## 3 Discussion  
In this study, we tried to clarify the cytotoxic mechanism resulting from excess SPD in _E. coli_. It is thought that cell toxicity is mainly caused by reactive oxygen species [38]. However, acrolein produced mainly from SPM is more toxic than reactive oxygen species [39; 40]. SPM is normally absent in most mesophilic bacteria such as _E. coli_, _Helicobacter pylori_, and _Pseudomonas_ species [41; 42]. Therefore, the cytotoxic mechanism resulting from accumulation of polyamines in bacteria has not been characterized in detail. We examined the cytotoxic mechanism of excessive accumulation of SPD using an _E. coli_ strain deficient in SPD acetyltransferase, which regulates polyamine concentrations. Interestingly, we found that cell growth and viability were reduced by excess polyamines through translational repression of the specific proteins encoded by polyamine modulon whose SD sequence in  
Figure 5: Analysis of the structural change of the bulged-out region at the initiation site of the _rmf_ mRNA by SPD using NMR. The predicted secondary structure of the designed RNA stem-loop for the initiation site of the _rmf_ mRNA. The RNA consists of the -1 to -41 nucleotides of the 5\({}^{\prime}\)-untranslated region from initiation codon AUG. The numbers show the position from the 5\({}^{\prime}\)-end of the nucleotide sequence. The bases shown in lowercase were necessary for in vitro transcription (**A**). NMR spectra in the imino proton region in the absence and presence of SPD (**B**). The chemical shift difference (ppm) induced by SPD is shown in (**C**). The residues whose chemical shift changes are shown in Figure 5C are indicated by a square in Figure 5A. the mRNA is located distant from initiation codon AUG. The synthesis of the specific proteins encoded by polyamine modulon that have a normal position of the SD sequence in the mRNA was not influenced by excess polyamines at the translational level. Among the identified polyamine modulon proteins, the synthesis of Fis and RMF was strongly inhibited by accumulated SPD (Figures 2 and 4). There was no change in intracellular magnesium with or without SPD (data not shown). The transformant of _E. coli_ CAG224 with pMWSAT recovered cell growth and viability with an increase in the translational level of Fis and RMF in the presence of excess SPD (Figure 4). Thus, the decreases in the proteins encoded by polyamine modulon by the accumulated SPD are probably one of the reasons for the decrease in cell growth and viability. In particular, when the cell viability in the event of polyamine deficiency and of excess polyamines was compared, that of excess polyamines decreased more rapidly [25]. Since synthesis of RMF was inhibited by the accumulated SPD, cell viability is strongly affected, and excess polyamines are more toxic to cells than polyamine deficiency. These results confirm that polyamines contribute to cell growth and viability of _E. coli_ under various stress conditions. Several reports showed that the effect of SPD is mainly caused by stabilization of the bulged-out region of the double-stranded RNA, whose structure is not stabilized by Mg\({}^{2+}\)[14; 43]. We have previously reported that two candidate SD sequences exist in the nucleotide sequence of the _rmf_ mRNA [25]. The first SD sequence (SD1) located at 11 nucleotides upstream of initiation codon AUG forms the bulged-out region, and the second SD sequence (SD2) located at 2 nucleotides upstream of initiation codon AUG forms the double-stranded RNA (Figure 5A). When the position of the SD1 of the _rmf_ mRNA was shifted to the more common position located at 8 nucleotides upstream of initiation codon AUG, the degree of polyamine stimulation was reduced, although the level of the RMF protein was increased in the cells cultured without polyamines. When the SD2 was modified together with the SD1, the level of the RMF protein was markedly increased in the cells cultured without polyamines because of the elimination of the SD2, which forms the double-stranded RNA.