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