and their role in the development of biofilm communities. Accordingly, this review focuses on recent advances in our understanding of bacterial biofilms.

Advances in techniques for the characterization of biofilms Much of our understanding of biofilm structure has been based on microscopic observation and on destructive taxonomic and biochemical analysis. Light microscopy is invaluable and can provide information on the extent of surface coverage, distribution of organisms, and physiological activity [1,2]. Its use is limited, however, w h e n studying maturing biofilms, where the three-dimensional nature of the community obscures observation of the constituent organisms. Electron microscopy has provided detailed information on the distribution of cells and their ultrastructure, but has the disadvantages that artifacts may be introduced during

specimen preparation and that living material cannot be observed in real time. The taxonomic and chemical compositions of biofilms can be analyzed, usually after removal of the biofilm from the surface. Even so, these are destructive techniques, which provide little information o n the structure of the biofilm, the distribution of organisms within it, or the identity of organisms that cannot be cultured. Only recently, has major progress b e e n made in the development of techniques that allow determination of the identity and distribution of non-culturable organisms in a biofilm, the architecture of communities within a biofilm, and the structure of the polymers that form the intercellular matrix. Major recent advances are the application of scanning confocal laser microscopy, molecular biology approaches, and microsensors.

Scanning confocal laser microscopy In scanning confocal laser microscopy (SCLM), the specimen is scanned in one plane by a laser beam, and the image is processed and analyzed by computer. Since the laser beam images only one point in the specimen at a time, stray light from above or below the focal plane, or even adjacent areas within the focal plane, is eliminated, resulting in a crisp image. Vertical and horizontal optical sections of the biofilm can be examined and recorded, and three-dimensional reconstructions can be made. Caldwell and co-workers [3,4,5°°,6 °°] have pioneered the application of SCLM to the study of biofilms, and, as access to this relatively expensive instrumentation increases, more workers are following this lead. The most significant finding of these studies is that, for the first time, it has been explicitly demonstrated that biofilms are structurally complex, with areas of both high and low cell density, uneven distributions

Abbreviations FITC---fluorescein isothiocyanate;MBC--minimum bactericidal concentration; MIC---minimum inhibitoryconcentration; SCLM---scanningconfocal lasermicroscopy; SRBs~sulfate-reducingbacteria. 302

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Bacterial biofilms and biofouling Fletcher 303 of extracellular polymer, and channels that penetrate the biofilm allowing movement of water deep into the biofilm matrix. Moreover, by the addition of fluorochromes (which are excluded from the cells but diffuse through the polymer matrix), Caldwell and co-workers [3,4] have demonstrated that even thick biofilms (>20 ~ ) can be penetrated by fluorophores of both low molecular weight, such as fluorescein (Mr 289), and higher molecular weight, such as fluorescein isothiocyanate (FITC)-conjugated dextrans (Mr 4400) [3,4]. SCLM studies have also demonstrated that the density of bacteria and their arrangement in a biofilm can be markedly influenced by bacterial behavior, such as motility [5"']. Biofilms composed of a motile Pseudomonas fluorescens strain (mot +) developed characteristic structural features, including channels, pores, and palisade layers, which did not occur in biofilms produced by a non-motile mutant (mot-) of the same strain. Apparently, motility is important for positioning of the bacteria in the biofilm. Films composed of mot + P. fluorescens were also thicker than those of the motstrain. In the thicker films, this ~esulted in the generation of chemical gradients, which did not develop in the thinner mot- biofilms [5"']. The SCLM method has also been used to study the influence of the type of primary substrate utilized by bacteria on biofilm structure. Using a consortium obtained from soil, the ultrastructure of biofilms developed in presence of a herbicide (diclofop) as the sole carbon and energy source was compared with the ultrastructure of biofilms developed in tryptic soy broth medium [6"']. Those grown on the herbicide developed heterogeneous structures, which included both conical bacterial consortia rising u p to 30 ~ above the biofilm and grape-like clusters of cocci within a matrix of perpendicularly oriented bacilli. The authors speculated that these unusual arrangements of organisms indicated the formation of consortia, which allowed utilization of the diclofop. Clearly, SCLM is an invaluable tool for gaining insights into the factors that determine biofilm development and structure.

Molecular biology approaches One of the greatest difficulties in determining the composition of biofilms is that most bacteria in natural environments cannot be cultured in the laboratory. Even so, considerable strides are now being made in the development of molecular methods that enable direct characterization o f organisms without their isolation and culture. Several recent studies illustrate the variety of molecular

approaches that can be used to assess the diversity and composition of biofilm communities. For example, hybridization with either DNA or 16S/23S rRNAdirected probes has been used to characterize bacteria in biofilms from drinking water [7], oil field production waters [8], and trickling filters [9"]. The use of hybridization probes allows characterization of in-

dividuals within a community, and if biofilm structure is preserved, distribution of taxonomic types can also be determined [9"]. Other approaches address overall community composition and diversity, such as those applied to a microbial mat of a hydrothermal vent system [10], microbial mats from sea sediments [11] and waste-water treatment reactors [11]. To study a hydrothermal vent biofilm, Moyer et aL [10] carried out a restriction fragment length polymorphism analysis of clones derived from a library of (eu)bacterial 16S rRNA genes, following tandem tetrameric site-specific restriction endonuclease treatment. They found twelve taxonomic units, and two of these comprised 73% of all of the 16S rRNA clones. Muyzer et al. [11] investigated microbial mats by denaturing gradient gel electrophoresis analysis of polymerase chain reaction amplified genes encoding 16S rRNA. A notable observation from their study w a s that sulfate-reducing bacteria (SRBs) were present in biofilms, even under aerobic conditions [11]. Some molecular techniques have targeted the physiological activity of the biofilm bacteria [12], including physiological changes that may have been induced by attachment to a surface. Two recent reports provide evidence that attachment to a surface can modulate the expression of genes. In the first study, Davies et al. [13"'] engineered a mucoid strain of Pseudomonas aeruginosa that contained an alginate algC promoter fused to a lacZreporter gene. Expression of algCcould then b e monitored by measurement of ~-galactosidase activity through use of chromogenic and fluorogenic substrates. They found that expression of algC in attached bacteria was increased compared with freeswimming cells. In the second report, Vandevivere and Kirchman [14"'] added sand to shake-flask cultures of six strains obtained from a subsurface environment to ascertain whether attachment affected exopolysaccharide synthesis. The results they obtained with one mucoid strain indicated that exopolysaccharide synthesis was increased in the presence of solid surfaces. Moreover, up-regulation was reversible, in that polysaccharide production decreased again w h e n the bacteria detached from the sand surfaces. Only in recent years has it become clear that attachment to surfaces can regulate expression of certain genes, but the proximate stimulus (or stimuli) for gene expression is still not known. It may be an environmental condition within the surface microenvironment, such as nutrient concentration, nutrient quality, electrolyte concentrations, or pH. Alternatively, the attached bacterium may be influenced directly if attraction forces, which are the basis of adhesion, influence cell wall components or receptors in some way that is yet to b e understood. If exopolysaccharide synthesis is stimulated by association with surfaces, what then was the consequent advantage and the selection pressure that led to its evolution? One suggestion is that nutrients are more abundant or accessible at surfaces than in the adjacent liquid phase, and that exopolymer production strength-

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Environmentalbiotechnology ens adhesion so that an attached bacterium becomes established in a more nutrient-rich environment. Alternatively, the biofilm environment, in which high densities of bacteria are retained within a potentially protective polymer matrix, may be more conducive to species survival than existence as an individual cell, thus resulting in the evolution of polymers that promote adhesion and lead to biofilm development [15-17]. Another explanation could be that evolution of these polymers occurred in soil environments, where desiccation was an environmental hazard, and exopolymers helped to protect the bacteria from desiccation [18,19]. A secondary consequence would be that these same polymers promoted attachment.

pepsin. The last two proteins also increased binding to polystyrene. These observations are somewhat unusual, as previous data indicated that surface adsorbed hydrated polymers (e.g. proteins) tend to inhibit attachment [23]. The fact that Azospirillum attaches preferentially to biological surfaces may reflect its evolutionary history, in that it is adapted to living in association with plant roots. In contrast, the aquatic bacteria that have been the subject of many previous studies [24], and which tend not to adhere rapidly to hydrated surfaces, have evolved to attach to non-biological surfaces with a wide range o f surface chemistries. In these circumstances, exclusion from water (i.e. hydrophobic interactions) may be the driving force behind initial attachment to available surfaces.

Microelectrodes Another type of technology that continues to provide information on biofilm structure and activity is that of microsensors. The precision of current microelectrodes is allowing spatial resolution of concentration profiles in biofilms of the order of 25-100 pan. Measured parameters include oxygen, sulfide, and pH [20]. Information on nutrient and environmental gradients obtained from microsensors (e.g. specific reaction rates) can be used to evaluate physiological processes. By combining biosensor measurements with subsequent analysis of community members, it becomes possible to relate the chemical microenvironment to the presence of specific taxonomic and physiological types of organisms. For example, a negative correlation between the vertical distribution of presumptive SRBs and measured oxygen was found in biofilms from a trickling filter of a sewage-treatment plant [9]. The oxygen, hydrogen sulfide, and pH gradients in the biofilms were first analyzed using microelectrodes. This was followed by freezing of biofilm samples with liquid nitrogen, sectioning, and probing the sections with fluorescently labeled phylogenetic 16S rRNA probes. The probes were specific for eukaryotes, eubacteria, SRBs, and Desulfobacter spp. (as well as a non-hybridizing controD.

Biofilm development Throughout the past twenty to thirty years, biofilm research has included studies on the attachment mechanisms of bacteria. It has been extremely difficult to identify general principles that describe bacterial adhesion because of the diversity of attachment mechanisms and the ability of microorganisms to change phenotypically with environmental conditions. Another complicating factor is that some, perhaps most, organisms are capable of more than one mechanism of attachment [21]. Such is the case with the attachment of Azospirillum brasilense to polystyrene and wheat roots [22°]. This organism attached more readily to root surfaces, and attachment was increased further with the addition of albumin, gelatin, fibrinogen, and

Resistance of bacteria in biofilms to biocides and antibiotics Bacterial biofouling is a serious problem not only because of its harmful effects, but also because biofilms are so difficult to remove. The treatments available for eradication of suspended bacteria, such as biocides and antibiotics, are much less effective against biofilms [25]. Almost always, some bacteria in biofilms survive, so that they can replenish the biofilm and disperse into the liquid phase after biocide or antibiotic levels have reduced. This enhanced resistance of biofilm bacteria leads to two primary questions: first, what is the mechanistic basis for the increased resistance of biofilm bacteria to chemical treatments; and second, what approaches can be taken to enhance the action of biocides and antibiotics on biofilm bacteria? Two reasons are most commonly cited to explain the increased resistance of biofilm bacteria. First, penetration of a chemical into the biofilm is reduced because of the diffusion barrier presented by the intercellular polymer matrix. Second, biofilm bacteria are less susceptible than suspended bacteria because they are in a less active physiological state. It is becoming increasingly clear that the polymer matrix between cells is not the diffusion barrier it was once considered to be. Recently, Dhnne et al. [26°] have used an equilibrium dialysis chamber to measure diffusion of vancomycin and rifampin (both singly and in combination) through a slime-producing Stapbyloccus epidermidis biofilm on the dialysis membrane. Although differences were detected in the diffusion kinetics of the antibiotics, in all cases, they eventually exceeded MBC and MIC concentrations on both sides of the biofilm-colonized membrane. Even so, viable bacteria were still present after 72 hours treatment. Using SCLM, Caldwell et al. [3] have provided further evidence for the ability of compounds to diffuse through biofilms, demonstrating that fluorophores, including FITC-conjugated dextrans (Mr 4400), could pass into biofilms of substantial thickness (>20 Ban) [3]. Thus, the basis for enhanced resistance is probably related to either the physiology of the

Bacterial biofilmsand biofouling Fletcher 305 organisms, or possibly the permeability characteristics of their cell envelopes [27,28].

2.

Schaule G, Flemming H-C, Ridgway HF: Use of 5-Cyano2,3-Ditolyl Tetrazolium Chloride for Quantifying Planktonic and Sessile Respiring Bacteria in Drinking Water. Appl Env Mtcrobiol 1993, 59:3850-3857.

3.

Caldwell DE, Korber DR, Lawrence JR: Imaging of Bacterial Cells by Fluorescence Exclusion using Scanning Confocal Laser Microscopy. J Microbtol Methods 1992, 15:249-261.

4.

Caldwell DE, Korber DR, Lawrence JR: Analysis of Biofilm Formation using 2D vs 3D Digital Imaging. J Appl Bactertol 1993, 74 (symp suppl):52S--66S.

Biofouling in aquatic environments Although an understanding of biofouling, and the ways in which it can be prevented, has long been the incentive for biofilm studies, progress in the development of new non-toxic strategies for prevention of biofouling has been disappointingly slow. Because macroorganisms, such as invertebrates, are often the most prominent constituents of biofilms, some researchers have attempted to find bacteria that could be used to produce biofilms that would prevent subsequent attachment of fouling invertebrates. For example, numerous bacterial strains have been found to inhibit subsequent settlement of barnacle (Balanus amphitrite) larvae [29,30]. In contrast, other earlier studies demonstrated that setdement of polychaetes [31] or oysters [32] is stimulated by certain bacteria. Furthermore, bacterial attachment mechanisms that are specific for invertebrate surfaces have been d o c u m e n t e d (i.e. the adhesion of Vibrio harveyi to invertebrate chitin [33"]). Such complex chemical cues and 'recognition' phenomena probably play a significant role in determining the composition and structure of marine biofouling communities. In the meantime, the search for bacteria that are potential sources of natural products that inhibit invertebrate fouling continues.

Conclusions Because of the structural complexity of biofilms and the diversity of microorganisms, much remains to be learned about bacterial attachment, biofilm development and structure, and the physiology of bacteria in biofilms. Even so, with the recent application of n e w technologies (e.g. SCLM, molecular biology, and microsensors) it is likely that knowledge will advance more rapidly in the coming years than it has up until now. Such progress may lead to n e w strategies for the control of biofouling, as well as insights into h o w biofilms may be manipulated for biotechnological applications. Moreover, it is certain to lead to an enhanced perception of the molecular and ecological diversity of biofilm composition and function.

5. •-

Korber DR, Lawrence JR, Hendry MJ, Caldwell DE: Analysis of Spatial Variability within MOT+ and MOT- Pseudomona$ fluorescens Biofilms using Representative Elements. Biofoultng 1993, 7:339-358. SCLM is used to determine the structure of biofilms formed by a motile P fluorescens strain and by a non-motile mutant. This is the first direct demonstration that motility can be an important factor determining distribution of bacteria within biofilms and overall biofilm architecture. 6. •.

Wolfaardt GM, Lawrence JR, Robarts RD, Caldwell SJ, Caldwell DE: Multicellular Organization in a Degradative Biofilm Community. Appl Env Mtcrobtol 1994, 60:43 a. a.d6. Using SCLM, these authors demonstrate a striking difference in the structures of biofilms that developed with a herbicide as the sole substrate and those that developed on tryptic soy broth. Direct visual evidence is provided that bacteria form structurally organized consortia that facilitate breakdown of the complex herbicide substrate. 7.

Manz W, Szewzyk U, Ericsson P, Amarm R, Schleifer K-H, Stenstr6m T-A: In Situ Identification of Bacteria in Drinking Water and Adjoining Biofilms by Hybridization with 16S and 23S rRNA-Directed Fluorescent Oligonucleotide Probes. Appl Env Microbtol 1993, 59:2293-2298.

8.

Voordouw G, Shen Y, Harrington CS, Telang AJ, Jack TR, Westlake DWS: Quantitative Reverse Sample Genome Probing of Microbial Communities and Its Application to Oil Field Production Waters. Appl Env Mfcrobtol 1993, 59:4101-4114.

9. •

Ramsing NB, Ktihl M, Jorgensen BB: Distribution of SulfateReducing Bacteria, O2, and H2S in Photosynthetic Biofilnm Determined by Ofigonucleotide Probes and Microelectrodes. Appl Env Mgcrobtol 1993, 59:3840-3849. This report demonstrates how microsensor data can be combined with molecular probing techniques to determine the distribution of different taxonomic groups in biofilms and correlate their presence with microenvironmental conditions. This type of approach may provide previously elusive information on the ecology of biofilm communities. 10.

Moyer CL, Dobbs FC, Karl DM: Estimation of Diversity and Community Structure through Restriction Fragment Length Polymorphism Distribution Analysis of Bacterial 16S rRNA Genes from a Microbial Mat at an Active, Hydrothermal Vent System, Loihi Seamount, Hawaii. Appl Env Microbtol 1994, 60:871-879.

11.

Muyzer G, de Waal EC, Uitterlinden AG: Profiling of Complex Microbial Populations by Denaturing Gradient Gel Electrophoresis Analysis of Polymerase Chain Reaction-Amplified Genes Coding for 16S rRNA. Appl Env Mtcrobtol 1993, 59:695-700.

12.

Poulsen LK, Ballard G, Stahl DA: Use of rRNA Fluorescence in Sltu Hybridization for Measuring the Activity of Single Cells in Young and Established Biofilms. Appl Env Mtcrobtol 1993, 59:1354-1360.

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13. •,

Davies DG, Chakrabarty AM, Geesey GG: Exopolysaccharide Production in Biofilms: Substratum Activation of Alginate Gene Expression by Pseudomonas aeruglnosa. Appl Env Microbiol 1993, 59:1181-1186. Describes an innovative approach, in which a mucoid strain of P. aerztginosa is engineered to contain an alginate promoter fused to a reporter gene. P. aerugtnosa typically produces alginate on sur-

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Environmental biotechnology faces, and accordingly, the authors demonstrate increased expression o f the alginate promoter in the recombinant bacteria following attachment. It m a y be possible to extend this approach to additional Pseudomonas spp. a n d to eventually identify the proximate stimuli that e n h a n c e polymer production o n surfaces. 14. •-

Vandevivere P, Kirchman DL: Attachment Stimulates Exopolysaccharide Synthesis by a Bacterium. Appl Env Microbfol 1993, 59:3280-3286. Describes a n elegantly simple study that demonstrates clearly that polymer synthesis can be increased as a result of exposure to solid surfaces. The results are convincing, a n d this is probably the first report demonstrating that ordinary isolates from a natural environment can respond to solid surfaces. 15.

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26. •

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29.

Holmstrom C, Rittschof D, Kjelleberg S: Inhibition of Settlem e n t by Larvae of Balanus amphitrite and Clona lntestln a l / s by a Surface-Colonizing Marine Bacterium. Appl Env Mtcrobtol 1992 58:2111-2115.

30.

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18.

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31.

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21.

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32.

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Bashan Y, Holguin G: Anchoring ofAzospirillum brasilense to Hydrophobic Polystyrene and W h e a t Roots. J Gen Microbtol 1993, 139:379-385. This is a comprehensive study of the attachment characteristics of A. brasilense. It is particularly important because it demonstrates the ability of this organism to adhere non-specifically to a wide range o f surfaces, yet preferentially to biological surfaces. This is different from m a n y previous reports, in which non-specific adhesion appeared to occur more readily to non-hydrated non-biological surfaces. 22. •

23.

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33. •

Montgomery MT, Kirchman DL: Role of Chitin-Binding Proteins in the Specific Attachment of the Marine Bacterium Vtbrlo harveyi to Chitin. Appl Env Mtcrobtol 1993, 59:373-379. This report is particularly interesting because it is an example of a 'specific' adhesion m e c h a n i s m in a marine bacterium. The vast majority of specific adhesion m e c h a n i s m s described, to date, have b e e n in bacteria colonizing m a m m a l s or plants in terrestrial environments. This report demonstrates that specificity may also have evolved in marine systems.

M Fletcher, Center of Marine Biotechnology, University of Maryland Biotechnology Institute, 600 East Lombard Street, Baltimore, Maryland 21202, USA.