Interactions in the competitive coexistence process of
Streptomyces sp. and Escherichia coli
Liyan Yu, Zhifei Hu, Zhijuan Hu, Zhongjun Ma
Contents
1. Competition ability evaluation of Streptomyces sp. and E.
coli
2. Structure determination of novel compound 5
3. structure determine data of compounds 1-4 and 6-9
4. Tables and Figures
Fig. S1. Routine coculture of Streptomyces sp. and E. coli: gram
staining of Streptomyces sp. single culture (A), gram staining of
Streptomyces sp. and E. coli coculture using a routine method,
broth was dominated by E. coli, Streptomyces sp. fragments was
sporadically scattered. (B). Competitive coculture process of
Streptomyces sp. and E. coli: single culture of Streptomyces sp. in
MM medium under static condition was a fermentation broth of
transparent culture medium with clustered mycelium sinking in the
bottom (C), after the adding of E. coli, the culture medium quickly
became an even muddy suspension of E. coli, the mycelium of
Streptomyces sp. floated to the medium surface (D), and developed
aerial hyphae (E), after 14 days of co-cultivation, the culture
medium became transparent again, with the E. coli sinking to the
bottom and Streptomyces sp. colonized and sporulated at the medium
surface (F).
Fig. S2. Secondary metabolites HPLC fingerprints of Streptomyces
sp. and E. coli competitive coculture medium (black) and
Streptomyces sp. floated mycelium (pink).
Fig. S3. Secondary metabolites of Streptomyces sp. cultured in
E. coli spent medium.
Fig. S4. Secondary metabolites of E. coli cultured in
Streptomyces sp. spent medium.
Fig. S5. Morphological changes and important interactions
happened during the competitive coexistence process of Streptomyces
sp. and E. coli.
Fig. S6. pH test paper in the enclosed air space of co-culture
medium. Left sample: co-culture medium of Streptomyces sp. with E.
coli. Right sample: co-culture medium of Streptomyces sp. with
another Streptomyces.
Fig. S7. Chemical structures of compounds 1-9 (A) and 1H-1H COSY
(bold bond) and key HMBC (arrow) correlations of 5 (B).
Fig. S8. UV spectrum of 6.
Fig. S9. HRESIMS spectrum of 6.
Fig. S10. IR spectrum of 6.
Fig. S11. 1H NMR spectrum of 6 (CDCl3, 400 MHz).
Fig. S12. 13C NMR spectrum of 6 (CDCl3, 400 MHz).
Fig. S13. HMQC spectrum of 6 (CDCl3).
Fig. S14. HMBC spectrum of 6 (CDCl3).
Fig. S15. 1H-1H COSY spectrum of 6 (CDCl3).
Table 1. 1H and 13C NMR data of compound 5 (CDCl3).
1. Competition ability evaluation of Streptomyces sp. and E.
coli
The competing ability of Streptomyces sp. and E. coli was
evaluated by co-culture using a routine culture method with some
modification (App Environ Microbiol 73:6159-6165; J Nat Prod
70:515-520). Specifically, Streptomyces sp. was first cultivated in
200 mL ISP2 medium (yeast extract 4 g, malt extract 10 g, glucose 4
g, 75% sea water 1L, pH 7.5) under 150 rpm for 3 days, then
different ratios of (0.01%, 0.1%, 1% v/v) E. coli suspension (OD600
0.5) was added at different time points (3 days, 7 days, 10 days),
and further co-cultivated in 28 oC for 7 days. The resulted
co-culture medium was then subjected to Gram staining analysis for
population evaluation.
2. Structure determination of novel compound 5
Structure elucidation of compound 5 was as follows: it was
isolated as orange solid (methanol), with UV absorptions maxima at
228, 282 and 327 nm. IR absorptions implied the presence of
secondary amino group or amide (3344 cm-1), saturated alkyl
hydrogen (2927 cm-1, 2855 cm-1), carbonyl (1710 cm-1), amide
carbonyl (1653 cm-1) and ortho-substituted phenyl (746 cm-1). The
negative-ion HR-ESI-MS showed [M - H]- ion at m/z 420.1351,
indicating a molecular formula of C26H19N3O3 ([M - H] calculated
420.1348). The 1H and 13C NMR data of 5 (Table S1) revealed two
2-substituted indoles [H 7.47 (2H, d, J = 7.9 Hz), H 7.04 (2H, t, J
= 7.9 Hz), H 7.19 (2H, t, J = 7.9 Hz), H 7.38 (2H, d, J = 7.9 Hz),
H 8.00 (2H, br.s), H 6.9 (2H, s)] (Veluri et al., 2003), and one
ortho-substituted phenyl [H 7.60 (1H, d, J = 8.3 Hz), H 6.88 (1H,
t, J = 8.3 Hz), H 7.26 (1H, t, J = 8.3 Hz), H 6.98 (1H, d, J = 8.3
Hz)]. HMBC correlations (Fig. S7B) showed that the two indoles were
connected by CH-8 (H 6.12, C 36.7). It also showed the remained
three tetrahedral carbons whose chemical shifts were more than 150,
among them, C-11 (C 168.5) was a carbonyl substituted at the
ortho-substituted phenyl, C-15a (C 156.8) belongs to
ortho-substituted phenyl, which suggests it was substituted by an
oxygen, so the remained carbonyl C-17 (C 158.0) links directly to
this C-17 and forms an ester. According to the HR-ESI-MS, the
remained NH was connected to C-11 and forms an amide. HMBC
correlations of H-9 to C-11 and C-17 suggesting the ester, amide
and CH were connected together to form a seven membered ring. Whats
more, 1H-1H COSY correlation of H-8 (H 6.12) and H-9 (H 6.90)
suggested these two CH were directly linked, so the whole structure
was determined as showed in Fig. S7A.
3. structure determine data of compounds 1-4 and 6-9
3-(hydroxymethyl)-4(1H)-quinolinone (1): 1H NMR (CDCl3, 400
MHz): 8.69 (1H, s), 8.28 (1H, t, J=4.2 Hz), 7.93 (1H, d, J=3.1 Hz),
7.46 (1H, t, J=4.2 Hz), 7.34 (2H, t, J=4.2 Hz), 4.79 (2H, s). 13C
NMR (CDCl3, 100 MHz): 193.2, 136.1, 130.5, 125.0, 124.1, 122.0,
114.4, 111.6, 65.4.
Halichrome A (2): 1H NMR (CDCl3, 400 MHz): 7.65 (1H, d, J=7.7
Hz), 6.85 (1H, t, J=7.4 Hz), 7.50 (1H, t, J=7.4 Hz), 6.93 (1H, d,
J=8.2 Hz), 2.30 (2H, m), 0.92 (3H, t, J=7.4 Hz), 8.16 (1H, br.s),
7.20 (1H, d, J=2.4 Hz), 7.49 (1H, d, J=8.7 Hz), 7.02 (1H, t, J=7.7
Hz), 7.16 (1H, t, J=7.7 Hz), 7.34 (1H, d, J=8.2 Hz). 13C NMR
(CDCl3, 100 MHz): 69.7, 203.2, 121.0, 125.0, 119.0, 137.4, 112.3,
160.8, 30.3, 8.0, 122.5, 115.0, 125.0, 120.0, 120.0, 122.5, 111.5,
136.8.
1,1,1-Tris (3-indolyl) methane (3): 1H NMR (CDCl3, 400 MHz):7.92
(3H, br.s), 6.96 (3H, br.s), 7.63 (2H, d, J=7.8 Hz), 7.09 (2H, t,
J=7.5 Hz), 7.19 (2H, t, J=7.5 Hz), 7.37 (2H, d, J=8.1 Hz), 4.25
(1H, s). 13C NMR (CDCl3, 100 MHz): 122.2, 119.1, 127.6, 119.2,
115.7, 121.9, 111.0, 136.5, 21.2.
Vibrindole A (4) : 1H NMR (CDCl3, 400 MHz):7.90 (2H, br.s), 6.94
(2H, d, J=1.6 Hz), 7.58 (2H, d, J=7.9 Hz), 7.04 (2H, t, J=7.5 Hz),
7.19 (2H, t, J=6.7 Hz), 7.36 (2H, d, J=8.2 Hz), 4.68 (1H, q, J=7.1
Hz), 1.81 (3H, d, J=7.1 Hz). 13C NMR (CDCl3, 100 MHz):121.2, 121.8,
126.9, 119.7, 119.0, 121.7, 111.0, 136.7, 28.2, 21.7.
Cyclo(Pro-Val) (6): 1H NMR (CDCl3, 400 MHz):3.58 (2H, m), 2.04
(2H, m), 2.14 (1H, m), 2.35 (1H, m), 4.12 (1H, t, J=6.5 Hz), 6.38
(1H, s), 4.02 (1H, dd, J=7.5 Hz, 2.8 Hz), 1.80 (1H, m), 1.92 (1H,
m), 1.54 (1H, m), 0.96 (3H, d, J=5.2 Hz), 1.00 (3H, d, J=5.3 Hz).
13C NMR (CDCl3, 100 MHz): 170.4, 45.5, 24.6, 28.1, 59.0, 166.3,
53.4, 38.6, 23.3, 21.3, 22.7.
Cyclo(Trp-Pro) (7): 1H NMR (CDCl3, 400 MHz):3.62 (2H, m), 1.90
(1H, m), 2.00 (2H, m), 2.33 (1H, m), 4.01 (1H, t, J=7.7 Hz), 5.79
(1H, s), 4.38 (1H, dd, J=10.8 Hz, 2.5 Hz), 3.76 (1H, dd, J=15.0 Hz,
3.6 Hz), 2.98 (1H, dd, J=15.0 Hz, 10.8 Hz), 7.11 (1H, s), 8.28 (1H,
s), 7.40 (1H, d, J=8.2 Hz), 7.24 (1H, t, J=7.4 Hz), 7.14 (1H, t,
J=7.7 Hz), 7.59 (1H, d, J=7.9 Hz). 13C NMR (CDCl3, 100 MHz): 169.4,
45.4, 22.6, 28.3, 59.2, 165.5, 54.6, 26.9, 110.0, 123.3, 136.7,
111.6, 122.8, 120.0, 118.5, 126.7.
Cyclo(Pro-Phe) (8): 1H NMR (CDCl3, 400 MHz):3.64 (1H, m), 3.57
(1H, m), 1.91 (1H, m), 2.02 (2H, m), 2.34 (1H, m), 4.08 (1H, t,
J=6.1 Hz), 5.63 (1H, s), 4.27 (1H, dd, J=8.4 Hz, 2.2 Hz), 3.64 (1H,
m), 2.78 (1H, dd, J=11.6 Hz, 8.5 Hz), 7.22 (2H, d, J=5.7 Hz), 7.35
(2H, t, J=6.0 Hz), 7.29 (1H, t, J=5.9 Hz). 13C NMR (CDCl3, 100
MHz): 165.1, 45.5, 22.6, 28.4, 59.1, 169.4, 56.2, 36.8, 135.9,
129.3, 129.1, 127.6.
Lumichrome (9): 1H NMR (DMSO, 400 MHz):11.68 (1H, br.s), 11.85
(1H, br.s), 7.71 (1H, s), 7.92 (1H, s), 2.46 (3H, s), 2.44 (3H, s).
13C NMR (DMSO, 100 MHz): 150.0, 160.6, 130.2, 138.3, 125.8, 144.6,
138.8, 128.7, 141.6, 146.4, 20.2, 19.6.
4. Figures
Fig. S1. Routine coculture of Streptomyces sp. and E. coli: gram
staining of Streptomyces sp. single culture (A), gram staining of
Streptomyces sp. and E. coli coculture using a routine method,
broth was dominated by E. coli, Streptomyces sp. fragments was
sporadically scattered. (B). Competitive coculture process of
Streptomyces sp. and E. coli: single culture of Streptomyces sp. in
MM medium under static condition was a fermentation broth of
transparent culture medium with clustered mycelium sinking in the
bottom (C), after the adding of E. coli, the culture medium quickly
became an even muddy suspension of E. coli, the mycelium of
Streptomyces sp. floated to the medium surface (D), and developed
aerial hyphae (E), after 14 days of co-cultivation, the culture
medium became transparent again, with the E. coli sinking to the
bottom and Streptomyces sp. colonized and sporulated at the medium
surface (F).
Fig. S2. Secondary metabolites HPLC fingerprints of Streptomyces
sp. and E. coli competitive coculture medium (black) and
Streptomyces sp. floated mycelium (pink).
Fig. S3. Secondary metabolites of Streptomyces sp. cultured in
E. coli spent medium. (alkaloids, tR 35.4, tR 46.9, tR 48.5, tR
57.5)
Fig. S4. Secondary metabolites of E. coli cultured in
Streptomyces sp. spent medium. (alkaloids, tR 35.4, tR 46.9, tR
48.5, tR 57.5)
Fig. S5. Morphological changes and important interactions
happened during the competitive coexistence process of Streptomyces
sp. and E. coli.
Fig. S6. pH test paper in the enclosed air space of co-culture
medium. Left sample: co-culture medium of Streptomyces sp. with E.
coli. Right sample: co-culture medium of Streptomyces sp. with
another Streptomyces.
Fig. S7. Chemical structures of compounds 1-9 (A) and 1H-1H COSY
(bold bond) and key HMBC (arrow) correlations of 5 (B)
Fig. S8. UV spectrum of 6.
Fig. S9. HRESIMS spectrum of 6.
Fig. S10. IR spectrum of 6.
Fig. S11. 1H NMR spectrum of 6 (CDCl3, 400 MHz).
Fig. S12. 13C NMR spectrum of 6 (CDCl3, 400 MHz).
Fig. S13. HMQC spectrum of 6 (CDCl3).
Fig. S14. HMBC spectrum of 6 (CDCl3).
Fig. S15. 1H-1H COSY spectrum of 6 (CDCl3).
Table 1. 1H and 13C NMR data of compound 5 (CDCl3).
C
H (J in Hz)
HMBC(CH)
1, 1-NH
8.00, 2H, br. s
2, 2
123.2
6.90, 2H, s
8
3, 3
117.7
4/4, 8, 9
3a,3a
126.8
5/5, 7/7 , 2/2, 8
4, 4
119.6
7.47, 2H, d (7.9)
6/6
5, 5
119.5
7.04, 2H, t (7.9)
7/7
6, 6
122.1
7.19, 2H, t (7.9)
4/4, 7/7
7, 7
111.2
7.38, 2H, d (7.9)
5/5
7a, 7a
136.6
4/4, 6/6, 2/2
8
36.7
6.12, 1H, s
2/2
9
113.2
6.90, 1H, s
8
10-NH
11
168.5
12, 9
11a
130.0
14
12
126.9
7.60 1H, d (8.3)
14
13
119.3
6.88, 1H, t (8.3)
15
14
131.5
7.26, 1H, t (8.3)
12
15
117.5
6.98, 1H, d (8.3)
13
15a
156.8
12, 14
16-O
17
158.0
8, 9
0.0
5.0
10.0
15.0
20.0
25.0
30.0
35.0
40.0
45.0
50.0
55.0
60.0
min
0
50
100
150
200
250
mAU
230nm,4nm (1.00)
0.05.010.015.020.025.030.035.040.045.050.055.060.0min
0
50
100
150
200
250
mAU
230nm,4nm (1.00)
0.0
5.0
10.0
15.0
20.0
25.0
30.0
35.0
40.0
45.0
50.0
55.0
60.0
min
0
100
200
300
400
500
600
mAU
230nm,4nm (1.00)
0.05.010.015.020.025.030.035.040.045.050.055.060.0min
0
100
200
300
400
500
600
mAU
230nm,4nm (1.00)
49f
nm
200220240260280300320340360380400
AU
0.0
5.0e-1
1.0
1.5
2.0
2.5
yly-49f 8061 (6.717) 2: Diode Array
3.151
228.04
282.04
327.04