title: Biosynthesis of Staphylococcus aureus autoinducing peptides by using the synechocystis DnaB mini-intein.
abstract: The Agr quorum-sensing system of Staphylococcus aureus modulates the expression of virulence factors in response to autoinducing peptides (AIPs). The peptides are seven to nine residues in length and have the C-terminal five residues constrained in a thiolactone ring. We have developed a new method to generate AIP structures using an engineered DnaB mini-intein from Synechocystis sp. strain PCC6803. In the method, an oligonucleotide encoding the AIP is ligated to the intein and the fusion protein is expressed and purified by affinity chromatography. To produce the correct AIP structure, intein splicing is interrupted, allowing the cysteine side chain to catalyze thiolactone ring formation and release AIP from the resin. The technique is simple and robust, and we have successfully produced the three main classes of AIPs using the intein system. The intein-generated AIPs possessed the correct thiolactone ring modification based on biochemical analysis, and, importantly, all the samples were bioactive against S. aureus. The AIP activity was confirmed through Agr interference and activation profiling with developed S. aureus reporter strains. The simplicity of the method, benefits of DNA encoding, and scalable nature enable the production of S. aureus AIPs for many biological applications.
authors: Department of Microbiology, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA.
authors: Cheryl L
authors: CL
authors: Malone
authors: Blaise R
authors: BR
authors: Boles
authors: Alexander R
authors: AR
authors: Horswill
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis*; D053488:DnaB Helicases / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D047668:Inteins / Q000235:genetics / Q000502:physiology*; D010455:Peptides / Q000378:metabolism*; D010456:Peptides, Cyclic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D013211:Staphylococcus aureus / Q000235:genetics / Q000378:metabolism*; D046939:Synechocystis / Q000737:chemistry*
chemical_list: C414532:AgrD protein, Staphylococcus; D001426:Bacterial Proteins; D010455:Peptides; D010456:Peptides, Cyclic; D011994:Recombinant Proteins; D053488:DnaB Helicases
references: Proc Natl Acad Sci U S A. 1989 Nov;86(21):8247-51
references: 2682640
references: J Bacteriol. 2004 Oct;186(20):6706-13
references: 15466021
references: Gene. 1991 Jun 15;102(1):75-8
references: 1840539
references: Methods Enzymol. 1991;204:587-636
references: 1658572
references: Infect Immun. 1992 Aug;60(8):3381-8
references: 1639506
references: J Biochem. 1992 May;111(5):638-42
references: 1639761
references: FEMS Microbiol Lett. 1992 Jul 1;73(1-2):133-8
references: 1521761
references: Biotechniques. 1994 Mar;16(3):410-2
references: 8185910
references: J Biol Chem. 1996 Sep 6;271(36):22159-68
references: 8703028
references: Nucleic Acids Res. 1997 Jun 1;25(11):2227-8
references: 9153325
references: Science. 1997 Jun 27;276(5321):2027-30
references: 9197262
references: Gene. 1997 Jun 19;192(2):271-81
references: 9224900
references: FEBS Lett. 1998 Mar 6;424(1-2):89-94
references: 9537521
references: Biochim Biophys Acta. 1998 Sep 8;1387(1-2):422-32
references: 9748659
references: Proc Natl Acad Sci U S A. 1999 Feb 16;96(4):1218-23
references: 9990004
references: J Biol Chem. 1999 Jun 25;274(26):18359-63
references: 10373440
references: Nat Biotechnol. 1999 Sep;17(9):889-92
references: 10471931
references: Proc Natl Acad Sci U S A. 2004 Nov 16;101(46):16168-73
references: 15528279
references: Proc Natl Acad Sci U S A. 2005 Feb 1;102(5):1691-6
references: 15665088
references: J Mol Biol. 2005 Mar 4;346(4):1095-108
references: 15701520
references: J Bacteriol. 2005 May;187(9):3139-50
references: 15838041
references: IUBMB Life. 2006 Jan;58(1):63
references: 16540435
references: Infect Immun. 2006 May;74(5):2947-56
references: 16622233
references: J Bacteriol. 2006 Nov;188(21):7686-8
references: 16936013
references: Mol Microbiol. 2007 Aug;65(3):780-98
references: 17608791
references: Proc Natl Acad Sci U S A. 1999 Nov 23;96(24):13638-43
references: 10570125
references: Proc Natl Acad Sci U S A. 2000 Jun 6;97(12):6640-5
references: 10829079
references: J Bacteriol. 2000 Nov;182(22):6517-22
references: 11053400
references: Mol Microbiol. 2001 Jul;41(1):145-54
references: 11454207
references: Chem Biol. 2001 Aug;8(8):801-15
references: 11514229
references: Infect Immun. 2002 Feb;70(2):631-41
references: 11796592
references: J Biol Chem. 2002 Mar 8;277(10):7790-8
references: 11742000
references: Mol Microbiol. 2002 Mar;43(6):1387-400
references: 11952893
references: Biochemistry. 2002 Aug 6;41(31):10095-104
references: 12146974
references: J Bacteriol. 2002 Oct;184(19):5457-67
references: 12218034
references: Mol Microbiol. 2003 Jun;48(6):1429-49
references: 12791129
references: J Biol Chem. 2003 Oct 3;278(40):39133-42
references: 12878593
references: Annu Rev Biochem. 2003;72:249-89
references: 12626339
references: J Bacteriol. 2004 Mar;186(6):1838-50
references: 14996815
references: J Biol Chem. 2004 Aug 20;279(34):35281-6
references: 15194682
references: Peptides. 2004 Sep;25(9):1389-403
references: 15374643
references: Gene. 1990 Nov 30;96(1):23-8
references: 2265755