## 2 Results
The synthesis of the proteins necessary for cell viability (RMF, RpoZ, and CpxR) encoded by members of polyamine modulon that have the position of the SD sequence distant from initiation codon AUG in their mRNAs was significantly inhibited by excess SPD (Figure 3C). However, the synthesis of the proteins (RRF, SpoT, UvrY, but not RpoS) encoded by members of polyamine modulon that have a normally positioned SD sequence were not influenced by excess SPD. The level of RpoS was decreased by SPD at both the logarithmic and the stationary phase in parallel. Thus, it is possible that excess polyamines reduce cell viability greatly by regulating synthesis of both ppGpp and RpoS, which inhibit RNA and protein synthesis. Figure 3: Inhibition of cell viability of _E. coli_ CAG2242 by excess SPD. _E. coli_ CAG2242 cells were cultured as described in the legend of Figure 1. Cell viability was determined by counting colony numbers in aliquots of the culture grown on LB-containing 1.5% agar plates at 37 \({}^{\circ}\)C. Values are means of triplicate determinations. Error bars are within the symbol size (**A**). Polyamine contents in the cells harvested at A\({}_{540}\) of 24 h and 36 h were measured as described under “Materials and Methods” (**B**). The levels of RMF, RpoZ (\(\omega\)), CpxR, RRF, SpoT, UvrY, and RpoS (\(\sigma^{38}\)) were measured together with a control protein, RpoD (\(\sigma^{70}\)), by Western blotting (**C**). 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, \(p\geq 0.05\); \({}^{\star}\), \(p<0.05\); \({}^{\star\star}\), \(p<0.01\); \({}^{\star\star}\), \(p<0.001\). Recovery of Cell Growth and Viability by Transformation of the Gene Encoding Spermidine Acetyltransferase  
The effect of SPD acetyltransferase on cell growth and viability was assessed. As shown in Figure 4A,B, the transformant of _E. coli_ CAG2242 with pMWSAT encoding the gene for SPD acetylcholinesteraseressrae recovered cell growth and viability in the presence of excess SPD. Polyamine contents in the cells grown with or without SPD were then measured at the logarithmic and stationary phases (Figure 4C). The level of SPD in cells was markedly decreased when pMWSAT was transformed. We previously reported that cell growth and viability of _E. coli_ MA261, a polyamine-requiring mutant, was reduced by depletion of polyamines. Under the depletion of polyamines, the transformant of _E. coli_ MA261 with _fis_ and _rmf_ genes with modifications in the position of the SD sequence to the normal position recovered cell growth and viability [20; 25]. Thus, the synthesis of Fis, a global regulator of transcription of some growth-related genes, including genes for rRNA and tRNA, was examined at the logarithmic phase, since Fis is an essential protein for cell growth. In addition, the synthesis of RMF was examined at the stationary phase, since RMF is an important protein for cell viability. With pMWSAT, Fis and RMF were synthesized efficiently even in the presence of SPD (Figure 4D). By the addition of excess SPD, cell growth and viability were enhanced in parallel with the increase in the levels of Fis and RMF (Figure 4A). These results indicate that cell growth and viability are reduced by excess polyamines through translational repression of the specific proteins encoded by polyamine modulon whose SD sequence in the mRNA is located distant from initiation codon AUG.  
Figure 4: Recovery of cell growth and viability by SPD acetyltransferase. _E. coli_ CAG2242 cells carrying pMWSAT or pMW119 (vector) were cultured with or without 4 mM SPD (**A**). Cell viability was determined by counting colony numbers in aliquots of the culture grown on LB-containing 1.5% agar plates at 37 \({}^{\circ}\)C (**B**). Polyamine contents in the cells harvested at A\({}_{540}\) of 0.2 and 24 h were measured as described under “Materials and Methods” (**C**).