Plasmids from freshwater environments capable of IncQ retrotransfer are diverse and include pQKH54, a new IncP-1 subgroup archetype

Abstract

Nine mercury-resistance plasmids isolated from river epilithon were assessed for their ability to retrotransfer the non-conjugative IncQ plasmid, R300B, derivatives of which have commercial uses that may result in accidental or deliberate release into the environment. Retrotransfer frequencies ranging from 2?1610 "4 to 1?75610 "5 were obtained for five of the nine plasmids -the remaining plasmids showed low or undetectable retrotransfer ability. The majority of the retrotransfer-proficient plasmids could not be classified by the tests used. Classical incompatibility testing with RP4 identified pQKH6, pQKH54 and pQM719 as IncP-1. Hybridization to replicon probes confirmed this for pQKH6 and pQM719 and added pQKH33. PCR with primers designed to amplify trfA and korA regions of IncP-1 plasmids did not identify any other plasmids. Plasmids pQKH6 and pQM719 but not pQKH54 produced similar SphI restriction profiles to the IncP-1b subgroup. The complete nucleotide sequence of pQKH54 was determined, revealing it to have a complete IncP-1 backbone but belonging to a new distinct subgroup which was designated IncP-1c. The results emphasize the ubiquity and diversity of IncP-1 plasmids in the environment but demonstrate that plasmids of as yet unknown groups are also able to retrotransfer IncQ plasmids efficiently.

Host range diversification within the IncP-1 plasmid group

“…This was observed in conjugation assays with a P. putida strain as plasmid donor (Haines et al, 2006). By comparing genomic signatures between whole plasmid genome sequences and bacterial chromosomes we previously showed that pQKH54 had the highest genomic signature similarity to three Pseudomonas chromosomes.…”

“…This 70 kb plasmid encodes mercury resistance as the only reported accessory trait and was captured from river epilithon in Wales by an exogenous plasmid isolation method (Haines et al, 2006). The authors defined the IncP-1c subgroup based on clear divergence of the plasmid's backbone genes from the wellcharacterized plasmids of the IncP-1a and IncP-1b subgroups (Haines et al, 2006;Norberg et al, 2011). Here we describe the first multi-drug resistance plasmid in this IncP-1c subgroup, pKS208, as well as the first cryptic plasmid, pMBUI1.…”

“…Plasmid pMBUI1 was obtained in a triparental mating between Escherichia coli and pond water in Moscow, Idaho, after selection for the ability to mobilize plasmid pBBR1-MCS . Recent phylogenetic analysis of their plasmid backbone genes indicated that both pKS208 and pMBUI1 belong to the IncP-1c subgroup, which until recently comprised only one completely sequenced plasmid, the archetype pQKH54 (Haines et al, 2006;Sen et al, 2013).…”

“…Since Yakobson & Guiney (1983) proposed to separate plasmids RP4 and R751 as different subgroups (a, b) based on the sequence similarity in the transfer origin (Yakobson & Guiney, 1983), six IncP-1 subgroups (a, b, c, d, e and j, see Fig. 1a) have been proposed based on the results of phylogenetic analysis for plasmid backbone genes (Jobanputra & Datta, 1974;Haines et al, 2006;Sen et al, 2010Sen et al, , 2013Bahl et al, 2007;Norberg et al, 2011). To date, more than 55 IncP-1 plasmid sequences have been deposited in the NCBI database.…”

“…To date, more than 55 IncP-1 plasmid sequences have been deposited in the NCBI database. Interestingly, the natural hosts of IncP-1c, IncP-1e and IncP-1j plasmids are unknown because these plasmids have been discovered solely by exogenous plasmid isolation, a method that captures plasmids by mating the bacterial community of an environmental sample with a laboratory strain (Haines et al, 2006;Norberg et al, 2011;Bahl et al, 2007;. A recent comparison of the genomic signatures of IncP-1 plasmid backbone genes with those of all available bacterial chromosome sequences suggested that plasmids from different IncP-1 subgroups had acquired signatures of hosts belonging to diverse taxonomic groups (Norberg et al, 2011).…”