Effects of pH and Lactate on Hydrogen Sulfide Production by Oral Veillonella spp.

Indigenous oral bacteria in the tongue coating such as Veillonella have been identified as the main producers of hydrogen sulfide (H₂S), one of the major components of oral malodor. However, there is little information on the physiological properties of H₂S production by oral Veillonella such as metabolic activity and oral environmental factors which may affect H₂S production. Thus, in the present study, the H₂S-producing activity of growing cells, resting cells, and cell extracts of oral Veillonella species and the effects of oral environmental factors, including pH and lactate, were investigated. Type strains of Veillonella atypica, Veillonella dispar, and Veillonella parvula were used. These Veillonella species produced H₂S during growth in the presence of l-cysteine. Resting cells of these bacteria produced H₂S from l-cysteine, and the cell extracts showed enzymatic activity to convert l-cysteine to H₂S. H₂S production by resting cells was higher at pH 6 to 7 and lower at pH 5. The presence of lactate markedly increased H₂S production by resting cells (4.5- to 23.7-fold), while lactate had no effect on enzymatic activity in cell extracts. In addition to H₂S, ammonia was produced in cell extracts of all the strains, indicating that H₂S was produced by the catalysis of cystathionine γ-lyase (EC 4.4.1.1). Serine was also produced in cell extracts of V. atypica and V. parvula, suggesting the involvement of cystathionine β-synthase lyase (EC 4.2.1.22) in these strains. This study indicates that Veillonella produce H₂S from l-cysteine and that their H₂S production can be regulated by oral environmental factors, namely, pH and lactate.     

Molecular Relationships in Biofilm Formation and the Biosynthesis of Exoproducts in Pseudoalteromonas spp.

Marine Biotechnology - Most members of the Pseudoalteromonas genus have been isolated from living surfaces as members of epiphytic and epizooic microbiomes on marine macroorganisms. Commonly...     

Biofilm Formation of Streptococcus equi ssp. zooepidemicus and Comparative Proteomic Analysis of Biofilm and Planktonic Cells

Streptococcus equi ssp. zooepidemicus (SEZ) is responsible for a wide variety of infections in many species, including pigs, horses and humans. Biofilm formation is essential for pathogenesis, and the ability to resist antibiotic treatment results in difficult-to-treat and persistent infections. However, the ability of SEZ to form biofilms is unclear. Furthermore, the mechanisms underlying SEZ biofilm formation and their attributes are poorly understood. In this study, scanning electron microscopy (SEM) demonstrated that SEZ strain ATCC35246 formed biofilms comprising a thick, heterogeneous layer with clumps on the coverslips when incubated for 24 h. In addition, we used a two-dimensional gel electrophoresis (2-DE) based approach to characterize differentially expressed protein in SEZ biofilms compared with their planktonic counterparts. The results revealed the existence of 24 protein spots of varying intensities, 13 of which were upregulated and 11 were downregulated in the SEZ biofilm compared with the planktonic controls. Most of proteins expressed during biofilm formation were associated with metabolism, adhesion, and stress conditions. These observations contribute to our understanding of the SEZ biofilm lifestyle, which may lead to more effective measures to control persistent SEZ infections.     

Physiological Aspects and Conservation of a Veillonella Strain Isolated From the Oral Cavity. Interaction with Streptococci

Bacteria of the genus Veillonella are anaerobic, Gram-negative cocci which are found as normal flora in the human oral cavity. Because it grows well with the lactate produced by streptococci, antinomyces and lactobacilli in dental plaque, they could play an anticariogenic role. The aim of the present study was to investigate the kinetics and viability of a Veillonella strain isolated from saliva and its interaction with oral streptococci. The growth rate was determined in Lactate broth measuring turbidity and CFU/mL over 64 h. Preservation media were tested at −70°C, −20°C and room temperature to strain conservation. To study bacterial interactions between veillonella and streptococci, an active culture of veillonella was spread on Mitis Salivarius Agar plates, which is a culture medium selective for oral streptococci. The replication time of this veillonella strain was 7.2 h in Lactate Broth and the specific growth rate (μ) was 0.096 h. The elected medium for conservation was Preservation Broth with 25% Glycerol at all temperatures. The Muñiz Maintenance Medium and Muñiz Maintenance Medium with Glycerol 25% media may be used at any temperature for short time periods. The interaction between veillonella and streptococci seems to be a result of nutritional factors.     

Multiple Streptococcus mutans Genes Are Involved in Biofilm Formation

Streptococcus mutans has been strongly implicated as the principal etiological agent in dental caries. One of the important virulence properties of these organisms is their ability to form biofilms known as dental plaque on tooth surfaces. Since the roles of sucrose and glucosyltransferases in S. mutans biofilm formation have been well documented, we focused our attention on sucrose-independent factors. We have initially identified several mutants that appear to be defective in biofilm formation on abiotic surfaces by an insertional inactivation mutagenesis strategy applied to S. mutans. A total of 27 biofilm-defective mutants were isolated and analyzed in this study. From these mutants, three genes were identified. One of the mutants was defective in the Bacillus subtilis lytR homologue. Another of the biofilm-defective mutants isolated was a yulF homologue, which encodes a hypothetical protein of B. subtilis whose function in biofilm formation is unknown. The vast majority of the mutants were defective in the comB gene required for competence. We therefore have constructed and examined comACDE null mutants. These mutants were also found to be attenuated in biofilm formation. Biofilm formation by several other regulatory gene mutants were also characterized using an in vitro biofilm-forming assay. These results suggest that competence genes as well as the sgp and dgk genes may play important roles in S. mutans biofilm formation.     

Involvement of NADH Oxidase in Biofilm Formation in Streptococcus sanguinis

Biofilms play important roles in microbial communities and are related to infectious diseases. Here, we report direct evidence that a bacterial nox gene encoding NADH oxidase is involved in biofilm formation. A dramatic reduction in biofilm formation was observed in a Streptococcus sanguinis nox mutant under anaerobic conditions without any decrease in growth. The membrane fluidity of the mutant bacterial cells was found to be decreased and the fatty acid composition altered, with increased palmitic acid and decreased stearic acid and vaccenic acid. Extracellular DNA of the mutant was reduced in abundance and bacterial competence was suppressed. Gene expression analysis in the mutant identified two genes with altered expression, gtfP and Idh, which were found to be related to biofilm formation through examination of their deletion mutants. NADH oxidase-related metabolic pathways were analyzed, further clarifying the function of this enzyme in biofilm formation.     

Inhibition of Streptococcus pyogenes Biofilm Formation by Coral-Associated Actinomycetes

Streptococcus pyogenes biofilms tend to exhibit significant tolerance to antimicrobials during infections. We screened coral-associated actinomycetes (CAA) for antibiofilm activity against different biofilm forming M serotype of Streptococcus pyogenes. Actinomycetes isolated from the mucus of the coral Acropora digitifera were screened for antibiofilm activity against S. pyogenes biofilms wherein several isolates clearly demonstrated antibiofilm activity. The biofilm inhibitory concentrations (BICs) and the sub-BICs (1/2 and 1/4 BIC) of the extracts significantly prevented biofilm formation up to 60–80%. The extract of Streptomyces akiyoshinensis (A3) displayed efficient antibiofilm activity against all the biofilm forming M serotypes. All the five extracts efficiently reduced the cell surface hydrophobicity (a crucial factor for biofilm formation in S. pyogenes) of three M types and thus may inhibit biofilm formation. CAA represent an interesting source of marine invertebrates-derived antibiofilm agents in the development of new strategies to combat Streptococcal biofilms.     

The Exopolysaccharide Matrix Modulates the Interaction between 3D Architecture and Virulence of a Mixed-Species Oral Biofilm

Virulent biofilms are responsible for a range of infections, including oral diseases. All biofilms harbor a microbial-derived extracellular-matrix. The exopolysaccharides (EPS) formed on tooth-pellicle and bacterial surfaces provide binding sites for microorganisms; eventually the accumulated EPS enmeshes microbial cells. The metabolic activity of the bacteria within this matrix leads to acidification of the milieu. We explored the mechanisms through which the Streptococcus mutans-produced EPS-matrix modulates the three-dimensional (3D) architecture and the population shifts during morphogenesis of biofilms on a saliva-coated-apatitic surface using a mixed-bacterial species system. Concomitantly, we examined whether the matrix influences the development of pH-microenvironments within intact-biofilms using a novel 3D in situ pH-mapping technique. Data reveal that the production of the EPS-matrix helps to create spatial heterogeneities by forming an intricate network of exopolysaccharide-enmeshed bacterial-islets (microcolonies) through localized cell-to-matrix interactions. This complex 3D architecture creates compartmentalized acidic and EPS-rich microenvironments throughout the biofilm, which triggers the dominance of pathogenic S. mutans within a mixed-species system. The establishment of a 3D-matrix and EPS-enmeshed microcolonies were largely mediated by the S. mutans gtfB/gtfC genes, expression of which was enhanced in the presence of Actinomyces naeslundii and Streptococcus oralis. Acidic pockets were found only in the interiors of bacterial-islets that are protected by EPS, which impedes rapid neutralization by buffer (pH 7.0). As a result, regions of low pH (<5.5) were detected at specific locations along the surface of attachment. Resistance to chlorhexidine was enhanced in cells within EPS-microcolony complexes compared to those outside such structures within the biofilm. Our results illustrate the critical interaction between matrix architecture and pH heterogeneity in the 3D environment. The formation of structured acidic-microenvironments in close proximity to the apatite-surface is an essential factor associated with virulence in cariogenic-biofilms. These observations may have relevance beyond the mouth, as matrix is inherent to all biofilms.     

牙菌斑链球菌与牙龈卟啉单胞菌相互作用的研究

牙菌斑生物膜是牙周病最主要的致病因素。早期定植菌链球菌与晚期定植菌牙龈卟啉单胞菌(P.gingivalis)的相互作用复杂多样,而牙龈卟啉单胞菌是重要的牙周致病菌,本文就链球菌与牙龈卟啉单胞菌的相互作用作一综述。     

Biofilm Formation on Reverse Osmosis Membranes Is Initiated and Dominated by Sphingomonas spp.

The initial formation and spatiotemporal development of microbial biofilm layers on surfaces of new and clean reverse osmosis (RO) membranes and feed-side spacers were monitored in situ using flow cells placed in parallel with the RO system of a full-scale water treatment plant. The feed water of the RO system had been treated by the sequential application of coagulation, flocculation, sand filtration, ultrafiltration, and cartridge filtration processes. The design of the flow cells permitted the production of permeate under cross-flow conditions similar to those in spiral-wound RO membrane elements of the full-scale system. Membrane autopsies were done after 4, 8, 16, and 32 days of flow-cell operation. A combination of molecular (fluorescence in situ hybridization [FISH], denaturing gradient gel electrophoresis [DGGE], and cloning) and microscopic (field emission scanning electron, epifluorescence, and confocal laser scanning microscopy) techniques was applied to analyze the abundance, composition, architecture, and three-dimensional structure of biofilm communities. The results of the study point out the unique role of Sphingomonas spp. in the initial formation and subsequent maturation of biofilms on the RO membrane and feed-side spacer surfaces.In the water production industry, reverse osmosis (RO) membrane technology is a durable, promising, and much-used separation method. Its application enables the efficient removal of a wide variety of contaminants (i.e., microbial constituents, total dissolved solids, and organic compounds). Feed streams of different qualities (e.g., raw, natural, chemically contaminated or brackish, and seawater) are used to produce high-purity water that is microbiologically safe and biologically stable (15, 25). However, the widespread application of this technology is limited because the current generation of RO filtration units experience biofouling problems (14). The design of so-called “spiral wound” membrane elements and the conditions at the membrane, feed-side spacer, and other internal surfaces within these RO filters make them prone to microbial attachment and the subsequent formation of biofilm layers. A variety of microorganisms are involved in the development of these surface-attached complex structures after prolonged operation of the RO system, depending on the type and concentration of contaminants in the feed water and the type of pretreatment (5, 6, 7, 32, 38). The biofilm occurrence is a principal problem for proper RO system performance. It can lead to blocking of the feed concentrate channel and to clogging of the membrane. Biofilm formation results in an increased energy requirement of the feed water pumps, a lower flux, and a decrease of permeate quality (14). Conventional prevention and/or management strategies of biofouling-caused problems require more frequent chemical cleanings, thereby leading to a shortened membrane life and, ultimately, to a loss of capacity of the water supply plant (3, 14). Finding more effective ways to deal with biofouling problems in the current RO systems still needs more fundamental investigations of all aspects of biofilm formation. Little is known about the microbial community that makes up the biofilm on the membranes. To diagnose biofouling and to choose the most appropriate pretreatment and cleaning strategies, the pressure difference between the inlet and outlet channels and microbial biomass concentrations can be determined (48). Additional microbiological research, such as total cell and heterotrophic plate counts, provides some basic information (12, 23). However, such experiments do not allow for a reliable evaluation of microbial abundance and diversity of species, because the majority of the microorganisms in ecosystems cannot be cultured (21). While knowledge of real biofilm microbial composition is essential in identifying the most effective cleaning protocols, only a few molecular-based microbial diversity studies on RO membrane surfaces are reported (5, 6, 7, 32). In addition, limited data about the formation and development of biofilms over time are available. What little is known comes from laboratory-controlled biofilm monitoring studies using one or a few bacterial strains for biofilm formation (18, 19). These studies, therefore, may not provide a true representation of the RO biofilm problem in situ.In this study, we investigated microbial biofilm formation in an experimental setup similar to an authentic RO system. Using stainless steel flow cells connected in parallel to the reverse osmosis system of a full-scale water treatment plant, the spatiotemporal development of microbial biofilms on the surfaces of new and clean reverse osmosis membranes and feed-side spacers was monitored. The bacteria responsible for the initial colonization and development of the biofilms were identified by various molecular and microscopic techniques.     

Streptococcus mutans Can Modulate Biofilm Formation and Attenuate the Virulence of Candida albicans

Streptococcus mutans and Candida albicans are found together in the oral biofilms on dental surfaces, but little is known about the ecological interactions between these species. Here, we studied the effects of S. mutans UA159 on the growth and pathogencity of C. albicans. Initially, the effects of S. mutans on the biofilm formation and morphogenesis of C. albicans were tested in vitro. Next, we investigate the influence of S. mutans on pathogenicity of C. albicans using in vivo host models, in which the experimental candidiasis was induced in G. mellonella larvae and analyzed by survival curves, C. albicans count in hemolymph, and quantification of hyphae in the host tissues. In all the tests, we evaluated the direct effects of S. mutans cells, as well as the indirect effects of the subproducts secreted by this microorganism using a bacterial culture filtrate. The in vitro analysis showed that S. mutans cells favored biofilm formation by C. albicans. However, a reduction in biofilm viable cells and inhibition of hyphal growth was observed when C. albicans was in contact with the S. mutans culture filtrate. In the in vivo study, injection of S. mutans cells or S. mutans culture filtrate into G. mellonella larvae infected with C. albicans increased the survival of these animals. Furthermore, a reduction in hyphal formation was observed in larval tissues when C. albicans was associated with S. mutans culture filtrate. These findings suggest that S. mutans can secrete subproducts capable to inhibit the biofilm formation, morphogenesis and pathogenicity of C. albicans, attenuating the experimental candidiasis in G. mellonella model.     

粪肠球菌生物被膜形成早期相关基因的表达研究

目的:以粪肠球菌为研究对象,探讨粪肠球菌基因srtA(转肽酶A编码基因)、esp(肠球菌表面蛋白)与粪肠球菌生物被膜形成早期的相关性。方法:用逆转录PCR与实时荧光定量PCR方法对生物被膜和浮游菌组细菌srtA、esp两种与生物被膜形成早期相关的基因其表达进行检测,并进行统计学分析。结果:srtA、esp基因与粪肠球生物被膜菌早期形成密切相关。生物被膜菌组srtA、esp表达量分别是浮游菌组的7.9与13.5倍。结论:srtA、esp基因与粪肠球生物被膜菌形成早期密切相关,可能是生物被膜早期形成的上调因子。     

Central Role of the Early Colonizer Veillonella sp. in Establishing Multispecies Biofilm Communities with Initial,Middle, and Late Colonizers of Enamel

Human dental biofilm communities comprise several species, which can interact cooperatively or competitively. Bacterial interactions influence biofilm formation, metabolic changes, and physiological function of the community. Lactic acid, a common metabolite of oral bacteria, was measured in the flow cell effluent of one-, two- and three-species communities growing on saliva as the sole nutritional source. We investigated single-species and multispecies colonization by using known initial, early, middle, and late colonizers of enamel. Fluorescent-antibody staining and image analysis were used to quantify the biomass in saliva-fed flow cells. Of six species tested, only the initial colonizer Actinomyces oris exhibited significant growth. The initial colonizer Streptococcus oralis produced lactic acid but showed no significant growth. The early colonizer Veillonella sp. utilized lactic acid in two- and three-species biofilm communities. The biovolumes of all two-species biofilms increased when Veillonella sp. was present as one of the partners, indicating that this early colonizer promotes mutualistic community development. All three-species combinations exhibited enhanced growth except one, i.e., A. oris, Veillonella sp., and the middle colonizer Porphyromonas gingivalis, indicating specificity among three-species communities. Further specificity was seen when Fusobacterium nucleatum (a middle colonizer), Aggregatibacter actinomycetemcomitans (a late colonizer), and P. gingivalis did not grow with S. oralis in two-species biofilms, but inclusion of Veillonella sp. resulted in growth of all three-species combinations. We propose that commensal veillonellae use lactic acid for growth in saliva and that they communicate metabolically with initial, early, middle, and late colonizers to establish multispecies communities on enamel.The human oral cavity contains a widely diverse community of resident bacteria composed of several hundred species (1, 18). They organize into multispecies communities through a recurrent sequence of colonization that occurs after each oral hygiene treatment; for example, dental plaque development on enamel starts with the initial colonizers streptococci and actinomyces (7, 15), which are followed by early-colonizing veillonellae (7, 11, 14), middle-colonizing porphyromonads (7) and fusobacteria (7, 10, 11), and late-colonizing aggregatibacters (9).During the initial stage of biofilm formation, streptococci and actinomyces bind to host-derived receptors in the salivary pellicle coating of enamel. In turn, other species bind to already-adherent cells, a process called coadhesion (2). This process and coaggregation (10), defined as specific cell-to-cell recognition between genetically distinct cells, as well as growth of adherent cells contribute to dental plaque development. While it is known that pure cultures of oral bacteria metabolize dietary sugars to lactic acid, little is known about the importance of lactic acid to community growth on saliva as a sole nutrient source. Most pure cultures and many combinations of species are unable to grow on whole saliva, which is a complex nutritional source. Growth might, in fact, require spatial organization and mutualistic interactions among selected species that collectively possess a combination of metabolic properties that are capable of converting latent nutrition into usable nutrition. In succession, groups of other selected species with other combined metabolic capabilities can further process this complex nutritional source, with a resultant assembling and disassembling of constantly changing oral biofilm communities.Streptococci make up 60 to 90% of the supragingival plaque biomass in the first 24 h of colonization (12, 15). They catabolize carbohydrates to short-chain organic acids, such as lactic acid and pyruvic acid (4). Veillonellae constitute as much as 5% of the initial plaque biomass but are unable to catabolize sugars. They rely on the fermentation of organic acids such as lactic acid (6) and thus set up a convenient metabolic food chain in dental plaque.In vivo studies using gnotobiotic rats demonstrated that veillonellae were unable to establish monoinfections. Yet when a strain of Veillonella was inoculated into rats already monoinfected with a strain of Streptococcus mutans that coaggregates with that Veillonella strain, the number of veillonellae on the teeth of the coinfected animals was 1,000-fold higher than the number when a noncoaggregating Veillonella strain was used (13). Also, in gnotobiotic rats, lower caries and plaque scores were obtained for two-species biofilms than for single-species colonization by streptococci, and inclusion of veillonellae reduced caries activity and demineralization of the enamel by streptococci (13). Streptococcus-Veillonella communities containing coaggregation partners were micromanipulated from 8-h human dental plaque, providing additional evidence of the close association of these two species in vivo (3). Further, Veillonella spp. are juxtaposed with coaggregation receptor polysaccharide-bearing streptococci in early communities in vivo, and a rapid succession of veillonella phylotypes occurs in these communities (16). These reports offer broad-based evidence that veillonellae and streptococci are linked in oral biofilms.The focus of the current investigation was to explore Veillonella-based mixed-species communities in saliva-fed flow cells. The concentration of lactic acid in the effluent of flow cells containing biofilm communities was determined. We hypothesize that spatiotemporal metabolic interactions and coaggregation of Veillonella sp. with Streptococcus oralis and early, middle, and late colonizers allow these organisms to form three-species biofilm communities. We show high specificity of community partnerships among the six species examined, suggesting that successions of species in naturally recurring dental plaque in vivo are centered on metabolic and physical interactions of the community participants which support the nonrandom sequential appearance of species in the development of oral biofilms.     

Chlorophyll Formation in Greening Bean Leaves during the Early Stages

In the evolution of the absorption spectrum of etiolated bean leaves (Phaseolus vulgaris L.) following illumination, a rapid photoconversion of 50% or more of the active protochlorophyllide at room temperature is followed by a shift of the chlorophyll(ide) absorption maximum: C₆₇₈→ →C₆₈₄→C_{672 nm}. Kinetic studies at 2 C and the absence of an isosbestic point provide evidence for an intermediate between C₆₇₈ and C₆₈₄. A dramatically different evolution is observed following the photoconversion of only 5 to 30% of the active protochlorophyllide at room temperature. C₆₇₂ appears within 30 seconds, and no subsequent dark shift occurs during the following 90 minutes. At 0 C, conversion of 5% of the active protochlorophyllide produces a new species, C₆₇₆, which converts progressively to C₆₇₂ within 10 minutes. We interpret the results in terms of two photochemical steps operating in series for the complete conversion of active protochlorophyllide. Furthermore, there appears to be competition between an irreversible, terminal dark shift and the second light reaction. We propose a scheme based on dimers of protochlorophyllide reduced stepwise to dimers of chlorophyllide in two successive light reactions. The intermediate mixed protochlorophyllide-chlorophyllide dimer absorbs at 676 nm and displays a much faster dissociation to monomers than does the chlorophyllide-chlorophyllide dimer.     

Effect of the Environment on Genotypic Diversity of Actinomyces naeslundii and Streptococcus oralis in the Oral Biofilm

The genotypic diversity of Actinomyces naeslundii genospecies 2 (424 isolates) and Streptococcus oralis (446 isolates) strains isolated from two sound approximal sites in all subjects who were either caries active (seven subjects) or caries free (seven subjects) was investigated by using the repetitive extragenic palindromic PCR. The plaque from the caries-active subjects harbored significantly greater proportions of mutans streptococci and lactobacilli and a smaller proportion of A. naeslundii organisms than the plaque sampled from the caries-free subjects. These data confirmed that the sites of the two groups of subjects were subjected to different environmental stresses, probably determined by the prevailing or fluctuating acidic pH values. We tested the hypothesis that the microfloras of the sites subjected to greater stresses (the plaque samples from the caries-active subjects) would exhibit reduced genotypic diversity since the sites would be less favorable. We found that the diversity of A. naeslundii strains did not change (χ2 = 0.68; P = 0.41) although the proportional representation of A. naeslundii was significantly reduced (P < 0.05). Conversely, the diversity of the S. oralis strains increased (χ2 = 11.71; P = 0.0006) and the proportional representation of S. oralis did not change. We propose that under these environmental conditions the diversity and number of niches within the oral biofilm that could be exploited by S. oralis increased, resulting in the increased genotypic diversity of this species. Apparently, A. naeslundii was not able to exploit the new niches since the prevailing conditions within the niches may have been deleterious and not supportive of its proliferation. These results suggest that environmental stress may modify a biofilm such that the diversity of the niches is increased and that these niches may be successfully exploited by some, but not necessarily all, members of the microbial community.     

内生菌与植物的相互作用：促生与生物薄膜的形成

植物内生菌由于其独特的生态学地位而广受关注,近年来有关植物内生菌与宿主相互作用的研究取得了很大进展.本文综述了植物内生菌通过分泌促生物质、拮抗病原菌等实现与宿主共生互作,同时植物为内生菌提供适宜的黏附表面,使其形成以生物薄膜(biofilm)为主要形式的多细胞聚集体结构以更好地适应周围的生存环境,从而更加高效地对植物产生促生作用.本文论述了内生菌在与植物的互作中形成的多细胞聚集结构在抵抗非生物胁迫方面的独特生理及生态学意义,结合水稻内生成团泛菌YS19形成多细胞聚集体symplasmata现象及其生物学效应,对未来有关植物内生菌的研究方向提出了一些看法.     

Protein Expression by Streptococcus mutans during Initial Stage of Biofilm Formation

Cells growing on surfaces in biofilms exhibit properties distinct from those of planktonic cells, such as increased resistance to biocides and antimicrobial agents. In spite of increased interest in biofilms, very little is known about alterations in cell physiology that occur upon attachment of cells to a surface. In this study we have investigated the changes induced in the protein synthesis by contact of Streptococcus mutans with a surface. Log-phase planktonic cells of S. mutans were allowed to adhere to a glass slide for 2 h in the presence of a ¹⁴C-amino acid mixture. Nonadhered cells were washed away, and the adhered cells were removed by sonication. The proteins were extracted from the nonadhered planktonic and the adhered biofilm cells and separated by two-dimensional gel electrophoresis followed by autoradiography and image analysis. Image analysis revealed that the relative rate of synthesis of 25 proteins was enhanced and that of 8 proteins was diminished ≥1.3-fold in the biofilm cells. Proteins of interest were identified by mass spectrometry and computer-assisted protein sequence analysis. Of the 33 proteins associated with the adhesion response, all but 10 were identified by mass spectrometry and peptide mass fingerprinting. The most prominent change in adhered cells was the increase in relative synthesis of enzymes involved in carbohydrate catabolism indicating that a redirection in protein synthesis towards energy generation is an early response to contact with and adhesion to a surface.