Mycobacteriophage D29 integrase-mediated recombination: specificity of mycobacteriophage integration

Mycobacteriophage D29 is a lytic phage that infects both fast- and slow-gro wing species of the mycobacteria. D29 forms clear plaques on lawns of Mycob acterium smegmatis and Mycobacterium bovis bacille Calmette-Guerin (BCG) in which a very high proportion of infected cells are killed. However, genomi c analysis of D29 demonstrates that it is a close relative of the temperate mycobacteriophage L5, and is presumably a non-temperate derivative of a te mperate parent. The D29 genome encodes a putative integrase protein with a primary amino acid sequence similar to that of the L5 integrase; the corres ponding int genes fall in colinear positions within the D29 and L5 genomes, immediately flanking and transcribed away from their associated attP sites . We show here that the D29 integrase is functional and catalyzes integrati ve recombination between the D29 attP site and the M. smegmatis attB site i n vitro in an mIHF-dependent manner. D29 integrase also mediates recombinat ion between the L5 attP site and attB DNA and, reciprocally, L5 integrase c atalyzes recombination with D29 attP DNA. However, in both in-vitro and in- vivo assays, the D29-encoded integrase recombines the D29 attP more efficie ntly than the L5 attP, and Vice versa, suggesting that each integration sys tem has evolved a degree of specificity of attP recognition. We also presen t the sequences of the putative attP site and integrase protein of the cryp tic prophage-like element phi Rv2, and compare them to those of mycobacteri ophages L5 and D29. (C) 1998 Elsevier Science B.V. All rights reserved.