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内容記述 |
Upon depletion of essential nutrients from the culture medium, the growth rate of bacteria slows down and eventually reaches zero. At this point the culture enters into the stationary phase, which has been operationally defined as the absence of increase in cell number. The transition from rapid growth, where cells grow with a generation time less than 20 min, into stationary phase is accompanied by adaptation of bacterial cells to the new state. In this period of the transition of Escherichia coli culture, a number of morphological and physiological changes take place, including a decrease of cell volume, an alteration of cell shape, a modulation of the nucleoid, an alteration in the components of cell wall, and an accumulation of several storage materials, and alteration of the transcription and the translation machineries. These changes are accompanied by changes in gene expression in such a way that the growth-coupled genes are mostly switched off while the stationary phase-specific genes are up-regulated. More than 100 stationary phase-specific genes have been identified, and these genes appear to be expressed sequentially in a definite order. These findings altogether mean that there is temporal alteration in the E.coli phenotype even after the cessation of cell growth.<br /> The morphological and physiological differentiation of E.coli during the growth transition was studied in terms of the global regulation of gene expression. Based on the transcriptome analysis by using a DNA microarray assay I identified more than 70 genes that were induced and other 70 repressed in the stationary-phase. Among the induced genes, those whose expression depends on the sigma S (RpoS) were identified by comparison of the transcriptome between the wild-type and a rpoS disruptant. To observe the changes in the promoter activities associated with these stationary-phase genes, a novel vector was constructed. It allows expression of two fluorescent proteins in different way: the green fluorescent protein under the control of a test promoter and the other, dsRed protein, under the control of reference promoter. Using this double-reporter vector, the levels and growth phase-dependent variations were determined by FACS for several representative promoters from the exponential phase- and stationary phase-specific genes. This analysis of the promoter activities indicated that the population heterogeneity of E.coli culture increases in the stationary phase.<br /> Attempts were then made to fractionate stationary-phase cultures into homogenous populations. Cultures of E.coli were separated into more than 15 cell populations, each forming a discrete band after centrifugation with Percoll gradient. The separation resulted from the difference in buoyant density but not the size difference. The cell density increased upon transition from exponential growth to stationary phase. Exponential-phase cultures formed at least 5 discrete bands with lower densities, whereas stationary-phase cultures formed more than 10 bands with higher densities. These findings altogether suggest that the growth phase-coupled transition of E.coli phenotype is discontinuous. Two molecular markers characterizing each cell population were identified: the functioning promoter species, as identified by measuring the expression of green fluorescent protein under the control of test promoters; and the expressed protein species, as monitored by quantitative-immunoblotting. The analysis of chemical composition revealed that significant increase was observed only for polysaccharides. In concert with this finding, glycogen granules were found to accumulate in the stationary-phase cells as revealed by thin section microscopy. This finding suggests that at least one component, which contributes the increase in cell density is polysacchatides.<br /> As an initial attempt to identify the gene or genes involved in each step of cell density increase, a random screening was performed, by analyzing the Percoll centrifugation pattern of a set of E.coli mutants, each with deletion of a large segment of the genome. Among of a total of mutant strains tested, the density increase stopped for mutants at specific steps, forming discrete intermediate bands along Percoll gradient. One or more genes within the deleted genome segments must be involved in the density shift during the growth transition of E. colt into stationary phase. In parallel, l also tested some of the known stationary-phase genes in the cell density shift. As an initial attempt, the role of RNA polymerase sigroa S. (RpoS) in the cell density shift was examined. The rpoS disruptant formed apparently a single low density band even in the stationary phase, and the growth phase-coupled density increase was small. Even after prolonged culture, no further increase in the cell density was observed for this rpoS disruptant. Thus I concluded that the growth phase-coupled increase in E.coli cell density ceased at an early stage for the rpoS disruptant. These findings of mutant studies indicate that a specific gene or a set of genes are involved in each step of the cell density increase during the transition period from exponential growth to stationary phase, and the RpoS sigma factor is one such factor that is needed at an early step of the cell density increase. |
要旨・審査要旨 / Abstract, Screening Result (293.0 kB)
