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PEPTIDES FOR INDUCING BACTERIOCIN SYNTHESIS AND METHODS TO IDENTIFY AND/OR SELECT AND/OR OPTIMIZE THE SAME

20220017573 · 2022-01-20

    Inventors

    Cpc classification

    International classification

    Abstract

    Described herein is a peptide or peptidomimetic with a length of at least 6 residues comprising, consisting essentially of, or consisting of the sequence motif Xaa.sub.1-Trp-Xaa.sub.2-Xaa.sub.3-Xaa.sub.4-Xaa.sub.5 (SEQ ID NO:1), wherein: Xaa.sub.1 represents an aromatic residue (Phe, Tyr, Trp, His), Cys or Ser; Xaa.sub.2, Xaa.sub.3 and Xaa.sub.4 represent any residue; and Xaa.sub.5 represents Gly, lie or Val.

    Claims

    1. A peptide or peptidomimetic able to induce bacteriocin production in a microbial organism with a length of at least 6 residues comprising the sequence motif Xaa.sub.1-Trp-Xaa.sub.2-Xaa.sub.3-Xaa.sub.4-Xaa.sub.5 (SEQ ID NO:1), wherein the sequence motif is located at the C-terminus of said peptide or peptidomimetic or located so that it is followed by 1, 2, 3, 4 or 5 additional C-terminal residues, and wherein: Xaa.sub.1 represents an aromatic residue (Phe, Tyr, Trp, His), Cys or Ser; Xaa.sub.2, Xaa.sub.3 and Xaa.sub.4 represent any residue; and Xaa.sub.5 represents Gly, Ile or Val.

    2. The peptide or peptidomimetic of claim 1, wherein the sequence motif is located at the C-terminus of said peptide or peptidomimetic.

    3. The peptide or peptidomimetic of claim 1, wherein at least one of the following conditions are fulfilled: the sequence motif is preceded immediately by a Pro residue; Xaa.sub.1 represents an aromatic residue (Phe, Tyr, Trp, His); and Xaa.sub.5 represents Gly.

    4. The peptide or peptidomimetic of claim 1, wherein the sequence motif is Phe-Trp-Leu-Val-Leu-Gly (SEQ ID NO: 2).

    5. The peptide or peptidomimetic of claim 1, wherein the peptide or peptidomimetic comprises one of the following sequences: Ala-Phe-Trp-Leu-Ile-Leu-Gly (SEQ ID NO: 3) Thr-Trp-Trp-Leu-Ile-Leu-Gly (SEQ ID NO: 4) Pro-Tyr-Trp-Leu-Gly-Leu-Gly (SEQ ID NO: 5) Pro-Trp-Trp-Val-Ser-Val-Gly (SEQ ID NO: 6) Pro-Phe-Trp-Leu-Ile-Leu-Gly (SEQ ID NO: 7) Pro-Tyr-Trp-Leu-Leu-Ile-Gly (SEQ ID NO: 8) Pro-Phe-Trp-Leu-Val-Leu-Gly (SEQ ID NO: 9) Pro-Phe-Trp-Val-Val-Ala-Gly (SEQ ID NO: 10) Pro-Phe-Trp-Leu-Ser-Val-Gly (SEQ ID NO: 11) Pro-Tyr-Trp-Leu-Asp-Met-Gly (SEQ ID NO: 12) Pro-Tyr-Trp-Val-Thr-Met-Gly (SEQ ID NO: 13) Pro-Tyr-Trp-Val-Val-Leu-Gly (SEQ ID NO: 14) Pro-Ser-Trp-Leu-Val-Val-Gly (SEQ ID NO: 15) Pro-His-Trp-Ile-Thr-Ile-Gly (SEQ ID NO: 16) Pro-His-Trp-Cys-Val-Leu-Gly (SEQ ID NO: 17) Pro-Phe-Trp-Leu-Ala-Leu-Gly (SEQ ID NO: 18) Pro-Phe-Trp-Cys-Val-Leu-Gly (SEQ ID NO: 19) Phe-Trp-Val-Glu-Leu-Gly (SEQ ID NO: 20) Tyr-Trp-Ala-Thr-Thr-Gly-Leu (SEQ ID NO: 21) Trp-Trp-Gly-Thr-Met-Ile (SEQ ID NO: 22) Pro-Tyr-Trp-Leu-Cys-Ile-Ile (SEQ ID NO: 23) Thr-Cys-Trp-Val-Cys-Ile-Val (SEQ ID NO: 24) Leu-Ala-Phe-Trp-Asp-Ser-Leu-Gly (SEQ ID NO: 749).

    6. The peptide or peptidomimetic according to of claim 1, wherein the peptide or peptidomimetic has a maximum length of 30 amino acids.

    7. The peptide or peptidomimetic according to of claim 1, wherein the peptide or peptidomimetic is able to induce bacteriocin production in a microbial organism without concomitantly inducing competence.

    8. A polypeptide able to induce bacteriocin production in a microbial organism, comprising the peptide or peptidomimetic of claim 1, wherein the peptide or peptidomimetic can be released from the polypeptide by natural, chemical or biological peptide hydrolysis.

    9. A culture medium comprising the peptide or peptidomimetic of claim 1.

    10. The culture medium of claim 9, further comprising a signaling molecule and/or a quenching molecule and/or an antimicrobial peptide and/or a bacteriocin.

    11. A composition for inducing bacteriocin production in a microbial organism, comprising the peptide or peptidomimetic of claim 1 and a solvent.

    12. The composition of claim 11, further comprising a signaling molecule and/or a quenching molecule and/or an antimicrobial peptide and/or a bacteriocin.

    13. A microbial organism able to produce and/or secrete the peptide or peptidomimetic of claim 1.

    14. A method for inducing bacteriocin production in a microbial organism, comprising: a) administering the peptide or peptidomimetic of claim 1 to the microbial organism; and/or b) culturing the microbial organism in a culture medium comprising the peptide or peptidomimetic of claim 1; and/or c) administering a composition comprising the peptide or peptidomimetic of claim 1 to the microbial organism.

    15. A method for inducing bacteriocin production in a first microbial organism, comprising contacting the first microbial organism with a second microbial organism comprising the microbial organism of claim 13 and/or co-culturing the first microbial organism with the second microbial organism comprising the microbial organism of claim 13.

    16. The method of claim 14, wherein the bacteriocin-producing microbial organism belongs to the microbiota.

    17. The method of claim 14, wherein the bacteriocin-producing microbial organism also produces a desired product.

    18. The method of claim 14, wherein the bacteriocin-producing microbial organism is a Gram-positive bacterium.

    19. The method of claim 14, wherein the bacteriocin production in the microbial organism neutralizes a second, undesired microbial organism.

    20. The method of claim 14, wherein the undesired microbial organism is a pathogenic microbial organism or a contaminant.

    21. A method for identifying, selecting and/or optimizing peptide ligands of peptide-responsive transcriptional regulators, comprising the following: a) generating a library of randomized genes, wherein said genes are operably linked to an inducible promoter, inducible by an inducer molecule; b) transforming the library into a microbial organism which comprises a nucleic acid encoding a selectable marker conferring resistance to a selection agent, operably linked to a promoter which is controlled by the peptide-responsive transcriptional regulator; and c) selecting or enriching positive clones by growing the microbial organism in the presence of the inducer molecule and the selection agent.

    22. The method of claim 21, wherein the nucleotide sequences of the randomized genes comprise both fixed-sequence codons and degenerate codons.

    Description

    DESCRIPTION OF THE FIGURES

    [0151] FIG. 1. Competence-Predation Dependencies of ComR Paralogs in S. salivarius

    [0152] (A) Scheme of genomic organization and transcriptional dependencies (dashed arrows) between competence activation (comX) and bacteriocins production (blpK, slvX, . . . ) in S. salivarius. Promoters are depicted with broken arrows. Regulators and the ComS pheromone are stained according to their encoding genes. The ComS precursor is produced (curled arrow) as an intracellular precursor (square) before secretion, maturation and import as an active pheromone (ellipses). The newly described two-Rgg system is shaded and the T arrow pinpoints the inhibitory role of SarF on ScuR.

    [0153] (B, C, D and E) Maximum luciferase activity/OD.sub.600 ratio (RLU/OD; logarithmic scale) of various competence or bacteriocin production-involved promoters fused to a luxAB reporter system in WT or overexpressing backgrounds. (B) Promoter activation of genes upon sComS addition (full bars) vs mock condition (striped bars) in WT (light grey bars) or scuR overexpression mutant (scuR.sup.++; dark grey bars). (C) Activity of sptA, comS and slvX promoters in WT strain, and scuR.sup.++ or sarF (sarF.sup.++) overexpression mutants. (D) Activity of sptA and comS promoters in WT and scuR.sup.++ mutant deleted or not for comR gene. (E) Activity of sptA and comS promoters in overexpression of scuR or sarF in a scuR locus (ΔscuR-sarF) deletion background. Experimental values represent the averages (with standard error of the mean, SEM) of at least three independent biological replicates.

    [0154] (F) Maximum luciferase activity/OD.sub.600 ratio (RLU/OD) of comS, blpK and slvX promoters fused to a luxAB reporter system in WT (open bars) or ΔscuR-sarF (grey bars) strain activated with sComS. Experimental values represent the averages (with standard error of the mean, SEM) of at least three independent biological replicates.

    [0155] FIG. 2. ScuR/SarF Activating Peptide Identification

    [0156] (A) Cartoon portraying the rational strategy for the peptide randomization-based screen. A library of randomized small genes under inducible promoter controls (P.sub.xyl) is transformed in a reporter strain in which the chloramphenicol resistance gene (cat) is translationally fused to sptA promoter. In absence of xylose or upon xylose induction of irrelevant peptides (square, hexagon and star), sptA promoter is maintained OFF and does not initiate cat transcription, causing cell sensitivity (Cm.sup.S) on chloramphenicol-supplemented media. The xylose-driven intracellular production of a cognate peptide (ellipse) promotes chloramphenicol resistance (Cm.sup.R), through P.sub.sptA activation by ScuR/SarF (dashed arrow).

    [0157] (B) Activity of sptA promoter in WT strains and various mutants expressing intracellularly activating peptides (cartoon) in medium supplemented with xylose (0.1% or 1%; grey bars) vs mock conditions (open bars). The BM 1 clone is an irrelevant peptide (negative control). Magnitude is expressed in percentage compared to the WT P.sub.sptA-luxAB reporter strain (Relative maximal luciferase activity). Experimental values represent the averages (with standard error of the mean, SEM) of at least three independent biological replicates.

    [0158] (C) Weighted consensus sequence for the suite of 22 activating peptides identified in the randomization-based screen. Randomized residues are crowned with a horizontal black bar while the non-variable amino acids are grey-coloured. The Bits represent the relative frequency of residues. Information content is plotted as a function of residues position and reckoned from the N-terminus (1 to 12) or the C-terminus (−1 to −12). The sequence logo image was generated using the WebLogo application (accessible on the world wide web at weblogo.berkeley.edu/logo.cgi).

    [0159] (D) Promoter activity of the sptA gene in response to the BI7 encoded peptide in various rgg mutant backgrounds. Media were supplemented with 0.1% xylose (open bars) or water (grey bars). Experimental values represent the averages (with standard error of the mean, SEM) of at least three independent biological replicates.

    [0160] FIG. 3. The ScuR/SarF System Responds to Exogenous Peptides

    [0161] (A) Cartoon depicting the ScuR/SarF-mediated activation of P.sub.sptA upon addition of exogenous synthetic peptides.

    [0162] (B) Fold increase in maximal P.sub.sptA activity upon addition of representative synthetic peptide (0.01 or 1 μM) vs mock conditions. Peptide sequences are correlated to peptide name and compared to the consensus motive (SEQ ID NO: 728) (open box). sBH6: SEQ ID NO: 729; sBI7: SEQ ID NO: 730; sBI10: SEQ ID NO: 731; sBJ1: SEQ ID NO: 732; sBK1: SEQ ID NO: 733; sBK3: SEQ ID NO: 734; sBK4: SEQ ID NO: 735; sBK8: SEQ ID NO: 736; sBO2: SEQ ID NO: 737. The crucial W and G residues are highlighted with grey boxes.

    [0163] (C) Maximal activity of P.sub.sptA exposed to sBI7 WT and mutant peptides (1 nM).

    [0164] (D) Dose response dot plot of sptA, slvX and comS promoter activity upon sBI7 induction at various concentrations (nM). Promoters were tested in WT strain and, ΔscuR or ΔsarF deletants.

    [0165] (B, C and D) Experimental values represent the averages (with standard error of the mean, SEM) of at least three independent biological replicates.

    [0166] (E) Maximal activity of PsptA (RLU/OD; logarithmic scale) exposed to WT and mutant sBI7 peptides (1 μM). Experimental values represent the averages (with standard error of the mean, SEM) of at least three independent biological replicates.

    [0167] (F) Activity of sptA, comS and slvX promoters (absolute maximal luciferase activity; ALU) in ΔscuR-sarF challenged with sBI7 synthetic peptide (grey bars) vs mock conditions (open bars). Experimental values represent the averages (with standard error of the mean, SEM) of at least three independent biological replicates.

    [0168] (G) Maximum luciferase activity/OD600 ratio (RLU/OD; logarithmic scale) of comS, slvX and sptA promoters fused to a luxAB reporter system in WT (open bars) or ΔcomR (grey bars) strain activated with the sBI7 synthetic peptide. Experimental values represent the averages (with standard error of the mean, SEM) of at least three independent biological replicates.

    [0169] FIG. 4. Singularities in Promoter Recognition of Rgg Paralogs

    [0170] (A) Mobility shift assays of comX, comS, slvX and sptA promoter probes conducted with purified Rgg paralogs and decreasing concentrations of their cognate peptide (grey triangles; 2:2 dilutions from 20 μM). Probes are 30 bp (or 40 bp for P.sub.sptA), were Cy3-conjugated and used at 40 ng. Protein concentration remains constant (grey boxes; 4 μM). Open triangles showcase ternary complexes (peptide-Rgg-DNA).

    [0171] (B) Nucleotide alignment of promoters of comX (SEQ ID NO: 738), comS (SEQ ID NO: 739), slvX (SEQ ID NO: 740) and sptA (SEQ ID NO: 741). The palindromic stretches (inverted arrows) and the sigma-bound DNA sequence (−10 boxes) are shaded in grey. Boxed nucleotides highlight the potential mismatches in the hairpin structure of P.sub.comX or P.sub.sptA that were substituted to restore a genuine dyad symmetry sequence (see FIGS. 4C and 4D). A and T represent the position and nature of single nucleotide insertion in the sptA promoter (see FIG. 4C).

    [0172] (C and D) Fold increase in maximal activity of WT and mutated promoters of sptA (C) or comX (D) exposed to sBI7 or sComS (1 μM). Nucleotides substitutions and insertions are disclosed in FIG. 4B. Experimental values represent the averages (with standard error of the mean, SEM) of at least three independent biological replicates.

    [0173] (E) Mobility shift assays of mutated comX promoter probes conducted with a unique concentration of ScuR or SarF (grey boxes; 4 μM) and decreasing concentrations of sBI7 peptide (grey triangles; 2:2 dilutions from 20 μM). Open triangles showcase ternary complexes (peptide-Rgg-DNA).

    [0174] (F) Mobility shift assays of comS, slvX and sptA promoter probes conducted with purified ScuR and decreasing concentrations of the non-cognate sComS peptide (grey triangles; 2:2 dilutions from 20 μM). Probes are 30 bp (or 40 bp for P.sub.sptA), were Cy3-conjugated and used at 40 ng. Protein concentration remains constant (grey boxes; 4 μM). Open triangle showcases binary complexes (ScuR-DNA).

    [0175] (G) Mobility shift assays of slvX and sptA promoter probes conducted with sBI7 peptide and decreasing concentrations of purified ScuR (grey triangles; 2:2 dilutions from 8 μM). Probes are 30 bp (P.sub.slvX) or 40 bp (P.sub.sptA), were Cy3-conjugated and used at 40 ng. Peptide concentration remains constant (grey boxes; 1 μM). Open triangles showcase ternary complexes (sBI7-ScuR-DNA).

    [0176] (H) Nucleotide alignment of bacteriocin-related gene promoters. P.sub.slvX: SEQ ID NO: 740; P.sub.00176: SEQ ID NO: 742; P.sub.01584: SEQ ID NO: 743; P.sub.blpK: SEQ ID NO: 744; P.sub.slvV: SEQ ID NO: 745; P.sub.blpG: SEQ ID NO: 746; P.sub.slvW: SEQ ID NO: 747; P.sub.slvY: SEQ ID NO: 748. The palindromic stretches (inverted arrows) and the sigma-bound DNA sequence (−10 boxes) are shaded in grey. The characteristic T-rich region is grey font.

    [0177] FIG. 5. Activated ScuR Drives Bacteriocin Secretion

    [0178] (A and B) Bacteriocin inhibition assay of S. salivarius WT and mutant derivatives. Indicator strains (L. lactis) were embedded in the top soft agar layer, while sBI7 was supplemented into the bottom agar layer as required. Producer strains were spotted on top of the two agar layers. (A) Killing properties of scuR or sarF overexpression mutants compared WT. (B) Effect of sBI7 addition (1 μM) on WT strain and scuR/sarF various mutants. scuR.sup.++ and bacteriocin null mutant (Δslv5) were used as positive and negative control, respectively.

    [0179] (C) Bacteriocin inhibition assay of S. salivarius WT and scuR/sarF mutant derivatives. Indicator strains (L. lactis) were embedded in the top soft agar layer, while sBI7 was supplemented or not into the bottom agar layer as stated. Producer strains were spotted on top of the two agar layers. scuR.sup.++ and bacteriocin null mutant (Δslv5) were used as positive and negative control, respectively.

    [0180] FIG. 6. Rgg Members Requisition Predation Control in S. salivarius

    [0181] (A) Conservation of ScuR locus, and BIpRH system across S. salivarius. Functional BIpRH pair, ScuR, SptA, SptB, and SarF were sought for homologs in various S. salivarius strains. The phylogenetic tree (100 bootstrap replicates) was adapted from (Yu et al., 2015). An empty box means that no functional ortholog was found in the species genome. Scale bar: 0.01 substitution per site.

    [0182] (B) Figurative illustration of RRNPPs vs two-component systems (TCSs) drift toward competence and predation regulation in paradigmatic streptococci (S. pneumoniae, S. mutans and S. salivarius).

    [0183] FIG. 7. Amino Acid Requirements for the sBI7-Mediated Effect

    [0184] Maximal activity of salivaricin promoters (RLU/OD; logarithmic scale) exposed to sBI7 (WT) (SEQ ID NO: 36) and mutant peptides (1 μM). Experimental values represent the averages (with standard error of the mean, SEM) of at least three independent biological replicates.

    [0185] FIG. 8. sBI7 Has No Effect on the comX Promoter Activation

    [0186] Maximal activity of P.sub.comX exposed to the sComS or sBI7 peptide (1 μM) (SEQ ID NO: 36) in WT strain and ΔscuR and/or ΔsarF mutants. Experimental values represent the averages (with standard error of the mean, SEM) of at least three independent biological replicates.

    [0187] FIG. 9. No Activation of comX or Late Competence Genes Under ComX Control by sBI7

    [0188] Maximal activity of P.sub.comX and ComX-dependent promoters exposed to the sComS (light grey) or sBI7 peptide (SEQ ID NO: 36) (dark grey) (1 μM) in comparison to basal activity (open box). Experimental values represent the averages (with standard error of the mean, SEM) of at least three independent biological replicates.

    [0189] FIG. 10. C-Terminal Tolerance of Synthetic Peptides

    [0190] (A) Maximal activity of P.sub.sptA exposed to LAFWDSLG (SEQ ID NO: 749), LAFWDSLGLLL (SEQ ID NO: 750) or the peptide sBI7 (SEQ ID NO: 36) (1 μM) in comparison to basal activity. Experimental values represent the averages (with standard error of the mean, SEM) of at least three independent biological replicates.

    [0191] (B) Mobility shift assays of the sptA promoter probe conducted with purified ScuR and decreasing concentrations of the peptides LAFWDSLG (SEQ ID NO: 749) or LAFWDSLGLLL (SEQ ID NO: 750) (grey triangles; 2:2 dilutions from 20 μM). The probe is 40 bp, Cy3-conjugated and used at 40 ng. Protein concentration remains constant (grey boxes; 4 μM). Open triangle showcases single or binary complexes (ScuR-DNA).

    EXAMPLES

    Experimental Procedures

    Bacterial Strains, Plasmids, Oligonucleotides, and Growth Conditions

    [0192] Bacterial strains, plasmids and oligonucleotides used in the Examples are listed and described in the tables below. Streptococcus salivarius HSISS4 and derivatives were grown at 37° C. without shaking in M17 (Difco Laboratories, Detroit, Mich.) or in CDM (Fontaine et al. Mol Microbiol 2013, 87:1113-1132) supplemented with 1% (w/v) glucose (M17G, CDMG, respectively). Escherichia coli TOP10 (Invitrogen) were cultivated with shaking at 37° C. in LB. Electrotransformation of E. coli was performed as previously described (Mignolet et al. Elife 2016, 5:e18647). Lactococcus lactis was grown in M17 broth with 1% glucose at 30° C. without shaking. We added 1.5% (w/v) agar into M17 and LB plates, and bacteriocin inhibition tests were assayed on M17 plates containing 0.2% agar. We added D-xylose (0.1 or 1%; w/v), ampicillin (250 μg/ml), spectinomycin (200 μg/ml), chloramphenicol (5 μg/ml; except if otherwise stated) or erythromycin (10 μg/ml), 5-FOA (1 mg/ml) (Melford Laboratories), as required. Synthetic peptides and sComS (purity of 95%; 1 μM, except if otherwise stated) were supplied by Peptide 2.0 Inc. (Chantilly, Va., USA) and resuspended in DMSO. Solid plates inoculated with streptococci cells were incubated anaerobically (BBL GasPak systems, Becton Dickinson, Franklin lakes, N.J.) at 37° C.

    TABLE-US-00011 TABLE 1 List of bacterial strains used in the Examples Characteristics Reference/source Escherichia coli TOP10 mcrA, Δ(mrr-hsdRMS-mcrBC), Phi80lacZ(del)M15, Invitrogen ΔlacX74, deoR, recA1, araD139, Δ(ara-leu)7697, galU, galK, rpsL(SmR), endA1, nupG Streptococcus salivarius HSISS4 Wild-type gastro-intestinal tract isolate (Van den Bogert et al., 2014) JM1004 HSISS4 ΔcomA::cat (Mignolet et al., 2018) JM1013 HSISS4 Δslv5 (Mignolet et al., 2018) JM1019 HSISS4 tRNA.sup.Thr::P.sub.comS-luxAB-cat (Mignolet et al., 2018) JM1020 HSISS4 tRNA.sup.Thr::P.sub.comX-luxAB-cat (Mignolet et al., 2018) JM1021 HSISS4 tRNA.sup.Thr::P.sub.blpK-luxAB-cat (Mignolet et al., 2018) JM1022 HSISS4 tRNA.sup.Thr::P.sub.HSISS4.sub.—.sub.00176-luxAB-cat (Mignolet et al., 2018) JM1023 HSISS4 tRNA.sup.Thr::P.sub.HSISS4.sub.—.sub.01584-luxAB-cat (Mignolet et al., 2018) JM1024 HSISS4 tRNA.sup.Thr::P.sub.slvV-luxAB-cat (Mignolet et al., 2018) JM1025 HSISS4 tRNA.sup.Thr::P.sub.blpG-luxAB-cat (Mignolet et al., 2018) JM1026 HSISS4 tRNA.sup.Thr::P.sub.slvW-luxAB-cat (Mignolet et al., 2018) JM1027 HSISS4 tRNA.sup.Thr::P.sub.slvX-luxAB-cat (Mignolet et al., 2018) JM1028 HSISS4 tRNA.sup.Thr::P.sub.slvY-luxAB-cat (Mignolet et al., 2018) JM1100 HSISS4 tRNA.sup.Thr::P.sub.sptA-luxAB-cat This work JM1101 HSISS4 tRNA.sup.Ser::P.sub.32-scuR-spec (scuR.sup.++) This work JM1102 HSISS4 tRNA.sup.Ser::P.sub.32-sarF-ST-spec (sarF-ST.sup.++) This work JM1103 JM1019 tRNA.sup.Ser::P.sub.32-scuR-spec This work JM1104 JM1020 tRNA.sup.Ser::P.sub.32-scuR-spec This work JM1105 JM1021 tRNA.sup.Ser::P.sub.32-scuR-spec This work JM1106 JM1022 tRNA.sup.Ser::P.sub.32-scuR-spec This work JM1107 JM1023 tRNA.sup.Ser::P.sub.32-scuR-spec This work JM1108 JM1024 tRNA.sup.Ser::P.sub.32-scuR-spec This work JM1109 JM1025 tRNA.sup.Ser::P.sub.32-scuR-spec This work JM1110 JM1026 tRNA.sup.Ser::P.sub.32-scuR-spec This work JM1111 JM1027 tRNA.sup.Ser::P.sub.32-scuR-spec This work JM1112 JM1028 tRNA.sup.Ser::P.sub.32-scuR-spec This work JM1113 JM1100 tRNA.sup.Ser::P.sub.32-scuR-spec This work JM1114 JM1019 tRNA.sup.Ser::P.sub.32-sarF-spec This work JM1115 JM1027 tRNA.sup.Ser::P.sub.32-sarF-spec This work JM1116 JM1100 tRNA.sup.Ser::P.sub.32-sarF-spec This work JM1117 JM1113 ΔcomR::ery This work JM1118 HSISS4 ΔscuR-sarF::ery This work JM1119 JM1113 ΔscuR-sarF::ery This work JM1120 JM1116 ΔscuR-sarF::ery This work JM1121 HSISS4 tRNA.sup.Thr::P.sub.sptA-cat-spec This work JM1122 HSISS4 tRNA.sup.Thr::P.sub.sptA-cat-lox72 This work JM1123 JM1122 tRNA.sup.Ser::P.sub.xyl1-comR-spec This work JM1124 JM1013 tRNA.sup.Thr::P.sub.sptA-cat-lox72 This work JM1125 JM1122 tRNA.sup.Ser::P.sub.xyl2-BH6-spec This work JM1126 JM1122 tRNA.sup.Ser::P.sub.xyl1-BI1-spec This work JM1127 JM1122 tRNA.sup.Ser::P.sub.xyl1-BI2-spec This work JM1128 JM1122 tRNA.sup.Ser::P.sub.xyl1-BI5-spec This work JM1129 JM1122 tRNA.sup.Ser::P.sub.xyl1-BI6-spec This work JM1130 JM1122 tRNA.sup.Ser::P.sub.xyl1-BI7-spec This work JM1131 JM1122 tRNA.sup.Ser::P.sub.xyl1-BI9-spec This work JM1132 JM1122 tRNA.sup.Ser::P.sub.xyl1-BI10-spec This work JM1133 JM1122 tRNA.sup.Ser::P.sub.xyl1-BI11-spec This work JM1134 JM1122 tRNA.sup.Ser::P.sub.xyl1-BI12-spec This work JM1135 JM1122 tRNA.sup.Ser::P.sub.xyl2-BJ1-spec This work JM1136 JM1122 tRNA.sup.Ser::P.sub.xyl1-BK1-spec This work JM1137 JM1122 tRNA.sup.Ser::P.sub.xyl1-BK2-spec This work JM1138 JM1122 tRNA.sup.Ser::P.sub.xyl1-BK3-spec This work JM1139 JM1122 tRNA.sup.Ser::P.sub.xyl1-BK4-spec This work JM1140 JM1122 tRNA.sup.Ser::P.sub.xyl1-BK5-spec This work JM1141 JM1122 tRNA.sup.Ser::P.sub.xyl1-BK6-spec This work JM1142 JM1122 tRNA.sup.Ser::P.sub.xyl1-BK7-spec This work JM1143 JM1122 tRNA.sup.Ser::P.sub.xyl1-BK8-spec This work JM1144 JM1122 tRNA.sup.Ser::P.sub.xyl1-BK9-spec This work JM1145 JM1124 tRNA.sup.Ser::P.sub.xyl1-BL1-spec This work JM1146 JM1124 tRNA.sup.Ser::P.sub.xyl1-BL2-spec This work JM1147 JM1124 tRNA.sup.Ser::P.sub.xyl1-BL3-spec This work JM1148 JM1124 tRNA.sup.Ser::P.sub.xyl2-BM1-spec This work JM1149 JM1124 tRNA.sup.Ser::P.sub.xyl1-BN1-spec This work JM1150 JM1124 tRNA.sup.Ser::P.sub.xyl1-BN2-spec This work JM1151 JM1124 tRNA.sup.Ser::P.sub.xyl1-BN3-spec This work JM1152 JM1124 tRNA.sup.Ser::P.sub.xyl1-BN4-spec This work JM1153 JM1122 tRNA.sup.Ser::P.sub.xyl2-BO1-spec This work JM1154 JM1122 tRNA.sup.Ser::P.sub.xyl2-BO2-spec This work JM1155 JM1122 tRNA.sup.Ser::P.sub.xyl1-BP1-spec This work JM1156 JM1100 tRNA.sup.Ser::P.sub.xyl1-BI5-spec This work JM1157 JM1100 tRNA.sup.Ser::P.sub.xyl1-BI7-spec This work JM1158 JM1100 tRNA.sup.Ser::P.sub.xyl1-BI10-spec This work JM1159 JM1100 tRNA.sup.Ser::P.sub.xyl2-BJ1-spec This work JM1160 JM1100 tRNA.sup.Ser::P.sub.xyl1-BK1-spec This work JM1161 JM1100 tRNA.sup.Ser::P.sub.xyl1-BK4-spec This work JM1162 JM1100 tRNA.sup.Ser::P.sub.xyl1-BK8-spec This work JM1163 JM1100 tRNA.sup.Ser::P.sub.xyl1-BK9-spec This work JM1164 JM1100 tRNA.sup.Ser::P.sub.xyl1-BL2-spec This work JM1165 JM1100 tRNA.sup.Ser::P.sub.xyl1-BL3-spec This work JM1166 JM1100 tRNA.sup.Ser::P.sub.xyl2-BM1-spec This work JM1167 JM1100 tRNA.sup.Ser::P.sub.xyl1-BN2-spec This work JM1168 JM1100 tRNA.sup.Ser::P.sub.xyl1-BN3-spec This work JM1169 JM1100 tRNA.sup.Ser::P.sub.xyl2-BO2-spec This work JM1170 JM1100 tRNA.sup.Ser::P.sub.xyl1-BP1-spec This work JM1171 JM1157 ΔscuR::ery This work JM1172 JM1157 ΔsarF::ery This work JM1173 JM1157 ΔscuR-sarF::ery This work JM1174 JM1157 ΔcomR::ery This work JM1175 JM1100 ΔscuR::ery This work JM1176 JM1100 ΔsarF::ery This work JM1177 JM1027 ΔscuR::ery This work JM1178 JM1027 ΔsarF::ery This work JM1179 JM1019 ΔscuR::ery This work JM1180 JM1019 ΔsarF::ery This work JM1181 HSISS4 tRNA.sup.Thr::P.sub.sptA.sup.CT.fwdarw.AC-luxAB-cat This work JM1182 HSISS4 tRNA.sup.Thr::P.sub.sptA.sup.+1-luxAB-cat This work JM1183 HSISS4 tRNA.sup.Thr::P.sub.sptA.sup.+A-luxAB-cat This work JM1184 HSISS4 tRNA.sup.Thr::P.sub.comX.sup.G.fwdarw.A-luxAB-cat This work JM1185 JM1101 ΔcomA::cat This work JM1186 JM1101 ΔsptA::cat This work JM1187 HSISS4 ΔscuR This work JM1188 HSISS4 ΔsarF This work JM1189 HSISS4 ΔsptA::cat This work JM1190 JM1019 ΔscuR-sarF::ery This work JM1191 JM1021 ΔscuR-sarF::ery This work JM1192 JM1027 ΔscuR-sarF::ery This work JM1193 JM1100 ΔscuR-sarF::ery This work Lactococcus lactis subsp. lactis IL1403 Laboratory strain (Chopin et al., 1984)

    TABLE-US-00012 TABLE 2 List of plasmids used in the Examples Characteristics Reference/source pGhostcre Thermosensitive replication origin vector in (Fontaine et al., 2010) S. salivarius, encoding the Cre recombinase; ery.sup.R pGIUD0855ery pUC18 derivative containing the erm gene (Fontaine et al., 2010) pSEUDO-P.sub.usp45-.sub.sfgfp(Bs) erm-oroP containing vector (Overkamp et al., 2013) pGILFspec pG.sup.+host9 derivative containing the spectinomycin (Haustenne et al., 2015) resistance cassette P.sub.spec-spec downstream of luxAB pJUDspecmut1 pGILFspec derivative in which a Spel restriction (Mignolet et al., 2018) site was mutated pJUDspecmut1- Terminator associated-gfp.sup.+ ORF cloned in (Mignolet et al., 2018) gfp.sup.+ter pJUDspecmut1 pNZ5319 pACYC184 derivative containing the cat gene (Lambert et al., 2007) under the control of the P32 constitutive promoter from Lactococcus lactis pJIMcat pG.sup.+host9 containing the luxAB genes of (Mignolet et al., 2018) Photorhabdus luminescens, a transcriptional terminator with a cat cassette pBAD-comR-ST pBADhisA derivative encoding ComR fused to a (Mignolet et al., 2018) C-terminal Streptagll pBAD-scuR-ST pBADhis4 derivative encoding ScuR fused to a This work C-terminal Streptagll pBAD-sarF-ST pBADhis4 derivative encoding SarF fused to a C- This work terminal Streptagll

    TABLE-US-00013 TABLE 3 List of oligonucleotides used in the Examples Names Sequences SEQ ID NO rggD_NcoI SS 5′-  88 AAAAAACCATGGCAGAAGATATTAAAA TCAAGA-3′ rggD_MunI 5′-  89 AAAAAACAATTGCTTTGACTCGTTACTT GTAT-3′ rggC_NcoI SS 5′-  90 AAAAAACCATGGCTGAAGATATTAAAA TCGAGA-3′ rggC_RI SS 5′-  91 AAAAAAGAATTCTTCTATATTTAAATCT TTTT-3′ Uplox66 5′-TAAGGAAGATAAATCCCATAAGG-3′  92 DNlox71 5′-TTCACGTTACTAAAGGGAATGTA-3′  93 lox66-ery 5′-  94 TAAGGAAGATAAATCCCATAAGGTACC TAATAATTTATCTACATTCC-3′ lox71-ery 5′-  95 TTCACGTTACTAAAGGGAATGTAAAAT GATACACCAATCAGTGC-3′ F_spec 5′-TAATAAGGCCGGCCAATAAA-3′  96 R_spec 5′-ATAGGATGAGAACTCCCATG-3′  97 UPery-oroP 5′-AAGGTTGATGTTACTGCTGATA-3′  98 DNery-oroP 5′-TGCTGACTTGCACCATATCATA-3′  99 UF_tRNAser 5′-CAAGATTAACCATGACCTTC-3′ 100 UR_tRNAser 5′-AGTAATTAAAAAGAAGATGG-3′ 101 DF_tRNAser 5′-TACCTAAAAAGTGTCCCTTC-3′ 102 DR2_tRNAser 5′-TTGGATAAGGTCTTGACTTC-3′ 103 UF_tRNAthr 5′-TGTCAAAGGATTAGGAAAAC-3′ 104 UR_tRNAthr 5′-TTGATTTATACCTCTCAATTT-3′ 105 DF_tRNAthr 5′-AAATCAACCTCTTTGAACATA-3′ 106 DR_tRNAthr 5′- 107 AAAAAAGAATTCATTCATGATGAGCGG GTTCGTGAGA-3′ F_pZX9 5′- 108 CCATCTTCTTTTTAATTACTTCTAGATT ATATATGATATGATC-3′ R_pZX9_ATG 5′-CATATTTACCTCCTTTGATTTA-3′ 109 F_luxAB_ATG 5′-ATGAAATTTGGAAACTTTTTGC-3′ 110 R_cat_tRNAthr 5′- 111 TATGTTCAAAGAGGTTGATTTCACGTT ACTAAAGGGAATGTA-3′ UFcomRJIM- 5′- 112 SS1-4 GCAGTACCACTCTATGCTAAATTTGCC AACTTTGA-3′ URcomRJIM- 5′- 113 SS1-4 CCTTATGGGATTTATCTTCCTTAGAGA CACTCCTTTATTTC-3′ DFcomRJIM- 5′- 114 SS1-4 TACATTCCCTTTAGTAACGTGAAAAAT GGTGGTGACATAAA-3′ DRcomRJIM- 5′- 115 SS1-4 TGACGTGATTTCACCAGTACGACGTGA ACTAAAGA-3′ Up_comR SS1- 5′-TTGCTTACAGTTGCTATGGT-3′ 116 4 Down_comR_ 5′-TCATCACAATGGTCACATCT-3′ 117 SS1-4 UFrggC JIM 5′- 118 AAAACTGCAAGTAGAGTCGCCGAATTA GAA-3′ URrggC_JIM 5′- 119 CCTTATGGGATTTATCTTCCTTAACATA ATTCCTTATGATTTAGA-3′ DFrggC JIM 5′- 120 TACATTCCCTTTAGTAACGTGAAGACA TTGATGTCCTTTTGA-3′ DRrggD SS 5′- 121 TAGCTTCATTCATGTCATGTGTCGTCA AAA-3′ Up_rggC JIM 5′-AAATATCGTCATTGCCAGTA-3′ 122 Down_rggD SS 5′-CTGAATAAGTTCAGCAGGTT-3′ 123 Down_rggD SS 5′- 124 TCACTTTGACTCGTTACTTGTGATTTTA ATATCTTCTGACAT-3′ rggD_S4_3 5′- 125 ATGTCAGAAGATATTAAAATCACAAGT AACGAGTCAAAGTGA-3′ rggD_S4_4 5′- 126 TATCAGCAGTAACATCAACCTTCATGT CATGTGTCGTCAAAA-3′ rggD_S4_5 5′- 127 TATGATATGGTGCAAGTCAGCATCATG AAGTCTCCTGTCTAT-3′ rggD_S4_6 5′-GGCTAGTACAGTAGCTGTAT-3′ 128 UFrggC SS 5′- 129 CTGTTTAGCCCTATCTTTGAGTTTATCA GT-3′ URrggD JIM 5′- 130 CCTTATGGGATTTATCTTCCTTATGTAT TCCCCTTGAGTTTT-3′ DFrggD JIM 5′- 131 TACATTCCCTTTAGTAACGTGAAGAAA ATATATCAGCAACAT-3′ DRrggC SS 5′- 132 CAGTGGTTTGACGTTGTTTTTGAATAC GGT-3′ Up_rggC SS 5′-AAGGTAGCCTAAACAACTCA-3′ 133 Down_rggC SS 5′-TTTATTGGTACCAAACGCCA-3′ 134 rggC_S4_2 5′- 135 CTATTCTATATTTAAATCTTTGATTTTAA TATCTTCAGACAT-3′ rggC_S4_3 5′- 136 ATGTCTGAAGATATTAAAATCAAAGATT TAAATATAGAATAG-3′ rggC_S4_4 5′- 137 TATCAGCAGTAACATCAACCTTGACGT TGTTTTTGAATACGGT-3′ rggC_S4_5 5′- 138 TATGATATGGTGCAAGTCAGCAACTAG ACATTCCTGAAGACT-3′ rggC_S4_6 5′-TCCGCTAGTAGGATAGCTTT-3′ 139 UF_PcomR_lux 5′-TAATTGAGGAGGTCTATGAG-3′ 140 AB UR_comA 5′- 141 CCTTATGGGATTTATCTTCCTTAATATG GATATTTTGACATGG-3′ DF_comA 5′- 142 TACATTCCCTTTAGTAACGTGAAGCTA ATTTCAATCCATTCCAG-3′ DR_comA 5′-ACAGTACTCTTTATTTGGTG-3′ 143 F_comR 5′- 144 CTAGAGGAGGAATTTAGATGAACATAA AAGACAGCATTG-3′ Down_PcomS_ 5′-GACAAAGTAGTCAAGACCGT-3′ 145 JIMSS1-4 UF_potA2 5′-ATACTATACCTTTCAATGTC-3′ 146 UR_potA2 5′- 147 CCTTATGGGATTTATCTTCCTTAATAAG GTTTGTCATATCTTG-3′ DF_potA2 5′- 148 TACATTCCCTTTAGTAACGTGAAGGAA AACTTAATGTTTAACC-3′ DR_potA2 5′-ACTGATCCCTGAAAGCATTG-3′ 149 Up_potA2 5′-AGAGTATACCTTAAATGACC-3′ 150 Down_potA2 5′-GATTTAAAGATTTCGTGAAC-3′ 151 F_PpotA2_ 5′- 152 tRNAthr AAATTGAGAGGTATAAATCAATCATTTT GGAAGCAAAATAC-3′ R_PpotA2_luxAB_ 5′- 153 ATG GCAAAAAGTTTCCAAATTTCATATCTTG ATTTCTCCAATTTG-3′ F_rggD 5′- 154 CTAGAGGAGGAATTTAGATGTCAGAA GATATTAAAATC-3′ R_rggD 5′- 155 TTTATTGGCCGGCCTTATTATCACTTT GACTCGTTACTTG-3′ F_rggC 5′- 156 CTAGAGGAGGAATTTAGATGTCTGAAG ATATTAAAATC-3′ R_StrepTag 5′- 157 TTTATTGGCCGGCCTTATTACTATTTCT CGAACTGCGG-3′ F_P32- 5′- 158 gfp + ter_spec CCATCTTCTTTTTAATTACTGTCCTCGG GATATGATAAG-3′ R_P32 5′-CATCTAAATTCCTCCTCTAG-3′ 159 F_cat_ATG 5′-ATGAACTTTAATAAAATTGATT-3′ 160 R_cat_(spec) 5′- 161 TTTATTGGCCGGCCTTATTATAAAAGC CAGTCATTAGGC-3′ R_PpotA2_cat_ 5′- 162 ATG AATCAATTTTATTAAAGTTCATATCTTG ATTTCTCCAATTTG-3′ Pxyl_seq 5′-TTGTTTATCCTCCTCTAGTC-3′ 163 spec2 5′-AACTCCTGATCCAAACATGTA-3′ 164 Cy3_F_PpotA2_ 5′-Cy3- 165 EMSA TAACGAGTCAAAGTGACATAGATGTCC TTTTGATTCGTTA-3′ R_PpotA2_ 5′- 166 EMSA TAACGAATCAAAAGGACATCTATGTCA CTTTGACTCGTTA-3′ Cy3_F_P01665_ 5′-Cy3- 167 EMSA CTCCATAGTGACATTTATGTCACTATTT TT-3′ R_P01665_ 5′- 168 EMSA AAAAATAGTGACATAAATGTCACTATG GAG-3′ Cy3_F_PcomS_ 5′-Cy3- 169 EMSA AATGGTGGTGACATAAATGTCACTACT TTT-3′ R_PcomS_ 5′- 170 EMSA AAAAGTAGTGACATTTATGTCACCACC ATT-3′ Cy3_F_PcomX_ 5′-Cy3- 171 EMSA TTTTATAGTGACATATATGTCGCTATTT TA-3′ R_PcomX_ 5′- 172 EMSA TAAAATAGCGACATATATGTCACTATA AAA-3′ Cy3_F_PcomX_ 5′-Cy3- 173 EMSArev TTTTATAGTGACATATATGTCACTATTT TA-3′ R_PcomX_ 5′- 174 EMSArev TAAAATAGTGACATATATGTCACTATAA AA-3′ F_PcomX_mut 5′-CATATATGTCACTATTTTATT-3′ 175 R_PcomX_mut 5′-AATAAAATAGTGACATATATG-3′ 176 F_PpotA2_mut2 5′-ACATAGATGTCACTTTGATTCGT-3′ 177 R_PpotA2_mut2 5′-ACGAATCAAAGTGACATCTATGT-3′ 178 F_PpotA2_mut + 5′-TGATTCGTTATTTTTTTTGTTT-3′ 179 1 R_PpotA2_mut 5′-AAACAAAAAAAATAACGAATCA-3′ 180 + 1 F_PpotA2_mut + 5′-CATAGATGTCACTTTTGATTC-3′ 181 A R_PpotA2_mut 5′-GAATCAAAAGTGACATCTATG-3′ 182 + A F_pept_xyl 5′- 183 TAAATCAAAGGAGGTAAATATGATCGC AATCCTANNNNNNNNNNNNNNNNNNN NNTGATAATAAGGCCGGCCAATAAA-3′

    TABLE-US-00014 TABLE 4 List of EMSA annealings, overlapping and cloning PCR subfragments amplified in this study PCR/annealing Primer 1 Primer 2 scuR amplification for pBAD-scuR-ST cloning rggD_Ncol SS rggD_Munl sarF amplification for pBAD-sarF-ST cloning rggC_Ncol SS rggC_Rl SS P.sub.32-cat cassette amplification Uplox66 DNlox71 Erm cassette amplification lox66-ery lox71-ery spec cassette amplification for tRNA.sup.Ser locus F_spec R_spec Random peptide gene and spec for tRNA.sup.Ser locus F_pept_xyl R_spec erm-oroP cassette amplification UPery-oroP DNery-oroP P.sub.xyl1 amplification F_pZX9 R_pZX9 ATG P.sub.xyl2 amplification F_pZX9 R_pZX9 ATG luxAB-cat amplification F_luxAB_ATG R_cat_tRNAthr P.sub.32 amplification F_P32-gfp + ter_spec R_P32 spec cassette amplification for tRNA.sup.Thr locus F_spec R_cat_tRNAthr Upstream homologous region of tRNA.sup.Ser locus UF_tRNAser UR_tRNAser Downstream homologous region of tRNA.sup.Ser locus DF_tRNAser DR2_tRNAser Upstream homologous region of tRNA.sup.Thr locus UF_tRNAthr UR_tRNAthr Downstream homologous region of tRNA.sup.Thr locus DF_tRNAthr DR_tRNAthr scuR amplification for P.sub.32-scuR fusion at tRNA.sup.Ser F_rggD R_rggD locus sarF-ST amplification for P.sub.32-sarF-ST fusion at F_rggC R_StrepTag tRNA.sup.Ser locus Promoter of sptA for luxAB fusion F_PpotA2_tRNAthr R_PpotA2_luxAB_ATG Promoter of sptA.sup.CT.fwdarw.AC for luxAB fusion F_PpotA2_mut2 R_PpotA2_mut2 Promoter of sptA.sup.+1 for luxAB fusion F_PpotA2_mut + 1 R_PpotA2_mut + 1 Promoter of sptA.sup.+A for luxAB fusion F_PpotA2_mut + A R_PpotA2_mut + A Promoter of comX.sup.G.fwdarw.A for luxAB fusion F_PcomX_mut R_PcomX_mut Promoter of sptA for cat fusion (screen) F_PpotA2_tRNAthr R_PpotA2_cat_ATG cat cassette amplification for P.sub.sptA fusion (screen) F_cat_ATG R_cat_(spec) Diagnostic PCR for random peptide sequencing Pxyl_seq spec2 PCR for random peptide backcross UF_tRNAser DR2_tRNAser PCR for tRNA.sup.Ser::P.sub.xyl1-comR-spec amplification UF_tRNAser DR2_tRNAser Upstream homologous region of scuR gene UFrggC JIM URrggC JIM Downstream homologous region of scuR gene DFrggC JIM DRrggD SS Upstream homologous region of scuR gene (in- UFrggC JIM rggD_S4_2 frame deletion) Downstream homologous region1 of scuR gene rggD_S4_3 rggD_S4_4 (in-frame deletion) Downstream homologous region2 of scuR gene rggD_S4_5 rggD_S4_6 (in-frame deletion) Diagnostic PCR for scuR deletion Up_rggC JIM Down_rggD SS Upstream homologous region of sarF gene UFrggC SS URrggD JIM Downstream homologous region of sarF gene DFrggD JIM DRrggC_SS Upstream homologous region of sarF gene (in- UFrggC SS rggC_S4_2 frame deletion) Downstream homologous region1 of sarF gene rggC_S4_3 rggC_S4_4 (in-frame deletion) Downstream homologous region2 of sarF gene rggC_S4_5 rggC_S4_6 (in-frame deletion) Diagnostic PCR for sarF deletion Up_rggC SS Down_rggC_SS Diagnostic PCR for scuR-sarF deletion Up_rggC JIM Down_rggC_SS Upstream homologous region of comR gene UFcomRJIM-SS1-4 URcomRJIM-SS1-4 Downstream homologous region of comR gene DFcomRJIM-SS1-4 DRcomRJIM-SS1-4 Diagnostic PCR for comR deletion Up_comR_SS1-4 Down_comR_SS1-4 Upstream homologous region of comA gene UF_PcomR_luxAB UR_comA Downstream homologous region of comA gene DF_comA DR_comA Diagnostic PCR for comA deletion F_comR Down_PcomS_JIMSS1-4 Upstream homologous region of sptA gene UF_potA2 UR_potA2 Downstream homologous region of sptA gene DF_potA2 DR_potA2 Diagnostic PCR for sptA deletion Up_potA2 Down_potA2 Promoter of sptA annealing for EMSA Cy3_F_PpotA2_EMSA R_PpotA2_EMSA Promoter of slvX annealing for EMSA Cy3_F_P01665_EMSA R_P01665_EMSA Promoter of comS annealing for EMSA Cy3_F_PcomS_EMSA R_PcomS_EMSA Promoter of comX annealing for EMSA Cy3_F_PcomX_EMSA R_PcomX_EMSA Promoter of comX.sup.G.fwdarw.A annealing for EMSA Cy3_F_PcomX_EMSArev R_PcomXE_EMSArev

    Randomized Peptide Screen

    [0193] To generate the two DNA libraries encoding randomized sequence of small peptides, we performed overlapping PCRs to graft fragments encompassing the follow features: (1) a 5′ recombination arm (for the ectopic tRNA.sup.ser locus); (2) the xylR gene that codes for the xylose responsive regulator; (3) either P.sub.xyl1 (library I) or P.sub.xyl2 (library II) translationally-fused to a 12 codon long gene, for which the last 7 are randomized; (4) the spec gene; (5) a 3′ recombination arm (for the ectopic tRNA.sup.ser locus). To obtain the randomized DNA stretch, we used a 78 nucleotide long primer degenerated on 21 contiguous positions. Next, we transformed these two libraries in strains containing the sptA promoter translationally-fused to the cat gene (chloramphenicol resistance) in which the associated spec gene was excised by the previously described cre-lox method. The initial background of these strains were either a comR-overexpressing (P.sub.xyl1-comR) or a salivaricin-deprived (Δslv5) strain. We plated transformed cells on solid medium supplemented with xylose (either 0.1 or 1%), chloramphenicol (2 mg/ml) and spectinomycin (200 mg/ml) and grew overnight. We restreaked single colonies on fresh chloramphenicol and spectinomycin solid medium supplemented or not with xylose. We finally collected clones that displayed an increased in growth on xylose vs non xylose medium (except for the clone BM1 that we used as a negative control).

    Mobility Shift Assays (EMSA)

    [0194] All double-stranded DNA fragments (30 or 40 bp) were obtained from annealing of single-stranded Cy3-labelled (at 5′ end) and unlabeled oligonucleotides. Primers used are listed in Table 3. Typically, a gel shift reaction (20 μl) was performed in a binding buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, 10% glycerol, 1 mg ml-1 BSA) and contained 150 ng labelled probe and 4 mM StrepTag proteins. When necessary, 8 mM of ComS peptides (unless otherwise stated) are added. The reaction is incubated at 37° C. for 10 min prior to loading of the samples on a native TBE 5% gel. The gel is next subjected to 80 V for approximately 1 h in TBE buffer. DNA complexes were detected by fluorescence on the Ettan DIGE Imager with bandpass excitation filters (nm): 540/25 (Cy3) or 635/30 (Cy5) and bandpass emission filters: 595/25 (Cy3) or 680/30 (Cy5) (GE Healthcare, Waukesha, Wis.).

    Bacteriocin Detection Assay

    [0195] The spot-on lawn (multilayer) detection method was performed as followed: 10 μl of overnight cultures of producer strains were diluted in fresh M17G medium and grown to reach mid-log phase (OD.sub.600=˜0.5). In parallel, we casted plates with a bottom feeding layer (M17G 1.5% agar) supplemented with a synthetic peptide where required. Next, we mixed 100 μl of an overnight culture of Lactococcus lactis IL1403 (indicator strain) in pre-warmed soft M17G medium (0.3% agar) and casted it as a top layer. Finally, we incubated mid-log phase cultures for 30 minutes with the corresponding synthetic peptides and spotted 3 μl of the producer strains on the top layer. Plates were incubated overnight before analysis of the inhibition zones surrounding the producer colonies.

    Competence Induction, Transformation Rate and Engineering of Mutants

    [0196] To induce competence, overnight CDMG precultures were diluted at a final OD.sub.600 of 0.05 in 300 μl (10 ml concerning the randomized peptide screen) of fresh CDMG and incubated 75 min at 37° C. Then, we added the pheromone sComS as well as DNA (overlapping PCRs or plasmids) and let the cells recover for 3 h at 37° C. before plating on M17G agar supplemented with antibiotics where required. Null-mutants were constructed by exchanging (double homologous recombination) the coding sequences (CDS) of target genes (sequence between start and stop codons) for either chloramphenicol or erythromycin resistance cassette. If stated, mutants were cleaned for the lox site-flanked resistance cassette, as previously described (Fontaine et al. Mol Microbiol 2010; 87:1113-1132). In case of deletion of multiple CDSs, the region between the start codon of the first CDS and the stop codon of the last CDS was deleted. Integration of the antibiotic resistance cassette at the right location was subsequently checked by PCR. The promoters of sptA genes was fused to the luxAB reporter genes and inserted with a chloramphenicol resistance cassette at the permissive tRNA threonine locus (HSISS4_r00061) by double homologous recombination. In case of ΔscuR and ΔsarF in-frame deletion, we used the two-step selection/counter-selection strategy previously described (Mignolet et al. Cell Rep 2018, 22:1627-1638). We transformed the wild-type strain with an overlapping PCR product composed of 4 fragments: (I) the upstream region of scuR or sarF genes, (II) the downstream region of scuR or sarF genes, (III) a cassette that includes the erythromycin resistance gene (erm) and a gene encoding the orotate transporter oroP, and finally (IV) the downstream region of scuR or sarF genes. We selected a first event of double recombination on medium supplemented with erythromycin. Next, we selected an intramolecular recombination between region (I) and (IV) that excises the erm-oroP cassette by growing cells on M17G supplemented with the toxic 5-fluoro-orotic acid (5-FOA) compound. In absence of oroP, 5-FOA is not able to cross the membrane and penetrate the cytoplasm where it is deleteriously incorporated in the nucleotide metabolic pathway (Overkamp et al., 2013). At final, we engineered an in-frame deletion mutant of scuR or sarF in which the first seven codons were fused to the last six codons without any cassette scar (see below for detailed cloning method).

    ComR, ScuR and SarF Purification

    [0197] The PCR-amplified scuR-StrepTag and gene sarF-StrepTag were cloned into the pBAD-comR-ST vector (see supplemental information). The ComR-StrepTag, ScuR-StrepTag and SarF-StrepTag recombinant proteins were overproduced in E. coli and purified as previously described (Fontaine et al., 2013) in standard native conditions on Strep-Tactin agarose beads (IBA).

    Measurements of Growth and Luciferase Activity

    [0198] Overnight precultures were diluted at a final OD.sub.600 of 0.05. A volume of 300 μl of culture samples was incubated in the wells of a sterile covered white microplate with a transparent bottom (Greiner, Alphen a/d Rijn, The Netherlands) for 75 min at 37° C. and then supplemented with synthetic peptides (1 μM, except if otherwise stated) or DMSO, and xylose where required. Growth (OD.sub.600) and luciferase (Lux) activity (expressed in relative light units) was monitored at 10 min intervals during 24 h in a multi-wells plate reader (Hidex Sense, Hidex, Turku, Finland) as previously described (Fontaine et al., 2013).

    Deep Sequencing (RNAseq) and Data Processing

    [0199] S. salivarius WT, ΔscuR, ΔsarF, scuR.sup.++ or ΔsarF-ST.sup.++ strains were pre-cultured overnight in CDMG at 37° C. They were resuspended in 50 ml of fresh pre-warmed CDMG to a final OD.sub.600 of 0.05 and grown for approximately 2 h 30 min (OD.sub.600=0.3) at 37° C. Cells were harvested by centrifugation (10 min; 4,050×g), the supernatant were discarded and the cell pellets were frozen with liquid nitrogen. Finally, RNA was extracted using the RiboPure bacteria kit (Ambion-Life Technologies) and the protocol provided by the manufacturer, with protocol changes to cell lysis and RNA precipitation. For lysis, cells were resuspended in RNAwiz buffer (Ambion-Life Technologies) supplemented with Zirconia beads and shaked for 40 sec (4 times) in a fastPrep homogenizer device (MP biomedicals). For RNA precipitation, a 1.25-ethanol volume (instead of 0.5) was added to partially purified RNAs. Total RNA was checked for quality on a RNA Nano chip (Agilent technologies) and concentration was measured using Ribogreen assay (Life technologies). rRNA depletion was performed on 2 μg total RNA with the Ribo-Zero rRNA removal kit for Gram-positive bacteria (Illumina) according to manufacturer's instructions. Total stranded mRNA libraries were prepped with the NEBNext Ultra Directional RNA Library Prep kit for Illumina (New England Biolabs). Library PCR was executed for 15 cycles. Quality of the libraries was evaluated with the use of a High sensitivity DNA chip (Agilent technologies) and concentrations were determined through qPCR according to Illumina protocol. Libraries were sequenced on a NextSeq 500 high-throughput run with 76 bp single reads. 2.3 pM of the library was loaded on the flowcell with a Phix spike-in of 5%. Sequenced mRNAs generated several million reads that were mapped on the WT S. salivarius chromosome and processed with both bowties V0.12.9 (http://bowtie-bio.sourceforge.net/bowtie2) and samtools V0.1.18 (http://samtools.sourceforge.net/) algorithms to yield BAM files containing the read coordinates. We imported these files into SeqMonk V0.23.0 (www.bioinformatics.babraham.ac.uk/projects/) to assess the total number of reads for each coding sequence (CDS). The dataset was exported into an excel file for further analyses. First, the dataset was standardized to CDS-mapped reads per million overall reads. Then, we estimated a ratio of CDS-mapped reads in mutants vs WT. All RNAseq data was deposited in the GEO database under accession number GSE120640.

    Plasmid and Linear DNA Fragment Constructions

    [0200] All DNA fragments were amplified by PCR using the Phusion high fidelity polymerase (www.thermoscientificbio.com/) following a protocol as recommended by the manufacturer. Overlapping PCR products were transferred in competence-induced HSISS4 derivatives (Mignolet at al. Genome Announc 2016, 4:e01637-01615). cat, erm, spec, erm-oroP, P.sub.xyl1, P.sub.xyl2, and luxAB-cat cassettes were amplified from pNZ5319, pGIUD0855ery, pJUDspecmut1-gfp.sup.+ter, pSEUDO-P.sub.usp45-sfgfp(Bs), pZX9, pZX10 and pJIMcat, respectively. comX and sptA mutated promoter were amplified from the WT comX and sptA luxAB-fused promoter strain, respectively. The sarF-ST allele was amplified from pBAD-sarF-ST. The full P.sub.xyl1-comR-spec at tRNAs.sup.Ser locus was amplified in one block from the strain tRNA.sup.Ser::P.sub.xyl1-comR-spec (Mignolet et al. Cell Rep 2018, 22:1627-1638). All the constructed plasmids were sequence-verified.

    [0201] pBAD-scuR-ST. The scuR-coding sequence was PCR amplified using the rggD_NcoI SS and rggD_MunI primers. This scuR fragment was digested with NcoI/MunI and cloned into NcoI/EcoRI-digested pBAD-comR-ST (Mignolet et al. Cell Rep 2018, 22:1627-1638).

    [0202] pBAD-sarF-ST. The sarF-coding sequence was PCR amplified using the rggC_NcoI SS and rggC_RI SS primers. This sarF fragment was digested with NcoI/EcoRI and cloned into NcoI/EcoRI-digested pBAD-comR-ST (Mignolet et al. Cell Rep 2018, 22:1627-1638).

    Example 1: Regulon Interweaving in ComR Paralogs

    [0203] The bacteriocin short-circuitry imposed by ComR in the S. salivarius species is startling and suggests a positive selection for species-specific strategies that participate in niche adaptation. Interestingly, the S. salivarius HSISS4 genome encodes five RRNPP transcriptional factors, including ComR. Besides it, the two regulators ScuR (HSISS4_01166; stands for salivaricins-competence uncoupling regulator) and SarF (HSISS4_01169; ScuR-associated Rgg factor) share a high level of similarity with ComR (64 and 63%, respectively). The residues involved in HTH sequestration and homodimerization of ComR are well-conserved in both ScuR and SarF, suggesting that they could display a similar mode of activation. Moreover, the paralogous ScuR and SarF proteins are highly identical (similarity of 91%). Strikingly, residue divergences are nearly all concentrated in only 3 amino-acids stretches, one of which overlaps the α-helices 13 and 14 that form a part of the peptide recognition pocket. This indicates that the two proteins are likely to homodimerize and could accommodate specific peptides. On the chromosome, the scuR and sarF genes are located in a unique locus and separated by two genes that code for two predicted subunits of an ABC transporter, SptA and SptB (for ScuR-promoted transporter A and B, respectively) (FIG. 1A). In contrast to characterized rgg/comR loci, no small coding sequence was distinguishable upstream or downstream of scuR and sarF genes, arguing for a different genomic coding topology of the communication system.

    [0204] Due to the huge conservation between ComR, ScuR and SarF, especially in the DNA binding domain, we questioned whether the two uncharacterized paralogs are capable to control competence and predation as well. Hence, we extracted mRNA of wild-type (WT) and engineered in-frame deletion mutants (ΔscuR and ΔsarF) and carried out a deep sequencing (RNAseq). With no hint about the genuine activating pheromones, we included in our high-throughput transcriptomic analyses overexpression mutants (scuR.sup.++ and sarF-ST.sup.++), in order to exacerbate the regulation phenotype. Indeed, a strong overproduction of ComR was reported to be sufficient for activation of its target promoters, even in absence of ComS (from endogenous production or synthetic peptide addition). Both deletion mutants did not dramatically affect the transcriptome compared to the WT strain, meaning that the function of ScuR and SarF is barely noticeable during standard growth conditions. However, the SarF loss slightly increased scuR expression, while the differential sptA and sptB mRNA level almost reached the arbitrary 5-fold induction cut-off, suggesting that SarF could be a repressor/antagonist of the ScuR-SptAB system. In contrast, the strong overexpression of scuR (28-fold) elicited a tremendous activation of the sptA-sptB operon (about 2000-fold). Furthermore, a second cluster of genes, all located inside salivaricin loci, was robustly influenced, even if with a lower magnitude of activation (ranging from 35- to 140-fold). Surprisingly, comX mRNA levels remained approximately stable in all mutants.

    [0205] Altogether, these results imply that the ComR, ScuR and SarF paralogs might shape overlapping but dedicated regulatory networks.

    Example 2: ScuR is an Alternative Self-Sufficient Pathway That Controls Salivaricin Production but Maintains the Competence Off

    [0206] In order to validate our transcript profile analyses, we performed promoter-probe assays, as previously described for ComR. We first expanded our collection of luciferase reporter strains (composed of comS, comX and bacteriocin gene promoters) to include and monitor the sptA promoter (P.sub.sptA) and, next transformed all of them with the scuR overexpression cassette. Finally, we measured promoter activity in presence or absence of sComS (synthetic peptide) during cell growth (FIG. 1B). In agreement with our RNA-seq data, sptA and bacteriocin promoters were all markedly up in the scuR.sup.++ strain, irrespective of the addition of sComS, and with no significant synergy. In addition, we observed no activation of the P.sub.comX due to ScuR accumulation, disabling ScuR as a trigger of competence state. Nonetheless, the P.sub.comS showed a 35-fold change in activity, suggesting that ScuR might modulate the ComR cell signaling. To complement our understanding of this bipartite system, we assessed the activity of P.sub.sptA, P.sub.comS and P.sub.slvX in a sarF-ST.sup.++ strain, and noticed that scuR and sarF overexpression governs P.sub.sptA activation amplitude in a similar range, while P.sub.comS and P.sub.slvX are irresponsive to SarF (FIG. 1C).

    [0207] Considering that the effects of transcriptional regulators could be indirect, we were prompted to inactivate one Rgg by gene deletion in our reporter strains and test the residual activity of the others. We discovered that ScuR still controls both P.sub.sptA and P.sub.comS in comR (FIG. 1D) or sarF deleted strains (FIG. 1E), while SarF is sufficient to activate P.sub.sptA even in absence of scuR (ΔscuR-sarF/sarF-ST.sup.++ mutant) (FIG. 1E). Finally, the sComS-mediated regulation of ComR is not crippled in absence of both ScuR and SarF (FIG. 1F), suggesting that each regulator can stand alone to fulfill its function and work in parallel.

    [0208] Taken together, our results suggest that the 3 transcriptional factors have only partly redundant functions, with regulatory network specificities, presumably to ensure a broader diversity of cellular response to environment stresses. ScuR and SarF, but not ComR, control the sptAB operon, while ScuR alone has a ComR-independent extra regulatory role on bacteriocin production. Even though ScuR raises ComS production, this regulator does not act on comX promoter and is likely to disconnect the competence-predation coupling compelled by ComR.

    Example 3: Randomization-Based Screen for Pheromone Identification

    [0209] Typically, the major challenge to address the transduction mechanism of cell-cell communication sensor is the identification of the ligand(s) or the perceived signal(s). As inspection of the genomic neighborhood did not reveal any small encoded peptide in the vicinity of scuR-sarF locus, we decided to conduct an empirical screen to unearth peptides able to activate the ScuR-SarF system (FIG. 2A). Thence, we constructed on one side a strain harboring a translational fusion of the ScuR/SarF-specific P.sub.sptA to a gene conferring resistance to chloramphenicol (cat). On the other side, we amplified a DNA fragment that allows recombination at a permissive locus (tRNA.sup.Ser) and encompasses, under xylose control, a 12 codons-long nucleotide sequence, the last 7 of which are randomized (see Experimental Procedures). We finally transformed this PCR product in the above-mentioned reporter mutant and selected clones on plates supplemented with chloramphenicol and xylose (0.1 or 1%). Note that, in order to increase the transformation rate or decrease the cytotoxicity due to concomitant bacteriocin production, we worked in comR overexpression (P.sub.syl1-comR) or salivaricin deprived (Δslv5) background, respectively. In total, over 9 runs of screen, we collected a hundred of clones that we streaked again on selective medium with and without xylose. We sent for sequencing clones that showed a clear improvement of growth due to xylose on chloramphenicol (Table 5). As a negative control, we included in our further analyses a clone (BM1) with a xylose-independent growth. Out of the 30 positive clones, 22 harbored a non-redundant peptide/nucleotide sequence.

    TABLE-US-00015 TABLE 5 Random Back- peptide Peptide SEQ SEQ Fusion ground promoter [xylose] Name sequence ID NO DNA sequence ID NO P.sub.sptA-cat (lox) P.sub.xyl1-COMR P.sub.xyl2 1% BH6 MIAILAFWLILG 26 ATGATCGCAATCCT 27 AGCGTTCTGGCTG ATCCTAGGTTGA P.sub.sptA-cat (lox) P.sub.xyl1-COMR P.sub.xyl1 0.1% B11 MIAILTWWLILG 28 ATGATCGCAATCCT 29 AACTTGGTGGTTAA TTCTGGGATGA P.sub.sptA-cat (lox) P.sub.xyl1-COMR P.sub.xyl1 0.1% B12 MIAILPYWLGLG 30 ATGATCGCAATCCT 31 ACCATACTGGTTAG GCCTAGGCTGA P.sub.sptA-cat (lox) P.sub.xyl1-COMR P.sub.xyl1 0.1% B15 MIAILPWWVSVG 32 ATGATCGCAATCCT 33 ACCTTGGTGGGTAT CAGTTGGTTGA P.sub.sptA-cat (lox) P.sub.xyl1-COMR P.sub.xyl1 0.1% B16 MIAILPYWLGLG 34 ATGATCGCAATCCT 35 ACCATACTGGTTAG GCCTAGGCTGA P.sub.sptA-cat (lox) P.sub.xyl1-COMR P.sub.xyl1 0.1% B17 MIAILPFWLILG 36 ATGATCGCAATCCT 37 ACCCTTTTGGCTCA TATTAGGCTGA P.sub.sptA-cat (lox) P.sub.xyl1-COMR P.sub.xyl1 0.1% B19 MIAILPFWLILG 38 ATGATCGCAATCCT 39 ACCCTTTTGGCTCA TATTAGGCTGA P.sub.sptA-cat (lox) P.sub.xyl1-COMR P.sub.xyl1 0.1% B110 MIAILPYWLLIG 40 ATGATCGCAATCCT 41 ACCATACTGGCTTC TCATAGGTTGA P.sub.sptA-cat (lox) P.sub.xyl1-COMR P.sub.xyl1 0.1% B111 MIAILPFWLVLG 42 ATGATCGCAATCCT 43 ACCTTTTTGGCTAG TCCTCGGATGA P.sub.sptA-cat (lox) P.sub.xyl1-COMR P.sub.xyl1 0.1% B112 MIAILPFWVVAG 44 ATGATCGCAATCCT 45 ACCGTTCTGGGTT GTCGCGGGCTGA P.sub.sptA-cat (lox) P.sub.xyl1-COMR P.sub.xyl2 0.1% BJ1 MIAILPFWLSVG 46 ATGATCGCAATCCT 47 ACCATTTTGGCTAA GCGTAGGCTGA P.sub.sptA-cat (lox) P.sub.xyl1-COMR P.sub.xyl1 1% BK1 MIAILPYWLDMG 48 ATGATCGCAATCCT 49 ACCATATTGGCTTG ATATGGGATGA P.sub.sptA-cat (lox) P.sub.xyl1-COMR P.sub.xyl1 1% BK2 MIAILPYWLDMG 50 ATGATCGCAATCCT 51 ACCATATTGGCTTG ATATGGGATGA P.sub.sptA-cat (lox) P.sub.xyl1-COMR P.sub.xyl1 1% BK3 MIAILWWGTMI 52 ATGATCGCAATCCT 53 ATGGTGGGGCACT ATGATCTAGTGA P.sub.sptA-cat (lox) P.sub.xyl1-COMR P.sub.xyl1 1% BK4 MIAILYWATTGL 54 ATGATCGCAATCCT 55 ATATTGGGCCACG ACTGGGTTATGA P.sub.sptA-cat (lox) P.sub.xyl1-COMR P.sub.xyl1 1% BKS MIAILPYWLCII 56 ATGATCGCAATCCT 57 ACCCTACTGGCTCT GCATTATATGA P.sub.sptA-cat (lox) P.sub.xyl1-COMR P.sub.xyl1 1% BK6 MIAILPYWVTMG 58 ATGATCGCAATCCT 59 ACCATATTGGGTCA CCATGGGTTGA P.sub.sptA-cat (lox) P.sub.xyl1-COMR P.sub.xyl1 1% BK7 MIAILPYWVVLG 60 ATGATCGCAATCCT 61 ACCTTACTGGGTAG TGCTAGGGTGA P.sub.sptA-cat (lox) P.sub.xyl1-COMR P.sub.xyl1 1% BK8 MIAILPYWVTMG 62 ATGATCGCAATCCT 63 ACCATATTGGGTCA CCATGGGTTGA P.sub.sptA-cat (lox) P.sub.xyl1-COMR P.sub.xyl1 1% BK9 MIAILPSWLVVG 64 ATGATCGCAATCCT 65 ACCAAGCTGGTTA GTTGTTGGCTGA P.sub.sptA-cat (lox) Δslv5 P.sub.xyl1 0.1% BL1 MIAILPHWITIG 66 ATGATCGCAATCCT 67 ACCACACTGGATCA CAATAGGCTGA P.sub.sptA-cat (lox) Δslv5 P.sub.xyl1 0.1% BL2 MIAILPYWLGLG 68 ATGATCGCAATCCT 69 ACCATACTGGTTAG GCCTAGGCTGA P.sub.sptA-cat (lox) Δslv5 P.sub.xyl1 0.1% BL3 MIAILPHWCVLG 70 ATGATCGCAATCCT 71 ACCACATTGGTGC GTGCTTGGCTGA P.sub.sptA-cat (lox) Δslv5 P.sub.xyl2 0.1% BM1 MIAILRPH 72 ATGATCGCAATCCT 73 ACGTCCACATTAAT AGTTCCACTGA P.sub.sptA-cat (lox) Δslv5 P.sub.xyl1 1% BN1 MIATLPFWLALG 74 ATGATCGCAACCCT 75 ACCTTTTTGGCTTG CCCTCGGATGA P.sub.sptA-cat (lox) Δslv5 P.sub.xyl1 1% BN2 MIAILTCWVCIV 76 ATGATCGCAATCCT 77 AACATGTTGGGTCT GCATAGTATGA P.sub.sptA-cat (lox) Δslv5 P.sub.xyl1 1% BN3 MIAILFWVELG 78 ATGATCGCAATCCT 79 ATTCTGGGTGGAAT TAGGATAATGA P.sub.sptA-cat (lox) Δslv5 P.sub.xyl1 1% BN4 MIAILTCWVCIV 80 ATGATCGCAATCCT 81 AACATGTTGGGTCT GCATAGTATGA P.sub.sptA-cat (lox) P.sub.xyl1-COMR P.sub.xyl2 0.1% B01 MIAILPFWCVLG 82 ATGATCGCAATCCT 83 ACCCTTCTGGTGTG TCCTTGGATGA P.sub.sptA-cat (lox) P.sub.xyl1-COMR P.sub.xyl2 0.1% B02 MIAILPFWCVLG 84 ATGATCGCAATCCT 85 ACCCTTCTGGTGTG TCCTTGGATGA P.sub.sptA-cat (lox) P.sub.xyl1-COMR P.sub.xyl1 1% BP1 MIAILPFWLVLG 86 ATGATCGCAATCCT 87 ACCTTTTTGGCTAG TCCTCGGATGA

    [0210] In order to discard clones with secondary mutations for which the survival phenotype was not related to the peptide nature, we amplified for each clone the full locus that encodes the small peptide and backcrossed it into WT or (Δslv5) backgrounds. We then confirmed on solid media that chloramphenicol resistance qualitatively increased upon xylose addition. We used the same PCR products to transform a strain bearing the P.sub.sptA-luxAB report fusion and quantitatively estimate the influence of peptide production. Again, we noticed that xylose addition potentiated the promoter activity with values ranging from 5 to 100 fold, while it has no effect on BM1 (negative control) and WT strains (FIG. 2B).

    [0211] We aligned the 22 unique peptide sequences to elicit common chemical properties (FIG. 2C). Strikingly, a tryptophan residue is highly conserved at position −5 from the C-terminus. On the top of this, the adjoined position (−6) is mainly occupied with an aromatic residue. Finally, the position −1 is preferentially a glycine. Positions −2, −3 and −4 are more erratic, even if we observed a tendency for hydrophobic amino acids. Given that it encodes a peptide (MIAILPFWLILG) that neatly mimics the consensus sequence (MIAILPFWLVLG), we decided to hereafter focus on clone BI7, and we disclosed that ScuR is specifically responsible for the xylose-driven phenotype. Indeed, neither comR nor sarF deletion has a dramatic effect, while ScuR loss annihilates both xylose induction and basal leaky expression (FIG. 2D).

    Example 4: Exogenous Pheromones Activate the ScuR-SarF Tandem

    [0212] We next checked whether we could, akin to ComS toward ComR, activate the system with synthetic peptides (FIG. 3A). We therefore selected a representative panel of peptides from our screen and ordered the synthesis of the last 8 amino acids and tested P.sub.sptA activation (FIG. 3B). Whatever their degree of kinship toward the consensus motif, the peptides were capable of inducing light production when supplemented to the medium. However, a weaker activation was displayed by the peptides that diverge the most from the paradigm such as sBK3, which does not harbor a C-terminus glycine, or sBK4, for which the tryptophan and glycine are shifted of one position (exacerbated effect at the non-saturating concentration of 0.01 μM). The huge variability in sequence and the similar amplitude of activation for all other peptides emphasizes that (1) the residues between the conserved tryptophan and glycine and the proline (position −7) are not essential for ScuR or SarF transactivation, while the position −6 tolerates substitutions as far as the peptide nature is aromatic. Moreover, rational mutations of the ultra-conserved tryptophan evidenced the strict requirement of the indole moiety, considering that neither alanine (sBI7.sup.W.fwdarw.A) nor phenylalanine (sBI7.sup.W.fwdarw.F) variants sustains luciferase transcription at low peptide concentration (0.001 μM, FIG. 3C). A similar strategy for the C-terminal glycine showed that its substitution by an alanine (sBI7.sup.G.fwdarw.A) decreases the P.sub.sptA response although to a lesser extent compare to the tryptophan (FIG. 3C). It is noteworthy that high concentration of sBI7.sup.W.fwdarw.F and sBI7.sup.G.fwdarw.A (but not sBI7.sup.W.fwdarw.A) can bypass the requirement of the tryptophan and glycine and activate P.sub.cat at a similar range than the WT sBI7 peptide (FIG. 3E), suggesting that the mutations do not totally abrogate the ScuR/SarF activation but rather modulate the kinetics of interaction.

    Example 5: Selective Recognition of Targeted Promoters

    [0213] To refine our view of the 2 Rgg-systems, P.sub.sptA, P.sub.comS and P.sub.slvX were challenged with increasing amount of sBI7 at low concentration in WT, ΔscuR or ΔsarF strains (FIG. 3D). In line with our overexpression data, P.sub.comS and P.sub.slvX were totally insensitive to SarF (no activity in ΔscuR), while both ScuR and SarF can turn on P.sub.sptA independently of each other. Furthermore, in WT backgrounds, we observed that all promoters were responsive to less than 1 nM of peptide, with the highest amplitude for P.sub.sptA and the lowest for P.sub.comS. Whereas activity of all promoters in ΔsarF were slightly up compared to the WT, supporting the notion that SarF might have a mild inhibitory effect on ScuR function. Expectedly, the induction provoked by sBI7 addition is utterly erased in a ΔscuR-SarF double mutant (FIG. 3F), however sBI7 is surprisingly able to induce the SarF-mediated P.sub.sptA response (FIG. 3D). This discrepancy in regard to the genome-encoded peptide results (FIG. 2D) might be due to inherent differences imposed by the screening method compared to the exogenous supplementation of a synthetic peptide (variable intracellular concentration, different peptide length, lower activation rate of SarF, . . . ). But it underlines that the peptide-binding pocket of both Rgg could accommodate a unique pheromone.

    [0214] Next, we carried out in vitro mobility shift assays to assess the direct interaction between proteins and promoter probes in absence or presence of decreasing concentration of synthetic peptides. We included a P.sub.comX probe as a negative control. Anew, we corroborated our promoter activity data at the magnitude and protein-peptide/DNA specificity level (FIG. 4A). The ScuR regulator displayed the weakest affinity for P.sub.comS, and the strongest one toward P.sub.sptA, starting from 0.08 nM of sBI7 peptide and complexing the full amount of probe at maximal concentrations (presence of a doublet presumably due to a second higher order of oligomerization) (FIG. 4A). We observed similar results with unique concentration of sBI7 and decreasing concentration of ScuR on P.sub.sptA and P.sub.slvX probe (FIG. 4G). Moreover, in comparison to the sComS-bound ComR, the ScuR.Math.sBI7 affinity for P.sub.comS and P.sub.slvX appears weaker with a less stable complex (probe smear) (FIG. 4A). Remarkably, the ScuR and SarF binding progressively and linearly increased with the amount of peptide, contrasting with ComR that showed a smaller interval between sub-activating and saturating concentrations of sComS. This suggests ScuR and SarF have a different dynamic of binding compared to ComR that might reflect the congruence between reactivity and physiological function. Finally, the ComR.Math.sComS pair can unexpectedly bind the P.sub.sptA probe, indicating that the sptA promoter topology, and presumably the distance between the palindrome center and the −10 box, might be crucial to dictate the specific ScuR/SarF-driven transactivation (FIG. 4A).

    [0215] The topology of ScuR, SarF or ComR responsive promoters appears extremely similar (FIGS. 4B and 4H). The architecture of every promoter includes a conserved nucleotide stretch of dyad symmetry and a T-rich spacer that separates it from the sigma-bound −10 box (exact same length in all promoters, P.sub.sptA apart). However, the core palindromic region of the ComR-specific P.sub.comX includes a mismatch, while the equivalent stretch in the ScuR- and SarF-specific P.sub.sptA is more extended, comprises 2 mismatches and is closer to the −10 box from one nucleotide (FIG. 4B). Considering the high degree of similitude between promoters at the primary sequence level (FIGS. 4B and 4H), we were prompted to address the nucleotide determinants that impose the protein-DNA selectivity. We therefore mutated comX and sptA promoters to sensitize them toward ScuR and ComR, respectively. In P.sub.comX, we substituted a guanosine for an adenosine (P.sub.comX.sup.G.fwdarw.A) to reconstitute the palindromic region observed in P.sub.comS and P.sub.slvX (FIG. 4B). Concerning P.sub.sptA, we performed three kinds of mutation (FIG. 4B). We reconstituted the symmetric region with 2 substitutions (P.sub.sptA.sup.CT.fwdarw.AC), we inserted a nucleoside in the T-rich stretch to redefine the same distance between palindrome center and −10 box (P.sub.sptA.sup.+1), and finally, with the mere insertion of an adenosine in the palindrome (P.sub.spTA.sup.+A), we redesigned both space and palindrome. Non-ambiguously, all these mutations impinged on selectivity at different extent, driving the engineered promoters sensitive to both pheromones (FIGS. 4C and 4D). In agreement with this, ScuR, but not SarF, is able to shift the P.sub.comX.sup.G.fwdarw.A probe (FIG. 4E). As competence entry hinders cell fitness whereas the mutation in comX promoter bolstered the ComRS-mediated activation (FIG. 4D), we suspect that evolution maintains a selective pressure to ensure an appropriate expression in time and scale of comX compatible with the bacterium life cycle.

    Example 6: Pheromone-Induced ScuR Promotes Bacteriocin Production

    [0216] The lower reactivity of salivaricin promoters toward ScuR (vs ComR) pheromone incited us to investigate the phenotypical output at the bacteriocin production level. Thence, we performed a standard bacteriocin test on soft overlay and showed that the scuR.sup.++ (but not sarF-ST.sup.++) overexpression mutant is able to produce a comA-dependent inhibitory halo in bacteriocin tests (FIG. 5A). Even if the overexpression of scuR is more potent, sBI7 induced a small halo of inhibition around the WT strain for concentration ranging from 1 nM to 1 μM (FIG. 5B). This effect is eradicated in single ΔscuR or double ΔscuR-sarF mutants, or in a strain deprived of bacteriocin genes (Δslv5), demonstrating that the toxicity is due to bacteriocins and mediated by ScuR (FIG. 5B).

    Example 7: Rgg Members Requisition Predation Control in S. salivarius

    [0217] ScuR tends to compensate for the loss/lack of a functional BIpRH system, as they usually do not co-exist in the same strain (FIG. 6A). Oddly, the direct control of competence and bacteriocin network drifts from a full TCS control in Streptococcus pneumoniae (ComCDE and BIpRHC) to a full Rgg regulation in S. salivarius (ComRS and ScuR), going through an hybrid mechanism in mutans, bovis and pyogenes streptococci groups (ComRS and BIpRHC) (FIG. 6B). Why opposing extracellular sensing vs intracellular sensing inside streptococci is a vast question. As a genuine member of the gastro-intestinal tract, S. salivarius is under a grueling selective pressure, competing for resources and territories in a constantly changing environment. The most tantalizing hypothesis is that a cytoplasmic receptor could be better protected from communication interferences. Indeed, quenching molecules extruded by competitors face the chemical selectivity of the semi-permeable cell membrane to penetrate the cell. A second hypothesis is that internal cues, such as starvation/lushness, might impinge on the nutritional oligopermease system (Opp) to globally modulate small peptide inward fluxes and make the cell transiently communication-less or superreactive to pheromones in particular stressful situations.

    [0218] All our phenotypical and molecular data coincide to conclude that ScuR is strictly devoted for predation in contrast to competence. Although highly similar to P.sub.comS and P.sub.slvX at primary sequence level, comX promoter cannot be occupied (FIG. 4A) nor activated (FIGS. 1B and 4D) by ScuR. Inconsistently, this regulator turns on P.sub.comS and should somehow modulate the ComRS activity through a flicking impetus in the positive feedback loop. An obvious reason for this discrepancy might be that the ScuR-driven expression of comS (FIGS. 1B and 3D) is weaker compared to our previous observations for ComR. Therefore it is not sufficient to reach the activation threshold, but it might ensure a basal production of ComA to extrude bacteriocins. Alternatively, we suspect that promoters are not responsive to ComR and ScuR pheromone with the same timeframe. In this case, ScuR might promote ComS production in a time interval during which the comX promoter is silenced or unreactive.

    Example 8: Induction of Bacteriocin Promoters, No Induction of Competence Genes

    [0219] Confirming the importance of the ultra-conserved tryptophane residue (see FIG. 3C), we observed similar trends for all bacteriocin gene promoters (FIG. 7), with a total inefficacy of the peptide sBI7.sup.W.fwdarw.A, and a low to high activation disability for peptides sBI7.sup.W.fwdarw.F and sBI7.sup.G.fwdarw.A.

    [0220] It was also confirmed that ScuR and SarF have no effect on the sComS-mediated competence entry (FIG. 8). Further confirming this finding, sBI7 cannot activate comX and late competence genes under direct ComX control (FIG. 9), and is ineffective to support natural transformation (Table 6).

    TABLE-US-00016 TABLE 6 Competence development (transformation frequency.sup.a) in S. salivarius HSISS4 derivatives Strains — sComS sBI7 Wild-type ND  1.1 (±0.08) E−03 ND ΔscuR ND 0.7 (±0.2) E−03 ND ΔsarF ND 1.5 (±0.5) E−03 ND P.sub.32-scuR ND NA NA P.sub.32-sarF ND NA NA .sup.acalculated as the ratio of transformants (chloramphenicol-resistant CFU) to the total CFU count per 0.1 ug of linear DNA. Transformation frequencies are expressed as the arithemtic mean of three independent experiments. Geometric means ± standard deviations are provided. ND: not detected (<1.0E−08), NA: not applicable.

    Example 9: C-Terminal Residues and Bacteriocin Induction Activity

    [0221] To evaluate whether the synthetic pheromones could tolerate amino acid addition at the C-terminus, we tested the close-to-consensus petide LAFWDSLG (SEQ ID NO: 749) as well as the peptide LAFWDSLGLLL (SEQ ID NO: 750) that harbors a triple leucine extension at the C-terminus. Strikingly, both peptides were able to induce the sptA promoter in vivo. However, the longer peptide was much more active at 1 μM concentration than the shorter one, with an induction magnitude comparable to the peptide sBI7 (FIG. 10A). By contrast, mobility shift assay performed with the very same peptides showed that ScuR binding to P.sub.sptA is more intense with LAFWDSLG (SEQ ID NO: 749), while it requires extreme concentration of the peptide LAFWDSLGLLL (SEQ ID NO: 750) to get activated (FIG. 10B). This suggests that LAFWDSLGLLL (SEQ ID NO: 750) can barely bind ScuR by itself but is processed in vivo, by one or more peptidases, to liberate an active peptide, presumably LAFWDSLG (SEQ ID NO: 749). It is thus clear that the peptides can tolerate amino acids at the C-terminus and still be active in vivo.

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