microbiology, Vol 140, 2109-2114, Copyright © 1994 by Society for General Microbiology
Cloning and DNA sequence analysis of the region containing attP of the temperate phage phi AR29 of Prevotella ruminicola AR29
K Gregg, BG Kennedy and AV Klieve
Institute of Biotechnology, University of New England, Armidale, NSW, Australia.
Phage phi AR29 was shown to exist as a prophage integrated into the
chromosome of Prevotella ruminicola AR29. By DNA hybridization studies, the
point of integrative recombination on the phage genome (attP) was located
on a 4.5 kb EcoRV fragment. After preliminary mapping with restriction
endonucleases, a 2.8 kb EcoRV/HindIII fragment was isolated, cloned in
Escherichia coli and sequenced. DNA hybridization localized the attP site
to the vicinity of an internal DraI site. Sequence analysis showed the
presence of several direct and inverted repeats around the attP site, with
consensus core sequences similar to the integrase binding sites of phage
lambda. Two open reading frames are present adjacent to attP (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 xis gene, then this
putative integration module would possess a notable difference from that of
other temperate phages in the inversion of the positions of int and xis
relative to attP. The proposed phi AR29 integration module is being used to
develop phage-based integrative vector systems for the genetic manipulation
of rumen bacteria.
