Characterization of five environmental phages infecting <i>Escherichia coli</i> K-12 isolated during a phage biology training course

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. <i>enterica</i> 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.IMPORTANCEBacterial 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.