Identification of phenolic compounds from pollen extracts using capillary electrophoresis-electrospray time-of-flight mass spectrometry.

In this work, a new, easy and rapid method of analyzing phenolic compounds in pollen extract, based on capillary electrophoresis coupled with electrospray ionization time-of-flight-mass spectrometry (CE-ESI-TOF-MS), has been developed. A systematic investigation of separation parameters has been performed with respect to resolution, sensitivity, analysis time and peak shape. The electrophoretic parameters and electrospray conditions must be optimized to obtain reproducible analyses. Using this method, several important phenolic compounds such as acetin-glucoside, 7-O-methylherbacetin-3-sophoroside, galloyl-glucose, quercetin-3-sophoroside, apigenin-6,8-di-C-glycoside, quercetin-3-rutinoside, genistein-7-O-beta-D: -glucoside, luteolin-7-O-glucoside, apigenin-7-O-glucoside and 2',4',6'-trihydroxy-3'-formyldihydrochalcone have been determined directly from pollen extract. The efficiency, the rapidity, the small amounts of sample required, and the high resolution of CE coupled with the sensitivity, the selectivity, the accurate masses and the true isotopic patterns obtained using TOF-MS point to the potential of this approach for identifying the phenolic compounds present in pollen.

Physical mapping of the conjugative plasmid pMB1-1 of Enterococcus faecalis.

The sexpheromone system of Enterococcus faecalis is a form of bacterial conjugation that plays an important role in the horizontal dissemination of genes. The ecological significance of 'sexual' plasmids is to permit a rapid mobilization of genes of interest for the species (e.g. those encoding haemolysins, bacteriocins or antibiotic resistance). The physical mapping of pMB1-1, a conjugative plasmid of Ent. faecalis that responds to cCF10 pheromone, has been undertaken. By means of hybridization with conserved sequences of pCF10 plasmid, the regions harbouring the genes responsible for the pheromone inhibitor and the aggregation and exclusion proteins of this plasmid have been identified. The results demonstrated that plasmids pMB1-1 and pCF10 only show homology in the region involved in the conjugative response, suggesting that this region may be transferred in an independent way to that of the rest of the plasmid.

Incidence of antibiotic resistance and sex pheromone response among enterococci isolated from clinical human samples and from municipal waste water.

The incidence of resistance to various antibiotics as well as the capacity to elicit aggregation response to sex pheromones have been investigated in strains of Enterococcus faecalis isolated from clinical and municipal waste waters (MWW). While clinical isolates showed a high incidence of antibiotic resistance (87%) and sex pheromone response (33%), these traits appeared with a much lower frequency in MWW isolates (12% and 4% respectively). The simultaneous incidence of both traits was of 52% and 0% for clinical and MWW isolates, respectively. The capacity to elicit a positive pheromone response as well as antibiotic resistance traits seemed to be strongly correlated with the presence of gelatinase activity among clinical isolates. Among MWW isolates, only sex pheromone response seemed to correlate with the presence of gelatinase activity.

Purification of sex pheromones specific for pMB1 and pMB2 plasmids of Enterococcus faecalis S-48.

The strain Enterococcus faecalis S-48 carries two large conjugative plasmids (pMB1 and pMB2) encoding for antagonistic substances. The pheromone response of these two plasmids was established by purifying the corresponding sex pheromones, using conventional reversed-phase columns. Plasmid pMB1 responds to pheromone cCF10. Plasmid pMB2 responds to a pheromone with an amino acid sequence identical to that of cPD1 (Phe-Leu-Val-Met-Phe-Leu-Ser-Gly). The two pheromone-responding plasmids coexist in a stable fashion in the wild-type strain E. faecalis S-48.

Bacteriocin plasmid pMB1 of Enterococcus faecalis: identification of the cell aggregation substance after induction by sex pheromone.

Strains of Enterococcus faecalis carrying the bacteriocinogenic plasmids pMB1 or pMB1.1 exhibit a clumping response to culture supernatants of different enterococcal strains. Antibodies raised against cells induced by a homologous pheromone recognize two surface proteins of 152 and 72.5 kDa (the second one is possibly the degradation product of the first), respectively. These antigens are very similar to those found in induced cells of E. faecalis OGIRF(pAM211) as shown by the cross-reaction of the immune sera obtained in this work. We propose that the 152-kDa protein corresponds to the aggregation substance coded by plasmids pMB1 and pMB1.1. Moreover, antibodies raised against induced cells are able to block the pheromone-induced clumping response. When the cells induced to form aggregates by pheromones were examined under a scanning electron microscope they showed a surface layer of hairlike structures.