A Sequence That Affects the Copy Number and Stability of pSW200 and ColE1

Abstract

Pantoea stewartii SW2 contains 13 plasmids. One of these plasmids, pSW200, has a replicon that resembles that of ColE1. This study demonstrates that pSW200 contains a 9-bp UP element, 5-AAGATCTTC, which is located immediately upstream of the ؊35 box in the RNAII promoter. A transcriptional fusion study reveals that substituting this 9-bp sequence reduces the activity of the RNAII promoter by 78%. The same mutation also reduced the number of plasmid copies from 13 to 5, as well as the plasmid stability. When a similar sequence in a ColE1 derivative, pYCW301, is mutated, the copy number of the plasmid also declines from 34 to 16 per cell. Additionally, inserting this 9-bp sequence stabilizes an unstable pSW100 derivative, pSW142K, which also contains a replicon resembling that of ColE1, indicating the importance of this sequence in maintaining the stability of the plasmid. In conclusion, the 9-bp sequence upstream of the ؊35 box in the RNAII promoter is required for the efficient synthesis of RNAII and maintenance of the stability of the plasmids in the ColE1 family.

Plasmid DNA launches live-attenuated Japanese encephalitis virus and elicits virus-neutralizing antibodies in BALB/c mice

“…However, the resulting plasmid was unstable in E. coli in the context of high-copy, kanamycin resistant pUC57-Kan plasmid backbone. The pUC family of plasmids has mutations, which increase plasmid copy number to approximately 500 copies per cell (Wu and Liu, 2010), which potentially can amplify effects of toxic translation products. Several putative bacterial promoters within NS1 have been identified in silico , and an additional synthetic intron was introduced into the JEV NS1 gene.…”

Antibiotic resistance due to an unusual ColE1-type replicon plasmid in Aeromonas salmonicida

“…In order to find an explanation for the copy number difference between pAsa2 and pAsa7, and since the respective rate of transcription for both RNAs will regulate the plasmid replication positively (in presence of more RNA II) and negatively (when there is more RNA I), we investigated the promoter regions (À35 and À10 boxes) and the sps sequence (Wu & Liu, 2010) for both plasmids. We found no difference directly in the promoter regions (Fig.…”

Mechanisms of Theta Plasmid Replication

“…The DNA duplex is opened in this case by transcription of a long (~600 bp) pre-primer called RNA II, which is transcribed from a constitutive promoter P2. Constitutive expression from this promoter is enhanced by a 9-bp motif 5’-AAGATCTTC, which is located immediately upstream of the −35 box (25). The 3’ end of the pre-primer RNA forms a stable hybrid with 5’ end of the lagging-strand DNA template of ori .…”