Prophage induction and inactivation by UV light

Barnhart, Benjamin J.; Cox, Summers H.; Jett, James H.

doi:10.1128/jvi.18.3.950-955.1976

Cited by 22 publications

(14 citation statements)

References 17 publications

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“…In the laboratory, the most commonly used method for induction is Mitomycin C (see protocol Raya & H’bert, 2009 ). Other common laboratory techniques include UV (e.g., Barnhart, Cox & Jett, 1976 ; Zhang et al, 2019 ), antibiotics (e.g., Shaikh & Tarr, 2003 ; Goerke, Köller & Wolz, 2006 ; Loś et al, 2009 ), and H 2 O 2 (e.g., Figueroa-Bossi & Bossi, 1999 ; Martín et al, 2009 ; Loś et al, 2009 ; Loś et al, 2010 ). A few studies have also employed changes in temperature (e.g., Kirby, Jacob & Goldthwait, 1967 ; Meijer et al, 1998 ; Lunde et al, 2005 ; Chu et al, 2011 ), nutrient availability (e.g., Wilson, Turner & Mann, 1998 ; Lunde et al, 2005 ; Williamson & Paul, 2006 ; Zeaki, Rådström & Schelin, 2015 ), and pH (e.g., Meijer et al, 1998 ; Lunde et al, 2005 ; Wallin-Carlquist et al, 2010 ; Choi, Kotay & Goel, 2010 ).…”

Section: Introductionmentioning

confidence: 99%

Mimicking prophage induction in the body: induction in the lab with pH gradients

Miller-Ensminger

Garretto

Stark

et al. 2020

PeerJ

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The majority of bacteria within the human body are lysogens, often harboring multiple bacteriophage sequences (prophages) within their genomes. While several different types of environmental stresses can trigger or induce prophages to enter into the lytic cycle, they have yet to be fully explored and understood in the human microbiota. In the laboratory, the most common induction method is the DNA damaging chemical Mitomycin C. Although pH has been listed in the literature as an induction method, it is not widely used. Here, we detail a protocol for prophage induction by culture under different pH conditions. We explored the effects of pH on prophage induction in bacterial isolates from the bladder, where the pH is well documented to vary significantly between individuals as well as between healthy individuals and individuals with urinary tract symptoms or disease. Using this protocol, we successfully induced phages from seven bladder E. coli strains. Testing conditions and stressors appropriate to the environment from which a lysogen is isolated may provide insight into community dynamics of the human microbiota.

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Section: Introductionmentioning

confidence: 99%

Mimicking prophage induction in the body: induction in the lab with pH gradients

Miller-Ensminger

Garretto

Stark

et al. 2020

PeerJ

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“…One wild-type (WT) phage from each source bacterium, 42argF and 52S, was isolated from each spot plaque by further plaque purification. Although not all prophages are inducible by mitomycin C, the methods for validation are well suited to work with any number of alternative induction strategies, including pH and temperature shifts, UV induction, spontaneous induction, and chemical induction ( 19 – 22 ).…”

Section: Resultsmentioning

confidence: 99%

Computational Basis for On-Demand Production of Diversified Therapeutic Phage Cocktails

et al. 2020

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New therapies are necessary to combat increasingly antibiotic-resistant bacterial pathogens. We have developed a technology platform of computational, molecular biology, and microbiology tools which together enable on-demand production of phages that target virtually any given bacterial isolate. Two complementary computational tools that identify and precisely map prophages and other integrative genetic elements in bacterial genomes are used to identify prophage-laden bacteria that are close relatives of the target strain. Phage genomes are engineered to disable lysogeny, through use of long amplicon PCR and Gibson assembly. Finally, the engineered phage genomes are introduced into host bacteria for phage production. As an initial demonstration, we used this approach to produce a phage cocktail against the opportunistic pathogen Pseudomonas aeruginosa PAO1. Two prophage-laden P. aeruginosa strains closely related to PAO1 were identified, ATCC 39324 and ATCC 27853. Deep sequencing revealed that mitomycin C treatment of these strains induced seven phages that grow on P. aeruginosa PAO1. The most diverse five phages were engineered for nonlysogeny by deleting the integrase gene (int), which is readily identifiable and typically conveniently located at one end of the prophage. The Δint phages, individually and in cocktails, killed P. aeruginosa PAO1 in liquid culture as well as in a waxworm (Galleria mellonella) model of infection. IMPORTANCE The antibiotic resistance crisis has led to renewed interest in phage therapy as an alternative means of treating infection. However, conventional methods for isolating pathogen-specific phage are slow, labor-intensive, and frequently unsuccessful. We have demonstrated that computationally identified prophages carried by near-neighbor bacteria can serve as starting material for production of engineered phages that kill the target pathogen. Our approach and technology platform offer new opportunity for rapid development of phage therapies against most, if not all, bacterial pathogens, a foundational advance for use of phage in treating infectious disease.

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“…no. CA34.1) Petri dishes, sterile Bacterial incubator Tabletop centrifuge and microcentrifuge 50-ml conical polypropylene tubes (e.g., BD Falcon) Additional reagents and equipment for titering bacteria (Basic Protocol 1) and phage (UNIT 4.4) Prophage induction by UV-light irradiation (Baluch and Sussman, 1978) By dosing bacteria with ultraviolet light, DNA is damaged and prophages are excised due to the formation of pyrimidine dimers (Takebe et al, 1967;Barnhart et al, 1976). After treatment with UV light followed by a period of outgrowth for phage release, determine whether phages have been induced by titering the supernatants on a phage-free (nonlysogenized) reporter strain.…”

Section: Methodsmentioning

confidence: 99%

Isolation of Bacteriophages from Environmental Sources, and Creation and Functional Screening of Phage DNA Libraries

Pelzek

Schuch

Schmitz

et al. 2008

CP Essential Lab Tech

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Bacteriophages (phages) have evolved specific classes of proteins such as adhesins and lysins to mediate specific bacterial host recognition and rapid and efficient lysis following infection. However, because many bacterial species, and thus phages, cannot be cultured in the laboratory, techniques for the efficient cloning and screening of phage gene libraries are required to aid in the discovery of novel phage proteins for use in diagnostic and therapeutic applications. This article contains protocols for the isolation of phages from environmental samples, enrichment of phages targeting host bacteria of interest, and induction of phages from lysogenized host strains. We also provide an optimized protocol for the creation and functional screening of phage DNA libraries derived from environmental samples. Curr. Protoc. Essential Lab. Tech. 7:13.3.1‐13.3.35. © 2013 by John Wiley & Sons, Inc.

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Prophage induction and inactivation by UV light

Cited by 22 publications

References 17 publications

Mimicking prophage induction in the body: induction in the lab with pH gradients

Mimicking prophage induction in the body: induction in the lab with pH gradients

Computational Basis for On-Demand Production of Diversified Therapeutic Phage Cocktails

Isolation of Bacteriophages from Environmental Sources, and Creation and Functional Screening of Phage DNA Libraries

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