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Applied and Environmental Microbiology, July 2008, p. 4119-4132, Vol. 74, No. 13
0099-2240/08/$08.00+0     doi:10.1128/AEM.00229-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Genomic and Functional Characterization of the Modular Broad-Host-Range RA3 Plasmid, the Archetype of the IncU Group ^,

Anna Kulinska,¹ Magdalena Czeredys,^1, Finbarr Hayes,² and Grazyna Jagura-Burdzy^1*

Department of Microbial Biochemistry, Institute of Biochemistry and Biophysics, PAS, 02-106 Warsaw, Poland,¹ Faculty of Life Sciences and Manchester Interdisciplinary Biocentre, The University of Manchester, Manchester M1 7DN, United Kingdom²

Received 25 January 2008/ Accepted 10 May 2008

IncU plasmids are a distinctive group of mobile elements with highly conserved backbone functions and variable antibiotic resistance gene cassettes. The IncU archetype is conjugative plasmid RA3, whose sequence (45,909 bp) shows it to be a mosaic, modular replicon with a class I integron different from that of other IncU replicons. Functional analysis demonstrated that RA3 possesses a broad host range and can efficiently self-transfer, replicate, and be maintained stably in alpha-, beta-, and gammaproteobacteria. RA3 contains 50 open reading frames clustered in distinct functional modules. The replication module encompasses the repA and repB genes embedded in long repetitive sequences. RepA, which is homologous to antitoxin proteins from alpha- and gammaproteobacteria, contains a Cro/cI-type DNA-binding domain present in the XRE family of transcriptional regulators. The repA promoter is repressed by RepA and RepB. The minireplicon encompasses repB and the downstream repetitive sequence r1/r2. RepB shows up to 80% similarity to putative replication initiation proteins from environmental plasmids of beta- and gammaproteobacteria, as well as similarity to replication proteins from alphaproteobacteria and Firmicutes. Stable maintenance functions of RA3 are most like those of IncP-1 broad-host-range plasmids and comprise the active partitioning apparatus formed by IncC (ParA) and KorB (ParB), the antirestriction protein KlcA, and accessory stability components KfrA and KfrC. The RA3 origin of transfer was localized experimentally between the maintenance and conjugative-transfer operons. The putative conjugative-transfer module is highly similar in organization and in its products to transfer regions of certain broad-host-range environmental plasmids.

Published ahead of print on 23 May 2008.

Supplemental material for this article may be found at http://aem.asm.org/.

Present address: Department of Molecular and Cell Neurobiology, Nencki Institute of Experimental Biology, 02-093 Warsaw, Poland.