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A viral protein antibiotic inhibits lipid II flippase activity


For bacteriophage infections, the cell walls of bacteria, consisting of a single highly polymeric molecule of peptidoglycan (PG), pose a major problem for the release of progeny virions. Phage lysis proteins that overcome this barrier can point the way to new antibacterial strategies1, especially small lytic single-stranded DNA (the microviruses) and RNA phages (the leviviruses) that effect host lysis using a single non-enzymatic protein2. Previously, the A2 protein of levivirus Qβ and the E protein of the microvirus ϕX174 were shown to be ‘protein antibiotics’ that inhibit the MurA and MraY steps of the PG synthesis pathway2,3,4. Here, we investigated the mechanism of action of an unrelated lysis protein, LysM, of the Escherichia coli levivirus M5. We show that LysM inhibits the translocation of the final lipid-linked PG precursor called lipid II across the cytoplasmic membrane by interfering with the activity of MurJ. The finding that LysM inhibits a distinct step in the PG synthesis pathway from the A2 and E proteins indicates that small phages, particularly the single-stranded RNA (ssRNA) leviviruses, have a previously unappreciated capacity for evolving novel inhibitors of PG biogenesis despite their limited coding potential.

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This work was supported by Public Health Service Grant GM27099 to R.Y., by NIH grant AI099144 and CETR U19 AI109764 to T.G.B., by NIH grant R01GM100951 to N.R., and by the American Heart Association under the award number 14POST18480014 to L.-T.S. Additional support for this work was provided by the Center for Phage Technology at Texas A&M University, jointly sponsored by Texas A&M AgriLife. The authors thank H. Rye and L. Kustigian of the Department of Biochemistry and Biophysics, Texas A&M University, for providing purified eGFP standards.

Author information

K.R.C., N.R., T.G.B. and R.Y. designed the study and the analysed results. K.R.C. performed genetic selections and microscopy, and constructed strains and plasmids. L.-T.S. performed ColM assays and performed the amJ rescue experiment. R.M.D. constructed various murJ haploid strains, performed their function and expression tests, and performed SCAM. L.M. performed lysis profiles of the LysM-resistant alleles and assisted in making the figures. H.C. constructed the E. coli multi-copy library. K.R.C., T.G.B. and R.Y. prepared the manuscript. N.R. and L.-T.S. edited the manuscript and provided text.

Competing interests

The authors declare no competing financial interests.

Correspondence to Thomas G. Bernhardt or Ry Young.

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Supplementary Information

Supplementary Tables 1–4, Supplementary Figures 1–12, Supplementary References.

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Fig. 1: The lysis protein of phage M blocks lipid II flipping.
Fig. 2: LysM-resistance changes map to TMD2 and TMD7 of MurJ.
Fig. 3: LysM induces conformational changes in MurJ.
Fig. 4: LysM blocks lipid II flipping.