Journal of Bacteriology, December 1999, p. 7291-7297, Vol. 181, No. 23
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Department of Microbiology, Technical University of Denmark, DK-2800 Lyngby, Denmark
Received 1 June 1999/Accepted 17 September 1999
In this work, the phage-encoded proteins involved in site-specific
excision of the prophage genome of the temperate lactococcal bacteriophage TP901-1 were identified. The phage integrase is required
for the process, and a low but significant frequency of excision is
observed when the integrase is the only phage protein present. However,
100% excision is observed when the phage protein Orf7 is provided as
well as the integrase. Thus, Orf7 is the TP901-1 excisionase, and it is
the first excisionase identified that is used during excisive
recombination catalyzed by an integrase belonging to the family of
extended resolvases. Orf7 is a basic protein of 64 amino acids, and the
corresponding gene (orf7) is the third gene in the early
lytic operon. This location of an excisionase gene of a temperate
bacteriophage has never been described before. The experiments are
based on in vivo excision of specifically designed excision vectors
carrying the TP901-1 attP site which are integrated into
attB on the chromosome of Lactococcus lactis. Excision of the vectors was investigated in the presence of different TP901-1 genes. In order to detect very low frequencies of excision, a
method for positive selection of loss of genetic material based upon
the upp gene (encoding uracil phosphoribosyltransferase) was designed, since upp mutants are resistant to
fluorouracil. By using this system, frequencies of excision on the
order of 10
5 per cell could easily be measured. The
described selection principle may be of general use for many organisms
and also for types of deletion events other than excision.
This article has been cited by other articles:
| Appl. Environ. Microbiol. | Infect. Immun. | Eukaryot. Cell |
|---|---|---|
| Mol. Cell. Biol. | J. Virol. | Microbiol. Mol. Biol. Rev. |
| ALL ASM JOURNALS |