## 2 Results
ns, \(p\geq 0.05\); \({}^{\star}\), \(p<0.05\); \({}^{\star\star}\), \(p<0.01\); \({}^{\star\star}\), \(p<0.001\). that cell toxicity caused by excess SPD was slightly increased at the elevated temperature. The synthesis of the proteins essential for cell growth under heat shock conditions [RpoE (\(\sigma^{24}\)), StpA] encoded by members of polyamine modulon that have the position of the SD sequence distant from initiation codon AUG in their mRNAs was slightly but significantly inhibited by excess SPD (Figure 2B). As shown in Figure 2C, when _E. coli_ CAG2242 was cultured in the presence of K\({}_{2}\)TeO\({}_{3}\), an inducer of oxidative stress, especially superoxide anion [33], the degree of cell toxicity by excess SPD for cell growth was greater than that in the cells cultured in the absence of K\({}_{2}\)TeO\({}_{3}\). The growth rate reduced, the lag phase was significantly prolonged, and the final yield also reduced to a large degree in the presence of 4 mM SPD under oxidative stress. Furthermore, growth of _E. coli_ CAG2242 in the presence of 4 mM SPD under oxidative stress was slower than under heat shock stress. Under these conditions, the syntheses of SoxR (redox systems) and EmrR were significantly inhibited by excess SPD (Figure 2D). These members of polyamine modulon have the position of the SD sequence distant from initiation codon AUG in their mRNAs. In contrast, the synthesis of GshA which has a normal position of the SD sequence was not influenced by excess SPD. However, the level of RpoS which has a normal position of the SD sequence was decreased by SPD under these conditions. It has also been reported that the stringent response factor, ppGpp, which is required for long-term survival [34], negatively regulates _rpoS_ transcription [35], and synthesis of ppGpp is enhanced by polyamines [36]. Moreover, it has been reported that _rpoE_ and _rpoS_ control transcription of polyamine catabolic enzymes (_patA_ and _patD_) [17]. Thus, these proteins encoded by polyamine modulon are at least partially involved in the decrease in cell growth under various stress conditions. Figure 2: Inhibition of cell growth of _E. coli_ CAG2242 by excess SPD under heat shock stress and oxidative stress conditions. _E. coli_ CAG2242 cells were cultured as described in the legend of Figure 1 at 42 \({}^{\circ}\)C (**A**) and in the presence of 0.6 uM K\({}_{2}\)TeO\({}_{3}\) (**C**) with or without 4 mM SPD. Values are means of triplicate determinations. Error bars are within the symbol size. The levels of RpoE (\(\sigma^{24}\)), StpA, SoxR, EmrR, and GshA were measured together with a control protein, RpoD (\(\sigma^{70}\)), by Western blotting (**B**,**D**). Values are means \(\pm\) S.E. of triplicate determinations. Student’s _t_-test was performed for the values obtained in the presence of SPD versus in the absence of SPD. ns, \(p\geq 0.05\); *, \(p<0.05\); **, \(p<0.01\); ***, \(p<0.001\). Correlation between the Decrease in Cell Viability and Translation of Proteins Encoded by Polyamine Modulon in Response to Excess Spermidine  
As shown in Figure 3A, cell viability of _E. coli_ CAG2242 with or without SPD was examined. Due to the accumulation of SPD, cell viability of _E. coli_ CAG2242 was decreased greatly at the stationary phase, but that of _E. coli_ C600 was not altered (Figure 3A). Polyamine contents in cells grown with or without SPD were then measured at the stationary phase (24 and 36 h) (Figure 3B). When _E. coli_ CAG2242 cells were grown without SPD, a small amount of SPD was detected in the cells. When the cells were grown with SPD, overaccumulated SPD was observed at the stationary phase. The accumulated SPD in the cells was approximately 6 times higher than that in the cells grown without SPD, whereas the level of PUT decreased over time. These results suggest that the concentration of SPD in the cells that could decrease cell viability of _E. coli_ CAG2242 was approximately 90 mM.