An Unusual Phage Repressor Encoded by Mycobacteriophage BPs

Villanueva, Valerie M.; Oldfield, Lauren M.; Hatfull, Graham F.

doi:10.1371/journal.pone.0137187

Cited by 14 publications

(13 citation statements)

References 23 publications

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“…Sequence-verified plasmid constructs were transformed into BL21 Star(DE3) cells, and single colonies were grown in LB medium supplemented with carbenicillin. Repressor expression was induced with 1 mM isopropyl-β- d -thiogalactopyranoside (IPTG) for 3 h, and cells were lysed by resuspension in lysis buffer (50 mM Tris [pH 8.0], 300 mM NaCl, 10% glycerol), treatment with 1 mg/ml lysozyme for 30 min on ice, and light sonication (54). C-terminally His-tagged Rep Trixie was purified using a nickel-nitrilotriacetic acid (NTA) matrix, dialyzed overnight with storage buffer (50 mM Tris [pH 8.0], 150 mM NaCl, 0.1 mM EDTA, 0.1 mM dithiothreitol [DTT], 50% glycerol), and quantified at ∼1 mg/ml using a NanoDrop instrument (Thermo Fisher).…”

Section: Methodsmentioning

confidence: 99%

“…DNA substrates for electrophoretic mobility shift assays (EMSAs) were designed to be 30 bp long, consisting of a 13-bp stoperator sequence flanked by 8 to 9 bp of sequence. Complementary 30-bp oligonucleotides were synthesized, radiolabeled at the 5′ end with γ- 32 P, and annealed (54). Oligonucleotides for each substrate in Fig.…”

Section: Methodsmentioning

confidence: 99%

“…For the 30-bp substrates in which the Trixie stoperator site is progressively converted to a Peaches stoperator site, the variable 13-bp sequence is flanked by invariable 8 to 9 bp derived from the Trixie substrate. EMSAs were performed with serially diluted Rep, which was electrophoresed on an 8% polyacrylamide gel and imaged, as previously described (54). The K D for each substrate was calculated with nonlinear regression in Prism software using the one-site-specific binding option and least-squares fit.…”

Section: Methodsmentioning

confidence: 99%

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Evolution of Superinfection Immunity in Cluster A Mycobacteriophages

Mavrich

Hatfull

2019

mBio

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Temperate phages encode an immunity system to control lytic gene expression during lysogeny. This gene regulatory circuit consists of multiple interacting genetic elements, and although it is essential for controlling phage growth, it is subject to conflicting evolutionary pressures. During superinfection of a lysogen, the prophage’s circuit interacts with the superinfecting phage’s circuit and prevents lytic growth if the two circuits are closely related. The circuitry is advantageous since it provides the prophage with a defense mechanism, but the circuitry is also disadvantageous since it limits the phage’s host range during superinfection. Evolutionarily related phages have divergent, orthogonal immunity systems that no longer interact and are heteroimmune, but we do not understand how immunity systems evolve new specificities. Here, we use a group of Cluster A mycobacteriophages that exhibit a spectrum of genetic diversity to examine how immunity system evolution impacts superinfection immunity. We show that phages with mesotypic (i.e., genetically related but distinct) immunity systems exhibit asymmetric and incomplete superinfection phenotypes. They form complex immunity networks instead of well-defined immunity groups, and mutations conferring escape (i.e., virulence) from homotypic or mesotypic immunity have various escape specificities. Thus, virulence and the evolution of new immune specificities are shaped by interactions with homotypic and mesotypic immunity systems. IMPORTANCE Many aspects regarding superinfection, immunity, virulence, and the evolution of immune specificities are poorly understood due to the lack of large collections of isolated and sequenced phages with a spectrum of genetic diversity. Using a genetically diverse collection of Cluster A phages, we show that the classical and relatively straightforward patterns of homoimmunity, heteroimmunity, and virulence result from interactions between homotypic and heterotypic phages at the extreme edges of an evolutionary continuum of immune specificities. Genetic interactions between mesotypic phages result in more complex mesoimmunity phenotypes and virulence profiles. These results highlight that the evolution of immune specificities can be shaped by homotypic and mesotypic interactions and may be more dynamic than previously considered.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Evolution of Superinfection Immunity in Cluster A Mycobacteriophages

Mavrich

Hatfull

2019

mBio

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“…The integration site on the phage genome (attP) resides within the ORF (open reading frame) of the repressor gene itself, and successful integration into the bacterial genome truncates the repressor gene and removes a proteolytic degradation signal from its C terminus, thus stabilizing the repressor protein and maintaining lysogeny ( Figure 2C). In the lytic cycle, the repressor and the integrase are degraded by cellular proteases, preventing lysogeny (Broussard et al, 2013;Villanueva et al, 2015).…”

Section: New Insights Into Phage Lysogenymentioning

confidence: 99%

Contemporary Phage Biology: From Classic Models to New Insights

Ofir

Sorek

2018

Cell

225

145

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Bacteriophages, discovered about a century ago, have been pivotal as models for understanding the fundamental principles of molecular biology. While interest in phage biology declined after the phage "golden era," key recent developments, including advances in phage genomics, microscopy, and the discovery of the CRISPR-Cas anti-phage defense system, have sparked a renaissance in phage research in the past decade. This review highlights recently discovered unexpected complexities in phage biology, describes a new arsenal of phage genes that help them overcome bacterial defenses, and discusses advances toward documentation of the phage biodiversity on a global scale.

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“…EMSAs were performed by established protocols [40,41]. DNA oligonucleotides were 5' radiolabelled with γ- 32…”

Section: Electrophoretic Mobility-shift Assaysmentioning

confidence: 99%

Brujita Integrase: A Simple, Arm-Less, Directionless, and Promiscuous Tyrosine Integrase System

Lunt

Hatfull

2016

Journal of Molecular Biology

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Mycobacteriophage Brujita is an unusual temperate phage in which establishment of superinfection immunity is dependent on chromosomal integration. Integration is mediated by a non-canonical tyrosine integrase (Int) lacking an N-terminal domain typically associated with binding to arm-type sites within the phage attachment site (attP). This raises the question as to how these Ints bind their DNA substrates, if they form higher-order protein DNA complexes, and how site selection and recombinational directionality are determined. Here we show that Brujita Int is a simple recombinase, whose properties more closely resemble those of FLP and Cre than it does the canonical phage Ints. Brujita Int uses relatively small DNA substrates, fails to discriminate between attP and attB, cleaves attachment site DNA to form a 6-base overlap region, and lacks directional control. Brujita Int also has an unusual pattern of binding to its DNA substrates. It binds to two half sites (B and B') at attB, although binding to the B half site is strongly dependent on occupancy of B'. In contrast, binding to the P half site is not observed, even when Int is bound at P'. However, an additional Int binding site (P1) is displaced to the left of the crossover site at attP, is required for recombination and is predicted to facilitate binding of Int to the P half site during synapsis. These simple phage Int systems may reflect ancestral states of phage evolution with the complexities of higher-order complex formation and directional control representing subsequent adaptations.

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An Unusual Phage Repressor Encoded by Mycobacteriophage BPs

Cited by 14 publications

References 23 publications

Evolution of Superinfection Immunity in Cluster A Mycobacteriophages

Evolution of Superinfection Immunity in Cluster A Mycobacteriophages

Contemporary Phage Biology: From Classic Models to New Insights

Brujita Integrase: A Simple, Arm-Less, Directionless, and Promiscuous Tyrosine Integrase System

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