Detection and characterization of broad-host-range plasmids in environmental bacteria by PCR

• Published in: Applied and Environmental Microbiology Vol. 62; no. 7; pp. 2621 - 2628
• Main Authors: Gotz, A. (Biologische Bundesanstalt fur Land- und Forstwirtschaft, Wageningen, The Netherlands.), Pukall, R, Smit, E, Tietze, E, Prager, R, Tschape, H, Elsas, J.D. van, Smalla, K
• Format: Journal Article
• Language: English
• Published: Washington, DC American Society for Microbiology 01.07.1996
• Subjects: ADN; BACTERIA; Bacteria - genetics; Bacteria - isolation & purification; Bacteriological methods and techniques used in bacteriology; Bacteriology; Base Sequence; Biological and medical sciences; CERDO; Conserved Sequence; Deoxyribonucleic acid; DNA; DNA Primers - genetics; DNA, Bacterial - genetics; Environmental Microbiology; Escherichia coli - genetics; ESTIERCOL; FLORA DEL SUELO; FLORE DU SOL; FUMIER; Fundamental and applied biological sciences. Psychology; Genes; HIBRIDACION DE ADN; HYBRIDATION D'ADN; Instituut voor Plantenziektenkundig Onderzoek; Microbiology; Molecular Sequence Data; PCR; PLASMIDE; PLASMIDIOS; Plasmids - genetics; Plasmids - isolation & purification; Polymerase Chain Reaction - methods; PORCIN; Replicon; Research Institute for Plant Protection; TECHNIQUE ANALYTIQUE; TECNICAS ANALITICAS; Plasmid; Polymerase chain reaction; Specificity; Primer; Bacteria; Environment; Method; Detection
• ISSN: 0099-2240; 1098-5336
• DOI: 10.1128/aem.62.7.2621-2628.1996

Primer systems for PCR amplification of different replicon-specific DNA regions were designed on the basis of published sequences for plasmids belonging to the incompatibility (Inc) groups IncP, IncN, IncW, and IncQ. The specificities of these primer systems for the respective Inc groups were tested with a collection of reference plasmids belonging to 21 different Inc groups. Almost all primer systems were found to be highly specific for the reference plasmid for which they were designed. In addition, the primers were tested with plasmids which had previously been grouped by traditional incompatibility testing to the IncN, IncW, IncP, or IncQ group. All IncQ plasmids gave PCR products with the IncQ primer systems tested. However, PCR products were obtained for only some of the IncN, IncP, and IncW group plasmids. Dot blot and Southern blot analyses of the plasmids revealed that PCR-negative plasmids also failed to hybridize with probes derived from the reference plasmids. The results indicated that plasmids assigned to the same Inc group by traditional methods might be partially or completely different from their respective reference plasmids at the DNA level. With a few exceptions, all plasmids related to the reference plasmid at the DNA level also reacted with the primer systems tested. PCR amplification of total DNA extracted directly from different soil and manure slurry samples revealed the prevalence of IncQ- and IncP-specific sequences in several of these samples. In contrast, IncN- and IncW-specific sequences were detected mainly in DNA obtained from manure slurries