Mn(III)-mediated bisphenol a degradation: Mechanisms and products.

Bisphenol A (BPA) is a high production volume chemical with potential estrogenic effects susceptible to abiotic degradation by MnO2. BPA transformation products and reaction mechanisms with MnO2 have been investigated, but detailed process understanding of Mn(III)-mediated degradation has not been attained. Rapid consumption of BPA occurred in batch reaction vessels with 1 mM Mn(III) and 63.9 ± 0.7% of 1.76 ± 0.02 µmol BPA was degraded in 1 hour at circumneutral pH. BPA was consumed at 1.86 ± 0.09-fold higher rates in vessels with synthetic MnO2 comprising approximately 13 mol% surface-associated Mn(III) versus surface-Mn(III)-free MnO2, and 10-35% of BPA transformation could be attributed to Mn(III) during the initial 10-min reaction phase. High-resolution tandem mass spectrometry (HRMS/MS) analysis detected eight transformation intermediates in reactions with Mn(III), and quantum calculations proposed 14 BPA degradation products, nine of which had not been observed during MnO2-mediated BPA degradation, suggesting mechanistic differences between Mn(III)- versus MnO2-mediated BPA degradation. The findings demonstrate that both Mn(III) and Mn(IV) can effectively degrade BPA and indicate that surface-associated Mn(III) increases the reactivity of synthetic MnO2, offering opportunities for engineering more reactive oxidized Mn species for BPA removal.

Metabolome patterns identify active dechlorination in bioaugmentation consortium SDC-9™.

Ultra-high performance liquid chromatography-high-resolution mass spectrometry (UPHLC-HRMS) is used to discover and monitor single or sets of biomarkers informing about metabolic processes of interest. The technique can detect 1000's of molecules (i.e., metabolites) in a single instrument run and provide a measurement of the global metabolome, which could be a fingerprint of activity. Despite the power of this approach, technical challenges have hindered the effective use of metabolomics to interrogate microbial communities implicated in the removal of priority contaminants. Herein, our efforts to circumvent these challenges and apply this emerging systems biology technique to microbiomes relevant for contaminant biodegradation will be discussed. Chlorinated ethenes impact many contaminated sites, and detoxification can be achieved by organohalide-respiring bacteria, a process currently assessed by quantitative gene-centric tools (e.g., quantitative PCR). This laboratory study monitored the metabolome of the SDC-9™ bioaugmentation consortium during cis-1,2-dichloroethene (cDCE) conversion to vinyl chloride (VC) and nontoxic ethene. Untargeted metabolomics using an UHPLC-Orbitrap mass spectrometer and performed on SDC-9™ cultures at different stages of the reductive dechlorination process detected ~10,000 spectral features per sample arising from water-soluble molecules with both known and unknown structures. Multivariate statistical techniques including partial least squares-discriminate analysis (PLSDA) identified patterns of measurable spectral features (peak patterns) that correlated with dechlorination (in)activity, and ANOVA analyses identified 18 potential biomarkers for this process. Statistical clustering of samples with these 18 features identified dechlorination activity more reliably than clustering of samples based only on chlorinated ethene concentration and Dhc 16S rRNA gene abundance data, highlighting the potential value of metabolomic workflows as an innovative site assessment and bioremediation monitoring tool.

Pseudomonas sp. Strain 273 Incorporates Organofluorine into the Lipid Bilayer during Growth with Fluorinated Alkanes.

Anthropogenic organofluorine compounds are recalcitrant, globally distributed, and a human health concern. Although rare, natural processes synthesize fluorinated compounds, and some bacteria have evolved mechanisms to metabolize organofluorine compounds. Pseudomonas sp. strain 273 grows with 1-fluorodecane (FD) and 1,10-difluorodecane (DFD) as carbon sources, but inorganic fluoride release was not stoichiometric. Metabolome studies revealed that this bacterium produces fluorinated anabolites and phospholipids. Mass spectrometric fatty acid profiling detected fluorinated long-chain (i.e., C12-C19) fatty acids in strain 273 cells grown with FD or DFD, and lipidomic profiling determined that 7.5 ± 0.2 and 82.0 ± 1.0% of the total phospholipids in strain 273 grown with FD or DFD, respectively, were fluorinated. The detection of the fluorinated metabolites and macromolecules represents a heretofore unrecognized sink for organofluorine, an observation with consequences for the environmental fate and transport of fluorinated aliphatic compounds.

Pseudomonas sp. Strain 273 Degrades Fluorinated Alkanes.

Fluorinated organic compounds have emerged as environmental constituents of concern. We demonstrate that the alkane degrader Pseudomonas sp. strain 273 utilizes terminally monofluorinated C7-C10 alkanes and 1,10-difluorodecane (DFD) as the sole carbon and energy sources in the presence of oxygen. Strain 273 degraded 1-fluorodecane (FD) (5.97 ± 0.22 mM, nominal) and DFD (5.62 ± 0.13 mM, nominal) within 7 days of incubation, and 92.7 ± 3.8 and 90.1 ± 1.9% of the theoretical maximum amounts of fluorine were recovered as inorganic fluoride, respectively. With n-decane, strain 273 attained (3.24 ± 0.14) × 107 cells per µmol of carbon consumed, while lower biomass yields of (2.48 ± 0.15) × 107 and (1.62 ± 0.23) × 107 cells were measured with FD or DFD as electron donors, respectively. The organism coupled decanol and decanoate oxidation to denitrification, but the utilization of (fluoro)alkanes was strictly oxygen-dependent, presumably because the initial attack on the terminal carbon requires oxygen. Fluorohexanoate was detected as an intermediate in cultures grown with FD or DFD, suggesting that the initial attack on the fluoroalkanes can occur on the terminal methyl or fluoromethyl groups. The findings indicate that specialized bacteria such as Pseudomonas sp. strain 273 can break carbon-fluorine bonds most likely with oxygenolytic enzyme systems and that terminally monofluorinated alkanes are susceptible to microbial degradation. The findings have implications for the fate of components associated with aqueous film-forming foam (AFFF) mixtures.

Discovery and Functional Characterization of a Yeast Sugar Alcohol Phosphatase.

Sugar alcohols (polyols) exist widely in nature. While some specific sugar alcohol phosphatases are known, there is no known phosphatase for some important sugar alcohols (e.g., sorbitol-6-phosphate). Using liquid chromatography-mass spectrometry-based metabolomics, we screened yeast strains with putative phosphatases of unknown function deleted. We show that the yeast gene YNL010W, which has close homologues in all fungi species and some plants, encodes a sugar alcohol phosphatase. We term this enzyme, which hydrolyzes sorbitol-6-phosphate, ribitol-5-phosphate, and (d)-glycerol-3-phosphate, polyol phosphatase 1 or PYP1. Polyol phosphates are structural analogs of the enediol intermediate of phosphoglucose isomerase (Pgi). We find that sorbitol-6-phosphate and ribitol-5-phosphate inhibit Pgi and that Pyp1 activity is important for yeast to maintain Pgi activity in the presence of environmental sugar alcohols. Pyp1 expression is strongly positively correlated with yeast growth rate, presumably because faster growth requires greater glycolytic and accordingly Pgi flux. Thus, yeast express the previously uncharacterized enzyme Pyp1 to prevent inhibition of glycolysis by sugar alcohol phosphates. Pyp1 may be useful for engineering sugar alcohol production.

Neurochemistry of Anesthetic States.

Anesthetic mechanisms that eliminate consciousness and perception of pain are products of the nervous system. Chemical approaches to the study of anesthetic mechanisms have the potential to serve as an ideal interface between basic and clinical neuroscience. There are disproportionately more basic neurochemical studies than clinical studies of anesthetic mechanisms. Even within neuroscience, the study of anesthetic mechanisms is sparse. The Society for Neuroscience hosts one of the world's largest and most vibrant scientific meetings, yet the content themes of that meeting do not include anesthesia. One goal of this chapter is to facilitate neurochemical studies of anesthetic mechanisms by outlining user-friendly descriptions of existing and emerging techniques. The introduction provides a context for chapter goals. The second portion of this chapter focuses on microdialysis methods that enable the humane acquisition of neurochemical samples from intact, behaving animals during anesthetic induction, maintenance, and emergence. No single neurotransmitter and no single brain region regulate the physiological and behavioral traits characteristic of any anesthetic state. This limitation is being addressed via application of new instrumentation and techniques in analytic chemistry. The final third of this chapter highlights selected omics approaches that are now being applied to the neurochemical study of anesthetic mechanisms. We hope that this brief chapter can stimulate basic and clinical metabolomic approaches aiming to elucidate the mechanisms of anesthetic action.

Thiobenzothiazole-modified Hydrocortisones Display Anti-inflammatory Activity with Reduced Impact on Islet ß-Cell Function.

Glucocorticoids signal through the glucocorticoid receptor (GR) and are administered clinically for a variety of situations, including inflammatory disorders, specific cancers, rheumatoid arthritis, and organ/tissue transplantation. However, glucocorticoid therapy is also associated with additional complications, including steroid-induced diabetes. We hypothesized that modification of the steroid backbone is one strategy to enhance the therapeutic potential of GR activation. Toward this goal, two commercially unavailable, thiobenzothiazole-containing derivatives of hydrocortisone (termed MS4 and MS6) were examined using 832/13 rat insulinoma cells as well as rodent and human islets. We found that MS4 had transrepression properties but lacked transactivation ability, whereas MS6 retained both transactivation and transrepression activities. In addition, MS4 and MS6 both displayed anti-inflammatory activity. Furthermore, MS4 displayed reduced impact on islet ß-cell function in both rodent and human islets. Similar to dexamethasone, MS6 promoted adipocyte development in vitro, whereas MS4 did not. Moreover, neither MS4 nor MS6 activated the Pck1 (Pepck) gene in primary rat hepatocytes. We conclude that modification of the functional groups attached to the D-ring of the hydrocortisone steroid molecule produces compounds with altered structure-function GR agonist activity with decreased impact on insulin secretion and reduced adipogenic potential but with preservation of anti-inflammatory activity.

Phaeobacter sp. strain Y4I utilizes two separate cell-to-cell communication systems to regulate production of the antimicrobial indigoidine.

The marine roseobacter Phaeobacter sp. strain Y4I synthesizes the blue antimicrobial secondary metabolite indigoidine when grown in a biofilm or on agar plates. Prior studies suggested that indigoidine production may be, in part, regulated by cell-to-cell communication systems. Phaeobacter sp. strain Y4I possesses two luxR and luxI homologous N-acyl-L-homoserine lactone (AHL)-mediated cell-to-cell communication systems, designated pgaRI and phaRI. We show here that Y4I produces two dominantAHLs, the novel monounsaturated N-(3-hydroxydodecenoyl)-L-homoserine lactone (3OHC(12:1)-HSL) and the relatively common N-octanoyl-L-homoserine lactone (C8-HSL), and provide evidence that they are synthesized by PhaI and PgaI, respectively.A Tn5 insertional mutation in either genetic locus results in the abolishment (pgaR::Tn5) or reduction (phaR::Tn5) of pigment production. Motility defects and denser biofilms were also observed in these mutant backgrounds, suggesting an overlap in the functional roles of these systems. Production of the AHLs occurs at distinct points during growth on an agar surface and was determined by isotope dilution high-performance liquid chromatography­tandem mass spectrometry (ID-HPLC-MS/MS) analysis.Within 2 h of surface inoculation, only 3OHC(12:1)-HSL was detected in agar extracts. As surface-attached cells became established (at approximately 10 h), the concentration of 3OHC(12:1)-HSL decreased, and the concentration of C8-HSL increased rapidly over 14 h.After longer (>24-h) establishment periods, the concentrations of the two AHLs increased to and stabilized at approximately 15 nM and approximately 600 nM for 3OHC12:1-HSL and C8-HSL, respectively. In contrast, the total amount of indigoidine increased steadily from undetectable to 642 Mby 48 h. Gene expression profiles of the AHL and indigoidine synthases (pgaI, phaI, and igiD) were consistent with their metabolite profiles. These data provide evidence that pgaRI and phaRI play overlapping roles in the regulation of indigoidine biosynthesis, and it is postulated that this allows Phaeobacter sp. strain Y4I to coordinate production of indigoidine with different growth-phase-dependent physiologies.

Quorum sensing signal production and microbial interactions in a polymicrobial disease of corals and the coral surface mucopolysaccharide layer.

Black band disease (BBD) of corals is a complex polymicrobial disease considered to be a threat to coral reef health, as it can lead to mortality of massive reef-building corals. The BBD community is dominated by gliding, filamentous cyanobacteria with a highly diverse population of heterotrophic bacteria. Microbial interactions such as quorum sensing (QS) and antimicrobial production may be involved in BBD disease pathogenesis. In this study, BBD (whole community) samples, as well as 199 bacterial isolates from BBD, the surface mucopolysaccharide layer (SML) of apparently healthy corals, and SML of apparently healthy areas of BBD-infected corals were screened for the production of acyl homoserine lactones (AHLs) and for autoinducer-2 (AI-2) activity using three bacterial reporter strains. AHLs were detected in all BBD (intact community) samples tested and in cultures of 5.5% of BBD bacterial isolates. Over half of a subset (153) of the isolates were positive for AI-2 activity. AHL-producing isolates were further analyzed using LC-MS/MS to determine AHL chemical structure and the concentration of (S)-4,5-dihydroxy-2,3-pentanedione (DPD), the biosynthetic precursor of AI-2. C6-HSL was the most common AHL variant detected, followed by 3OC4-HSL. In addition to QS assays, 342 growth challenges were conducted among a subset of the isolates, with 27% of isolates eliciting growth inhibition and 2% growth stimulation. 24% of BBD isolates elicited growth inhibition as compared to 26% and 32% of the bacteria from the two SML sources. With one exception, only isolates that exhibited AI-2 activity or produced DPD inhibited growth of test strains. These findings demonstrate for the first time that AHLs are present in an active coral disease. It is possible that AI-2 production among BBD and coral SML bacteria may structure the microbial communities of both a polymicrobial infection and the healthy coral microbiome.

Structure and stability of phenoxide and fluorophenoxide anions investigated with infrared multiple-photon dissociation and detachment spectroscopy and tandem mass spectrometry.

The gas-phase infrared multiple-photon dissociation and detachment (IRMPD) vibrational action spectra of the unsubstituted phenoxide anion and a series of fluorine- and trifluoromethyl-substituted phenoxide anions in the spectral region between 600 and 1800 cm(-1) are presented along with density functional theory (DFT) harmonic vibrational frequency calculations to establish the characteristic vibrations of the phenoxide functionality. The fluorophenoxide anions studied include the conjugate bases of o-, m-, and p-fluorophenol (C6H4FO(-)) as well as o-, m-, and p-α,α,α-trifluorocresol (CF3C6H4O(-)). The influence of the substituent on the characteristic vibrational frequencies is interpreted in terms of inductive and resonance shifts. In addition to the dissociation induced by infrared multiple-photon excitation, the electron detachment is also shown to play an important role in the decomposition of the unsubstituted phenoxide. It is demonstrated that the amount of electron detachment relative to dissociation is strongly mitigated by fluorination, and interpretations aided by DFT energy calculations suggest this is primarily due to the increased availability of low-energy dissociation pathways in the substituted phenoxides. Collision-induced dissociation (CID) mass spectrometry of the parent ions is used to estimate relative energies of the dissociation processes, and particular fragmentation motifs are elucidated. In particular, overall HF and CO losses provide facile decomposition pathways, yielding interesting fragment ions such as C6H(-) or C3H2FO(-) from the CF3C6H4O(-) parent anions.

Phage infection of an environmentally relevant marine bacterium alters host metabolism and lysate composition.

Viruses contribute to the mortality of marine microbes, consequentially altering biological species composition and system biogeochemistry. Although it is well established that host cells provide metabolic resources for virus replication, the extent to which infection reshapes host metabolism at a global level and the effect of this alteration on the cellular material released following viral lysis is less understood. To address this knowledge gap, the growth dynamics, metabolism and extracellular lysate of roseophage-infected Sulfitobacter sp. 2047 was studied using a variety of techniques, including liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based metabolomics. Quantitative estimates of the total amount of carbon and nitrogen sequestered into particulate biomass indicate that phage infection redirects ∼75% of nutrients into virions. Intracellular concentrations for 82 metabolites were measured at seven time points over the infection cycle. By the end of this period, 71% of the detected metabolites were significantly elevated in infected populations, and stable isotope-based flux measurements showed that these cells had elevated metabolic activity. In contrast to simple hypothetical models that assume that extracellular compounds increase because of lysis, a profile of metabolites from infected cultures showed that >70% of the 56 quantified compounds had decreased concentrations in the lysate relative to uninfected controls, suggesting that these small, labile nutrients were being utilized by surviving cells. These results indicate that virus-infected cells are physiologically distinct from their uninfected counterparts, which has implications for microbial community ecology and biogeochemistry.

Evolution of the toxins muscarine and psilocybin in a family of mushroom-forming fungi.

Mushroom-forming fungi produce a wide array of toxic alkaloids. However, evolutionary analyses aimed at exploring the evolution of muscarine, a toxin that stimulates the parasympathetic nervous system, and psilocybin, a hallucinogen, have never been performed. The known taxonomic distribution of muscarine within the Inocybaceae is limited, based only on assays of species from temperate regions of the northern hemisphere. Here, we present a review of muscarine and psilocybin assays performed on species of Inocybaceae during the last fifty years. To supplement these results, we used liquid chromatography-tandem mass spectrometry (LC-MS/MS) to determine whether muscarine was present in 30 new samples of Inocybaceae, the majority of which have not been previously assayed or that originated from either the tropics or temperate regions of the southern hemisphere. Our main objective is to test the hypothesis that the presence of muscarine is a shared ancestral feature of the Inocybaceae. In addition, we also test whether species of Inocyabceae that produce psilocybin are monophyletic. Our findings suggest otherwise. Muscarine has evolved independently on several occasions, together with several losses. We also detect at least two independent transitions of muscarine-free lineages to psilocybin-producing states. Although not ancestral for the family as a whole, muscarine is a shared derived trait for an inclusive clade containing three of the seven major lineages of Inocybaceae (the Inocybe, Nothocybe, and Pseudosperma clades), the common ancestor of which may have evolved ca. 60 million years ago. Thus, muscarine represents a conserved trait followed by several recent losses. Transitions to psilocybin from muscarine-producing ancestors occurred more recently between 10-20 million years ago after muscarine loss in two separate lineages. Statistical analyses firmly reject a single origin of muscarine-producing taxa.

Production of the antimicrobial secondary metabolite indigoidine contributes to competitive surface colonization by the marine roseobacter Phaeobacter sp. strain Y4I.

Members of the Roseobacter lineage of marine bacteria are prolific surface colonizers in marine coastal environments, and antimicrobial secondary metabolite production has been hypothesized to provide a competitive advantage to colonizing roseobacters. Here, we report that the roseobacter Phaeobacter sp. strain Y4I produces the blue pigment indigoidine via a nonribosomal peptide synthase (NRPS)-based biosynthetic pathway encoded by a novel series of genetically linked genes: igiBCDFE. A Tn5-based random mutagenesis library of Y4I showed a perfect correlation between indigoidine production by the Phaeobacter strain and inhibition of Vibrio fischeri on agar plates, revealing a previously unrecognized bioactivity of this molecule. In addition, igiD null mutants (igiD encoding the indigoidine NRPS) were more resistant to hydrogen peroxide, less motile, and faster to colonize an artificial surface than the wild-type strain. Collectively, these data provide evidence for pleiotropic effects of indigoidine production in this strain. Gene expression assays support phenotypic observations and demonstrate that igiD gene expression is upregulated during growth on surfaces. Furthermore, competitive cocultures of V. fischeri and Y4I show that the production of indigoidine by Y4I significantly inhibits colonization of V. fischeri on surfaces. This study is the first to characterize a secondary metabolite produced by an NRPS in roseobacters.

Stability of gas-phase tartaric acid anions investigated by quantum chemistry, mass spectrometry, and infrared spectroscopy.

In an effort to understand the chemical factors that stabilize dianions, experimental and theoretical studies on the stability of the tartrate dianion were performed. Quantum chemical calculations at the coupled cluster level reveal only a metastable state with a possible decomposition pathway (O(2)C-CH(OH)-CH(OH)-CO(2))(2-) → (O(2)C-CH(OH)-CH(OH))(•-) + CO(2) + e(-) explaining the observed gas-phase instability of this dianion. Further theoretical data were collected for the bare dianion, this molecule complexed to water, sodium, and a proton, in both the meso and l forms as well as for the uncomplexed radical anion and neutral diradical. The calculations suggest that the l-tartrate dianion is more thermodynamically stable than the dianion of the meso stereoisomer and that either dianion can be further stabilized by association with a separate species that can help to balance the charge of the molecular complex. Mass spectrometry was then used to measure the energy needed to initiate collisionally induced dissociation of the racemic tartrate dianion and for the proton and sodium adducts of both the racemic and meso form of this molecule. Infrared action spectra of the dianion stereoisomers complexed with sodium were also acquired to determine the influence of the metal ion on the vibrations of the dianions and validate the computationally predicted structures. These experimental data support the theoretical conclusions and highlight the instability of the bare tartrate dianion. From the experimental work, it could also be concluded that the pathway leading to dissociation is under kinetic control because the sodium adduct of the racemic stereoisomer dissociated at lower collisional energy, although it was calculated to be more stable, and that decomposition proceeded via C-C bond dissociation as computationally predicted. Taken together, these data provide insight into the gas-phase stability of the tartrate dianion and highlight the role of adducts in stabilizing this species.

Detection and quantitation of bacterial acylhomoserine lactone quorum sensing molecules via liquid chromatography-isotope dilution tandem mass spectrometry.

A range of acylhomoserine lactones (AHLs) are used as intraspecies quorum sensing signals by Gram-negative bacteria, and the detection and quantitation of these molecules is of interest. This manuscript reports a liquid chromatographic-isotope dilution tandem mass spectrometric method for the quantitation of these molecules. A divergent solid-phase synthesis of stable-isotope-labeled AHLs suitable for use as an internal standard is reported. This route relies on the biomimetic conversion of a dideuterated methionine equivalent, N-Fmoc-(4,4-(2)H(2))methionine, to the desired labeled AHL, and a representative series of eight of these molecules was produced in >95% purity and yields up to ~50%. The representative AHL internal standards were then used to develop an optimized liquid chromatography-tandem mass spectrometric (LC-MS/MS) separation and detection protocol for these molecules, which relies on a high-efficiency C18 core-shell column to minimize the time necessary for separation. The addition of internal standards at different steps during sampling was also found to affect the analysis for hydrophobic AHLs with addition prior to cell removal giving the most accurate results. Taken together, the use of the internal standards and separation method reported herein provides a rapid and quantitative method for the study of AHL production in bacteria.

Quorum sensing control of phosphorus acquisition in Trichodesmium consortia.

Colonies of the cyanobacterium Trichodesmium are abundant in the oligotrophic ocean, and through their ability to fix both CO(2) and N(2), have pivotal roles in the cycling of carbon and nitrogen in these highly nutrient-depleted environments. Trichodesmium colonies host complex consortia of epibiotic heterotrophic bacteria, and yet, the regulation of nutrient acquisition by these epibionts is poorly understood. We present evidence that epibiotic bacteria in Trichodesmium consortia use quorum sensing (QS) to regulate the activity of alkaline phosphatases (APases), enzymes used by epibionts in the acquisition of phosphate from dissolved-organic phosphorus molecules. A class of QS molecules, acylated homoserine lactones (AHLs), were produced by cultivated epibionts, and adding these AHLs to wild Trichodesmium colonies collected at sea led to a consistent doubling of APase activity. By contrast, amendments of (S)-4,5-dihydroxy-2,3-pentanedione (DPD)-the precursor to the autoinducer-2 (AI-2) family of universal interspecies signaling molecules-led to the attenuation of APase activity. In addition, colonies collected at sea were found by high performance liquid chromatography/mass spectrometry to contain both AHLs and AI-2. Both types of molecules turned over rapidly, an observation we ascribe to quorum quenching. Our results reveal a complex chemical interplay among epibionts using AHLs and AI-2 to control access to phosphate in dissolved-organic phosphorus.

Establishing a quantitative definition of quorum sensing provides insight into the information content of the autoinducer signals in Vibrio harveyi and Escherichia coli.

Extracellular autoinducer concentrations in cultures of Vibrio harveyi and Escherichia coli were monitored by liquid chromatography-tandem mass spectrometry to test whether a quantitative definition of quorum sensing could help decipher the information content of these signals. Although V. harveyi was able to keep the autoinducer-2 to cell number ratio constant, the ratio of signal to cell number for V. harveyi autoinducer-1 and E. coli autoinducer-2 varied as the cultures grew. These data indicate that V. harveyi uses autoinducer-2 for quorum sensing, while the other molecules may be used to transmit different information or are influenced by metabolic noise.

Direct quantitation of the quorum sensing signal, autoinducer-2, in clinically relevant samples by liquid chromatography-tandem mass spectrometry.

Quorum Sensing is a type of bacterial cell-to-cell signaling that allows for cell density dependent regulation of gene expression. Many of the behaviors mediated by quorum sensing are critical for bacterial colonization or infection, and autoinducer-2 has been proposed as a universal interspecies signaling molecule that allows multispecies colonies of bacteria, e.g., biofilms or dental plaque, to behave as pseudomulticellular organisms. However, the direct detection of autoinducer-2 has been difficult, leaving the in vivo relevance of this signal in question. Herein we report a liquid chromatography-tandem mass spectrometric technique that enables reproducible, quantitative, and sensitive measurement of the concentration of autoinducer-2 from a variety of sources. This technique was applied to the detection of autoinducer-2 from Escherichia coli and Vibrio harveyi in proof-of-concept studies and was then used to directly measure the concentration of the signal produced by oral bacteria in human saliva.