The initial aim of this project was to characterise the integrative recombination mechanism of bacteriophage ÖAR29 , 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 ÖAR29 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 ÖAR29 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 ÖAR29 attP was shown to include a 16-bp att core region, 117 bp upstream of the previously suggested location. Integration of ÖAR29 was found to occur at the 3end 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 ÖAR29 prophage, providing an improved host for integrative plasmids, and for detailed studies of AR29 physiology and ÖAR29 life cycles.
| Identifer | oai:union.ndltd.org:ADTP/221727 |
| Date | January 2005 |
| Creators | shawnseet@gmail.com, Shawn Ginn Ming Seet |
| Publisher | Murdoch University |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | http://www.murdoch.edu.au/goto/CopyrightNotice, Copyright Shawn Ginn Ming Seet |
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