ABSTRACT
We identified a genetic context encoding a transcriptional regulator of the Rgg family and a small hydrophobic peptide (SHP) in nearly all streptococci and suggested that it may be involved in a new quorum-sensing mechanism, with SHP playing the role of a pheromone. Here, we provide further support for this hypothesis by constructing a phylogenetic tree of the Rgg and Rgg-like proteins from Gram-positive bacteria and by studying the shp/rgg1358 locus of Streptococcus thermophilus LMD-9. We identified the shp1358 gene as a target of Rgg1358, and used it to confirm the existence of the steps of a quorum-sensing mechanism including secretion, maturation and reimportation of the pheromone into the cell. We used surface plasmon resonance to demonstrate interaction between the pheromone and the regulatory protein and performed electrophoretic mobility shift assays to assess binding of the transcriptional regulator to the promoter regions of its target genes. The active form of the pheromone was identified by mass spectrometry. Our findings demonstrate that the shp/rgg1358 locus encodes two components of a novel quorum-sensing mechanism involving a transcriptional regulator of the Rgg family and a SHP pheromone that is detected and reimported into the cell by the Ami oligopeptide transporter.
Subject(s)
Bacterial Proteins/metabolism , Pheromones/metabolism , Quorum Sensing , Streptococcaceae/metabolism , Trans-Activators/metabolism , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , DNA-Binding Proteins , Electrophoretic Mobility Shift Assay , Gene Expression Regulation, Bacterial , Hydrophobic and Hydrophilic Interactions , Mass Spectrometry , Phylogeny , Promoter Regions, Genetic , Sequence Analysis, DNA , Signal Transduction , Streptococcaceae/chemistry , Streptococcaceae/genetics , Surface Plasmon Resonance , Trans-Activators/chemistry , Trans-Activators/genetics , Transcription, GeneticABSTRACT
In anesthetized, normotensive beta-blocked rats, the alpha 1-adrenoceptor blocking drug, AR-C 239 (300 micrograms/kg, i.v.) induced a bradycardic effect related to a central increase in the vagal tone. This bradycardia was inhibited by previous administration of naloxone, intravenously (1 mg/kg) or centrally (100 micrograms/kg, i.c.) injected. Naloxone, by itself did not change the heart rate. In brainstem membranes from normotensive rats, AR-C 239 did not influence the stereoselective binding of [3H]naloxone. In spontaneously hypertensive (SH) rats, naloxone peripherally or centrally administered did not influence the activation of the vagal tone induced by AR-C 239, in beta-blocked animals. These results suggest the possible involvement of opiate release in the AR-C 239-induced vagal bradycardia, in normotensive rats. They also afford new arguments for the existence of close interactions between central alpha-adrenergic and opiate systems in the cardiovascular regulation. The possible participation of kappa-receptors in this effect is discussed. In addition, such an opiate mechanism triggered by central alpha 1-adrenoceptor blockade seems to be either absent or inactive in SH rats.
