49NISR Research GRANT
2009 Young Investigator Research Grant

Study on Aerial Hyphae Formation Mechanism of Siderophorein the Cell of Streptomyces coelicolor
Shinya KODANI
Graduate school of science and technology, Shizuoka University

Research aims

Methods

Since iron acquisition is a vital process for bacteria tosurvive, more than 100 enzymes functioning in primary andsecondary metabolism possess iron-containing cofactors
such as iron-sulfur clusters or heme groups. In aqueoussolution, iron exists as ferrous ion Fe (II), or ferric ion Fe(III). The ferric ion is constantly insufficient in natural environments because the solubility of ferric ion is as low as
1018 mM in water of biological pH, while optimum growthof one bacterial cell needs 1 mM of iron1). In order to utilize
such a low concentration of ferric ion in natural habitats,bacteria excrete low molecular compounds calledsiderophores, which have the ability to chelate ferric ion.
After chelating ferric ion, the ferric siderophore is taken up
into cells via receptors located across the cell membrane2).
Iron uptake systems of pathogenic bacteria using
siderophores have been well studied for the sake of establishing efficient clinical treatments because iron acquisitionis critical process for pathogen to survive in host cells. Infact, the secretion of some siderophores was reported to enhance the virulence of the pathogenic bacteria3). Due tothese biological significances, many siderophores includingpyochelin4), yersiniabactin5), petrobactin6), and mycobactin7)
were isolated from pathogenic bacteria. Regarding uniquesiderophore of Streptomyces, Umezawa et al.8) reported
about isolation of foroxymithine from S. nitrosporeus in1985 and recently a related peptide siderophore coelichelin
was found in S. coelicolor by combination of genome-mining technique and chemical analysis9,10).
Streptomyces scabies is a plant pathogenic bacterium
which causes serious damage to potato plants. Therefore
many studies about this bacterium, including the wholegenome sequencing project, have been performed fordecades to understand the infection mechanism11, 12). The
phytotoxin named thaxtomin A was isolated from S.
scabies. This toxin was shown to be a direct virulence factor to plants13) and was recently reported to cause apoptosis
in plant cells14). The necrogenetic factor, nec1, was also distributed in S. scabies and related strains15–17).
It was reported that the presence of deferoxiamine enhanced the production of antibiotics in Streptomyces
griseus. In order to clarify the role of siderophore in thebacterial cell, we attempted to isolate siderophore-binding
proteins from S. coelicolor using deferoxiamine, a commercially available siderophore, as a probe.

The affinity column was prepared by coupling deferoxiamine (Sigma-Aldrich) to the Affi-Gel affinity supports(Bio-Rad) in MOPS buffer at pH 7.5. The bacterium S.
coelicolor was cultured in ISP2 liquid media at 27°C for 5days. The cells were harvested by centrifugation, andwashed with distilled water. Cellular proteins were extracted from the washed cells by subjecting the cells to ultrasonication in 10% SDS-containing buffer. The proteinsin the cell extract were precipitated with trichloroaceticacid. The precipitated proteins were dissolved in Tris
buffer. The protein solution was loaded onto the affinity
column, followed by elution of putative deferoxiaminebinding proteins with distilled water and 0.1 N NaOH solution. Each fraction was analysed by SDS-PAGE.
Results
The result of SDS-PAGE was shown in Fig. 1. As shownby the arrow, a thick protein band (approximately 50 kDa)
was observed in the fraction eluted with 0.1 N NaOH. In
order to identify the protein, the mass-fingerprinting
method was applied after SDS-PAGE. The Fig. 2 illustrates
the MOF-MS spectrum of fragments generated by trypsindigestion of the protein. As a result of MASCOT web-Fig. 1. SDS-PAGE analysis of protein fractions.
Lane 1, cellular protein fraction; lane 2, flowthrough fraction; lane 3, protein fraction eluted with distilled water; lane 4, protein fraction eluted with
0.1 N NaOH.

50References
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Fig. 2. TOF-MS spectrum of peptides recovered after tryptic digestionof the 50 kDa protein.

9)10)

search, the protein was identified as the dihydrolipoamide
dehydrogenase of S. coelicolor.
It has been reported that the S. coelicolor dihydrolipoamide dehydrogenase had the ability to catalyze oxidation of dihydrolipoamide in an NAD dependent manner.
In this study, we attempted to isolate deferoxiamine-binding
proteins in S. coelicolor by using the deferoxiamine-immobilized affinity column. As a result, we obtained the dihydrolipoamide dehydrogenase of S. coelicolor as a putative
binding protein for deferoxiamine. However, this was most
likely due to the structural similarity between deferoxiamine and dihydrolipoamide.

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