Detection of quorum-sensing N-acyl homoserine lactone signal molecules by bacterial biosensors

Many Gram-negative bacteria use N-acyl homoserine lactones (AHLs) as quorum-sensing (QS) signal molecules. AHL QS has been the subject of extensive investigation in the last decade and has become a paradigm for bacterial intercellular signaling. Research in AHL QS has been considerably aided by simple methods devised to detect AHLs using bacterial biosensors that phenotypically respond when exposed to exogenous AHLs. This article reviews and discusses the currently available bacterial biosensors which can be used in detecting and studying the different AHLs.     

Regulation of LuxPQ receptor activity by the quorum-sensing signal autoinducer-2

The extracellular signaling molecule autoinducer-2 (AI-2) mediates quorum-sensing communication in diverse bacterial species. In marine vibrios, binding of AI-2 to the periplasmic receptor LuxP modulates the activity of the inner membrane sensor kinase LuxQ, transducing the AI-2 information into the cytoplasm. Here, we show that Vibrio harveyi LuxP associates with LuxQ in both the presence and absence of AI-2. The 1.9 A X-ray crystal structure of apoLuxP, complexed with the periplasmic domain of LuxQ, reveals that the latter contains two tandem Per/ARNT/Simple-minded (PAS) folds. Thus, although many prokaryotic PAS folds themselves bind ligands, the LuxQ periplasmic PAS folds instead bind LuxP, monitoring its AI-2 occupancy. Mutations that disrupt the apoLuxP:LuxQ interface sensitize V. harveyi to AI-2, implying that AI-2 binding causes the replacement of one set of LuxP:LuxQ contacts with another. These conformational changes switch LuxQ between two opposing enzymatic activities, each of which conveys information to the cytoplasm about the cell density of the surrounding environment.     

Rapid screening of quorum-sensing signal N-acyl homoserine lactones by an in vitro cell-free assay

A simple, sensitive, and rapid cell-free assay system was developed for detection of N-acyl homoserine lactone (AHL) autoinducers involved in bacterial quorum sensing (QS). The present approach improves upon previous whole-cell biosensor-based approaches in its utilization of a cell-free assay approach to conduct bioassays. The cell-free assay was derived from the AHL biosensor bacterium Agrobacterium tumefaciens NTL4(pCF218)(pCF372), allowing the expression of beta-galactosidase upon addition of exogenous AHLs. We have shown that beta-galactosidase expression is possible in cell-free solution [lysate from Agrobacterium tumefaciens NTL4(pCF218)(pCF372) culture]. Assay detection limits with the use of chromogenic substrate X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) ranged from approximately 100 nM to 300 nM depending on the specific AHL. Replacement (of X-Gal) with the luminescent substrate Beta-Glo increased sensitivity to AHLs by 10-fold. A major advantage of the cell-free assay system is elimination of time-consuming steps for biosensor cell culture conditioning, which are required prior to whole-cell bioassays. This significantly reduced assay times from greater than 24 h to less than 3 h, while maintaining high sensitivity. Assay lysate may be prepared in bulk and stored (-80 degrees C) over 6 months for future use. Finally, the present protocol may be adapted for use with other biosensor strains and be used in high-throughput AHL screening of bacteria or metagenomic libraries.     

Detection of different quorum-sensing signal molecules in a virulent Edwardsiella tarda strain LTB-4

Aims:  The aim of this study was to elucidate the potential quorum-sensing (QS) signal molecules of an emerging pathogen ( Edwardsiella tarda strain LTB-4) of cultured turbot ( Scophthalmus maximus ).
Methods and Results:  A sensitive and rapid double-layer plate method using biosensor strain Agrobacterium tumefaciens KYC55 was developed to detect the N-acylhomoserine lactone (AHL)-related compounds in bacteria. LTB-4 was found to have two QS systems, one was based on the AHLs and the other was based on the autoinducer-2 (AI-2). The AI-2 activity produced by LTB-4 was growth phase dependent and topped at OD₆₀₀ of 1·0. The protocol to detect cholerae autoinducer 1 (CAI-1) activity in bacteria was modified, lowering the background luminescence of biosensor strain Vibrio harveyi JAF375. CAI-1 activity could not be detected in LTB-4.
Conclusion:  Edwardsiella tarda LTB-4 produced at least four kinds of AHLs during its whole growth phase. In comparison with the AHL-inducing QS, AI-2 may be the first predominant signal, functioning at early exponential phase. LTB-4 did not produce any CAI-1 activity.
Significance and Impact of the Study:  Different QS signal molecules of Edw. tarda LTB-4 were clarified by improved bioassays. In contrast to earlier studies detecting two types of AHLs, strain LTB-4 produced at least four kinds of AHLs, which seemed to be C₄-HSL, C₆-HSL, 3-oxo-C₆-HSL and an uncharacterized AHL molecule.     

Salmonella typhimurium recognizes a chemically distinct form of the bacterial quorum-sensing signal AI-2

Bacterial populations use cell-cell communication to coordinate community-wide regulation of processes such as biofilm formation, virulence, and bioluminescence. This phenomenon, termed quorum sensing, is mediated by small molecule signals known as autoinducers. While most autoinducers are species specific, autoinducer-2 (AI-2), first identified in the marine bacterium Vibrio harveyi, is produced and detected by many Gram-negative and Gram-positive bacteria. The crystal structure of the V. harveyi AI-2 signaling molecule bound to its receptor protein revealed an unusual furanosyl borate diester. Here, we present the crystal structure of a second AI-2 signal binding protein, LsrB from Salmonella typhimurium. We find that LsrB binds a chemically distinct form of the AI-2 signal, (2R,4S)-2-methyl-2,3,3,4-tetrahydroxytetrahydrofuran (R-THMF), that lacks boron. Our results demonstrate that two different species of bacteria recognize two different forms of the autoinducer signal, both derived from 4,5-dihydroxy-2,3-pentanedione (DPD), and reveal new sophistication in the chemical lexicon used by bacteria in interspecies signaling.     

The LuxS family of bacterial autoinducers: biosynthesis of a novel quorum-sensing signal molecule

Many bacteria control gene expression in response to cell population density, and this phenomenon is called quorum sensing. In Gram-negative bacteria, quorum sensing typically involves the production, release and detection of acylated homoserine lactone signalling molecules called autoinducers. Vibrio harveyi, a Gram-negative bioluminescent marine bacterium, regulates light production in response to two distinct autoinducers (AI-1 and AI-2). AI-1 is a homoserine lactone. The structure of AI-2 is not known. We have suggested previously that V. harveyi uses AI-1 for intraspecies communication and AI-2 for interspecies communication. Consistent with this idea, we have shown that many species of Gram-negative and Gram-positive bacteria produce AI-2 and, in every case, production of AI-2 is dependent on the function encoded by the luxS gene. We show here that LuxS is the AI-2 synthase and that AI-2 is produced from S-adenosylmethionine in three enzymatic steps. The substrate for LuxS is S-ribosylhomocysteine, which is cleaved to form two products, one of which is homocysteine, and the other is AI-2. In this report, we also provide evidence that the biosynthetic pathway and biochemical intermediates in AI-2 biosynthesis are identical in Escherichia coli, Salmonella typhimurium, V. harveyi, Vibrio cholerae and Enterococcus faecalis. This result suggests that, unlike quorum sensing via the family of related homoserine lactone autoinducers, AI-2 is a unique, 'universal' signal that could be used by a variety of bacteria for communication among and between species.     

FRET-based analysis of protein-nucleic acid interactions by genetically incorporating a fluorescent amino acid

Protein–nucleic acid interaction is an important process in many biological phenomena. In this study, a fluorescence resonance energy transfer (FRET)-based protein–DNA binding assay has been developed, in which a fluorescent amino acid is genetically incorporated into a DNA-binding protein. A coumarin-containing amino acid was incorporated into a DNA-binding protein, and the mutant protein specifically produced a FRET signal upon binding to its cognate DNA labeled with a fluorophore. The protein–DNA binding affinity was then measured under equilibrium conditions. This method is advantageous for studying protein-nucleic acid interactions, because it is performed under equilibrium conditions, technically easy, and applicable to any nucleic acid-binding protein.     

Genes of Rhodobacter sphaeroides 2.4.1 regulated by innate quorum-sensing signal, 7,8-cis-N-(tetradecenoyl) homoserine lactone

The free-living photoheterotrophic Gram-negative bacterium Rhodobacter sphaeroides possesses a quorumsensing (QS) regulatory system mediated by CerR-CerI, a member of the LuxR-LuxI family. To identify the genes affected by the regulatory system, random lacZ fusions were generated in the genome of R. sphaeroides strain 2.4.1 using a promoter-trapping vector, pSG2. About 20,000 clones were screened and 23 showed a significantly different level of beta- gal activities upon the addition of synthetic 7,8-cis-Ntetradecenoyl- homoserine lactone (RAI). Among these 23 clones, the clone showing the highest level of induction was selected for further study, where about a ten-fold increase of beta-gal activity was exhibited in the presence of RAI and induction was shown to be required for cerR. In this clone, the lacZ reporter was inserted in a putative gene that exhibited a low homology with catD. A genetic analysis showed that the expression of the catD homolog was initiated from a promoter of another gene present upstream of the catD. This upstream gene showed a strong homology with luxR and hence was named qsrR (quorum-sensing regulation regulator). A comparison of the total protein expression profiles for the wild-type cells and qsrR-null mutant cells using two-dimensional gel electrophoresis and a MALDI-TOF analysis allowed the identification of sets of genes modulated by the luxR homolog.     

Real-time measurement of myosin-nucleotide noncovalent complexes by electrospray ionization mass spectrometry

Nanoelectrospray ionization mass spectrometry has been used to measure the binding of ATP and ADP to the active site of rabbit skeletal myosin-S1. Increases in the molecular mass of myosin-S1 of 425 +/- 10 Da were obtained with the binding of ADP to the active site and by 530 +/- 10 Da with either ATP or hydrolysis products ADP and phosphate. Active site titrations of myosin-S1 with ADP gave a stoichiometry of approximately 1 ADP/S1 with an affinity in the micromolar range. The binding of ATP to myosin-S1 could be observed in the presence of up to 60 muM of excess MgATP without nonspecific binding of MgATP to the myosin. Conversion of the nucleotide complex containing an equilibrium mixture of ATP and ADP-Pi bound to myosin-S1 to one containing only bound ADP occurs at a rate consistent with that of the known steady-state rate of ATP hydrolysis. We expect this method to be of considerable use in the analysis of ligand binding and hydrolysis by the active sites of expressed myosin and myosin subfragments, which are not available in sufficient quantities for conventional methods of measurement of ligand binding.     

Antibacterial and hemolytic activities of linenscin OC2, a hydrophobic substance produced by Brevibacterium linens OC2

Linenscin OC2 is an antibacterial substance produced by the orange cheese coryneform bacterium Brevibacterium linens OC2. It inhibits the growth of Gram-positive bacteria but it is inactive against Gram-negative bacteria. The intact outer membrane of Gram-negative bacteria was shown to be an effective permeability barrier against linenscin OC2. At high dosage the effect of linenscin OC2 was bacteriolytic on Listeria innocua. Bacteriostasis was observed at low dosage and peptidoglycan biosynthesis was affected at an early step upstream of the UDP-N-acetylglucosamine. Hemolytic activity of this substance on sheep erythrocytes suggested a common mode of action on prokaryotic and eukaryotic cells. It also suggested that the cytoplasmic membrane might be the primary target of linenscin OC2.     

Real-time measurement of PMA-induced cellular alterations by microelectrode array-based impedance spectroscopy

For a feasible and cost-effective impedance measurement of cellular alterations in real-time, we combined commercially available microelectrode arrays (MEAs), consisting of 60 microelectrodes, with a conventional impedance analyzer. For proof of principle, a breast carcinoma cell line (MCF-7) was cultured on MEAs, and cellular alterations were measured by impedance spectroscopy at a frequency ranging from 10 Hz to 1 MHz. Cells were stimulated with phorbol 12-myristate 13-acetate (PMA) at different concentrations to activate protein kinase C (PKC)-mediated extra- and intracellular changes. By addition of 0.03 microM PMA, an increase of the relative impedance (Z(rel)) was observed after 10 min with a maximum at 1 kHz. Moreover a gradual elevation of the impedance was measured 60 min after stimulation with PMA. If 0.3 microM PMA was applied, the maximal amplitude of the relative impedance after 60 min shifted from 1 kHz (0.03 microM PMA) to 150 Hz. Subsequently, the impedance was further increased up to 90 min after PMA application, after which the impedance reduced after 240 min. Since we could use MEAs for at least 10 times without affecting the sensitivity, our study revealed that commercially available MEAs comprising nanocolumnar titanium nitrite electrodes are suitable microstructures for a highly reproducible and cost-effective multisite measurement of intracellular processes by impedance spectroscopy.     

Inhibition of Candida albicans biofilm formation by farnesol,a quorum-sensing molecule

Farnesol is a quorum-sensing molecule that inhibits filamentation in Candida albicans. Both filamentation and quorum sensing are deemed to be important factors in C. albicans biofilm development. Here we examined the effect of farnesol on C. albicans biofilm formation. C. albicans adherent cell populations (after 0, 1, 2, and 4 h of adherence) and preformed biofilms (24 h) were treated with various concentrations of farnesol (0, 3, 30, and 300 micro M) and incubated at 37 degrees C for 24 h. The extent and characteristics of biofilm formation were then assessed microscopically and with a semiquantitative colorimetric technique based on the use of 2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide. The results indicated that the effect of farnesol was dependent on the concentration of this compound and the initial adherence time, and preincubation with 300 micro M farnesol completely inhibited biofilm formation. Supernatant media recovered from mature biofilms inhibited the ability of planktonic C. albicans to form filaments, indicating that a morphogenetic autoregulatory compound is produced in situ in biofilms. Northern blot analysis of RNA extracted from cells in biofilms indicated that the levels of expression of HWP1, encoding a hypha-specific wall protein, were decreased in farnesol-treated biofilms compared to the levels in controls. Our results indicate that farnesol acts as a naturally occurring quorum-sensing molecule which inhibits biofilm formation, and we discuss its potential for further development and use as a novel therapeutic agent.     

Improvement of a FRET-based indicator for cAMP by linker design and stabilization of donor-acceptor interaction

F?rster resonance energy transfer (FRET) technology has been used to develop genetically encoded fluorescent indicators for a variety of intracellular molecular events. Often, however, the poor dynamic range of such reporters prevents detection of subtle but physiologically relevant signals. Here we present a strategy for improving FRET efficiency between donor and acceptor fluorophores in a green fluorescent protein (GFP)-based protein indicator for cAMP. Such indicator is based on protein kinase A (PKA) and was generated by fusion of CFP and YFP to the regulatory and catalytic subunits of PKA, respectively. Our approach to improve FRET efficiency was to perform molecular dynamic simulations and modelling studies of the linker peptide (L11) joining the CFP moiety and the regulatory subunit in order to define its structure and use this information to design an improved linker. We found that L11 contains the X-Y-P-Y-D motif, which adopts a turn-like conformation that is stiffly conserved along the simulation time. Based on this finding, we designed a new linker, L22 in which the YPY motif was doubled in order to generate a stiffer peptide and reduce the mobility of the chromophore within the protein complex, thus favouring CFP/YFP dipole-dipole interaction and improving FRET efficiency. Molecular dynamic simulations of L22 showed, unexpectedly, that the conformational behaviour of L22 was very loose. Based on the analysis of the three principal conformational states visited by L22 during the simulation time, we modified its sequence in order to increase its rigidity. The resulting linker L20 displayed lower flexibility and higher helical content than L22. When inserted in the cAMP indicator, L20 yielded a probe showing almost doubled FRET efficiency and a substantially improved dynamic range.     

Real-time measurement of glucose using chrono-impedance technique on a second generation biosensor

Chrono-impedance technique (CIT) was implemented as a new transduction method for real time measurement of glucose in a biosensor system based in carbon paste (CP)/Ferrocene (FC)/glucose oxidase (GOx). The system presents high selectivity because the optimal stimulation signal composed by a 165mV DC potential and 50mV(RMS) AC signal at 0.4Hz was used. The low DC potential used decreased the interfering species effect and the biosensor showed a linear impedance response toward glucose detection at concentrations from 0mM to 20mM,with 0.9853 and 0.9945 correlation coefficient for impedance module (|Z|) and phase (Φ), respectively. The results of quadruplicate sets reveal the high repeatability and reproducibility of the measurements with a relative standard deviation (RSD) less than 10%. CIT presented good accuracy (within 10% of the actual value) and precision did not exceed 15% of RSD for high concentration values and 20% for the low concentration ones. In addition, a high correlation coefficient (R(2)=0.9954) between chrono-impedance and colorimetric methods was obtained. On the other hand, when two samples prepared at the same conditions were measured in parallel with both methods (the measurement was repeated four times), it should be noticed that student's t-test produced no difference between the two mentioned methods (p=1). The biosensor system hereby presented is highly specific to glucose detection and shows a better linear range than the one reported on the previous article.     

Real-time fluorescence measurement of cell-free endosome fusion: regulation by second messengers.

A quantitative real-time assay of cell-free endosomal vesicle fusion was developed and applied to study fusion mechanisms in endosomes from baby hamster kidney (BHK-21) cells. The assay is based on an irreversible approximately 10-fold increase in BODIPY-avidin fluorescence on binding of biotinylated conjugates. BODIPY-avidin and biotin-dextran were internalized for 10 min at 37 degrees C into separate populations of BHK-21 cells, and endosome fractions were prepared. Postnuclear supernatant fractions underwent ATP- and temperature-dependent fusion, as measured in a sensitive custom-built microfluorimeter by the continuous increase in BODIPY-avidin fluorescence. Fusion processes of efficiency > 2.5% could be detected with 200-ms time resolution in sample volumes of 50 microL containing endosomes derived from approximately 4 x 10(4) cells. The fusion time course consisted of a distinct lag phase (up to 10 min) in which little fusion occurred, followed by an approximately exponential rise (t 1/2 10-30 min; fusion efficiency approximately 15%). The lag phase was reduced by preincubation of separate endosome fractions with ATP at 37 degrees C and by coincubation of endosomes at 22 degrees C before the assay, suggesting a rate-limiting step involving binding of a soluble protein to the endosome membrane. Endosome fusion was strongly inhibited by GTP gamma S, N-ethylmaleimide, and AIF4-. Endosome fusion was not affected by phorbol myristate acetate but was significantly inhibited by cAMP and bovine brain calmodulin. The results establish a sensitive real-time fluorescence assay to quantify the kinetics and extent of endosome fusion in a cell-free system and demonstrate regulation of early endosome fusion by cytosolic second messengers.