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THE DISCOVERY OF QUORUM QUENCHING SOIL MICROBES FOR THE DEVELOPMENT OF ANTIMICROBIAL COMPOUNDS

BACTERIAL SILENCING:. THE DISCOVERY OF QUORUM QUENCHING SOIL MICROBES FOR THE DEVELOPMENT OF ANTIMICROBIAL COMPOUNDS. Sonia Rao. Quorum Quenching. Enzymes interfere with Quorum sensing, inhibiting virulence New class of pharmaceuticals Increase in antibiotic resistant. Lactonases.

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THE DISCOVERY OF QUORUM QUENCHING SOIL MICROBES FOR THE DEVELOPMENT OF ANTIMICROBIAL COMPOUNDS

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  1. BACTERIAL SILENCING: THE DISCOVERY OF QUORUM QUENCHING SOIL MICROBES FOR THE DEVELOPMENT OF ANTIMICROBIAL COMPOUNDS Sonia Rao

  2. Quorum Quenching • Enzymes interfere with Quorum sensing, inhibiting virulence • New class of pharmaceuticals • Increase in antibiotic resistant

  3. Lactonases • Removes lactone ring from the AHL molecule • AiiA gene - Erwinia carotovora , Pseudomonas aeruginosa

  4. Acylases • Detach nitrogen bond to form a fatty acid chain and homoserine lactone • PvdQ and QuiP - P. aeruginosa

  5. Hypothesis • Soil communities would contain quorum quenching isolates • Late successional communities would contain greater number of quorum quenching isolates than early successional communities

  6. Variables • Independent - Successional stage - Competitive nature • Dependent -Quorum quenching isolates -Frequency of natural communities to produce quorum quenchers • Constant: - Incubation time for soil samples and isolates - Amount of x-gal

  7. Method • C.violaceum 12472, 31532, CV026 • A.tumefaciens A136, KYC6 • X-gal, antibiotics

  8. Method • 1 gram of 10 soil sample cultured -R2A broth, vortex • 0.1 ml added to R2A plate

  9. Method • C.violaceum 12472 next to isolate • A.tumefaciens A136 • TraR gene detects AHL C6-C14 • lacZ gene which expresses β-galactosidase

  10. Method • A.tumefaciens A136 bioassay • 4.5 mg/ml of tetracycline and 50 mg/ ml of spectinomycin • 50 microliters x-gal (5-bromo-4-chloro-3-indolyl-galactopyranoside) • 3 replicates • Frozen in glycerin

  11. Method • CV026 and 31532 • A136 and KYC6

  12. Method • Gene sequencing • Cells suspended in 10 µl of sterile water • 10 µl of sterile water, 2 µl of 27 F primer, 2 µl of 1392 primer, 4 µl of a master mix, 10 µl of cells culture PCR program: 95 °C – 5.5 min 58 °C – 30 sec 72 °C – 6.5 min

  13. Method • Gel electrophoresis • 2 µl Ethidium bromide • 2 µl added to eppendorf tubes-2nd PCR

  14. Method • Gene sequencing lab • BLAST ( Basic Local Alignment Search Tool) Database

  15. Summation of Method Culturing of Soil Samples and isolates C.Violaceum 12472 bioassay A.tumefaciens A136 bioassay Gene Sequencing and BLAST

  16. Results • 10 Isolates • Late Successional communities have greater number of quorum quenching isolates than early succession communities (p = 0.03). • Gene sequencing- inconclusive

  17. Results

  18. Results

  19. Conclusions • Soil isolates that produce quorum quenching enzymes was supported • Late succession communities would have a greater number of isolates that quorum quench than early succession communities was supported (p = 0.03).

  20. Applications • Sources for quorum quenching microbes • Increase in antibiotics resistant bacteria

  21. Future Studies • Identifying the quorum quenching enzyme • Inducing E.coli to produce the enzyme

  22. Acknowledgments • Chris Reeves and Kim Griggs • Professor Robert McLean • Professor Dave Westenberg • Dr. Chris Waters • Diane Ayers

  23. Bibliography • Bjarnsholt T. and Givskov M. 2007. Quorum-sensing blockade as a strategy for enhancing host defences against bacterial pathogens. Philosophical Transactions of the Royal Society B. 362: 1213-1222.  • Blosser R.S. and Gray K.M. 2000. Extraction of violacein from Chromobacterium violacein provides a new quantitative bioassay for N-acyl homoserine lactone autoinducers. Journal of Microbiological Methods. 40(1): 47-55. • Cámara M. et al. 2002. Controlling infection by tuning in and turning down the volume of bacterial small-talk. Lancet Infectious Diseases. 2(11): 667-676.  • Christensen et al. 2003. Quorum-sensing-directed protein expression in Serratia proteamaculans B5a. Microbiology. 149: 471-483.  • Chu W et al. 2009. Bioassay of quorum sensing compounds using Chromobacterium violacein. Methods in Molecular Biology. Chapter two  • Clarridge J.E. 2004. Impact of 16S rRNA gene sequence analysis for identification of bacteria on Clinical microbiology and infectious disease. Clinical Microbiological Reviews. 17(4):840- 862.  • Czajkowski and Jafra. 2008. Quenching of acyl-homoserine lactone-dependent quorum sensing by enzymatic disruption of signal molecules. Acta Biochimica Polonica Journal. 56(1):1-16.  de Kievit T.R. and Iglewski B. 2000. Bacterial Quorum Sensing in pathogenic Relationships. Infection and Immunity. 68(9): 4839-4849  • de Nys et al. 1993. New halogenated furanones from the marine alga Delisea pulchra. Tetrahedron. 49: 11213-11220.   • Defoirdt T. et al. 2008. Quorum sensing and quorum quenching in Vibrio harveyi: lessons learned in vivo work. International Society for Microbial Ecology Journal. 2:19-26.

  24. Bibliography • Dong Y.H. et al. 2001. Identification of quorum-quenching N-acyl homoserine lactonases from Bacillus species. Applied and Environmental Microbiology. 68(4):1754-1759.  • Dong Y.H et al. 2001. Quenching quorum-sensing-dependent bacterial infection by an N-acyl homoserine lactone. Nature. 411:813-817.  • Dong Y.H. and Zhang L.H. 2005. Quorum sensing and Quorum-Quenching Enzymes. Journal of Microbiology. 43:101-109. • Dong Y.H. et al. 2007. Quorum-quenching microbial infections: mechanisms and implications. Philosophical Transactions of the Royal Society B. 362:1201-1211.  • Dong Y.H. et al. 2000. AiiA, enzyme that inactivates the acylhomoserine lactone quorum- sensing signal and attenuates the virulence of Erwinia carotovora. Proceedings of the National Academy of Sciences. 97(7):3526-31. • Drancourt M et al. 2000. 16S ribosomal DNA sequence analysis of a large collection of environmental and clinical unidentifiable bacterial isolates. Journal of Clinical Microbiology. 38(10): 3623-3630.   • Fox G.E. et al. 1992. How close is close: 16S rRNA sequence identity may not be sufficient to guarantee species identity. Journal of Systemic Bacteriology. 42: 166-170.  • Gee J.E. et al. 2003. Use of 16S rRNA gene sequencing for rapid identification and differentiation of Burkholderia pseudomallei and B. mallei. Journal of Clinical Microbiology. 41(10): 4647-4654. • González J.E. and Keshavan N.D. 2006. Messing with Bacterial Quorum Sensing.Microbiology and Molecular Biology Reviews. 70(4): 859-875.

  25. Bibliography • Gram et al. 2002. Production of acylated homoserine lactones by psychrotrophic members of the Enterobacteriaceae isolated from foods. Applied environmental Microbiology. 65:3458-3463.  • Hentzer M.2002. Inhibition of quorum sensing in Pseudomonas aeruginosa biofilms bacteria by a halogenated furanone compound. Microbiology. 148: 87-102.  • Kjelleberg S et al. 2008. Quorum-sensing Inhibition. . In: Winans S.C., Bassler B.L., editors. Chemical Communications among Bacteria. American Society for Microbiology. p. 393-416.  • Lin Y.H. et al. 2003. Acyl-homoserine lactone aclyase from Ralstonia strain XJ12B represents a novel and potent class of quorum-quenching enzymes. Molecular Microbiology. 47: 849-860. •  Manefield M et al, 1999. Evidence that halogenated furanones from Delisea pulchra inhibit acylated homoserine lactone (AHL)-mediated gene expression by displacing the AHL signal from its receptor protein. Microbiology. 145:283-291.   • McClean K.H. et al. 1997. Quorum sensing and Chromobacterium violaceum: exploitation of violacein production and inhibition for the detection of N-acyl homoserine lactones. Microbiology 143:3703-3711.   • McLean RJ et al. 2004. A simple screening protocol for the identification of quorum signal antagonists. Journal of Microbiological Methods. 58(3): 351-360. • McLean R.J. et al. 1997. Evidence of autoinducer activity in naturally occuring biofilms. FEMS Microbiology letters. 154: 259-263.   • Molina L et al. 2003. Degradation of pathogen quorum-sensing molecules by soil bacteria: a preventive and curative biological control mechanism. Microbiology Ecology. 45(1): 71-81. 

  26. Bibliography • Park S.Y. et al. 2003. AhlD, an N-acylhomoserine lactone in Arthrobacter sp., and predicted homologues in other bacteria. Microbiology. 149: 1541-1550. • Rao. S. 2009. The Effect of Elevated Temperature on Quorum Sensing and Biofilm Formation in Pseudomonas aeruginosa. • Rasmussen T.B. et al. 2005. Screening for Quorum-Sensing Inhibitors (QSI) by Use of a Novel Genetic System, the QSI Selector. Journal of Bacteriology. 187(5): 1799-1814. • Ravn L et al. 2001. Methods for detecting acylated homoserine lactones produced by gram- negative bacteria and their application in studies of AHL-production kinetics. Journal of Microbiological Methods. 44(3): 239-251.   • Ren D. et al. 2008. Quorum sensing inhibitory compounds. In: Balaban N, editor. Control of Biofilm infections by Signal Manipulations. Springer publishing co. p. 51-79.  • Roche D.M. et al. 2004. Communications blackout? Do N-acylhomoserine-lactone-degrading enzymes have any role in quorum sensing? Micobiology. 150: 2023-2028.  • Shaw P.D. et al. 1997. Detecting and characterizing N-acyl homoserine lactone signal molecules by thin-layer chromatography. Proceedings of the National Academy of Sciences. 94(12): 6036-6041.   • Sio C.F. et al. 2006. Quorum quenching by an N-acyl-homoserine lactone acylase from Pseudomonas aeruginosa PAO1. Journal of Infection and Immunity. 74(3): 1673-82.  • Smith et al. 2003. Induction and inhibition of Pseudomonas aeruginosa quorum sensing by synthetic autoinducer analogs. Chemistry and Biology. 10(1): 81-89.  • Stickler D.J. et al. 1998. Biofilm on Indwelling Urethral Catheters produce Quorum-sensing signal molecules In Situ and In Vitro. Applied and Environmental Microbiology. 64(9):3486- 3490.

  27. Bibliography • Ulrich R.L. 2004. Quorum Quenching: Enzymatic Disruption of N-acylhomoserine lactone- mediated bacterial communication in Burkholderia thailandensis. Journal of Applied and Environmental Microbiology. 70(10): 6173-6180. • Uroz S. et al. 2003. Novel bacteria degrading N-acylhomoserine lactones and their use as quenchers of quorum-sensing-regulated functions of plant-pathogenic bacteria. Microbiology. 149: 1981-1989.  • Wang L.H. et al. 2008. Quorum Quenching: Impact and mechanisms. In: Winans S.C., Bassler B.L., editors. Chemical Communications among Bacteria. American Society for Microbiology. P379-392.   • Want LH et al. 2002. Specificity and enzyme kinetics of the quorum-quenching N-Acyl homoserine lactone lactonase (AHL-lactonase). Journal of Biological Chemistry. Vol 279: 13645-13651.  • Waters C.M and Bassler B.L. 2005. QUORUM SENSING: Cell-to-Cell Communication in bacteria. Annual Review of Cell and Development Biology. 21: 319-346.  • Weber M. et al. 2010. A previously uncharacterized gene, yjfO (bsmA), influences Escherichia coli biofilm formation and stress response. Microbiology. 156.   • Whitehead N.A. et al. 2002. The regulation of virulence in phytopathogenic Erwinia species: Quorum sensing, antibiotics, and ecological considerations. Antonie Van Leeuwenhoek. 81: 223-231.  • Williams P. et al. 2007. Look who’s talking: communication and quorum sensing in the bacterial world. Philosophical Transactions of the Royal Society B. 362(1483): 1119-1134.   • Xu et al. 2003. Degradation of N-acylhomoserine lactones, the bacterial quorum-sensing molecules, by acylase. Journal of Biotechnology. 101(1): 89-96.

  28. Bibliography • Zhang H.B. et al. 2007. Detection and analysis of quorum-quenching enzymes against acyl homoserine lactone quorum-sensing signals. Current Protocols in Microbiology. Chapter 1:Unit 1C.3.   • Zhang H.B. et al. 2002. Genetic control of quorum-sensing signal turnover in Agrobacterium tumefaciens. Proceedings of the National Academy of Sciences. 99:4638-4643.  • Zhang L.H. and Dong Y.H. 2004. Quorum sensing and signal interference: diverse implications. Molecular Microbiology. 53(6):1563-1571.  • Zhang L.H. 2003. Quorum quenching and proactive host defense. Trends in Plant Science. 8(5):238-244.

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