60NISR Research GRANT
2008 Young Investigator Research Grant

Exploration of novel transporter proteins for oligosaccharides
using ATPase (MsiK)
Akihiro SAITO
Graduate School of Advanced Integration Science, Chiba University

Research aims
Streptomycetes are saprophytic soil bacteria which decompose biopolymers (protein, starch, cellulose, hemicelluloses, and chitin). To deepen our knowledge of recognitionand decomposition systems for biopolymers in streptomycetes, we have investigated a degradation system forchitin, which is an N-acetylglucosamine polymer, in Streptomyces coelicolor A3(2). The bacterium possesses at least
13 (putative) chitinase genes (chi) in its genome, and thetranscription of 8 among the 13 chi genes is induced in thepresence of chitin and its degradation product N, N-diacetylchitobiose [(GlcNAc)2]1–3). (GlcNAc)2 uptake is supposed to be required for transcriptional induction of the chigenes, as the msiK gene, encoding an ATP-hydrolyzing
component of (GlcNAc)2 transporters, is essential for induction of chitinase production4). Hurtubise et al.5) have already demonstrated that uptake of cellobiose and xhlobiose
is essential for induction of cellulase and xylanase production, respectively, in Streptomyces lividans. Uptake of disaccharides is thus assumed to be a prerequisite for induction of corresponding polysaccharide-hydrolases.
In streptomycetes, uptake systems for maltose, cellobiose, xylobiose, and (GlcNAc)2 have been identified as
disaccharide transporters6–10). All of these uptake systems
are, interestingly, ABC (ATP-binding cassette) transporters
and supposed to act with the above-mentioned MsiK protein4,11). msiK seems to be transcribed constitutively,
whereas malE, cebE, bxlE, and dasA, encoding sugar-binding proteins of ABC transporters, are induced by corresponding sugars; i.e. maltose, cellobiose, xylobiose, and(GlcNAc)2, respectively4).
There are 37 sets of genes for (putative) ABC transporters for OSPs (oligosaccharides and polyols) on the S.
coelicolor A3(2) genome (ABCISSE database). It is noteworthy that in streptomycetes, in contrast to other bacteria,a gene for the ATP-hydrolyzing component is not locatedamong or with each of the 37 gene clusters, which encode
the other components (a solute-binding protein and twomembrane proteins). MsiK is, thus, supposed to act withthose components for OSP transporters as a common component. However, transport substrates for 28 of the 37 transporters remain, up to now, unclear. This study aims to investigate novel ABC transporter components for OSP,
whose uptake systems are unidentified, by obtaining proteins that interact with MsiK in the presence of correspon-ding transport substrate. We report here the development ofa system to obtain proteins interacting with MsiK.
Methods
msiK and its flanking region (including the putative promoter sequence) were amplified by using primers to produce C-terminally His-tagged MsiK protein under the control of the putative native promoter. The amplified DNAfragment (1295 bp) was cloned and its sequence was confirmed to be identical to that registered in the genome database (http://www.sanger.ac.uk/Projects/S_coelicolor/). The
cloned fragment was integrated into a multi-copy plasmid
vector pWHM3 to obtain the resulting plasmid pCMK02.
The msiK-null mutant ASC34) was transformed with
pWHM3 or pCMK02. To investigate the localization ofMsiK, S. coelicolor A3(2) transformants were cultivatedaccording to a method described previously3), with somemodifications. Cytoplasmic and envelope fractions wereprepared from the harvested mycelia by sonication,centrifugation, and ultracentrifugation. Nickel affinity column chromatography was done according to a describedmethod 12).
Results
In S. coelicolor A3(2) ASC3 (pMCK02), a protein bandcorresponding to 45 kDa was detected by immunoblot
analyses using anti-MsiK antiserum or anti-His-tag antibodies, while no band was detected with the antiserum or
the antibodies in its vector control strain ASC3 (pWHM3).
In contrast, using the anti-MsiK antiserum, a protein band(40 kDa) was observed in the wild type strain carrying theplasmid vector pWHM3. The data demonstrated that Histagged MsiK protein (MsiKHis) was successfully producedin the strain ASC3 (pCMK02). Irrespective of the kind ofsugar in the culture media, MsiK protein was found in thecell envelope fraction of S. coelicolor A3(2) wild typestrain M145. MsiKHis was also detected in the envelope
fraction of the strain ASC3 (pCMK02). The data impliedthat MsiK protein, regardless of the C-terminal His-tag, interacted with membrane proteins, which may act as transporters. MsiKHis was obtained by nickel affinity column
chromatography from the cell lysate of ASC3 (pCMK02),
although other proteins were co-eluted. To investigate proteins interacting with MsiKHis, the composition of eluted

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proteins was compared by SDS-PAGE among M145
(pWHM3), ASC3 (pWHM3), and ASC3 (pCMK02). Someprotein bands were specifically detected in ASC3(pCMK02). It was thus concluded that proteins interactingwith MsiKHis were gained by the chromatography.

4)5)Conclusion
We successfully produced C-terminally His-tagged MsiKprotein (MsiKHis) in the S. coelicolor A3(2) msiK-null mutant carrying pCMK02. MsiKHis was detected in cell-envelope fraction of the transformant, as native MsiK protein in
the wild type strain M145. The data suggested that MsiKHis
interacted with membrane proteins as does native MsiK. By
obtaining MsiKHis from the S. coelicolor A3(2) transformant using nickel affinity column chromatography, someproteins, which would interact with MsiKHis in vivo, wereobtained. We shall explore novel sugar transporter proteins
acting with MsiK, by identifying the probable MsiK-interacting proteins.

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References
1)

Saito, A., Fujii, T., and Miyashita, K. Distribution and evolution ofchitinase genes in Streptomyces species: involvement of gene-duplication and domain-deletion. Antonie van Leeuwenhoek. 84, 7–15
(2003)
2) Saito, A., Fujii, T., Yoneyama, T., Redenbach, M., Ohno, T., Watanabe, T., and Miyashita, K. High-multiplicity of chitinase genes in
Streptomyces coelicolor A3(2). Biosci. Biotech. Biochem. 63,710–718 (1999)
3) Saito, A., Ishizaka, M., Francisco, P. B. Jr., Fujii, T., and Miyashita,
K. Transcriptional co-regulation of five chitinase genes scattered on
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12)

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