A pair of PCR primers for IncP-9 plasmids

Greated, Alicia; Thomas, Christopher M.

doi:10.1099/00221287-145-11-3003a

Cited by 27 publications

(25 citation statements)

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“…The investigation of prevalence of plasmids belonging to the IncP‐9 group was carried out for samples originating from a range of environments, in most cases characterised by the presence of strong selective pressure, caused by antibiotics, metals and organic pollutants. To search for IncP‐9‐like plasmids we used the rep primer pair that had already been tested [4] as well as new pairs of oligonucleotides (Table 2), that amplified short segments of genes across the core replication and partitioning region exhibiting over 85% sequence identity between pMT2 and pWW0. The two pairs mpfA 1Fa– korA 2Ra and korA 3Fa– rep 3Rc that appear to be efficient for analysis of IncP‐9 isolates were not considered appropriate for screening of community DNA since the size of fragments they amplify would limit detection to intact DNA which is, however, likely to be sheared during extraction.…”

Section: Resultsmentioning

confidence: 99%

PCR primers for detection and characterisation of IncP-9 plasmids

Krasowiak

Smalla²,

Sokolov

et al. 2002

FEMS Microbiology Ecology

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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.

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Section: Resultsmentioning

confidence: 99%

PCR primers for detection and characterisation of IncP-9 plasmids

Krasowiak

Smalla²,

Sokolov

et al. 2002

FEMS Microbiology Ecology

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“…Nevertheless, our analysis revealed a number of genes which seem likely to be largely responsible for the replication and stable maintenance of IncP-9 plasmids. PCR primers designed on the basis of the putative rep gene found in pMT2 (Greated & Thomas, 1999) also gave a product for another IncP-9 plasmid, pWW0, suggesting that this replication system is an element of other IncP-9 plasmids. Since IncP-9 plasmids have been mainly isolated from Pseudomonas species, the temperature sensitivity in enteric bacteria may simply reflect the lack of selection to retain a functional rep system for these species.…”

Section: Discussionmentioning

confidence: 99%

“…The sequence and associated analysis should allow development of tools to monitor IncP-9 plasmids in the environment (Greated & Thomas, 1999).…”

Section: Discussionmentioning

confidence: 99%

The replication and stable-inheritance functions of IncP-9 plasmid pM3 The GenBank accession number for the sequence reported in this paper is AF078924.

et al. 2000

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Little is known of the transfer and maintenance machinery of the IncP-9 plasmids, which are found in Pseudomonas spp. and include both degradative and resistance plasmids. One such plasmid, pM3, which confers resistance to streptomycin and tetracycline, was found repeatedly in Pseudomonas species from numerous locations in Belarus. pM3 has a broad host range, but is unable to replicate in enterobacteria at 37 SC and above. A mini derivative, pMT2, was constructed by partial PstI digestion and ligation with a fragment encoding Km R . The complete sequence of pMT2 was determined. Analysis of its 8526 bp of pM3 DNA revealed several ORFs whose predicted polypeptide products were found to have similarity to previously analysed proteins involved in plasmid replication (rep gene), transfer (mpf ; mating-pair formation gene) and stable maintenance (par, mrs genes). The organization of these genes showed similarity to several plasmid systems including the Ti and pSYM plasmids as well as IncP-1 plasmids. Subcloning narrowed down the region required for replication, and identified the putative rep gene and putative par promoter region as able to express incompatibility. rep deletion mutants were lost from the cell line, and expression of the rep gene was shown to be controlled by negative autoregulation. A pMT2 derivative with an insertion between the rep and par genes showed very weak, if any, ability to replicate autonomously, suggesting that plasmid maintenance may depend on a close interaction of rep and par functions.

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“…21 Further analysis of the pDTG1 repA gene showed that, unlike the other IncP-9 plasmids from which the PCR primers were designed, these DNA regions contained three mismatches where the forward IncP-9 rep PCR primer bound and two mismatches where the reverse IncP-9 rep PCR primer bound. Even though the pDTG1 repA gene sequence is highly homologous to that encoded on other IncP-9 plasmids, in our hands, using variations in PCR annealing temperatures from 45 -65 8C and modifications to PCR reaction mixtures with DMSO or betaine, we were unable to generate this PCR product (data not shown).…”

Section: Plasmid Replication Control Functions Show Highest Similaritmentioning

confidence: 99%