Identification of sortase gene

A nucleic acid sequence encoding a substantially purified sortase-transamidase enzyme from a Gram-positive bacterium, the enzyme having a molecular weight of about 29,076 daltons and catalyzing a reaction that covalently cross-links the carboxyl terminus of a protein having a sorting signal to the peptidoglycan of a Gram-positive bacterium, the sorting signal having:

• (1) a motif of NPQ/KTN/G therein;
• (2) a substantially hydrophobic domain of at least 31 amino acids carboxyl to the motif;
• and (3) a charged tail region with at least two positively charged residues carboxyl to the substantially hydrophobic domain, at least one of the two positively charged residues being arginine, the two positively charged residues being located at residues 31-33 from the motif, wherein the nucleic acid sequence includes therein a sequence selected from the group consisting of:
• 10 (1) AAAAACCCTTGTGGTGTCACTGTACCTGATAAAGATTCAGCAACTTTCATGTTTATT (SEQ ID NO:
• 40) TCAAAAACTTCTTGCGCGTATGCGATAATTTGCTGATCTAATCTTGCCGGTTCAATT GCAAATAATTGTGTAATTACAATTCCACTTTGATAAGCTTCTTCAATTAAATGCACAC CTTCAATTAAAGCTAATCCAGTTTTATCCCTCTCACGTTTCTTTTTTAGCTTGTTCGC TTGTTTAATTCTATTATTTTGTGCAGAAGTAATTTGTTCCATTGATAGCTCCTCGCTT TATTTTTAAAAATAAAAATATTAATCATTAATAAGATGAAAACATTTGATTGTATAGTT AATATTAATTAATCGCTTTTATCACTCATAATATTTCAAATTGTATAAATTTCTTTTAT CGATACTACTACTATAAATCATACGCCCCAAAATATCATTATTAATTCTTTTCTTCTT CAAAATAAATCAAAATGATATAATTGATGATTATTTTCAAAGCACATTCAAATCAAAC TATGTTTTAGCAATTTGTTGTTAGCATGTTTGTGTTCATTAAAAAAACGACCATCATC GGTATCATGTATGGTCGTTACAAAAGCTAACAATACCAATTGTCATAACAAGTACTG CAACCTCTTTAAATTCAATTATTTCATGTAACTATAGCCTATATCATATGTAATTACTT TGTTATTTATAATCGGGCTACTTTCATCTTCATTTTTACTTCTAACATGTTTATGCGC TGCTTTAAAGACATCAGATTTTAACCAATCCGTAAAAGCTTGCTTTGATTTCCAAAC TGTTAAAATTTTCACTTCATCAAAATCTTCTTGTTCTAAAGTTTGTGTAACAAACATG CCATCAAAGCCTTCTAATGTTTCAATCCCATGTCTCGTGTAAAATCGTTCTATAATAT CTTTTGCTGTTCCTTTTGTTAACGTCAGCCTATTTTCTGCCATAAATTTCATAATTAT CCTCTTTTCTGTTTAACTTACCTTAATTATTTTTGCGACAACAACAATTCTTTTCGTC GTTTCACTATATGCATCTTCGCACGTTGATAAAGTCATTATTCTATCTTTTACCGTTA CATTAACATCTGAATTAATTACAGATTTACGTTTTGTCTCATCTAAAAATTGTTGATA ATCTTGATCATTTTCAAAATCTGTACGTATGTAATTATCTTTAGTAGTAGTTTTATATG CACTAAATACTTGCAATTGATATTTACCATATTTATTGTCAAATTCAATTATCTTGTGT TTTTCATAAAACGATTGGTTTAAATAATCTTCTAACACATCAAACATCGTATTATCAC CGACATGGTGCCCGTATAAAATAGTATTATGATTTAAATTCTTCAATTCATTTCTAAA ATCCATAAAAATACTACCTTTACGTCGATGTTCTCGCTCAAAATCTAAATTTAAATAA TCGTGATTTGTCTTACCTTGTAGTACTGGATAATTTAATGATGTTCCTGATAATTTTA TCCATCCAACAATGTCTTTATTTATTTTTTCAAGTGATTCAAATTGTGGTCTCACATG TTCTTGATGTTTGCTCATCAGCATTTGAAATTTTTGTTGTAATTTCTCATAATTTGGG CGTTCTTGCTTGTCTTCAATATATGTTTGAACAATTTTGTAACCAAAAATGATAATAA TTACAACCAATAAAATTTGTACAATAGTTAAAAATCGCTTCATTCTCATAAAAATCCT CTTTTATTAACGACGTTTCTTCAGTCATCACTAAACCAGTTGTTGTACCGTTTTAGAT TCGATTTCGTTGACTTTGACAAATTAAGTAAATTAGCATTGGACCACCGACAATCAT TAAAATAGCATTGGGTGGAATTTCTAAAGGAGGCTGTATCACTCGTCCTAATAAATC AGCCACTAACAATAGCCATGCACCAATAACTGTAGAAAACGGAATAAGTACTCTGT AATTGCCCCCAACTAGCTTTCTAACCACATGTGGCACAATAATACCTAAAAAGGCTA GTTGTCCAACAATCGCAACAGTTGCACTTGCTAAAAATACTGCTAATAAACCTGTTA ACCATCTGTAACGATCAATATTAAAACCGATACTTCGCGCTTGTATGTCGTCTAAAT TTAGTAAATTCAATTTAGGGGACAATAGTAATGTTAATATTAATCCCAATAATGCTGA TACTGCTAATATGTATACGTGGCTCCATATTTTCATTGTTAAGCCTTGAGGAATTTTC ATTAAAGGGTTTTGAGTTAAAATTTCTAAAACACCATTTAATAATACGAATAACGCAA CACCTACTAATATCATACTTACAGCATTGAATCTAAATTTAGAATGCAACAATATAAT TATTAAAAATGGTATTAAACCTCCAATAAAACTTAATAATGGTAAGTAAAAGTACAAT TGTGGAATAAACAACATACAAAGTGCTCTCATTATAAGTGCACCTGAGGAAACGCC AATGATATTCGCCTCTGCCAAAGGATTTTGTAGTGCTGCTTGTAATAATGCTCCAGA AACTGCTAACATTGCGCCAACCATCAATGCAATTAATATACGTGGCAATCGCAAAT CAATGATTGAATCCACTGCTTCATTGCTACCAGTTGTAAATTTTGTAAATAGGTCATT AAATGACAATTTAATTGTACCGGTTACAAACGAAATATAAGCAGTTGCGATTAAAAT GACTAACAAACATAAAAA (SEQ ID NO:
• 37);
• or (2) a sequence complementary to SEQ ID NO:
• 37 .

A nucleic acid sequence encoding a substantially purified sortase-transamidase enzyme from a Gram-positive bacterium, the enzyme having a molecular weight of about 29,076 daltons and catalyzing a reaction that covalently cross-links the carboxyl terminus of a protein having a sorting signal to the peptidoglycan of a Gram-positive bacterium, the sorting signal having (1) a motif of NPQ/KTN/G therein;

• (2) a substantially hydrophobic domain of at least 31 amino acids carboxyl to the motif;
• and (3) a charged tail region with at least two positively charged residues carboxyl to the substantially hydrophobic domain, at least one of the two positively charged residues being arginine, the two positively charged residues being located at residues 31-33 from the motif, wherein the nucleic acid sequence hybridizes with a sequence selected from the group consisting of:
• 11 (1) AAAAACCCTTGTGGTGTCACTGTACCTGATAAAGATTCAGCAACTTTCATGTTTATT (SEQ ID NO:37) TCAAAAACTTCTTGCGCGTATGCGATAATTTGCTGATCTAATCTTGCCGGTTCAATT GCAAATAATTGTGTAATTACAATTCCACTTTGATAAGCTTCTTCAATTAAATGCACAC CTTCAATTAAAGCTAATCCAGTTTTATCCCTCTCACGTTTCTTTTTTAGCTTGTTCGC TTGTTTAATTCTATTATTTTGTGCAGAAGTAATTTGTTCCATTGATAGCTCCTCGCTT TATTTTTAAAAATAAAAATATTAATCATTAATAAGATGAAAACATTTGATTGTATAGTT AATATTAATTAATCGCTTTTATCACTCATAATATTTCAAATTGTATAAATTTCTTTTAT CGATACTAGTACTATAAATCATACGCCCCAAAATATCATTATTAATTCTTTTCTTCTT CAAAATAAATCAAAATGATATAATTGATGATTATTTTCAAAGCACATTCAAATCAAAC TATGTTTTAGCAATTTGTTGTTAGCATGTTTGTGTTCATTAAAAAAACGACCATCATC GGTATCATGTATGGTCGTTACAAAAGCTAACAATACCAATTGTCATAACAAGTACTG CAACCTCTTTAAATTCAATTATTTCATGTAACTATAGCCTATATCATATGTAATTACTT TGTTATTTATAATCGGGCTACTTTGATCTTCATTTTTACTTCTAACATGTTTATGCGC TGCTTTAAAGACATCAGATTTTAACCAATCCGTAAAAGCTTGCTTTGATTTCCAAAC TGTTAAAATTTTCACTTCATCAAAATCTTCTTGTTCTAAAGTTTGTGTAACAAACATG CCATCAAAGCCTTCTAATGTTTCAATCCCATGTCTCGTGTAAAATCGTTCTATAATAT CTTTTGCTGTTCCTTTTGTTAAGGTCAGCCTATTTTCTGCCATAAATTTCATAATTAT CCTCTTTTCTGTTTAACTTACCTTAATTATTTTTGCGACAAGAACAATTCTTTTCGTC GTTTCACTATATGCATCTTCGCACGTTGATAAAGTCATTATTCTATCTTTTACCGTTA CATTAACATCTGAATTAATTACAGATTTACGTTTTGTCTCATCTAAAAATTGTTGATA ATCTTGATCATTTTCAAAATCTGTACGTATGTAATTATCTTTAGTAGTAGTTTTATATG CACTAAATACTTGCAATTGATATTTACCATATTTATTGTCAAATTCAATTATCTTGTGT TTTTCATAAAACGATTGCTTTAAATAATCTTCTAACACATCAAACATCGTATTATCAC CGACATGGTGCCCGTATAAAATAGTATTATGATTTAAATTCTTCAATTCATTTCTAAA ATCCATAAAAATACTACCTTTACGTCGATGTTCTCGCTCAAAATCTAAATTTAAATAA TCGTGATTTGTCTTACCTTGTAGTACTGGATAATTTAATGATGTTCCTGATAATTTTA TCCATCCAACAATGTCTTTATTTATTTTTTCAAGTGATTCAAATTGTGGTCTCACATG TTCTTGATGTTTGCTCATCAGCATTTGAAATTTTTGTTGTAATTTCTCATAATTTGCG CGTTCTTGCTTGTCTTCAATATATGTTTGAACAATTTTGTAACCAAAAATGATAATAA TTACAACCAATAAAATTTGTACAATAGTTAAAAATCGCTTCATTCTCATAAAAATCCT CTTTTATTAACGACGTTTCTTCAGTCATCACTAAACCAGTTGTTGTACCGTTTTAGAT TCGATTTCGTTGACTTTGACAAATTAAGTAAATTAGCATTGGACCACCGACAATCAT TAAAATAGCATTGGCTGGAATTTCTAAAGGAGGCTGTATCACTCGTCCTAATAAATC AGCCACTAACAATAGCCATGCACCAATAACTGTAGAAAACGGAATAAGTACTCTGT AATTGCCCCCAACTAGCTTTCTAACCACATGTGGCAGAATAATACCTAAAAAGGCTA GTTGTCCAACAATCGCAACAGTTGCACTTGCTAAAAATACTGCTAATAAACCTGTTA ACCATCTGTAACGATCAATATTAAAACCGATACTTCGCGCTTGTATGTCGTCTAAAT TTAGTAAATTCAATTTAGGGGACAATAGTAATGTTAATATTAATCCCAATAATGCTGA TACTGCTAATATGTATACGTCGCTCCATATTTTCATTGTTAAGCCTTGAGGAATTTTC ATTAAAGGGTTTTGAGTTAAAATTTCTAAAACACCATTTAATAATACGAATAACGCAA CACCTACTAATATCATACTTACAGCATTGAATCTAAATTTAGAATGCAACAATATAAT TATTAAAAATGGTATTAAACCTCCAATAAAACTTAATAATGGTAAGTAAAAGTACAAT TGTGGAATAAACAACATAGAAAGTGCTCTCATTATAAGTGCACCTGAGGAAACGCC AATGATATTCGCCTCTGCCAAAGGATTTTGTAGTGCTGCTTGTAATAATGCTCCAGA AACTGCTAACATTGCGCCAACCATCAATGCAATTAATATACGTGGCAATCGCAAAT CAATGATTGAATCCACTGCTTCATTGCTACCAGTTGTAAATTTTGTAAATAGGTCATT AAATGACAATTTAATTGTACCGGTTACAAACGAAATATAAGCAGTTGCGATTAAAAT GACTAACAAACATAAAAA (SEQ ID NO:37), or (2) a sequence complementary to SEQ ID NO:
• 37, with no greater than about a 15% mismatch, under stringent conditions.

A method for screening a compound for anti-sortase-transamidase activity comprising the steps of:

• a) providing an active fraction of sortase-transamidase enzyme from a Gram-positive bacterium;
• b) performing an assay for sortase-transamidase in the presence and in the absence of the compound;
• and
• c) comparing the activity of the sortase-transamidase enzyme in the presence and in the absence of the compound to screen the compound for sortase-transamidase activity.

A method for displaying a polypeptide on the surface of a Gram-positive bacterium comprising the steps of:

• a) cloning a nucleic acid segment encoding a chimeric protein into a Gram-positive bacterium to generate a cloned chimeric protein including therein a carboxyl-terminal sorting signal, the chimeric protein including the polypeptide to be displayed, the sorting signal having:
• (1) a motif of NPQ/KTN/G (SEQ ID NO:
• 41) therein;
• (2) a substantially hydrophobic domain of at least 31 amino acids carboxyl to the motif;
• and (3) a charged tail region with at least two positively charged residues carboxyl to the substantially hydrophobic domain, at least one of the two positively charged residues being arginine, the two positively charged residues being located at residues 31-33 from the motif;
• b) growing the bacterium into which the nucleic acid segment has been cloned to express the cloned chimeric protein to generate a chimeric protein including therein a carboxyl-terminal sorting signal;
• and
• c) binding the polypeptide covalently to the cell wall by the enzymatic action of a sortase-transamidase expressed by the Gram-positive bacterium involving cleavage of the chimeric protein within the NPQ/KTN/G (SEQ ID NO:
• 41) motif so that the polypeptide is displayed on the surface of the Gram-positive bacterium in such a way that the polypeptide is accessible to a ligand.

A method for screening for expression of a cloned polypeptide comprising the steps of:

• a) cloning a nucleic acid segment encoding a chimeric protein into a Gram-positive bacterium to generate a cloned chimeric protein including therein a carboxyl-terminal sorting signal, the chimeric protein including the polypeptide whose expression is to be screened, the sorting signal having:
• (1) a motif of NPQ/KTN/G (SEQ ID NO:
• 41) therein;
• (2) a substantially hydrophobic domain of at least 31 amino acids carboxyl to the motif;
• and (3) a charged tail region with at least two positively charged residues carboxyl to the substantially hydrophobic domain, at least one of the two positively charged residues being arginine, the two positively charged residues being located at residues 31-33 from the motif;
• b) growing the bacterium into which the nucleic acid segment has been cloned to express the cloned chimeric protein to generate a chimeric protein including therein a carboxyl-terminal sorting signal;
• c) binding the polypeptide covalently to the cell wall by the enzymatic action of a sortase-transamidase expressed by the Gram-positive bacterium involving cleavage of the chimeric protein within the NPQ/KTN/G (SEQ ID NO:
• 41) motif so that the polypeptide is displayed on the surface of the Gram-positive bacterium in such a way that the polypeptide is accessible to a ligand;
• and
• d) reacting the displayed polypeptide with a labeled specific binding partner to screen the chimeric protein for reactivity with the labeled specific binding partner.

A method for the diagnosis or treatment of a bacterial infection caused by a Gram-positive bacterium comprising the steps of:

• a) conjugating an antibiotic or a detection reagent to a protein including therein a carboxyl-terminal sorting signal to produce a conjugate, the carboxyl-terminal sorting signal having:
• (1) a motif of NPQ/KTN/G (SEQ ID NO:
• 41) therein;
• (2) a substantially hydrophobic domain of at least 31 amino acids carboxyl to the motif;
• and (3) a charged tail region with at least two positively charged residues carboxyl to the substantially hydrophobic domain, at least one of the two positively charged residues being arginine, the two positively charged residues being located at residues 31-33 from the motif;
• and
• b) introducing the conjugate to an organism infected with a Gram-positive bacterium in order to cause the conjugate to be sorted and covalently cross-linked to the cell walls of the bacterium in order to treat or diagnose the infection.

A conjugate comprising an antibiotic or a detection reagent covalently conjugated to a protein including therein a carboxyl-terminal sorting signal to produce a conjugate, the carboxyl-terminal sorting signal having:

• (1) a motif of NPQ/KTN/G (SEQ ID NO:
• 41) therein;
• (2) a substantially hydrophobic domain of at least 31 amino acids carboxyl to the motif;
• and (3) a charged tail region with at least two positively charged residues carboxyl to the substantially hydrophobic domain, at least one of the two positively charged residues being arginine, the two positively charged residues being located at residues 31-33 from the motif.