Phage ΦAR29 was shown to exist as a prophage integrated into the chromosome of AR29. By DNA hybridization studies, the point of integrative recombination on the phage genome (P) was located on a 4.5 kb RV fragment. After preliminary mapping with restriction endonucleases, a 2.8 kb RV/dlll fragment was isolated, cloned in and sequenced. DNA hybridization localized the P site to the vicinity of an internal l site. Sequence analysis showed the presence of several direct and inverted repeats around the P site, with consensus core sequences similar to the integrase binding sites of phage λ. Two open reading frames are present adjacent to P (ORF1 and ORF2). The predicted polypeptide product of ORF1 has a region of structural similarity to known integrases. Although the predicted product of ORF2 shows at best weak homology with known excisionases, no other ORFs occur in the sequence upstream from ORF1, leaving ORF2 as the most likely candidate for this role. However, if ORF2 does represent an gene, then this putative integration module would possess a notable difference from that of other temperate phages in the inversion of the positions of and relative to P. The proposed ΦAR29 integration module is being used to develop phage-based integrative vector systems for the genetic manipulation of rumen bacteria.


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