Autoinducer 2 (AI-2) is the only species-nonspecific autoinducer known in bacteria and is produced by both gram-negative and gram-positive organisms. Consequently, it is proposed to function as a universal quorumsensing signal for interaction between bacterial species. AI-2 is produced as the by-product of a metabolic transformation carried out by the LuxS enzyme. To separate the metabolic function of the LuxS enzyme from the signaling role of AI-2, we carried out a global transcriptome analysis of a luxS null mutant culture of Streptococcus mutans UA159, an important cariogenic bacterium and a crucial component of the dental plaque biofilm community, in comparison to a luxS null mutant culture supplemented with chemically pure 4,5-dihydroxy-2,3-pentanedione, the precursor of AI-2. The data revealed fundamental changes in gene expression affecting 585 genes (30% of the genome) which could not be restored by the signal molecule AI-2 and are therefore not caused by quorum sensing but by lack of the transformation carried out by the LuxS enzyme in the activated methyl cycle. All functional classes of enzymes were affected, including genes known to be important for biofilm formation, bacteriocin synthesis, competence, and acid tolerance. At the same time, 59 genes were identified whose transcription clearly responded to the addition of AI-2. Some of them were related to protein synthesis, stress, and cell division. Three membrane transport proteins were upregulated which are not related to any of the known AI-2 transporters. Three transcription factors were identified whose transcription was stimulated repeatedly by AI-2 addition during growth. Finally, a global regulatory protein, the ␦ subunit of the RNA polymerase (rpoE), was induced 147-fold by AI-2, representing the largest differential gene expression observed. The data show that many phenotypes related to the luxS mutation cannot be ascribed to quorum sensing and have identified for the first time regulatory proteins potentially mediating AI-2-based signaling in gram-positive bacteria.Streptococcus mutans is an important component of the dental plaque biofilm community and plays a key role in the development of caries, the potential result of a complex succession process beginning with the attachment of the bacteria to the tooth surface, followed by the development of a mixedspecies biofilm (17). Tooth decay is triggered by a local reduction of pH following the fermentation of available carbohydrates. The main virulence factors of S. mutans (adhesion, acid tolerance, and acidogenicity) work coordinately to alter dental plaque ecology. The selection for a cariogenic flora increases the magnitude of the drop in pH and the rate of enamel demineralization (32).Cell-cell communication is thought to play a large role in controlling the species composition and virulence properties of the dental plaque community (33, 34). In S. mutans, a signaling peptide (competence-stimulating peptide [CSP]) typical of quorum-sensing circuits in gram-positive bacteria regulates com...

In the human mouth, fungi and several hundred species of bacteria coexist. Here we report a case of interkingdom signaling in the oral cavity: A compound excreted by the caries bacterium Streptococcus mutans inhibits the morphological transition from yeast to hyphae, an important virulence trait, in the opportunistic fungus Candida albicans. The compound excreted by S. mutans was originally studied because it inhibited signaling by the universal bacterial signal autoinducer-2 (AI-2), determined by the luminescence of a Vibrio harveyi sensor strain. The inhibitor was purified from cell-free culture supernatants of S. mutans guided by its activity. Its chemical structure was elucidated by using NMR spectroscopy and GC-MS and proved to be trans-2-decenoic acid. We show that trans-2-decenoic acid does not inhibit AI-2-specific signaling, but rather the luciferase reaction used for its detection. A potential biological role of trans-2-decenoic acid was then discovered. It is able to suppress the transition from yeast to hyphal morphology in the opportunistic human pathogen Candida albicans at concentrations that do not affect growth. The expression of HWP1, a hyphal-specific signature gene of C. albicans, is abolished by trans-2-decenoic acid. trans-2-Decenoic acid is structurally similar to the diffusible signal factor (DSF) family of interkingdom-signaling molecules and is the first member of this family from a Gram-positive organism (Streptococcus DSF, SDSF). SDSF activity was also found in S. mitis, S. oralis, and S. sanguinis, but not in other oral bacteria. SDSF could be relevant in shaping multispecies Candida bacteria biofilms in the human body.

In this study, the combination of culture enrichments and molecular tools was used to identify bacterial guilds, plasmids and functional genes potentially important in the process of petroleum hydrocarbon (PH) decontamination in mangrove microniches (rhizospheres and bulk sediment). In addition, we aimed to recover PHdegrading consortia (PHDC) for future use in remediation strategies. The PHDC were enriched with petroleum from rhizosphere and bulk sediment samples taken from a mangrove chronically polluted with oil hydrocarbons. Southern blot hybridization (SBH) assays of PCR amplicons from environmental DNA before enrichments resulted in weak positive signals for the functional gene types targeted, suggesting that PH-degrading genotypes and plasmids were in low abundance in the rhizosphere and bulk sediments. However, after enrichment, these genes were detected and strong microniche-dependent differences in the abundance and composition of hydrocarbonoclastic bacterial populations, plasmids (IncP-1a, IncP1b, IncP-7 and IncP-9) and functional genes (naphthalene, extradiol and intradiol dioxygenases) were revealed by in-depth molecular analyses [PCR-denaturing gradient gel electrophoresis and hybridization (SBH and microarray)]. Our results suggest that, despite the low abundance of PH-degrading genes and plasmids in the environmental samples, the original bacterial composition of the mangrove microniches determined the structural and functional diversity of the PHDC enriched.

Autoinducer-2 (furanosyl borate diester) is a biologically active compound whose role as a universal bacterial signalling molecule is currently under intense investigation. Because of its instability and the low concentrations of it found in biological samples, its detection relies at present on a bioassay that measures the difference in the timing of the luminescence of the Vibrio harveyi BB170 sensor strain with and without externally added AI-2. Here we systematically investigated which parameters affected the fold induction values of luminescence obtained in the bioassay and developed a modified protocol. Our experiments showed that growth and luminescence of V. harveyi BB170 are strongly influenced by trace elements. In particular, addition of Fe3+ within a certain concentration range to the growth medium of the preinoculum culture improved the reproducibility and reduced the variance of the bioassay. In contrast, trace elements and vitamins introduced directly into the bioassay caused inhibitory effects. The initial density and luminescence of the sensor strain are very important and the values required for these parameters were defined. Borate interferes with the detection of AI-2 by giving false positive results. The response of V. harveyi BB170 to chemically synthesized AI-2 in the bioassay is nonlinear except over a very small concentration range; it is maximum over three orders of magnitude and shows inhibition above 35 mu M. Based on the modified protocol, we were able to detect AI-2 in the absence of inhibitors with maximum fold induction values for the positive control (chemically synthesized AI-2) of > 120 with a standard deviation of similar to 30% in a reliable and reproducible way

SENSING THE SIGNAL: A gas chromatography-mass spectrometry (GC-MS) method for the analysis of the quorum-sensing autoinducer-2 is described. It allows, for the first time, the direct analysis and accurate determination of this highly water soluble signaling compound, which exists in complex equilibria. The application on the caries-causing bacterium Streptococcus mutans is described. Autoinducer-2 (AI-2) is an important, small extracellular signaling molecule that is used by many bacteria. It is part of the AI-2 pool, a group of equilibrium-connected compounds derived from (S)-4,5-dihydroxy-2,3-pentanedione [(S)-DPD, 1]. Currently, these compounds are analyzed by indirect methods relying on the luminescence of sensor strains, the fluorescence of receptor proteins modified with fluorophores, or by isolation procedures not practical for quantitative analysis. Herein, we report a direct analytical procedure that allows for the unambiguous identification and quantification of molecular species by mass spectrometry. Phenylenediamine reacts readily and quantitatively with 1 to form the quinoxalinediol 12 under aqueous conditions. The extraction and silylation of this compound results in the formation of a silyl ether (13), which is amenable for analysis by gas chromatography-mass spectrometry. The use of an isotopically labeled variant (16) of 12 as an internal standard opens the possibility for the accurate quantification of samples containing AI-2 or its equilibrium products. The analysis of cell-free culture supernatants of Vibrio harveyi and Streptococcus mutans allowed for the accurate quantification of the AI-2 concentration above the limit of detection (0.7 ng mL(-1)). No compounds were detected in mutants lacking the capability to produce AI-2. In addition, the absolute configuration of 1 can be analyzed using the derivative 13 by chiral gas chromatography.