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Control of phage Bxb1 excision by a novel recombination directionality factor.

ABSTRACT: Mycobacteriophage Bxb1 integrates its DNA at the attB site of the Mycobacterium smegmatis genome using the viral attP site and a phage-encoded integrase generating the recombinant junctions attL and attR. The Bxb1 integrase is a member of the serine recombinase family of site-specific recombination proteins and utilizes small (<50 base pair) substrates for recombination, promoting strand exchange without the necessity for complex higher order macromolecular architectures. To elucidate the regulatory mechanism for the integration and excision reactions, we have identified a Bxb1-encoded recombination directionality factor (RDF), the product of gene 47. Bxb1 gp47 is an unusual RDF in that it is relatively large (approximately 28 kDa), unrelated to all other RDFs, and presumably performs dual functions since it is well conserved in mycobacteriophages that utilize unrelated integration systems. Furthermore, unlike other RDFs, Bxb1 gp47 does not bind DNA and functions solely through direct interaction with integrase-DNA complexes. The nature and consequences of this interaction depend on the specific DNA substrate to which integrase is bound, generating electrophoretically stable tertiary complexes with either attB or attP that are unable to undergo integrative recombination, and weakly bound, electrophoretically unstable complexes with either attL or attR that gain full potential for excisive recombination.

SUBMITTER: Ghosh P

PROVIDER: S-EPMC1470463 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:UnlabelledPathogenicity islands (PAIs) are mobile integrated genetic elements (MIGEs) that contain a diverse range of virulence factors and are essential in the evolution of pathogenic bacteria. PAIs are widespread among bacteria and integrate into the host genome, commonly at a tRNA locus, via integrase-mediated site-specific recombination. The excision of PAIs is the first step in the horizontal transfer of these elements and is not well understood. In this study, we examined the role of recombination directionality factors (RDFs) and their relationship with integrases in the excision of two PAIs essential for Vibrio cholerae host colonization: Vibrio pathogenicity island 1 (VPI-1) and VPI-2. VPI-1 does not contain an RDF, which allowed us to answer the question of whether RDFs are an absolute requirement for excision. We found that an RDF was required for efficient excision of VPI-2 but not VPI-1 and that RDFs can induce excision of both islands. Expression data revealed that the RDFs act as transcriptional repressors to both VPI-1- and VPI-2-encoded integrases. We demonstrated that the RDFs Vibrio excision factor A (VefA) and VefB bind at the attachment sites (overlapping the int promoter region) of VPI-1 and VPI-2, thus supporting this mode of integrase repression. In addition, V. cholerae RDFs are promiscuous due to their dual functions of promoting excision of both VPI-1 and VPI-2 and acting as negative transcriptional regulators of the integrases. This is the first demonstration of cross talk between PAIs mediated via RDFs which reveals the complex interactions that occur between separately acquired MIGEs.ImportanceDeciphering the mechanisms of pathogenicity island excision is necessary for understanding the evolution and spread of these elements to their nonpathogenic counterparts. Such mechanistic insight would assist in predicting the mobility of uncharacterized genetic elements. This study identified extensive RDF-mediated cross talk between two nonhomologous VPIs and demonstrated the dual functionality of RDF proteins: (i) inducing PAI excision and (ii) acting as transcriptional regulators. Findings from this study may be implicated in determining the mobilome contribution of other bacteria with multiple MIGEs.

Project description:The serine integrase of phage A118 catalyzes integrative recombination between attP on the phage and a specific attB locus on the chromosome of Listeria monocytogenes, but it is unable to promote excisive recombination between the hybrid attL and attR sites found on the integrated prophage without assistance by a recombination directionality factor (RDF). We have identified and characterized the phage-encoded RDF Gp44, which activates the A118 integrase for excision and inhibits integration. Gp44 binds to the C-terminal DNA binding domain of integrase, and we have localized the primary binding site to be within the mobile coiled-coil (CC) motif but distinct from the distal tip of the CC that is required for recombination. This interaction is sufficient to inhibit integration, but a second interaction involving the N-terminal end of Gp44 is also required to activate excision. We provide evidence that these two contacts modulate the trajectory of the CC motifs as they extend out from the integrase core in a manner dependent upon the identities of the four att sites. Our results support a model whereby Gp44 shapes the Int-bound complexes to control which att sites can synapse and recombine.IMPORTANCE Serine integrases mediate directional recombination between bacteriophage and bacterial chromosomes. These highly regulated site-specific recombination reactions are integral to the life cycle of temperate phage and, in the case of Listeria monocytogenes lysogenized by A118 family phage, are an essential virulence determinant. Serine integrases are also utilized as tools for genetic engineering and synthetic biology because of their exquisite unidirectional control of the DNA exchange reaction. Here, we identify and characterize the recombination directionality factor (RDF) that activates excision and inhibits integration reactions by the phage A118 integrase. We provide evidence that the A118 RDF binds to and modulates the trajectory of the long coiled-coil motif that extends from the large carboxyl-terminal DNA binding domain and is postulated to control the early steps of recombination site synapsis.

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Control of phage Bxb1 excision by a novel recombination directionality factor.

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