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Molecular analysis of RepHI1 A, a minimal replicon of the IncHI1 plasmid R27
Author(s) -
Newnham P. J.,
Taylor D. E.
Publication year - 1994
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.1994.tb00353.x
Subject(s) - replicon , biology , plasmid , genetics , subcloning , open reading frame , transcription (linguistics) , microbiology and biotechnology , gene , peptide sequence , linguistics , philosophy
Summary R27, a large conjugative plasmid of the HI incompatibility group, was subjected to a subcloning analysis which revealed the presence of a Poll‐independent replicon and determinants contributing to incompatibility within a 2.7 kb Sal I/Xbal fragment. The DNA sequence of the minimal replicon revealed the presence of a large open reading frame (ORF) as well two sets of 19 bp repeated oligonucleotides (iterons), In addition to characteristic Escherichia coli origin elements. The protein encoded by the ORF possesses homology with replication initiator proteins encoded by a number of plasmids from different incompatibility groups. Deletion analysis suggested that the iterons are responsible for incompatibility reactions. Dissection of the replicon confirmed this and defined a minimal origin of 230 bp. The putative replication initiator was expressed in an in vitro transcription‐translation system, and the 5’end of the mRNA encoding its synthesis was identified. Transcriptional fusion of the repA promoter to lacZ demonstrated an auto‐regulatory function of RepA. A series of iterons present downstream of the RepA coding sequence are dispensable but are responsible for copy‐number control. The minimal replicon appears to be partition‐defective.