Sage Journals: Discover world-class research

Abstract

Biofilm control is fundamental to oral health. Existing oral prophylactic measures, however, are insufficient. The main reason is probably because the micro-organisms involved organize into complex biofilm communities with features that differ from those of planktonic cells. Micro-organisms have traditionally been studied in the planktonic state. Conclusions drawn from many of these studies, therefore, need to be revalidated. Recent global approaches to the study of microbial gene expression and regulation in non-oral micro-organisms have shed light on two-component and quorum-sensing systems for the transduction of stimuli that allow for coordinated gene expression. We suggest interference with two-component and quorum-sensing systems as potential novel strategies for the prevention of oral diseases through control of oral biofilms. Information is still lacking, however, on the genetic regulation of oral biofilm formation. A better understanding of these processes is of considerable importance.

(1) Introduction

Oral diseases, such as dental caries and periodontal disease, should be considered as consequences of ecologically driven imbalances of oral microbial biofilms (Marsh, 1994). Both diseases are caused by micro-organisms belonging to the resident oral microflora rather than by classic microbial pathogens. Thus, most individuals harbor the micro-organisms involved in these diseases. In the case of dental caries, a low pH environment caused by microbial fermentation of carbohydrates selects a population of acid-tolerant and acid-producing strains like mutans streptococci and lactobacilli. This in turn increases acid formation that may cause demineralization. Mixed anaerobic micro-organisms are involved in periodontal disease, which develops when the plaque community equilibrium is altered and inflammation is induced. The environment is altered by an increased flow of gingival crevicular fluid, increased nutrients, and pH rise that favors growth of periodontal pathogens which may contribute to periodontal destruction (Marsh, 1994).

Control of oral biofilms is fundamental to the maintenance of oral health and to the prevention of dental caries, gingivitis, and periodontitis. However, oral biofilms are not easily controlled by mechanical means and represent difficult targets for chemical control (Socransky, 2002). Apart from chlorhexidine and fluorides, only a few of the existing oral prophylactic agents have significant effects (Petersen and Scheie, 1998; Wu and Savitt, 2002; Scheie, 2003). One probable explanation for this low efficacy is the fact that the micro-organisms involved organize into complex biofilm communities with features that differ from those of planktonic cells, whereas micro-organisms have traditionally been studied in a planktonic state. Conclusions drawn from many of these studies, therefore, need to be revalidated. Recent global approaches to the study of microbial gene expression and regulation in non-oral micro-organisms have shed light on two-component and quorum-sensing systems for the transduction of stimuli that allow for coordinated gene expression. Development of novel strategies to prevent and treat diseases caused by oral biofilms might benefit from these recent studies to understand microbial gene expression and regulation in oral biofilms.

(2) The Biofilm Life Style

The biofilm mode of growth seems to be advantageous for micro-organisms. Biofilm micro-organisms form distinct three-dimensional structured communities with fluid channels for transport of substrate, waste products, and signal molecules (Costerton, 1999). The matrix that holds the biofilm together is a mixture of polysaccharides, proteins, and DNA secreted by the cells (Sutherland, 2001; Whitchurch et al., 2002). It was recently discovered that micro-organisms like Pseudomonas aeruginosa actively maintain their three-dimensional biofilm structure by releasing surfactant molecules that prevent other micro-organisms from clogging the channel systems. Those cells that were unable to produce the surfactant were also unable to maintain the characteristic biofilm architecture necessary for a functional biofilm (Davey et al., 2003).

When organized in biofilms, the micro-organisms are less susceptible to anti-microbials and more resistant to immune defense mechanisms (Mah and O’Toole, 2001; Stewart and Costerton, 2001; Davies, 2003). The concentration of an agent which kills planktonic micro-organisms might have to be increased by 10–1000 times to have the same efficacy on micro-organisms in a biofilm (Lewis, 2001; Mah and O’Toole, 2001; Davies, 2003). This relative resistance to anti-microbial agents partly explains why many oral prophylactic agents predicted to be efficacious in in vitro assays show only marginal clinical effects. Retarded or incomplete penetration of the agent into the biofilm, or reduced growth rate of the micro-organisms due to nutrient limitations, has been considered a reason for lack of efficacy. Another likely explanation is the fact that micro-organisms within a biofilm are physiologically altered due to differential gene expression (Mah and O’Toole, 2001; Lewis, 2001; Davies, 2003).

(2.1) Adaptation to life in a biofilm

Micro-organisms undergo a wide range of physiological and morphological adaptations in response to environmental changes. In biofilms, different gradients of chemicals, nutrients, and oxygen create micro-environments to which micro-organisms must adapt to survive. The perception and processing of chemical information from the environment form a central part of the regulatory control of these adaptive responses. Adaptation to a biofilm life style involves regulation of a vast set of genes, and the micro-organisms are thus able to optimize phenotypic properties for the particular environment. Consequently, biofilm micro-organisms differ phenotypically from their planktonic counterparts. For instance, the enzyme urease in Streptococcus salivarius was found to be differentially regulated during biofilm growth (Li et al., 2000). Also, in Streptococcus mutans, glucosyltransferase and fructosyltransferase genes are differentially expressed in mature biofilm compared with initial biofilms (Burne et al., 1997). Compared with its planktonic counterpart, approximately 20% of the proteins that are expressed in Streptococcus mutans biofilms were shown to be either up- or down-regulated (Svensäter et al., 2001), whereas the level of secreted proteins is altered in biofilms formed by Actinobacillus actinomycetemcomitans (Fletcher et al., 2001). In P. aeruginosa, differences in protein expression as large as 50% were observed between biofilm and planktonic cells and up to 40% between consecutive stages of biofilm development (Sauer et al., 2002).

Biofilm formation is a step-wise process (Fig. 1a) which commences by adhesion of planktonic micro-organisms to a surface. Further steps involve colonization and co-adhesion, growth and maturation, and finally detachment of some micro-organisms. Evidence is emerging that expression of genes required during the various stages is well-regulated (Davies et al., 1998; Pratt and Kolter, 1999; Sauer et al., 2002; Stoodley et al., 2002).

The coordinated gene expression is regulated through various signal transduction systems that induce cascades of reactions, leading to the induction or inhibition of gene transcription. The external stimulus is in some cases inherent in the environment, although the molecules involved are mostly unknown. For other systems, the stimulus represents known molecules. The so-called two-component regulatory systems are frequently involved in the control of gene expression in response to various stimuli. Two-component regulatory systems include a histidine kinase and a response regulator (Fig. 2). Several such two-component systems exist in Gram-positive and Gram-negative micro-organisms (Dunny and Leonard, 1997; Fabret et al., 1999; Hoch, 2000; Miller and Bassler, 2001). They play important roles in signal transduction and may be essential sensors for adaptation to a biofilm life. Examples of two-component systems involved in biofilm formation include the GacA/GacS in P. aeruginosa (Parkins et al., 2001) and the HK11/RR11 in S. mutans (Li et al., 2002b), as well as the ComD/ComE in S. mutans and S. gordonii (Loo et al., 2000; Li et al., 2001a,b , 2002a; Yoshida and Kuramitsu, 2002).

The external stimulus is sensed by the transmembrane histidine kinase receptor that then catalyzes an intracellular ATP-dependent autophosphorylation (Fig. 2) (Fabret et al., 1999; Hoch, 2000). The phosphoryl group is subsequently transferred to a conserved aspartate residue of the regulatory domain of its cognate response regulator. In the most sophisticated systems, activation of the response regulator occurs through multistep phospho-relay cascades. The phosphorylated form of the response regulator will influence transcription by binding to the promoter sequences of genes under its control, resulting in gene activation or repression.

Quorum-sensing signaling represents a signaling pathway that is activated as a response to cell density (Dunny and Leonard, 1997; Bassler, 1999; de Kievit and Iglewski, 2000; Miller and Bassler, 2001). Such systems are found in both Gram-positive and Gram-negative micro-organisms. The stimuli of quorum-sensing systems are signal molecules, called autoinducers. The autoinducers are produced at a basal constant level, and the concentration thus is a function of microbial density. Perception of the signal occurs at a concentration threshold. The term “quorum” is used to describe this kind of signal system, since a certain number of micro-organisms must be present for the signal to be sensed and for the population to respond to the signal.

Gram-positive bacteria usually produce oligopeptide signals which are recognized by two-component signal transduction systems (Fig. 3a) (Dunny and Leonard, 1997; de Kievit and Iglewski, 2000). In Gram-negative organisms, the signal molecule belongs to a group of homoserine lactones. These molecules usually diffuse into the cells and bind directly to a response regulator (Fig. 3b) (Bassler, 1999; de Kievit and Iglewski, 2000). As micro-organisms release the signal molecules when approaching a surface, the signal molecule concentration increases in the area between the micro-organism and the surface, due to limited diffusion. The micro-organisms will perceive this concentration increase and thus sense the presence of the surface they are colonizing (Costerton, 1999).

The signal molecules of quorum-sensing systems are often highly specific. Quorum-sensing signaling thus serves intra-species communication purposes. More recently, a second communication system, the so-called autoinducer system 2 (AI-2), has been described. An important aspect is that, in contrast to most other quorum-sensing systems which are used for single-species communication, AI-2 allows for interspecies communication (Bassler, 1999).

AI-2 was first found to stimulate bioluminescence in the marine bacterium Vibrio harveyi (Bassler et al., 1994). The role of AI-2 as a density-dependent signal for regulating V. harveyi bioluminescence is now well-established, and evidence for AI-2 signal production by other micro-organisms has been obtained by assessment of their ability to activate the bioluminescence response in V. harveyi (Bassler et al., 1997; Frias et al., 2001; Miller and Bassler, 2001). Evidently, AI-2 signals are released by many micro-organisms, including Gram-positive and Gram-negative species. In V. harveyi, the AI-2 signal molecule is a furanosyl borate di-ester which bears no resemblance to previously characterized bacterial quorum-sensing signals. In most cases, however, the signal molecule involved is unknown (Chen et al., 2002), but its synthesis depends on the luxS gene product (Schauder et al., 2001; Winans and Bassler, 2002). It is not clear whether all AI-2 producers use this molecule as a true quorum-sensing signal. The possibility that AI-2 may represent a metabolic waste product has been raised (Winzer et al., 2002). Further research into the actual nature and function of the AI-2-like molecules in various micro-organisms is therefore needed.

(2.2) Communication in oral biofilms

Biofilms are likely to represent a natural scenario for bacterial communication (Davey and O’Toole, 2000; Kolenbrander et al., 2002). The ability to communicate through quorum-sensing has been shown in some oral streptococci and some periodontal pathogens (Table). For most oral biofilm micro-organisms, however, the presence and function of signal transduction pathways and quorum-sensing communication remain to be clarified. Evidence for the involvement of two-component signal transduction systems in oral biofilm formation was first found in Streptococcus gordonii (Loo et al., 2000). Biofilm-formation-deficient S. gordonii mutants resulted from the disruption of comD, which encodes the histidine kinase receptor for the competence stimulating signal peptide (CSP). This quorum-sensing peptide was originally described as an inducer of competence for natural transformation (Håvarstein et al., 1996 , 1997). The competent state permits the binding, uptake, and integration of extracellular DNA to occur. It is thought that this system influences the ability of streptococci to adapt to the environment by allowing for the acquisition of new genetic traits from other bacteria. Also in S. mutans, inactivation of the gene encoding the CSP and other competence-related genes, including comC/comE, resulted in biofilm formation deficiency or altered biofilm architecture (Bhagwat et al., 2001; Li et al., 2002a; Yoshida and Kuramitsu, 2002). Although the CSP was first ascribed a role in natural transformation, it has recently been suggested that the main function of the competence system is to sense and elicit responses to environmental stress conditions (Yother et al., 2002). Accordingly, the competence-related signaling system in S. mutans has been related to the ability of these cells to adapt to acidic conditions (Li et al., 2001b).

Several putative periodontopathogenic micro-organisms possess autoinducer-like activities (Frias et al., 2001). AI-2 is produced by Porphyromonas gingivalis, Prevotella intermedia, and Fusobacterium nucleatum (Frias et al., 2001). More recently, DNA sequence analysis has confirmed the presence of highly conserved homologues of the AI-2 synthase gene, luxS (Table), in S. mutans (Wen and Burne, 2002; Merritt et al., 2003), S. gordonii (McNab et al., 2003), P. gingivalis (Chung et al., 2001; Frias et al., 2001; Burgess et al., 2002), and A. actinomycetemcomitans (Fong et al., 2001 , 2003). Inactivation of the homologous AI-2 synthase gene in S. mutans (Wen and Burne, 2002; Merritt et al., 2003) and S. gordonii (McNab et al., 2003) did not seem to affect the amount of single-species biofilm. It was recently shown, however, that inactivation of the S. mutans AI-2 synthase gene affected the structure of biofilm when grown in the presence of sucrose (Merritt et al., 2003). In S. gordonii, such mutants show altered expression of genes involved in carbohydrate metabolism. In P. gingivalis, AI-2 deficiency was associated with altered expression of putative virulence genes such as those encoding products involved in hemagglutinin and protease activities (Chung et al., 2001; Burgess et al., 2002). In A. actinomycetemcomitans, AI-2 stimulated leukotoxin production (Fong et al., 2001) and was involved in adaptation to iron limitation (Fong et al., 2001 , 2003). This occurred through altered expression of genes encoding various iron transport systems and iron storage proteins.

Since the AI-2 response system can be used for interspecies communication, investigation of its role in mixed biofilm communities is of particular interest. It was recently demonstrated, for instance, that in mixed biofilms of S. gordonii and P. gingivalis, inactivation of the AI-2 synthase gene of both strains impaired binding of P. gingivalis to S. gordonii biofilms (McNab et al., 2003).

Bacteriocins might also function as quorum-sensing signals and have a direct effect on biofilm composition. The pre-peptide sequence of the bacteriocin mutacin IV produced by S. mutans (Qi et al., 2001) contains a double glycine-containing leader peptide, suggesting an export and processing pathway similar to that observed for the CSP and other bacteriocins that function as signals in two-component systems. Recently, a link between transformation and bacteriocin production was suggested (Yother et al., 2002). Disruption of S. gordonii genes involved in the early stages of competence development for transformation resulted in mutants deficient in their ability to produce two bacteriocins, STH₁ and STH₂. This link suggests that killing micro-organisms by bacteriocin could serve dual functions: to liberate DNA for uptake by the competent bacteriocin producers, and to prevent colonization by competing micro-organisms. Sensing of the bacteriocin salivaricin A by S. salivarius probably involves a two-component system comprised of the histidine kinase SalK and the cognate response regulator SalR. Interestingly, S. salivarius can sense the salivaricin A homologue produced by Streptococcus pyogenes (Upton et al., 2001). This clearly illustrates a mechanism of interspecies communication not commonly observed via oligopeptide quorum signals.

(3) Prospects for Prevention

The recent emergence and spread of multi-resistant micro-organisms and refractory biofilm-induced infections have prompted an intense search for novel antibiotics that inhibit pathogenic micro-organisms through novel targets. Presently available microbial and human genomic sequence data allow for the prediction of potential targets that are unique to the micro-organisms. Targeted agents are thus expected to be highly specific, to pose an insignificant resistance development problem, and to have minimal effects on vital human cell functions.

For therapeutic purposes, it is necessary to attack the established biofilm. Therefore, genes essential for viability represent the traditional targets for anti-microbial drug design. Potential agents include, among others, microbial fatty acid biosynthesis inhibitors, bacteriophages, and anti-microbial peptides (Hancock, 1999; Payne et al., 2001; Sulakvelidze and Morris, 2001).

For prophylactic purposes, it seems reasonable to target processes involved in the actual biofilm formation of single- or mixed-bacterial communities that have the potential to cause or favor disease, without perturbing the balance of the normal flora. In this respect, two-component systems and quorum-sensing seem to represent promising future targets. One could envision that interference with such systems in the oral microflora might be used to secure an ecologic balance that promotes the persistence of a healthy flora. The facts that two-component systems are present in most micro-organisms and that signal transduction in mammalian cells is mediated through different mechanisms provide the rationale for using signal transduction systems as targets for prophylactic purposes.

It is likely that the mature oral biofilm is the result of a well-regulated series of processes that could represent potential targets for biofilm control. In the following section, prospects for signal transduction interference and its potential for prophylactic intervention will be discussed. We will also briefly discuss possible strategies for immunization, replacement therapy, and surface modification in view of the novel biofilm concept of dental plaque. Not surprisingly, certain traditional anti-microbial compounds that affect bacterial growth and survival also interfere with bacterial regulatory mechanisms, including the two-component systems. Since their mechanism of action is not specific for two-component systems, they will not be considered here.

(3.1) Signal transduction interference

(3.1.1) Two-component systems

Two-component signal transduction systems and histidine kinases represent potential prophylactic targets (Barrett and Hoch, 1998; Matsushita and Janda, 2002; Stephenson and Hoch, 2002). The histidine kinases and response regulators of the two-component systems exhibit both conserved and variable domains (Fig. 2). This characteristic can be used for the development of inhibitors with species-specific or broad-spectrum activity. The stimulus input domain in the histidine kinases is the most variable, which reflects the wide variety of stimuli or signal molecules that may be sensed by two-component systems. The possibility of interfering with recognition of a stimulus can be achieved by the use of signal analogues, as discussed in the following section for the Gram-positive oligopeptide quorum-sensing system. In this case, the analogues are likely to have a narrow spectrum of activity. Notably, it is not always the fact that inhibition of histidine kinases is sufficient to prevent response regulators from receiving phosphate from other sources. Therefore, the response regulator itself may represent the best target for the inhibition of two-component systems. In most response regulators, conserved and variable residues are found to interact with the histidine kinase. Targeting the conserved residues may therefore interfere with several response regulators in the same organism. Structural similarities of regulators in different microbial species may also allow for the production of cross-species inhibitors based on this principle.

The recent indications that two-component systems are involved in oral streptococcal biofilm formation and acid tolerance (Li et al., 2001b , 2002a,Li et al., b; Loo et al., 2000) support the possibility for interference with oral biofilms through two-component signal transduction.

(3.1.2) Quorum-sensing

The Australian red macro algae Delisea pulchra avoids bacterial colonization through the production of halogenated furanones as secondary metabolites (Givskov et al., 1996). Halogenated furanones are similar in structure to the homoserine lactones that are used as quorum-sensing signal molecules by some Gram-negative micro-organisms. Synthetic halogenated furanones were shown to inhibit homoserine lactone-mediated quorum-sensing and biofilm formation in P. aeruginosa (Hentzer et al., 2002). Initially, furanones were thought to compete with homoserine lactone signal molecules for binding to the transcriptional activator (Gram et al., 1996). More recent evidence, however, suggests that these compounds act by accelerating degradation of the transcriptional regulator that binds to the signal (Manefield et al., 2002). Several systems that disrupt homoserine lactone-mediated quorum-sensing systems are also found in nature, such as those produced by bacillus and variovorax species (Dong et al., 2000 , 2001; Leadbetter and Greenberg, 2000).

De-acylation of the signal molecule is another way of interfering with quorum-sensing signaling. Porcine kidney acylase I was recently shown to de-acylate both N-butyryl-homoserine lactone and N-octanoyl-homoserine lactone (Xu et al., 2003), two representatives of homoserine lactone molecules of Gram-negative micro-organisms. The de-acylation resulted in reduced biofilm formation by aquatic micro-organisms. Synthetic signal analogues might also be used to interfere with quorum-sensing. Potent agonists and antagonists of homoserine lactones have been reported (Olsen et al., 2002; Reverchon et al., 2002; Smith et al., 2003). Staphylococcus aureus produces thiolactone oligopeptide signals that act as both activators and inhibitors of quorum-sensing. Naturally occurring truncated forms of the thiolactone peptides may reduce S. aureus virulence (Lyon et al., 2000).

Interestingly, the broad-spectrum anti-microbial bis-phenol triclosan, frequently used in antiseptic soaps and toothpastes, may inhibit the synthesis of precursors of homoserine lactones (Hoang and Schweizer, 1999). The search for homoserine lactone-mediated quorum-sensing, however, has failed to identify this system in oral bacteria (Whittaker et al., 1996; Frias et al., 2001; Burgess et al., 2002). The quorum-sensing systems found in oral bacteria include the AI-2 and streptococcal CSP signaling pathways (Table). As a potential universal quorum-sensing signal used by many Gram-positive and Gram-negative cells, AI-2 may represent an attractive novel target for prophylactic purposes. The finding that AI-2 influences binding of P. gingivalis to S. gordonii biofilms supports the assumption that AI-2 may serve as a communication molecule (McNab et al., 2003). Further elucidation of the role of AI-2 in single- and mixed-bacterial communities is necessary for full appreciation of the AI-2 signaling system as a potential oral prophylactic target.

Interference with the CSP signaling mechanism could provide another way of combatting oral-biofilm-related diseases. The ability of S. gordonii and S. mutans to form biofilms, for instance, could be impaired by inhibiting signal transduction through this system. This pathway should be further explored to ascertain whether early streptococcal colonizers such as Streptococcus oralis, Streptococcus sanguis, and Streptococcus mitis, known to be induced to competence by specific CSPs, also use this communication system for biofilm formation. It would also be interesting to investigate whether the addition of synthetic CSPs interferes with biofilm formation when the required microbial quorum is not present. Such information may be used not only to develop strategies to inactivate the signal or its transmission, but also to favor micro-organisms that can oppose the colonization of more virulent species. Since involvement of most two-component and quorum-sensing systems in biofilm formation seems to depend on environmental conditions, it is important to determine whether such mechanisms are also active under relevant in vivo conditions.

(3.2) Other strategies

(3.2.1) Surface modification

The strategy of surface modification involves altering the tooth surface or the salivary pellicle to impede bacterial colonization (Fig. 1b). It is well-known that the salivary pellicle provides binding sites for the oral bacteria through a complex array of specific and non-specific binding mechanisms. Thus, the composition of the pellicle may modulate bacterial adhesion events. One approach is to change the surface characteristics by manipulating the protein film on the enamel, thereby reducing bacterial adhesion. Several routes of surface modification have been investigated. Functional groups like phosphate and phosphonate may be used to anchor water-soluble, protein-repelling substances to the mineral surface (Olsson, 1998). It has been shown in vitro that the combination of an alkylphosphate and a non-ionic surfactant alters the surface characteristics of the tooth, making it less attractive for micro-organisms. Unfortunately, the clinical efficacy of such coating agents has been low, probably because of difficulties in securing persistent coating with the active component (Olsson, 1998). If these problems are resolved, a future approach to reduce colonization could be to coat the tooth surface with agents that interfere with two-component signal transduction in oral micro-organisms.

(3.2.2) Replacement therapy

Replacement therapy has been suggested as a strategy to replace potential pathogenic micro-organisms with genetically modified organisms that are less virulent (Fig. 1b). The requirements for this type of therapy are, first, that there be a definite pathogen to replace. The replacement organism must not cause disease itself, it must colonize persistently, it must replace the pathogen effectively, and it must possess a high degree of genetic stability. DNA technology has made it possible to produce potential candidates for replacement therapy in caries prevention. Among those is a super-colonizing strain of S. mutans. This strain produces mutacin, which enables it to replace wild-type strains efficiently. It lacks the enzyme lactate dehydrogenase and therefore does not form lactate (Hillman et al., 2000). Other recombinant strains which are ureolytic have also been constructed (Clancy et al., 2000). These strains hydrolyze urea to ammonia, thereby counteracting acidification of the environment.

Animal experiments involving lactate-dehydrogenase-deficient and ureolytic S. mutans strains have shown promising results in caries prevention (Clancy et al., 2000). In the former case, the approach is directed against single microbial species. However, one should not disregard the role of other micro-organisms, such as non-mutans acidogenic streptococci, in the caries process in humans.

A possible future approach is to use genetically modified micro-organisms to deliver tailored molecules that could interfere with adaptive pathways such as two-component signal transduction or quorum-sensing systems. However, one cannot exclude the possibility that a genetically modified replacement strain might later undergo transformation in oral biofilms and then become an opportunistic pathogenic strain.

(3.2.3) Immunization

Immunization against oral diseases—particularly dental caries, but also periodontal disease—has been a central research topic in recent decades (Koga et al., 2002; Smith, 2002). The aim is to inhibit adhesion or reduce the virulence of putative microbial etiologic agents. Several molecules involved in the various stages of both dental caries and periodontal disease pathogenesis would be susceptible to immune intervention and could function as vaccine targets. Micro-organisms could, for instance, be cleared from the oral cavity by antibodies prior to colonization, antibodies could block adhesins or receptors involved in adhesion, or modify metabolically important functions or virulence factors (Fig. 1b). Efforts have been made to immunize both actively and passively. In active immunization, an antigen which will elicit a protective immune response is administered. In passive immunization, the antibody itself is administered.

Animal studies using either active or passive immunization approaches have been successful. There are also data to show that passive immunization in humans impedes recolonization of selected target micro-organisms in both caries (Koga et al., 2002; Smith, 2002) and periodontal disease (Booth et al., 1996). As vehicles for passive immunization, both milk from immunized cows (Shimazaki et al., 2001) and transgenic plants (Ma et al., 1998) have been tested, with encouraging results. Likewise, chimeric recombinant microbial vectors that are avirulent but which express antigens from S. mutans (Huang et al., 2001; Taubman et al., 2001) or P. gingivalis (Sharma et al., 2001) have been shown to provide protection against dental caries and alveolar bone loss, respectively, in experimental animals. However, no vaccination scheme in humans is yet clinically applicable. The question that still needs to be resolved is whether the systemic or the mucosal immune system needs to be stimulated. If an anti-caries vaccine becomes available, it is most likely that it will be administered via the oral route and be based on the induction of the common mucosal immune system. A vaccine against periodontal disease should probably involve both systemic and mucosal immunity.

A major problem is that immunization approaches are generally directed against single bacterial species epitopes, whereas both dental caries and periodontal disease are ecologically driven multi-microbial diseases (Marsh, 1994). Furthermore, since micro-organisms have the ability to form biofilms and to adapt and undergo transformation that may lead to altered antigenicity, it is questionable whether immunization will provide lasting protection.

(4) Concluding Remarks and Future Directions

Developing oral prophylactic strategies through interference with two-component systems or quorum-sensing of biofilm micro-organisms represents an interesting future challenge. Unlike strategies that target microbial viability, such approaches may interfere with microbial adaptive pathways without killing the micro-organisms. Therefore, resistance development would probably represent a minor problem.

While the stages of biofilm formation seem to follow basically the same model in various micro-organisms, the biofilm architecture and molecular mechanisms involved in biofilm formation appear to differ. The mechanisms involved in biofilm formation by P. aeruginosa are some of the best-characterized and have served as a model for new hypotheses on mechanisms used by other micro-organisms. Information on the genetic regulation of oral biofilm formation, however, is still lacking. A better understanding of these processes is necessary to the development of novel strategies for oral disease prevention and control based on interference of two-component signal transduction systems or quorum-sensing. Since the systems contain both conserved and variable components, both broad- and narrow-spectrum responses may be available. This could allow for tailoring of prophylactic measures based on individual oral health status and risk assessment.

TABLE
Quorum-sensing in Oral Bacteria

Signal Bacterium Sequence of the Mature Signal Phenotype Reference

^a CSP = Competence Stimulating Peptide encoded by comC.

^b AI-2 = Autoinducer 2, hypothetically synthesized by luxS.

^c SalA, Salivaricin A, bacteriocin encoded by salA.

Streptococcus mutans SGSLSTFFRLFNRSFTQALGK Biofilm formation, natural transformation, acid tolerance, cell separation Li et al., 2001a,b, 2002a; Petersen and Scheie, 2000; Yoshida and Kuramitsu, 2002

Streptococcus gordonii SQKGVYASQRSFVPSWFRKIFRN Biofilm formation, natural transformation Håvarstein et al., 1996, 1997; Loo et al., 2000

DVRSNKIRLWWENIFFNKK

DIRHRINNSIWRDIFLKRK

Streptococcus mitis EIRQTHNIFFNFFKRR Natural transformation Håvarstein et al., 1997; Whatmore et al., 1999

ESRLPKIRFDFIFPRKK

CSP^a EMRKSNNNFFHFLRRI

EMRKPDGALFNLFRRR

ESRVSRIILDFLFQRKK

ESRISDILLDFLFQRKK

Streptococcus oralis DWRISETIRNLIFPRRK Håvarstein et al., 1997; Whatmore et al., 1999

DKRLPYFFKHLFSNRTK

EMRLPKILRDFIFPRKK

Streptococcus sanguis DLRGVPNPWGWIFGR Håvarstein et al., 1997

Streptococcus crista DLRNIFLKIKFKKK Håvarstein et al., 1997

Streptococcus anginosus DSRIRMGFDFSKLFGK Håvarstein et al., 1997

Streptococcus intermedius Håvarstein et al., 1997

Streptococcus constellatus

Streptococcus mutans Natural transformation, regulation of the stress response or homeostasis, biofilm structure Yother et al., 2002; Merritt et al., 2003

Streptococcus gordonii Mixed-species biofilm formation, metabolism McNab et al., 2003

Porphyromonas gingivalis Mixed-species biofilm formation, protease and hemagglutinin activities Burgess et al., 2002; Chung et al., 2001; Frias et al., 2001; McNab et al., 2003

AI-2^b Unknown

Fusobacterium nucleatum Unknown Frias et al., 2001

Prevotella intermedia Unknown Frias et al., 2001

Actinobacillus actinomycetemcomitans Adaptation to iron-limited conditions, leukotoxin production Fong et al., 2001, 2003; Frias et al., 2001

SalA^c Streptococcus salivarius KRGSGWIATITDDCPNSVFVCC Bacteriocin production Upton et al., 2001

Figure 1.
Oral biofilm formation (a) and prospects for future intervention (b).

Figure 2.
In a typical two-component signal transduction system, the transmembrane domain of the histine kinase recognizes a stimulus. Upon recognition, the histidine kinase catalyzes ATP-dependent autophosphorylation of a conserved histidine residue in the dimerization domain. The phosphoryl group is subsequently transferred to a conserved aspartate residue in the regulatory domain of the cognate response regulator. Phosphorylation activates the effector domain of the response regulator. The activated response regulator will then activate or repress transcription of specific target gene(s), promoting a specific response.

Figure 3a.
Quorum-sensing systems in Gram-positive micro-organisms. The oligopeptide signal precursor () is processed and exported by ABC-transporters. When a threshold level is reached, the extracellular peptides () bind to a histidine kinase receptor (HK) in the cell membrane, resulting in autophosphorylation of the histidine phosphotransferase domain. The phosphate (P) is transferred to the cognate response regulator (RR), which can then bind to target DNA and alter gene expression.

Figure 3b.
Quorum-sensing systems in Gram-negative micro-organisms. The signal molecules ()produced by the cells diffuse freely through the cell envelope. When a threshold level is reached, the signal molecules bind to and activate the transcriptional activator (TA). The signal-TA complex binds to target DNA and alters gene expression.

Signal	Bacterium	Sequence of the Mature Signal	Phenotype	Reference
^a CSP = Competence Stimulating Peptide encoded by comC.
^b AI-2 = Autoinducer 2, hypothetically synthesized by luxS.
^c SalA, Salivaricin A, bacteriocin encoded by salA.
	Streptococcus mutans	SGSLSTFFRLFNRSFTQALGK	Biofilm formation, natural transformation, acid tolerance, cell separation	Li et al., 2001a,b, 2002a; Petersen and Scheie, 2000; Yoshida and Kuramitsu, 2002
	Streptococcus gordonii	SQKGVYASQRSFVPSWFRKIFRN	Biofilm formation, natural transformation	Håvarstein et al., 1996, 1997; Loo et al., 2000
		DVRSNKIRLWWENIFFNKK
		DIRHRINNSIWRDIFLKRK
	Streptococcus mitis	EIRQTHNIFFNFFKRR	Natural transformation	Håvarstein et al., 1997; Whatmore et al., 1999
		ESRLPKIRFDFIFPRKK
CSP^a		EMRKSNNNFFHFLRRI
		EMRKPDGALFNLFRRR
		ESRVSRIILDFLFQRKK
		ESRISDILLDFLFQRKK
	Streptococcus oralis	DWRISETIRNLIFPRRK		Håvarstein et al., 1997; Whatmore et al., 1999
		DKRLPYFFKHLFSNRTK
		EMRLPKILRDFIFPRKK
	Streptococcus sanguis	DLRGVPNPWGWIFGR		Håvarstein et al., 1997
	Streptococcus crista	DLRNIFLKIKFKKK		Håvarstein et al., 1997
	Streptococcus anginosus	DSRIRMGFDFSKLFGK		Håvarstein et al., 1997
	Streptococcus intermedius			Håvarstein et al., 1997
	Streptococcus constellatus
	Streptococcus mutans		Natural transformation, regulation of the stress response or homeostasis, biofilm structure	Yother et al., 2002; Merritt et al., 2003
	Streptococcus gordonii		Mixed-species biofilm formation, metabolism	McNab et al., 2003
	Porphyromonas gingivalis		Mixed-species biofilm formation, protease and hemagglutinin activities	Burgess et al., 2002; Chung et al., 2001; Frias et al., 2001; McNab et al., 2003
AI-2^b		Unknown
	Fusobacterium nucleatum		Unknown	Frias et al., 2001
	Prevotella intermedia		Unknown	Frias et al., 2001
	Actinobacillus actinomycetemcomitans		Adaptation to iron-limited conditions, leukotoxin production	Fong et al., 2001, 2003; Frias et al., 2001
SalA^c	Streptococcus salivarius	KRGSGWIATITDDCPNSVFVCC	Bacteriocin production	Upton et al., 2001

References

Barrett JF, Hoch JA (1998). Two-component signal transduction as a target for microbial anti-infective therapy. Antimicrob Agents Chemother 42:1529–1536.

Bassler BL (1999). How bacteria talk to each other: regulation of gene expression by quorum sensing. Curr Opin Microbiol 2:582–587.

Bassler BL, Wright M, Silverman MR (1994). Multiple signalling systems controlling expression of luminescence in Vibrio harveyi: sequence and function of genes encoding a second sensory pathway. Mol Microbiol 13:273–286.

Bassler BL, Greenberg EP, Stevens AM (1997). Cross-species induction of luminescence in the quorum-sensing bacterium Vibrio harveyi. J Bacteriol 179:4043–4045.

Bhagwat SP, Nary J, Burne RA (2001). Effects of mutating putative two-component systems on biofilm formation by Streptococcus mutans UA159. FEMS Microbiol Lett 205:225–230.

Booth V, Ashley FP, Lehner T (1996). Passive immunization with monoclonal antibodies against Porphyromonas gingivalis in patients with periodontitis. Infect Immun 64:422–427.

Burgess NA, Kirke DF, Williams P, Winzer K, Hardie KR, Meyers NL, et al. (2002). LuxS-dependent quorum sensing in Porphyromonas gingivalis modulates protease and haemagglutinin activities but is not essential for virulence. Microbiology 148(Pt 3):763–772.

Burne RA, Chen YY, Penders JE (1997). Analysis of gene expression in Streptococcus mutans in biofilms in vitro. Adv Dent Res 11:100–109.

Chen X, Schauder S, Potier N, Van Dorsselaer A, Pelczer I, Bassler BL, et al. (2002). Structural identification of a bacterial quorum-sensing signal containing boron. Nature 415:545–549.

10.

Chung WO, Park Y, Lamont RJ, McNab R, Barbieri B, Demuth DR (2001). Signaling system in Porphyromonas gingivalis based on a LuxS protein. J Bacteriol 183:3903–3909.

11.

Clancy KA, Pearson S, Bowen WH, Burne RA (2000). Characterization of recombinant, ureolytic Streptococcus mutans demonstrates an inverse relationship between dental plaque ureolytic capacity and cariogenicity. Infect Immun 68:2621–2629.

12.

Costerton JW (1999). Introduction to biofilm. Int J Antimicrob Agents 11:217–221; discussion 237–239.

13.

Davey ME, O’Toole GA (2000). Microbial biofilms: from ecology to molecular genetics. Microbiol Mol Biol Rev 64:847–867.

14.

Davey ME, Caiazza NC, O’Toole GA (2003). Rhamnolipid surfactant production affects biofilm architecture in Pseudomonas aeruginosa PAO1. J Bacteriol 185:1027–1036.

15.

Davies D (2003). Understanding biofilm resistance to antibacterial agents. Nat Rev Drug Discov 2:114–122.

16.

Davies DG, Parsek MR, Pearson JP, Iglewski BH, Costerton JW, Greenberg EP (1998). The involvement of cell-to-cell signals in the development of a bacterial biofilm. Science 280:295–298.

17.

de Kievit TR, Iglewski BH (2000). Bacterial quorum sensing in pathogenic relationships. Infect Immun 68:4839–4849.

18.

Dong YH, Xu JL, Li XZ, Zhang LH (2000). AiiA, an enzyme that inactivates the acylhomoserine lactone quorum-sensing signal and attenuates the virulence of Erwinia carotovora. Proc Natl Acad Sci USA 97:3526–3531.

19.

Dong YH, Wang LH, Xu JL, Zhang HB, Zhang XF, Zhang LH (2001). Quenching quorum-sensing-dependent bacterial infection by an N-acyl homoserine lactonase. Nature 411:813–817.

20.

Dunny GM, Leonard BA (1997). Cell-cell communication in Gram-positive bacteria. Annu Rev Microbiol 51:527–564.

21.

Fabret C, Feher V, Hoch JA (1999). Two-component signal transduction in Bacillus subtilis: how one organism sees its world. J Bacteriol 181:1975–1983.

22.

Fletcher JM, Nair SP, Ward JM, Henderson B, Wilson M (2001). Analysis of the effect of changing environmental conditions on the expression patterns of exported surface-associated proteins of the oral pathogen Actinobacillus actinomycetemcomitans. Microb Pathog 30:359–368.

23.

Fong KP, Chung WO, Lamont RJ, Demuth DR (2001). Intra- and interspecies regulation of gene expression by Actinobacillus actinomycetemcomitans LuxS. Infect Immun 69:7625–7634.

24.

Fong KP, Gao L, Demuth DR (2003). luxS and arcB control aerobic growth of Actinobacillus actinomycetemcomitans under iron limitation. Infect Immun 71:298–308.

25.

Frias J, Olle E, Alsina M (2001). Periodontal pathogens produce quorum sensing signal molecules. Infect Immun 69:3431–3434.

26.

Givskov M, de Nys R, Manefield M, Gram L, Maximilien R, Eberl L, et al. (1996). Eukaryotic interference with homoserine lactone-mediated prokaryotic signalling. J Bacteriol 178:6618–6622.

27.

Gram L, de Nys R, Maximilien R, Givskov M, Steinberg P, Kjelleberg S (1996). Inhibitory effects of secondary metabolites from the red alga Delisea pulchra on swarming motility of Proteus mirabilis. Appl Envir Microbiol 62:4284–4287.

28.

Hancock RE (1999). Host defence (cationic) peptides: what is their future clinical potential? Drugs 57:469–473.

29.

Håvarstein LS, Gaustad P, Nes IF, Morrison DA (1996). Identification of the streptococcal competence-pheromone receptor. Mol Microbiol 21:863–869.

30.

Håvarstein LS, Hakenbeck R, Gaustad P (1997). Natural competence in the genus Streptococcus: evidence that streptococci can change pherotype by interspecies recombinational exchanges. J Bacteriol 179:6589–6594.

31.

Hentzer M, Riedel K, Rasmussen TB, Heydorn A, Andersen JB, Parsek MR, et al. (2002). Inhibition of quorum sensing in Pseudomonas aeruginosa biofilm bacteria by a halogenated furanone compound. Microbiology 148(Pt 1):87–102.

32.

Hillman JD, Brooks TA, Michalek SM, Harmon CC, Snoep JL, van Der Weijden CC (2000). Construction and characterization of an effector strain of Streptococcus mutans for replacement therapy of dental caries. Infect Immun 68:543–549.

33.

Hoang TT, Schweizer HP (1999). Characterization of Pseudomonas aeruginosa enoyl-acyl carrier protein reductase (FabI): a target for the antimicrobial triclosan and its role in acylated homoserine lactone synthesis. J Bacteriol 181:5489–5497.

34.

Hoch JA (2000). Two-component and phosphorelay signal transduction. Curr Opin Microbiol 3:165–170.

35.

Huang Y, Hajishengallis G, Michalek SM (2001). Induction of protective immunity against Streptococcus mutans colonization after mucosal immunization with attenuated Salmonella enterica serovar typhimurium expressing an S. mutans adhesin under the control of in vivo-inducible nirB promoter. Infect Immun 69:2154–2161.

36.

Koga T, Oho T, Shimazaki Y, Nakano Y (2002). Immunization against dental caries. Vaccine 20:2027–2044.

37.

Kolenbrander PE, Andersen RN, Blehert DS, Egland PG, Foster JS, Palmer RJ Jr (2002). Communication among oral bacteria. Microbiol Mol Biol Rev 66:486–505.

38.

Leadbetter JR, Greenberg EP (2000). Metabolism of acyl-homoserine lactone quorum-sensing signals by Variovorax paradoxus. J Bacteriol 182:6921–6926.

39.

Lewis K (2001). Riddle of biofilm resistance. Antimicrob Agents Chemother 45:999–1007.

40.

Li YH, Chen YY, Burne RA (2000). Regulation of urease gene expression by Streptococcus salivarius growing in biofilms. Environ Microbiol 2:169–177.

41.

Li YH, Lau PC, Lee JH, Ellen RP, Cvitkovitch DG (2001a). Natural genetic transformation of Streptococcus mutans growing in biofilms. J Bacteriol 183:897–908.

42.

Li YH, Hanna MN, Svensäter G, Ellen RP, Cvitkovitch DG (2001b). Cell density modulates acid adaptation in Streptococcus mutans: implications for survival in biofilms. J Bacteriol 183:6875–6884.

43.

Li YH, Tang N, Aspiras MB, Lau PC, Lee JH, Ellen RP, et al. (2002a). A quorum-sensing signaling system essential for genetic competence in Streptococcus mutans is involved in biofilm formation. J Bacteriol 184:2699–2708.

44.

Li YH, Lau PC, Tang N, Svensäter G, Ellen RP, Cvitkovitch DG (2002b). Novel two-component regulatory system involved in biofilm formation and acid resistance in Streptococcus mutans. J Bacteriol 184:6333–6342.

45.

Loo CY, Corliss DA, Ganeshkumar N (2000). Streptococcus gordonii biofilm formation: identification of genes that code for biofilm phenotypes. J Bacteriol 182:1374–1382.

46.

Lyon GJ, Mayville P, Muir TW, Novick RP (2000). Rational design of a global inhibitor of the virulence response in Staphylococcus aureus, based in part on localization of the site of inhibition to the receptor-histidine kinase, AgrC. Proc Natl Acad Sci USA 97:13330–13335.

47.

Ma JK, Hikmat BY, Wycoff K, Vine ND, Chargelegue D, Yu L, et al. (1998). Characterization of a recombinant plant monoclonal secretory antibody and preventive immunotherapy in humans. Nat Med 4:601–606.

48.

Mah TF, O’Toole GA (2001). Mechanisms of biofilm resistance to antimicrobial agents. Trends Microbiol 9:34–39.

49.

Manefield M, Rasmussen TB, Henzter M, Andersen JB, Steinberg P, Kjelleberg S, et al. (2002). Halogenated furanones inhibit quorum sensing through accelerated LuxR turnover. Microbiology 148(Pt 4):1119–1127.

50.

Marsh PD (1994). Microbial ecology of dental plaque and its significance in health and disease. Adv Dent Res 8:263–271.

51.

Matsushita M, Janda KD (2002). Histidine kinases as targets for new antimicrobial agents. Bioorg Med Chem 10:855–867.

52.

McNab R, Ford SK, El-Sabaeny A, Barbieri B, Cook GS, Lamont RJ (2003). LuxS-based signaling in Streptococcus gordonii: autoinducer 2 controls carbohydrate metabolism and biofilm formation with Porphyromonas gingivalis. J Bacteriol 185:274–284.

53.

Merritt J, Qi F, Goodman SD, Anderson MH, Shi W (2003). Mutation of luxS affects biofilm formation in Streptococcus mutans. Infect Immun 71:1972–1979.

54.

Miller MB, Bassler BL (2001). Quorum sensing in bacteria. Annu Rev Microbiol 55:165–199.

55.

Olsen JA, Severinsen R, Rasmussen TB, Hentzer M, Givskov M, Nielsen J (2002). Synthesis of new 3- and 4-substituted analogues of acyl homoserine lactone quorum sensing autoinducers. Bioorg Med Chem Lett 12:325–328.

56.

Olsson J (1998). Inhibition of dental plaque by chemical surface modification. In: Oral biofilms and plaque control. Busscher HJ, Evans LV, editors. Amsterdam: Harwood Academic Publisher, pp. 295–309.

57.

Parkins MD, Ceri H, Storey DG (2001). Pseudomonas aeruginosa GacA, a factor in multihost virulence, is also essential for biofilm formation. Mol Microbiol 40:1215–1226.

58.

Payne DJ, Warren PV, Holmes DJ, Ji Y, Lonsdale JT (2001). Bacterial fatty-acid biosynthesis: a genomics-driven target for antibacterial drug discovery. Drug Discov Today 6:537–544.

59.

Petersen FC, Scheie AAa (1998). Chemical plaque control: a comparison of oral health care products. In: Oral biofilms and plaque control. Busscher HJ, Evans LV, editors. Amsterdam: Harwood Academic Publisher, pp. 277–293.

60.

Petersen FC, Scheie AAa (2000). Genetic transformation in Streptococcus mutans requires a peptide secretion-like apparatus. Oral Microbiol Immunol 15:329–334.

61.

Pratt LA, Kolter R (1999). Genetic analyses of bacterial biofilm formation. Curr Opin Microbiol 2:598–603.

62.

Qi F, Chen P, Caufield PW (2001). The group I strain of Streptococcus mutans, UA140, produces both the lantibiotic mutacin I and a nonlantibiotic bacteriocin, mutacin IV. Appl Environ Microbiol 67:15–21.

63.

Reverchon S, Chantegrel B, Deshayes C, Doutheau A, Cotte-Pattat N (2002). New synthetic analogues of N-acyl homoserine lactones as agonists or antagonists of transcriptional regulators involved in bacterial quorum sensing. Bioorg Med Chem Lett 12:1153–1157.

64.

Sauer K, Camper AK, Ehrlich GD, Costerton JW, Davies DG (2002). Pseudomonas aeruginosa displays multiple phenotypes during development as a biofilm. J Bacteriol 184:1140–1154.

65.

Schauder S, Shokat K, Surette MG, Bassler BL (2001). The LuxS family of bacterial autoinducers: biosynthesis of a novel quorum-sensing signal molecule. Mol Microbiol 41:463–476.

66.

Scheie AAa (2003). The role of antimicrobials. In: Dental caries. The disease and its clinical management. Fejerskov O, Kidd E, editors. Oxford: Blackwell Munksgaard, pp. 179–189.

67.

Sharma A, Honma K, Evans RT, Hruby DE, Genco RJ (2001). Oral immunization with recombinant Streptococcus gordonii expressing Porphyromonas gingivalis FimA domains. Infect Immun 69:2928–2934.

68.

Shimazaki Y, Mitoma M, Oho T, Nakano Y, Yamashita Y, Okano K, et al. (2001). Passive immunization with milk produced from an immunized cow prevents oral recolonization by Streptococcus mutans. Clin Diagn Lab Immunol 8:1136–1139.

69.

Smith DJ (2002). Dental caries vaccines: prospects and concerns. Crit Rev Oral Biol Med 13:335–349.

70.

Smith KM, Bu Y, Suga H (2003). Induction and inhibition of Pseudomonas aeruginosa quorum sensing by synthetic autoinducer analogs. Chem Biol 10:81–89.

71.

Socransky S (2002). Dental biofilms: difficult therapeutic targets. Periodontol 2000 28:12–15.

72.

Stephenson K, Hoch JA (2002). Histidine kinase-mediated signal transduction systems of pathogenic micro-organisms as targets for therapeutic intervention. Curr Drug Targets Infect Disord 2:235–246.

73.

Stewart PS, Costerton JW (2001). Antibiotic resistance of bacteria in biofilms. Lancet 358:135–138.

74.

Stoodley P, Sauer K, Davies DG, Costerton JW (2002). Biofilms as complex differentiated communities. Annu Rev Microbiol 56:187–209.

75.

Sulakvelidze A, Morris JG Jr (2001). Bacteriophages as therapeutic agents. Ann Med 33:507–509.

76.

Sutherland IW (2001). Biofilm exopolysaccharides: a strong and sticky framework. Microbiology 147:3–9.

77.

Svensäter G, Welin J, Wilkins JC, Beighton D, Hamilton IR (2001). Protein expression by planktonic and biofilm cells of Streptococcus mutans. FEMS Microbiol Lett 205:139–146.

78.

Taubman MA, Holmberg CJ, Smith DJ (2001). Diepitopic construct of functionally and epitopically complementary peptides enhances immunogenicity, reactivity with glucosyltransferase, and protection from dental caries. Infect Immun 69:4210–4216.

79.

Upton M, Tagg JR, Wescombe P, Jenkinson HF (2001). Intra- and interspecies signaling between Streptococcus salivarius and Streptococcus pyogenes mediated by SalA and SalA1 lantibiotic peptides. J Bacteriol 183:3931–3938.

80.

Wen ZT, Burne RA (2002). Functional genomics approach to identifying genes required for biofilm development by Streptococcus mutans. Appl Environ Microbiol 68:1196–203.

81.

Whatmore AM, Barcus VA, Downson CG (1999). Genetic diversity of the streptococcal competence (com) gene locus. J Bacteriol 181:3144–3154.

82.

Whitchurch CB, Erova TE, Emery JA, Sargent JL, Harris JM, Semmler AB, et al. (2002). Phosphorylation of the Pseudomonas aeruginosa response regulator AlgR is essential for type IV fimbria-mediated twitching motility. J Bacteriol 184:4544–4554.

83.

Whittaker CJ, Klier CM, Kolenbrander PE (1996). Mechanisms of adhesion by oral bacteria. Annu Rev Microbiol 50:513–552.

84.

Winans S, Bassler BL (2002). Mob psychology. Meeting report. J Bacteriol 184:873–883.

85.

Winzer K, Hardie KR, Williams P (2002). Bacterial cell-to-cell communication: sorry, can’t talk now—gone to lunch! Curr Opin Microbiol 5:216–222.

86.

Wu C, Savitt E (2002). Evaluation of the safety and efficacy of over-the-counter oral hygiene products for the reduction and control of dental plaque and gingivitis. Periodontol 2000 28:91–105.

87.

Xu F, Byun T, Dussen HJ, Duke KR (2003). Degradation of N-acylhomoserine lactones, the bacterial quorum-sensing molecules, by acylase. J Biotechnol 101:89–96.

88.

Yoshida A, Kuramitsu HK (2002). Multiple Streptococcus mutans genes are involved in biofilm formation. Appl Environ Microbiol 68:6283–6291.

89.

Yother J, Trieu-Cuot P, Klaenhammer TR, De Vos WM (2002). Genetics of streptococci, lactococci, and enterococci: review of the sixth international conference. J Bacteriol 184:6085–6092.