| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2005: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 2004: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Research Abstract |
We found that four chitinases (ChiA, ChiB, ChiC, and ChiD), three β-N-acetylglucosaminidases (GlcNAcases A, B, and C), a transglycosylative enzyme (Hex99), a chitin-binding protein (Cbp1), a serine protease (AprIV) and a metalloprotease (MprIII) were involved in chitin degradation system of Pseudoalteromonas sp. strain O-7. AprIV and chitinases were induced in the presence of N-acetylglucosamine (GlcNAc). These results suggest that AprIV and chitinases might be coordinately controlled by the same regulatory system. Recently, we have found that ArcA, which is a response regulator of ArcB/ArcA two-component signal transduction system, binds to the upstream region of aprIV gene. The arcA and arcB genes were cloned and expressed in Escherichia coli. The ArcA protein as a response regulator was transphosphorylated by the ArcB protein as a sensor kinase. The ArcA protein was also phosphorylated by acetylphosphate which is an indicator of nutritive conditions of cells. Transphosphorylation fr
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om ArcB to ArcA was promoted by GlcNAc which is a component of chitin. Generally ArcA-ArcB is a two-component signal transduction system which facilitates adaptation to anoxia. However, our research indicated that ArcA plays an important role even in aerated conditions. Gel retardation assay and BIAcore analysis demonstrated that phosphorylated ArcA (ArcA-P) more tightly bound to the upstream region of aprIV and chiD genes compared to those of chiA, chiB, chiC, and cpb1 genes. Furthermore, footprint analyses indicate that ArcA-P binds to the consensus sequence, 5'-ACAATTAGANNNNNACTAAAACA-3'. The consensus sequence was found near the promoter region of each gene. These results suggest that ArcB/ArcA two-component signal transduction system negatively regulates the expression of genes involved in chitin degradation system of the strain.
On the other hand, Streptomyces thermoviolaceus OPC-520 produced four chitinases (Chi40, Chi35, Chi30, Chi25) in the presence of chitin. These genes contain a common similar pair of direct repeat sequences in the promoter regions, which have been suggested to be involved in both chitin induction and glucose repression. We found that regulatory gene involved in induction existed in the upstream region of genes encoding chitobiose transporter. We are doing the cloning and expression of the regulatory gene to examine whether the gene product binds to the direct repeat sequences of chitinase genes. Less
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