In many organisms, population-density sensing and sexual attraction rely on small-molecule-based signalling systems1,2. In the nematode Caenorhabditis elegans, population density is monitored through specific glycosides of the dideoxysugar ascarylose (the `ascarosides') that promote entry into an alternative larval stage, the non-feeding and highly persistent dauer stage3,4. In addition, adult C. elegans males are attracted to hermaphrodites by a previously unidentified small-molecule signal5,6. Here we show, by means of combinatorial activity-guided fractionation of the C. elegans metabolome, that the mating signal consists of a synergistic blend of three dauer-inducing ascarosides, which we call ascr#2, ascr#3 and ascr#4. This blend of ascarosides acts as a potent male attractant at very low concentrations, whereas at the higher concentrations required for dauer formation the compounds no longer attract males and instead deter hermaphrodites. The ascarosides ascr#2 and ascr#3 carry different, but overlapping, information, as ascr#3 is more potent as a male attractant than ascr#2, whereas ascr#2 is slightly more potent than ascr#3 in promoting dauer formation7. We demonstrate that ascr#2, ascr#3 and ascr#4 are strongly synergistic, and that two types of neuron, the amphid single-ciliated sensory neuron type K (ASK) and the male-specific cephalic companion neuron (CEM), are required for male attraction by ascr#3. On the basis of these results, male attraction and dauer formation in C. elegans appear as alternative behavioural responses to a common set of signalling molecules. The ascaroside signalling system thus connects reproductive and developmental pathways and represents a unique example of structure- and concentration-dependent differential activity of signalling molecules.
Lantibiotics are polycyclic peptides containing unusual amino acids, which have binding specificity for bacterial cells, targeting the bacterial cell wall component lipid II to form pores and thereby lyse the cells. Yet several members of these lipid II-targeted lantibiotics are too short to be able to span the lipid bilayer and cannot form pores, but somehow they maintain their antibacterial efficacy. We describe an alternative mechanism by which members of the lantibiotic family kill Gram-positive bacteria by removing lipid II from the cell division site (or septum) and thus block cell wall synthesis.
Elucidation of the composition of chemical-biological samples is a main focus of systems biology and metabolomics. Due to the inherent complexity of these mixtures, reliable, efficient, and potentially automatable methods are needed to identify the underlying metabolites and natural products. Because of its rich chemical information content, nuclear magnetic resonance (NMR) spectroscopy has a unique potential for this task. Here we present a generalization and application of a recently introduced NMR data collection, processing, and analysis strategy that circumvents the need for extensive purification and hyphenation prior to analysis. It uses covariance TOCSY NMR spectra measured on a 1-mm high-temperature cryogenic probe that are analyzed by a spectral trace clustering algorithm yielding 1D NMR spectra of the individual components for their unambiguous identification. The method is demonstrated on a metabolic model mixture and is then applied to the unpurified venom mixture of an individual walking stick insect that contains several slowly interconverting and closely related metabolites.
Mutacin 1140 is a member of a family of ribosomally synthesized peptide bacteriocins called lantibiotics (lanthionine-containing antibiotics) and is produced by the Gram-positive bacterium Streptococcus mutans. Mutacin 1140 has been shown to be effective against a broad array of Gram-positive bacteria. Chromatography and mass spectroscopy data suggested that mutacin 1140 forms a small compact structure. Nuclear magnetic resonance (NMR) data and restrained molecular dynamics simulations showed that mutacin 1140 interconverts between multiple structures. Calculations of scalar (J) coupling constants showed the best agreement with experimental values when the entire population-weighted ensemble of structures was used, providing independent support for the ensemble. Representative structures from each major group in the ensemble had a common feature in which they are all kinked around the hinge region forming a horseshoe-like shape, and the regions of flexibility of the molecule were limited and well-defined. The structures determined in this study provide a starting point for modeling the mutacin 1140-membrane interactions and pore formation.
Lithium has been used clinically in the treatment of manic depression. However, its pharmacologic mode of action remains unclear. Characteristics of Li ± interactions in red blood cells (RBCs) have been identified. We investigated Li + interactions on human neuroblastoma SH-SY5Y cells by developing a novel 7Li NMR method that provided a clear estimation of the intra-and extracellular amounts of LV in the presence of the shift reagent thulium-1,4,7,10tetrazacyclododecane-N , N', N", N"-tetramethylene phosphonate (HTmDOTP4). The first-order rate constants of Li + influx and efflux for perfused, agarose-embedded SH-SY5Y cells in the presence of 3 mM HTmDOTP4 were 0.055 ±0.006 (n = 4) and -0.025 ±0.006 min~1(n = 3), respectively. Significant increases in the rate constants of Li + influx and efflux in the presence of 0.05 mM veratridine indicated the presence of Nachannel-mediated Litransport in SH-SY5Y cells. 7Li NMR relaxation measurements showed that Li~is immobilized more in human neuroblastoma SH-SY5Y cells than in human RBCs. A 0, starting area of the intracellular resonance of Li * -loaded cells in Li + efflux measurements; A~, intracellular resonance area at time t; AA, atomic absorption; DIDS, 4,4 '-diisothiocyanatostilbene-2,2 '-disulfonic acid; DMEM, Dulbecco's modified Eagle's medium; HTmDOTP 4~, thulium-1,4,7,1 0-tetrazacyclododecane-N, N', N", N"-tetramethylenephosphonate; PCr, phosphocreatine; P,, inorganic phosphorus; ppm, parts per million; RBC, red blood cell; T,, spin-lattice relaxation value; T 2, spin-spin relaxation value; v0, initial rate of Li * influx.
Conformational properties of several similar FMRFamide-like neuropeptides from mollusks were investigated by nuclear magnetic resonance (NMR) spectroscopy. It was found that amino acid substitutions in the N-terminal variable regions of the peptides had dramatic effects on the populations of reverse turns in solution. The populations of turns, as measured by two independent NMR parameters, were found to be highly correlated (r(2) = 0.93 and 0. 82) with IC(50) values using receptor membrane preparations from Helix aspersa (Payza, 1987; Payza et al., 1989). These results suggest that the amount of turn in the free peptide can influence the receptor binding affinities of that peptide. On the basis of these observations, a model was developed in which only a single species from a conformational ensemble of an unbound peptide will bind to a particular receptor. Thus, the conformational ensemble reduces the effective concentration of a particular peptide with respect to a particular receptor.
Theory indicates that at least some proteins will undergo a rapid and unimpeded collapse, like a disorganized hydrophobic chain, prior to folding. Yet experiments continue to find signs of an organized, or barrier-limited, collapse in even the fastest (approximately mus) folding proteins. Does the kinetic barrier represent a signature of the equilibrium "foldability" of these molecules? We have measured the rate of chain contraction in two nonfolding analogs of a very fast-collapsing protein. We find that these chains contract on the same time scale (approximately 10(-5)s) as the natural protein, and both pass over an energetic barrier at least as large as that encountered by the protein. The equilibrium foldability of the native structure therefore does not alone determine the dynamics of collapse; even the disordered chains contract approximately 1000x slower than expected for an ideal chain.
Caenorhabditis elegans, a bacterivorous nematode, lives in complex rotting fruit, soil, and compost environments, and chemical interactions are required for mating, monitoring population density, recognition of food, avoidance of pathogenic microbes, and other essential ecological functions. Despite being one of the best-studied model organisms in biology, relatively little is known about the signals that C. elegans uses to chemically interact with its environment or as defense. C. elegans exudates were analyzed using several analytical methods and found to contain 36 common metabolites including organic acids, amino acids and sugars, all in relatively high abundance. Furthermore, the concentrations of amino acids in the exudates were dependent on developmental stage. The C. elegans exudates were tested for bacterial chemotaxis using Pseudomonas putida (KT2440), a plant growth promoting rhizobacterium, Pseudomonas aeruginosa (PAO1), a soil bacterium pathogenic to C. elegans, and E. coli (OP50), a non-motile bacterium tested as a control. The C. elegans exudates attracted the two Psuedomonas species, but had no detectable antibacterial activity against P. aeruginosa. To our surprise, the exudates of young adult and adult life stages of C. elegans exudates inhibited quorum sensing in the reporter system based on the LuxR bacterial quorum sensing (QS) system, which regulates bacterial virulence and other factors in Vibrio fischeri. We were able to fractionate the QS inhibition and bacterial chemotaxis activities, demonstrating that these activities are chemically distinct. Our results demonstrate that C. elegans can attract its bacterial food and has the potential of partially regulating the virulence of bacterial pathogens by inhibiting specific QS systems.
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