THE DISCOVERY OF QUORUM QUENCHING SOIL MICROBES FOR THE DEVELOPMENT OF
ANTIMICROBIAL COMPOUNDS
Sonia Rao
BACTERIAL SILENCING:
• Enzymes interfere with Quorum sensing, inhibiting virulence
• New class of pharmaceuticals
• Increase in antibiotic resistant
Quorum Quenching
• Removes lactone ring from the AHL molecule• AiiA gene - Erwinia carotovora , Pseudomonas
aeruginosa
Lactonases
• Detach nitrogen bond to form a fatty acid chain and homoserine lactone
• PvdQ and QuiP - P. aeruginosa
Acylases
• Soil communities would contain quorum quenching isolates
• Late successional communities would contain greater number of quorum quenching isolates than early successional communities
Hypothesis
• 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
Variables
• C.violaceum 12472, 31532, CV026• A.tumefaciens A136, KYC6• X-gal, antibiotics
Method
Method
• 1 gram of 10 soil sample cultured -R2A broth, vortex
• 0.1 ml added to R2A plate
• C.violaceum 12472 next to isolate
• A.tumefaciens A136
Method
• TraR gene detects AHL C6-C14
• lacZ gene which expresses β-galactosidase
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
• CV026 and 31532
Method
• A136 and KYC6
• Gene sequencing
• Cells suspended in 10 µl of sterile water
Method
• 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 min58 °C – 30 sec72 °C – 6.5 min
• Gel electrophoresis
Method
• 2 µl Ethidium bromide
• 2 µl added to eppendorf tubes-2nd PCR
• Gene sequencing lab
• BLAST ( Basic Local Alignment Search Tool) Database
Method
Summation of Method
Culturing of Soil Samples and isolates
C.Violaceum 12472 bioassay
A.tumefaciens A136 bioassay
Gene Sequencing and BLAST
• 10 Isolates
• Late Successional communities have greater number of quorum quenching isolates than early succession communities (p = 0.03).
• Gene sequencing- inconclusive
Results
Comparison of quourum quenchers by early and late succesion
0
1
2
3
4
5
6
7
8
Early Succession Late succession
Soil Community Stage
Ave
rag
e %
of
Qu
oru
m q
uen
cher
s
Results
% of Inhibitors, Possible quorum quenchers, and Verified Quorum quenchers out of total # of Isolates
0
5
10
15
20
25
30
35
40
Inhibitors Possible quorum quenchers Verified quorum quenchers
Perc
enta
ge o
f Iso
late
s fr
om to
tal #
of
isol
ates
Results
• 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).
Conclusions
• Sources for quorum quenching microbes
• Increase in antibiotics resistant bacteria
Applications
• Identifying the quorum quenching enzyme
• Inducing E.coli to produce the enzyme
Future Studies
• Chris Reeves and Kim Griggs
• Professor Robert McLean
• Professor Dave Westenberg
• Dr. Chris Waters
• Diane Ayers
Acknowledgments
• 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 proteamaculansB5a. 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.
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 ofenvironmental 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 toguarantee 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 ClinicalMicrobiology. 41(10): 4647-4654.
• González J.E. and Keshavan N.D. 2006. Messing with Bacterial Quorum Sensing. Microbiologyand Molecular Biology Reviews. 70(4): 859-875.
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 forMicrobiology. 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 signalfrom 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.
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 NovelGenetic 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 ofBiofilm 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 bysynthetic 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.
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 forMicrobiology. 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. Vol279: 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 bacterialworld. 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.
Bibliography
• Zhang H.B. et al. 2007. Detection and analysis of quorum-quenching enzymes against acylhomoserine lactone quorum-sensing signals. Current Protocols in Microbiology. Chapter1: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.
Bibliography