| Budget Amount *help |
¥19,890,000 (Direct Cost: ¥15,300,000、Indirect Cost: ¥4,590,000)
Fiscal Year 2010: ¥4,550,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥1,050,000)
Fiscal Year 2009: ¥4,160,000 (Direct Cost: ¥3,200,000、Indirect Cost: ¥960,000)
Fiscal Year 2008: ¥11,180,000 (Direct Cost: ¥8,600,000、Indirect Cost: ¥2,580,000)
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| Research Abstract |
Selenomonas ruminantium, which is a strictly anaerobic, Gram-negative bacterium has neither free nor bound forms of lipoprotein which plays an important role of the maintenance of the structural integrity of the cell surface in general Gram-negative bacteria. Instead, it has cadaverine, which links covalently to the pepticloglycan as a pivotal constituent for the cell devision. In S. rmuinantimn cadaverine is synthesized constitutively from L-lysine by lysine decarboxylase (LDC) and transferred to the peptidoglycan as an essential constituent for normal cell growth. Furthermore, S. rwninantuan LDC has two unique characteristics ; (1) it has decarboxylase activity towards both L-lysine and L-ornithine. (2) biochemical future is similar to that of eukaryotic ornithine. Here, we found the following evidences, i.e.
(1) S. rtm inanluon LDC was dramatically degraded at the early stationary phase by ATP -dependent serine protease(s). This degradation proteolysis was required a degradation fact
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or, which was named as P22. P22 was induced by the accumulation of putrescine in S. raminanlium cells, and it bound tightly to LDC with a K_D) of 8.5X10^<-11> M. P22 seems to have similar biochemical and biophysical characteristics to those of an antizynte (AZ), which have been reported only in mammalian cells. P22 was identified as a ribosomal protein LI0 of S. rimliuunlimn. The binding of L10 to LDC for forming LDC-L10 complex may cause a conformational change(s) of LDC subunit, which allow it to be attacked by ATP-dependent serine protease(s), and the LDC would be broken down to small peptides. We also identified the two regions, A region (K^<101>NKLD^<105>) and B region (G^<106>VIRNAVYVLD^<170>) in L-10 molecule essential for its binding to LDC.
(2) We identified the ATP-dependent serine protease(s), which is involved in the degradation of S. ruminantiian LDC. There were three genes for ATP-dependent protease chromosomal in S. ruminantium and we focused on one of them, which belongs to C IpXP protease(s) since they require an adapter protein. CIpXP protease consists of the CIpX ATPase and the CIpP peptidase. In S. rmninantitan, L10 may work as an adapter protein and carry the LDC to CIpXP protease. We cloned C1pP gene from S. ruminantiuni into pET15b and expressed it in E, coli BL21 (DE3) and purified.
(3) Cadaverine covalently linked to the peptidoglycan is required for the interaction between the peptidoglycan and the S-layer homologous (SLH) domain of the major outer membrane protein, Mep45. Here, using a series of diamines with a general structure of NH_3^+(CH_2)_nNH_3^+, n=3-6), we found that cadaverine (n=5) specifically serves as the most efficient constituent of the peptidoglycan to acquire the high resistance of the cell to the external damage agents, and is required for the effective interaction between the SLH domain of Mep45 and the peptidoglycan facilitating the correct anchoring of the outer membrane to the peptidoglycan. Less
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