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Birmingham-group IncP-1α plasmids revisited: RP4, RP1 and RK2 are identical and their remnants can be detected in
Vuong Van Hung Le1,2,3, Zhuang Gong3, Lorrie Maccario3
1Living Systems Institute, University of Exeter, Exeter, UK.
Abstract:
RP4, RP1, RK2 and R68 were isolated from the multidrug-resistant bacterial wound isolates in 1969 in the Birmingham Accident Hospital, Birmingham, England, and collectively called Birmingham-group IncP-1α plasmids. These plasmids have been widely used as models to study different aspects of plasmid biology, develop genetic delivery systems and design plasmid vectors. Early studies showed that these plasmids conferred the same antibiotic resistance profile, had a similar size and were undistinguishable from each other using DNA heteroduplex electron microscopy and restriction endonuclease analyses. These observations have led to the widely held assumption that they are identical, although there has been no conclusive supporting evidence. In this work, we sequenced the plasmids RP1 and RP4 from our laboratory strain collection and compared these new sequences with the plasmids RP4 and RK2 assembled from a publicly available sequencing database, showing that the RP1, RP4 and RK2 plasmids are 60 095 bp in length and identical at the nucleotide resolution. Noteworthily, the plasmid sequence is highly conserved despite having been distributed to different labs over 50 years and propagated in different bacterial hosts, strengthening the previous observation that the bacterial host adapts to the RP4/RP1/RK2 plasmid rather than the opposite. In the updated RP4/RP1/RK2 sequence, we found a fusion gene, called pecM-orf2, that was formed putatively by a genetic deletion event. By searching for pecM-orf2 in the National Center for Biotechnology Information database, we detected remnants of the RP4/RP1/RK2 plasmid that carry features of laboratory-engineered vectors in bacterial environmental isolates, either in their chromosome or as a plasmid. This suggests a leak of these plasmids from the laboratory into the environment, which may subsequently impact bacterial evolution and raises concerns about the biocontainment of engineered plasmids when being handled in laboratory settings.
Insights
The Birmingham-group IncP-1α plasmids RP1, RP4, and RK2 are identical at the nucleotide level. Remnants of these lab-engineered plasmids were found in environmental bacteria, suggesting potential laboratory containment breaches.
Area of Science:
- Molecular Biology
- Microbiology
- Genetics
Background:
- The Birmingham-group IncP-1α plasmids (RP4, RP1, RK2, R68) were isolated in 1969 and widely used as model systems in plasmid biology.
- Previous studies indicated these plasmids were similar, leading to the assumption of their identity, but conclusive evidence was lacking.
Purpose of the Study:
- To definitively determine the nucleotide sequence identity of RP1, RP4, and RK2 plasmids.
- To investigate the conservation of these plasmids over 50 years of use.
- To identify potential environmental dissemination of these laboratory-derived plasmids.
Main Methods:
- Whole-genome sequencing of RP1 and RP4 plasmids from laboratory strains.
- Comparison of sequenced plasmids with publicly available RP4 and RK2 sequences.
- Bioinformatic analysis of the National Center for Biotechnology Information database for plasmid remnants.
Main Results:
- RP1, RP4, and RK2 plasmids were confirmed to be identical at the nucleotide level (60,095 bp).
- The plasmid sequence demonstrated remarkable conservation despite decades of propagation in diverse hosts.
- A novel fusion gene, pecM-orf2, was identified in the updated sequence.
- Evidence of RP4/RP1/RK2 plasmid features was found in environmental bacterial isolates, suggesting laboratory plasmid leakage.
Conclusions:
- RP1, RP4, and RK2 are evolutionarily conserved and effectively identical plasmids.
- The bacterial host adapts to these plasmids, rather than vice versa.
- The presence of laboratory-engineered plasmid remnants in the environment raises concerns about biocontainment and potential impacts on bacterial evolution.
