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Characterization of five environmental phages infecting Escherichia coli K-12 isolated during a phage biology training course.

Shyrokova, Lena LU orcid ; Egorov, Artyom A LU orcid ; Cole, Astrid ; Duque-Pedraza, Julian J LU ; Tyagi, Anu LU ; Ernits, Karin LU ; Mets, Toomas LU ; Kurata, Tatsuaki LU ; Juozapaitis, Jonas and Yang, Alessio Ling Jie , et al. (2025) In Microbiology spectrum
Abstract

Phage collections are essential tools for discovering and dissecting bacterial anti-phage defense systems. Here, we report the isolation, characterization, and annotated genome sequences of five environmental Escherichia coli-infecting phages, obtained during the 2023 Fundamentals of Basic and Applied Phage Biology course at Lund University. The phages were isolated using a motile E. coli K-12 BW25113 strain, whose motility is conferred by an IS5 insertion upstream of the flhDC operon, the master regulator of flagellar synthesis. The isolated Escherichia phages include Lubas (LuPh1) and Lucat (LuPh2) of the genus Tequatrovirus; Lupin (LuPh3) and Lucris (LuPh4) of the genus ... (More)

Phage collections are essential tools for discovering and dissecting bacterial anti-phage defense systems. Here, we report the isolation, characterization, and annotated genome sequences of five environmental Escherichia coli-infecting phages, obtained during the 2023 Fundamentals of Basic and Applied Phage Biology course at Lund University. The phages were isolated using a motile E. coli K-12 BW25113 strain, whose motility is conferred by an IS5 insertion upstream of the flhDC operon, the master regulator of flagellar synthesis. The isolated Escherichia phages include Lubas (LuPh1) and Lucat (LuPh2) of the genus Tequatrovirus; Lupin (LuPh3) and Lucris (LuPh4) of the genus Tequintavirus; and Kompetensportalen (LuPh5) of the genus Chivirus. Transmission electron microscopy confirmed myovirus and siphovirus morphologies typical of these genera. Consistent with its classification in the flagellotropic Chivirus genus, LuPh5 did not infect a poorly motile BW25113 strain lacking the IS5 element upstream of flhDC, but did infect the motile laboratory Salmonella enterica subsp. enterica serovar Typhimurium strain 12023s. By testing a panel of eight previously described anti-phage defense systems, we found that LuPh1 and LuPh2 are inhibited by the toxin-antitoxin-chaperone CmdTAC system; LuPh5 is inhibited by both the restriction-modification system EcoRI and the abortive infection reverse transcriptase AbiK; and all five phages are sensitive to the hybrid artificial CmdTA-HigC system. Collectively, our findings expand the toolkit for probing phage-host interactions and underscore the pedagogical value of incorporating phage isolation into practical training for emerging researchers.

IMPORTANCE Bacterial anti-phage defense systems are central to microbiology and have applications in biotechnology and medicine. Phage collections are key tools for uncovering and studying these systems. By isolating and characterizing five E. coli K12-infecting phages, including three newly identified species, this work expands resources for probing phage-host interactions. It also shows that hands-on phage isolation in educational settings can contribute meaningfully to both training and research.

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Contribution to journal
publication status
epub
subject
in
Microbiology spectrum
article number
e0227425
publisher
American Society for Microbiology
external identifiers
  • pmid:41196041
ISSN
2165-0497
DOI
10.1128/spectrum.02274-25
project
Anti-phage defense by abortive infection reverse transcriptase systems
Phage sensing and defence by NAD+-depleting immunity systems
Experimental exploration of bacterial toxin-antitoxin systems
language
English
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yes
id
d702b88d-6ad2-4c5c-bc16-a5f78e16b760
date added to LUP
2025-11-12 10:19:18
date last changed
2025-11-19 13:24:16
@article{d702b88d-6ad2-4c5c-bc16-a5f78e16b760,
  abstract     = {{<p>Phage collections are essential tools for discovering and dissecting bacterial anti-phage defense systems. Here, we report the isolation, characterization, and annotated genome sequences of five environmental <i>Escherichia coli</i>-infecting phages, obtained during the 2023  <i>Fundamentals of Basic and Applied Phage Biology</i> course at Lund University. The phages were isolated using a motile  <i>E. coli</i> K-12 BW25113 strain, whose motility is conferred by an IS5 insertion upstream of the  <i>flhDC</i> operon, the master regulator of flagellar synthesis. The isolated  <i>Escherichia</i> phages include Lubas (LuPh1) and Lucat (LuPh2) of the genus  <i>Tequatrovirus</i>; Lupin (LuPh3) and Lucris (LuPh4) of the genus  <i>Tequintavirus</i>; and Kompetensportalen (LuPh5) of the genus  <i>Chivirus</i>. Transmission electron microscopy confirmed myovirus and siphovirus morphologies typical of these genera. Consistent with its classification in the flagellotropic  <i>Chivirus</i> genus, LuPh5 did not infect a poorly motile BW25113 strain lacking the IS5 element upstream of  <i>flhDC</i>, but did infect the motile laboratory  <i>Salmonella enterica</i> subsp.  enterica serovar Typhimurium strain 12023s. By testing a panel of eight previously described anti-phage defense systems, we found that LuPh1 and LuPh2 are inhibited by the toxin-antitoxin-chaperone CmdTAC system; LuPh5 is inhibited by both the restriction-modification system EcoRI and the abortive infection reverse transcriptase AbiK; and all five phages are sensitive to the hybrid artificial CmdTA-HigC system. Collectively, our findings expand the toolkit for probing phage-host interactions and underscore the pedagogical value of incorporating phage isolation into practical training for emerging researchers.</p><p>IMPORTANCE Bacterial anti-phage defense systems are central to microbiology and have applications in biotechnology and medicine. Phage collections are key tools for uncovering and studying these systems. By isolating and characterizing five <i>E. coli </i>K12-infecting phages, including three newly identified species, this work expands resources for probing phage-host interactions. It also shows that hands-on phage isolation in educational settings can contribute meaningfully to both training and research. </p>}},
  author       = {{Shyrokova, Lena and Egorov, Artyom A and Cole, Astrid and Duque-Pedraza, Julian J and Tyagi, Anu and Ernits, Karin and Mets, Toomas and Kurata, Tatsuaki and Juozapaitis, Jonas and Yang, Alessio Ling Jie and Ibarra Chavez, Rodrigo and Graversgaard Henriksen, Frederik Oskar and Lassen, Swenja and Atkinson, Gemma C and Hauryliuk, Vasili and Johansson, Marcus J O}},
  issn         = {{2165-0497}},
  language     = {{eng}},
  month        = {{11}},
  publisher    = {{American Society for Microbiology}},
  series       = {{Microbiology spectrum}},
  title        = {{Characterization of five environmental phages infecting <i>Escherichia coli</i> K-12 isolated during a phage biology training course.}},
  url          = {{http://dx.doi.org/10.1128/spectrum.02274-25}},
  doi          = {{10.1128/spectrum.02274-25}},
  year         = {{2025}},
}