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Genome sequence of bacteriophage ΦAR29: a basis for integrative plasmid vectors

Seet, Shawn Ginn Ming (2005) Genome sequence of bacteriophage ΦAR29: a basis for integrative plasmid vectors. PhD thesis, Murdoch University.

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The initial aim of this project was to characterise the integrative recombination mechanism of bacteriophage ΦAR29 , to provide a better understanding for development of the shuttle plasmid pBA as a site-specific Bacteroides integration vector. RT-PCR showed that the previously identified ΦAR29 recombination genes, integrase (Int) and excisionase (Xis), were transcribed from pBA in E. coli SCS110, B. thetaiotaomicron AR29 and B. uniformis AR20. In silico derived amino acid sequences from both genes showed only very low levels of similarity to other known Int and Xis in GenBank. To improve understanding of the phage recombination system, the ΦAR29 genome was sequenced. This revealed a 35,558 bp double-stranded DNA genome with GC content of 39.11%. Bioinformatic analysis identified 53 open reading frames (>30 codons) and gene promoters and terminators that allowed the genome arrangement to be compared with other phages. Comparison of deduced gene products with proteins from other phages identified 6 reading frames, allowed tentative identification of 7 others, but left 40 ORFs unidentified. Those with strong homology to known genes were: large terminase subunit (44.66 kDa), dnaC (27.94 kDa), helix-turn-helix (HTH) transcription regulator (14.69 kDa), cI repressor (26.48 kDa), amidase (18.42kDa) and a novel integrase (54.22 kDa). The integrase gene is located 162 base-pairs downstream of the phage attachment (attP) core site, rather than the previously suggested location upstream of the integration site. The ΦAR29 attP was shown to include a 16-bp att core region, 117 bp upstream of the previously suggested location. Integration of ΦAR29 was found to occur at the 3'end of an arg-tRNA gene on the AR29 genome (attB). Imperfect direct repeats with a consensus sequence (ANGTTGTGCAA) were found surrounding the attP core. A review of pBA sequence showed that only the 5' end (435 bp) of the newly identified Int gene was cloned in pBA. Despite this, PCR analysis revealed integration of pBA into the AR29 genome. Serial subculturing of pBA transformed AR29 was able to cure AR29 of the ΦAR29 prophage, providing an improved host for integrative plasmids, and for detailed studies of AR29 physiology and ΦAR29 life cycles.

Item Type: Thesis (PhD)
Murdoch Affiliation(s): School of Engineering Science
Supervisor(s): Gregg, Keith
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