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 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.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 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.