Supplementary Information. StrainsDescriptionSources E. coli strains BGL1HArb + Sal + (33)...
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Transcript of Supplementary Information. StrainsDescriptionSources E. coli strains BGL1HArb + Sal + (33)...
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 Sources
E. 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
Lo
g P
FU
/ml
0
2
4
6
8
50a + Glucose 50a + Arbutin Arbutin
ND
Time (in hours)
0 20 40 60 80
Lo
g P
FU
/ml
0
2
4
6
8
AK1 + Salicin AK102 + Glucose AK102 +Salicin 50a + Glucose 50a + Salicin Salicin
ND
ND
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
Wo
rms
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
Ch
emo
tax
is In
de
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 NA
2 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 NA
8 26 21 0 0 NA
9 26 21 0 9 NA
10 22 14 0 1 NA
11 23 15 0 1 NA
12 23 18 1 0 NA
13 23 20 0 0 NA
14 30 20 6 0 NA
15 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 NA
32 30 12 16 NA NA
33 22 0 22 NA NA
34 26 0 26 NA NA
35 31 4 27 NA NA
36 21 18 0 NA NA
37 33 18 9 NA NA
38 24 0 24 NA NA
39 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
Days
0 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
wo
rms
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 Concentration
28mM 20mM 10mM 4mM 1mM
% O
ccu
pan
cy
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
cc
up
an
cy
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
Ch
emo
taxi
s In
dex
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
Nu
mb
er o
f n
emat
od
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 6L
og
CF
U/m
l
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.