Microb. Resour. Syst. 36
Dec.
（2）
：39
2020─ 48, 2020

Vol. 36, No. 2

Antimicrobial activities and natural products ofcellulolytic myxobacteria isolated from soils in Japan
Yoshio Kimura＊, Yurika Yoshida, Yuu Shikata, Miri Maeda, and Yumi Mori
Department of Applied Biological Science, Faculty of Agriculture, Kagawa University
2393 Ikenobe, Miki-cho, Kagawa 761-0795, Japan
Cellulolytic myxobacteria cultured using filter paper as the only carbon source were isolated from soils in Japan. To
purify isolated strains, fruiting bodies of cellulolytic myxobacteria formed on Stanier agar medium were incubated at
60℃ for 1-7 days and/or at room temperature for 3-6 months. Purified 120 strains were cultured in P- and E-liquid
media for 4-6 weeks at 28℃. Approximately 75% of the strains were found to produce metabolites with antimicrobialactivity, and 63% of extracts showed growth inhibitory activity against Aspergillus niger. Based on LC-MS analysis, weestimated that 34 known natural products of the genus Sorangium were produced by 100 isolates, including spirodienal,
ambruticin, disorazole, and epothilone. Also, many products whose molecular weights did not match those of known
products from the genus Sorangium were detected.

Key words: a ntimicrobial activity, isolation and purification methods, natural products, Sorangium, cellulolytic
myxobacteria

Myxobacteria are Gram-negative, unicellular, aero-ered myxobacterial secondary metabolites are pro-bic, gliding bacteria with relatively large rod-shaped

duced by different strains of this species (Reichen-vegetative cells and a complex life cycle (Dworkin,bach, 2001; Gerth et al., 2003; Bode & Müller, 2006).

1996; Shimkets et al., 2006). They are isolable from

However, there are very few reports on comprehen-soils, animal dung, and decaying plant materials

sive analysis of antimicrobial activities and natural(Reichenbach & Dworkin, 1992; Reichenbach, 1999).

products in cellulolytic myxobacteria (Gerth et al.,Under starvation conditions, vegetative cells aggre-

2003), and on isolation of cellulolytic myxobacteriagate into fruiting bodies and differentiate into desic-

from Japan (Komaki et al., 2008).
We isolated cellulolytic myxobacteria from soil

cation-resistant spores (Dawid, 2000).

samples in Japan in order to obtain useful com-

Myxobacteria fall into either bacteriolytic and cellulolytic nutritional subgroups.

Most species ofpounds from myxobacteria. In this study, antimicro-

myxobacteria are bacteriolytic, lysing dead organic

bial activities of 120 isolates were determined by thematter as well as dead and living microorganisms

paper disc assay, and natural products of the isolateswith a host of excreted enzymes. Myxobacterial

were analyzed by liquid chromatography-mass spec-

genera, Sorangium and Byssovorax, are the members

trometry (LC-MS).

of the taxon which decompose cellulosic materials

Approximately 2,000 soils in Japan except Hokkaido(Gaspari et al., 2005; Reichenbach et al., 2006). Ge-

were collected and approximately 1 g of each wasnome sizes of myxobacteria range from 9.2 to 14.8

placed on filter paper (70 mm diameter, Advantec

Mbp, and among myxobacteria, Sorangium cellu︲

No. 1) placed on Stanier agar medium (0.1% KNO3,

losum has the largest bacterial genome (Schneiker etal., 2007; Han et al., 2013). Almost half of the discovCorresponding author＊

E-mail: kimura.yoshio@kagawa-u.ac.jp
Accepted: June 16, 2020

0.02% K2HPO4, 0.01% MgSO4, 0.01% CaCl2, 0.002%

FeCl3, 0.1% standard trace element solution (0.1 g

MnCl2, 0.02 g CoCl2, 0.01 g CaSO4, 0.01 g NaMoO4,

0.02 g ZnCl2, 0.005 g LiCl, 0.005 g SnCl2, 0.01 g H3BO3,

0.02 g KBr, 0.02 g KI per liter) and 1% agar; Stanier,1942) and incubated for 2 to 4 weeks at room tem-─ 39 ─

Cellulolytic myxobacteria in Japan

Kimura et al.

perature. The spot where the paper was decom-

pended in 1 ml P-medium containing 7 antibiotics at

posed was observed with an optical microscope, and50 μg ml-1 each: nystatin, penicillin G, fradiomycin,

it was confirmed that the fruiting bodies of genera

gentamycin, carbenicillin, ampicillin, and kanamycin

Sorangium or Byssovorax were formed. Many cellu-

or 4 antibiotics at 50 μg ml-1 each: oleandomycin, spi-

lolytic myxobacteria strains on the filter paper pro-ramycin, lincomycin, and rifampicin. After incuba-

duced an orange to brown color pigment (Reichen-tion for 1 to 2 weeks at room temperature, fruiting

bach et al., 2006; Mohr et al., 2018), and then strainsbodies were inoculated on filter papers, and then theformed red-brown, sometimes yellow or black fruit-

filter papers were placed on Stanier agar medium.

ing bodies. In approximately 20% of the plates, fruit-

Fruiting bodies formed on filter paper were checkeding bodies of cellulolytic myxobacteria strains werefor purity by the method described above. In thisobserved on the filter paper, and 360 isolates wereexperiment, approximately 20% of the subjects wereobtained from 2,000 soil samples.

purified by the antibiotic treatment. Other methods

To purify the isolates, the appearance of fruitingfor purifying cellulolytic myxobacteria fruiting bodies

bodies on the filter papers was checked by micro-

contaminated with bacteria were attempted, includ-

scope, and then transferred to fresh Stanier plates

ing ultrasonic treatment using a sonifier (Branson)(55 mm diameter) that had filter papers (35 mm di-for 2.5-5 min, washing treatment with 1.5 l of steril-

ameter) on top using a needle. After several weeks

ized water using a membrane (10 μm pore size) filtra-of incubation, formed fruiting bodies were picked uption apparatus, and incubation with 1-5% sodium do-with a needle and transferred into sterilized 1.5 ml

decyl sulfate, 2-3 M NaCl, 10-20% dimethyl sulfoxide

tubes. The tubes containing fruiting bodies of cellu-

or 6-18 mM H2O2 for 7-14 days; however, other bac-lolytic myxobacteria were placed in an oven at 60℃

teria were not removed by these treatments. The

for 1 to 7 days for heat treatment, and then at room

purification ratio of isolates was approximately 50%

temperature for 3 to 6 months for drying treatment.

in this study, and the fruiting bodies of purified 178

After these incubations, the fruiting bodies in thestrains were stocked in P-medium with 20% glycerol

tubes were inoculated on filter paper that wereand stored at −80℃.

placed on Stanier agar medium and incubated at

Isolated cellulolytic myxobacteria strains were pre-28℃ for 2-4 weeks. To check for purity of culture,

cultured in 40 ml of P-medium for 1 week at 30℃.

fruiting bodies of approximately 5×5 mm2 that

Following this, precultures were inoculated into 500-

formed on the filter paper were inoculated into 3 ml

ml Erlenmeyer flasks containing 250 ml of P-medium

P-liquid medium (Gerth et al., 1984), cultured for 3

or 250 ml of E-medium (0.4% skim milk powder, 0.4%

days at 30℃, and whether other bacteria were grow-soybean flour, 1% corn starch, 0.2% yeast extracts,
0.5% glycerol, 0.1% CaCl2･2H2O, 0.1% MgSO4･7H2O,

ing in the P-medium was verified using a microAs a simple determination method, we

0.008% Na-Fe-EDTA, and 1.2% HEPES, pH 7.2) andjudged whether or not the liquid medium became

3.5 g of absorber resin XAD-16 (Sigma Aldrich), andclouded by growth of other bacteria over the coursegrown at 30℃ on a rotary shaker at 100 rpm for 4 orof 3 days’ incubation, since the growth of cellulolytic

6 weeks, respectively. The resin and microbial cells

myxobacteria is comparatively very slow with gen-were harvested separately and extracted with 60-

eration times approaching 16 h (Reichenbach, 1999).

100 ml of acetone. Extracts were concentrated using

If isolate was not purified by this treatment, thisan evaporator to 5 ml and analyzed using antimicro-

treatment was repeated, and many purified strainsbial activity analysis and LC-MS.

scope.

were obtained by this method.

We first analyzed antimicrobial activities of ex-

In contaminated samples, we attempted purification

tracts from 120 isolated strains. Antimicrobial activ-

using various antibiotics, which do not kill spores of

ity was examined by paper disc assay against threecellulolytic myxobacteria. Contaminated fruiting

bacteria, two yeasts, and two molds as indicator mi-bodies of cellulolytic myxobacteria strains were sus-

croorganisms. Micrococcus luteus NBRC 3333 and─ 40 ─

Microb. Resour. Syst. Dec. 2020

Vol. 36, No. 2

Corynebacterium glutamicum NBRC 12168, and Gor︲

MS. Fifty μl of the extracts was concentrated under

donia rubripertincta NBRC 101908 were used as tar-a vacuum at 28℃, and then redissolved in 500 μl ofgets of bacteria and actinomycete, respectively. Can︲

acetonitrile:methanol 1:1 (v/v) for LC-MS analysis

dida albicans NBRC 1385 and Wickerhamomyces(Shimadzu LCMS-2020). A 2 μl aliquot was injected

anomalus NBRC 10213 were used as representative

into a Kinetex 5 μm C18 100A column (2.1 mm×100

yeast. These were prepared as overnight cultures inmm, particle size 5 μm, Phenomenex) with a guard

20 ml medium consisting of 1% peptone, 0.2% yeast

column (SecurityGuard ULTRA, Phenomenex). Mobile

extract, and 0.1% MgSO4 (Bacterial medium) or YM

phase A was water and mobile phase B was acetoni-broth (Difco Laboratories), respectively. One ml oftrile, where both phases contained 0.1% formic acid.

each culture was mixed with 20 ml of melted Bacte-

The gradient elution at a flow rate of 0.4 ml min-1

rial agar medium or YM agar, and they were used in

was performed as follows: 0-22 min 10-90% B andantimicrobial activity evaluations. Meanwhile, Asper︲

22-25 min 90% B. Mass spectra were obtained in

gillus niger NBRC 33023 and Mucor hiemalis NBRC

both positive and negative reflector ion mode in the9404 were grown on potato dextrose agar (Difco

range of m/z 200-1200. The mass spectrometer fit-

Laboratories). One cm3 of each was transferred into

ted with an electrospray ionization (ESI) source wasa tube, and agar containing hyphae was crushed

used for analysis. It was operated the following pa-

with a platinum loop, followed with glass beads in a

rameters: probe voltage +4.5 kV (+ESI) / −3.5 kV

vortexer, and then mixed with 20 ml of melted pota-(−ESI), nebulizer gas flow 1.5 l min-1, drying gas flow

to dextrose agar. Ten μl from the acetone extracts

15.0 l min-1, block heater 400℃, DL temperature

were spotted onto sterile filter paper discs (8 mm in

250℃.

diameter; Advantec) and allowed to dry. These discs

Of the natural products isolated from the genus

were placed on plates prepared with the above de-Sorangium, 241 are registered in Dictionary of Natu-scribed targets and incubated for 48 h at 30℃. Fol-ral Products 29.1. Gerth et al. have reported that dis-lowing this, the diameters of inhibition zones wereorazole, icumazole, spirangien, tartrolon, soraphen,measured.

ambruticin, chivosazol, and epothilone were com-

Among 120 isolates, 91 exhibited antimicrobial ac-

monly found in 1,700 S. cellulosum isolates from Ger-

tivities against at least one of seven indicator micro-

many (Gerth et al., 2003). From m/z values obtainedorganisms (Table 1). Gerth et al. have reported thatfrom LC-MS analysis, metabolites from 100 cellulo-

approximately 90% of S. cellulosum isolates pro-lytic myxobacteria strains yielded 34 known prod-

duced some biologically active compounds (Gerth etucts (Tables 1 and 2), and spirodienal, ambruticin, di-al., 2003). 63% and 35% of extracts showed inhibitorysorazole, epothilone, pellasoren, microsclerodermin M,

activity against A. niger and M. hiemalis, respective-

and spirangien were major known products of cellu-ly (Table 1). Eight strains (No. 102, 162, 202, 250, 280,

lolytic myxobacteria isolates from Japan (Table 2).

290, 301, and 396) had broad-spectrum antimicrobial

These are natural polyketides, with potent antifungal

activity against bacteria, yeasts, and molds. Howev-

activity and cytotoxicity against human tumor cells.

er, most of extracts from isolated strains (70%) did

Approximately 80% of spirodienal-, ambruticin-, dis-

not show activity against the three bacteria (M. lute︲orazole-, and spirangien-producing strains had anti-us, C. glutamicum, and G. rubripertincta), indicating

microbial activities against A. niger (Table 1). In ad-that secondary metabolites of cellulolytic myxobacte-dition, precursors of epothilones (for examples,

ria have mainly antifungal activity. The extracts

5,13-dihydroxy-2,4,6,14-tetramethyl-15-(2-methyl-

prepared from cultures in E-medium showed stron-

4-thiazolyl)-10,14-pentadecadien-3-one, 11-hy-

ger antibiotic activity compared to those prepareddroxy-4,12-dimethyl-13-(2-methyl-4-thiazolyl)-8,12-tri-

from P-medium, likely because many isolates grew

decadien-3-one, and 3,5-dihydroxy-6-methyl-7-(2-more readily in E-medium (data not shown).

methyl-4-thiazolyl)-6-heptenoic acid), were frequently

We next analyzed the products of isolates by LC-

detected.
─ 41 ─

Meanwhile, icumazole was rarely ob-

Cellulolytic myxobacteria in Japan

Table 1
Strain
No.
58
113
124
128
150
165
169
192
205
206
214
221
223
236
239
240
245
247
248
259
262
272
303
319
321
322
327
339
373
175
249
130
155
313
127
135
151
157
357
233
243
104
105
156
199
201
207
217
220
224
235
238
252
266
278
291
305
311
342
258
283
299
164

Kimura et al.

Antimicrobial activities of isolates against seven indicator microorganisms, and known natural products
produced by isolates
Antimicrobial activity against

MIa
+
+
+
-

Cgb
+
+
+
+

Grc
+
+
+
-

Cad
-

Wae
+
+
+
+
+
-

Mhf
+
+
+
-

Ang
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+

Chemical name
Disorazole I
Soraphen E, Epothilone D or G2
Spirodienal, Ambruticin VS2, Ripostatin
Spirodienal, Sorazolone E2
Spirodienal, Ambruticin VS2
Ambruticin VS2Spirodienal, Microsclerodermin M
Ambruticin VS2, Jerangolide D
Pellasoren A

Tuscoron B, Phoxalone, Epothilone N, Disorazole B2
Spirodienal, Ambruticin VS2
Disorazole F2
Pellasoren A
Tuscoron B, Phoxalone
Soraphen E, Disorazole F2
Tuscoron B
Ambruticin VS2, Epothilone D or G2
Spirodienal
Spirodienal, Microsclerodermin M
Spirodienal, Disorazole A2
Microsclerodermin M
Disorazole A2Spirodienal, Pellasoren A
Ambruticin VS3
Microsclerodermin M
Spirodienal, Ambruticin VS2
Epothilone B
Spirodienal, Chivorazole E, Disorazole F2Spirodienal, Soraphen E
Ambruticin VS3
Spirodienal, Microsclerodermin M
Disorazole A
Tuscoron B, Chivorazole A
Microsclerodermin M
Spirodienal
Chivorazole A
Pellasoren A, Soraphen A
Ambruticin VS2
Pellasoren A
Spirangien B
Pellasoren A
Ambruticin VS2, Tuscoron B
Spirangien Q
Phoxalone, Epothilone A1 or Chlorotonil A
Spirodienal
Spirodienal, Phoxalone─ 42 ─

Microb. Resour. Syst. Dec. 2020

Vol. 36, No. 2

Table 1
Strain
No.
154
181
200
213
329
371
273
123
57
136
161
50
107
125
129
131
144
145
166
212
215
264
328
121
115
148
211
142
158
114
216
307
103
116
126
139
172
230
254
286
304
225
295
138
141
237
101
118
203
102
250
301
202
162
280
396
290

Continued

Antimicrobial activity against
MIa
+
+
+
+
+
+

Cgb
+
+
+
+
+
+
+
+
+

Grc
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+

Cad
+
+
+
+
+
+
+
+
+
+
+

Wae
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+

Mhf
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+

Ang
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+

42

75

No. of active strains against indicator strains
9

13

27

11

35

Chemical name

Spirangien A, Spirangien B
Disorazole A
Spirodienal, Tuscoron B, Soraphen E, Epothilone D or G2
Epothilone E or A9 or M, Phoxalone, Ambruticin VS1
Spirangien A, Spirangien B
Spirangien A, Spirangien B
Ambruticin VS2, Disorazole F2, Soraphen E, Chivosazole E
Pellasoren A
Ambruticin VS2, Disorazole I
Ambruticin VS5
Pellasoren A
Disorazole A, Pellasoren A
Disorazole A, Pellasoren A
Phoxalone, Chivorazole A
Disorazole A
Disorazole A
Pellasoren A
Spirodienal, Pellasoren A, Microsclerodermin M
Disorazole A
Spirodienal, Ambruticin VS2, Tuscoron B, Epothilone D or G2, Epothilone N
Ambruticin VS2, Soraphen E
Ambruticin VS3
Ambruticin VS2, Epothilone D or G2
Spirodienal, Ambruticin VS2, Tuscoron B, Phoxalone, Spirangien Q
Spirodienal, Ambruticin VS2, Microsclerodermin M
Pellasoren A
Ambruticin VS2, Tuscoron B
Spirodienal, Ambruticin VS2, Disorazole F2Spirodienal, Soraphen E
Spirangien Q
Spirodienal, Ambruticin VS2, Disorazole F2, Microsclerodermin M, Chivosazole E
Disorazole A
Disorazole A, Pellasoren A
Spirodienal, Soraphen E, Epothilone E or A9 or M, Chivorazole E
Spirodienal, Ambruticin VS2, Disorazole F2, Epothilone D or G2
Spirodienal, Disorazole F2, Soraphen E, Microsclerodermin M
Spirodienal, Disorazole F2, Spirangien Q, Microsclerodermin M
Pellasoren A
Disorazole A, Phoxalone, Chivorazole A, Tuscoron B
Icumazole A
Spirodienal, Sorazolone E2, Disorazole F2, Epothilone D or G2
Spirodienal, Disorazole A
Spirodienal, Ambruticin VS2, Disorazole F2, Epothilone E or A9 or M, Spirangien Q
Spirodienal
Spirodienal, Chivosazole E, Tuscoron B, Microsclerodermin M
Spirodienal, Soraphen E, Disorazole A2Spirodienal, Ambruticin VS2, Chivosazole E, Microsclerodermin M
Spirodienal, Ambruticin VS2
Pellasoren A, Spirangien A, Spirangien B, Tuscoron A or Epothilone I3
Spirodienal, Ambruticin VS2
Tuscoron B, Chivotriene, Eliamid, Epothilone D or G2, Microsclerodermin M
Epothilone E or A9 or M
Spirodienal, Epothilone E or A9 or M, Microsclerodermin M
Spirodienal, Ambruticin VS2, Ambruticin VS5, Soraphen E, Secoepothilone A Me ester
or Soraphen M

M. luteus; bC. glutamicum; cG. rubripertincta; dC. albicans; eW. anomalus; fM. hiemalis; g A. niger. +: active against indicator microorganism;-: non-active against indicator microorganisma─ 43 ─

Cellulolytic myxobacteria in Japan

Kimura et al.

Table 2
(M+H)
m/z
552
433
553
730
425
826
708
703
502
422
546
355
390
512
568
780
526

+

RT
min
7.2
4.5
4.4
5.2
3.8
8.5
4.3
18.9
15.5
4.9
3.5
10.1
17.0
5.6
20.0
4.4
3.2

a

Known natural products produced by isolated strains

No. of (M+H)+
strainsm/z
Spirodienal
39
530
Pellasoren A
16
887
Tuscoron B
12538
Disorazole F2
11759
Phoxalone8491
Chivosazole E
6511
Spirangien Q
5717
Spirangien A
4866
Ambruticin VS3
3
746
Sorazolone E22489
Epothilone N
2758
Chivotriene
1360
Eliamid1
496
Soraphen A
1
517
Icumazole A
1
639
Disorazole B2
1
480
Secoepothilone A Me ester or sora1535
phen M
Chemical name

RTa
min
6.4
7.4
6.8
16.5
6.0
8.6
20.7
6.6
3.3
11.0
5.2
3.3
4.6
11.3
6.6
3.5
10.0

No. ofstrains
Ambruticin VS225
Microsclerodermin M
15
Soraphen E
11
Disorazole A
10
Epothilone D or G2
8
Epothilone E or A9 or M
5
Spirangien B
5
Chivosazole A
4
Disorazole A2
3
Ambruticin VS5
2
Disorazole I
2
Jerangolide D
1
Ripostatin
1
Ambruticin VS 1
1
Epothilone B
1Epothilone A1 or Chlorotonil A
1
Tuscoron A or Epothilone I3
1
Chemical nameretention timea

Table 3

(M+H)
m/z

RT
min

255
273
282
329
330
374

6.9
8.5
8.0
8.7
8.4
4.4

394
400
417

3.9
5.1
3.9

439
459

5.7
4.9

560
572
652
659
698
741
747
755
821
889
997
1000
1124

3.4
5.8
4.5
3.4
4.8
5.1
7.9
6.3
6.8
6.2
4.0
8.6
6.2

+

a

Major unidentified products from isolates

Strain No.
105, 125, 127, 142, 144, 172, 175, 200, 213, 238, 245, 266, 272, 273, 278, 301, 303, 305
124, 138, 203, 249
57, 250, 291, 301, 371
57, 103, 114, 286
57, 101, 103, 203, 249, 280, 286, 342, 357
57, 101, 102, 103, 104, 114, 124, 128, 129, 130, 131, 138, 139, 141, 151, 154, 157, 158, 161, 175, 192, 199, 203,
205, 206, 207, 212, 217, 220, 223, 225, 230, 233, 235, 236, 237, 239, 249, 250, 252, 254, 262, 283, 286, 290, 311,
321, 327, 329, 342, 357, 396
212, 235, 238, 252
125, 131, 166, 213, 220, 238, 313
57, 102, 103, 104, 113, 114, 118, 124, 138, 139, 141, 151, 158, 202, 203, 216, 220, 230, 233, 237, 247, 250, 254,
258, 259, 280, 286, 291, 299, 307, 313, 319, 321, 329, 342, 371, 373, 396
103, 113, 114, 115, 139, 158, 161, 192, 216, 237, 248, 254, 258, 259, 264, 283, 286, 290, 321, 328, 339, 371
50, 113, 115, 118, 151, 158, 162, 169, 199, 202, 203, 216, 247, 249, 259, 280, 286, 291, 307, 313, 319, 321, 357,
373, 396
114, 151, 328
128, 158, 252, 264, 373
113, 115, 130, 145, 148, 166, 192, 202, 203, 225, 235, 252, 259, 264, 299, 327, 328, 329, 339
162, 166, 169, 207, 216, 223, 225, 286, 291, 311, 327, 328, 339, 357
131, 162, 175, 225, 239, 327
138, 139, 216, 237, 254
114, 135, 148, 150, 157, 175
118, 130, 139, 199, 207, 214, 216, 224, 240, 254, 286, 328, 329, 339, 357
148, 192, 202, 207, 211, 252, 264
135, 139, 157, 158, 165, 206, 207, 211, 214, 216, 237, 295, 311, 329, 339, 357
104, 175, 235, 259, 327, 329
220, 252, 299
130, 211, 311, 339
a─ 44 ─

retention timeMicrob. Resour. Syst. Dec. 2020

Vol. 36, No. 2served. Frequently, genus Sorangium strains pro-ions at m/z 255, 374, 417, 439, 459, and 652 that wereduce several chemically completely different com-frequently found (Table 3).

pounds (Gerth et al., 2003). Six strains (No. 103, 148,

Twenty-two purified cellulolytic myxobacteria

162, 237, 264, and 290) and 12 strains (No. 57, 102,

strains were used for 16S rRNA gene sequence anal-

118, 138, 139, 225, 230, 247, 254, 286, 301, and 329) iso-ysis. For genomic DNA extraction, fruiting bodies

lated from soil in Japan produced five and four dif-on the filter paper from 22 isolated strains were

ferent known compounds, respectively (Table 1).

transferred into 1.5-ml tubes, and 50 μl of 50 mM

Most of them exhibited antimicrobial activity against

NaOH was added. After incubation at 80℃ for 5

multiple indicator microorganisms. On the othermin, 11 μl of 1 M Tris-HCl (pH 7.0) was added, andhand, 18 isolates without antimicrobial activity pro-the tubes were centrifuged at 12,000 rpm for 3 min.

duced known compounds, suggesting that these iso-The supernatant was used as template for 16S rRNA

lates may have antimicrobial activity as they only

gene sequence analysis. The 16S rRNA gene fromproduced low amounts of known compounds in theisolates was amplified via PCR using the universal

cultures.

primer set 27F (5’-AGAGTTTGATCCTGGCTCAG-3’)

In the analysis, many products, which did not

and 1492R (5’-GGCTACCTTGTTACGACTT-3’). PCRmatch the molecular weights of known products

products were purified by PEG precipitation. The

from the genus Sorangium, were found in the 120

sequencing reaction was performed with the same

isolate cultures, including some showing protonatedprimers using BigDye Terminator v3.1 Cycle Se-

Fig. 1 Neighbor-joining tree based on partial 16S rRNA gene sequences (1,300 bp) showing relationships among 22 isolated strains, 8Sorangium strains, and Byssovorax cruenta. Jahnella thaxteri(Sorangiineae, Polyangiaceae) was used to root the tree. Bar, 0.05
substitutions per nucleotide position. Numbers at the forks represent bootstrap values (%) out of 1,000 replicates.
─ 45 ─

Cellulolytic myxobacteria in Japan

Kimura et al.

Table 4 Highest sequence similarities between isolates and reference strains in 16S rRNA gene sequences, andknown products and antimicrobial activities of isolates
StrainClosest species
No.

% of
similarity

126
127
136
154
156
162

S. ambruticinum
S. ambruticinum
S. ambruticinum
S. ambruticinum
S. ambruticinum
S. ambruticinum

99.86
99.91
99.86
99.36
99.86
99.62

164
166
201
225
114
123
148
206
245
262
105
155
240
157
165
172

S. ambruticinum
S. ambruticinum
S. ambruticinum
S. ambruticinum
S. arenae
S. arenae
S. arenae
S. arenae
S. arenae
S. arenae
S. cellulosumb
S. cellulosumc
S. cellulosumb
S. kenyense
S. kenyense
S. kenyense

99.86
99.52
99.70
99.86
99.78
99.72
99.72
99.79
99.64
99.59
99.65
99.78
99.77
99.79
99.41
99.51

Antimicrobialactivitya

Known products
Disorazole A, Pellasoren A
Ambruticin VS3
Pellasoren A
None
Disorazole A
Tuscoron B, Epothilone D/G2, Chivotriene, Microsclerodermin M,
Eliamid
None
Pellasoren A
None
Disorazole A, Tuscoron B, Phoxalone, Chivosazole A
Spirodienal, Ambruticin VS2, Disorazole F2Spirangien A, Spirangien B
Spirodienal, Ambruticin VS2, Tuscoron B, Phoxalone, Spirangien Q
None
None
None
None
Disorazole A2
None
Epothilone B
None
None

Antimicrobial activity against A: actinomycete, B: bacterium, Y: yeast, M: mold. bS. cellulosum Soce 56.
Soce 1871T

a

Y, M
Y
Y, M
A, M
M
A, Y, M
B, M
M
M
B, Y, M
A, Y, MY
B, A, M
None
None
None
M
A
None
Y
None
Y, M
S. cellulosumc

CONCLUSION

quencing Kit (Applied Biosystems).
Phylogenetic tree was constructed by the neighbor-

We isolated cellulolytic myxobacteria from soils in

joining method (Saitou & Nei, 1987) using the Clust-Japan, and these strains could be isolated from ap-

alX program, and the resulting neighbor-joining tree

proximately 20% of soil samples. Purification of thebased on 16S rRNA gene sequences is shown in Fig.

isolates was mainly performed by heating and drying

1. The 16S rRNA gene sequences (approximately

the fruiting bodies. Approximately 80% of isolates

1,300-1,400 bp in length) were analyzed using an Ez-

were found to produce metabolites with antimicrobi-

BioCloud database. The 16S rRNA gene sequences

al activity, and they had mainly antifungal activity.

of all strains showed more than 99% sequence iden-

Based on LC-MS analysis of extracts from 120 iso-tity with the cellulose-decomposing genus Sorangi︲

lates, 34 known natural products of the genus Soran︲um. As shown in Table 4, 10 out of the 22 strains

gium were produced by 100 isolates.

had the highest 16S rRNA gene similarity with
Sorangium ambruticinum (Soce 176T), and the other

ACKNOWLEDGMENTS

strains had the highest similarity with Sorangium
arenae (Soce 1078 ), S. cellulosum, (Soce 56 or Soce
T

1871T) or Sorangium kenyense (Soce 375T) (Mohr etThis work was supported by a general grant frominstitute for fermentation, Osaka.

al., 2018). The isolates with the highest similarity to

REFERENCES

S. ambruticinum produced mainly pellasoren A andB o d e , H . B . & M ü l l e r , R . 2006. A n a l y s i s o f

disorazole A, and had antifungal activities.

myxobacterial secondary metabolism goesmolecular. J. Ind. Microbiol. Biotechnol. ₃₃: 577─ 46 ─

Microb. Resour. Syst. Dec. 2020

Vol. 36, No. 2

arenae sp. nov., Sorangium bulgaricum sp. nov.,

588.
Dawid, W. 2000. Biology and global distribution of

Sorangium dawidii sp. nov., Sorangium kenyense

myxobacteria in soils. FEMS Microbiol. Rev. ₂₄:

sp. nov., Sorangium orientale sp. nov. and403-427.

Sorangium reichenbachii sp. nov. Int. J. Syst. Evol.
Microbiol. ₆₈: 3576-3586.

Dworkin, M. 1996. Recent advances in the social anddevelopmental biology of the myxobacteria.

R e i c h e n b a c h , H . 1999. T h e e c o l o g y o f t h emyxobacteria. Environ. Microbiol. ₁: 15-21.

Microbiol. Rev. ₆₀: 70-102.
Gaspari, F., Paitan, Y., Mainini, D., Losi, D., Ron, E.Z.

Reichenbach, H. 2001. Myxobacteria, producers of& Marinelli, F. 2005. Myxobacteria isolated in

novel bioactive substances. J. Ind. Microbiol.
Biotechnol. ₂₇: 149-156.

Israel as potential source of new anti-infectives. J.

R e i c h e n b a c h , H . & D w o r k i n , M . 1992. T h eAppl. Microbiol. ₉₈: 429-439.
Gerth, K., Pradella, S., Perlova, O., Beyer, S. &myxobacteria, In Trüper, H.G., Dworkin, M.,M ü l l e r , R . 2003. M y x o b a c t e r i a : p r o f i c i e n t

Harder, W. & Schleifer, K.-H. (eds.), The

producers of novel natural products with various

Prokaryote, second edition. p. 3418-3487, SprigerVerlag, Berlin.

biological activities-past and future

Reichenbach, H., Lang, E., Schumann, P. & Spröer, C.

biotechnological aspects with the focus on the2006. Byssovorax cruenta gen. nov., sp. nov., nom.

genus Sorangium. J. Biotechnol. ₁₀₆: 233-253.
Gerth, K., Trowitzsch, W., Piehl, G., Schultze, R. &rev., a cellulose-degrading myxobacterium:

Lehmann, J. 1984. Inexpensive media for mass

rediscovery of ‘Myxococcus cruentus’ Thaxter

cultivation of myxobacteria. Appl. Microbiol.

1897. Int. J. Syst. Evol. Microbiol. ₅₆: 2357-2363.
Saitou, N. & Nei, M. 1987. The neighbor-joining

Biotechnol. ₁₉: 23-28.

m et h od : a n ew m et h od f or r econ s t r u c t in gHan, K., Li, Z., Peng, R., Zhu, L., Zhou, T., Wang, L.,phylogenetic trees. Mol. Biol. Evol. ₄: 406-425.

Li, S., Zhang, X., Hu, W., Wu, Z., Qin, N. & Li Y.
2013. Extraordinary expansion of a Sorangium

Schneiker, S., Perlova, O., Kaiser, O., Gerth, K., Alici,

cellulosum genome from an alkaline milieu. Sci.

A., Altmeyer, M.O., Bartels, D., Bekel, T., Beyer, S.,

Rep. ₃: 2101. doi:10.1038/srep02101.

Bode, E. & other 49 authors 2007. Complete

Komaki, H., Fudou, R., Iizuka, T., Nakajima, D.,genome sequence of the myxobacterium

Okazaki, K., Shibata, D., Ojika, M. & Harayama, S.

Sorangium cellulosum. Nat. Biotechnol. ₂₅: 1281-

2008. PCR detection of type I polyketide synthase1289.

genes in myxobacteria. Appl. Environ. Microbiol.

Shimkets, L.J., Dworkin, M. & Reichenbach, H. 2006.
The myxobacteria, In Dworkin, M., Falkow, S.,

₇₄: 5571-5574.
Mohr, K.I., Wolf, C., Nübel, U., Szafranska, A.K.,

Rosenberg, E., Schleifer, K.-H. & Stackebrandt, E.

Steglich, M., Hennessen, F. Gemperlein, K.,(eds.), The Prokaryotes vol. 7, p. 31-115, Springer,
Berlin.

Kampfer, P., Martin, K., Müller, R. & Wink, J. 2018.
A polyphasic approach leads to seven new species

S t a n i e r , R . Y . 1942. T h e c y t o p h a g a g r o u p : aof the cellulose-decomposing genus Sorangium,

contribution to the biology of myxobacteria.

Sorangium ambruticinum sp. nov., Sorangium

Bacteriol. Rev. ₆: 143-196.

─ 47 ─

Cellulolytic myxobacteria in Japan

Kimura et al.

日本の土壌から分離されたセルロース分解性粘液細菌の抗菌活性と天然物質
木村義雄，吉田有梨花，四方遊，前田美璃，森裕美

香川大学農学部応用生物科学科
セルロース分解性粘液細菌は細菌のなかで最も大きなゲノムを有し，新規な有用物質を生産すると考えられるが，日本ではほとんど分離されていない．日本の土壌を 2000 ヵ所から採取し，ろ紙を置いた培地に接種後，室温で 30 日程度培養すると 2 割程度のプレートでセルロース分解性粘液細菌の子実体が観察され，主に 60℃の高温処理と室温での乾燥により純化を行った．純化菌 22 株の 16S rRNA の塩基配列を決定すると，Sorangium ambruticinum, S. arenae, S. cellulosum や S.
kenyense と 99％以上の相同性が見られた．120 株の純化菌を XAD-16 樹脂を含む P 培地と E 培地に 30 日ほど振とう培養後，生産物をアセトン抽出し，微生物の生育阻害を見たところ，約 6 割の抽出物において Aspergillus niger の生育阻害が見られ，分離株は主に真菌に対する抗菌物質を生産していた．また，120 株の抽出物の LC/MS 解析より spirodienal, ambruticin, disorazole および epothilone など 34 の Sorangium 属由来の既知化合物が 100 株の抽出物より同定されたが，Sorangium 属由来の既知物質と分子量が異なる化合物も多く見出され，未知の有益物質を生産している可能性が示唆された．

─ 48 ─