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Phage single-gene lysis: Finding the weak spot in the bacterial cell wall

  • Karthik Chamakura
    Affiliations
    Department of Biochemistry and Biophysics, Texas A&M AgriLife Research, Texas A&M University, College Station, Texas 77843-2128

    the Center for Phage Technology, Texas A&M AgriLife Research, Texas A&M University, College Station, Texas 77843-2128
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  • Ry Young
    Correspondence
    To whom correspondence should be addressed: Dept. of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843-2128. Tel.:979-845-2087
    Affiliations
    Department of Biochemistry and Biophysics, Texas A&M AgriLife Research, Texas A&M University, College Station, Texas 77843-2128

    the Center for Phage Technology, Texas A&M AgriLife Research, Texas A&M University, College Station, Texas 77843-2128
    Search for articles by this author
Open AccessPublished:November 12, 2018DOI:https://doi.org/10.1074/jbc.TM118.001773
      In general, the last step in the vegetative cycle of bacterial viruses, or bacteriophages, is lysis of the host. dsDNA phages require multiple lysis proteins, including at least one enzyme that degrades the cell wall (peptidoglycan (PG)). In contrast, the lytic ssDNA and ssRNA phages have a single lysis protein that achieves cell lysis without enzymatically degrading the PG. Here, we review four “single-gene lysis” or Sgl proteins. Three of the Sgls block bacterial cell wall synthesis by binding to and inhibiting several enzymes in the PG precursor pathway. The target of the fourth Sgl, L from bacteriophage MS2, is still unknown, but we review evidence indicating that it is likely a protein involved in maintaining cell wall integrity. Although only a few phage genomes are available to date, the ssRNA Leviviridae are a rich source of novel Sgls, which may facilitate further unraveling of bacterial cell wall biosynthesis and discovery of new antibacterial agents.

      Introduction to small lytic phages and “single-gene lysis”

      By definition, the lytic bacteriophages encode proteins for disruption of the host envelope. The large dsDNA phages, the Caudovirales, have multiple lysis proteins, including holins, endolysins, and spanins, targeting the cytoplasmic or inner membrane (IM),
      The abbreviations used are: IM
      inner membrane
      PG
      peptidoglycan
      OM
      outer membrane
      MurNAc
      N-acetylmuramic acid
      CTD
      C-terminal domain
      NTD
      N-terminal domain
      TMD
      transmembrane domain
      gRNA
      guide RNA
      SCAM
      substituted-cysteine accessibility method
      PDB
      Protein Data Bank
      mDAP
      meso diaminopimelic acid.
      peptidoglycan (PG), and outer membrane (OM), respectively, as well as multiple proteins that regulate the lysis process (
      • Young R.
      Phage lysis: do we have the hole story yet?.
      ,
      • Young R.
      Phage lysis: three steps, three choices, one outcome.
      ). In contrast, the small lytic phages of Gram-negative hosts, comprising the ssDNA (Microviridae) and ssRNA (Leviviridae), achieve host lysis by a single gene, encoding a protein lacking any PG-degrading activity (
      • Bernhardt T.G.
      • Wang I.N.
      • Struck D.K.
      • Young R.
      Breaking free: “protein antibiotics” and phage lysis.
      ). This review exclusively focuses on these single-gene lysis (Sgl) proteins of small lytic phages.

      Bacterial cell wall structure and biosynthesis

      An exploration of Sgl mechanisms requires a brief review of the structure of the Gram-negative cell wall and its biosynthesis. The key to the structure and shape of the cell envelope is the PG layer, consisting of 2–3 layers of glycan strands made up of repeating disaccharide units of MurNAc-pentapeptide and GlcNAc, cross-linked by peptide bridges between pentapeptide side chains of MurNAcs of adjacent strands (Fig. 1A) (
      • Vollmer W.
      • Blanot D.
      • de Pedro M.A.
      Peptidoglycan structure and architecture.
      ,
      • Typas A.
      • Banzhaf M.
      • Gross C.A.
      • Vollmer W.
      From the regulation of peptidoglycan synthesis to bacterial growth and morphology.
      • Silhavy T.J.
      • Kahne D.
      • Walker S.
      The bacterial cell envelope.
      ). The PG has considerable tensile strength (3–300 megapascals), allowing the cell to tolerate high internal osmotic pressures (3–10 atmospheres) while maintaining shape (
      • Thwaites J.J.
      • Mendelson N.H.
      Mechanical properties of peptidoglycan as determined from bacterial thread.
      ,
      • Stock J.B.
      • Rauch B.
      • Roseman S.
      Periplasmic space in Salmonella typhimurium and Escherichia coli.
      ). The entire PG of a cell can be isolated as a single complex polymer, the sacculus, studies of which have revealed that the glycan chains run almost perpendicular to the long axis of the cell (
      • Gan L.
      • Chen S.
      • Jensen G.J.
      Molecular organization of Gram-negative peptidoglycan.
      ). In most Gram-negative bacteria, the PG layer is covalently linked through >105 peptide linkages to the C-terminal Lys residue of the major lipoprotein, Lpp; the PG-linked Lpp is anchored almost exclusively in the inner leaflet of OM (
      • Typas A.
      • Banzhaf M.
      • Gross C.A.
      • Vollmer W.
      From the regulation of peptidoglycan synthesis to bacterial growth and morphology.
      ,
      • Braun V.
      Covalent lipoprotein from the outer membrane of Escherichia coli.
      ,
      • Cowles C.E.
      • Li Y.
      • Semmelhack M.F.
      • Cristea I.M.
      • Silhavy T.J.
      The free and bound forms of Lpp occupy distinct subcellular locations in Escherichia coli.
      ).
      Figure thumbnail gr1
      Figure 1The PG biosynthesis pathway and the Sgl system. A, the PG precursor pathway, from cytoplasmic UDP-GlcNAc to periplasmic lipid II, with known targets of “protein antibiotics” indicated. B, genome organization in ssDNA (ϕ X174) and ssRNA (Qβ, AP205, MS2, PhiCb5, and M) phages. In ssRNA phages, mat encodes the maturation protein responsible for adsorption to the receptor pilus, coat encodes the capsid protein, and rep encodes the replicase. In Qβ, the mat gene is named A2 and has the additional function of inducing host lysis. This research was originally published in Nature Microbiology. Chamakura, K. R., Sham, L. T., Davis, R. M., Min, L., Cho, H., Ruiz, N., Bernhardt, T. G., and Young, R. A viral protein antibiotic inhibits lipid II flippase activity. Nat. Microbiol. 2017; 2:1480–1484. © Nature Research.
      Biosynthesis of the PG can be divided into cytoplasmic, membrane, and periplasmic stages. There are seven enzymatic steps in the cytoplasm, beginning with the transfer of an enolpyruvyl moiety from PEP to UDP-GlcNAc, catalyzed by MurA (Fig. S1) (
      • Brown E.D.
      • Vivas E.I.
      • Walsh C.T.
      • Kolter R.
      MurA (MurZ), the enzyme that catalyzes the first committed step in peptidoglycan biosynthesis, is essential in Escherichia coli.
      ). After reduction of the enolpyruvyl moiety by MurB to create UDP-MurNAc, the next five enzymes are involved in adding amino acids that form the pentapeptide (l-Ala, d-Glu, m-Dap, d-Ala, and d-Ala) to the lactyl group, resulting in the final soluble intermediate UDP-MurNAc-pep5 (Fig. 1A) (
      • Typas A.
      • Banzhaf M.
      • Gross C.A.
      • Vollmer W.
      From the regulation of peptidoglycan synthesis to bacterial growth and morphology.
      ,
      • Lovering A.L.
      • Safadi S.S.
      • Strynadka N.C.
      Structural perspective of peptidoglycan biosynthesis and assembly.
      ). The first membrane-linked step in PG synthesis begins with the transfer of this sugar nucleotide pentapeptide to the lipid carrier undecaprenyl phosphate (C55-P or UndP). This reaction is catalyzed on the cytoplasmic face of the IM by the integral membrane protein MraY to generate a monosaccharide–lipid compound, lipid I (Fig. 1A and Fig. S2A). MurG then catalyzes the addition of a second sugar moiety (UDP-GlcNAc), resulting in the final precursor, lipid II (Fig. 1A). The last step of the membrane phase is the flipping of lipid II so that its disaccharide pentapeptide moiety is on the periplasmic face of the membrane. The enzyme “flippase” that effects this transfer has been controversial until very recently. Although FtsW was shown to flip lipid II in vitro (
      • Mohammadi T.
      • van Dam V.
      • Sijbrandi R.
      • Vernet T.
      • Zapun A.
      • Bouhss A.
      • Diepeveen-de Bruin M.
      • Nguyen-Distèche M.
      • de Kruijff B.
      • Breukink E.
      Identification of FtsW as a transporter of lipid-linked cell wall precursors across the membrane.
      ,
      • Mohammadi T.
      • Sijbrandi R.
      • Lutters M.
      • Verheul J.
      • Martin N.I.
      • den Blaauwen T.
      • de Kruijff B.
      • Breukink E.
      Specificity of the transport of lipid II by FtsW in Escherichia coli.
      ), several lines of evidence now support MurJ as being the essential lipid II flippase (
      • Ruiz N.
      Bioinformatics identification of MurJ (MviN) as the peptidoglycan lipid II flippase in Escherichia coli.
      • Sham L.T.
      • Butler E.K.
      • Lebar M.D.
      • Kahne D.
      • Bernhardt T.G.
      • Ruiz N.
      Bacterial cell wall. MurJ is the flippase of lipid-linked precursors for peptidoglycan biogenesis.
      ,
      • Meeske A.J.
      • Sham L.T.
      • Kimsey H.
      • Koo B.M.
      • Gross C.A.
      • Bernhardt T.G.
      • Rudner D.Z.
      MurJ and a novel lipid II flippase are required for cell wall biogenesis in Bacillus subtilis.
      ,
      • Kuk A.C.
      • Mashalidis E.H.
      • Lee S.Y.
      Crystal structure of the MOP flippase MurJ in an inward-facing conformation.
      ,
      • Bolla J.R.
      • Sauer J.B.
      • Wu D.
      • Mehmood S.
      • Allison T.M.
      • Robinson C.V.
      Direct observation of the influence of cardiolipin and antibiotics on lipid II binding to MurJ.
      • Zheng S.
      • Sham L.T.
      • Rubino F.A.
      • Brock K.P.
      • Robins W.P.
      • Mekalanos J.J.
      • Marks D.S.
      • Bernhardt T.G.
      • Kruse A.C.
      Structure and mutagenic analysis of the lipid II flippase MurJ from Escherichia coli.
      ). The extracellular steps of PG biosynthesis utilize the energy stored in the phosphodiester–muramic acid bond of the flipped lipid II and in the d-Ala–d-Ala peptide bond to drive the glycosyltransferase and cross-linking reactions, respectively (
      • Lovering A.L.
      • Safadi S.S.
      • Strynadka N.C.
      Structural perspective of peptidoglycan biosynthesis and assembly.
      ). These steps are carried out by mono- or bifunctional penicillin-binding proteins, and recently, RodA, a member of the SEDS superfamily, was shown to catalyze glycosyltransferase reactions (
      • Meeske A.J.
      • Riley E.P.
      • Robins W.P.
      • Uehara T.
      • Mekalanos J.J.
      • Kahne D.
      • Walker S.
      • Kruse A.C.
      • Bernhardt T.G.
      • Rudner D.Z.
      SEDS proteins are a widespread family of bacterial cell wall polymerases.
      ,
      • Cho H.
      • Wivagg C.N.
      • Kapoor M.
      • Barry Z.
      • Rohs P.D.
      • Suh H.
      • Marto J.A.
      • Garner E.C.
      • Bernhardt T.G.
      Bacterial cell wall biogenesis is mediated by SEDS and PBP polymerase families functioning semi-autonomously.
      • Sjodt M.
      • Brock K.
      • Dobihal G.
      • Rohs P.D.A.
      • Green A.G.
      • Hopf T.A.
      • Meeske A.J.
      • Srisuknimit V.
      • Kahne D.
      • Walker S.
      • Marks D.S.
      • Bernhardt T.G.
      • Rudner D.Z.
      • Kruse A.C.
      Structure of the peptidoglycan polymerase RodA resolved by evolutionary coupling analysis.
      ).

      The first Sgl: Protein E from microvirus ϕX174

      Famous phage, famous gene

      ϕX174 is the founding member and genetic paradigm of the Microviridae, which are nearly as widespread as the Caudovirales (
      • Kim M.S.
      • Park E.J.
      • Roh S.W.
      • Bae J.W.
      Diversity and abundance of single-stranded DNA viruses in human feces.
      ). It was the first gDNA to be completely sequenced and also to be synthesized in vitro (
      • Sanger F.
      • Air G.M.
      • Barrell B.G.
      • Brown N.L.
      • Coulson A.R.
      • Fiddes C.A.
      • Hutchison 3rd, C.A.
      • Slocombe P.M.
      • Smith M.
      Nucleotide sequence of bacteriophage ϕX174 DNA.
      ,
      • Smith H.O.
      • Hutchison 3rd, C.A.
      • Pfannkoch C.
      • Venter J.C.
      Generating a synthetic genome by whole genome assembly: ϕX174 bacteriophage from synthetic oligonucleotides.
      ). The 10 genes include three that are embedded out-of-frame in essential cistrons (Fig. 1B). One of these embedded genes is E, encoding the Sgl protein in the +1 reading frame of the essential scaffolding gene D (Fig. 1B). E is famous not only for being the first embedded gene to be discovered but also the first gene to be subjected to site-directed mutagenesis (
      • Sanger F.
      • Air G.M.
      • Barrell B.G.
      • Brown N.L.
      • Coulson A.R.
      • Fiddes C.A.
      • Hutchison 3rd, C.A.
      • Slocombe P.M.
      • Smith M.
      Nucleotide sequence of bacteriophage ϕX174 DNA.
      ,
      • Hutchison 3rd, C.A.
      • Phillips S.
      • Edgell M.H.
      • Gillam S.
      • Jahnke P.
      • Smith M.
      Mutagenesis at a specific position in a DNA sequence.
      ). More important here is the fact that it is the only DNA virus Sgl, and it was the first Sgl gene for which the lytic mechanism was firmly established. The methods for working out its functional pathway have been replicated for all of the other Sgl systems and thus will be reviewed here in some detail.

      Genetics clarifies E function

      The E gene was cloned into a medium copy expression plasmid and shown to support lysis after induction (
      • Henrich B.
      • Lubitz W.
      • Plapp R.
      Lysis of Escherichia coli by induction of cloned ϕX174 genes.
      ,
      • Young K.D.
      • Young R.
      Lytic action of cloned ϕX174 gene E.
      ). Despite this early focus and the availability of the cloned E gene, the lysis mechanism of E remained controversial for 2 decades (
      • Bernhardt T.G.
      • Wang I.N.
      • Struck D.K.
      • Young R.
      Breaking free: “protein antibiotics” and phage lysis.
      ). Early transmission EM studies showed that cells infected with ϕX174 lysed as a result of septal blebs in dividing cells, generating a morphology that was remarkably similar to penicillin-mediated lysis (
      • Bradley D.E.
      • Dewar C.A.
      • Robertson D.
      Structural changes in Escherichia coli infected with a ϕX174 type bacteriophage.
      ,
      • Ciak J.
      • Hahn F.E.
      Penicillin-induced lysis of Escherichia coli.
      ). Reproduction of this morphology after induction of the cloned E led to the general model that E interfered with PG biosynthesis (
      • Young K.D.
      • Young R.
      Lytic action of cloned ϕX174 gene E.
      ,
      • Bläsi U.
      • Henrich B.
      • Lubitz W.
      Lysis of Escherichia coli by cloned ϕX174 gene E depends on its expression.
      ). However, based on physiological and scanning-EM studies, other groups proposed that E functioned either by the activation of unspecified autolytic functions (
      • Lubitz W.
      • Plapp R.
      Stimulation of autolysis by adsorption of bacteriophage ϕX174 to isolated cell walls.
      ,
      • Bläsi U.
      • Halfmann G.
      • Lubitz W.
      Induction of autolysis of Escherichia coli by ϕX174 gene E product.
      • Lubitz W.
      • Halfmann G.
      • Plapp R.
      Lysis of Escherichia coli after infection with ϕX174 depends on the regulation of the cellular autolytic system.
      ) or by the formation of polymeric “transmembrane tunnels” that opened the cytoplasm directly to the medium (
      • Witte A.
      • Wanner G.
      • Bläsi U.
      • Halfmann G.
      • Szostak M.
      • Lubitz W.
      Endogenous transmembrane tunnel formation mediated by ϕX174 lysis protein E.
      ). This profusion of models painted a confusing picture for the mechanism of E lysis, primarily because all lacked genetic evidence.
      Mutational and deletion analysis of E revealed that the lytic function requires only the first 34 residues; lytic function was retained without the C-terminal 57 residues, a highly basic, Pro-rich segment, as long it was replaced by a stable cytoplasmic domain, even β-gal (
      • Maratea D.
      • Young K.
      • Young R.
      Deletion and fusion analysis of the ϕX174 lysis gene E.
      ,
      • Buckley K.J.
      • Hayashi M.
      Lytic activity localized to membrane-spanning region of ϕX174 E protein.
      ). The first genetic approach to Sgl function to be repeated successfully for several other Sgl systems was done by selecting spontaneous host mutants resistant to plasmid-borne expression of E (
      • Maratea D.
      • Young K.
      • Young R.
      Deletion and fusion analysis of the ϕX174 lysis gene E.
      ). The first E-resistant mutations turned out to be in a single locus, designated as slyD (“sensitivity to lysis”), which encodes a FKBP-type PPIase (peptidylprolyl-cis-trans-isomerase) (
      • Roof W.D.
      • Horne S.M.
      • Young K.D.
      • Young R.
      slyD, a host gene required for ϕX174 lysis, is related to the FK506-binding protein family of peptidyl-prolyl cis-trans-isomerases.
      ). Indeed, ϕX174 infections of slyD knockout mutants proceed normally in every respect, except lysis never occurs and virions hyperaccumulate. Purified SlyD was shown to accelerate folding of proteins limited by cis-trans peptidylprolyl isomerization steps, but its function in vivo is not known (
      • Kay J.E.
      Structure-function relationships in the FK506-binding protein (FKBP) family of peptidylprolyl cis-trans isomerases.
      ). However, E was found to be highly unstable in a slyD background, suggesting that SlyD has a chaperone role, probably in folding the C-terminal domain (CTD), which is rich in Pro residues (
      • Bernhardt T.G.
      • Roof W.D.
      • Young R.
      The Escherichia coli FKBP-type PPIase SlyD is required for the stabilization of the E lysis protein of bacteriophage ϕX174.
      ).
      To continue pursuit of the E target, bypass suppressor mutations were isolated as rare plaque formers on a slyD lawn (
      • Bernhardt T.G.
      • Roof W.D.
      • Young R.
      The Escherichia coli FKBP-type PPIase SlyD is required for the stabilization of the E lysis protein of bacteriophage ϕX174.
      ). These Epos (plates on slyD) were found to be missense alleles at the N terminus of E, each of which resulted in a ~10-fold increase in the biosynthesis rate, thereby compensating for the proteolytic instability of E. This proved to be a key technological advance. Spontaneous “Eps” host mutants (E-pos sensitivity) that were resistant to induction of the plasmid-borne Epos allele were selected and cross-streaked for sensitivity to the phage. Of ~2000 survivors, all but three retained ϕX174 sensitivity and presumably were defective in E expression or plasmid copy number. Subsequent genetic mapping and sequencing revealed that the mutations mapped to residues (Pro-172 and Phe-288) in TMD5 and -9 of MraY (see Fig. 3). Labeling experiments with [3H]mDAP showed that E blocked cell wall synthesis ~20 min before lysis, and TLC chromatography revealed depletion of lipid-linked label and the accumulation of UDP-GlcNAc, confirming the inhibition of MraY (
      • Bernhardt T.G.
      • Struck D.K.
      • Young R.
      The lysis protein E of ϕX174 is a specific inhibitor of the MraY-catalyzed step in peptidoglycan synthesis.
      ). Based on the mutational data and membrane localization of both proteins, Bernhardt et al. (
      • Bernhardt T.G.
      • Struck D.K.
      • Young R.
      The lysis protein E of ϕX174 is a specific inhibitor of the MraY-catalyzed step in peptidoglycan synthesis.
      ,
      • Bernhardt T.G.
      • Roof W.D.
      • Young R.
      Genetic evidence that the bacteriophage ϕX174 lysis protein inhibits cell wall synthesis.
      ) proposed that E interacted with MraY through TMD–TMD interactions that were disrupted in the mutant alleles (Fig. S2B).
      Figure thumbnail gr3
      Figure 3The structure of Qβ bound to MurA. A, structure of Qβ bound to MurA, colored as follows: coat proteins (blue), A2 (hot pink), gRNA (yellow), and MurA (orange). B, a 90° turn and cutaway view of Qβ shows MurA bound to the maturation protein with same color scheme as in A except in the case of coat proteins (radially colored from light blue to blue) and extra protein density (green). C, the ribbon model of A2 bound to MurA with uridine diphosphate GlcNAc (UDP-GlcNAc) in the active site (cornflower blue). D, the ribbon model of MurA viewed from the MurA–A2 interface. The point mutations that render MurA resistant to A2 are labeled and shown as red stick models. Locations of the catalytic loop and the UDP-GlcNAc are indicated by black arrows. E, ribbon model of A2 as viewed from the MurA–A2 interface. The region interacting with MurA, encompassing the N-terminal β-sheet region of residues 30–120, is outlined by a black lasso. The N and C termini are indicated by black arrows. This research was originally published in Proceedings of the National Academy of Sciences of the United States of America. Cui, Z., Gorzelnik, K. V., Chang, J. Y., Langlais, C., Jakana, J., Young, R., and Zhang, J. Proc. Natl. Acad. Sci. U.S.A. Structures of Qβ virions, virus-like particles, and the Qβ-MurA complex reveal internal coat proteins and the mechanism of host lysis. 2017; 114:11697–11702. © United States National Academy of Sciences.

      In vitro analysis of E-mediated inhibition of MraY

      Initial attempts to overexpress full-length E failed due to its inherent lethality, but by doing inductions in the presence of the heterologous MraY from B. subtilis, a His-tagged full-length E protein was purified with a yield of 27 μg of EHis6 per liter of culture (
      • Zheng Y.
      • Struck D.K.
      • Young R.
      Purification and functional characterization of ϕX174 lysis protein E.
      ). Using this as an immunoblot standard, these workers determined that E was produced at ~500 molecules/cell at the time of lysis, in agreement with estimates from radiolabeling and E-LacZ enzyme assays (
      • Maratea D.
      • Young K.
      • Young R.
      Deletion and fusion analysis of the ϕX174 lysis gene E.
      ,
      • Pollock T.J.
      • Tessman E.S.
      • Tessman I.
      Identification of lysis protein E of bacteriophage ϕX174.
      ). An assay based on UDP-MurNAc-pentapeptide DNS, a fluorescent analogue of UDP-MurNAc-pentapeptide, and phytol-P, a 20-carbon analogue of C55-P, was developed to determine kinetic parameters in the presence and absence of E. For both substrates, the addition of purified E had no effect on the apparent Km but reduced the Vmax, indicating that E is a noncompetitive inhibitor of MraY (
      • Zheng Y.
      • Struck D.K.
      • Young R.
      Purification and functional characterization of ϕX174 lysis protein E.
      ). This was further supported by the observation that overexpression of either WT or the catalytically inactive allele of mraYD267N protects cells from E-mediated lysis, presumably by titrating out E (
      • Bernhardt T.G.
      • Struck D.K.
      • Young R.
      The lysis protein E of ϕX174 is a specific inhibitor of the MraY-catalyzed step in peptidoglycan synthesis.
      ,
      • Zheng Y.
      • Struck D.K.
      • Bernhardt T.G.
      • Young R.
      Genetic analysis of MraY inhibition by the ϕX174 protein E.
      ).

      The E–MraY interaction

      Some details of the interaction between E and MraY have been determined by assessing lysis kinetics in bulk culture in the context of mutant E alleles, both from selections and site-directed mutagenesis and both WT and catalytically inactive MraY (
      • Bernhardt T.G.
      • Struck D.K.
      • Young R.
      The lysis protein E of ϕX174 is a specific inhibitor of the MraY-catalyzed step in peptidoglycan synthesis.
      ,
      • Bernhardt T.G.
      • Roof W.D.
      • Young R.
      Genetic evidence that the bacteriophage ϕX174 lysis protein inhibits cell wall synthesis.
      • Zheng Y.
      • Struck D.K.
      • Young R.
      Purification and functional characterization of ϕX174 lysis protein E.
      ,
      • Zheng Y.
      • Struck D.K.
      • Bernhardt T.G.
      • Young R.
      Genetic analysis of MraY inhibition by the ϕX174 protein E.
      ). Despite nearly saturating mutagenesis, mutations in only five mraY positions yielded E-insensitive phenotypes: one in TMD5 (G186S), two in TMD9 (F288L and V291M), and two (P170L and ΔL172) in the periplasmic loop above TMD5 (
      • Zheng Y.
      • Struck D.K.
      • Bernhardt T.G.
      • Young R.
      Genetic analysis of MraY inhibition by the ϕX174 protein E.
      ). By testing these alleles in the context of a catalytically inactive MraYD267N for the ability to protect against E lysis in an mraY+/mraYD267N merodiploid, the alleles were grouped into three classes of apparent affinity for E in vivo, with G186S and V291M ranking with the WT at the highest affinity, F288L and the B. subtilis version of MraY the lowest, and the P170L and ΔP172 alleles intermediate (
      • Zheng Y.
      • Struck D.K.
      • Bernhardt T.G.
      • Young R.
      Genetic analysis of MraY inhibition by the ϕX174 protein E.
      ). This classification was reflected in the in vitro inhibition assays (
      • Zheng Y.
      • Struck D.K.
      • Young R.
      Purification and functional characterization of ϕX174 lysis protein E.
      ). Based on the crystal structure of MraY from Aquifex aeolicus, the TMD5 and TMD9 sites are clustered on the same outside face of the molecule within the periplasmic leaflet of the IM (Fig. 2) (
      • Chung B.C.
      • Zhao J.
      • Gillespie R.A.
      • Kwon D.Y.
      • Guan Z.
      • Hong J.
      • Zhou P.
      • Lee S.Y.
      Crystal structure of MraY, an essential membrane enzyme for bacterial cell wall synthesis.
      ). TMD9 is split into two helical fragments: the N-terminal 9a, within which both E-insensitivity mutations map, and the C-terminal 9b, which does not pack in the helical bundle but is splayed out nearly laterally in the bilayer. The loop mutations map to a β hairpin structure, with one directly above TMD5 (Leu-172) and the other just above TMD9 (Pro-170). Assuming these mutations reflect critical contacts in the E binding site, the spatial arrangement indicates that the N-terminal segment of the TMD of E makes those contacts. This notion is consistent with the results from the Clemons group, who constructed a large set of alanine and leucine substitutions as well as truncation alleles, focused on the TMD of E (
      • Tanaka S.
      • Clemons Jr., W.M.
      Minimal requirements for inhibition of MraY by lysis protein E from bacteriophage ϕX174.
      ). This collection was analyzed by induction in vivo, monitoring the kinetics of lysis and the accumulation of the E protein in the membrane fraction. Mutations that affected lysis without affecting accumulation mapped to a single face of the TMD, suggesting that this face interacts with the two TMDs of MraY that housed the E-resistant changes. Also, the TMD as a whole appears to be relatively insensitive to changes in its C-terminal segment, which can be replaced by a homopolymer of Leu residues. Moreover, Phe substitutions at Leu-19 and Leu-23, both of which are on the same helix face as the inactivating mutations and one of which (L19F) corresponds to an original Epos mutation, enhance lytic function without enhancing membrane insertion. Using immobilized metal-affinity chromatography and Western blotting, complexes of E and MraY could be demonstrated. However, the efficiency of MraY recovery did not correlate well with lytic function of the E allele used, indicating that simple binding of E to MraY is not sufficient for inhibition. Overall, the details of how E binds MraY and interferes with lipid I synthesis will likely require a structure of the E–MraY complex.
      Figure thumbnail gr2
      Figure 2The E-resistant E. coli mraY alleles mapped on Aquifex aeolicus MraYAA. The E. coli mraY alleles resistant to E are mapped on the structure of MraY from Aquifex aeolicus (MraYAA) (PDB code 4J72). The structure is shown from the face that forms the putative E–MraY interface, and the domains of the interface are colored as follows: the periplasmic beta hairpin (yellow), the loop connecting PB with TMD5 (purple), TMD5 (magenta), and TMD9 (9a and 9b) (gray). The homologous E-resistant residues in MraYAA are shown as spheres, and the catalytically important aspartate (red) and histidine (blue) residues are shown as stick projections.

      Overview of Sgl genes in ssRNA phages

      The Sgl proteins undoubtedly evolved because of the extremely constrained size of the phage genome; compared with the Microviridae, this constraint is even worse for the ssRNA phages (the Leviviridae, or the leviviruses), which have ~4-kb gRNAs and only three core genes (Fig. 1B). Lacking tail structures, leviviruses exploit retractable pili to initiate infection. By far the best studied leviviruses are MS2 and Qβ, both specific for the canonical F conjugation pilus; many other F-specific leviviruses that are related to these two paradigms have been studied (
      • Inokuchi Y.
      • Jacobson A.B.
      • Hirose T.
      • Inayama S.
      • Hirashima A.
      Analysis of the complete nucleotide sequence of the group IV RNA coliphage SP.
      • Inokuchi Y.
      • Takahashi R.
      • Hirose T.
      • Inayama S.
      • Jacobson A.B.
      • Hirashima A.
      The complete nucleotide sequence of the group II RNA coliphage GA.
      ,
      • Inokuchi Y.
      • Hirashima A.
      • Watanabe I.
      Comparison of the nucleotide sequences at the 3′-terminal region of RNAs from RNA coliphages.
      ,
      • Stewart J.R.
      • Vinjé J.
      • Oudejans S.J.
      • Scott G.I.
      • Sobsey M.D.
      Sequence variation among group III F-specific RNA coliphages from water samples and swine lagoons.
      ,
      • Friedman S.D.
      • Cooper E.M.
      • Casanova L.
      • Sobsey M.D.
      • Genthner F.J.
      A reverse transcription-PCR assay to distinguish the four genogroups of male-specific (F+) RNA coliphages.
      • Adhin M.R.
      • Hirashima A.
      • van Duin J.
      Nucleotide sequence from the ssRNA bacteriophage JP34 resolves the discrepancy between serological and biophysical classification.
      ). However, seven other distinct leviviruses are known, each targeting a different retractable pilus (Table S1) (
      • Kazaks A.
      • Voronkova T.
      • Rumnieks J.
      • Dishlers A.
      • Tars K.
      Genome structure of Caulobacter phage phiCb5.
      • Ruokoranta T.M.
      • Grahn A.M.
      • Ravantti J.J.
      • Poranen M.M.
      • Bamford D.H.
      Complete genome sequence of the broad host range single-stranded RNA phage PRR1 places it in the Levivirus genus with characteristics shared with Alloleviviruses.
      ,
      • Rumnieks J.
      • Tars K.
      Diversity of pili-specific bacteriophages: genome sequence of IncM plasmid-dependent RNA phage M.
      ,
      • Klovins J.
      • Overbeek G.P.
      • van den Worm S.H.
      • Ackermann H.W.
      • van Duin J.
      Nucleotide sequence of a ssRNA phage from Acinetobacter: kinship to coliphages.
      ,
      • Olsthoorn R.C.
      • Garde G.
      • Dayhuff T.
      • Atkins J.F.
      • Van Duin J.
      Nucleotide sequence of a single-stranded RNA phage from Pseudomonas aeruginosa: kinship to coliphages and conservation of regulatory RNA structures.
      • Kannoly S.
      • Shao Y.
      • Wang I.N.
      Rethinking the evolution of single-stranded RNA (ssRNA) bacteriophages based on genomic sequences and characterizations of two R-plasmid-dependent ssRNA phages, C-1 and Hgal1.
      ). The Sgl genes have been identified in eight of the nine distinct leviviruses by showing that, as for E, induction of a plasmid-borne clone is necessary and sufficient for lysis. Importantly, all cause disruption of the cell wall by accessing different cellular targets, suggesting that in each case, a new Sgl was evolved after radiation to a new retractable pilus, no doubt facilitated by the extremely high mutation rate of the RNA-dependent replicase (
      • Domingo E.
      • Holland J.J.
      RNA virus mutations and fitness for survival.
      ). This means that even with the low total genomic database of less than 50,000 bases of unique Leviviridae genomes, there are multiple Sgl systems that might be exploited for probing the biosynthesis and dynamic homeostasis of the cell wall. In the following, the Sgl systems of Qβ, M, and MS2 will be reviewed. The order is not chronological but makes sense in that the targets of the first two Sgl proteins have been identified, whereas the MS2 Sgl system is an enduring mystery.

      The “protein antibiotic”: A2 from Qβ

      Finding rat mutants

      The A2 protein has multiple functions during Qβ infection; it functions in virion assembly (
      • Cui Z.
      • Gorzelnik K.V.
      • Chang J.Y.
      • Langlais C.
      • Jakana J.
      • Young R.
      • Zhang J.
      Structures of Qβ virions, virus-like particles, and the Qβ-MurA complex reveal internal coat proteins and the mechanism of host lysis.
      ), is bound to and provides protection for the gRNA against RNase degradation (
      • Weber K.
      • Konigsberg W.
      Proteins of the RNA phages.
      ), mediates interaction with the F-pilus, and is internalized into the host cytoplasm along with the genomic RNA (
      • Cui Z.
      • Gorzelnik K.V.
      • Chang J.Y.
      • Langlais C.
      • Jakana J.
      • Young R.
      • Zhang J.
      Structures of Qβ virions, virus-like particles, and the Qβ-MurA complex reveal internal coat proteins and the mechanism of host lysis.
      ,
      • Weber K.
      • Konigsberg W.
      Proteins of the RNA phages.
      • Kozak M.
      • Nathans D.
      Fate of maturation protein during infection by coliphage MS2.
      ). Remarkably, A2 also functions as the Sgl protein. The lytic activity of A2 was first demonstrated in 1983 by Winter and Gold (
      • Winter R.B.
      • Gold L.
      Overproduction of bacteriophage Qβ maturation (A2) protein leads to cell lysis.
      ), who showed that induction of A2 cloned on a medium-copy plasmid is necessary and sufficient to cause lysis.
      To identify the target of A2, the same method was used as for ϕX174 E, selecting for host mutants that survived induction of a plasmid-borne A2 gene, followed by screening survivors by cross-streaking with Qβ phage, and the mutants that passed selection/screen were designated as rat (resistant to A-two) mutants (
      • Bernhardt T.G.
      • Wang I.N.
      • Struck D.K.
      • Young R.
      A protein antibiotic in the phage Qβ virion: diversity in lysis targets.
      ). Genetics and sequence analyses revealed a single missense change in murA, L138Q. As with E, the incorporation of [3H]mDAP label into PG was blocked at least 20 min before the onset of lysis in cells induced for A2. Biochemical analysis of the sugar nucleotide pool from A2-inhibited cells revealed that UDP-GlcNAc was elevated, confirming MurA as the target. In vitro inhibition of MurA by purified A2 could not be demonstrated, mainly because overexpressed A2 was insoluble. However, in what seems to be the only instance of using virions for enzyme inhibition, it was shown that the catalytic activity of MurA, but not MurAL138Q, in crude extracts could be blocked by the addition of highly purified Qβ virions. Later experiments done with purified MurA and Qβ particles confirmed these results (
      • Reed C.A.
      • Langlais C.
      • Kuznetsov V.
      • Young R.
      Inhibitory mechanism of the Qβ lysis protein A2.
      ). Based on the turnover number of MurA and the number of purified virions needed to block its enzymatic activity, the MurA–A2 dissociation constant was determined to be ~10 nm (
      • Bernhardt T.G.
      • Wang I.N.
      • Struck D.K.
      • Young R.
      A protein antibiotic in the phage Qβ virion: diversity in lysis targets.
      ).

      The A2–MurA interaction

      In addition to in vitro inhibition assays, direct protein–protein interaction between A2–MurA and A2–MurAL138Q was also demonstrated by yeast two-hybrid analysis, with the latter pair displaying a weaker signal, suggesting that the mutant allele weakens A2 binding (
      • Reed C.A.
      • Langlais C.
      • Kuznetsov V.
      • Young R.
      Inhibitory mechanism of the Qβ lysis protein A2.
      ). To probe the interaction of MurA with A2 in vitro, the well-characterized catalytic pathway of MurA was exploited. The MurA reaction is well-ordered, with UDP-GlcNAc binding in the catalytic cleft associated with a dramatic shift from open to closed conformation, which then allows PEP binding and catalysis. Interaction studies were done using a soluble MBP–A2 fusion protein and various forms of MurA, including the original rat allele, MurAL138Q, and MurAD305A, which is disabled for catalysis but not substrate binding, and with various combinations of the substrates and the suicide inhibitor fosfomycin. The results clearly showed that A2 preferentially binds to the UDP-GlcNAc–liganded, closed form MurA, preventing PEP from binding. Details of the binding surface were obtained by site-directed substitutions of amino acids in the area around Leu-138, yielding a cluster of new rat alleles that blocked Qβ plaque formation and clearly defined an interaction surface surrounding the catalytic loop, including the catalytic domain, CTD, and the catalytic loop (Fig. S3).
      A2 differs significantly from the Mat proteins of MS2 and related Leviviridae, especially in the N terminus; deletion analysis confirmed that the lytic function is fully defined in the first 180 residues. To map the interaction domain, A2por (plates on rat) suppressor alleles were isolated and mapped to three positions (Leu-28, Asp-52, and Glu-125) in the N-terminal domain (
      • Reed C.A.
      • Langlais C.
      • Wang I.N.
      • Young R.
      A2 expression and assembly regulates lysis in Qβ infections.
      ). However, none of the por alleles were lytic when cloned and induced in the rat1 host. Immunoblot analysis revealed that A2por mutant levels increased much more rapidly than the parental A2 during infection, resulting in early lysis and reduced yield of progeny virions in the WT host. Inspection of the sequence around the por sites confirmed that the mutations disrupt significant RNA structures that repress translational initiation in the viral RNA, thus bypassing the reduced A2–MurAL138Q affinity by increasing the quantity of the phage protein.
      The interaction interface was recently resolved in asymmetric cryo-EM structures of Qβ particles in complex with UDP-GlcNAc–liganded MurA or fosfomycin-liganded MurA (
      • Cui Z.
      • Gorzelnik K.V.
      • Chang J.Y.
      • Langlais C.
      • Jakana J.
      • Young R.
      • Zhang J.
      Structures of Qβ virions, virus-like particles, and the Qβ-MurA complex reveal internal coat proteins and the mechanism of host lysis.
      ) (Fig. 3, A and B). The cryo-EM structures validated the interaction interface on MurA inferred from the various rat alleles and also confirmed that the NTD of A2 is in contact with MurA (Fig. 3, C–E).

      lysM: New target and settling a debate

      The lysis gene (lysM) of phage M has evolved completely embedded in the +1 reading frame of the rep gene, and it encodes a 37-amino acid protein with a single TMD (
      • Rumnieks J.
      • Tars K.
      Diversity of pili-specific bacteriophages: genome sequence of IncM plasmid-dependent RNA phage M.
      ). The functional LysM-eGFP fusion suggests an N-out and C-in membrane topology (
      • Chamakura K.R.
      • Sham L.T.
      • Davis R.M.
      • Min L.
      • Cho H.
      • Ruiz N.
      • Bernhardt T.G.
      • Young R.
      A viral protein antibiotic inhibits lipid II flippase activity.
      ). Early insights into the molecular mechanism of LysM lethality came from the observations that lysis proceeded through septal catastrophes, like A2 and E, suggesting that LysM might be an inhibitor of cell wall biosynthesis (
      • Bradley D.E.
      • Dewar C.A.
      • Robertson D.
      Structural changes in Escherichia coli infected with a ϕX174 type bacteriophage.
      ,
      • Bernhardt T.G.
      • Wang I.N.
      • Struck D.K.
      • Young R.
      A protein antibiotic in the phage Qβ virion: diversity in lysis targets.
      ,
      • Chamakura K.R.
      • Sham L.T.
      • Davis R.M.
      • Min L.
      • Cho H.
      • Ruiz N.
      • Bernhardt T.G.
      • Young R.
      A viral protein antibiotic inhibits lipid II flippase activity.
      ). The identity of MurJ as the molecular target of LysM was revealed in multicopy suppression experiments, where plasmids carrying murJ in random fragments of Escherichia coli genome–suppressed lysM lethality. Furthermore, isolation of nine spontaneous lysM-resistant mutants that mapped to two of the 14 TMDs of MurJ suggested a possible interaction interface and a plausible mechanism (Fig. 4). Additionally, it was shown that LysM was specific to MurJ, and the cells can be rescued from LysM lethality by the expression of heterologous lipid II flippase Amj from B. subtilis.
      Figure thumbnail gr4
      Figure 4LysM-resistance mutations map to TMD2 and TMD7 of MurJ. The amino acid changes in E. coli MurJ (MurJEC) resulting in LysM resistance were mapped onto the structure of MurJ from Thermosipho africanus (MurJTA) (PDB code 5T77). A, cytoplasmic-open conformation. B, model of the periplasmic-open conformation (
      • Kuk A.C.
      • Mashalidis E.H.
      • Lee S.Y.
      Crystal structure of the MOP flippase MurJ in an inward-facing conformation.
      ). The TMDs that line the central hydrophilic cavity are colored: TMD2 (light blue) and TMD7 (magenta). Left, lateral view; right, periplasmic view. The changes in MurJEC and homologous amino acids in MurJTA are shown on the right. This research was originally published in Nature Microbiology. Chamakura, K. R., Sham, L. T., Davis, R. M., Min, L., Cho, H., Ruiz, N., Bernhardt, T. G., and Young, R. A viral protein antibiotic inhibits lipid II flippase activity. Nat. Microbiol. 2017; 2:1480–1484. © Nature Research.
      To address the conformational state in which LysM binds to MurJ, a substituted-cysteine accessibility method (SCAM) was used (
      • Chamakura K.R.
      • Sham L.T.
      • Davis R.M.
      • Min L.
      • Cho H.
      • Ruiz N.
      • Bernhardt T.G.
      • Young R.
      A viral protein antibiotic inhibits lipid II flippase activity.
      ,
      • Butler E.K.
      • Davis R.M.
      • Bari V.
      • Nicholson P.A.
      • Ruiz N.
      Structure-function analysis of MurJ reveals a solvent-exposed cavity containing residues essential for peptidoglycan biogenesis in Escherichia coli.
      ). In the presence of LysM, SCAM analysis showed that five TMD positions showed altered SCAM patterns, which suggested that LysM binding locks MurJ into one of the two conformations proposed to constitute the lipid II–flipping cycle. The SCAM labeling pattern is consistent with MurJ being locked in a “periplasmic open” conformation, which would lead to an accumulation of lipid-linked PG precursors in the inner leaflet of IM and a corresponding decrease on the periplasmic side. Both predictions were confirmed by in vivo flippase assays, strongly indicating that LysM blocks MurJ's activity (
      • Chamakura K.R.
      • Sham L.T.
      • Davis R.M.
      • Min L.
      • Cho H.
      • Ruiz N.
      • Bernhardt T.G.
      • Young R.
      A viral protein antibiotic inhibits lipid II flippase activity.
      ). Moreover, in the presence of the LysM-resistant murJ alleles, the precursor levels were restored to normal. The fact that LysM only targets MurJ and causes lysis strongly suggests that MurJ is the only active lipid II flippase in E. coli. However, the interpretation of the LysM-resistant murJ alleles and the interaction interface would greatly benefit from the structure of the MurJ–LysM complex.

      MS2 lysis: To L and back

      L: The first autolysin?

      The L gene was not recognized as a gene in MS2 until the isolation of a plaque-forming defective nonsense mutant that belonged to a complementation group distinct from mat, coat, and rep (
      • Model P.
      • Webster R.E.
      • Zinder N.D.
      Characterization of Op3, a lysis-defective mutant of bacteriophage f2.
      ). Subsequent radioactive labeling experiments established L as the fourth gene of MS2, encoding a 75-amino acid polypeptide (
      • Beremand M.N.
      • Blumenthal T.
      Overlapping genes in RNA phage: a new protein implicated in lysis.
      ). As had been done with E and A2, a plasmid clone of L was shown to cause lysis after induction, and the L protein was shown to be associated with the membrane fraction (
      • Beremand M.N.
      • Blumenthal T.
      Overlapping genes in RNA phage: a new protein implicated in lysis.
      ,
      • Coleman J.
      • Inouye M.
      • Atkins J.
      Bacteriophage MS2 lysis protein does not require coat protein to mediate cell lysis.
      ). Opposite to E, it is the 39-residue CTD of L that accounts for membrane localization and lytic function, with the N-terminal 36 highly basic residues shown to be dispensable for lysis (Fig. S4) (
      • Berkhout B.
      • de Smit M.H.
      • Spanjaard R.A.
      • Blom T.
      • van Duin J.
      The amino terminal half of the MS2-coded lysis protein is dispensable for function: implications for our understanding of coding region overlaps.
      ). A key experiment was that, after induction, net murein synthesis, as assessed by [3H]mDAP into SDS-insoluble material, was unaffected prior to the onset of lysis (
      • Holtje J.V.
      • van Duin J.
      MS2 phage induced lysis of E. coli depends upon the activity of the bacterial autolysins.
      ). This clearly differentiates L action from the E, A2, and LysM Sgl proteins, all of which cause cessation of cell wall synthesis by interrupting the supply of lipid II to the PG machinery (
      • Bernhardt T.G.
      • Struck D.K.
      • Young R.
      The lysis protein E of ϕX174 is a specific inhibitor of the MraY-catalyzed step in peptidoglycan synthesis.
      ,
      • Bernhardt T.G.
      • Wang I.N.
      • Struck D.K.
      • Young R.
      A protein antibiotic in the phage Qβ virion: diversity in lysis targets.
      ,
      • Chamakura K.R.
      • Sham L.T.
      • Davis R.M.
      • Min L.
      • Cho H.
      • Ruiz N.
      • Bernhardt T.G.
      • Young R.
      A viral protein antibiotic inhibits lipid II flippase activity.
      ). To these workers, the most significant finding was that induced L lysis was severely compromised in acidic (pH 5.5) conditions, despite normal accumulation of L, raising a compelling analogy to penicillin-induced autolysis, which is also blocked under these conditions (
      • Walderich B.
      • Ursinus-Wösner A.
      • van Duin J.
      • Höltje J.V.
      Induction of the autolytic system of Escherichia coli by specific insertion of bacteriophage MS2 lysis protein into the bacterial cell envelope.
      ). This led to a general model in which L effected lysis by inducing autolysis, although the precise definition thereof was not provided. In immuno-EM experiments, L was shown to preferentially localize to apparent zones of adhesion between the IM and OM (
      • Walderich B.
      • Höltje J.V.
      Specific localization of the lysis protein of bacteriophage MS2 in membrane adhesion sites of Escherichia coli.
      ). This association with adhesion zones was emphasized by the fact that L lysis is also compromised in cells that lacked the periplasmic osmoprotectant membrane-derived oligosaccharide (
      • Höltje J.V.
      • Fiedler W.
      • Rotering H.
      • Walderich B.
      • van Duin J.
      Lysis induction of Escherichia coli by the cloned lysis protein of the phage MS2 depends on the presence of osmoregulatory membrane-derived oligosaccharides.
      ). These cells were shown to have many fewer adhesion zones and a much wider periplasm, and in this case, L appeared to be subject to degradation. Furthermore, a synthetic polypeptide corresponding to the C-terminal 25 amino acids of L was shown to permeabilize both liposomes and inverted membrane vesicles, leading the authors to invoke induction of autolysis after membrane permeabilization (
      • Goessens W.H.F.
      • Driessen A.J.M.
      • Wilschut J.
      • van Duin J.
      A synthetic peptide corresponding to the C-terminal 25 residues of phage MS2-coded lysis protein dissipates the proton-motive force in Escherichia coli membrane vesicles by generating hydrophilic pores.
      ). However, these experiments lacked a negative polypeptide control, and the experiments were done at peptide/vesicle ratios in excess of 1000; moreover, permeabilization and depolarization does not result in rapid autolysis in E. coli, so the physiological relevance of these experiments is questionable.

      Back to L: Genetic and molecular analysis

      The consignment of MS2 L to the role of phage-encoded autolysin seemed to end further interest in its function, despite the likelihood that a critical component of cell wall homeostasis was targeted. However, over the next decades, a few new Leviviridae were characterized, many of which shared the same genetic architecture as MS2, despite no significant nucleotide sequence similarity (
      • Olsthoorn R.C.
      • Garde G.
      • Dayhuff T.
      • Atkins J.F.
      • Van Duin J.
      Nucleotide sequence of a single-stranded RNA phage from Pseudomonas aeruginosa: kinship to coliphages and conservation of regulatory RNA structures.
      ,
      • Kannoly S.
      • Shao Y.
      • Wang I.N.
      Rethinking the evolution of single-stranded RNA (ssRNA) bacteriophages based on genomic sequences and characterizations of two R-plasmid-dependent ssRNA phages, C-1 and Hgal1.
      ) (Table S1). This included not only new leviviruses specific for the F pilus but also against the conjugational pilus of several R-factor plasmids and the polar pilus of Pseudomonas (Fig. S5) (
      • Ruokoranta T.M.
      • Grahn A.M.
      • Ravantti J.J.
      • Poranen M.M.
      • Bamford D.H.
      Complete genome sequence of the broad host range single-stranded RNA phage PRR1 places it in the Levivirus genus with characteristics shared with Alloleviviruses.
      ,
      • Rumnieks J.
      • Tars K.
      Diversity of pili-specific bacteriophages: genome sequence of IncM plasmid-dependent RNA phage M.
      ,
      • Olsthoorn R.C.
      • Garde G.
      • Dayhuff T.
      • Atkins J.F.
      • Van Duin J.
      Nucleotide sequence of a single-stranded RNA phage from Pseudomonas aeruginosa: kinship to coliphages and conservation of regulatory RNA structures.
      ,
      • Kannoly S.
      • Shao Y.
      • Wang I.N.
      Rethinking the evolution of single-stranded RNA (ssRNA) bacteriophages based on genomic sequences and characterizations of two R-plasmid-dependent ssRNA phages, C-1 and Hgal1.
      ). We noticed that the L proteins, although unrelated in terms of sequence, shared an apparent domain organization with L: domain 1, N-terminal, highly charged; domain 2, very hydrophobic and lacking charged residues; domain 3, a central Leu-Ser dipeptide; and domain 4, a variable CTD (
      • Chamakura K.R.
      • Edwards G.B.
      • Young R.
      Mutational analysis of the MS2 lysis protein L.
      ). The conserved architecture suggested that L-like Sgl systems widespread among Gram-negative bacteria were all targeting the same host function.
      Two genetics-based approaches were mounted, the first aimed at identifying host factors, using, as before, inducible plasmid-based clones of L (
      • Chamakura K.R.
      • Edwards G.B.
      • Young R.
      Mutational analysis of the MS2 lysis protein L.
      ,
      • Chamakura K.R.
      • Tran J.S.
      • Young R.
      MS2 lysis of Escherichia coli depends on host chaperone DnaJ.
      ). To avoid mutations that reduced the copy number or L transcription, a blue/white reporter plasmid was constructed, and from hundreds of colonies surviving L induction from this construct, two blue colonies were identified and designated as ill (insensitive to L lysis) mutants (
      • Chamakura K.R.
      • Tran J.S.
      • Young R.
      MS2 lysis of Escherichia coli depends on host chaperone DnaJ.
      ). Surprisingly, the ill mutations mapped to dnaJ, which encodes a widely conserved chaperone involved in the heat shock response (
      • Qiu X.B.
      • Shao Y.M.
      • Miao S.
      • Wang L.
      The diversity of the DnaJ/Hsp40 family, the crucial partners for Hsp70 chaperones.
      ). Analysis revealed that, in both, a P330Q missense change in dnaJ accounted for the Ill phenotype and abolished MS2 plaque formation, with both phage and survival phenotypes recessive. The Pro-330 residue is the most conserved residue in the CTD of DnaJ, which is clearly a conserved segment, although its function is unclear. The P330Q change was found to preserve the heat shock function of DnaJ but abrogates the ability of DnaJ to form complexes with L. The L suppressors, designated as Lodj (overcomes dnaJ) alleles, were isolated as mutants that allowed lysis in dnaJP330Q background; these proved to be deletions of the dispensable NTD of L. Isogenic inductions of the parental and Lodj alleles revealed that lysis was much earlier with the truncations. These results led to a model in which the NTD of L has a regulatory, lysis-delay function that blocks the interaction of L with its target; in this model, DnaJ is required for relief of this steric block (Fig. S6).
      To identify key functional elements of L itself, a nearly saturated mutational analysis of L generated a collection of 103 alleles with single codon changes conferring absolute lysis defects (Fig. S4) (
      • Chamakura K.R.
      • Edwards G.B.
      • Young R.
      Mutational analysis of the MS2 lysis protein L.
      ). The mutational distribution validated the proposed four domain structure of the L Sgl proteins. Domain 1, comprising the dispensable, highly basic region, gave rise to only one nonlytic allele (Q33H). Domain 3 containing the LS dipeptide motif and the adjacent segments of domains 2 and 4 had the most missense changes conferring nonlytic character. All of the missense alleles tested were genetically recessive and generated membrane-associated products of parental size. In addition, several of the inactivating missense changes (i.e. L44V, F47L, F47Y, S49T, F51L, and L56F) were conservative, suggesting that the L protein makes specific heterotypic protein–protein contacts in the membrane.
      Taken together, the isolation of dnaJP330Q and mutational analysis of L both suggest that L targets a host membrane protein; that the interaction is through the mutationally sensitive residues in domains 2, 3, and 4; and that, like SlyD and -E, a host chaperone is involved in regulating L function. Obviously, further investigation into the host factors involved in L lysis is needed to understand the mechanistic details of L function.

      What's next?

      The premise of this review was that the study of the Sgl systems of small lytic phages would be interesting and lead to a better understanding of the bacterial cell wall biosynthesis and homeostasis. To summarize what has been discussed, four Sgl systems have been studied in depth, one from the microvirus ϕX174 and three from the Leviviridae. In three cases, the Sgl proteins turned out to be “protein antibiotics,” specific inhibitors of different enzymes of the highly conserved PG biosynthesis pathway (
      • Bernhardt T.G.
      • Wang I.N.
      • Struck D.K.
      • Young R.
      Breaking free: “protein antibiotics” and phage lysis.
      ,
      • Chamakura K.R.
      • Sham L.T.
      • Davis R.M.
      • Min L.
      • Cho H.
      • Ruiz N.
      • Bernhardt T.G.
      • Young R.
      A viral protein antibiotic inhibits lipid II flippase activity.
      ,
      • Hatfull G.F.
      Microbiology: the great escape.
      ). Consideration of the available genetic and biochemical data has already increased our understanding of how these important enzymes function and, in the case of LysM, settled the identification of the flippase that exports lipid II to the periplasm. The next level of understanding will come from detailed structural information about the Sgl–enzyme inhibition complexes. The fourth case, the L protein of MS2, has not yet been fully characterized but appears to be the prototype of an Sgl type that has evolved multiple times in Leviviridae, infecting a wide range of Gram-negative bacteria (
      • Chamakura K.R.
      • Edwards G.B.
      • Young R.
      Mutational analysis of the MS2 lysis protein L.
      ). Genetic and biochemical evidence was cited showing that L does not inhibit any of the steps that lead to externalized lipid II and its incorporation into existing PG and suggests that it targets a host protein. There is no conceivable answer to the L target mystery that would not be important, possibly identifying conserved proteins that are essential for proper coordination or localization of cell wall synthesis machineries or are involved in the control of powerful autolytic enzymes.
      Even with the L story still incomplete, this seems like a pretty good haul of information from the study of four small genes, starting with very “low-tech,” old-fashioned and simple genetic selections. The shocking thing is that this wealth of molecular information is derived from the study of only nine distinct Leviviridae (Table S1), comprising a total of <50 kb of total genomic information. Despite the low number, these nine phages segregate into five different phage types based on where the Sgl evolved. Listed from 5′ to 3′ of the gRNA, they are as follows: AP205 (5′ of mat), Qβ (mat = A2), MS2 (overlapping end of coat and beginning of rep), phiCb5 (middle of rep), and M (near 3′ end of rep) (Fig. 2B). The diverse location of the Sgl genes suggests that they have evolved more than once and probably as a late addition to the genome after speciation to different pili or hosts (
      • Rumnieks J.
      • Tars K.
      Diversity of pili-specific bacteriophages: genome sequence of IncM plasmid-dependent RNA phage M.
      ,
      • Kannoly S.
      • Shao Y.
      • Wang I.N.
      Rethinking the evolution of single-stranded RNA (ssRNA) bacteriophages based on genomic sequences and characterizations of two R-plasmid-dependent ssRNA phages, C-1 and Hgal1.
      ). (This includes all of the L-like genes; none of the 6 L-type Sgls has any detectable sequence identity other than the LS dipeptide sequence.) Given the diversity of Sgl systems and the existence of multiple protein targets in PG biosynthesis and maintenance, it is not difficult to imagine the existence of Sgl inhibitors for every known step in PG biosynthesis and, if L is any indicator, possibly uncharacterized components critical for dynamic cell wall homeostasis.
      Taken together, it seems obvious that it would be useful to identify new Sgl genes, and the old-fashioned “phage hunt” is a reliable approach. RNA phage hunts have so far been done for five conjugational pili, resulting in two protein antibiotic Sgls (A2 and LysM) and four unrelated L-type Sgls (LMS2, LHGAL, LC1, and LPRR1). Only three nonconjugational pili (Caulobacter, Acinetobacter, and Pseudomonas) have been targeted, resulting in two L-type Sgls and one, Lys of Caulobacter phage phiCB5, that does not have an L-type domain structure but does have a single N-terminal TMD, resembling both E and LysM. Considering the existence of many more retractable pili systems, there is a clear rationale to conduct RNA phage hunts in many other systems with retractable pili, especially in pathogenic bacteria.
      Metagenomics is also having an impact. A recent survey of publicly available RNA-inclusive metagenomes and RNA virome studies of invertebrate species led to the identification of ~200 new ssRNA phage genomes (
      • Krishnamurthy S.R.
      • Janowski A.B.
      • Zhao G.
      • Barouch D.
      • Wang D.
      Hyperexpansion of RNA bacteriophage diversity.
      ,
      • Shi M.
      • Lin X.D.
      • Tian J.H.
      • Chen L.J.
      • Chen X.
      • Li C.X.
      • Qin X.C.
      • Li J.
      • Cao J.P.
      • Eden J.S.
      • Buchmann J.
      • Wang W.
      • Xu J.
      • Holmes E.C.
      • Zhang Y.Z.
      Redefining the invertebrate RNA virosphere.
      ). Although most of the new leviviral genomes are partial, ~80 are either complete or nearly so, with all three core genes annotated. Only one (AVE017) of these ~200 genomes had an annotated Sgl gene, being a close relative (38% sequence identity) of MS2 L (
      • Krishnamurthy S.R.
      • Janowski A.B.
      • Zhao G.
      • Barouch D.
      • Wang D.
      Hyperexpansion of RNA bacteriophage diversity.
      ). Given their small size, predilection for being embedded in the core genes, and extreme sequence diversity at the protein level, finding Sgl candidates in these genomes poses unique challenges to the traditional gene annotation tools. Moreover, currently, there is no direct way to sort out these leviviral genomes to a particular bacterial host or, more specifically, to a particular retractable pilus. Nevertheless, the promise of more intriguing Sgl proteins targeting novel components of the bacterial cell wall machinery surely makes our current effort, which involves identifying potential Sgl ORFs and characterizing them one by one for the ability to support lysis after induction of synthetic clones, worth doing.

      Acknowledgments

      The following figures and figure legends were reproduced verbatim from published references: Figs. 1, 3, and 4 and Figs. S3–S6. We thank past and current members of the Young laboratory for advice. We also thank Ing-Nang Wang, Kaspar Tars, and Rene Olsthoorn for providing ssRNA phage resources and expertise. The clerical assistance of Daisy Wilbert is highly appreciated.

      Supplementary Material

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