FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2166131252> ?p ?o ?g. }

• W2166131252 endingPage "10409" @default.
• W2166131252 startingPage "10403" @default.
• W2166131252 abstract "Quorum sensing mediated by specific signal compounds (autoinducers) allows bacteria to monitor their cell density and enables a synchronized regulation of target gene sets. The best studied group of autoinducers are the acylhomoserine lactones (AHSLs), which are central to the regulation of virulence in many plant and animal pathogens. Variation of the acyl side chain of the AHSLs underlies the observed species specificity of this communication system. Here we show that even different strains of the plant pathogen Erwinia carotovora employ different dialects of this language and demonstrate the molecular basis for the acyl chain length specificity of distinct AHSL synthases. Under physiological concentrations, only the cognate AHSL with the “right” acyl chain is recognized as a signal that will switch on virulence genes. Mutagenesis of the AHSL synthase gene expISCC1 identified the changes M127T and F69L as sufficient to effectively alter ExpISCC1 (an N-3-oxohexanoyl-l-homoserine lactone producer) substrate specificity to that of an N-3-oxooctanoyl-l-homoserine lactone producer. Our data identify critical residues that define the size of the substrate-binding pocket of the AHSL synthase and will help in understanding and manipulating this bacterial language. Quorum sensing mediated by specific signal compounds (autoinducers) allows bacteria to monitor their cell density and enables a synchronized regulation of target gene sets. The best studied group of autoinducers are the acylhomoserine lactones (AHSLs), which are central to the regulation of virulence in many plant and animal pathogens. Variation of the acyl side chain of the AHSLs underlies the observed species specificity of this communication system. Here we show that even different strains of the plant pathogen Erwinia carotovora employ different dialects of this language and demonstrate the molecular basis for the acyl chain length specificity of distinct AHSL synthases. Under physiological concentrations, only the cognate AHSL with the “right” acyl chain is recognized as a signal that will switch on virulence genes. Mutagenesis of the AHSL synthase gene expISCC1 identified the changes M127T and F69L as sufficient to effectively alter ExpISCC1 (an N-3-oxohexanoyl-l-homoserine lactone producer) substrate specificity to that of an N-3-oxooctanoyl-l-homoserine lactone producer. Our data identify critical residues that define the size of the substrate-binding pocket of the AHSL synthase and will help in understanding and manipulating this bacterial language. Bacteria are able to monitor their cell density by producing and detecting specific signal compounds commonly referred to as autoinducers. This quorum sensing allows bacteria to respond to fluctuations in their numbers and enables synchronous regulation of target gene sets when living in a community. The best studied group of autoinducers are the acylhomoserine lactones (AHSLs), 1The abbreviations used are: AHSL, acylhomoserine lactone; ACP, acyl-carrier protein; Cel, cellulase; HSL, homoserine lactone; 3-oxo-C6-HSL, N-3-oxohexanoyl-l-HSL; 3-oxo-C8-HSL, N-3-oxooctanoyl-l-HSL; LB, Luria-Bertani (medium).1The abbreviations used are: AHSL, acylhomoserine lactone; ACP, acyl-carrier protein; Cel, cellulase; HSL, homoserine lactone; 3-oxo-C6-HSL, N-3-oxohexanoyl-l-HSL; 3-oxo-C8-HSL, N-3-oxooctanoyl-l-HSL; LB, Luria-Bertani (medium). which are central to the regulation of virulence in Gram-negative plant and animal (including human) pathogens (1Whitehead N.A. Barnard A.M. Slater H. Simpson N.J.L. Salmond G.P.C. FEMS Microbiol. Rev. 2001; 25: 365-404Crossref PubMed Google Scholar, 2Fuqua C. Greenberg E.P. Nat. Rev. Mol. Cell Biol. 2002; 3: 685-695Crossref PubMed Scopus (812) Google Scholar, 3Loh J. Pierson E.A. Pierson III, L.S. Stacey G. Chatterjee A. Curr. Opin. Plant Biol. 2002; 5: 285-290Crossref PubMed Scopus (149) Google Scholar, 4Taga M.E. Bassler B.L. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 14549-14554Crossref PubMed Scopus (332) Google Scholar, 5Zhang L. Trends Plant Sci. 2003; 8: 238-244Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar) and have been shown to control processes as diverse as bioluminescence in the marine organism Vibrio fischeri (6Engebrecht J. Nealson K.H. Silverman M. Cell. 1983; 32: 773-781Abstract Full Text PDF PubMed Scopus (588) Google Scholar), biofilm formation and virulence in the opportunistic human pathogen Pseudomonas aeruginosa (7Parsek M.R. Greenberg E.P. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8789-8793Crossref PubMed Scopus (466) Google Scholar, 8Singh P.K. Schaefer A.L. Parsek M.R. Moninger T.O. Welsh M.J. Greenberg E.P. Nature. 2000; 407: 762-764Crossref PubMed Scopus (1190) Google Scholar), Ti plasmid conjugal transfer in Agrobacterium tumefaciens (9Piper K.R. Beck von Bodman S. Farrand S.K. Nature. 1993; 362: 448-450Crossref PubMed Scopus (380) Google Scholar, 10Zhang L. Murphy P.J. Kerr A. Tate M.E. Nature. 1993; 362: 446-448Crossref PubMed Scopus (354) Google Scholar), production of the exopolysaccharide stewartan acting as a virulence factor in the maize pathogen Pantoea stewartii (11Beck von Bodman S. Majerczak D.R. Coplin D.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7687-7692Crossref PubMed Scopus (214) Google Scholar), and production of carbapenem antibiotics as well as plant cell wall-degrading extracellular enzymes in the plant pathogen Erwinia carotovora (12Bainton N.J. Stead P. Chhabra S.R. Bycroft B.W. Salmond G.P.C. Stewart G.S. Williams P. Biochem. J. 1992; 288: 997-1004Crossref PubMed Scopus (222) Google Scholar, 13Pirhonen M. Flego D. Heikinheimo R. Palva E.T. EMBO J. 1993; 12: 2467-2476Crossref PubMed Scopus (353) Google Scholar). The chain length (C4–C18) and the oxidative status of the acyl side chain of the AHSLs vary and reflect the observed species-specificity of this communication system (2Fuqua C. Greenberg E.P. Nat. Rev. Mol. Cell Biol. 2002; 3: 685-695Crossref PubMed Scopus (812) Google Scholar, 4Taga M.E. Bassler B.L. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 14549-14554Crossref PubMed Scopus (332) Google Scholar).However different these processes are, the general mechanism of autoinducer-mediated quorum sensing is similar and involves an AHSL synthase and a sensor protein binding to the cognate autoinducer and regulating target gene expression. The I-protein produces basal levels of a specific AHSL, which passes through bacterial cell membranes by diffusion and/or active transport (2Fuqua C. Greenberg E.P. Nat. Rev. Mol. Cell Biol. 2002; 3: 685-695Crossref PubMed Scopus (812) Google Scholar, 5Zhang L. Trends Plant Sci. 2003; 8: 238-244Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). After the AHSL concentration reaches a critical threshold level corresponding to a certain quorum of bacteria, the AHSL interacts with the cognate R-protein and controls the expression of quorum sensing-regulated target genes (1Whitehead N.A. Barnard A.M. Slater H. Simpson N.J.L. Salmond G.P.C. FEMS Microbiol. Rev. 2001; 25: 365-404Crossref PubMed Google Scholar, 2Fuqua C. Greenberg E.P. Nat. Rev. Mol. Cell Biol. 2002; 3: 685-695Crossref PubMed Scopus (812) Google Scholar, 14Whitehead N.A. Byers J.T. Commander P. Corbett M.J. Coulthurst S.J. Everson L. Harris A.K.P. Pemberton C.L. Simpson N.J.L. Slater H. Smith D.S. Welch M. Williamson N. Salmond G.P.C. Antonie Van Leeuwenhoek. 2002; 81: 223-231Crossref PubMed Scopus (95) Google Scholar). At present, >70 AHSL synthases (LuxI-like I-proteins) and sensors (LuxR-like R-proteins) are known (4Taga M.E. Bassler B.L. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 14549-14554Crossref PubMed Scopus (332) Google Scholar). In E. carotovora, the production of both carbapenem antibiotics and extracellular enzyme (cellulase, pectate lyase, polygalacturonase, and protease) virulence factors is controlled by AHSLs and, hence, requires a functional I-protein known as CarI or ExpI, respectively (1Whitehead N.A. Barnard A.M. Slater H. Simpson N.J.L. Salmond G.P.C. FEMS Microbiol. Rev. 2001; 25: 365-404Crossref PubMed Google Scholar, 13Pirhonen M. Flego D. Heikinheimo R. Palva E.T. EMBO J. 1993; 12: 2467-2476Crossref PubMed Scopus (353) Google Scholar, 14Whitehead N.A. Byers J.T. Commander P. Corbett M.J. Coulthurst S.J. Everson L. Harris A.K.P. Pemberton C.L. Simpson N.J.L. Slater H. Smith D.S. Welch M. Williamson N. Salmond G.P.C. Antonie Van Leeuwenhoek. 2002; 81: 223-231Crossref PubMed Scopus (95) Google Scholar). Although carbapenem synthesis in E. carotovora ATT39048 is positively regulated by an R-protein (CarR) (1Whitehead N.A. Barnard A.M. Slater H. Simpson N.J.L. Salmond G.P.C. FEMS Microbiol. Rev. 2001; 25: 365-404Crossref PubMed Google Scholar, 15Welch M. Todd D.E. Whitehead N.A. McGowan S.J. Bycroft B.W. Salmond G.P.C. EMBO J. 2000; 19: 631-641Crossref PubMed Scopus (150) Google Scholar), the cognate AHSL sensor protein and the mechanism for extracellular enzyme regulation is not known (1Whitehead N.A. Barnard A.M. Slater H. Simpson N.J.L. Salmond G.P.C. FEMS Microbiol. Rev. 2001; 25: 365-404Crossref PubMed Google Scholar, 14Whitehead N.A. Byers J.T. Commander P. Corbett M.J. Coulthurst S.J. Everson L. Harris A.K.P. Pemberton C.L. Simpson N.J.L. Slater H. Smith D.S. Welch M. Williamson N. Salmond G.P.C. Antonie Van Leeuwenhoek. 2002; 81: 223-231Crossref PubMed Scopus (95) Google Scholar, 16Andersson R.A. Eriksson A.R.B. Heikinheimo R. Mäe A. Pirhonen M. Kõiv V. Hyytiäinen H. Tuikkala A. Palva E.T. Mol. Plant-Microbe Interact. 2000; 13: 384-393Crossref PubMed Scopus (99) Google Scholar). For the actual change in target protein levels, several other factors might be important, including small regulatory RNA chaperones influencing RNA stability (e.g. Hfq of Vibrio harveyi and RsmA in E. carotovora (17Lenz D.H. Mok K.C. Lilley B.N. Kulkarni R.V. Wingreen N.S. Bassler B.L. Cell. 2004; 118: 69-82Abstract Full Text Full Text PDF PubMed Scopus (713) Google Scholar, 18Chatterjee A. Cui Y. Chatterjee A.K. J. Bacteriol. 2002; 184: 4089-4095Crossref PubMed Scopus (57) Google Scholar) and proteins binding to R-proteins (e.g. TraM of A. tumefaciens) (19Vannini A. Volpari C. Di Marco S. J. Biol. Chem. 2004; 279: 24291-24296Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar).The enzymatic reaction mechanism of AHSL synthesis involves the substrates S-adenosyl-l-methionine and an acyl-acyl carrier protein (acyl-ACP) (20More M.I. Finger L.D. Stryker J.L. Fuqua C. Eberhard A. Winans S.C. Science. 1996; 272: 1655-1658Crossref PubMed Scopus (309) Google Scholar, 21Val D.L. Cronan Jr., J.E. J. Bacteriol. 1998; 180: 2644-2651Crossref PubMed Google Scholar, 22Parsek M.R. Val D.L. Hanzelka B.L. Cronan Jr., J.E. Greenberg E.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4360-4365Crossref PubMed Scopus (406) Google Scholar). The crystal structure of EsaI, the I-protein of P. stewartii, revealed a high similarity of the AHSL synthase to N-acetyltransferases and allowed modeling of the 3-oxo-C6-phosphopantetheine part of acyl-ACP in the active cavity of EsaI. Mutation analysis was used to demonstrate the importance of several residues for the activity of EsaI, thereby confirming the model (23Watson W.T. Minogue T.D. Val D.L. Beck von Bodman S. Churchill M.E.A. Mol. Cell. 2002; 9: 685-694Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). Additional conserved residues essential for activity have been identified in LuxI (24Hanzelka B.L. Stevens A.M. Parsek M.R. Crone T.J. Greenberg E.P. J. Bacteriol. 1997; 179: 4882-4887Crossref PubMed Google Scholar) and RhlI (25Parsek M.R. Schaefer A.L. Greenberg E.P. Mol. Microbiol. 1997; 26: 301-310Crossref PubMed Scopus (72) Google Scholar). In EsaI, the substrate acyl-ACP selection seems to be due to binding to a hydrophobic pocket. Indeed, changing of the conserved Thr-140 to Ala resulted in the production of fully reduced homoserine lactones instead of N-3-oxohexanoyl-l-homoserine lactone (3-oxo-C6-HSL), which has been explained as a stabilizing effect of a favorable hydrogen bond between the C3 carbonyl and the hydroxy group of Thr (23Watson W.T. Minogue T.D. Val D.L. Beck von Bodman S. Churchill M.E.A. Mol. Cell. 2002; 9: 685-694Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). The recently described crystal structure of LasI from P. aeruginosa seems to indicate that different bacteria might have different mechanisms for substrate selection. Here, a tunnel with no apparent restriction on acyl chain length passing through the enzyme can accommodate the acyl chain of the acyl-ACP substrate (26Gould T.A. Schweizer H.P. Churchill M.E.A. Mol. Microbiol. 2004; 53: 1135-1146Crossref PubMed Scopus (118) Google Scholar).It has been speculated (2Fuqua C. Greenberg E.P. Nat. Rev. Mol. Cell Biol. 2002; 3: 685-695Crossref PubMed Scopus (812) Google Scholar, 23Watson W.T. Minogue T.D. Val D.L. Beck von Bodman S. Churchill M.E.A. Mol. Cell. 2002; 9: 685-694Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar) that the C terminus of I-proteins should contain residues responsible for specificity to a substrate with a certain acyl side chain length, but the actual residues and structural prerequisites determining this specificity have not yet been identified. Here, we describe the specificity of AHSL signaling in E. carotovora and demonstrate the molecular basis for the substrate chain length specificity of the AHSL synthase ExpI. The changes M127T and F69L in ExpI resulted in a product shift from 3-oxo-C6-HSL to N-3-oxooctanoyl-l-homoserine lactone (3-oxo-C8-HSL). Additional multiple amino acid changes restored production of the changed product 3-oxo-C8-HSL to wild type levels.EXPERIMENTAL PROCEDURESBacterial Strains and Mutagenesis—The E. carotovora strains SCC1 and SCC3193 originate from potato field collections near the Viikki Campus of the University of Helsinki (27Pirhonen M. Palva E.T. Mol. Gen. Genet. 1988; 214: 170-172Crossref Scopus (29) Google Scholar). The expISCC1 gene was amplified from E. carotovora SCC1 DNA by PCR with 5′-CGGGATCCATGTTAGAGATATTTG-3′ (5′-CGGGATCCTTAGAGATATTTGATGTAAATC-3′ without ATG for cloning into pQE30 (Qiagen)) and 5′-GGAAGCTTTCAAGCCTGTGCAATAG-3′ cloned into BamHI/HindIII sites of pBluescript II SK or pQE30 and transformed into Escherichia coli DH5α and JM109. ExpISCC1 (GenBank™ accession number AY507108) is >99% identical to CarI and 70% identical to ExpISCC3193. Mutations were created using the Stratagene QuikChange site-directed mutagenesis kit with plasmids containing expISCC3193 (13Pirhonen M. Flego D. Heikinheimo R. Palva E.T. EMBO J. 1993; 12: 2467-2476Crossref PubMed Scopus (353) Google Scholar) or expISCC1. Each mutation was confirmed by sequencing, and plasmids were transformed by electroporation to an AHSL synthase-deficient (ExpI-) transposon mutant of E. carotovora without detectable AHSL production (SCC3065), which is derived from strain SCC3193 (13Pirhonen M. Flego D. Heikinheimo R. Palva E.T. EMBO J. 1993; 12: 2467-2476Crossref PubMed Scopus (353) Google Scholar, 28Pirhonen M. Saarilahti H. Karlsson M.-B. Palva E.T. Mol. Plant-Microbe Interact. 1991; 4: 276-283Crossref Scopus (108) Google Scholar).Plant Inoculations and Cellulase Assays—For plant inoculations, E. carotovora was grown overnight in LB medium (29Elbing K. Brent R. Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley and Sons, New York2002: 1.1.1-1.1.7Google Scholar) at 28 °C, centrifuged, and washed with 0.9% sodium chloride. 5 μl of bacterial suspension containing 106 colony-forming units was used to infect sliced, 5-mm thick, and surface-sterilized (70% ethanol) potato tubers (Van Gogh), which were kept at 28 °C in Petri dishes with wet filter paper. Cellulase assays were performed with 10-μl supernatants of overnight grown cultures on cellulose indicator plates (carboxymethylcellulose) using Congo Red to determine cellulose digestion as described (13Pirhonen M. Flego D. Heikinheimo R. Palva E.T. EMBO J. 1993; 12: 2467-2476Crossref PubMed Scopus (353) Google Scholar).Determination of AHSL Content and Structure Elucidation—AHSLs were extracted twice with equal volumes of ethyl acetate from 2-ml E. carotovora or E. coli culture supernatants grown in LB or minimal M9 medium (29Elbing K. Brent R. Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley and Sons, New York2002: 1.1.1-1.1.7Google Scholar) containing 0.4% sucrose or from 20 μl of macerated potato tuber tissue diluted in 500 μl of water. After evaporating to dryness in a SpeedVac, samples were dissolved in 20 μl (potato) or 200 μl (LB and M9) of 50% acetonitrile in 0.1% formic acid. 5 μl of the dissolved samples were injected for liquid chromatography-mass spectroscopy analysis by an Agilent 1100 series high performance liquid chromatography system (Agilent Technologies) equipped with a Luna (Phenomenex) C18 column (100 × 4.6-mm inner diameter, 3-μm particle size) at a flow rate of 0.5 ml min-1 with 45 or 70% acetonitrile in 0.1% formic acid coupled without split to a quadrupole/time-of-flight mass spectrometer (Q-TOF Micro, Micromass Instruments). The positive electrospray ionization conditions included a capillary voltage of 3.2 kV, a cone voltage of 20 V, 5 eV ionization, a source temperature of 110 °C, and a desolvation temperature of 400 °C. Tandem mass spectrometry spectra were generated with a collision energy of 12 eV with argon. AHSL standards were purchased from Sigma-Aldrich (C7-, C8-, 3-oxo-C6-HSL) or synthesized (C4-, C6-, C9-, 3-oxo-C8-HSL) as described (10Zhang L. Murphy P.J. Kerr A. Tate M.E. Nature. 1993; 362: 446-448Crossref PubMed Scopus (354) Google Scholar). 3-OH-C6- and 3-OH-C8-HSL have been reduced from the corresponding 3-oxo-derivatives by stirring at room temperature with equimolar amounts of sodium borohydride in methanol for 15 min followed by purification over silica gel with chloroform/methanol. 3-Oxo-C5-, 3-oxo-C7-, 3-oxo-C9, 3-oxo-C10-, C5-, and C10-HSL have been identified by their high performance liquid chromatography retention time as well as by tandem mass spectrometry analysis.Immunoblot Analysis—For Western blot hybridization, E. coli JM109 was grown overnight in triplicate, diluted into fresh LB medium, and grown until the A600 was 0.7. Expression was induced with 1 mm isopropyl-β-d-thiogalactopyranoside and, after 4 h, 1 ml of culture was collected. After an additional 2 h of growth, 2-ml supernatants of the same cultures were collected for AHSL measurement as described above. The pellet collected at 4 h was suspended in 100 μl of 6× PAGE sample buffer, and 10 μl were loaded to the gel. Protein extracts were separated on a 12.5% SDS-polyacrylamide gel and transferred with a buffer tank blotting system (Hoefer, Amersham Biosciences) to nitrocellulose membrane (Nitro Bind, Micron Separations Inc.) using standard protocols (29Elbing K. Brent R. Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley and Sons, New York2002: 1.1.1-1.1.7Google Scholar). As the primary antibody, an anti-His5 monoclonal antibody (Qiagen) was used at a 1:2000 dilution according to manufacturer's instructions. Signals were visualized with rabbit anti-mouse IgG alkaline phosphatase-conjugated secondary antibody (Pierce Biotechnology) and alkaline phosphatase conjugate substrate kit (Bio-Rad).Figure Preparation—Homology modeling was performed with Deep-View and SwissModel at swissmodel.expasy.org (30Schwede T. Kopp J. Guex N. Peitsch M.C. Nucleic Acids Res. 2003; 31: 3381-3385Crossref PubMed Scopus (4417) Google Scholar). Structure figures were prepared with Protein Explorer (molvis.sdsc.edu/protexpl/frntdoor.htm), ISISDraw2.4, and Rasmol 2.5.RESULTSSpecificity of AHSL Sensing in E. carotovora—Most of the E. carotovora, Erwinia chrysanthemi, and P. stewartii strains characterized appear to produce and recognize the 3-oxo-C6-HSL autoinducer (1Whitehead N.A. Barnard A.M. Slater H. Simpson N.J.L. Salmond G.P.C. FEMS Microbiol. Rev. 2001; 25: 365-404Crossref PubMed Google Scholar, 14Whitehead N.A. Byers J.T. Commander P. Corbett M.J. Coulthurst S.J. Everson L. Harris A.K.P. Pemberton C.L. Simpson N.J.L. Slater H. Smith D.S. Welch M. Williamson N. Salmond G.P.C. Antonie Van Leeuwenhoek. 2002; 81: 223-231Crossref PubMed Scopus (95) Google Scholar, 12Bainton N.J. Stead P. Chhabra S.R. Bycroft B.W. Salmond G.P.C. Stewart G.S. Williams P. Biochem. J. 1992; 288: 997-1004Crossref PubMed Scopus (222) Google Scholar, 23Watson W.T. Minogue T.D. Val D.L. Beck von Bodman S. Churchill M.E.A. Mol. Cell. 2002; 9: 685-694Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 31Swift S. Winson M.K. Chan P.F. Bainton N.J. Birdsall M. Reeves P.J. Rees C.E. Chhabra S.R. Hill P.J. Throup J.P. Bycroft B.W. Salmond G.P. Williams P. Stewart G.S. Mol. Microbiol. 1993; 10: 511-520Crossref PubMed Scopus (169) Google Scholar, 32Nasser W. Bouillant M.L. Salmond G.P. Reverchon S. Mol. Microbiol. 1998; 29: 1391-1405Crossref PubMed Scopus (149) Google Scholar). Accordingly, an AHSL synthase-deficient transposon mutant (expI-; SCC3065) (13Pirhonen M. Flego D. Heikinheimo R. Palva E.T. EMBO J. 1993; 12: 2467-2476Crossref PubMed Scopus (353) Google Scholar, 28Pirhonen M. Saarilahti H. Karlsson M.-B. Palva E.T. Mol. Plant-Microbe Interact. 1991; 4: 276-283Crossref Scopus (108) Google Scholar) of E. carotovora wild type strain SCC3193 exhibits strongly reduced virulence and extracellular enzyme production, which can be rescued by exogenous addition of this autoinducer (13Pirhonen M. Flego D. Heikinheimo R. Palva E.T. EMBO J. 1993; 12: 2467-2476Crossref PubMed Scopus (353) Google Scholar). However, the substrate specificity of the AHSL synthases and the AHSL recognition specificity of the R-proteins is not absolute but may also include structurally similar AHSLs (15Welch M. Todd D.E. Whitehead N.A. McGowan S.J. Bycroft B.W. Salmond G.P.C. EMBO J. 2000; 19: 631-641Crossref PubMed Scopus (150) Google Scholar, 22Parsek M.R. Val D.L. Hanzelka B.L. Cronan Jr., J.E. Greenberg E.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4360-4365Crossref PubMed Scopus (406) Google Scholar). To assess the specificity of the autoinducer communication system in SCC3193, we compared the ability of AHSLs with different chain lengths and with or without a 3-oxo-group at the C3 position of the acyl chain to restore cellulase (Cel) production in SCC3065. To our surprise, <0.05 μm 3-oxo-C8-HSL is required to restore Cel production; this is >200 times less than the concentration of 3-oxo-C6-HSL needed to achieve the same effect (Fig. 1). The AHSLs with fully reduced acyl chains have intermediate activity with N-octanoyl-l-homoserine lactone (C8-HSL) as the most active compound (Fig. 1B). Obviously, the recognition of AHSLs in SCC3065 (and SCC3193) seems to be optimized for 3-oxo-C8-HSL rather than for 3-oxo-C6-HSL.This finding prompted us to characterize the AHSL profile of SCC3193 with liquid chromatography-mass spectrometry analysis (Fig. 2A and Table I). Indeed, the main AHSL in SCC3193 is 3-oxo-C8-HSL, and only traces of 3-oxo-C6-HSL can be detected. An inverse AHSL profile can be observed in another E. carotovora strain (SCC1) (27Pirhonen M. Palva E.T. Mol. Gen. Genet. 1988; 214: 170-172Crossref Scopus (29) Google Scholar) with 3-oxo-C6-HSL as the main component. The type of AHSL produced is dependent on the corresponding I-protein (22Parsek M.R. Val D.L. Hanzelka B.L. Cronan Jr., J.E. Greenberg E.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4360-4365Crossref PubMed Scopus (406) Google Scholar, 23Watson W.T. Minogue T.D. Val D.L. Beck von Bodman S. Churchill M.E.A. Mol. Cell. 2002; 9: 685-694Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). The expI mutant SCC3065, which itself produces no detectable amounts of AHSLs, produces either 3-oxo-C6- or 3-oxo-C8-HSL after the introduction of expISCC1 or expISCC3193, respectively (Fig. 2, A and B). A mixed culture of SCC3065 + expISCC1 and SCC3065 + expISCC3193 results in the concomitant production of both 3-oxo-C6- and 3-oxo-C8-HSL. E. coli JM109 containing expISCC1 or expISCC3193 produces similar profiles with 3-oxo-C6- or 3-oxo-C8-HSL as the main components (data not shown).Fig. 2Strain- and ExpI-specific AHSL production in E. carotovora. A, AHSL content in bacterial cultures (in LB) 14 h post inoculation (n = 3). B, AHSL content in macerated potato tuber tissue 24 h post inoculation with 106 colony-forming units of E. carotovora (n = 3). C, potato tuber slices and the mean diameter of the macerated area (n = 10) 48 h post inoculation with 106 colony-forming units of E. carotovora. Bacterial strains and plasmids used in panels A, B, and C are indicated at the bottom of panel C. b. d., below detection limit; n. d., not detectable due to lack of maceration.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IAcyl-HSL production by E. carotovora SCC1 and SCC319314 h of growth in LB24 h p.i.aPost inoculation with 106 colony forming units in potato tubersEcbEc, E. carotovora SCC1EcbEc, E. carotovora SCC3193EcbEc, E. carotovora SCC1EcbEc, E. carotovora SCC3193nm ± S.E.nm ± S.E.3-Oxo-C5-HSL16.7 ± 7.6BDcBelow detection limitBDcBelow detection limitBDcBelow detection limitC5-HSL3.8 ± 2.0BDcBelow detection limitBDcBelow detection limitBDcBelow detection limit3-Oxo-C6-HSL864.4 ± 109.818.9 ± 1.02028.7 ± 406.475.1 ± 65.1C6-HSL51.0 ± 4.03.5 ± 0.8177.6 ± 12.7BDcBelow detection limit3-Oxo-C7-HSL70.1 ± 33.532.3 ± 9.8BDcBelow detection limitBDcBelow detection limitC7-HSL7.8 ± 2.918.6 ± 10.7BDcBelow detection limitBDcBelow detection limit3-Oxo-C8-HSL3.5 ± 1.0679.2 ± 22.07.9 ± 6.82402.3 ± 479.1C8-HSLBDcBelow detection limit94.7 ± 6.0BDcBelow detection limit169.1 ± 31.93-OH-C8-HSLBDcBelow detection limit6.2 ± 0.9BDcBelow detection limitBDcBelow detection limit3-Oxo-C9-HSLBDcBelow detection limit15.1 ± 4.2BDcBelow detection limitBDcBelow detection limitC9-HSLBDcBelow detection limit21.3 ± 13.0BDcBelow detection limitBDcBelow detection limit3-Oxo-C10-HSLBDcBelow detection limit9.3 ± 0.6BDcBelow detection limit15.8 ± 13.7C10-HSLBDcBelow detection limit0.5 ± 0.2BDcBelow detection limitBDcBelow detection limita Post inoculation with 106 colony forming unitsb Ec, E. carotovorac Below detection limit Open table in a new tab Interestingly, a series of AHSLs with unusual acyl chains having odd carbon numbers can be detected in bacterial cultures grown in rich medium (LB) but are undetectable in infected potato tissue or in cultures grown in a minimal medium (Table I and Supplemental Table I, available in the on-line version on this article). It seems that in the more stringent in planta environment only a few AHSLs have a role in determining virulence, as indicated by the poorer AHSL profile produced by E. carotovora in planta (Supplemental Table I). The specificity of SCC3193 for 3-oxo-C8-HSL in planta is also demonstrated by the inability of the 3-oxo-C6-HSL-producing ExpISCC1 to complement the avirulent phenotype of SCC3065 in potato (Fig. 2C). This lack of complementation is not due to the inability of ExpISCC1 to direct the synthesis of 3-oxo-C6-HSL in SCC3065 in planta as revealed by mixed infection with SCC3065 + expISCC1 and SCC3065 + expISCC3193 (Fig. 2B). Taken together, our results demonstrate that the two different strains of E. carotovora use different dialects of the same AHSL language in their control of virulence and that these dialects are strain-specific at the physiological concentrations of the respective AHSLs.Mutation Analysis Reveals Critical Residues for the Substrate Chain Length Specificity of the AHSL Synthase—The molecular basis for the distinct acyl chain length specificity of different autoinducer synthases, essential for the specificity of bacterial communication, has not been elucidated. The alignment of closely related ExpI-proteins with different chain length specificities augmented by the crystal structure of EsaI allows us now to identify candidates for the critical residues determining acyl chain length specificity. A comparison of the amino acid sequences of the relatively closely related I-proteins EsaI, CarI, ExpISCC1, and ExpI3937 from E. chrysanthemi (32Nasser W. Bouillant M.L. Salmond G.P. Reverchon S. Mol. Microbiol. 1998; 29: 1391-1405Crossref PubMed Scopus (149) Google Scholar) (all producers of 3-oxo-C6-HSL) with ExpISCC3193 and ExpICFBP 6272 (33Carlier A. Uroz S. Smadja B. Fray R. Latour X. Dessaux Y. Faure D. Appl. Environ. Microbiol. 2003; 69: 4989-4993Crossref PubMed Scopus (159) Google Scholar) from another C8-producing E. carotovora with high homology to ExpISCC3193 revealed residues within close proximity of the putative acyl moiety-binding pocket (23Watson W.T. Minogue T.D. Val D.L. Beck von Bodman S. Churchill M.E.A. Mol. Cell. 2002; 9: 685-694Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar) that are conserved in the C6 producers but different in the C8 producers (Fig. 3). These residues in ExpISCC1 are Leu-67 (Val in EsaI and ExpIEch), Phe-69, Leu-123, and Met-127; the corresponding residues in ExpISCC3193 are Ser-67, Leu-69, Phe-123, and Thr-127 (numbering according to ExpISCC1). The identity of residue 126 does not correlate with C6 or C8 production, as it is Ser-126 in CarI and ExpISCC1 but Ala-126 in all other sequences. It was included in the subsequent studies because of its vicinity to the other residues of interest.Fig. 3Sequence comparison of I-proteins. Protein sequences are f" @default.
• W2166131252 created "2016-06-24" @default.
• W2166131252 creator A5002909703 @default.
• W2166131252 creator A5014233814 @default.
• W2166131252 creator A5040483663 @default.
• W2166131252 creator A5074117103 @default.
• W2166131252 creator A5080035391 @default.
• W2166131252 date "2005-03-01" @default.
• W2166131252 modified "2023-09-27" @default.
• W2166131252 title "Altering Substrate Chain Length Specificity of an Acylhomoserine Lactone Synthase in Bacterial Communication" @default.
• W2166131252 cites W1502461125 @default.
• W2166131252 cites W1508017654 @default.
• W2166131252 cites W1555657499 @default.
• W2166131252 cites W1726719959 @default.
• W2166131252 cites W1783481228 @default.
• W2166131252 cites W1882810357 @default.
• W2166131252 cites W191752848 @default.
• W2166131252 cites W1945743597 @default.
• W2166131252 cites W1970582294 @default.
• W2166131252 cites W1975327876 @default.
• W2166131252 cites W1991095439 @default.
• W2166131252 cites W1994080414 @default.
• W2166131252 cites W1999388135 @default.
• W2166131252 cites W2002317558 @default.
• W2166131252 cites W2014197955 @default.
• W2166131252 cites W2015038267 @default.
• W2166131252 cites W2015272204 @default.
• W2166131252 cites W2035999312 @default.
• W2166131252 cites W2036553709 @default.
• W2166131252 cites W2042544884 @default.
• W2166131252 cites W2045154470 @default.
• W2166131252 cites W2045909145 @default.
• W2166131252 cites W2047630852 @default.
• W2166131252 cites W2060809301 @default.
• W2166131252 cites W2061579546 @default.
• W2166131252 cites W2062438057 @default.
• W2166131252 cites W2070879436 @default.
• W2166131252 cites W2074687201 @default.
• W2166131252 cites W2080003611 @default.
• W2166131252 cites W2091710634 @default.
• W2166131252 cites W2103559990 @default.
• W2166131252 cites W2107304641 @default.
• W2166131252 cites W2113490404 @default.
• W2166131252 cites W2120473134 @default.
• W2166131252 cites W2130360393 @default.
• W2166131252 cites W2135162586 @default.
• W2166131252 cites W2142596421 @default.
• W2166131252 cites W2144150209 @default.
• W2166131252 cites W2162771732 @default.
• W2166131252 cites W2171248714 @default.
• W2166131252 cites W2320616319 @default.
• W2166131252 cites W298464754 @default.
• W2166131252 cites W4247831389 @default.
• W2166131252 doi "https://doi.org/10.1074/jbc.m408603200" @default.
• W2166131252 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/15634689" @default.
• W2166131252 hasPublicationYear "2005" @default.
• W2166131252 type Work @default.
• W2166131252 sameAs 2166131252 @default.
• W2166131252 citedByCount "47" @default.
• W2166131252 countsByYear W21661312522012 @default.
• W2166131252 countsByYear W21661312522013 @default.
• W2166131252 countsByYear W21661312522014 @default.
• W2166131252 countsByYear W21661312522015 @default.
• W2166131252 countsByYear W21661312522017 @default.
• W2166131252 countsByYear W21661312522018 @default.
• W2166131252 countsByYear W21661312522019 @default.
• W2166131252 countsByYear W21661312522021 @default.
• W2166131252 countsByYear W21661312522022 @default.
• W2166131252 crossrefType "journal-article" @default.
• W2166131252 hasAuthorship W2166131252A5002909703 @default.
• W2166131252 hasAuthorship W2166131252A5014233814 @default.
• W2166131252 hasAuthorship W2166131252A5040483663 @default.
• W2166131252 hasAuthorship W2166131252A5074117103 @default.
• W2166131252 hasAuthorship W2166131252A5080035391 @default.
• W2166131252 hasBestOaLocation W21661312521 @default.
• W2166131252 hasConcept C112243037 @default.
• W2166131252 hasConcept C121332964 @default.
• W2166131252 hasConcept C1276947 @default.
• W2166131252 hasConcept C181199279 @default.
• W2166131252 hasConcept C185592680 @default.
• W2166131252 hasConcept C18903297 @default.
• W2166131252 hasConcept C199185054 @default.
• W2166131252 hasConcept C2776612806 @default.
• W2166131252 hasConcept C2777289219 @default.
• W2166131252 hasConcept C2994592520 @default.
• W2166131252 hasConcept C55493867 @default.
• W2166131252 hasConcept C71240020 @default.
• W2166131252 hasConcept C86803240 @default.
• W2166131252 hasConceptScore W2166131252C112243037 @default.
• W2166131252 hasConceptScore W2166131252C121332964 @default.
• W2166131252 hasConceptScore W2166131252C1276947 @default.
• W2166131252 hasConceptScore W2166131252C181199279 @default.
• W2166131252 hasConceptScore W2166131252C185592680 @default.
• W2166131252 hasConceptScore W2166131252C18903297 @default.
• W2166131252 hasConceptScore W2166131252C199185054 @default.
• W2166131252 hasConceptScore W2166131252C2776612806 @default.
• W2166131252 hasConceptScore W2166131252C2777289219 @default.
• W2166131252 hasConceptScore W2166131252C2994592520 @default.

• next