Armshaw_2012_functional.pdf (72.12 MB)
Functional genomic analysis of the UV-inducible ‘cytotoxic’ gene from the ICE R391, an archetypal member of the SXT/R391 family of integrative conjugative elements
thesisposted on 2022-10-05, 10:10 authored by Patricia Armshaw
The enterobacterial mobile element, R391, is a prototype member of the SXT/R391 family of Integrative Conjugative Elements (ICEs). SXT/R391-like ICEs are sitespecific genome-integrating mobile elements, frequently isolated from human pathogens. They encode a range of known adaptive traits, including multiple antibiotic resistance determinants and DNA repair mechanisms. The functionality of a large percentage of SXT/R391-like ICEs genes is unknown (31% of predicted ORFs of R391). These currently cryptic genes may encode as yet uncharacterised adaptive traits. Unusually, SXT/R391-like ICEs have also been found to exhibit an apparently disadvantageous recA-dependent UV-inducible cell sensitising phenotype. The identification and characterisation of the R391 ICE gene(s) responsible for this unusual cell sensitising, cytotoxic effect was investigated. A comparative bioinformatic analysis of SXT/R391-like ICEs was performed, which predicted that 49 putative core genes were shared amongst all ICE members. Construction of a complete R391 gene deletion library implicated three of these core genes, orf90, orf91 and orf43, in the sensitising phenotype. Cloning of orfs90/91 and orf43, precise deletion mutant construction and RT-PCR analysis determined that orf90, orf91 and orf43 are UV-inducible and that orfs90/91 putatively encode a transcriptional activator complex which regulates orf43 transcription. Functional analysis utilising recombinant expression, TEM, laser scanning confocal microscopy and site-directed mutagenesis of orf43 which encodes a putative TraV homolog (TraVR391), functioning as an outer membrane-associated pore-forming protein, determined that expression of orf43 (TraVR391) was cytotoxic to host cells by destabilising cell integrity. Results suggested that orf43 (TraVR391) expression alone was responsible for the cell sensitisation phenotype and that UV was a facilitator of this overexpression. A hypothesis was proposed and supporting evidence presented suggesting that orf43 may function as a stress inducible ICE ‘trap door’ escape mechanism for SXT/R391- like ICEs. Hence conservation of this UV-inducible ‘cytotoxic’ gene may aid element survival and be advantageous under stress conditions.
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First supervisorPembroke, Tony J.
Other Funding informationIRC
Department or School
- Chemical Sciences