Genome Sequencing of dsDNA-Containing Bacteriophages Directly from a Single Plaque

Kot, Witold

doi:10.1007/978-1-4939-7343-9_13

Cited by 5 publications

(3 citation statements)

References 11 publications

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“…The phages’ DNA was extracted using the protocol described by Kot (2018) [ 37 ]. Briefly, in a 1.5 mL tube, 90 μL of phage lysate was filtered through a 0.45 μm ultrafiltration spin-column and then mixed with 10 μL of DNase I buffer and 5 U of DNase I.…”

Section: Methodsmentioning

confidence: 99%

Isolation and Characterization of the Acadevirus Members BigMira and MidiMira Infecting a Highly Pathogenic Proteus mirabilis Strain

da Silva,

Bens,

Santos

et al. 2023

Microorganisms

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Proteus mirabilis is an opportunistic pathogen and is responsible for more than 40% of all cases of catheter-associated urinary tract infections (CAUTIs). Healthcare-associated infections have been aggravated by the constant emergence of antibiotic-resistant bacterial strains. Because of this, the use of phages to combat bacterial infections gained renewed interest. In this study, we describe the biological and genomic features of two P. mirabilis phages, named BigMira and MidiMira. These phages belong to the Acadevirus genus (family Autographiviridae). BigMira and MidiMira are highly similar, differing only in four missense mutations in their phage tail fiber. These mutations are sufficient to impact the phages’ depolymerase activity. Subsequently, the comparative genomic analysis of ten clinical P. mirabilis strains revealed differences in their antibiotic resistance profiles and lipopolysaccharide locus, with the latter potentially explaining the host range data of the phages. The massive presence of antimicrobial resistance genes, especially in the phages’ isolation strain P. mirabilis MCS, highlights the challenges in treating infections caused by multidrug-resistant bacteria. The findings reinforce BigMira and MidiMira phages as candidates for phage therapy purposes.

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Section: Methodsmentioning

confidence: 99%

Isolation and Characterization of the Acadevirus Members BigMira and MidiMira Infecting a Highly Pathogenic Proteus mirabilis Strain

da Silva,

Bens,

Santos

et al. 2023

Microorganisms

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“…Phage DNA originating from a single plaque was directly sequenced as described elsewhere, 16 on an Illumina NextSeq platform with the Mid Output Kit v2 (300 cycles).…”

Section: Dna Isolation and Genome Sequencingmentioning

confidence: 99%

A Rare, VirulentClostridium perfringensBacteriophage Susfortuna Is the First Isolated Bacteriophage in a New Viral Genus

Pedersen

Kot

Plöger

et al. 2020

PHAGE

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Background: Clostridium perfringens is a well known swine pathogen. C. perfringens type A is considered the causative agent of enteric diseases in neonatal and weaned piglets. Phage therapy using C. perfringens phages in vivo has previously proved effective. Materials and Methods: Pig fecal samples were used to isolate phages, with Clostridium perfringens type A as host. Complete genome sequencing, comparative genomics, a proteome analysis and electron microscopy were used to characterize the phage. Results: Clostridium phage Susfortuna has a double-stranded DNA genome of 19,046 bp with a G+C% content of 29.2, inverted terminal repeats and 28 predicted coding sequences (CDSs). Putative functions could not be assigned to most of the CDSs (64.3%). Transmission electron microscopy of phage Susfortuna revealed an isometric head and a short protruding tail stub resembling the structure of the Podoviridae family. A proteome analysis of phage Susfortuna identified seven structural proteins, but only one could be assigned with a putative function. Conclusions: Based on the morphology, the inverted terminal repeats and the small genome size, phage Susfortuna belongs to subfamily Picovirinae within the Podoviridae family in the order Caudovirales. Together with C. perfringens bacteriophage CPD7, phage Susfortuna represent a new genus of bacteriophages with very limited DNA sequence similarity to other known C. perfringens phages. Despite the limited DNA sequence similarity, the gene synteny among putative structural genes of phage Susfortuna is conserved among several C. perfringens bacteriophages belonging to the Podoviridae family indicating a common ancestor.

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“…Genome sequences were obtained by a combination of methods. Two well-separated plaques were subjected to direct sequencing using a Nextera kit (8). Phage genomic DNA was also purified from high-titer stocks prepared from webbed plates using the Promega Wizard DNA cleanup system followed by Nextera tagmentation.…”

mentioning

confidence: 99%

Complete Genome Sequences of Two Vibrio natriegens Bacteriophages

Harris

Fruchtman

et al. 2020

Microbiol Resour Announc

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Genome Sequencing of dsDNA-Containing Bacteriophages Directly from a Single Plaque

Abstract: The sequencing of phage genomes has become a routine procedure for phage characterization. The protocol presented here allows rapid isolation of DNA from a single phage plaque followed by building ready-to-sequence Illumina-compatible library.

Cited by 5 publications

References 11 publications

Isolation and Characterization of the Acadevirus Members BigMira and MidiMira Infecting a Highly Pathogenic Proteus mirabilis Strain

Isolation and Characterization of the Acadevirus Members BigMira and MidiMira Infecting a Highly Pathogenic Proteus mirabilis Strain

A Rare, VirulentClostridium perfringensBacteriophage Susfortuna Is the First Isolated Bacteriophage in a New Viral Genus

Complete Genome Sequences of Two Vibrio natriegens Bacteriophages

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