Control of Recombination Directionality by the Listeria Phage A118 Protein Gp44 and the Coiled-Coil Motif of Its Serine Integrase

Abstract

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.KEYWORDS site-specific DNA recombination, serine integrase, recombination directionality factor, Listeria monocytogenes T emperate phage genomes integrate into and excise out of bacterial chromosomes using phage-specific site-specific recombination systems. Many phage integrases are members of a subfamily of serine recombinases (SRs) that are known as large SRs due to their very long C-terminal DNA binding regions relative to other SRs (1-4). In the few serine integrase systems that have been studied, phage-encoded recombination

Control of serine integrase recombination directionality by fusion with the directionality factor

“…There are several reasons for this. First, relatively few RDFs have been characterized compared to the large number of known serine integrases, as they are very diverse proteins that are much more difficult to identify than integrases ( 2 , 21 ). Second, attL × attR recombination requires the presence of integrase, RDF and substrate in appropriate stoichiometric ratio, complicating its use in vivo or in vitro .…”

Recombination directionality factor gp3 binds ϕC31 integrase via the zinc domain, potentially affecting the trajectory of the coiled-coil motif

“…Mandali et al. also found that the coiled-coil motif was required for the interaction between A118 integrase and its RDF gp44 ( 19 ) but they did not identify specific residues.…”

“…The RDF binds integrase and acts stoichiometrically to promote attL × attR recombination ( 7 , 8 , 19 ). Recently, Mandali et al.…”

“…Recently, Mandali et al. proposed that the RDF for Listeria phage A118, gp44, acts by binding to the base of the coiled-coil motif, perhaps altering the trajectory of the motif and enabling the inter-dimer contacts required for formation of a synaptic complex ( 19 , 28 ). As RDFs are extremely diverse, a question remains as to whether this mechanism is general.…”

New candidates for regulated gene integrity revealed through precise mapping of integrative genetic elements

“…Promiscuity has also emerged among the serine recombinases, all members of which share a core catalytic domain; members can be further classified as either having a second larger C-terminal domain associated with IGE integrases (S-Ints) or lacking it (S-Cores) (20). This second domain has complex-stabilizing coiled-coil motifs that control S-Int recombination directionality, i.e., integration vs. excision (21). The simpler S-Core proteins were traditionally not known as integrases, but instead as resolvases and DNA invertases.…”