Streptobactin, a tricatechol-type siderophore from marine-derived streptomyces sp. YM5-799

Article abstract of DOI:10.1021/np200290j

A new catechol-type siderophore, streptobactin (1), was isolated from a culture broth of the marine-derived actinomycete Streptomyces sp. YM5-799. The structure of streptobactin was determined by NMR and MS analyses and ESIMS/MS experiments to be a cyclic trimer of benarthin. A dibenarthin (2), a tribenarthin (3), and benarthin (4) were also obtained. The production of 1 was regulated by an iron concentration in the culture. The iron-chelating activity of the compounds was evaluated by the chrome azurol sulfonate assay.

Full text of DOI:10.1021/np200290j

Article
pubs.acs.org/jnp
Streptobactin, a Tricatechol-Type Siderophore from Marine-Derived
Streptomyces sp. YM5-799
Yoshihide Matsuo,† Kaneo Kanoh,‡ Jae-Hyuk Jang,§ Kyoko Adachi,⊥ Satoru Matsuda,∥ Osamu Miki,‡
Toshiaki Kato,*,‡ and Yoshikazu Shizuri∇
†Suntory Holdings Ltd., Yamazaki, Shimamoto, Mishima, Osaka 618-0001, Japan
‡Nippon Steel Corporation, 20-1 Shintomi, Futtsu, Chiba 293-8511, Japan
§Chemical Biology Research Center, Bio-Therapeutics Research Institute, Korea Research Institute of Bioscience and Biotechnology,
125 Gwahak-ro, Yuseong-gu, Daejeon, Korea
⊥Nippon Steel Chemical Co., Ltd., 1 Tsukiji, Kisaradu, Chiba 292-0835, Japan
∥Department of Bioresource Engineering, section of Bioprocess Engineering, Yamagata University, Wakaba-machi 1-23, Tsuruoka-shi,
Yamagata 997-8555, Japan
∇Marine Biotechnology Institute Co., Ltd., 3-75-1 Heita, Kamaishi, Iwate 026-0001, Japan
S
* Supporting Information
ABSTRACT: A new catechol-type siderophore, streptobactin
(1), was isolated from a culture broth of the marine-derived
actinomycete Streptomyces sp. YM5-799. The structure of
streptobactin was determined by NMR and MS analyses and
ESIMS/MS experiments to be a cyclic trimer of benarthin. A
dibenarthin (2), a tribenarthin (3), and benarthin (4) were
also obtained. The production of 1 was regulated by an iron
concentration in the culture. The iron-chelating activity of the
compounds was evaluated by the chrome azurol sulfonate
assay.
ron is an essential element for most organisms because of its
various roles in important biological processes, such as
streptobactin1 (1) and its related compounds (2, 3) together
with benarthin2 (4). Benarthin was isolated from the culture
broth of Streptomyces xanthophaeus MJ244-SF1 as an inhibitor
of pyroglutamyl peptidase and determined as L-N-(2,3-
dihydroxybenzoyl)arginyl-L-threonine.2a
I
nucleic acid synthesis and repair, respiration, photosynthetic
transport, nitrate reduction, nitrogen fixation, and detoxification
of free radicals.3 In spite of its high abundance in Earth’s crust,
the dissolved iron concentration is particularly low (<0.4 μM)
in the surface waters of the open oceans.4 Under such iron-
limited conditions, marine bacteria utilize siderophores,
relatively low molecular weight compounds that have a high-
affinity iron-chelating ability, for their vital activity.5 A wide
range of structures have been reported for the siderophores
produced by terrestrial and enteric bacteria.6 Several side-
rophores have recently been isolated from marine-derived
bacteria including actinomycetes.7 We have also been collecting
bacteria from various marine environments, particularly those
producing siderophores detectable by the chrome azurol
sulfonate (CAS) assay,8 and have reported new catechol-type
siderophores, pseudoalterobactins A and B, from the marine
bacterium Pseudoalteromonas sp. KP20-49 and hydroxamate
siderophores, tenacibactins A−D, from Tenacibaculum sp. A4K-
17.10 During our subsequent screening for siderophores from
marine bacteria, we found that the marine-derived actinomycete
Streptomyces sp. YM5-799 produced a new catechol-type
siderophore along with its related compounds. Herein we
describe the fermentation, isolation, structural determination
including absolute configuration, and iron-binding activity of
RESULTS AND DISCUSSION
■
Strain YM5-799, producing siderophores 1−3 and benarthin
(4), was isolated from the surface of algae collected from
Hokkaido in north Japan. Morphological, physiological, and
phylogenetic properties enabled YM5-799 to be identified as
the genus Streptomyces. The siderophores, produced in the
culture supernatant of Streptomyces sp. YM5-799, were isolated
by chromatography on a porous-polymer resin Diaion HP20
column with a stepwise gradient of aqueous MeOH under acidic
conditions. Using CAS assay-guided fractionation, the active
fractions were further purified by preparative RP-HPLC to afford
three new siderophores, streptobactin (1), dibenarthin (2), and
tribenarthin (3), and a known compound, benarthin (4).
Streptobactin (1) was obtained as a colorless gum, the
molecular formula being established as C51H69N15O18 on the
basis of HR-FABMS data [found m/z 1180.5004, calcd for
Received: April 11, 2011
Published: October 20, 2011
© 2011 American Chemical Society and
American Society of Pharmacognosy
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Chart 1
1
[C51H69N15O18 + H]+ 1180.5023]. The H and 13C NMR
spectra of compound 1 were uncomplicated considering its
molecular weight. Thus compound 1 was presumed to be a
structure with high symmetry. The 13C NMR and HSQC
spectra revealed the presence of 17 carbon signals. Therefore,
compound 1 should have a C3 symmetry axis in its structure,
an amide proton in Thr (δ 8.79) to the carbonyl carbon in Arg
(δ 172.3), and an amide proton in Arg (δ 8.80) to the carbonyl
carbon in DHBA (δ 168.10 or 168.07), were revealed as a
partial structure (-Thr-Arg-DHBA). Compound 1 has the
formula C51H69N15O18 and a C3 symmetry axis. Furthermore, a
partial structure (-Thr-Arg-DHBA) has the formula
C17H23N5O6; therefore, streptobactin possessed a catechol-
bonded arginine-substituted siderophore framed on a cyclic
triester backbone of threonine. NMR data of streptobactin (1)
and benarthin (4), isolated in this study, are summarized in
Table 1. The spectroscopic data of 4 corresponded well with
literature data2b and also streptobactin (1), except for 1H NMR
suggesting a trimer of formula C17H23N5O6. The H and 13C
1
NMR and HSQC spectra revealed the presence of one methyl
(δC 16.2, δH 1.15), three broad methylene (δC 24.9, δH 1.58
and 1.63; δC 28.9, δH 1.80 and 1.90; δC 40.6, δH 3.10 and
3.20), six methine (δC 52.6, δH 4.78; δC 55.4, δH 4.88; δC 70.8,
δH 5.43; δC 118.2, δH 6.70; δC 118.6, δH 6.93; δC 118.8, δH
7.40), and seven quaternary carbons (δ C 116.5; 145.9; 147.8;
156.7; 168.07; 168.10; 172.3). These physiological properties
and NMR data suggested that 1 was related to a catechol-type
siderophore such as enterobactin,11 bacillibactin,12 vanchro-
bactin,13 or JBIR-16.14
A partial structure was elucidated by the interpretation of
data from 1H−1H COSY and 1H−13C HMBC experiments (see
SI, Figure SI-1). A threonine moiety was determined by the
spin network from the methyl to the amide proton (δ H 8.79).
The COSY and HMBC signals also revealed the 2,3-
dihydroxybenzoyl (DHBA) part. The COSY spectrum did
not sufficiently provide signals derived from three broad
methylenes, but the distinctive quaternary carbon signal (δ C
156.7), NH-substituted methylene carbon signal (δ C 40.6) and
its spin network (−NH−CH2−), and methine-NH spin signal
(−NH−CH<) suggested the existence of an arginine moiety in
the structure of 1. Connectivities of the moieties were
determined by HMBC correlations. The HMBC signals from
1
signals in relation to the Thr moiety. The H NMR signals of
C-2a (δ 4.88), C-2a-NH (δ 8.80), and C-3a (δ 4.53) of 1 were
shifted downfield compared to 2 at δ 4.23, 7.95, and 4.17,
respectively. These downfield shifts indicated that 1 has a cyclic
triester-bonded backbone similar to that in the enterobactin11b
and dimeric 2,3-dihydroxybenzoylserine case.8b
Compound 2 was obtained as a colorless gum, the molecular
formula being determined as C34H48N10O13 on the basis of HR-
FABMS data [found m/z 805.3454 [M + H]+, calcd 805.3481].
The 13C NMR spectrum showed 32 resolved signals correspond-
ing to 34 carbons. The molecular formula of compound 2
suggested that two benarthin units are combined via an ester bond
[2(C17H25N5O7)−H2O].
A partial structure of compound 2 was elucidated by the
interpretation of data from COSY, TOCSY, and HMBC
experiments (see SI, Figure SI-9). In the same manner as for
streptobactin (1), two benarthin units were constructed from
1
2D NMR analysis. As mentioned above, the H NMR signals
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Table 1. NMR Data (750 MHz, DMSO-d6) of Streptobactin
(1) and Benarthin (4)
Table 2. NMR Data (750 MHz, DMSO-d6) of Dibenarthin
(2)
a
b
c
streptobactin (1)
benarthin (4)
position
Thr-a
13C,
m
1H (J in Hz)
a
b
c
a
b
c
position
13C,
m
1H (J in Hz)
13C,
171.8, C
4.88, d (10.0) 57.5, CH 4.23, dd (3.2, 8.7)
m
1H (J in Hz)
1a
170.5, C
Thr-a
1a
2a
54.8, CH
4.64, dd (2.4, 8.9)
8.42, br d (9.0)
5.32, m
168.10 or
168.07 , C
55.4, CH
f
2a-NH
3a
2a
70.5, CH
16.2, CH3
2a-NH
3a
8.79, br
5.43, m
7.95, d (8.7)
66.2, CH 4.17, m
4a
1.15, d (6.1)
70.8, CH
16.2, CH3
Arg-a
1′a
e
4a
1.15, d (5.8) 20.3, CH3 1.18, d (6.4)
171.7 or 171.9, C
52.5, CH
Arg-a
1′a
2′a
4.70, m
e
172.3, C
52.6, CH
171.5, C
2′a-NH
3′a
8.79 or 8.81, d (8.0)
1.75, 1.86, m
1.58, m
e
2′a
4.78, m
8.80, br
52.4, CH 4.68, m
8.79, d (8.0)
29.0 or 29.1, CH2
2′a-NH
3′a
4′a
25.2 CH2
e
28.9, CH2
24.9, CH2
40.6, CH2
1.80, 1.90, m 28.9, CH2 1.73, 1.86, m
1.58, 1.63, m 25.1, CH2 1.57, m
3.10, 3.20, m 40.4, CH2 3.13, m
5′a
40.4 or 40.5, CH2
3.14, m
e
4′a
5′a-NH
6′a
7.50, m or 7.52, t (5.8)
5′a
156.7, C
d
5′a-NH
6′a
7.47, t (5.3)
7.47, t (5.5)
DHBA -a
1″a
e
156.7, C
156.6, C
168.5, C
168.3 or 168.6, C
d
e
DHBA -a
2″a
115.9 or 116.1, C
e
1″a
168.10 or
f
3″a
148.2 or 148.4, C
e
168.07, C
4″a
145.99 or 146.03, C
e
2″a
116.5, C
115.9, C
148.4, C
146.0, C
5″a
118.7 or 118.8, CH
6.94, dd (1.6, 8.0)
6.71, dd (7.7, 8.0)
7.40, dd (1.6, 8.0)
3″a
147.8, C
6″a
118.1, CH
e
4″a
145.9, C
7″a
118.5 or 118.6, CH
e
5″a
118.6, CH
118.2, CH
118.8, CH
6.93, d (7.7) 118.7, CH 6.93, dd (1.6, 8.0)
6.70, t (8.0) 118.1, CH 6.70, dd (8.0, 8.0)
7.40, d (8.0) 118.3, CH 7.38, dd (1.6, 8.0)
3″a-OH
4″a-OH
Thr-b
1b
11.65 or 11.80, br
6″a
9.36, br
7″a
3″a-OH
4″a-OH
11.55, br
11.80, s
169.3, C
57.2, CH
e
e
nd
nd
2b
4.36, dd (2.9, 9.0)
8.08, d (8.7)
4.29, m
a
b
c
d
δ in ppm (187.5 MHz). Multiplicity. δ in ppm (750 MHz). 2,3-
2b-NH
3b
e
f
Dihydroxybenzoic acid. Not detected. Indistinguishable.
66.2, CH
20.1, CH3
4b
1.08, d (6.1)
Arg-b
1′b
1
e
around the ester bond were deshielded. The H NMR signals
171.7 or 171.9, C
of C-2a (δ 4.64), C-2a-NH (δ 8.42), and C-3a (δ 5.32) of 2
were shifted downfield compared to C-2b (δ 4.36), C-2b-NH
(δ 8.08), and C-3b (δ 4.29), respectively. Therefore, a hydroxy
group at C-3a should be connected to a carboxylic group
at C-1b by an ester bond (see SI, Figure SI-9-b). ESIMS/
MS analysis of compound 2 supported the structure (see SI).
The 1H and 13C NMR assigned data are summarized in
Table 2.
2′b
52.5, CH
4.70, m
e
2′b-NH
3′b
8.79 or 8.81, d (8.0)
e
29.0 or 29.1, CH2
1.75, 1.86, m
1.58, m
4′b
25.2, CH2
e
5′b
40.4 or 40.5, CH2
3.14, m
e
5′b-NH
6′b
7.50, m or 7.52, t (5.8)
156.7, C
d
DHBA -b
e
1″b
168.3 or 168.6, C
Compound 3 was also isolated as a colorless gum, the
molecular formula being determined as C51H71N15O19 on the
basis of HR-FABMS data [found m/z 1198.5100 [M + H]+,
calcd 1198.5129]. This formula indicates that three benarthin
units are linearly connected via two ester bonds [3-
e
2″b
115.9 or 116.1, C
e
3″b
148.2 or 148.4, C
e
4″b
145.99 or 146.03, C
e
5″b
118.7 or 118.8, CH
6.94, dd (1.6, 8.0)
6.71, dd (7.7, 8.0)
7.40, dd (1.6, 8.0)
1
6″b
118.1, CH
(C17H25N5O7)−2H2O]. The H NMR, COSY, and TOCSY
e
7″b
118.5 or 118.6, CH
spectra revealed three overlapped DHBAs, three overlapped
arginines, and three threonine moieties (see SI, Figure SI-18).
Because of sample limitations, the 13C NMR spectra only
permitted assignment of chemical shifts estimated from HSQC
and HMBC spectra. As for dibenarthin (2), a C-3a hydroxy
group should be connected to a C-1b carboxylic group, and a
C-3b hydroxy group should be connected to a C-1c carboxylic
group via two ester bonds. ESIMS/MS analysis of compound 3
also supported the linear structure of tribenarthin (see SI).
The ESIMS/MS fragmentation spectrum is similar to that of
e
3″b-OH
4″b-OH
11.65 or 11.80, br
9.36, br
a
b
c
d
δ in ppm (187.5 MHz). Multiplicity. δ in ppm (750 MHz). 2,3-
e
Dihydroxybenzoic acid. Indistinguishable.
The absolute configuration of streptobactin (1), dibenarthin
(2), and benarthin (4) was determined by Marfey’s analysis.15
HPLC analysis of the FDAA derivatives of the acid hydrolysates
of 1, 2, and 4 revealed that the constituent amino acids of these
compounds were L-Arg and L-Thr.
trivanchrobactin.13 The H and 13C NMR assigned data are
1
summarized in Table 3.
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Table 3. NMR Data (750 MHz, DMSO-d6) of Tribenarthin (3)
a
bg
,
c
a
bg
,
c
position
Thr-a
13C,
m
1H (J in Hz)
position
13C,
m
1H (J in Hz)
d
DHBA -b
e
g
g
g
g
g
1a
nd
1″b
169.7,
116.4,
149.0,
146.4,
C
C
C
C
g
2a
54.6, CH
4.56, m
8.44, m
5.31, m
2″b
2a-NH
3a
3″b
g
70.2, CH
4″b
g
4a
15.9, CH3
1.08, d (6.0)
5″b
118.0, CH
6.93, m
6.70, m
7.39, m
g
Arg-a
1′a
6″b
118.2, CH
e
g
nd
7″b
118.7, CH
g
f
2′a
52.1, CH
4.76, m
3″b-OH
11.57 or 11.66 or 11.81, br
f
2′a-NH
3′a
8.76, m
9.30 or 9.36 or 9.38, br
g
29.0, CH2
1.74, 1.85, m
1.57, m
Thr-c
1c
g
e
4′a
24.4, CH2
nd
g
g
5′a
40.1, CH2
3.12, m
2c
56.9, CH
4.36, dd (3.0, 9.0)
8.06, d (9.0)
4.25, m
5′a-NH
6′a
7.48, m
2c-NH
3c
g
g
157.3,
C
66.0, CH
d
g
DHBA -a
1″a
4c
20.0, CH3
1.08, d (6.0)
g
g
g
g
g
169.7,
116.4,
149.0,
146.4,
C
C
C
C
Arg-c
1′c
e
2″a
nd
g
3″a
2′c
52.1, CH
4.76, m
4″a
2′c-NH
3′c
8.76, m
g
5″a
118.0, CH
6.93, m
6.70, m
7.39, m
29.0, CH2
1.74, 1.85, m
1.57, m
g
g
6″a
118.2, CH
4′c
24.4, CH2
g
g
7″a
118.7, CH
5′c
40.1, CH2
3.12, m
f
3″a-OH
4″a-OH
Thr-b
1b
11.57 or 11.66 or 11.81, br
5′c-NH
6′c
7.48, m
f
g
9.30 or 9.36 or 9.38, br
157.3,
C
d
DHBA -c
e
g
g
g
g
g
nd
1″c
169.7,
116.4,
149.0,
146.4,
C
C
C
C
g
2b
54.3, CH
4.76, dd (3.0, 9.0)
8.52, d (9.0)
5.41, m
2″c
2b-NH
3b
3″c
g
70.2, CH
4″c
g
4b
15.9, CH3
1.15, d (6.8)
5″c
118.0, CH
6.93, m
6.70, m
7.39, m
g
Arg-b
1′b
6″c
118.2, CH
e
g
nd
7″c
118.7, CH
g
f
2′b
52.1, CH
4.76, m
3″c-OH
4″c-OH
11.57 or 11.66 or 11.81, br
f
2′b-NH
3′b
8.76, m
9.30 or 9.36 or 9.38, br
g
29.0, CH2
1.74, 1.85, m
1.57, m
g
4′b
24.4, CH2
g
5′b
40.1, CH2
3.12, m
5′b-NH
6′b
7.48, m
g
157.3,
C
a
b
c
d
e
f
g
δ in ppm (187.5 MHz). Multiplicity. δ in ppm (750 MHz). 2,3-Dihydroxybenzoic acid. Not detected. Indistinguishable. Estimated values
from HSQC and HMBC spectra.
Streptobactin (1), dibenarthin (2), and benarthin (4)
possessed Fe-chelating activity comparable to that of deferox-
amine mesylate by a CAS assay. The ED50 values (quad-
ruplicate mean values) for streptobactin (1) and dibenarthin
(2) were 156 and 117 μM, slightly stronger than that of
deferoxamine mesylate (ED50 = 195 μM), while benarthin (4)
showed a weak color change (ED50 = 937 μM) under our assay
conditions. These siderophores are considered to have played
an important role in the survival strategy of Streptomyces sp.
strain YM5-799 under iron-limited conditions.
Patzer and Braun recently reported on the MS detection of
“griseobactin” as a cyclic trilactone of DHBA-arginine-
threonine from the unpurified fraction of Streptomyces sp.
ATCC 700974.16 They reported that analysis of related
biosynthetic gene clusters of the strain ATCC 700974 and
some S. griseus species indicated that streptobactin and
“griseobactin” may be the same compound.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured with a Horiba SEPA-300 digital polarimeter. UV spectra
were recorded with a Beckman DU 640 spectrometer and IR spectra
with a JASCO FT/IR-430 instrument. The H, 13C, and all 2D NMR
1
spectra were recorded with a Varian Unity INOVA 750 instrument.
Chemical shifts are referenced to the solvent peaks of δH 2.49 and δC
39.5 for DMSO-d6. Low- and high-resolution MS data were obtained
on a JEOL JMS700 spectrometer. ESIMS/MS spectra were measured
with a ThermoFinigan LCQ Advantage instrument.
Producing Microorganism and Taxonomy. The producing
microorganism, strain YM5-799, was isolated from a brown alga
[Analipus japonicus (Harvey) Wynne] collected off Charatsunai beach
(Muroran, Japan, 42°18′18″ N, 140°59′16″ E) in June 2003. Detailed
strain isolation procedures were previously described.17 The algal
sample was repeatedly washed with sterile seawater and homogenized
with a glass rod in 5 mL of sterile seawater. A 50 μL sample of
homogenate was plated onto 1/10 marine agar plates (10% marine
broth 2216 (Becton Dickinson, MD) in seawater, solidified with 1.5%
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agar) containing 50 mg/L of cycloheximide and incubated at 25 °C for
2 weeks. The bacterial colonies that had developed on the 1/10 marine
agar plates were further purified by single colony isolation. A pure
culture of strain YM5-799 was preserved in sterile seawater containing
10% glycerol at −80 °C. The strain was grown at 30 °C for 7 days on
MB2216 agar and ISP medium No. 2 plate for maintenance and
taxonomic characterization. Strain YM5-799 has branched aerial
mycelia, forming spores and shapes brownish striated-banded, 1−2
mm in diameter colonies on ISP medium No. 2 plate. Strain YM5-799
produces grayish aerial and substrate mycelia on ISP media No. 3,
No. 4, and No. 5 media. Strain YM5-799 has moderate salt tolerance
capabilities, and thereby this strain has been fully acclimated to the
marine environment. Other taxonomic characteristic features of this
strain are summarized in Table 4. Genomic DNA was purified from
and 10 mM FeCl3 (10 μL/L, final concentration of 0.1 μM) at pH 6.8
(before autoclaving) for 72 h at 30 °C with rotary shaking at 100 rpm.
Extraction and Isolation of Siderophores. The culture broth
(5 L) was centrifuged at 8000g for 15 min at 4 °C. The resulting
supernatant was acidified to pH 3 with 6 N HCl and applied to a
column of Diaion HP20 (Mitsubishi Chemical Co.). The column was
eluted with a stepwise gradient of aqueous MeOH (H2O, 50% MeOH,
and 100% MeOH, pH 3). The Fe-binding fraction was eluted with
50% MeOH using the CAS assay as the index. The 50% MeOH
fraction was purified by HPLC (TSK gel ODS 80Ts, 2.0 × 25 cm,
10 mL/min, UV detection at 210 nm) with 5% MeCN in H2O for
5 min and then a linear gradient up to 50% over 50 min containing
0.1% TFA to furnish four active fractions. The final purification was
performed by RP-HPLC (TSK gel ODS 80Ts, ϕ2.0 × 25 cm, 10 mL/
min, UV detection at 210 nm) eluted with a linear gradient from 20%
to 50% aqueous MeCN containing 0.1% TFA in 30 min to yield pure
benarthin (4, 3.4 mg, tR = 8 min), benarthin-linear-dimer (2, 2.5 mg,
tR = 11 min), linear benarthin-linear-trimer (3, 0.4 mg, tR = 14 min),
and streptobactin (1, 18.8 mg, tR = 16 min).
Table 4. Taxonomic Characteristics of Streptomyces sp. YM5-
799
strain YM5-799
Streptobactin (1): colorless gum; [α]23 +16 (c 0.1, MeOH); UV
D
liquefaction of gelatin
hydrolysis of starch
nitrate reduction
−
+
λmax (MeOH) (log ε) 246 (3.08), 320 (2.62), 357 (1.98) nm; IR
1
(KBr) νmax 3422, 1751, 1675, 1543, 1265, 1203, 1137 cm−1; H and
+
13C NMR data (DMSO-d6), see Table 1; HR-FABMS m/z 1180.5004
peptonization of skim milk
coagunation of skim milk
growth temperature range
salt tolerance
−
−
(calcd for [C51H69N15O18 + H]+ 1180.5023).
Dibenarthin (2): colorless gum; [α]23 +13 (c 0.1, MeOH); UV
D
20 °C (+), 25 °C (+), 30 °C (+), 37 °C (−)
1% (+), 2% (+), 3% (+), 4% (+), 5% (−)
λmax (MeOH) (log ε) 247 (3.99), 317 (3.65), 357 (3.44) nm; IR
1
(KBr) νmax 3423, 1638, 1543, 1386, 1262, 1205, 1132 cm−1; H and
13C NMR data (DMSO-d6), see Table 2; HR-FABMS m/z 805.3454
Carbon Utilization
(calcd for [C34H48N10O13 + H]+ 805.3481).
ISP medium No. 9 (negative
control)
−
Tribenarthin (3): colorless gum; [α]23 +29 (c 0.03, MeOH); UV
D
glucose (positive control)
L-rhamnose
+
λmax (MeOH) (log ε) 247 (2.91), 317 (2.63), 359 (2.40) nm; IR
1
(KBr) νmax 3422, 1629, 1459, 1358, 1206, 1124 cm−1; H and 13C
−
+
NMR data (DMSO-d6), see Table 3; HR-FABMS m/z 1198.5100
D-mannitol
(calcd for [C51H71N15O19 + H]+ 1198.5129).
D-fructose
+
CAS Assay. Siderophore activity was evaluated by the chrome
azurol sulfonate assay.20 The principle of the assay is based on the
color change of CAS from blue to orange, resulting from the removal
of iron from CAS. Standard curves relating the CAS reactivity to iron-
binding ligands were determined by using deferoxamine mesylate. The
ED50 value is defined as the concentration of a compound that reduced
the absorbance at 630 nm of the CAS solution by 50% in 4 h under
our assay conditions.
L-arabinose
−
−
−
−
−
raffinose
sucrose
D-xylose
inositol
Melanin Formation
ISP medium No. 6
ISP medium No. 7
+
Marfey’s Analysis.15 Approximately 0.2 mg of 1 was hydrolyzed
with 100 μL of 6 N HCl at 110 °C for 14 h. The acid hydrolysate was
freeze-dried and dissolved in 100 μL of 0.1 N HCl. To 50 μL of the
acidic solution were added 20 μL of 1 N NaHCO3 and 100 μL of a 1%
1-fluoro-2,4-dinitrophenyl-5-L-alaninamide (FDAA, Marfey’s reagent,
Pierce) solution in acetone, and the mixture was heated at 40 °C for
1 h. The mixture was cooled to room temperature, neutralized with
1 N HCl (20 μL), and evaporated to dryness. The residue was
dissolved in 100 μL of MeCN, and 20 μL of the FDAA derivatives was
analyzed by HPLC. The HPLC conditions were as follows: Develosil
C30-UG5 column (ϕ4.6 × 250 mm, Muromachi Chemical Co.)
maintained at 30 °C, photodiode array detector (primary UV
detection at 340 nm), flow rate of 1.0 mL/min, linear gradient elution
from 20% MeOH−0.1% TFA containing 10% MeCN to 20%
MeOH−0.08% TFA containing 50% MeCN for 25 min. The FDAA
derivatives of the acid hydrolysate were identified by comparing the
retention time with FDAA derivatized from authentic amino acids.
Compounds 2 and 4 were also analyzed in the same procedures.
The retention times of the acid hydrolysate were 16.85 (L-Arg; D-Arg,
15.79 min) and 19.12 (L-Thr; L-allo-Thr, 20.93; D-allo-Thr, 22.53, and
D-Thr, 23.89 min).
−
+: utilization, −: no utilization
the strain YM5-799 by using the QIAGEN Genomic-tip and buffer set
(Qiagen). The 16S rRNA gene fragment was amplified by using
universal primers corresponding to positions 8−27 as the forward
primer and 1492−1510 as the reverse primer.18 On the basis of the
1462 bp long 16S rRNA gene sequences, phylogenetically related
bacteria were searched for by using the BLAST program19 against the
GenBank/EMBL/DDBJ database. The 16S rDNA sequence analysis
enabled YM5-799 to be identified as the genus Streptomyces with high
similarity, and the strain was tentatively designated as Streptomyces sp.
YM5-799 [GenBank/EMBL/DDBJ accession number for the 16S
rRNA gene sequence is AB534176].
Fermentation of Streptomyces sp. YM5-799. The culture for
the production of siderophores was carried out under iron-limited
conditions. The strain YM5-799 produced siderophores in ASG
medium containing 0.1 μM FeCl3, but no production was observed in
ASG medium containing 100 μM FeCl3 or in marine broth 2216 (ca.
400 μM as FeCl3) (data not shown). All glassware used for the culture
and isolation of the siderophore was washed with 6 N HCl and then
rinsed with Milli Q H2O to avoid any iron contamination. The strain
YM5-799 was cultured in 5 L of an ASG medium containing casamino
acid (5 g/L), glycerol (3 g/L), glycerophosphate (0.1 g/L), NaCl
(15.5 g/L), KCl (0.8 g/L), MgSO4·7H2O (12.4 g/L), CaCl2·2H2O
(2.9 g/L), NH3Cl (1.0 g/L), HEPES (2.6 g/L), NaHCO3 (0.17 g/L),
ASSOCIATED CONTENT
■
S
* Supporting Information
This material is available free of charge via the Internet at
http://pubs.acs.org.
2375
dx.doi.org/10.1021/np200290j|J. Nat. Prod. 2011, 74, 2371−2376

Journal of Natural Products
Article
(15) Marfey, P.; Ottesen, M. Carlsberg Res. Commun. 1984, 49, 585−
AUTHOR INFORMATION
■
590.
Corresponding Author
*Tel: +81-439-80-3071. Fax: +81-439-80-2745. E-mail: kato.
toshiaki@nsc.co.jp.
(16) Patzer, S. I.; Braun, V. J. Bacteriol. 2010, 192, 426−435.
(17) (a) Nakanishi, K.; Nishijima, M.; Nishimura, M.; Kuwano, K.;
Saga, N. J. Phycol. 1996, 32, 479−482. (b) Manh, H. D.; Matsuo, Y.;
Katsuta, A.; Matsuda, S.; Shizuri, Y.; Kasai, H. Int. J. Syst. Evol.
Microbiol. 2008, 58, 1660−1664. (c) Hayakawa, Y.; Shirasaki, S.; Shiba,
S.; Kawasaki, T.; Matsuo, Y.; Adachi, K.; Shizuri, Y. J. Antibiot. 2007,
60 (3), 196−200.
ACKNOWLEDGMENTS
■
We thank Professor R. Sakai of Hokkaido University for the
HR-FABMS measurements. We also thank Professor T.
Motomura of Hokkaido University for the sample collection
and identification. This work was performed as part of the
project entitled “Constructing the Genetic Resource Library of
Unidentified Microbes Based on Genome Information”
supported by New Energy and Industrial Technology Develop-
ment Organization of Japan (NEDO).
(18) Weisburg, W. G.; Barns, S. M.; Pelletier, D. A.; Lane, D. J.
J. Bacteriol. 1991, 173, 697−703.
(19) Altsuchul, S. F.; Gish, W.; Miller, W.; Myers, E. W.; Lipman, D. J.
J. Mol. Biol. 1990, 215, 403−410.
(20) Schwyn, B.; Neilands, J. B. Anal. Biochem. l987, 160, 47−56.
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