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京都大学 / 新制・課程博士 / 博士(理学) / 甲第23682号 / 理博第4772号 / 新制||理||1684(附属図書館) / 京都大学大学院理学研究科数学・数理解析専攻 / (主査)准教授 矢野 孝次, 教授 泉 正己, 教授 日野 正訓 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DFAM

<p>The sigma S subunit (RpoS) of RNA polymerase acts as the master regulator of stress in <em>Escherichia coli</em>, allowing adaptation and survival under unfavourable conditions such as nutrient deprivation. RpoS regulates and integrates the signals of hundreds of genes (about 10% of the genome) organized into complex networks and modules that govern the response to stress and entry of the cell into stationary phase. Although microarray studies have been performed on starvation models in <em>E. coli</em>, the expression of the RpoS regulon has not been studied in long-term cultures due to the difficulty of isolating RNA from starving cells. In this study, the development of a protocol for isolating RNA from stationary phase cells, employing hot acid phenol without the use of DNase, is described. In addition, the expression of several genes during different phases of growth is analyzed by RT-qPCR in order to validate preliminary microarray data obtained from 24 and 48 hour-old cultures. Although the results obtained by RT-qPCR agree well with the literature, they do not corroborate preliminary microarray data at the 24 and 48 hour timepoint.</p> / Master of Science (MSc)

<p><em>Escherichia coli </em>cells undergo many morphological and physiological changes to survive in late stationary phase due, in part, to the exhaustion of nutrients and accumulation of inhibitory metabolites. Previous microarray data in our lab provided the general profile of gene expression from exponential phase to 48 h of incubation. The goal of this study is to use qPCR to validate the preferentially expressed genes in late stationary phase determined by the microarray data. The expression of three genes (<em>hha, tomB </em>and <em>emrK</em>) with increased expression levels from 24 h to 48 h of incubation and another two RpoS-dependent genes (<em>bolA</em> and <em>osmY</em>) with decreased expression from 24 h to 48 h of incubation were chosen as the target genes. RNA was first extracted from exponential phase, early stationary phase, 24 h and 48 h bacterial cultures. Hot phenol-chloroform and a commercial kit were used to isolate RNA. All these methods recovered low RNA in late stationary phase. qPCR partly confirmed the previous microarray data. The expression of <em>hha</em>, <em>tomB</em> and <em>emrK</em> was validated to increase from 24 h to 48 h of incubation. The discordant results between qPCR and microarray data may be due to the low transcript abundance of target genes and genomic DNA contamination in RNA extracted from 48 h bacterial cultures.</p> / Master of Science (MSc)

Escherichia coli survives in both nutrient rich nutrient-limited environments. As such, understanding the gene and protein level activity that occurs during stationary phase is considered an important aspect of bacterial survival. Escherichia coli has been studied for decades providing substantial insight into gene expression profiles in exponential phase and recently, during adaptation to stationary phase. This led to the discovery of RpoS as a growth phase-dependent sigma factor. Further studies indicated that there are many genes that are expressed in an RpoS-independent but stationary phase-specific manner. However, proteins represent the functional molecules of the cell. Additionally, protein expression does not always correlate with the corresponding gene expression patterns. Therefore, to obtain an in depth understanding of the proteins that play a role in long-term growth in E. coli, TMT- (Tandem Mass Tags) based quantitative proteomic analysis was performed to identify proteins that are preferentially expressed during prolonged starvation. We identified proteins that were both positively and negatively regulated by RpoS during stationary phase, such as GadA and TnaA, respectively. RpoS levels peaked during early stationary phase and declined thereafter. However, proteins that were RpoS-dependent continued to increase during prolonged stationary phase. Additionally, we identified proteins that were expressed in an RpoS-independent manner during stationary phase. This suggests that protein expression during early stationary phase is distinct from prolonged stationary phase. Furthermore, RpoS-independent proteins may also play an important role during long-term growth. / Thesis / Master of Science (MSc) / Escherichia coli adapts to shifts in nutrient availability using the alternative sigma factor RpoS which controls morphological and physiological changes. Although gene expression during growth has been extensively studied, comparable information regarding changes in protein abundance during prolonged incubation is not available. We employed a quantitative proteomics approach to identify proteins that are preferentially expressed during stationary phase in E. coli. We identified classes of proteins that are upregulated and downregulated by RpoS in addition to proteins regulated independently of RpoS. Global analysis of protein expression during growth can aid in understanding the adaptation of E. coli under starvation conditions.