Unusual enantiomeric D,L-N-acyl homoserine lactones in Pectobacterium atrosepticum and Pseudomonas aeruginosa.

Quorum Sensing allows bacteria to sense their population density via diffusible N-acyl homoserine lactone (N-HL) signaling molecules. Upon reaching a high enough cell density, bacteria will collectively exhibit a phenotype. Until recently, methods used for detection of N-HLs have not considered the chirality of these molecules and it was assumed that only the L-enantiomer was produced by bacteria. The production and effects of D-N-HLs have rarely been studied. In this work, the temporal production of D-N-HLs by the plant pathogen Pectobacterium atrosepticum and the human pathogen Pseudomonas aeruginosa are reported. Both bacteria produced D-N-HLs in significant amounts and in some cases their concentrations were higher than other low abundance L-N-HLs. Previously unreported D-enantiomers of N-3-oxoacyl and N-3-hydroxyacyl homoserine lactones were detected in P. atrosepticum. Interestingly, L-N-HLs produced in the lowest concentrations had relatively higher amounts of their corresponding D-enantiomers. Potential sources of D-N-HLs and their significance are considered.

Investigating chirality in quorum sensing by analysis of Burkholderia cepacia and Vibrio fischeri with comprehensive chiral LC-MS/MS and GC-MS/MS methods.

N-acyl homoserine lactones (N-HLs) are signaling molecules used by Gram-negative bacteria in a phenomenon called quorum sensing. Bacteria will detect N-HLs as a way of monitoring their population which, upon reaching a critical level, will express a specific phenotype. An example is the expression of bioluminescence by Vibrio fischeri. Most studies have not considered the chirality of these molecules nor have they used highly sensitive detection methods. Here, the production of d,l-N-HLs are monitored for V. fischeri, Burkholderia cepacia, Pseudomonas fluorescens, and P. putida, using highly sensitive tandem mass spectrometry analysis. Novel N-HLs are reported for both V. fischeri and B. cepacia, including a plethora of previously unknown d-N-HLs, including the first d-N-HLs containing oxo and hydroxy functionalities. Anomalously, N-HLs were not detected in any cultures of P. fluorescens and P. putida, which are species that previously were reported to produce N-HLs. However, it is apparent that differences in the reported occurrence and levels of N-HLs can result from (a) different strains of bacteria, (b) different growth media and environmental conditions, and (c) sometimes false-positive results from detection methodologies. Time studies of V. fischeri suggest the possibility that separate synthetic and elimination pathways exist between d- and l-N-HLs. Possible biological processes that could be the source of d-N-HL production are considered.

Comprehensive chiral GC-MS/MS and LC-MS/MS methods for identification and determination of N-acyl homoserine lactones.

N-acyl homoserine lactones (N-HLs) are signaling molecules synthesized by gram-negative bacteria to communicate in a process called quorum sensing. Most reported methods for the analysis of N-HLs, which are chiral molecules, do not distinguish between enantiomers. Typical examples include biosensors, liquid chromatography with UV detection, gas chromatography coupled with a mass spectrometer (GC-MS) and liquid chromatography coupled with mass spectrometer (LC-MS). Recently, the production of both D,L-N-HLs have been reported in Vibrio fischeri and Burkholderia cepacia. Concentrations of the D-N-HLs were found at the limit of quantification for the employed method. Therefore, for further studies of the role of the D-N-HLs in bacterial physiology, more sensitive, reliable, and selective analytical methods are necessary. In this work, such comprehensive chiral analytical methods for the identification and determination of 18 N-HLs using solid phase extraction followed by GC-MS/MS and LC-MS/MS analyses were developed. Extraction recoveries for the more hydrophilic C4 N-HLs were <10% of all other N-HLs, thus offering a possible explanation as to their lack of detection in previous studies. The chiral separations of all 18 N-HLs derivatives were accomplished by the complementary GC-MS/MS and LC-MS/MS methods. The limit of detection for LC-MS/MS method was as low as 1 ppb. The limit of detection for the GC-MS/MS method was found to be one to three orders of magnitude higher than the LC-MS/MS method. Due to the high extraction recovery and a preconcentration factor of 100, concentrations as low as 10 ppt can be detected by LC-MS/MS in biological samples. The LC-MS/MS approach provided greater enantioselectivity for the larger, more hydrophobic N-HLs while GC-MS/MS provided better enantioselectivity for the smaller N-HLs.

Production of both l- and d- N-acyl-homoserine lactones by Burkholderia cepacia and Vibrio fischeri.

Quorum sensing (QS) is a complex process in which molecules, such as l-N-acyl-homoserine lactones (l-AHLs), are produced as essential signaling molecules allowing bacteria to detect and respond to cell population density by gene regulation. Few studies have considered the natural production and role of the opposite enantiomers, d-AHLs. In this work, production of d,l-AHLs by Burkholderia cepacia and Vibrio fischeri was monitored over time, with significant amounts of d-AHLs detected. Bioluminescence of V. fischeri was observed with maximum bioluminescence correlating with the maximum concentrations of both l- and d- octanoyl-homoserine lactones (l- and d-OHL). l-Methionine, a precursor to l-AHLs, was examined via supplementation studies conducted by growing three parallel cultures of B. cepacia in M9 minimal media with added l-, d-, or d,l-methionine and observing their effect on the production of d,l-AHL by B. cepacia. The results show that addition of any methionine (l-, d-, or d,l-) does not affect the overall ratio of l- to d-AHLs, that is d-AHL production was not selectively enhanced by d-methionine addition. However, the overall AHL (l- and d-) concentration does increase with the addition of any methionine supplement. These findings indicate the possibility of a distinct biosynthetic pathway for d-AHL production, possibly exposing a new dimension within bacterial communication.

Headspace study of chiral interconversion of N-acetyl-homocysteine thiolactones.

The rate constants for (L)-N-acetyl homocysteine thiolactone enantiomerization have been obtained from batch-wise studies and by dynamic gas chromatography of racemic mixtures. Results from the batch-wise experiments show that the kinetics of racemization at 150 °C is the same for vials made of glass, silanized glass or Teflon-coated glass so that the vial surface exhibited no effect on the kinetics of racemization. From the temperature dependence of the rate constants the preexponential factor, activation energy, the activation Gibbs energy and activation entropy have been obtained from transition state theory. The catalytic effect of G-DP, G-BP and B-DP GC chiral stationary phases on racemization has been observed and quantified by the values of rate constants; B-DP exhibited the greatest activity. The Eyring activation parameters obtained from batch-wise experiment were compared with theoretical values acquired from quantum chemical modelling. Agreement between the experimental and calculated values of activation Gibbs energy, activation enthalpy and activation entropy is good. The dynamic gas chromatography of racemic mixture on chiral B-DP, G-DP and G-BP capillary columns indicate that the rate constants of forward and reverse reactions are different in chiral environments. The greatest accelerating effect in the process of enantiomerization has been identified for G-BP both in the batch-wise experiments and by the dynamic gas chromatography.

Evaluation of the Edman degradation product of vancomycin bonded to core-shell particles as a new HPLC chiral stationary phase.

A modified macrocyclic glycopeptide-based chiral stationary phase (CSP), prepared via Edman degradation of vancomycin, was evaluated as a chiral selector for the first time. Its applicability was compared with other macrocyclic glycopeptide-based CSPs: TeicoShell and VancoShell. In addition, another modified macrocyclic glycopeptide-based CSP, NicoShell, was further examined. Initial evaluation was focused on the complementary behavior with these glycopeptides. A screening procedure was used based on previous work for the enantiomeric separation of 50 chiral compounds including amino acids, pesticides, stimulants, and a variety of pharmaceuticals. Fast and efficient chiral separations resulted by using superficially porous (core-shell) particle supports. Overall, the vancomycin Edman degradation product (EDP) resembled TeicoShell with high enantioselectivity for acidic compounds in the polar ionic mode. The simultaneous enantiomeric separation of 5 racemic profens using liquid chromatography-mass spectrometry with EDP was performed in approximately 3 minutes. Other highlights include simultaneous liquid chromatography separations of rac-amphetamine and rac-methamphetamine with VancoShell, rac-pseudoephedrine and rac-ephedrine with NicoShell, and rac-dichlorprop and rac-haloxyfop with TeicoShell.