A resolvase-like protein is required for the site-specific integration of the temperate lactococcal bacteriophage TP901-1

Abstract

The integration system of the temperate lactococcal phage TP901-1 was characterized in Lactococcus lactis subsp. cremoris LM0230 and MG1363 with the use of deletion derivatives of the integration vector pBC143 (B. Christiansen, M. G. Johnsen, E. Stenby, F. K. Vogensen, and K. Hammer, J. Bacteriol. 176:1069-1076, 1994). The phage-encoded elements necessary for integration were localized on a 2.8-kb NsiI-EcoRI fragment including the phage attachment site, attP. This fragment was DNA sequenced, and sequence analysis revealed three putatively expressed open reading frames, Orf1, Orf2, and Orf3. By the introduction of mutations within the orf1, orf2, and orf3 genes, it was shown that only Orf1 was necessary for the integration process. Furthermore, it was found that Orf1, attP, and a 425-bp region upstream of the orf1 gene are sufficient for integration. Orf1 contains 485 amino acids and is located just upstream of attP. The N-terminal 150 to 180 amino acids of Orf1 showed 38 to 44% similarity to the resolvase group of site-specific integrases, while no similarity to known proteins was found in the C-terminal end. Bacteriophage TP901-1 therefore contains a unique integration system that does not resemble the Int class of site-specific integrases usually found in temperate bacteriophages. The constructed integration vector, pBC170, integrates into the chromosomal attachment site very efficiently and forms stable transformants with a frequency corresponding to 20% of the transformation efficiency.Most temperate bacteriophages from gram-negative and gram-positive bacteria, including the well-known bacteriophage, integrate their DNA into the host chromosome when they enter the lysogenic cycle. The integration often occurs site specifically and has, in these cases, been found to be mediated by an integrase (Int) belonging to the Int class of site-specific recombinases (1, 24).TP901-1 is a pac-type, lactococcal temperate bacteriophage with a small isometric head and a long noncontractile tail. It belongs to a group of temperate bacteriophages which show a high degree of homology to a group of virulent bacteriophages, represented by the type bacteriophage P335 (3, 15). It is likely that TP901-1 is identical to TP936-1 and C3-T1 on the basis of DNA restriction patterns and hybridizations (5, 16). We have previously described the site-specific integration of the temperate lactococcal bacteriophage TP901-1 and the construction of a TP901-1-based integration vector (5). The phage attachment site (attP) and the chromosomal attachment site (attB) are unrelated to those found in other phages of lactic acid bacteria and their host strains, e.g., adh, mv4, LC3, Tuc2009, and BK5-T (2,8,10,21,36).This communication contains a further characterization of the integration system from TP901-1 by deletion analysis, DNA sequencing, and mutational analysis. The data presented show that the integration system from TP901-1 is very different from the bacteriophage integration systems reported so far, since Orf1, which is identified to be necess...

In vitro site-specific integration of bacteriophage DNA catalyzed by a recombinase of the resolvase/invertase family

“…The C31 integrase is a member of a new subfamily of resolvase͞invertases that have, on the basis of amino acid sequence comparisons, the resolvase͞invertase catalytic domain at their N termini but that are much larger than the typical members of the family (13)(14)(15)(16)(17)(18)(19)(20). Consistent with this classification is the observation that mutation of the putative nucleophile, Ser-12, of C31 integrase completely abolished recombination both in vivo and in vitro (Fig.…”

“…2 and 21). This subgroup includes enzymes involved in excision of DNA during spore (13) or heterocyst (14) development, excision of transposons (15, 16), or integration and excision of bacteriophage genomes (17)(18)(19)(20). The ability of some of this group of enzymes to catalyze integration, i.e., the integrases from the lactococcal phage TP901-1, and actinophages R4 and C31, is a significant departure from the usual activities of the resolvase͞invertases (2).…”

“…[13][14][15][16][17][18][19][20] than, and extremely diverged from, the typical enzymes of the family (Ϸ20 kDa; refs. 2 and 21).…”

Integration and excision of the Mycobacterium tuberculosis prophage‐like element, φRv1

“…Currently, 11 other serine integrases associated with phage or phage-like elements have been described, six of which (encoded by fC31, TP901-1, R4, A118, SPbc2 and fFC1) have been shown to be active either in vitro and/or in vivo (Kuhstoss and Rao, 1991;Christiansen et al, 1996;Matsuura et al, 1996;Thorpe and Smith, 1998;Lazarevic et al, 1999;Loessner et al, 2000;Breuner, 2001;Yang et al, 2002). All of the members of this class of integrases contain related ~140-residue N-terminal segments common to resolvases and DNA invertases, but the C-terminal domains are highly diverse in their lengths and sequences.…”

In vivo and in vitro characterization of site-specific recombination of actinophage R4 integrase

“…The tyrosine integrases such as λ, Cre, and FLP integrases utilize tyrosine as their active site nucleophile and the recombination reaction proceeds through a Holliday junction intermediate (Enquist et al, 1979;Hsu and Landy, 1984). On the other hand, serine integrases use a serine-catalyzed reaction mechanism (Christiansen et al, 1996;Matsuura et al, 1996;Thorpe and Smith, 1998). Serine family members include the resolvases of γδ and Tn3 transposons, the invertase of Gin and the phage-encoded integrases of ΦC31, R4 and TP901-1 (Groth and Calos, 2004;Smith and Thorpe, 2002).…”