Supplementary Information

Supplementary Methods

Lawn Avoidance Assay: Lawn avoidance assay was conducted as described by Pradel et al (12). Overnight-grown 50a (Bgl+) and OP50 (Bgl-) cultures were spotted on NGM plates without cholesterol containing glucose or salicin. The plates were incubated for 24hrs and 20 young adults were added on the spots. The number of worms inside the spot was counted in time interval of 4 hrs for 24 hrs and plotted as percentage occupancy which is

% occupancy = Number of worms inside the spot

Total number of worms added

Organic analysis of bacterial supernatant: Bacterial supernatant (grown M9 medium with

salicin) was collected after removing the cells by centrifugation. Thin Layer Chromatography

(TLC) of the supernatant was performed by manually spotting it on a pre-coated silica gel

TLC plate. Samples were partitioned using a 2% methanol in chloroform solvent system. For

NMR, the supernatant was mixed with dichloromethane solvent, and the organic layer was

separated using a separating funnel. The solvent was removed with the help of a Rota

Vaporiser and the resultant crystals were analyzed by NMR spectroscopy. 1H NMR and 13C

NMR spectra were recorded using a Bruker 400 MHz instrument. Chemical shifts were given

in parts per million and coupling constants (J) in Hertz

× 100

Strains Description SourcesE. coli strains

BGL1H Arb+Sal+ (33)

JM-chb1 Arb-Sal- (34)

Natural Isolates of Enterobacteriaceae

MS201 Arb+Sal+ (35)

Table S1: Additional strains of bacteria used in the study

Fig S1: Effect of β-glucoside metabolism on Dictyostelium discoideum a) Duplicate plot of NC4 viability experiment shown in Figure 1.b) Viability of amoebae plotted as number of Ax2 plaques formed on SM + bacterial lawn at different time points of incubation in SM/5 medium containing 55mM glucose and 50a (Bgl+) [50a + Glucose], 36mM arbutin and 50a (Bgl+) [50a + Arbutin], 36mM arbutin [Arbutin]. “Not detected (ND) “ indicates the absence of plaque formation at subsequent time points when similar dilution in other conditions showed detectable plaque formation

Time (in hours)

0 10 20 30 40 50 60

Log

PFU

/ml

0

2

4

6

8

50a + Glucose 50a + Arbutin Arbutin

ND

Time (in hours)

0 20 40 60 80

Log

PFU

/ml

0

2

4

6

8

AK1 + Salicin AK102 + Glucose AK102 +Salicin 50a + Glucose 50a + Salicin Salicin

NDND

a b

Experimental Conditions Plaque formation

AK1(Bgl-) + AX2 +

AK1 + AX2 + 35mM Salicin +

AK102(Bgl+) + AX2 +

AK102 + AX2 + 35mM Salicin -

Klebsiella (Bgl+) + AX2 +

Klebsiella + AX2 + 35mM Salicin -

MS201(Bgl+) + AX2 +

MS201+ AX2 + 35mM Salicin -

50a(Bgl+) + AX2 +

50a + AX2 + 35mM Salicin -

Klebsiella (Bgl+) + AX2 + 0.4% Bgl+ Supernatant* -

Table S2 Dictyostelium discoideum Ax2 plaque assay

(+) indicates plaque formation or presence of viable amoeba and (-) indicates no plaques or absence viable amoeba. n=3 * Bgl+ supernatant was obtained by growing Bgl+ bacteria in 36 mM salicin for 36hrs followed by concentrating the supernatant to solid form.

Experimental Conditions Strains Plaques

SM AK1(Bgl-) +

Klebsiella(Bgl+) +

36mM Arbutin + SM# AK1 +

Klebsiella -

14mM Salicin + SM# AK1 +

Klebsiella -

29mM Esculin + SM# AK1 +

Klebsiella +

ASE (3mM each) + SM# AK1 +

Klebsiella -

Table S3 : Effect of utilization of multiple β-glucosides on the viability of Ax2 ASE= Arbutin Salicin Esculin, SM# = SM without glucose n=3

Table S4. Lethal effect of salicin metabolism on C elegans

Eggs were exposed to the test environment (NGM + salicin + Bgl+ bacteria). L2*= L2 of the second generation. Bacteria were grown overnight at 37oC as spots in different test environments prior to the addition of N2 eggs. Subsequent incubations after addition of eggs were at 160C. n=3

Experimental Conditions Eggs

added

Stage of

lethality

AK1+ Glucose + 35mMSalicin 20 No lethality

AK102 + Glucose 20 No lethality

AK102 + 17.5mM Salicin 20 L2*

50a + Glucose 20 No lethality

50a + 17.5mM Salicin 20 L2*

MS201 + Glucose 20 No Lethality

MS201 + 17.5mM Salicin 20 L2*

Klebsiella aerogenes + Glucose 20 No Lethality

K. aerogenes + 17.5mM salicin 20 L2*

Time (in days)

0 2 4 6 8

% D

ead

Wor

ms

0

20

40

60

80

100

NGM +OP50 + 0.4% Bgl+ Supernatant NGM+ OP50

Fig S2: Effect of addition of concentrated culture supernatant of Bgl+ bacteria grown on salicin on N2 nematodes Bgl+ bacteria were grown on 35 mM salicin for 36hrs and the culture supernatant was concentrated to solid form. The concentrated supernatant was added to NMG agar at a final concentration of 0.4%. Young adult N2 worms were transferred to the plate seeded with OP50 and their survival was plotted over several days. The control plates had N2 worms on NGM + OP50 without the supernatant. n=3

Figure S3. Detection of saligenin (aglycone part of salicin)

Thin Layer Chromatogram of supernatants of bacteria grown in the presence salicin. Organic extracts of Bgl+ (lane 2-4) and Bgl- (lane 5) culture supernatants were fractionated by TLC. Their migration rates were compared with that of pure saligenin (lane 1) indicated by the arrow.

Fig. S 4a. 1H NMR spectra of crystals obtained from supernatants of bacteria grown in the presence of salicin. 1H NMR (400 MHz, CDCl3): δ 7.30 (s, 1H), 7.22-7.19 (t, J = 8 Hz, 1H), 7.04-7.02 (d, J= 8 Hz, 1H), 6.89-6.87 (d, J= 8 Hz, 1H), 6.87-6.83 (t, J= 8 Hz, 1H), 4.86 ( s, 2H), 2.35 (s, 1H)

Fig. S4b. 13C NMR spectra of crystals obtained from supernatants of bacteria grown in the presence of salicin. 13C NMR (400 MHz, CDCl3): δ 156.05, 129.51,127.81, 124.61, 120.07, 116.53, 64.64.

50a-Arbutin vs OP50-Arbutin

32mM Saligenin vs Solvent

Che

mot

axis

Inde

x

0.0

0.2

0.4

0.6

0.8

Figure S5a. Effect of arbutin metabolism and saligenin on worm behavior

Chemotaxis assay was performed as described in Materials and Methods. 50-Arbutin and 32mM Saligenin were the test conditions.

Figure S5b. Effect of salicin metabolism on worm behavior

Lawn avoidance assay of N2 (See Supplementary Methods for details). n=5. (Error bars indicate SD)

Time (in hours)

0 5 10 15 20 25 30

% O

ccup

ancy

0

20

40

60

80

100

120

OP50 + Salicin 50a + Glucose 50a + Salicin

N2 + 40 mM Saligenin dop-1 + 40 mM Saligenin

Figure S5c. Effect of salicin metabolism on worm behavior

Anesthetic assay of wild type N2 and the dop-1 mutant. One day adult worms were suspended in 40mM saligenin and their spontaneous thrashing movement was monitored after 20 minutes. Motile worms here means worms with spontaneous thrashing movement. Approximately 20 worms were used for each experiment. N=5

SoilSample

Bacterial colonies patched on

MacConkey agar

Bgl‑ Bgl+ Nema-todes

Amoeba

1 30 0 4 2 NA2 30 0 20 7 +3 30 4 0 8 -4 30 0 11 3 +5 30 21 0 2 -6 30 0 9 2 +7 23 19 0 1 NA8 26 21 0 0 NA9 26 21 0 9 NA10 22 14 0 1 NA11 23 15 0 1 NA12 23 18 1 0 NA13 23 20 0 0 NA14 30 20 6 0 NA15 26 10 0 8 +16 28 9 0 5 -17 25 0 7 4 +18 26 0 9 2 +19 28 2 2 5 +20 23 8 3 6 -21 24 15 0 11 +22 23 3 15 8 +23 26 16 0 4 +

24 26 13 3 30 +25 22 13 0 7 +26 23 14 0 22 -27 22 12 0 4 +28 24 0 12 14 +29 23 4 11 4 +30 21 4 2 8 +31 27 0 27 NA NA32 30 12 16 NA NA33 22 0 22 NA NA34 26 0 26 NA NA35 31 4 27 NA NA36 21 18 0 NA NA37 33 18 9 NA NA38 24 0 24 NA NA39 25 21 0 2 +40 26 10 11 1 +41 25 9 13 3 +42 26 7 10 4 +43 26 8 12 2 +44 26 12 13 1 -45 25 6 5 3 -46 26 25 0 4 -47 26 8 15 2 +48 25 3 14 1 +

Table S5: Frequency of occurrence of cellular slime moulds, nematodes and Enterobacteriaceae (Sal+/Sal-) in soil samples.+, - and NA represents presence, absence and not analyzed respectively in the case of amoeba.

Bgl+

Nematode Amoeba

3 17 65

Bgl-

AmoebaNematode

8

6

17

Bgl+

Nematode

Amoeba

11 Bgl-

Fig S6: Distribution of bacteria and their predators extrapolated from Table S5. Total soil samples considered=31

Isolate number Genera Identified

Isolate 1 Mesorhabditis sp.

Isolate 2 Oscheius tipulae

Isolate 3 Oscheius tipulae

Isolate 4 Oscheius tipulae

Isolate 5 Oscheius tipulae

Isolate 6 Rhabditis rainai

Table S6: Soil nematodes isolated in this study*

* Typed by using 18S rDNA sequencing (36)

Strain SourceCaenorhabditis elegansJU263 C. elegans wild isolate (From Le Blanc (Indre,

France). From a vegetable garden garbage pile.Caenorhabditis remaneiPB206 Associated with terrestrial isopod, Trachelipus

rathkii, Wright State University Biology Preserve, Dayton

Caenorhabditis angariaPS1010 Associated with palm and sugarcane weevils, in Trinidad and Florida

Caenorhabditis brenneriCB5161 Found in decaying, compost-like plant material in Sumatra, India, Trinidad, Costa Rica, and Guadeloupe

Caenorhabditis briggsaeDH1300 Wild isolate

Table S7: List of wild Caenorhabditis strains with habitats

Days0 2 4 6 8

% li

ve w

orm

s

0

20

40

60

80

100

120

0 mM saligenin 4 mM saligenin 10 mM saligenin 20 mM saligenin 28 mM saligenin

Figure S7. Viability assay of Mesorhabditis sp with different concentrations of saligenin

Survival of soil nematodes (plotted as percentage live worms versus time) was monitored on NGM plates containing different concentrations of saligenin and OP50. ~20 adult nematodes were used in each experiment. n=3

Time (in days)

0 2 4 6 8 10 12 14

% L

ive

wor

ms

0

20

40

60

80

100

120

140

C. angaria C. brenneri C. remanei C. elegansJU263 N2

Figure S8. Effect of saligenin on the viability of different wild Caenorhabditis strains

Survival of different Caenorhabditis species, plotted as percentage live worms versus time, was monitored on NGM plates containing 28mM saligenin and OP50. ~20 adult worms were used in each experiment. n=3

Saligenin Concentration28mM 20mM 10mM 4mM 1mM

% O

ccup

ancy

0

20

40

60

80

100

120

NGM Saligenin

Fig S9: Determination of minimum concentration of saligenin sensed and avoidedby Mesorhabditis sp.

Avoidance assay was performed as described before except multiple concentrations of saligenin were used here. ~ 20 adults were used for each experiment. n=5, Error bars indicate SD.

Test Condition

Hydroquinone Salicin

% O

ccup

ancy

0

20

40

60

80

100

120

NGM Test Condition

Fig S10: Avoidance assay performed with hydroquinone and salicin

Avoidance assay was performed as described before. Members of both Oscheius and Mesorhabditis were considered for the assay. ~ 20 adults were used for each experiment. The plot is representative of independent experiments performed eitherwith Oscheius or Mesorhabditis strains showing similar trend. n=5, Error bars indicate SD.

N2

C . elegans JU263

C. brenneri

C. remanei

Che

mot

axis

Inde

x

0.0

0.1

0.2

0.3

0.4

0.5

0.6

Chemotaxis Index

Fig S11: Chemotaxis assay performed with wild isolates of Caenorhabditis

Assay was performed as described before. N2 was used as control. n=5, Error bars indicate SD.

N2 Oscheius tipulae

Num

ber o

f nem

atod

es

0

50

100

150

200

250

AK1 + Soil + Salicin AK102 + Soil AK102 + Soil + Salicin Soil

Fig S12: Effect of salicin metabolism on nematodes in soil environment

The soil microcosm experiment was performed as described in Materials and Methods ~ 108 cellsof either AK102 (Bg+) or AK1 (Bgl-) bacteria were added in soil along with ~ 100 nematodes. Number of adult nematodes extracted after 10 days were plotted. n=3. Error bars here represent SD

Time (in days)

0 1 2 3 4 5 6

Log

CFU

/ml

0

2

4

6

8

AK102 + Buffer AK102 + Dead N2 AK102 + dead Ax2

Figure S13a,b. Growth of Shigella sonnei strain AK102 (Bgl+) on saligenin-killed amoebae (Ax2) and nematodes (N2).

Growth of AK102 in KK2 buffer supplemented with dead predators N2 (AK102 + N2), Ax2 (AK102 + Ax2) or without predators (AK102 + Buffer) was monitored and plotted as log CFU versus time. Both N2 and Ax2 were killed by incubating them with 32mM saligenin for 48hrs. The plots are representative of three independent experiments showing a similar trend

Time (in days)0 1 2 3 4 5 6

Log

CFU

/ml

0

2

4

6

8

AK102 + Buffer AK102 + Dead N2 AK102 + Dead Ax2

a b

Bet

a- G

al a

ctiv

ity (i

n M

iller

Uni

ts)

0

20

40

60

80

100

120

140

CHB1 + Buffer CHB1 + AX2 cells

Bet

a- G

al a

ctiv

ity (i

n M

iller

Uni

ts)

0

50

100

150

200

250

Fig S14: Activity of the bgl promoter in the presence of Ax2 cells or Ax2 culture supernatant. The strains BGL1H (Bgl+) and JM-chb1 (Bgl-) harboring bgl-lacZ and chb-lacZ transcriptional fusions respectively were cultured in the presence or absence of Ax2 cells or Ax2-culture supernatant. Beta-galactosidase activity in the two strains was measured after 6 hrs as described by Miller (37). n = 4. Error bar indicates SD. ** indicates p≤ .01 by Student’s t-test.

**

Supplementary references

33) Madan R, Moorthy S, Mahadevan S. 2008 Enhanced expression of the bgl operon of Escherichia coli in the stationary phase. FEMS Microbiol Lett 288: 131–139

34) Plumbridge, J. & Pellegrini O 2004 Expression of the chitobiose operon of Escherichia coli is regulated by three transcription factors: NagC, ChbR and CAP. Mol Microbiol 52: 437-449

35) Kachroo, A. H., A. K. Kancherla, N. S. Singh, U. Varshney & S. Mahadevan, 2007 Mutations that alter the regulation of the chb operon of Escherichia coli allow utilization of cellobiose. Mol Microbiol 66: 1382-1395.

36) Haber, M., Schungel, M., Putz, A., Muller, S., Hasert, B., and Schulenburg, H. 2005. Evolutionary history of Caenorhabditis elegans inferred from microsatellites: Evidence for spatial and temporal genetic differentiation and the occurrence of outbreeding. Mol. Biol. Evol. 22, 160-173

37) Miller JH 1992 A short course in bacterial genetics. A laboratory manual and handbook for Escherichia coli and related bacteria. Cold Spring Harbor Laboratory Press, Cold Spring Harbor,NY, USA.