Introduction of pAM beta 1 into Listeria monocytogenes by conjugation and homology between native L. monocytogenes plasmids

Abstract

The broad host range antibiotic resistance plasmid pAM,B1 was transferred from Streptococcus faecalis to 9 of 15 Listeria monocytogenes strains by conjugation. L. monocytogenes transconjugates could transfer the plasmid either among L. monocytogenes strains or back to S. faecalis. Transfer between the various strains occurred without any detectable plasmid DNA rearrangements. The pAMP1 replicon was stable in L. monocytogenes-it was retained without antibiotic selection when the bacteria were grown in culture media or passed in mice-and the presence of pAMP1 had no major effect on L. monocytogenes virulence. These data suggest that pAM,B1 or its derivatives might serve as useful L. monocytogenes cloning vehicles. The data presented also demonstrate that pAMP1 is compatible with two different native L. monocytogenes plasmids and that Listeria species harbor native plasmids in addition to the 38.5-megadalton plasmid pRYC16 previously reported by Perez-Diaz et al. (J. C. Perez-Diaz, M. F. Vicente, and F. Banquero, Plasmid 8:112-118, 1982). Of 29 L. monocytogenes strains screened, 7 contained plasmid DNA. Four strains had similar if not identical plasmids that were 34 megadaltons in size, whereas three other strains contained either a 53-, 44-, or 32-megadalton plasmid; none of these plasmids has the same restriction pattern as pRYC16. DNA homology experiments indicate that the various plasmids are related and suggest that there may be a common set of sequences present in all of the plasmids examined.

Differentiation of Brucella species by Random Amplified Polymorphic DNA analysis

“…Total DNA was extracted from the strains using two methods, one including a phenolization step (Flamm et al 1984) and the other not (Pitcher et al 1989). The extracted DNA was always treated with RNAse (Boehringer, Mannheim, Germany), according to the description of Pitcher et al (1989).…”

Repetitive element sequence-based PCR for species and strain discrimination in the genus Listeria

“…Genomic DNA was extracted by a modified version of the method of Flamm (1984) as follows. Bacterial cells from 2 ml of culture were pelleted by centrifugation at 13 000 g for 2 min, washed in 1 ml of 1 x SSC (0.15 rnol 1-' NaCl; 0.015 rnol I-' sodium citrate), suspended in 100 pl of lysozyme solution (0.01 mol I-' sodium phosphate, 20% sucrose, lysozyme 4 mg ml-I, Boehringer Mannheim, Mannheim, Germany) and incubated for 45 min at 37°C.…”

A combined PCR and selective enrichment method for rapid detection of Listeria monocytogenes

“…Genomic DNA of Listeria species listed in Table 1 was extracted by the method of Flamm et al (1984). DNA from non-Listeria species was extracted by conventional methods.…”