FoliaMicrobiol.47 (5), 493-498 (2002) h t t p ://www. biomed, c a s .

c z / m b u / f o l i a /

Isolation and Identification of Streptomyces sp.

and Assay of Its Exocellular Water-Soluble Blue Pigments
LINGLU, H.-L. CUt, Y.-N. CHEN, S. YUAN
Collegeof Life Sciences.NanjingNormal University,Nanjing,210 097, People"sRepublicof China
e-mail linglu@njnu.edu.cn

Received21 January2002

ABSTRACT. A bacterial strain producing a great amount of blue pigment during submerse fermentation was
isolated and identified. Based on morphological characteristics, cell-wall chemotype and sequence of 16S
rRNA gene, the strain should belong to the genus Streptomyces; it had 99.4 % homology of 16S rRNA gene
sequence with that ofStreptomyces indigocolor. The pigment production by the strain was affected by carbon
and nitrogen sources. The main components of the pigment mixture (detected by HPLC and TLC) were
tentatively classified as actinorhodin-related compounds. The pigment was relatively stable against light and
higher temperature but was sensitive to low pH. The preliminary acute-toxicity determination showed that the
pigment was nontoxic (LD50 > 15 mg/g).

Water-soluble pigments from microbial sources are not common but are of great biotechnological
interest. The current antagonism toward synthetic colors has invited a great deal of research into the potential
of microorganisms to provide suitable water-soluble pigments as colorants in food industry (Margalith 1992).
Streptomyces strains produce many kinds of soluble pigments; the diffusible ones and their pH sensitivity have
also been regarded as useful taxonomic characters (Arai and Mikami 1972). Some of these pigments are con-
sidered to possess antibiotic properties (Donadio et al. 1991; Zotchev et al. 2000). Although in Streptomyces
coelicolor biosynthetic pathways were studied as model systems (Rudd and Hopwood 1979; Hobbs et al.
1990; Bystrykh et al. 1996; Hopwood 1997; Kormanec et al. 2000; Stoytcheva et al. 2000) data on charac-
terization and practical application of isolated pigments are relatively scant.
Here we report the isolation and identification of the strain which produces relatively large amounts
of water-soluble, stable and nontoxic pigment(s).

MATERIALS AND METHODS

Strain LS-1 was isolated from samples collected from soil in Nanjing (Jiangsu Province, China);
Streptomyces coelicolor strain CGMCC AS4-863 was from the China General Microbiological Culture Col-
lection Center (Beijing, China) and Streptomyces microflavus ACCC 40027 from China Agricultural Culture
Collection Center (Beijing, China),
Media and culture conditions. The strains were cultured in Gause's medium containing (in g/L):
starch 20, K N O 3 1, NaC10.5, MgSO4-7H20 0.5, K2HPO3-3H20 0.5, FeSO4"7H20 0.01; pH 7.4-7.6. Cultures
were grown in tube slopes on the above medium for 1 d at 30 ~ then transferred to 250-mL flasks containing
100 mL medium and cultivated on an orbital shaker (3.3 Hz, 30 ~ The submerse cultivation was done in an
automatic Vir-Tis 3L fermentor (Virtis Comp., USA) (5 Hz, 30 ~ the initiatial inoculum was 10 % (V/V); all
inocula were derived from a single-spore stock; fresh spore suspension was used to minimize the lag phase.
Morphological, physiological and biochemical tests. The morphological examination was carried out
in Gause's synthetic medium at 28 ~ for 7 d. Characteristics were examined according to Locci (1989). To
investigate whether pigment production is affected by different nitrogen and carbon sources, we used 0.1%
(W/W) NH4NO3, NH4C1 and KNO3, respectively, as nitrogen sources. Other components were the same as in
Gause's medium. With potassium nitrate (as the principal source of nitrogen) the effect of carbon sources (glu-
cose, sucrose and starch; 2 %, W/W) was examined.
Analysis of fatty acids. The cells were harvested by centrifugation (4000 g, 10 rain), washed three
times with distilled water and freeze-dried. Dry biomass (0.5 g) was degraded at 55 ~ for 4 h with
a mixture of 6 mL methanol and sulfuric acid (1 : 10, V/V). After the reaction, 6 mL cyclohexane was added;
then the sample was vigorously shaken and centrifuged at 4000 g. The cyclohexane phase was collected, dried
 494 LINGLU et al. Vol. 47

using 2 g disodium sulfate, filtered and subjected to GC (whole-cell fatty acid-methyl ester analysis was done
using the HPSF-1890 gas chromatograph equipped with 30 m bonded-crossed polyethylene-glycol silica-co-
lumn; HP Comp., China); standards were obtained from Sigma.
Analysis of cell-wall chemical composition. Cell walls were purified and analyzed according to Le-
chevalier and Lechevalier (1980); the whole-cell chemical composition was determined according to Hase-
gawa et al. (1983).
16S rRNA sequencing and analysis. Genomic DNA was extracted and used for PCR amplification of
16S rRNA genes. Reaction mixture containing 1 x PCR buffer, primer A (5'-AGA GTT TGA TCC TGG CTC
AG-3') and primer H (5'-AAG GAG GTG ATC CAG CCG CA-3'). The final volume of the PCR mixture was
adjusted to 50 ~tL by adding extra pure H20. Thermal cycle was performed with the PTC-200 Peltier Thermal
Cycler DNA Engine (MJ Research, USA). The samples were subjected to an initial denaturing step (5 min,
95 ~ The thermal profile used was 30 cycles (40 s denaturation at 95 ~ 2 rain primer annealing and
extension at 68 ~ a final extension step was 8 min at 72 ~ Amplified DNA was detected by agarose gel
electrophoresis and visualized by UV fluorescence after ethidium bromide staining. The PCR products were
purified and concentrated using a PCR products purification kit and directly sequenced. The sequencing
primers were A, H and M (5'-AGG GTT GCG CTC GTT G-3'). A model 310 Prism Automatic DNA
Sequencer (PE-AB1, USA) detected the sequencing reaction mixtures. The 16S rRNA gene sequence
was aligned using Clustal X program (Thompson et al. 1997). Phylogenetic tree was constructed from the
evolutionary distances using molecular-evolutionary genetic-analysis software MEGA 2.0 (Sudhir et al.
1993). Bootstrap analysis was used to evaluate the tree topology of the neighbor-joining date by performing
1000 samplings (Saitou and Nei 1987). The 16S rRNA gene sequences obtained (Table I) were deposited in
DDBJ/EMBL/GenBank data library.
Pigment quantification. At appropriate time periods, 10-20 mL samples were taken to determine the
A590 of the supernatant (2max of absorption spectrum of fermentation liquid; Horinouchi and Beppu 1984). The
results are means from three experiments.
Pigment extraction. Culture supematants were acidified to pH 2-3 with HC1. A few crystals of pepsin
were added and the solution was incubated at room temperature for 30 min. After centrifugation
(25 000 g, 10 min), the pellet was suspended in water and pH adjusted with 1 mol/L NaOH to 7-8; then it
was dried to obtain dry matter (Bystrykh et al. 1996).
Analysis of pigments. Silica-coated glass plates (Bystrykh et al. 1996) were used for TLC assay. The
RP-HPLC assay was done on a chromatography column with Zorbax ODS C18 (250 x 4.6 mm, 5 ~tm;mobile
phase methanol-water 1 : 1, flow rate 1 mL/min; detection at 532 nm) at 25 ~
The acute toxicity tests. The pigment was orally administered to mice and LD50 was determined
(Forbes 1998; Fawell et al. 1999).

RESULT AND DISCUSSION

Isolation and identification. Based on morphological, physiological and biochemical tests (Table II)
the strain LS-1 was found to be consistent with the genus Streptomyces. Its 16S rRNA gene sequence was
determined and filed into database under the Accession no. AF275257. The phylogenetic tree was constructed
with reported sequence data of other members of some typical strains of the genus Streptomyces to examine
the phylogenetic position (Fig. 1). Based on the morphological characteristics, cell-wall chemotype and 16S
rRNA sequence, the strain LS-1 was classified as Streptomyces. On the phylogenetic tree, the blue
pigment-producing streptomycetes form two distinct clusters - one encompasses Streptomyces indigocolor
and related species, the other includes S. coelicolor and allied taxa. The highest percentage (99.4 %) of 16S
rRNA gene sequence homology was observed between strain LS- 1 and S. indigocolor; it suggests that strain
LS-1 is identical with or belongs to strains closely related to S. indigocolor.
Effect of carbon and nitrogen source on the blue pigment production. When the nitrogen source was
KNO 3, pigment was produced and accumulated. No pigment production was observed on NH4NO 3 or NI-I4CI
(this could indicate that the synthesis of the pigment is inhibited by ammonium nitrogen). Glucose was the best
carbon source (Fig. 2). In submerse fermentation the pigment production lagged for 46 h, then sharply
increased and reached a maximum after 4 d (Fig. 2). The amount of pigment in a 3-L fermentor was about
three times higher than in flask cultures and the cultivation time was by about 60 h shorter; this can be
explained, e.g., by higher aeration during oxygen-accelerated pigment production in the fermentor.
2002 EXOCELLULAR BLUE-PIGMENT PRODUCTION BY Streptomyces sp. 495

Table I. Strains used in the 16S rDNA sequence analysis

Species Strain number Length, bp Accession no.

Actinoalloteichus cyanogriseus IFO 14455 1468 AB 006178

Actinomyces graevenitzii CCUG 27294 1360 Y 09589
neuii 97/90 1487 X 7186 i
Escherichia coli ATCC 43895 1525 Z 83205
Itrasporangium calvum IFO 12989 1475 D 85486
Kineosporia rhamnosa JCM 9953 1469 AB 003934
succinea JCM 9957 1476 AB 003932
Nocardia beijingensis AS 4.1521T 1422 AF 154129
vaccinii DSM 43285 1472 Z 36927
Nocardioides albus KCTC 9186 1470 AF 004988
jensenii KCTC 9134 1473 AF 005006
Sporichthya polymorpha IFO 12702 1472 AB 025317
Streptomyces albidoflavus DSM 46452 1476 Z 76685
albulus MF861-C4 1488 AB 024443
aureofaciens NRRL 2209 1469 Y 15504
bellus ISP 5185 1441 AJ 399476
bicolor ISP 5140 1448 AJ 276569
bluensis ISP 5564 1520 X 79324
caelestis ISP 5084 1448 AJ 399467
canescens DSM 40001T 1476 Z 76684
cinnabarinus ISP 5467 1446 AJ 399487
coelicolor A3 (2) 1526 Y 00411
coeruleofuscus ISP 5144 1447 AJ 399473
coerulescens ISP 5146 1450 AJ 399462
cyaneus ISP 5108 1448 AJ 399460
griseus ATCC 10137 1478 Y 15501
indigocolor ISP 5432 1448 AJ 399464
lazureus ISP 5433 1517 AB 037564
lividans TK 21 1521 X 86354
longisporus ISP 5166 1302 AJ 399475
lydicus ATCC 25470 1481 Y 15507
macrosporus DSM 41449 1484 Z 68099
megasporus DSM 41476 1490 Z 68100
thermodiastaticus DSM 40573 1483 Z 68101
thermoviolaceus DSM 40443 1483 Z 68096
violatus ISP 5209 1448 AJ 399480
Streptomycoides glaucoflavus IFO 14668 1472 AB 006155
Our isolate LS-I 1475 AF 275257

Table II. Identification o f strain LS-1 by morphological, physiological and biochemical tests

Test Result Test Result

Aerial mycelium +, blue Utilization o f arabinose +

Substrate mycelium blue sucrose +
Spore shape spheroid Sugars in whole-cell hydrolysate glucose, ribose
chains 5-7, spiral Antimicrobial activity against
surface smooth B. subtilis +
Coagulation of milk E. coli
Peptonization o f milk + S. aureus +
Hydrolysis o f starch + M. luteus +
gelatin + S. cerevisiae
Production of H2S A. niger
Growth in cellulose Fatty acids a 14:0 2.11
Nitrate reduction + iso-14:0 3.68
Utilization o f glucose + 15:0 5.82
fructose + anteiso-15:0 23.2
xylose + 16:0 17.2
raffinose + iso-16:0 21.8
rbamnose + 16:1 14.2
myo-inositol + 17:0 10.9
mannitol + anteiso-I 7:0 1.15

aIn %.
496 LING LU et aL Vol. 47

10~0~ ' Streptomyces lividans

Streptomyces lazureus
3 5 ' r ~ --
_. d "- Streptomyces coelicolor
u~dt'-- Streptomyces caelestis
2alL[ Streptomyces bellus
g| ~ Streptomyces coerulescens
93 I I- Streptomyces violatus
Streptomyces albidoflavus
81 I 1~00 ' Streptomyces canescens
1 ~ " Streptomyces coeruleofuscus
I [ " ' - Streptomyces bluensis
38 9-~._{__Streptomyces thermoviolaceus
- g9 Streptomyces thermodiastaticus
Streptomyces albulus
t_. Streptomyces lydicus
'2 3~.U-"--- Streptomyces griseus
" ~ ~ Streptomyces aureofaciens
99 ~ [ - ' - Streptomyces longisporus
30 Streptomyces bicoTor
'~ cmnabarinus
72~- Streptomyces cyaneus
30l~ LSreltomyce s indigocolor
f"" Streptomyces megasporus
100 ' - - Streptomyces macrosporus
1oo Kineosporia succinea
I Kineosporia rhamnosa
. . . . Intrasporangium calvum
Sporichthya
_ _. l o oq ~ Actinomyces graevenitzii
-~ i ~ - - Actinomyces neuii
. . . . . Streptomycoides glaucoflavus
11~4LI 9 7 [ 100 - - Nocardioidesalbus
' " Nocardioidesjensenii
Actinoalloteichus
~oo I Nocardia beijingensis
Nocardia vaccinii
Escherichia coli
I
0.05

Fig. 1. Phylogenetic position of blue pigment-producing strain LS- 1; based on 16S rDNA sequence; b a r - 0.05 mutational
events per site.

3 I I I 1.8

A590 A590

2 - 1.2

m
- 0.6

0 i-i
0
6
0 100 h 200
A590
4

I I
50 100 h 150

Fig. 2. Pigment production by strain LS-1; above: effect of carbon (left; glucose - squares, s u c r o s e -
diamonds, starch - triangles) and nitrogen (right; NH4CI - squares, NH4NO3 - diamonds, KNO3 -
triangles) sources (batch cultures grown in 250-mL conical flasks containing 100 mL of medium); below:
submerged fermentation in a fermentor.
 2002 EXOCELLULAR BLUE-PIGMENTPRODUCTION BY Streptomyces sp. 497

2 6 pH 10
1 20 I I I I 120
% A B %

80 - 80

40 - 40

0 I I I 0
600 I I 120
Zmax
580 D %
80
560

540 40

520
I
2 6 pH 10 0 60 120
*C
Fig, 3, Effect of pH, light and temperatureon 2max and stability of pigment solution.
A: after 20 d under sunlight (% of control); B: after30 min under UV light (% of control);
C: )'maxof absorption spectra at pH 3-9; D: stabilityafter I h incubation (% ofcontroI),

Pigment characteristic and preliminary test of safety. The absorption spectra of pigment solution
were measured over a broad pH range (3-9) (Fig. 3); the 2 of the main peak of pigment spectra increased
with increasing pH; the pigment possessed some characteristics of red-blue pH indicator. We also found that
the pigment was more stable against visible light in the alkaline range than in the acidic one; no distinct effect
of UV light on pigment stability at different pH values was observed. Temperature-stability measurements
(20-100 ~ showed that after a 1-h incubation at 100 ~ 86 % of the pigment remains. The preliminary result
of acute toxicity tests showed that the pigment was nontoxic (LDs0 > 15 mg/g).
Although the LS-1 strain was not shown to be identical with S. coelicolor and the phylogenetic
distance (Fig. 1) was relatively large we could assume that the pigments produced by strain LS-I are similar to
actinorhodin-related compounds described earIier by Bystrykh et al. (I996). When both pigment mixtures
were compared on TLC (Fig. 4A), mutually corresponding zones (according to RF) were found; intensity of

A B

"I---" T F"
30
2.07 1.58
mV

' -II !j 28

a--
26 2.10

1.63
24

22
_Z
AS 4-863 LS-1 0 2 4 0 2 min 4

Fig. 4. Extracellularblue pigments of strains AS 4-863 and LS-1. A: TLC; a - the mixture of a-, 13-,6- and/or e-actinorhodins,
b - 7-actinorhodin; B: HPLC;numbers atpeaks - retention time (rain).
498 LING LU et al. Vol. 47

both main zones indicated the opposite relative content of two main components (y-actinorhodin and the
mixture of 0r-, 13-,~i- and/or e-actinorhodins). Similar results were obtained with HPLC - two main components
of the pigment mixture were shown to have identical retention times (Fig. 4B) but represent different
production levels. From the above data we may consider the mixture of pigments produced by the LS-1 strain
to be closely related to actinorhodin-related blue pigments produced by S. coelicolor.

This study was supported by the research grant BJ97123 of Jiangsu Province (People's Republic of China).

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