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