BACTERIAL ATTRACTION AND QUORUM SENSING INHIBITIONIN CAENORHABDITIS ELEGANS EXUDATES
SUPPORTING ONLINE MATERIAL
SUPPORTING METHODS
Pyocyanin Assays. The pyocyanin quantification assay was performed by monitoring the absorbance of pyocyanin at 520 nm in acidic solution (Kurachi, 1958a, b; MacDonald, 1967). PAO1 bacteria, grown overnight at 37oC in LB broth with shaking, was used as an inoculum. For the assays, PAO1 inoculum was added to give a final OD600 of 0.04 to 3 ml of LB broth by supplementing with or without 100 µl of filter-sterilized exudate (3000 worm equivalents: WE) and allowed to grow 24 h at 37oC with shaking. After 24 h, the culture was extracted with 3 mlof chloroform. The chloroform phase was washed with 1 ml of 0.2 N HCL to give a pink to deep red solution. Concentrations, expressed as µg/ ml of pyocyanin produced, were determined by multiplying the absorption at 520 nm by 17.072(Kurachi, 1958a, b). Three independent experiments, each with 3 replications, were done.
Elastase Assays. Elastolytic activity in PAO1 culture fluids was determined by elastin congo red (ECR) assays(Bjorn et al., 1979) with modifications. Briefly, PAO1 cells from mid-log-phase cultures in PTSB were washed and resuspended with PTSB to an OD600 of 0.05, by supplementing with or without 100 µl of filter-sterilized exudate (3000 WE). After 21 h at 37oC with shaking, culture supernatants were filter-sterilized (0.45 µm pore-size filter). Triplicate 100 µl samples of culture filtrates were added to tubes containing 20 mg of ECR and 1 ml of buffer (phosphate buffer, pH 7.0). Samples were incubated for 18 h at 37oC with shaking, centrifuged to remove excess ECR, and the OD495 was measured. Absorption due to pigments produced by PAO1 was corrected for by subtracting the OD495 of each sample that had been incubated in the absence of ECR. Three independent experiments, each with 3 replications, were done.
Protease Assay. To determine protease activity, we followed the method described by Greene et al. (1989). Briefly, triplicate 100 µl aliquots of exudate(3000 W.E.) were added to the reaction mixture containing 0.8 % azocaesin in 500 µl of 50mM phosphate buffer (pH 7.0). Reaction mixtures were incubated at 25oC for 3 h. The reaction was stopped by adding 0.5 ml of 1.5 M HCL, and the mixture was placed on ice for 30 minutes and then centrifuged. After addition of 0.5 ml of 1 N NaOH the OD440 was measured.Three independent experiments, each with 3 replications, were done.
SUPPORTING FIGURES and TABLES
Fig. S1 Effect of Caenorhabditiselegans exudates on Pseudomonas aeruginosa QS regulated virulence factors. (a) Small increase in pyocyanin content using PAO1 cells in response to C. elegans secretions. Error bars indicate standard deviations. Different letters on the top of the bar indicates significance (b) at Pvalue 0.05 compared with control (a). The results represented are the mean of three experiments in triplicates each (N=9). L3, L4, YA (young adult), A (adult) exudates. (b) No change in elastase and protease activity in response to worm exudates. Spectrometric assays of total protease and elastase activities were performed using the substrates azocaesin and elastin-congo red as described in Materials and Methods. Error bars indicate standard deviations. The results represented are the mean of three experiments in triplicates each (N=9).
Fig.S2 Covariance TOCSY spectrum and 1D traces of young adult Caenorhabditiselegans exudates. The experimental DemixC traces are shown above the TOCSY spectrum. The automatically identified compounds represent, with a two exceptions, the top database matches (Supp Table 1). The exceptions are leucine and trehalose. Leucine was the number 3 match due to failure of the 0.03 ppm clustering threshold to incorporate the shoulder of the 1.5 ppm signal in trace 6 into the rest of the signal. The position of the shoulder is then identified by the third scoring algorithm and penalized. The return of trehalose second to glucose illustrates an interesting point in the nature of the method. As the DemixC protocol deconvolutes TOCSY spectra by selecting traces, good traces then belong to a single spin system of the target compound. Monosaccharides like Glucose exist in equilibrium between their alpha and beta states in solution. For trace 2 both trehalose and glucose could be the right answerbecause trehalose is a disaccharide with an alpha 1, 1 glycosidic bond, so trehalose would have a similar spectrum to alpha glucose, but not beta-glucose.
Table S1 KEY FOR SEMI-AUTOMATED CHEMICAL IDENTIFICATION USING DEMIXC TRACES OF TOCSY OF YOUNG ADULT Caenorhabditis elegans EXUDATES
Compound / Forward assignment / Reverse assignment / Weighted Matching / Average Penalty ValueTrace 1
Glucose / 1 / 1 / 1 / 0.019Glucose-6-Phosphate / 2 / 2 / 2 / 0.03
Xylose / 4 / 3 / 3 / 0.045
Trace 2
Glucose / 1 / 2 / 5 / 0.077Trehalose / 4 / 1 / 11 / 0.093
Arabinose / 5 / 4 / 8 / 0.092
Trace 3
Glutamic Acid / 3 / 1 / 3 / 0.038Arginine Glutamate / 2 / 10 / 2 / 0.054
Acetyl Glutamine / 6 / 3 / 6 / 0.061
Trace 5
Isoleucine / 1 / 1 / 1 / 0.018Propionic Acid / 4 / 2 / 4 / 0.2
Valine / 3 / 3 / 5 / 0.2
Trace 6
Isoleucine / 4 / 3 / 3 / 0.17Lysine / 7 / 4 / 6 / 0.21
Leucine / 1 / 1 / 27 / 0.25
Trace 7
Lysine / 2 / 1 / 2 / 0.089dTMP / 1 / 4 / 1 / 0.098
Citrulline / 5 / 2 / 3 / 0.16
Trace 8
R-Lactate / 1 / 1 / 1 / 0.0017L-Lactic Acid / 2 / 2 / 2 / 0.0028
Acetyl Alanine / 3 / 3 / 3 / 0.014
Trace 9
Valine / 1 / 2 / 1 / 0.03Isoleucine / 3 / 5 / 3 / 0.06
Leucine / 5 / 3 / 9 / 0.17
Table S2 CHROMATOGRAPHIC CONDITIONS USED ON THE SUNFIRE COLUMN FOR AMINO ACID ANALYSIS
Time / Flow(ml/min) / %A / %B / %C1 / %D / Curve
1 / 1.0 / 0.0 / 0.0 / 100.0 / 0.0
2 / 0.50 / 1.0 / 0.0 / 0.0 / 100.0 / 0.0 / 11
3 / 22.50 / 1.0 / 0.0 / 3.5 / 96.5 / 0.0 / 6
4 / 40.12 / 1.0 / 0.0 / 3.5 / 26.5 / 70.0 / 6
5 / 46.00 / 1.0 / 0.0 / 5.0 / 95.0 / 0.0 / 6
6 / 48.00 / 1.0 / 0.0 / 9.0 / 91.0 / 0.0 / 6
7 / 66.00 / 1.0 / 0.0 / 17.0 / 83.0 / 0.0 / 6
8 / 80.00 / 1.0 / 40.0 / 60.0 / 0.0 / 0.0 / 6
9 / 86.00 / 1.0 / 0.0 / 0.0 / 100.0 / 0.0 / 6
Chromatographic conditions developed in-house, with eluent C1 adopted from Cohen and Michaud (Cohen and Michaud, 1993).
Table S3 CHROMATOGRAPHIC CONDITIONS USED ON THE NOVA-PAK COLUMN FOR AMINO ACID ANALYSIS
Time / Flow(ml/min) / %A / %B / %C2 / %D / Curve
1 / 1.0 / 0.0 / 0.0 / 90.0 / 10.0
2 / 0.50 / 1.0 / 0.0 / 1.0 / 89.0 / 10.0 / 6
3 / 17.00 / 1.0 / 0.0 / 2.0 / 88.0 / 10.0 / 6
4 / 24.00 / 1.0 / 0.0 / 5.0 / 86.0 / 9.0 / 6
5 / 32.00 / 1.0 / 0.0 / 12.0 / 63.0 / 25.0 / 6
6 / 33.50 / 1.0 / 0.0 / 12.5 / 0.0 / 87.5 / 6
7 / 33.80 / 1.3 / 0.0 / 12.5 / 22.0 / 65.5 / 6
8 / 37.00 / 1.3 / 0.0 / 13.0 / 22.0 / 65.0 / 6
9 / 48.00 / 1.3 / 0.0 / 15.0 / 22.0 / 63.0 / 6
10 / 50.00 / 1.3 / 40.0 / 60.0 / 0.0 / 0.0 / 6
11 / 55.00 / 1.3 / 40.0 / 60.0 / 0.0 / 0.0 / 11
12 / 57.00 / 1.0 / 0.0 / 0.0 / 90.0 / 10.0 / 6
Chromatographic conditions as recommended by Waters, except that Nova-Pak column replaced AccQ•Tag column.
REFERENCES
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GREENE, R. V., COTTA, M. A., and GRIFFIN, H. L. 1989. A novel, symbiotic bacterium isolated from marine shipworm secretes proteolytic activity. Curr. Microbiol. 19:353-356.
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MACDONALD, J. C. (ed.) 1967. Pyocyanine, Sringer-Verlag, New York.