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Abstract
Plasmid pSW200 from Pantoea stewartii contains 41 copies of 15-bp repeats and has a replicon that is homologous to that of ColE1. Although deleting the repeats (pSW207) does not change the copy number and stability of the plasmid. The plasmid becomes unstable and is rapidly lost from the host when a homoplasmid with the repeats (pSW201) is present. Deleting the repeats is found to reduce the transcriptional activity of RNAIp and RNAIIp by about 30%, indicating that the repeats promote the transcription of RNAI and RNAII, and how the RNAI that is synthesized by pSW201 inhibits the replication of pSW207. The immunoblot analysis herein demonstrates that RNA polymerase β subunit and σ70 in the lysate from Escherichia coli MG1655 bind to a biotin-labeled DNA probe that contains the entire sequence of the repeat region. Electrophoretic mobility shift assay also reveals that purified RNA polymerase shifts a DNA probe that contains four copies of the repeats. These results thus obtained reveal that RNA polymerase holoenzyme binds to the repeats. The repeats also exchange RNA polymerase with RNAIp and RNAIIp in vitro, revealing the mechanism by which the transcription is promoted. This investigation elucidates a mechanism by which a plasmid prevents the invasion of an incompatible plasmid and maintains its stability in the host cell during evolution.
Highlights
Stability is the most important determinant of the survival of a plasmid during evolution [1]
After E. coli HB101 was cotransformed with pSW252 and pSW207, 94% of the colonies were found to be resistant to kanamycin, and 6% were resistant to tetracycline (Table 2), verifying that the repeats in pSW252 are responsible for plasmid competition
The results revealed that RNA polymerase bound to the direct repeats (DR) probe was exchanged with the RNAIp and RNAIIp probes
Summary
Stability is the most important determinant of the survival of a plasmid during evolution [1]. A plasmid with a low copy number, such as F or P1, uses complex segregation systems to ensure it is precisely segregated into daughter cells following plasmid replication and cell division [2,3,4]. Post-segregational killing (PSK) systems ensure that all of the living daughter cells receive a plasmid following cell division [5]. Precise control of the copy number ensures that a constant number of plasmids are maintained in a cell [6,10]. Incompatibility is another factor that is likely to threaten the stability of a plasmid [11]. The plasmid that invades the cell may multiply and cause the loss of the original resident plasmid
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