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PMID: 2038309 Published · ppublish English Comparative Study Journal Article Research Support, Non-U.S. Gov't

Structural and functional analysis of the origin of conjugal transfer of the broad-host-range IncW plasmid R388 and comparison with the related IncN plasmid R46.

Molecular & general genetics : MGG ·Vol. 226 ·No. 3 ·1991-05-00 ·Pages 473-83

Llosa M, Bolland S, de la Cruz F

Abstract

We cloned and sequenced a 402 bp DNA segment containing the origin of conjugal transfer (oriT) of the IncW plasmid R388. Progressive deletions from each end of the sequence were assayed for oriT activity. Stepwise reductions in mobilization frequencies, representing the loss of functional elements, correlated with deletion of structural motifs in the sequence. A sequence of 330 bp of oriT was sufficient for efficient mobilization. The first 86 bp of the sequence contains five tandemly repeated DNA sequences of 11 bp, followed by a 10 bp perfect inverted repeat. Deletion of the first 95 bp reduced the frequency of transfer by a hundred-fold. The sequence between bp 183 and 218 was necessary and sufficient for low frequency mobilization and, thus, it was assumed to contain the nick site. This basis core was cloned as a 60 bp segment (from bp 176-236) that could be mobilized at low frequency. It includes two inverted repeats and a perfect integration host factor (IHF) consensus binding site. A third functionally important segment in oriT was located between bp 260 and 330. The DNA sequence of the oriT of R388 could be aligned with that of the broad-host-range IncN plasmid R46. Moreover, the relative positions of the three inverted repeats are also conserved. Overall sequence similarity was 52%, but was significantly higher in particular regions, which coincided with the functionally important segments mapped by deletion analysis. Conservation of these segments provided independent support for their essential role in oriT function.

Related Genes
oriT
MeSH Terms
Base Sequence Conjugation, Genetic DNA/genetics Escherichia coli/genetics Genes, Bacterial Molecular Sequence Data Nucleic Acid Conformation Plasmids Sequence Homology, Nucleic Acid
Chemicals
DNA
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Llosa M
Departamento de Biología Molecular, Universidad de Cantabria, Santander, Spain.
Bolland S
de la Cruz F
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Article Info
Journal
Molecular & general genetics : MGG
Abbr.
Mol Gen Genet
ISSN
0026-8925
DOI
10.1007/BF00260661
Published
1991-05-00
Pages
473-83
Language
English
Region
Germany
NLM ID
0125036
Subset
IM
Databases
GENBANK
X51705
External Links
PubMed source DOI: 10.1007/BF00260661
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