Effects of polyamines on protein synthesis and growth of Escherichia coli

The polyamines (PA) putrescine, spermidine, and spermine have numerous roles in the growth of both prokaryotic and eukaryotic cells. For example, it is well known that putrescine and spermidine are strongly involved in proliferation and viability of Escherichia coli cells. Studies of polyamine functions and distributions in E. coli cells have revealed that polyamines mainly exist as an RNA–polyamine complex. Polyamines stimulate the assembly of 30S ribosomal subunits and thereby increase general protein synthesis 1.5- to 2.0-fold. Moreover, these studies have shown that polyamines stimulate synthesis of 20 different proteins at the level of translation, which are strongly involved in cell growth and viability. The genes encoding these 20 different proteins were termed as the “polyamine modulon.” We here review the mechanism of activation of 30S ribosomal subunits and stimulation of specific proteins. Other functions of polyamines in E. coli are also described.

The polyamines, consisting of putrescine (NH 2 (CH 2 ) 4 NH 2 ), spermidine (NH 2 (CH 2 ) 3 NH(CH 2 ) 4 NH 2 ), and spermine (NH 2 (CH 2 ) 3 NH(CH 2 ) 4 NH(CH 2 ) 3 NH 2 ), have been implicated in numerous growth processes in Escherichia coli and mammalian cells (1)(2)(3)(4)(5)(6). E. coli is useful for studying the functions of polyamines, because various mutants are available, and cloning of genes is relatively easy. Polyamine content in E. coli consisting of putrescine and spermidine is adjusted by biosynthesis (7,8), uptake (9), and excretion (9). Dr. Tabor's lab isolated a polyamine-deficient E. coli mutant HT283, in which the rate of cell growth is about 30% that of cells cultured with polyamine addition (7,10). They reported that polyamines are involved in some aspects of protein synthesis (7,10) and that polyamines are necessary for efficient translation of amber codons in cells containing bacteriophage T7 carrying an amber mutation in gene 1 (11). Our group also studied the effect of polyamines on protein synthesis in a cell-free system or using a polyaminerequiring mutant MA261 (speB, agmatine ureohydrolase, and speC, ornithine decarboxylase-deficient mutant) (8), and the results are summarized below.

Effect of polyamines on protein synthesis in E. coli cellfree systems and on the assembly of ribosomal subunits
The effects of polyamines were studied using polyamine-free dialyzed ribosomes and Sephadex G-50 -treated supernatant at various Mg 2ϩ concentrations (12). As shown in Fig. 1A, addition of 2-4 mM spermidine or 15-25 mM putrescine with various concentrations of Mg 2ϩ increased poly(U)-directed polyphenylalanine synthesis up to twice that synthesized at an optimal Mg 2ϩ concentration (13 mM) in the absence of polyamines. Spermine exists together with putrescine and spermidine in mammalian cells (13). The effective concentration of spermine for stimulation of protein synthesis was approximately one-fifth that of spermidine (14). Thus, the effects of polyamines on protein synthesis have a rank order of potency spermine Ͼ spermidine Ͼ putrescine. These results show that polyamines not only have a sparing effect on the Mg 2ϩ requirement for polyphenylalanine synthesis but also a stimulatory effect, which cannot be fulfilled by Mg 2ϩ in the absence of polyamines. Then, the degree of stimulation of polypeptide synthesis by spermidine was examined using various synthetic mRNAs, and it was found that stimulation of polypeptide synthesis by spermidine depends on the uracil content in mRNAs (15). Polyphenylalanine synthesis was also stimulated when [ 14 C]Phe-tRNA was used instead of [ 14 C]Phe, indicating that stimulation by polyamines is based on stimulation at the level of ribosomal polypeptide synthesis. Next, the effect of polyamines on Q␤ RNA-directed synthesis of proteins was tested. It was found that only Q␤ RNA-directed RNA replicase synthesis was stimulated by polyamines (16). In the case of T7-DNAdirected protein synthesis, synthesis of T7 RNA polymerase and the 42-kDa protein, but not the 13.5-kDa protein, were stimulated by spermidine mainly at the level of translation (17). The results confirm that specific types of protein synthesis are stimulated by polyamines.
It also became clear that the assembly of 30S ribosomal subunits in E. coli was stimulated by polyamines (18,19). Stimulation of the assembly of 30S ribosomal subunits, consisting of 16S rRNA and 21 ribosomal proteins (S1 to S21), by polyamines occurred by polyamine stimulation of enzymatic methylation of two adjacent adenine residues near the 3Ј-end of 16S rRNA (Fig. 1B) (20). Translation efficiency increased 1.5-to 2.0-fold due to stimulation of the assembly of the 30S ribosomal subunit

Polyamine distribution in E. coli
To confirm that polyamines also function at the level of translation in vivo, polyamine distribution in E. coli was estimated by measuring the binding constants of polyamines for the target molecules and the concentration of the target molecules in E. coli W3110 harvested at logarithmic phase (21). Intracellular water space of E. coli was estimated to be 2.9 l of cell volume/mg of protein (22). As shown in Fig. 2A, the total spermidine and putrescine concentrations in E. coli were 6.88 and 32 mM, respectively, each mainly existing as a polyamine-RNA complex, supporting the idea that major polyamines function at the level of translation. It has been reported that the yeast tRNA Phe crystal structure includes two molecules of spermine (23), which is close to our estimation for polyamine binding to RNA (24).

Polyamine stimulation of synthesis of specific proteins at the level of translation in E. coli
It has been examined as to what kinds of protein synthesis are stimulated by polyamines using a polyamine-requiring mutant of E. coli MA261 (8). Cell growth of E. coli MA261 slows down in the absence of polyamines. When putrescine is added to the culture medium, it is taken up into the cells by polyamine transporters consisting of PotA-D proteins (spermidine-preferential uptake system) and PotF-I proteins (putrescine-specific uptake system) (9), and spermidine is synthesized from putrescine. Accordingly, cell growth of E. coli MA261 was stimulated ϳ3-fold by the addition of putrescine (0.6 mM) to the medium.
A set of genes whose expression is enhanced by polyamines at the level of translation was classified as a "polyamine modulon" (25,26). In E. coli, we have thus far identified 20 kinds of genes as components of the polyamine modulon (5,27). There are several mechanisms underlying polyamine stimulation of the synthesis of various members of the polyamine modulon (Fig.  2B). First, polyamine stimulation of protein synthesis can occur when a Shine-Dalgarno (SD) 2 sequence (28) in the mRNA is obscure or is distant from the initiation codon AUG. Polyamines cause structural changes of a region of the SD sequence and the initiation codon AUG facilitating formation of the initiation complex. This is the case for oppA, fecI ( 18 ), fis, rpoN ( 54 ), hns, rpoE ( 24 ), stpA, emrR, rmf, rpoZ (), cpxR, and soxR. Proteins anno- 2 The abbreviations used are: SD, Shine-Dalgarno; RMF, ribosome modulation factor; RRF, ribosome recycling factor.  7 split proteins S1, S2, S3, S5, S9, S10, S14 THEMATIC MINIREVIEW: Stimulation of protein synthesis by PA tated in red in Fig. 2B are involved in cell growth during the logarithmic phase, and those shown in blue are involved in cell viability at the stationary phase. In a second mechanism, polyamines enhance the inefficient initiation codon UUG-or GUG-dependent fMet-tRNA binding to cya, cra, spoT, uvrY, frr (RRF), or gshA mRNA-ribosome complex. In a third mechanism, polyamines stimulate read-through of the amber codon UAG by Gln-tRNA SupE on ribosome-associated rpoS mRNA or stimulate a ϩ1 frameshifting at the 26th UGA codon of prfB mRNA encoding RF2 (translation releasing factor 2). Bound %* *"Bound %" indicates "bound mol of polyamine per 100 mol of phosphates or 100 mol of ATP".

THEMATIC MINIREVIEW: Stimulation of protein synthesis by PA
Because the synthesis of many transcription factors was stimulated by polyamines, transcription profiles of the logarithmic phase culture of E. coli MA261 with or without putrescine were determined by a two-color (Cy3 and Cy5) cDNA microarray analysis (29,30). Expression of 2,742 genes was detected in cells cultured with or without putrescine. Among these, 309 genes were up-regulated more than 2-fold by polyamines (Fig.  2C). In 309 up-regulated genes, 240 kinds of mRNA were under the control of nine transcription factors mainly existing in logarithmic phase and two transcription factors mainly existing in stationary phase (see Fig. 4

Molecular mechanism of polyamine stimulation of EmrR, SoxR, and GshA synthesis
Under oxidative stress conditions caused by 0.6 M K 2 TeO 3 , an inducer of oxidative stress (31,32), synthesis of SoxR, EmrR, GshA, and RpoS was stimulated by polyamines (27). Because RpoS has been already identified as a polyamine modulon protein through polyamine stimulation of suppression of an amber termination codon in the ORF (33), the mechanism of polyamine stimulation of EmrR, SoxR, and GshA was studied by measuring protein and mRNA levels by Western blotting (34) and dot blotting (35), respectively. As shown in Fig. 3, stimulation of the synthesis of EmrR and GshA proteins occurred at the level of translation. The nucleotide sequence of emrR and gshA mRNAs was then determined. The SD sequence of emrR mRNA was 11 nucleotides upstream of the initiation codon AUG, whereas the initiation codon of gshA mRNA was UUG instead AUG. When the position of the SD sequence of emrR mRNA was shifted to a normal position (8 nucleotides upstream of the initiation codon AUG), the synthesis of an EmrR-␤-gal fusion protein measured in the absence of polyamines increased, and the degree of polyamine stimulation was reduced (Fig. 3A). In soxR mRNA, the SD sequence is also distant from the initiation codon AUG. So, SoxR synthesis was stimulated by polyamines. Stimulation of EmrR and SoxR syntheses by polyamines was due to a selective structural change of the bulged-out region of dsRNA in the initiation region of mRNA by spermidine, similar to stimulation of OppA synthesis by polyamines (36,37). When the initiation codon UUG of gshA mRNA was changed to AUG, synthesis of GshA-␤-gal fusion protein in the absence of polyamines increased, and the degree THEMATIC MINIREVIEW: Stimulation of protein synthesis by PA of polyamine stimulation was reduced (Fig. 3B). These results indicate that EmrR and SoxR syntheses are enhanced by polyamines due to the unusual position of the SD sequence and that GshA synthesis is due to the presence of the inefficient initiation codon UUG instead of AUG. The results indicate that polyamines stimulate protein synthesis encoded by inefficient mRNAs.

Role of polyamines at various cell growth conditions
Members of the polyamine modulon were classified according to the growth conditions (Fig. 4). At the logarithmic phase, 10 members of the polyamine modulon were identified as transcriptional and translational factors. Another member of the polyamine modulon is OppA, which is involved in the transport of oligopeptides (38). Thus, polyamines greatly contribute to cell growth at the logarithmic phase. At the stationary phase, six kinds of protein synthesis were stimulated by polyamines. UvrY and CpxR are response regulators in two-component signaltransducing systems, which are involved in biofilm formation (39,40). SpoT and RpoZ are proteins involved in the function of ppGpp, a stringent response factor, i.e. a regulating factor of transcription (41,42). RRF and ribosome modulation factor (RMF) increased cell viability through adjusting protein synthesis (39,43). Under conditions of oxidative stress, synthesis of two transcription factors (SoxR and EmrR) and a GSH synthetic enzyme (GshA) was stimulated by polyamines (27). Because SoxR is a transcription factor for expression of the superoxide response regulon, it stimulated the transcription of the sodA gene encoding a superoxide dismutase (Fig. 4C). Furthermore, the level of glutathione (GSH) was increased through poly-amine stimulation of the synthesis of EmrR and GshA (Fig. 4C). GshA directly increases GSH synthesis. Stimulation of EmrR synthesis probably causes the inhibition of the efflux of many metabolites with small molecular weight, which includes GSH. Thus, hydrogen peroxide is detoxified effectively by GSH peroxidase through the increase in GSH. It was also shown that transcription of genes for katG and katE encoding catalases is enhanced through polyamine stimulation of RpoS ( 38 ) synthesis (27). It has been reported that RpoS also induced the stimulation of gadE (a regulator of GDAR (glutamate-dependent acid resistance) system at acidic pH (44)), which is important for survival under acidic conditions. The results confirm that polyamines strongly contribute to cell growth and viability of E. coli under various conditions.

Factors involved in the decrease in toxicity of spermidine accumulation
Although it is normally difficult for spermidine to overaccumulate in E. coli due to the existence of spermidine acetyltransferase (45) as well as spermidine excretion protein complex (MdtJI) (46), the mechanism of detoxification of excessive spermidine levels was studied (47). E. coli CAG2242 cells are deficient in the speG gene encoding spermidine acetyltransferase (48). When these cells were cultured in the presence of 0.5 to 4 mM spermidine, their viability was greatly decreased through inhibition of protein synthesis by overaccumulation of spermidine. When these CAG2242 cells were cultured with a high concentration of spermidine (4 mM), a revertant strain was obtained. We found that a 55-kDa protein, glycerol kinase, was overexpressed in the revertant and that synthesis of RMF and  THEMATIC MINIREVIEW: Stimulation of protein synthesis by PA the RNA polymerase 38 subunit (RpoS), important factors for cell viability, were increased in the revertant. Levels of L-glycerol 3-phosphate were also increased in the revertant. Transformation of glpFK, which encodes a glycerol diffusion facilitator (glpF) and glycerol kinase (glpK), to E. coli CAG2242 partially prevented the cell death caused by accumulation of spermidine. It was also found that L-glycerol 3-phosphate inhibited spermidine binding to ribosomes and attenuated the inhibition of protein synthesis caused by high concentrations of spermidine. These results indicate that L-glycerol 3-phosphate reduces the binding of excess amounts of spermidine to ribosomes so that protein synthesis is recovered.

Role of EF-P and eIF5A in protein synthesis
Eukaryotic initiation factor 5A (eIF5A) is the only protein containing hypusine (N ⑀ -(4-amino-2-hydroxybutyl)-lysine) derived from spermidine at Lys-50 in eukaryotic cells (49) and is essential for cell growth through stimulation of protein synthesis containing polyproline peptides (50). EF-P in E. coli is posttranslationally modified by a hydroxylated ␤-lysine attached to Lys-34 and is essential for the synthesis of a subset of proteins containing proline stretches (51). So, EF-P functions similarly to eukaryotic eIF5A in E. coli, although spermidine is not involved in the activation of EF-P.

Other functions of polyamines in E. coli
In addition to polyamine effects at the level of translation, polyamines function in the following aspects.
1) Polyamines protect E. coli cells from the toxic effect of oxygen, i.e. when E. coli cell growth was inhibited in the presence of 95% O 2 , 5% CO 2 , cell growth was recovered by the addition of 0.1 mM putrescine, spermidine, or cadaverine (52).
3) Polyamines stabilize bacterial spheroplasts and protoplasts from osmotic shock (55). 4) Polyamines may stimulate B to Z conversion of DNA (56,57), although the interaction between polyamines and DNA is weak (see Fig. 2). 5) Polyamine content in E. coli is partially regulated by the transporter systems (9), and spermidine uptake is catalyzed by PotA-D proteins (58). Among the four proteins, PotD protein is a substrate-binding protein existing in the periplasm. When spermidine is present in excess in cells, the PotD precursor protein binds to the transcriptional initiation site of the potA-BCD operon, and the transcription of the operon was inhibited (59). This contributes to maintenance of optimal spermidine concentration in E. coli.

Conclusions
Based on the idea that "there is no function without interaction with targeted molecules," the physiological functions of polyamines were studied at the level of RNA, because polyamines mainly exist as a polyamine-RNA complex. In E. coli, we found that assembly of 30S ribosomal subunits is stimulated by polyamines and that 20 kinds of protein syntheses encoded by members of the polyamine modulon are stimulated by poly-amines at the level of translation. These polyamine functions in E. coli strongly contribute to the stimulation of cell growth and the increase in cell viability by polyamines. The concept of the "polyamine modulon" is applicable to eukaryotic cells (5,60). Other polyamine functions except protein synthesis were also mentioned.