Delineation of the integrase-attachment and origin-of-transfer regions of the symbiosis island ICEMlSymR7A

Callum J Verdonk; John T Sullivan; Kate M Williman; Leila Nicholson; Tahlia R Bastholm; Michael F Hynes; Clive W Ronson; Charles S Bond; Joshua P Ramsay

doi:10.1016/j.plasmid.2019.102416

Delineation of the integrase-attachment and origin-of-transfer regions of the symbiosis island ICEMlSym^R7A

Plasmid. 2019 Jul:104:102416. doi: 10.1016/j.plasmid.2019.102416. Epub 2019 May 9.

Authors

Callum J Verdonk¹, John T Sullivan², Kate M Williman², Leila Nicholson², Tahlia R Bastholm³, Michael F Hynes⁴, Clive W Ronson², Charles S Bond⁵, Joshua P Ramsay⁶

Affiliations

¹ School of Molecular Sciences, University of Western Australia, Perth, WA, Australia; School of Pharmacy and Biomedical Sciences and Curtin Health Innovation Research Institute, Curtin University, Perth, WA, Australia.
² Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand.
³ School of Pharmacy and Biomedical Sciences and Curtin Health Innovation Research Institute, Curtin University, Perth, WA, Australia.
⁴ Department of Biological Sciences, University of Calgary, Calgary, Canada.
⁵ School of Molecular Sciences, University of Western Australia, Perth, WA, Australia.
⁶ School of Pharmacy and Biomedical Sciences and Curtin Health Innovation Research Institute, Curtin University, Perth, WA, Australia. Electronic address: josh.ramsay@curtin.edu.au.

PMID: 31078551
DOI: 10.1016/j.plasmid.2019.102416

Abstract

Integrative and conjugative elements (ICEs) are chromosomally-integrated mobile genetic elements that excise from their host chromosome and transfer to other bacteria via conjugation. ICEMlSym^R7A is the prototypical member of a large family of "symbiosis ICEs" which confer upon their hosts the ability to form a nitrogen-fixing symbiosis with a variety of legume species. Mesorhizobial symbiosis ICEs carry a common core of mobilisation genes required for integration, excision and conjugative transfer. IntS of ICEMlSym^R7A enables recombination between the ICEMlSym^R7A attachment site attP and the 3' end of the phe-tRNA gene. Here we identified putative IntS attP arm (P) sites within the attP region and demonstrated that the outermost P1 and P5 sites demarcated the minimal region for efficient IntS-mediated integration. We also identified the ICEMlSym^R7A origin-of-transfer (oriT) site directly upstream of the relaxase-gene rlxS. The ICEMlSym^R7A conjugation system mobilised a plasmid carrying the cloned oriT to Escherichia coli in an rlxS-dependent manner. Surprisingly, an in-frame, markerless deletion mutation in the ICEMlSym^R7A recombination directionality factor (excisionase) gene rdfS, but not a mutation in intS, abolished mobilisation, suggesting the rdfS deletion tentatively has downstream effects on conjugation or its regulation. In summary, this work defines two critical cis-acting regions required for excision and transfer of ICEMlSym^R7A and related ICEs.

Keywords: Excisionase; ICEMlSym(R7A); Integrase; Integrative and conjugative element; Origin of transfer.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Base Sequence
Binding Sites
Cloning, Molecular
Conjugation, Genetic*
DNA Nucleotidyltransferases
DNA Transposable Elements*
Gene Order
Gene Transfer, Horizontal
Genomic Islands*
Integrases / metabolism*
Nucleotide Motifs
Protein Binding
Recombination, Genetic
Replication Origin*
Symbiosis
Viral Proteins

Substances

DNA Transposable Elements
Viral Proteins
DNA Nucleotidyltransferases
Integrases
excisionase