| Budget Amount *help |
¥3,800,000 (Direct Cost: ¥3,800,000)
Fiscal Year 2006: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 2005: ¥3,000,000 (Direct Cost: ¥3,000,000)
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| Research Abstract |
The metabolism of bacteria in stationary phase is significantly different from that in exponential phase. The complex of σS and RNA polymerase core mainly functions in gene expression during stationary phase in Escherichia coll. Drastic alteration in metabolism occurs at the transition point from exponential phase to stationary phase, where more than 90% cells become living but non-culturable. We discovered that σE expression causes cell lysis of such living but non-culturable cells at the transition period, and proposed the lysis as σE-directed programmed cell death (PCD). However, the trigger for the PCD and the genes related to the PCD were still unknown. This study focused stresses causing the PCD and the σE regulon genes involved in the PCD. The results obtained are follows.
1) The results with a knock-out mutant of the σS gene revealed that its regulon genes suppress the change of living cells to non-culturable cells. Consistent with this, the katE-disrupted mutant showed the simi
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lar effect of σS gene-disrupted mutant, suggesting that the oxidative stress is one of the trigger to cause living but non-culturable cells.
2) With the transient expression system of the σE gene, DNA microarray was performed. From the results, many new σE regulon genes were found. Many up-regulated and down-regulated genes by σE were listed. Out of up-regulated genes, 4 proteases were found, which might be involved in the PCD. Out of down-regulated genes, most of Omp genes were found.
3) About 20, 000 transposon-inserted mutants were constructed and subjected to screening of PCD-suppressed mutants. 5 such mutants were isolated but found to be leaky. While, PCD-suppressed mutants isolated from mutagenesis showed the strong suppression. These mutants exhibited Omp proteins in the extent similar to that of the wild type.
4) Since many Omp genes were down-regulated under the increased expression of the σE gene, Mg 2+ may hardly be imported into cells. Addition of Mg2+ protected cell lysis, σE-directed PCD. The effect of Mg2+ was also examined by DNA microarray and many genes were found to be up-regulated.
From this study, it was found that the oxidative stress is one of the triggers to cause living but non-culturable cells. The increased expression of the σE gene reduced the expression of the Omp genes, which in turn reduced Mg2+ uptake. As the result, PCD was induced. Along this model, further experiments are required. Less
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