PCR primers for detection and characterisation of IncP-9 plasmids

Abstract

IncP-9 plasmids are best known as the vehicles for spreading biodegradation functions among Pseudomonas species but can also carry resistance determinants. New PCR primer systems targeting different replicon-specific regions were designed to allow detection of IncP-9 plasmids. Their specificity was checked against a range of IncP-9 plasmids as well as representatives of incompatibility groups IncFI, IncFII, IncN, IncQ, IncP-1alpha, IncP-1beta, IncP-2, IncP-7, IncP-13, IncW, IncU, IncX and IncZ. Products obtained for plasmids assigned to IncP-9 group by traditional incompatibility testing varied in size and restriction pattern suggesting diversity in the 'core' sequence among related replicons. Specific primer pairs were applied to community DNA extracted from a range of environments including those subject to strong selective pressure, caused by antibiotics, metals and organic pollutants. Abundant products were observed in manure and sewage, independently of the presence of antibiotics and metals, but could also be detected in coastal water and streptomycin-treated soil. Community DNA from faeces of piglets treated and non-treated with Zn gave particularly strong PCR product with IncP-9 rep primers. Therefore, an attempt was made to isolate bacteria carrying the IncP-9-like plasmids, but this was not successful. The results of application of these newly designed primer pairs to plasmid isolates as well as community DNA indicate that the IncP-9-related plasmids are a diverse family prevalent in various environments and widely distributed geographically.

Diversity of IncP-9 plasmids of Pseudomonas

“…When sequences of IncP-9 plasmids pMT2, pWW0, pDTG1 and NAH7 became available the relationships between them were estimated by sequence comparison and phylogenetic analysis (Dennis & Zylstra, 2004; Dennis, 2005; Greated & Thomas, 1999; Greated et al , 2002; Sota et al , 2006). PCR primers that target the basic replicon functions of IncP-9 have facilitated studies on the diversity and prevalence of naturally occurring IncP-9 plasmids (Greated & Thomas, 1999; Krasowiak et al , 2002). Thus, the genetic diversity of a limited number of phenotypically different IncP-9 plasmids (Krasowiak et al , 2002) and large collections of IncP-9 Nah replicons (Izmalkova et al , 2006; Leuchuk et al , 2006) have been evaluated by RFLP analysis of replication/maintenance loci, selected nah determinants or complete plasmid genomes.…”

“…PCR primers that target the basic replicon functions of IncP-9 have facilitated studies on the diversity and prevalence of naturally occurring IncP-9 plasmids (Greated & Thomas, 1999; Krasowiak et al , 2002). Thus, the genetic diversity of a limited number of phenotypically different IncP-9 plasmids (Krasowiak et al , 2002) and large collections of IncP-9 Nah replicons (Izmalkova et al , 2006; Leuchuk et al , 2006) have been evaluated by RFLP analysis of replication/maintenance loci, selected nah determinants or complete plasmid genomes. Environmental studies indicate a wide distribution of IncP-9-like replicons in manure, soils and coastal waters and their involvement in natural HGT of the naphthalene-degradation trait (Herrick et al , 1997; Krasowiak et al , 2002; Smalla et al , 2000).…”

Occurrence of Plasmids in the Aromatic Degrading Bacterioplankton of the Baltic Sea

“…Although degradative and drug resistant plasmids from the plasmid families IncP-1, IncP-2, IncP-4 (IncQ), IncP-7 and IncP-9 are of obvious significance in different environments [2,5,6,10], little is known about the plasmid pool of marine ecosystems. Only a few studies revealed that 19%–30% of isolated seawater bacterial strains may carry plasmids [8,11,12].…”

“…, man-made chemical compounds is often encoded by IncP-1 representatives [1,3,4]. It has been shown also that the resistance to antibiotics in Pseudomonas species is associated with the IncP-9 plasmids [5,6]. …”

“…Additionally, a few certain types of atypical IncP-9 plasmids are exempted from comparative phylogenetic analyses, revealing high level of sequence diversity among the IncP-9 plasmid family [6]. Wide geographical spread of IncP-9 plasmids appears to be strongly correlated with the existence of strong selective pressure [5]. …”