Most bacteria release extracellular molecules into the surrounding medium to self-regulate expression of specific sets of genes in response to their own population density, once a critical concentration ("quorum") of the signalling molecules has been reached. The process is known as "quorum sensing" (QS) and is used by bacteria to monitor population density and to change bacterial gene expression in order to compete and persist in nature or to colonize a particular environment. QS is also involved in inter-species communications, like that of higher plants with bacteria. In Gram negative bacteria, QS is mediated mostly by N-acylhomoserine lactones (AHSs). The chemical structure of AHLs comprises a homoserine lactone moiety linked to an acyl side chain, varying in length and nature of the substituent (i.e. oxo or hydroxy group). In addition, the acyl side chain can vary with regard to the presence of double bonds; although most AHLs have fully saturated acyl side chains. This communication reports the results of a study performed to identify the N-acylhomoserine lactone signalling molecules released by several nitrogenfixing bacteria such as Azospirillum brasilense, Herbaspirillum seropedicae, Burkholderia ambifaria, and Gluconacetobacter diazotrophicus, which are of interest in plant microbiology to study pathogenic or symbiotic interactions of bacteria with plant hosts. The AHLs, extracted from cell-free spent culture supernatants of the selected bacteria, have been separated ad identified by HPLC-ESI-MS, using a narrow bore reversed phase column. The HPLC method has been developed by a Quality-by-Design approach using the chromatographic modelling software DryLab® to optimize gradient time, column temperature, composition and pH of the eluent, flow rate, and start and end concentration of the gradient. The optimized method has been validated in terms of linearity of calibration graphs, limits of detection, limits of quantification, repeatability and accuracy, which has been evaluated by a recovery study.
Narrow-borrow RP-HPLC of N-acylhomoserine lactone (AHL) quorum sensing signaling molecules released by gramnegative bacteria
Isabella Nicoletti;Danilo Corradini
2014
Abstract
Most bacteria release extracellular molecules into the surrounding medium to self-regulate expression of specific sets of genes in response to their own population density, once a critical concentration ("quorum") of the signalling molecules has been reached. The process is known as "quorum sensing" (QS) and is used by bacteria to monitor population density and to change bacterial gene expression in order to compete and persist in nature or to colonize a particular environment. QS is also involved in inter-species communications, like that of higher plants with bacteria. In Gram negative bacteria, QS is mediated mostly by N-acylhomoserine lactones (AHSs). The chemical structure of AHLs comprises a homoserine lactone moiety linked to an acyl side chain, varying in length and nature of the substituent (i.e. oxo or hydroxy group). In addition, the acyl side chain can vary with regard to the presence of double bonds; although most AHLs have fully saturated acyl side chains. This communication reports the results of a study performed to identify the N-acylhomoserine lactone signalling molecules released by several nitrogenfixing bacteria such as Azospirillum brasilense, Herbaspirillum seropedicae, Burkholderia ambifaria, and Gluconacetobacter diazotrophicus, which are of interest in plant microbiology to study pathogenic or symbiotic interactions of bacteria with plant hosts. The AHLs, extracted from cell-free spent culture supernatants of the selected bacteria, have been separated ad identified by HPLC-ESI-MS, using a narrow bore reversed phase column. The HPLC method has been developed by a Quality-by-Design approach using the chromatographic modelling software DryLab® to optimize gradient time, column temperature, composition and pH of the eluent, flow rate, and start and end concentration of the gradient. The optimized method has been validated in terms of linearity of calibration graphs, limits of detection, limits of quantification, repeatability and accuracy, which has been evaluated by a recovery study.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
