New Applications for Phage Integrases

Fogg, Paul C. M.; Colloms, Sean D.; Rosser, Susan J.; Stark, Marshall W.; Smith, Margaret C. M.

doi:10.1016/j.jmb.2014.05.014

Cited by 169 publications

(174 citation statements)

References 132 publications

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“…Serine integrases are being used in a variety of genetic engineering and synthetic biology applications (15)(16)(17)(18)(19)(20)(21)(22). A major attribute of serine integrases is that they perform unidirectional recombination, as regulated by their dedicated RDF.…”

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confidence: 99%

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

et al. 2017

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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

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confidence: 99%

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

et al. 2017

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“…Given the specificity of the 289 integrase (Fogg et al, 2014), this difference could explain why AsaGEI1 and AsaGEI2 are 290 inserted at different sites on the chromosome (see below). The structures of the AsaGEIs can 291 be divided into two parts, with a sudden shift of transcriptional orientation between orf34 and 292 orf35, a featured shared by the various forms of the AsaGEIs (green diamond in Figure 3D).…”

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Variants of a genomic island in Aeromonas salmonicida subsp. salmonicida link isolates with their geographical origins

Emond-Rheault

Vincent

Trudel

et al. 2015

Veterinary Microbiology

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“…55 The development of multiple non-compatible attB/attP pairs, 56, 57 as well as non-overlapping attachment sites for different integrase systems are a harbinger of their utility in multiplex genome engineering of actinomycetes. 45, 53 …”

Section: Advances In Genetic Engineering Of Actinomycetesmentioning

confidence: 99%

Engineering microbial hosts for production of bacterial natural products

et al. 2016

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Microbial fermentation provides as an attractive alternative to chemical synthesis for the production of structurally complex natural products. In most cases, however, production titers are low and need to be improved for compound characterization and/or commercial production. Owing to advances in functional genomics and genetic engineering technologies, microbial hosts can be engineered to overproduce a desired natural product, greatly accelerating the traditionally time-consuming strain improvement process. This review covers recent developments and challenges in the engineering of native and heterologous microbial hosts for production of bacterial natural products, focusing on the genetic tools and strategies for strain improvement. Special emphasis is placed on bioactive secondary metabolites from actinomycetes. The considerations for the choice of host systems will also be discussed in this review.

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New Applications for Phage Integrases

Cited by 169 publications

References 132 publications

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

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

Variants of a genomic island in Aeromonas salmonicida subsp. salmonicida link isolates with their geographical origins

Engineering microbial hosts for production of bacterial natural products

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