Coaggregation among nonflocculating bacteria isolated from activated sludge

Thirty-two strains of nonflocculating bacteria isolated from sewage-activated sludge were tested by a spectrophotometric assay for their ability to coaggregate with one other in two-membered systems. Among these strains, eight showed significant (74 to 99%) coaggregation with Acinetobacter johnsonii S35 while only four strains coaggregated, to a lesser extent (43 to 65%), with Acinetobacter junii S33. The extent and pattern of coaggregation as well as the aggregate size showed good correlation with cellular characteristics of the coaggregating partners. These strains were identified by sequencing of full-length 16S rRNA genes. A. johnsonii S35 could coaggregate with strains of several genera, such as Oligotropha carboxidovorans, Microbacterium esteraromaticum, and Xanthomonas spp. The role of Acinetobacter isolates as bridging organisms in multigeneric coaggregates is indicated. This investigation revealed the role of much-neglected nonflocculating bacteria in floc formation in activated sludge.     

Intergeneric coaggregations among Oligotropha carboxidovorans and Acinetobacter species present in activated sludge

The coaggregation traits of two pairs of sewage sludge bacteria were tested and characterized. Oligotropha carboxidovorans S23 coaggregated with two strains of the genus Acinetobacter viz. Acinetobacter junii S33 (56%) and Acinetobacter johnsonii S35 (99%). Coaggregates of O. carboxidovorans S23 and A. junii S33 were small (20-40 microm), weak and susceptible to EDTA and a commercial protease (Actinase E). Actinase/periodate pretreatment of the partners prior to coaggregation revealed that interaction in this case was mediated by protein surface components. Coaggregates of O. carboxidovorans S23 and A. johnsonii S35 were large (above 100 microm), strong and not deflocculated by EDTA or Actinase E. Only periodate pretreatment of A. johnsonii S35 prevented this coaggregation indicating a role for a carbohydrate-containing moiety without the involvement of protein components. The potential mechanisms and strength of bacterial coaggregations seem to be pair dependent.     

Effect of cultivation time and growth medium on the coaggregation capability of <Emphasis Type="Italic">Acinetobacter johnsonii</Emphasis> S35

The effects of different cultivation periods and growth media on the cell surface hydrophobicity and coaggregation ability of Acinetobacter johnsonii S35 with Oligotropha carboxidovorans S23 (both sewage sludge isolates) were examined. It was observed that when cultivated in polypeptone medium, A. johnsonii S35 shows high hydrophobicity and strong coaggregation (98% aggregation index) with O. carboxidovorans S23, which remain unaltered after a 1-day, 3-day or 7-day cultivation period. When A. johnsonii S35 was cultivated with different carbon substrates, large coaggregates and a high aggregation index were observed using acetate-grown cells (96%), followed by ethanol-(83%) and vegetable oil-grown cells (72%). Coaggregates were small and narrow in ethanol-grown cells and still smaller in case of the vegetable oil-grown cells. Although no direct correlation could be observed, more hydrophobic cells showed a higher aggregation index. None of the culture supernatants of A. johnsonii S35 showed emulsification of hexadecane or a significant ability to aggregate O. carboxidovorans S23 cells, indicating that the coaggregation factor of A. johnsonii S35 is cell-bound. These results demonstrate that the coaggregation ability of A. johnsonii S35 is constitutive and is not lost under diverse cultivation conditions.     

Effect of surfactants on stability of Acinetobacter johnsonii S35 and Oligotropha carboxidovorans S23 coaggregates

The effect of anionic (sodium dodecyl sulphate or SDS) and cationic (cetyltrimethylammonium bromide or CTAB) surfactants on the stability of binary bacterial coaggregates comprising Acinetobacter johnsonii S35 and Oligotropha carboxidovorans S23 (both sewage sludge isolates) was studied and compared with that on the complex sewage sludge flocs. Both SDS and CTAB enhanced the bacterial coaggregation at their lower concentrations of 0.2 and 0.07 mg ml(-1), respectively. However, complete deflocculation of coaggregates was observed at 1 mg ml(-1) SDS and 0.3 mg l(-1) CTAB concentrations. Further, sewage sludge flocs did not deflocculate in the presence of CTAB, although a concentration-dependent deflocculation was observed in the presence of SDS. A. johnsonii S35 and O. carboxidovorans S23 cells were separately pretreated (prior to coaggregation) with the surfactants. In spite of the partial (complete) loss of viability during SDS (CTAB) pretreatment, washed cells still retained hydrophobic character and displayed significant coaggregation (aggregation index ranging from 84% to 97% in comparison to 96% in the case of non-treated cells), demonstrating reversibility of the surfactant induced deflocculation. Further, when exposed to lower concentration of surfactants (0.2 mg ml(-1) SDS), coaggregates were more resistant (76% viability) as compared to the individual partner (S35: 52%; S23: 39% viability). Since the coaggregates are stable and provide protection from surfactants at lower concentrations (those normally expected in the sewage treatment plants), their presence as well as a sustained role in the sewage sludge bioflocculation is evident.     

Novobiocin-resistant mutants of Streptococcus sanguis with reduced cell hydrophobicity and defective in coaggregation

Mutants of Streptococcus sanguis resistant to novobiocin (NovR-mutants) were isolated after mutagenesis of strain Challis with ethyl methanesulphonate. The resistance phenotype was transferred by DNA-mediated transformation back into the parent strain at high frequency suggesting resistance was due to mutation(s) in a single gene or in closely-linked genes. Cells of NovR-mutants had normal morphology and secreted similar proteins to the wild-type strain. However, mutant cultures had slower growth rates, the mutant cells had reduced hydrophobicity, and they showed a reduced degree of coaggregation with Actinomyces viscosus and Actinomyces naeslundii. Cell envelopes prepared from NovR-mutants differed from wild-type cell envelopes in that they (a) were impaired in ability to coaggregate with A. viscosus cells, and (b) had altered protein composition as detected by SDS-PAGE. The results suggest that hydrophobic proteins in the cell envelope of S. sanguis may be necessary for coaggregation of this bacterium with actinomycetes.     

Expression of the surface properties of the fibrillar Streptococcus salivarius HB and its adhesion deficient mutants grown in continuous culture under glucose limitation

Streptococcus salivarius HB and four adhesion deficient mutants, HB-7, HB-V5, HB-V51 and HB-B, were grown in continuous culture in a defined medium under glucose limitation over a range of growth rates from 0.1 to 1.1 h-1. The ability to coaggregate with Veillonella parvula V1 cells and the ability to adhere to buccal epithelial cells did not alter with increasing growth rate. Cell surface hydrophobicity decreased markedly with increasing growth rate for the non-fibrillar non-adhesive mutant HB-B but not for the other four strains which all carry different combinations of fibril classes. The thickness of the ruthenium red staining layer (RRL) also varied with growth rate for strain HB-B, ranging from 19.5 +/- 3.8 nm at high growth rate to a minimum of 12.3 +/- 4.8 nm at low growth rate. Low cell surface hydrophobicity correlated with a thicker RRL for strain HB-B. Strains HB-V5 and HB-7 also showed a significant increase in RRL thickness at high growth rates although to a lesser degree than HB-B. SDS-PAGE revealed a large number of protein bands common to all strains at all growth rates, with the major common protein occurring at 15.6 kDa. Protein bands at 70, 56, 40.5 and 39 kDa appeared stronger at high growth rates than at low. A protein band at 82 kDa showed strongly only at low growth rates. Therefore, adhesion and coaggregation are not phenotypically variable with increasing growth rate but RRL thickness, hydrophobicity and cell surface proteins may be phenotypically variable depending on the strain.     

Coaggregation among Nonflocculating Bacteria Isolated from Activated Sludge

Thirty-two strains of nonflocculating bacteria isolated from sewage-activated sludge were tested by a spectrophotometric assay for their ability to coaggregate with one other in two-membered systems. Among these strains, eight showed significant (74 to 99%) coaggregation with Acinetobacter johnsonii S35 while only four strains coaggregated, to a lesser extent (43 to 65%), with Acinetobacter junii S33. The extent and pattern of coaggregation as well as the aggregate size showed good correlation with cellular characteristics of the coaggregating partners. These strains were identified by sequencing of full-length 16S rRNA genes. A. johnsonii S35 could coaggregate with strains of several genera, such as Oligotropha carboxidovorans, Microbacterium esteraromaticum, and Xanthomonas spp. The role of Acinetobacter isolates as bridging organisms in multigeneric coaggregates is indicated. This investigation revealed the role of much-neglected nonflocculating bacteria in floc formation in activated sludge.     

Role of hydrogen peroxide in competition and cooperation between Streptococcus gordonii and Actinomyces naeslundii

In dental plaque alpha-haemolytic streptococci, including Streptococcus gordonii, are considered beneficial for oral health. These organisms produce hydrogen peroxide (H(2)O(2)) at concentrations sufficient to kill many oral bacteria. Streptococci do not produce catalase yet tolerate H(2)O(2). We recently demonstrated that coaggregation with Actinomyces naeslundii stabilizes arginine biosynthesis in S. gordonii. Protein arginine residues are sensitive to oxidation by H(2)O(2). Here, the ability of A. naeslundii to protect S. gordonii against self-produced H(2)O(2) was investigated. Coaggregation with A. naeslundii enabled S. gordonii to grow in the absence of arginine, and promoted survival of S. gordonii following growth with or without added arginine. Arginine-replete S. gordonii monocultures contained 20-30 microM H(2)O(2) throughout exponential growth. Actinomyces naeslundii did not produce H(2)O(2) but synthesized catalase, removed H(2)O(2) from coaggregate cultures and decreased protein oxidation in S. gordonii. On solid medium, S. gordonii inhibited growth of A. naeslundii; exogenous catalase overcame this inhibition. In coaggregate cultures, A. naeslundii cell numbers were >90% lower than in monocultures after 24 h. These results indicate that coaggregation with A. naeslundii protects S. gordonii from oxidative damage. However, high cell densities of S. gordonii inhibit A. naeslundii. Therefore, H(2)O(2) may drive these organisms towards an ecologically balanced community in natural dental plaque.     

Fimbria-associated proteins of Bacteroides loescheii PK1295 mediate intergeneric coaggregations

Bacteroides loescheii PK1295 serves as a coaggregation bridge between Streptococcus sanguis 34 and Actinomyces israelii PK14, two gram-positive oral bacteria that are otherwise unable to coaggregate. Whereas coaggregation with S. sanguis 34 is inhibited by lactose, no simple sugar was found that inhibited coaggregation with A. israelii PK14. Coaggregation-defective (Cog-) mutants of B. loescheii PK1295 were isolated for the purpose of identifying the surface components responsible for the interaction with each coaggregation partner. Selection for spontaneously occurring Cog- mutants gave rise to two phenotypic classes of mutants. Type I lost the ability to coaggregate with S. sanguis 34, whereas type II failed to coaggregate with either S. sanguis 34 or A. israelii PK14. Purified fimbriae from the parent agglutinated cells of both partners, and agglutination with S. sanguis 34 was inhibited by lactose. Denaturing polyacrylamide gel electrophoresis and immunoblot analysis demonstrated the presence of both a 75- and a 43-kilodalton (kDa) protein associated with parental fimbriae, but only a 43-kDa protein was seen with fimbriae prepared from the type I mutant. Neither polypeptide was found in similar preparations from the type II mutants. Our data suggest that coaggregation of B. loescheii PK1295 with both gram-positive partners is mediated by fimbria-associated proteins present on the surface of the gram-negative organism and that the 75- and 43-kDa polypeptides are responsible for the recognition of S. sanguis 34 and A. israelii PK14 cells, respectively.     

Influence of growth environment on coaggregation between freshwater biofilm bacteria

AIM: To characterize the expression of coaggregation between Blastomonas natatoria 2.1 and Micrococcus luteus 2.13 following growth in liquid culture, on agar and in an artificial biofilm matrix composed of poloxamer hydrogel. METHODS AND RESULTS: The ability of B. natatoria 2.1 and M. luteus 2.13 to coaggregate with one another was assessed following growth in liquid culture as colonies on agar or within a poloxamer hydrogel matrix. In all these environments a cycle of gain and loss of coaggregation occurred when the two cell types were aged simultaneously, with optimum expression occurring in early stationary phase. Blastomonas natatoria 2.1 cells only coaggregated maximally after entry into stationary phase. Conversely, M. luteus 2.13 cells only coaggregated in exponential phase and early stationary phase and coaggregation ability was lost in late stationary phase. Maximal coaggregation therefore only occurred between the two strains if both were in early stationary phase, when the surface properties of the two cell types were optimal for coaggregation. CONCLUSION: In addition to occurring between cells grown in liquid culture, coaggregation between aquatic bacteria occurs after growth as a biofilm on agar and in an artificial biofilm matrix in poloxamer. Under all conditions, the B. natatoria 2.1 coaggregation adhesin and complementary receptor on M. luteus 2.13 were only expressed simultaneously during early stationary phase.     

Physicochemical parameters influencing coaggregation between the freshwater bacteria Sphingomonas natatoria 2.1 and Micrococcus luteus 2.13

The aim of this study was to explore the physicochemical parameters that influence coaggregation between the freshwater bacteria Sphingomonas natatoria 2.1 and Micrococcus luteus 2.13. Using visual coaggregation assays, the effect of different buffers, solutions of differing ionic strength, pH, temperature, and viscosity on the degree of coaggregation was assessed. Coaggregation occurred maximally in distilled water but was inhibited when coaggregates were suspended in a commonly-used oral bacterial coaggregation buffer, saline solutions, and Tris-Cl buffers. Coaggregation was weakly expressed in standard laboratory buffers. The ionic strength of inorganic salt solutions required to inhibit coaggregation depended upon the inorganic salt being tested. Coaggregation occurred at a pH of 3-10, between 5 and 80°C and was inhibited in solutions with a viscosity of 22.5 centipoises at 20°C. Inhibition of coaggregation with NaCl impaired biofilm development. When developing buffers to test for coaggregation, the natural liquid environment should be considered. Coaggregation between S. natatoria 2.1 and M. luteus 2.13 is only affected by physicochemical conditions beyond those typically found in natural freshwater ecosystems. Such a robust ability to coaggregate may enhance the ability of S. natatoria 2.1 and M. luteus 2.13 to develop a niche in freshwater biofilms.     

Physicochemical parameters influencing coaggregation between the freshwater bacteria Sphingomonas natatoria 2.1 and Micrococcus luteus 2.13

The aim of this study was to explore the physicochemical parameters that influence coaggregation between the freshwater bacteria Sphingomonas natatoria 2.1 and Micrococcus luteus 2.13. Using visual coaggregation assays, the effect of different buffers, solutions of differing ionic strength, pH, temperature, and viscosity on the degree of coaggregation was assessed. Coaggregation occurred maximally in distilled water but was inhibited when coaggregates were suspended in a commonly-used oral bacterial coaggregation buffer, saline solutions, and Tris-Cl buffers. Coaggregation was weakly expressed in standard laboratory buffers. The ionic strength of inorganic salt solutions required to inhibit coaggregation depended upon the inorganic salt being tested. Coaggregation occurred at a pH of 3–10, between 5 and 80°C and was inhibited in solutions with a viscosity of 22.5 centipoises at 20°C. Inhibition of coaggregation with NaCl impaired biofilm development. When developing buffers to test for coaggregation, the natural liquid environment should be considered. Coaggregation between S. natatoria 2.1 and M. luteus 2.13 is only affected by physicochemical conditions beyond those typically found in natural freshwater ecosystems. Such a robust ability to coaggregate may enhance the ability of S. natatoria 2.1 and M. luteus 2.13 to develop a niche in freshwater biofilms.     

Gene disruption identifies a 290 kDa cell-surface polypeptide conferring hydrophobicity and coaggregation properties in Streptococcus gordonii

The C-terminal coding region of the gene (denoted cshA) encoding a high-molecular-mass (290 kDa) cell-surface polypeptide in the oral bacterium Streptococcus gordonii was cloned and sequenced. Insertion of ermAM into the S. gordonii chromosome at the 3' end of the coding region of cshA led to the production of isogenic mutants that secreted a truncated form (260 kDa) of the CshA polypeptide into the growth medium. Mutants had reduced cell-surface hydrophobicity and were impaired in their ability to coaggregate with oral actinomyces. The results identify a carboxyl terminus-anchored cell-surface protein determinant of hydrophobicity and coaggregation in S. gordonii.     

Proteome and membrane fatty acid analyses on Oligotropha carboxidovorans OM5 grown under chemolithoautotrophic and heterotrophic conditions

Oligotropha carboxidovorans OM5 T. (DSM 1227, ATCC 49405) is a chemolithoautotrophic bacterium able to utilize CO and H(2) to derive energy for fixation of CO(2). Thus, it is capable of growth using syngas, which is a mixture of varying amounts of CO and H(2) generated by organic waste gasification. O. carboxidovorans is capable also of heterotrophic growth in standard bacteriologic media. Here we characterize how the O. carboxidovorans proteome adapts to different lifestyles of chemolithoautotrophy and heterotrophy. Fatty acid methyl ester (FAME) analysis of O. carboxidovorans grown with acetate or with syngas showed that the bacterium changes membrane fatty acid composition. Quantitative shotgun proteomic analysis of O. carboxidovorans grown in the presence of acetate and syngas showed production of proteins encoded on the megaplasmid for assimilating CO and H(2) as well as proteins encoded on the chromosome that might have contributed to fatty acid and acetate metabolism. We found that adaptation to chemolithoautotrophic growth involved adaptations in cell envelope, oxidative homeostasis, and metabolic pathways such as glyoxylate shunt and amino acid/cofactor biosynthetic enzymes.     

Cell-surface proteins of Streptococcus sanguis associated with cell hydrophobicity and coaggregation properties

Incubating cells of Streptococcus sanguis with sodium lauroyl sarcosinate, under conditions that did not cause lysis, solubilized material comprising 5-8% of the cell dry weight. The treatment reduced cell hydrophobicity, and reduced the ability of the cells to coaggregate with Actinomyces spp. The extract contained about 20 polypeptides and these were identified as being cell-surface components on the basis of one or more of the following criteria: being degraded when cells were incubated with protease; being labelled when cells were iodinated using a lactoperoxidase-catalysed reaction; reacting with antibodies raised to fixed whole cells. Eight of the polypeptides accounted for more than 70% of the total protein extracted, and one component (molecular mass 16 kDa) was hydrophobic. The cell-surface proteins described are implicated in cell hydrophobicity and coaggregation.     

Evaluation of Adherence,Hydrophobicity, Aggregation,and Biofilm Development of <Emphasis Type="Italic">Flavobacterium johnsoniae</Emphasis>-Like Isolates

Flavobacterium spp. isolates have been identified in diverse biofilm structures, but the mechanism of adherence has not been elucidated. The absence of conventional biofilm-associated structures such as fimbriae, pili, and flagella suggest that surface hydrophobicity, and/or autoaggregation and coaggregation may play an important role in adherence and biofilm formation. The biofilm-forming capacity of 29 Flavobacterium johnsoniae-like isolates obtained from South African aquaculture systems was assessed using microtiter plate assays. The role of hydrophobicity [salting aggregation test (SAT) and bacterial adherence to hydrocarbons (BATH) assays], autoaggregation, and coaggregation on biofilm formation by Flavobacterium spp. was also investigated, while biofilm structure was examined using flow cells and microscopy. All isolates displayed a hydrophilic nature, but showed varying levels of adherence in microtiter assays. Significant negative correlations were observed between adherence and biofilm-forming capacity in nutrient-poor medium at 26°C and BATH hydrophobicity and motility, respectively. Isolates displayed strain-to-strain variation in their autoaggregation indices and their abilities to coaggregate with various Gram-negative and Gram-positive organisms. Microcolony and/or biofilm development were observed microscopically, and flavobacterial isolates displayed stronger biofilm structures and interaction with a Vibrio spp. isolate than with an Aeromonas hydrophila isolate. The role of extracellular polysaccharides and specific outer membrane proteins will have to be examined to reveal mechanisms of adherence and coaggregation employed by biofilm-forming F. johnsoniae-like strains.     

Characterization of vaginal lactobacilli coaggregation ability with Escherichia coli

The coaggregation abilities of probiotic strains might enable it to form a barrier that prevents colonization by pathogenic bacteria. In the present study, the characterization of the coaggregation ability of 19 vaginal lactobacilli was studied. Coaggregation ability of all lactobacilli with Escherichia coli ATCC 11229 was positive. Only the highest coaggregation percentage of Lactobacillus acidophilus S1 was obtained with E. coli ATCC 11229 under both aerobic (71%) and anaerobic conditions (62%). The coaggregation abilities of strains occurred higher at acidic pH than at basic pH values. Moreover, the coaggregation abilities of tested strains against E. coli decreased after heat treatment (70 or 85 °C). Also, the relationship between hydrophobicity and coaggregation of strains was found to be significant. The effect of sonication, some enzymes (lipase and pepsin) and sodium periodate on coaggregation ability of L. acidophilus S1, which is one of the highest potentials on coaggregation ability, was investigated. Sodium periodate did not have a significant effect on coaggregation ability of L. acidophilus S1. The sonicated cell showed lower coaggregation than the control, the supernatant fluid of this sonicated cells showed similar coaggregation ability to the control. Coaggregation abilities of bacteriotherapeutic lactobacilli with pathogenic bacteria can be used for preliminary screening in order to identify potentially probiotic bacteria suitable for human use against urogenital tract infections.     

Localization of the Fusobacterium nucleatum T18 adhesin activity mediating coaggregation with Porphyromonas gingivalis T22.

Adherence of pathogenic bacteria is often an essential first step in the infectious process. The ability of bacteria to adhere to one another, or to coaggregate, may be an important factor in their ability to colonize and function as pathogens in the periodontal pocket. Previously, a strong and specific coaggregation was demonstrated between two putative periodontal pathogens, Fusobacterium nucleatum and Porphyromonas gingivalis. The interaction appeared to be mediated by a protein adhesin on the F. nucleatum cells and a carbohydrate receptor on the P. gingivalis cells. In this investigation, we have localized the adhesin activity of F. nucleatum T18 to the outer membrane on the basis of the ability of F. nucleatum T18 vesicles to coaggregate with whole cells of P. gingivalis T22 and the ability of the outer membrane fraction of F. nucleatum T18 to inhibit coaggregation between whole cells of F. nucleatum T18 and P. gingivalis T22. Proteolytic pretreatment of the F. nucleatum T18 outer membrane fraction resulted in a loss of coaggregation inhibition, confirming the proteinaceous nature of the adhesin. The F. nucleatum T18 outer membrane fraction was found to be enriched for several proteins, including a 42-kDa major outer membrane protein which appeared to be exposed on the bacterial cell surface. Fab fragments prepared from antiserum raised to the 42-kDa outer membrane protein were found to partially but specifically block coaggregation. These data support the conclusion that the 42-kDa major outer membrane protein of F. nucleatum T18 plays a role in mediating coaggregation with P. gingivalis T22.     

Coaggregation between Prevotella nigrescens and Prevotella intermedia with Actinomyces naeslundii strains

Abstract Using a visual coaggregation assay, 43% (6 of 14) of Prevotella nigrescens and 50% (4 of 8) of Prevotella intermedia strains coaggregated with Actinomyces naeslundii strains which represented the six Actinomyces coaggregation groups (A to F). For both species, coaggregation occurred most frequently with A. naeslundii strains from coaggregation groups C, D and E. No coaggregation was observed with Actinomyces israelii , Actinomyces odontolyticus or six oral Streptococcus species. Coaggregation was not inhibited by lactose, saliva or serum. Pretreatment of Prevotella strains with heat, SDS and proteinase K abolished coaggregation when the treated cells were added to untreated Actinomyces strains. The same pretreatment of the Actinomyces strains had no effect on their ability to coaggregate with untreated Prevotella strains. Pretreatment of all coaggregating P. nigrescens strains with trypsin abolished coaggregation, whereas the coaggregation ability of the P. intermedia and Actinomyces strains was resistant to trypsin pretreatment. Pretreatment of the strains of both Prevotella species and the Actinomyces with periodate abolished coaggregation in all cases. These results suggest that the Prevotella strains each possess a protein coaggregation adhesin, which for the P. intermedia strains is resistant to trypsin, that interacts with a non-protein receptor on the A. naeslundii strains.     

Characterization of a Streptococcus sp.-Veillonella sp. community micromanipulated from dental plaque

Streptococci and veillonellae occur in mixed-species colonies during formation of early dental plaque. One factor hypothesized to be important in assembly of these initial communities is coaggregation (cell-cell recognition by genetically distinct bacteria). Intrageneric coaggregation of streptococci occurs when a lectin-like adhesin on one streptococcal species recognizes a receptor polysaccharide (RPS) on the partner species. Veillonellae also coaggregate with streptococci. These genera interact metabolically; lactic acid produced by streptococci is a carbon source for veillonellae. To transpose these interactions from undisturbed dental plaque to an experimentally tractable in vitro biofilm model, a community consisting of RPS-bearing streptococci juxtaposed with veillonellae was targeted by quantum dot-based immunofluorescence and then micromanipulated off the enamel surface and cultured. Besides the expected antibody-reactive cell types, a non-antibody-reactive streptococcus invisible during micromanipulation was obtained. The streptococci were identified as Streptococcus oralis (RPS bearing) and Streptococcus gordonii (adhesin bearing). The veillonellae could not be cultivated; however, a veillonella 16S rRNA gene sequence was amplified from the original isolation mixture, and this sequence was identical to the sequence of the previously studied organism Veillonella sp. strain PK1910, an oral isolate in our culture collection. S. oralis coaggregated with S. gordonii by an RPS-dependent mechanism, and both streptococci coaggregated with PK1910, which was used as a surrogate during in vitro community reconstruction. The streptococci and strain PK1910 formed interdigitated three-species clusters when grown as a biofilm using saliva as the nutritional source. PK1910 grew only when streptococci were present. This study confirms that RPS-mediated intrageneric coaggregation occurs in the earliest stages of plaque formation by bringing bacteria together to create a functional community.