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Excessive accumulation of polyamines causes cytotoxicity, including inhibition of cell growth and a decrease in viability. We investigated the mechanism of cytotoxicity caused by spermidine accumulation under various conditions using an Escherichia coli strain deficient in spermidine acetyltransferase (SAT), a key catabolic enzyme in controlling polyamine levels. Due to the excessive accumulation of polyamines by the addition of exogenous spermidine to the growth medium, cell growth and viability were markedly decreased through translational repression of specific proteins [RMF (ribosome modulation factor) and Fis (rRNA transcription factor) etc.] encoded by members of polyamine modulon, which are essential for cell growth and viability. In particular, synthesis of proteins that have unusual locations of the Shine-Dalgarno (SD) sequence in their mRNAs was inhibited. In order to elucidate the molecular mechanism of cytotoxicity by the excessive accumulation of spermidine, the spermidine-dependent structural change of the bulged-out region in the mRNA at the initiation site of the rmf mRNA was examined using NMR analysis. It was suggested that the structure of the mRNA bulged-out region is affected by excess spermidine, so the SD sequence of the rmf mRNA cannot approach initiation codon AUG.

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., (© 2018 Igarashi and Kashiwagi.)

The effects of spermidine analogues [norspermidine (NSPD, 33), spermidine (SPD, 34), homospermidine (HSPD, 44) and aminopropylcadaverine (APCAD, 35)] on cell growth were studied using Escherichia coli polyamine-requiring mutant MA261. Cell growth was compared at 32°C, 37°C, and 42°C. All four analogues were taken up mainly by the PotABCD spermidine-preferential uptake system. The degree of stimulation of cell growth at 32°C and 37°C was NSPD ≥ SPD ≥ HSPD > APCAD, and SPD ≥ HSPD ≥ NSPD > APCAD, respectively. However, at 42°C, it was HSPD » SPD > NSPD > APCAD. One reason for this is HSPD was taken up effectively compared with other triamines. In addition, since natural polyamines (triamines and teteraamines) interact mainly with RNA, and the structure of RNA is more flexible at higher temperatures, HSPD probably stabilized RNA more tightly at 42°C. We have thus far found that 20 kinds of protein syntheses are stimulated by polyamines at the translational level. Among them, synthesis of OppA, RpoE and StpA was more strongly stimulated by HSPD at 42°C than at 37°C. Stabilization of the initiation region of oppA and rpoE mRNA was tighter by HSPD at 42°C than 37°C determined by circular dichroism (CD). The degree of polyamine stimulation of OppA, RpoE and StpA synthesis by NSPD, SPD and APCAD was smaller than that by HSPD at 42°C. Thus, the degree of stimulation of cell growth by spermidine analogues at the different temperatures is dependent on the stimulation of protein synthesis by some components of the polyamine modulon.

Spermidine acetyltransferase (SAT) from Escherichia coli, which catalyses the transfer of acetyl groups from acetyl-CoA to spermidine, is a key enzyme in controlling polyamine levels in prokaryotic cells. In this study, we determined the crystal structure of SAT in complex with spermidine (SPD) and CoA at 2.5Å resolution. SAT is a dodecamer organized as a hexamer of dimers. The secondary structural element and folding topology of the SAT dimer resemble those of spermidine/spermine N(1)-acetyltransferase (SSAT), suggesting an evolutionary link between SAT and SSAT. However, the polyamine specificity of SAT is distinct from that of SSAT and is promiscuous. The SPD molecule is also located at the inter-dimer interface. The distance between SPD and CoA molecules is 13Å. A deep, highly acidic, water-filled cavity encompasses the SPD and CoA binding sites. Structure-based mutagenesis and in-vitro assays identified SPD-bound residues, and the acidic residues lining the walls of the cavity are mostly essential for enzymatic activities. Based on mutagenesis and structural data, we propose an acetylation mechanism underlying promiscuous polyamine recognition for SAT., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Members of polyamine modulon whose synthesis is enhanced at the level of translation were looked for under oxidative stress conditions caused by 0.6 μM K2TeO3. When an Escherichia coli polyamine-requiring mutant MA261 was cultured in the presence of K2TeO3, the degree of polyamine stimulation of cell growth was greater than in cells cultured in the absence of K2TeO3. Under these conditions, synthesis of SoxR, a transcriptional factor for expression of the superoxide response regulon, EmrR, a negative transcriptional factor for expression of the genes for drug excretion proteins, EmrA and EmrB, and of GshA, γ-glutamylcysteine synthetase necessary for glutathione (GSH) synthesis, were stimulated by polyamines at the level of translation. Polyamine stimulation of SoxR and EmrR synthesis was dependent on the existence of an unusually located Shine-Dalgarno (SD) sequence in soxR and emrR mRNAs. Polyamine stimulation of GshA synthesis was due to the existence of the inefficient initiation codon UUG instead of AUG. Polyamine stimulation of the synthesis of EmrR was mainly observed at the logarithmic phase of growth, while that of the synthesis of SoxR and GshA was at the stationary phase. These results strongly suggest that polyamines are involved in easing of oxidative stress through stimulation of synthesis of SoxR, EmrR and GshA together with RpoS, previously found as a member of polyamine modulon at the stationary phase.

Polyamines are ubiquitous small basic molecules that play important roles in cell growth and viability. Since polyamines mainly exist as a polyamine-RNA complex, we looked for proteins whose synthesis is preferentially stimulated by polyamines at the level of translation, and thus far identified 17 proteins in Escherichia coli and 6 proteins in eukaryotes. The mechanisms of polyamine stimulation of synthesis of these proteins were investigated. In addition, the role of eIF5A, containing hypusine formed from spermidine, on protein synthesis is described. These results clearly indicate that polyamines and eIF5A contribute to cell growth and viability through modulation of protein synthesis., (© 2015 International Union of Biochemistry and Molecular Biology.)

Properties of putrescine uptake by PotFGHI and PuuP and their physiological significance were studied using a polyamine biosynthesis and uptake deficient Escherichia coli KK3131 transformed with pACYC184 containing potFGHI or puuP. Putrescine uptake activity of E. coli KK3131 transformed with pACYC184-PotFGHI was higher than that of E. coli 3131 transformed with pACYC-PuuP when cells were cultured in the absence of putrescine. Putrescine uptake by PotFGHI was both ATP and membrane potential dependent, while that by PuuP was membrane potential dependent. Feedback inhibition by polyamines occurred at the PotFGHI uptake system but not at the PuuP uptake system. Expression of PuuP was reduced in the presence of PuuR, a negative regulator for PuuP, and expression of PuuR was positively regulated by glucose, which reduces the level of cAMP. The complex of cAMP and CRP (cAMP receptor protein) inhibited the expression of PuuR in the absence of glucose. Thus, the growth rate of E. coli KK3131 in the presence of both 0.4% (22.2 mM) glucose and 10 mM putrescine was in the order of cells transformed with pACYC-PotFGHI > pACYC-PuuP > pACYC-PuuP + PuuR, which was parallel with the polyamine content in cells. The results indicate that PotFGHI is necessary for rapid cell growth in the presence of glucose as an energy source. When glucose in medium was depleted, however, PuuP was absolutely necessary for cell growth in the presence of putrescine, because accumulation of putrescine to a high level by PuuP was necessary for utilization of putrescine as an energy source.

The spermidine acetyltransferase (SAT) from Escherichia coli catalyses the transfer of acetyl groups from acetyl-CoA to spermidine. SAT has been expressed and purified from E. coli. SAT was crystallized by the sitting-drop vapour-diffusion method to obtain a more detailed insight into the molecular mechanism. Preliminary X-ray diffraction studies revealed that the crystals diffracted to 2.5 Å resolution and belonged to the cubic space group P23, with unit-cell parameters a = b = c = 148.7 Å. They contained four molecules per asymmetric unit.

The structure and function of a cadaverine-lysine antiporter CadB and a putrescine-ornithine antiporter PotE in Escherichia coli were evaluated using model structures based on the crystal structure of AdiC, an agmatine-arginine antiporter, and the activities of various CadB and PotE mutants. The central cavity of CadB, containing the substrate binding site, was wider than that of PotE, mirroring the different sizes of cadaverine and putrescine. The size of the central cavity of CadB and PotE was dependent on the angle of transmembrane helix 6 (TM6) against the periplasm. Tyr(73), Tyr(89), Tyr(90), Glu(204), Tyr(235), Asp(303), and Tyr(423) of CadB, and Cys(62), Trp(201), Glu(207), Trp(292), and Tyr(425) of PotE were strongly involved in the antiport activities. In addition, Trp(43), Tyr(57), Tyr(107), Tyr(366), and Tyr(368) of CadB were involved preferentially in cadaverine uptake at neutral pH, while only Tyr(90) of PotE was involved preferentially in putrescine uptake. The results indicate that the central cavity of CadB consists of TMs 2, 3, 6, 7, 8, and 10, and that of PotE consists of TMs 2, 3, 6, and 8. These results also suggest that several amino acid residues are necessary for recognition of cadaverine in the periplasm because the level of cadaverine is much lower than that of putrescine in the periplasm at neutral pH. All the amino acid residues identified as being strongly involved in both the antiport and uptake activities were located on the surface of the transport path consisting of the central cavity and TM12.

Polyamine content in cells is regulated by biosynthesis, degradation, and transport. With regard to transport, uptake and excretion proteins exist in Escherichia coli and Saccharomyces cerevisiae. In E. coli, the uptake systems comprise a spermidine-preferential uptake system consisting of the PotA, B, C, and D proteins, and a putrescine-specific uptake system consisting of the PotF, G, H, and I proteins. Two other proteins, PotE and CadB, each containing 12 transmembrane segments, function as antiporters (putrescine-ornithine and cadaverine-lysine) and are important for cell growth at acidic pH. MdtJI was identified as a spermidine excretion system. When putrescine was used as energy source, PuuP functioned as a putrescine transporter. In S. cerevisiae, DUR3 and SAM3, containing 16 or 12 transmembrane segments, are the major polyamine uptake proteins, whereas TPO1 and TPO5, containing 12 transmembrane segments, are the major polyamine excretion proteins, and UGA4 is a putrescine transporter on the vacuolar membrane. The activities of DUR3 and TPO1 are regulated by phosphorylation of serine/threonine residues. The identification and assay procedures of these transporters are described in this chapter.

We searched for proteins whose synthesis is enhanced by polyamines at the stationary phase of cell growth using an Escherichia coli polyamine-requiring mutant in which cell viability is greatly decreased by polyamine deficiency. The synthesis of ribosome modulation factor (RMF) was strongly enhanced by polyamines at the level of translation at the stationary phase of cell growth. In rmf mRNA, a Shine-Dalgarno (SD) sequence is located 11 nucleotides upstream of the initiation codon AUG. When the SD sequence was moved to the more common position 8 nucleotides upstream of the initiation codon, the degree of polyamine stimulation was reduced, although the level of RMF synthesis was markedly increased. Polyamine stimulation of RMF synthesis was found to be caused by a selective structural change of the bulged-out region of the initiation site of rmf mRNA. The decrease in cell viability caused by polyamine deficiency was prevented by the addition of a modified rmf gene whose synthesis is not influenced by polyamines. The results indicate that polyamines enhance cell viability of E. coli at least in part by enhancing RMF synthesis.

Polyamine content in cells is regulated by biosynthesis, degradation and transport. In Escherichia coli, there are two polyamine uptake systems, namely spermidine-preferential (PotABCD) and putrescine-specific (PotFGHI), which belong to the family of ATP binding cassette transporters. Putrescine-ornithine and cadaverine-lysine antiporters, PotE and CadB, each consisting of 12 transmembrane segments, are important for cell growth at acidic pH. Spermidine excretion protein (MdtJI) was also recently identified. When putrescine was used as energy source, PuuP functioned as a putrescine transporter. In Saccharomyces cerevisiae, there are four kinds of polyamine uptake proteins (DUR3, SAM3, GAP1 and AGP2), consisting of either 12 or 16 transmembrane segments. Among them, DUR3 and SAM3 mostly contribute to polyamine uptake. There are also five kinds of polyamine excretion proteins (TPO1-5), consisting of 12 transmembrane segments. Among them, TPO1 and TPO5 are the most active proteins. Since a polyamine metabolizing enzyme, spermidine/spermine N(1)-acetyltransferase, is not present in yeast, five kinds of excretion proteins may exist. The current status of polyamine transport in mammalian and plant cells are reviewed., (2010 Elsevier Masson SAS. All rights reserved.)

Proteins whose synthesis is enhanced by polyamines at the level of translation were identified with a polyamine-requiring mutant cultured in the presence of 0.1% glucose and 0.02% glutamate at 42 degrees C. Polyamines had a greater effect on cell growth at 42 degrees C than at 37 degrees C. At 42 degrees C, the synthesis of RpoE (sigma(24)) and StpA, which are involved in the transcription of a number of heat shock response genes, was stimulated by polyamines at the level of translation. In the rpoE and stpA mRNAs, a Shine-Dalgarno (SD) sequence is located at 13 and 12 nucleotides, respectively, upstream of the initiation codon AUG. When the SD sequences were moved to the more common position 7 nucleotides upstream of the initiation codon AUG, the degree of polyamine stimulation was reduced, although the level of RpoE and StpA synthesis was markedly increased. The mechanism underlying polyamine stimulation of RpoE synthesis was then studied. Polyamine stimulation of RpoE synthesis was reduced by changing the bulged-out structure in the initiation site of rpoE mRNA, although the level of RpoE synthesis increased. A selective structural change of this bulged-out region induced by spermidine at 42 degrees C was observed by circular dichroism. Polyamine stimulation of fMet-tRNA binding to ribosomes at 42 degrees C also disappeared by changing the bulged-out structure in the initiation site of rpoE mRNA. The results suggest that polyamines enhance the synthesis of RpoE by changing the bulged-out structure in the initiation site of rpoE mRNA.

A spermidine excretion protein in Escherichia coli was looked for among 33 putative drug exporters thus far identified. Cell toxicity and inhibition of growth due to overaccumulation of spermidine were examined in an E. coli strain deficient in spermidine acetyltransferase, an enzyme that metabolizes spermidine. Toxicity and inhibition of cell growth by spermidine were recovered in cells transformed with pUCmdtJI or pMWmdtJI, encoding MdtJ and MdtI, which belong to the small multidrug resistance family of drug exporters. Both mdtJ and mdtI are necessary for recovery from the toxicity of overaccumulated spermidine. It was also found that the level of mdtJI mRNA was increased by spermidine. The spermidine content in cells cultured in the presence of 2 mM spermidine was decreased, and excretion of spermidine from cells was enhanced by MdtJI, indicating that the MdtJI complex can catalyze excretion of spermidine from cells. It was found that Tyr4, Trp5, Glu15, Tyr45, Tyr61, and Glu82 in MdtJ and Glu5, Glu19, Asp60, Trp68, and Trp81 in MdtI are involved in the excretion activity of MdtJI.

Proteins whose synthesis is enhanced by polyamines at the level of translation were identified in a polyamine-requiring mutant cultured in the presence of 0.1% glucose and 0.02% glutamate instead of 0.4% glucose as an energy source. Under these conditions, enhancement of cell growth by polyamines was almost the same as that in the presence of 0.4% glucose. It was found that synthesis of RpoN, Cra, and H-NS was enhanced by polyamines at the level of translation at the early logarithmic phase of growth (A(540) of 0.15). The effects of polyamines on synthesis of RpoN, H-NS, and Cra were due to the existence of unusual Shine-Dalgarno sequences (RpoN and H-NS) and an inefficient GUG initiation codon (Cra) in their mRNAs. Thus, rpoN, cra, and hns genes were identified as new members of the polyamine modulon. Because most of the polyamine modulon genes thus far identified encode transcription factors (RpoS [sigma(38)], Cya, FecI [sigma(18)], Fis, RpoN [sigma(54)], Cra, and H-NS), DNA microarray analysis of mRNA expressed in cells was performed. At the early logarithmic phase of growth, a total of 97 species of mRNAs that were up-regulated by polyamines more than twofold were under the control of seven polyamine modulon genes mentioned above.

Amino acid residues involved in cadaverine uptake and cadaverine-lysine antiporter activity were identified by site-directed mutagenesis of the CadB protein. It was found that Tyr(73), Tyr(89), Tyr(90), Glu(204), Tyr(235), Asp(303), and Tyr(423) were strongly involved in both uptake and excretion and that Tyr(55), Glu(76), Tyr(246), Tyr(310), Cys(370), and Glu(377) were moderately involved in both activities. Mutations of Trp(43), Tyr(57), Tyr(107), Tyr(366), and Tyr(368) mainly affected uptake activity, and Trp(41), Tyr(174), Asp(185), and Glu(408) had weak effects on uptake. The decrease in the activities of the mutants was reflected by an increase in the K(m) value. Mutation of Arg(299) mainly affected excretion, suggesting that Arg(299) is involved in the recognition of the carboxyl group of lysine. These results indicate that amino acid residues involved in both uptake and excretion, or solely in excretion, are located in the cytoplasmic loops and the cytoplasmic side of transmembrane segments, whereas residues involved in uptake were located in the periplasmic loops and the transmembrane segments. The SH group of Cys(370) seemed to be important for uptake and excretion, because both were inhibited by the existence of Cys(125), Cys(389), or Cys(394) together with Cys(370). The relative topology of 12 transmembrane segments was determined by inserting cysteine residues at various sites and measuring the degree of inhibition of transport through crosslinking with Cys(370). The results suggest that a hydrophilic cavity is formed by the transmembrane segments II, III, IV, VI, VII, X, XI, and XII.

We have recently proposed an idea to explain how polyamines enhance cell growth in Escherichia coli. Since most polyamines exist as polyamine-RNA complexes, our idea is that polyamines stimulate several kinds of protein synthesis which are important for cell growth at the level of translation. We found that synthesis of oligopeptide binding protein (OppA), which is important for nutrient supply, adenylate cyclase (Cya), RNA polymerase sigma(38) subunit (RpoS), transcription factor of iron transport operon (FecI), and transcription factor of growth-related genes including rRNA and some kinds of tRNA synthesis (Fis) was enhanced by polyamines at the level of translation. We proposed that a group of genes whose expression is enhanced by polyamines at the level of translation be referred to as a "polyamine modulon." By DNA microarray, we found that 309 of 2,742 mRNA species were up-regulated by polyamines. Among the 309 up-regulated genes, transcriptional enhancement of at least 58 genes might be attributable to increased levels of the transcription factors Cya, RpoS, FecI, and Fis. This unifying molecular mechanism is proposed to underlie the physiological role of polyamines in controlling the growth of Escherichia coli.

We reported previously that the synthesis of specific proteins such as OppA, Cya, and RpoS (sigma(38)), which are important for cell growth and viability, is stimulated by polyamines at the level of translation. In this study we found that the synthesis of FecI and Fis was also stimulated by polyamines at the level of translation. The FecI and Fis proteins enhance the expression of mRNAs that are involved in iron uptake and energy metabolism and the expression of rRNA and some tRNAs. The Shine-Dalgarno (SD) sequence of their mRNAs was not obvious or was not located at the usual position. When the SD sequences were created at the normal position on these mRNAs, protein synthesis was no longer influenced by polyamines. Thus, the common characteristic of these mRNAs was to have a weak or ineffective SD sequence. We propose that a group of genes whose expression is enhanced by polyamines at the level of translation be referred to as a "polyamine modulon." By DNA microarray, we found that 309 of 2,742 mRNA species were upregulated by polyamines. Among the 309 up-regulated genes, transcriptional enhancement of at least 58 genes might be attributable to increased levels of the transcription factors Cya, RpoS, FecI, and Fis, which are all organized in the polyamine modulon. This unifying molecular mechanism is proposed to underlie the physiological role of polyamines in controlling the growth of Escherichia coli.

The functions of the putative cadaverine transport protein CadB were studied in Escherichia coli. CadB had both cadaverine uptake activity, dependent on proton motive force, and cadaverine excretion activity, acting as a cadaverine-lysine antiporter. The Km values for uptake and excretion of cadaverine were 20.8 and 303 microM respectively. Both cadaverine uptake and cadaverine-lysine antiporter activities of CadB were functional in cells. Cell growth of a polyamine-requiring mutant was stimulated slightly at neutral pH by the cadaverine uptake activity and greatly at acidic pH by the cadaverine-lysine antiporter activity. At acidic pH, the operon containing cadB and cadA, encoding lysine decarboxylase, was induced in the presence of lysine. This caused neutralization of the extracellular medium and made possible the production of CO(2) and cadaverine and aminopropylcadaverine instead of putrescine and spermidine. The induction of the cadBA operon also generated a proton motive force. When the cadBA operon was not induced, the expression of the speF-potE operon, encoding inducible ornithine decarboxylase and a putrescine-ornithine antiporter, was increased. The results indicate that the cadBA operon plays important roles in cellular regulation at acidic pH.

In a speG-disrupted Escherichia coli mutant, which cannot metabolize spermidine to acetylspermidine, addition of spermidine to the medium caused a decrease in cell viability at the late stationary phase of growth. There were parallel decreases in the levels of ribosome modulation factor (RMF), the sigma(38) subunit of RNA polymerase, and the outer membrane protein C (OmpC). To clarify that these three proteins are strongly involved in cell viability, the rmf, rpoS (encoding sigma(38)), and ompC genes were disrupted. Viability of the triple mutant decreased to less than 1% of normal cells. The triple mutant had a reduced cell viability compared to any combination of double mutants, which also had a reduced cell viability. The single rmf and rpoS, but not ompC, mutant only slightly reduced cell viability. The results indicate that cooperative functions of these three proteins are necessary for cell viability at the late stationary phase. The triple mutant had a reduced level of ribosomes and of intracellular cations.

The mechanisms by which polyamines stimulate synthesis of the RNA polymerase sigma(38) subunit in Escherichia coli were studied. Polyamine stimulation was observed only in strains in which the 33rd codon of RpoS mRNA is a UAG termination codon instead of a CAG codon for glutamine in wild-type E. coli. Readthrough of the termination codon by Gln-tRNA(supE) was stimulated by polyamines. This stimulation was found to be caused by an increase in both the level of suppressor tRNA(supE) and the binding affinity of Gln-tRNA(supE) for ribosomes. The stimulatory effect was observed with a UAG termination codon but not with UGA and UAA codons. Readthrough of the UAG termination codon at the 270th amino acid position of RpoS mRNA was also stimulated by polyamines, indicating that polyamines stimulate readthrough of a UAG codon regardless of its location within the RpoS mRNA. When cell viability of an E. coli strain having a termination codon in the 33rd position of RpoS mRNA was compared using cells cultured with or without putrescine, it was higher in cells cultured with putrescine than in cells cultured without putrescine. The level of sigma(38) subunit in the cells cultured with putrescine was higher than that in cells cultured without putrescine on days 2, 4, and 8, but the level of sigma(70) subunit was almost the same in cells cultured with or without putrescine. These results confirm that elevated expression of the rpoS gene is important for cell viability at late stationary phase.

Escherichia coli CAG2242 cells are deficient in the speG gene encoding spermidine acetyltransferase. When these cells were cultured in the presence of 0.5 to 4 mM spermidine, their viability was greatly decreased through the inhibition of protein synthesis by overaccumulation of spermidine. When the 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 a ribosome modulation factor and the RNA polymerase sigma(38) subunit, factors important for cell viability, was increased in the revertant. Levels of L-glycerol 3-phosphate 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.

The effects of polyamines on the synthesis of various final sigma subunits of RNA polymerase were studied using Western blot analysis. Synthesis of final sigma(28) was stimulated 4.0-fold and that of final sigma(38) was stimulated 2.3-fold by polyamines, whereas synthesis of other final sigma subunits was not influenced by polyamines. Stimulation of final sigma(28) synthesis was due to an increase in the level of cAMP, which occurred through polyamine stimulation of the synthesis of adenylate cyclase at the level of translation. Polyamines were found to increase the translation of adenylate cyclase mRNA by facilitating the UUG codon-dependent initiation. Analysis of RNA secondary structure suggests that exposure of the Shine-Dalgarno sequence of mRNA is a prerequisite for polyamine stimulation of the UUG codon-dependent initiation.

The polyamine content of cells is regulated by biosynthesis, degradation, and transport. In Escherichia coli, the genes for three different polyamine transport systems have been cloned and characterized. Two uptake systems (putrescine-specific and spermidine-preferential) are ABC transporters, each consisting of a periplasmic substrate binding protein, two transmembrane proteins, and a membrane-associated ATPase. The third transport system, catalyzed by PotE, mediates both uptake and excretion of putrescine. In this article, the properties of the first two polyamine uptake systems are reviewed in detail.

The PotE protein can catalyze both uptake and excretion of putrescine. The K(m) values of putrescine for uptake and excretion are 1.8 and 73 microm, respectively. Uptake of putrescine is dependent on the membrane potential, whereas excretion involves putrescine-ornithine antiporter activity. Amino acids involved in both activities were identified using mutated PotE proteins. It was found that Cys(62), Trp(201), Trp(292), and Tyr(425) were strongly involved in both activities, and that Tyr(92), Cys(210), Cys(285), and Cys(286) were moderately involved in the activities. Mutations of Tyr(78), Trp(90), and Trp(422) mainly affected uptake activity, and the K(m) values for putrescine uptake by these PotE mutants increased greatly, indicating that these amino acids are involved in the high affinity uptake of putrescine by PotE. Mutations of Lys(301) and Tyr(308) mainly affected excretion activity (putrescine-ornithine antiporter activity), and excretion by these mutants was not stimulated by ornithine, indicating that these amino acids are involved in the recognition of ornithine. It was found that the putrescine and ornithine recognition site on PotE is located at the cytoplasmic surface and the vestibule of the pore consisting of 12 transmembrane segments. Based on the results of competition experiments with various putrescine analogues and the disulfide cross-linking of PotE between cytoplasmic loops and the COOH terminus, a model of the putrescine recognition site on PotE consisting of the identified amino acids is presented.

Accumulation of spermidine in Escherichia coli causes a decrease in cell viability at the late stationary phase of cell growth. The mechanism underlying this effect has been studied. Spermidine accumulation caused an increase in the level of ppGpp and a decrease in ribosome modulation factor (RMF) and stationary phase-specific sigma factor sigma(S), both of which are believed to be involved in cell viability. Transformation of E. coli with the gene for stringent factor, which synthesizes ppGpp, also caused a significant decrease in the levels of RMF and sigma(S) factor and a decrease in cell viability. The results strongly suggest that the accumulation of ppGpp is also involved in the decrease in cell viability and that the sigma(S) factor assists the function of RMF in cell viability., (Copyright 1999 Academic Press.)

Ribosome modulation factor (RMF) is involved in stabilization of ribosomes during the transition from exponential growth to the stationary growth phase in Escherichia coli. A deficiency of RMF is known to reduce cell viability. Overaccumulation of spermidine also leads to a decrease in cell viability and to a decrease in the synthesis of RMF and of the cation-selective porin OmpC. Thus, a decrease in RMF levels may be involved in the decreased cell viability caused by excess spermidine. Because spermidine also influences the expression of OmpC, we examined whether OmpC deficiency enhances the cell death caused by RMF deficiency. The ompC mutant by itself did not affect protein synthesis or cell viability, but the double rmf ompC mutant produced a much larger decrease in protein synthesis and cell viability than did the single rmf mutant. There was also a decrease in the amount of ribosomes and in the Mg2+ content in the double rmf ompC mutant, and cell viability could be partially restored by the addition of Mg2+ to the growth medium. RMF deficiency was found to inhibit the synthesis of another cation-selective porin OmpF. Thus, the double rmf ompC mutant is deficient in both OmpC and OmpF, which probably accounts for the pronounced decrease in Mg2+ uptake in this mutant. The results indicate that both RMF and Mg2+, acting through stabilization of ribosomes, are important for cell viability at the stationary growth phase., (Copyright 1998 Academic Press.)

Changes in the amount of oligopeptide binding protein (OppA) in spontaneous kanamycin-resistant mutants of Escherichia coli were investigated. Among 20 colonies obtained from 10(8) cells cultured in the presence of 20 microgram of kanamycin/ml, 1 colony had no detectable OppA and 7 colonies were mutants with reduced amounts of OppA. Sensitivity of wild-type cells to kanamycin increased slightly by transformation of the oppA gene, but the sensitivity of the mutants increased greatly by the transformation. A mutant with no OppA was found to be a nonsense mutant of the oppA gene at amino acid position 166. In a mutant having a reduced level of OppA, the reduction was due to the decrease in OppA synthesis at the translational level. These mutants were also resistant to other aminoglycoside antibiotics, including streptomycin, neomycin, and isepamicin. Isepamicin uptake activities decreased greatly in these two kinds of mutants. The results support the proposition that aminoglycoside antibiotics are transported into cells by the oligopeptide transport system, and that transport is an important factor for spontaneous resistance to aminoglycoside antibiotics.

PotF protein is a periplasmic substrate-binding protein of the putrescine transport system in Escherichia coli. We have determined the crystal structure of PotF protein in complex with the substrate at 2.3-A resolution. The PotF molecule has dimensions of 54 x 42 x 30 A and consists of two similar globular domains. The PotF structure is reminiscent of other periplasmic receptors with a highest structural homology to another polyamine-binding protein, PotD. Putrescine is tightly bound in the deep cleft between the two domains of PotF through 12 hydrogen bonds and 36 van der Waals interactions. The comparison of the PotF structure with that of PotD provides the insight into the differences in the specificity between the two proteins. The PotF structure, in combination with the mutational analysis, revealed the residues crucial for putrescine binding (Trp-37, Ser-85, Glu-185, Trp-244, Asp-247, and Asp-278) and the importance of water molecules for putrescine recognition.

The primary receptor (PotF) of the putrescine transport system in E. coli has been crystallized by the hanging-drop vapor-diffusion technique. The crystals belong to the space group P21212 with unit-cell dimensions a = 269.4, b = 82.33 and c = 93.74 A. The crystals diffract beyond 2.2 A with a rotating-anode X-ray source. A complete data set from the native crystals has been collected and processed at 2.3 A resolution. Two heavy-atom derivatives have been prepared from the same Pt compound at 293 and 277 K. The difference Patterson maps revealed completely different major heavy-atom sites between these two derivatives.

The structure and function of the polyamine transport protein PotE was studied. Uptake of putrescine by PotE was dependent on the membrane potential. In contrast, the putrescine-ornithine antiporter activity of PotE studied with inside-out membrane vesicles was not dependent on the membrane potential (Kashiwagi, K., Miyamoto, S., Suzuki, F., Kobayashi, H., and Igarashi, K. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 4529-4533). The Km values for putrescine uptake and for putrescine-ornithine antiporter activity were 1.8 and 73 microM, respectively. Uptake of putrescine was inhibited by high concentrations of ornithine. This effect of ornithine appears to be due to putrescine-ornithine antiporter activity because it occurs only after accumulation of putrescine within cells and because ornithine causes excretion of putrescine. Thus, PotE can function not only as a putrescine-ornithine antiporter to excrete putrescine but also as a putrescine uptake protein. Both the NH2 and COOH termini of PotE were located in the cytoplasm, as determined by the activation of alkaline phosphatase and beta-galactosidase by various PotE-fusion proteins. The activities of putrescine uptake and excretion were studied using mutated PotE proteins. It was found that glutamic acid 207 was essential for both the uptake and excretion of putrescine by the PotE protein and that glutamic acids 77 and 433 were also involved in both activities. These three glutamic acids are located on the cytoplasmic side of PotE, and the function of these three residues could not be replaced by other amino acids. Putrescine transport activities did not change significantly with mutations at the other 13 glutamic acid or aspartic acid residues in PotE.

The PotD protein from Escherichia coli is one of the components of the polyamine transport system present in the periplasm. This component specifically binds either spermidine or putrescine. The crystal structure of the E. coli PotD protein complexed with spermidine was solved at 1.8 A resolution and revealed the detailed substrate-binding mechanism. The structure provided the detailed conformation of the bound spermidine. Furthermore, a water molecule was clearly identified in the binding site lying between the amino-terminal domain and carboxyl-terminal domain. Through this water molecule, the bound spermidine molecule forms two hydrogen bonds with Thr 35 and Ser 211. Another periplasmic component of polyamine transport, the PotF protein, exhibits 35% sequence identity with the PotD protein, and it binds only putrescine, not spermidine. To understand these different substrate specificities, model building of the PotF protein was performed on the basis of the PotD crystal structure. The hypothetical structure suggests that the side chain of Lys 349 in PotF inhibits spermidine binding because of the repulsive forces between its positive charge and spermidine. On the other hand, putrescine could be accommodated into the binding site without any steric hindrance because its molecular size is much smaller than that of spermidine, and the positively charged amino group is relatively distant from Lys 349.

Spermidine-binding sites on PotD protein, substrate-binding protein in periplasm, in the spermidine-preferential uptake system in Escherichia coli were studied by measuring polyamine transport activities of right-side-out membrane vesicles with mutated PotD proteins prepared by site-directed mutagenesis of the potD gene and by measuring polyamine binding activities of these mutated PotD proteins. Polyamine transport activities of the mutated PotD proteins paralleled their polyamine binding activities. It was found that Trp-34, Thr-35, Glu-36, Tyr-37, Ser-83, Tyr-85, Asp-168, Glu-171, Trp-229, Trp-255, Asp-257, Tyr-293, and Gln-327 of PotD protein were involved in the binding to spermidine. When spermidine uptake activities were measured in intact cells expressing the mutated PotD proteins, it was found that Glu-171, Trp-255, and Asp-257 were more strongly involved in the binding of spermidine to PotD protein than the other amino acids listed above. The dissociation constants of spermidine for the mutated PotD proteins at Glu-171, Trp-255, and Asp-257 increased greatly in comparison with those for the other mutated PotD proteins. Since these three amino acids clearly interact with the diaminopropane moiety of spermidine, the results are in accordance with the finding that PotD protein has a higher affinity for spermidine than for putrescine. Putrescine was found to bind at the position of the diaminobutane moiety of spermidine.

PotD protein is a periplasmic binding protein and the primary receptor of the polyamine transport system, which regulates the polyamine content in Escherichia coli. The crystal structure of PotD in complex with spermidine has been solved at 2.5-A resolution. The PotD protein consists of two domains with an alternating beta-alpha-beta topology. The polyamine binding site is in a central cleft lying in the interface between the domains. In the cleft, four acidic residues recognize the three positively charged nitrogen atoms of spermidine, while five aromatic side chains anchor the methylene backbone by van der Waals interactions. The overall fold of PotD is similar to that of other periplasmic binding proteins, and in particular to the maltodextrin-binding protein from E. coli, despite the fact that sequence identity is as low as 20%. The comparison of the PotD structure with the two maltodextrin-binding protein structures, determined in the presence and absence of the substrate, suggests that spermidine binding rearranges the relative orientation of the PotD domains to create a more compact structure.

The polyamine content in cells is regulated by both polyamine biosynthesis and its transport. We recently obtained and characterized three clones of polyamine transport genes (pPT104, pPT79 and pPT71) in Escherichia coli. The system encoded by pPT104 was the spermidine-preferential uptake system and that encoded by pPT79 the putrescine-specific uptake system. Furthermore, these two systems were ABC (ATP binding cassette) transporters consisting of four kinds of proteins: pPT104 clone encoded PotA, -B, -C, and -D proteins and pPT79 clone encoded PotF, -G, -H, and I proteins. PotD and -F proteins were periplasmic substrate binding proteins and PotA and -G proteins membrane associated proteins having the nucleotide binding site. PotB and -C proteins, and PotH and -I proteins were transmembrane proteins probably forming channels for spermidine and putrescine, respectively. Their amino acid sequences in the corresponding proteins were similar to each other. The functions of PotA and -D proteins in the spermidine-preferential uptake system encoded by pPT104 clone were studied in detail through a combined biochemical and genetic approach. In contrast, the putrescine transport system encoded by pPT71 consisted of one membrane protein (PotE protein) having twelve transmembrane segments, and was active in both the uptake and excretion of putrescine. The uptake was dependent on the membrane potential, and the excretion was due to the exchange reaction between putrescine and ornithine. In mouse mammary carcinoma FM3A cells, it was shown that the antizyme, which negatively regulates the amount of ornithine decarboxylase, also negatively regulates the activity of polyamine transport.

PotA protein, one of the components of the spermidine-preferential uptake system in Escherichia coli, was purified to homogeneity, and some of its properties were examined. PotA protein showed Mg(2+)-and SH-dependent ATPase activity. The specific activity was approximately 400 nmol/min/mg of protein and the Km value for ATP was 385 microM. The nature of the ATP binding site was explored by identification of the amino acid residue photoaffinity-labeled with 8-azido-ATP. It was found that 8-azido-ATP was attached to cysteine 26. In the spermidine transport-deficient mutant E. coli NH1596, valine 135 of PotA protein, which is located between two consensus amino acid sequences for nucleotide binding (50-57 and 168-173), was replaced by methionine (Kashiwagi, K., Miyamoto, S., Nukui, E., Kobayashi, H., and Igarashi, K. (1993) J. Biol. Chem. 268, 19358-19363). This mutated PotA protein could be labeled with 8-azido-ATP, but showed very low ATPase activity. To identify which cysteine is involved in the function of potA protein, cysteines 26, 54, and 276 were replaced by alanine, threonine, and alanine, respectively. Among the three mutated PotA proteins, the mutated PotA protein C54T only lost both ATPase and spermidine uptake activities. The results taken together indicate that the adenine portion of ATP interacts with a domain close to the NH2-terminal end of PotA protein, and active centers of ATP hydrolysis are located both within and between the two consensus amino acid sequences for nucleotide binding. Association of PotA protein with membranes was strengthened by the existence of channel forming PotB and PotC proteins. ATPase of PotA protein was inhibited by spermidine, suggesting that uptake inhibition by spermidine may function during this process.

Physiological functions of spermidine acetyltransferase in Escherichia coli have been studied using the spermidine acetyltransferase (speG) gene-deficient mutant CAG2242 and the cloned speG gene. The growth of E. coli CAG2242 in the defined M9 medium was normal in the presence and absence of 0.5mM spermidine. However, cell viability of E. coli CAG2242 at 48 h after the onset of growth decreased greatly by the addition of 0.5 mM spermidine. The amount of spermidine accumulated in the cells was approximately 3-fold that in the cells grown in the absence of spermidine. Transformation of the cloned speG gene to E. coli CAG2242 recovered the cell viability. Decreased in cell viability of E. coli CAG2242 was observed even when 0.5mM spermidine was added at 24 h after the onset of growth. The results indicate that accumulated spermidine functions at the late stationary phase of growth. The accumulation of spermidine caused a decrease in protein synthesis but not in DNA and RNA synthesis at 28 h after the onset of growth. The synthesis of several kinds of proteins was particularly inhibited. They included ribosome modulation factor and OmpC protein. Since the ribosome modulation factor is essential for cell viability at the stationary phase of growth (Yamagishi, M., Matsushima, H., Wada, A., Sakagami, M., Fujita, N., and Ishihama, A. (1993) EMBO J. 12, 625-630), the decrease in the protein was thought to be one of the reasons for the decrease in cell viability. The decrease in the ribosome modulation factor mainly occurred at the translational level.

Spermidine acetyltransferase (SAT) from Escherichia coli was purified about 40,000-fold. The molecular mass of native SAT was 95 kDa, and it consisted of four identical subunits. The products formed from the reaction of acetyl-CoA with spermidine by SAT were N1- and N8-acetylspermidine. The Km values for acetyl-CoA, spermidine, and spermine were 2 microM, 1.29 mM, and 220 microM, respectively. The enzymatic activity increased by 2.5-3.5-fold under the condition of poor nutrition but not in response to cold shock or high pH. By using synthetic oliogonucleotides deduced from amino acid sequences of the peptides in SAT, a polymerase chain reaction product with a length of 250 nucleotides was obtained. Using this polymerase chain reaction product, the gene encoding SAT (speG) was cloned and mapped at 35.6 min in the E. coli chromosome. E. coli cells transformed with the cloned speG gene increased SAT activity by 8-40-fold. The gene encoded a 186-amino acid protein, but SAT consisted of 185 amino acids because the initiator methionine was liberated from the protein. Thus, the predicted molecular mass was 21,756 Da. Significant similarity to aminoglycoside acetyltransferase and peptide N-acetyltransferase was observed in the amino acid sequence 87-141, and some similarity with spermidine-preferential binding protein (potD protein) in the spermidine-preferential uptake system was observed in the amino acid sequence 122-141. The results suggest that the active center of SAT may be located in the COOH-terminal portion.

Since the speF-potE operon (pPT71 clone) encoding inducible ornithine decarboxylase (iODC) and polyamine transport potE protein is inducible at acidic pH, a gene encoding a protein involved in the enhancement of expression of the operon was searched for. Using the fused gene containing the upstream sequence of the speF-potE operon and the open reading frame of beta-galactosidase as a reporter gene, a clone (pPTS23) which causes the increase of beta-galactosidase activity at acidic pH was isolated. The clone also increased iODC activity at acidic pH and was identified as a gene encoding RNase III. This is the first example that RNase III increases the translational efficiency of mRNA derived from Escherichia coli gene by cutting the 5'-untranslated region of mRNA.

The gene encoding a Verotoxin 2 variant, VTvp1, was mutated by oligonucleotide-directed site-specific mutagenesis. Among 6 mutant toxins encoded by the mutated genes, E167Q-R170L (glutamic acid at position 167 and arginine at position 170 from N-terminus of the A subunit were replaced by glutamine and leucine, respectively) was found to have markedly decreased activities; inhibition of protein synthesis, Vero cell cytotoxicity and mouse lethality of the purified E167Q-R170L were 1/1,900, 1/125,000 and 1/2,000, respectively, of those of the purified wild-type VT2vp1. Since the antigenic property of the E167Q-R170L was demonstrated to be similar to that of the wild-type VT2vp1 by Ouchterlony double gel diffusion test and by neutralization test of Vero cell cytotoxicity of the VT2vp1, a possibility to use the mutant VT2vp1, E167Q-R170L, as a toxoid is discussed.

Functions of potA and -D proteins in the spermidine-preferential uptake system, which consists of potA, -B, -C, and -D proteins, were studied. Spermidine uptake activity was lost when the gene for potA or potD protein was disrupted, and transformation of the cells with potA or potD gene recovered the uptake activity. PotD protein was found to bind spermidine with a 3.2 microM dissociation constant. Spermidine uptake by membrane vesicles prepared from Escherichia coli DR112 containing the genes for potA, -B, and -C proteins was strongly dependent on the addition of potD protein, and its optimal concentration was 5 microM when 10 microM spermidine was used as substrate. The ATP dependence of spermidine uptake was examined with the atp mutant of E. coli. The uptake was completely dependent on ATP. When the membrane potential was extinguished by carbonyl cyanide m-chlorophenylhydrazone, the uptake activity was decreased by 60% even if ATP existed. This suggests that the membrane potential is also involved in the spermidine uptake. ATP was found to bind to potA protein. In the spermidine transport-deficient mutant E. coli NH1596, valine 135 of potA protein, which is located between two consensus amino acid sequences for nucleotide binding, was replaced by methionine. Although the amount of mutated potA protein expressed in E. coli cells was the same as that of normal potA protein and the mutated protein was membrane-associated, no significant spermidine uptake was observed. The results taken together indicate that potA and -D proteins are absolutely necessary for spermidine uptake in conjunction with the two channel forming proteins (potB and -C).

The nucleotide sequence of the operon for the putrescine transport system that maps at 19 min on the Escherichia coli chromosome was determined. It contained four open reading frames encoding potF, -G, -H, and -I proteins. The potF protein (M(r) = 38,000) was inferred to be a putrescine-specific binding protein existing in a periplasmic fraction from the results of Western blot analysis of the cell fractions and from measurements of polyamine binding to the protein. The potG protein (M(r) = 45,000) had consensus amino acid sequences for the nucleotide-binding site. The potH (M(r) = 35,000) and potI (M(r) = 31,000) proteins consisted of six putative transmembrane-spanning segments linked by hydrophilic segments of variable length as shown by hydropathy profiles. The spermidine-putrescine transport system, which is mainly involved in spermidine transport, consisted of potA, -B, -C, and -D proteins (Furuchi, T., Kashiwagi, K., Kobayashi, H., and Igarashi, K. (1991) J. Biol. Chem. 266, 20928-20933). The homologies of the corresponding two proteins between those two systems, F and D, G and A, H and B, and I and C, were 35, 42, 37, and 36%, respectively. The initiation point of the transcription of the operon for the putrescine transport system was determined by primer extension and S1 nuclease mapping. Transcription started from the T residue located either 149 or 150 nucleotides upstream from the initiator AUG codon of potF protein mRNA. By making several subclones and a mutant lacking the potF gene, we showed that the expression of all four proteins was necessary for maximal putrescine transport activity. These results indicate that the putrescine transport system can also be defined as a bacterial periplasmic transport system.

To estimate the polyamine distribution in Escherichia coli, the binding constants (K) for DNA, RNA, phospholipids, and ATP were calculated under the condition of 10 mM Tris-HCl, pH 7.5, 150 mM K+, and 10 mM Mg2+. The binding constants of spermidine for E. coli DNA, E. coli 16S rRNA, phospholipids in E. coli membrane, and ATP were 0.015, 0.066, 0.028, and 0.081 mM-1, respectively. Similarly, those of putrescine were 0.010, 0.010, 0.007, and 0.037 mM-1, respectively. The concentrations of putrescine, spermidine, and ATP and phosphates in DNA, RNA, and phospholipids in E. coli harvested at A600 = 0.3 were 32.2, 6.88, and 2.66 and 96.4, 436, and 57.2 mM, respectively. Accordingly, the percentage of spermidine bound to DNA, RNA, phospholipids, and ATP and that of free spermidine were 5.1, 90, 0.7, 0.8, and 3.8%, respectively. The percentage of putrescine bound to DNA, RNA, phospholipids, and ATP and that of free putrescine were 9.3, 48, 1.4, 2.6, and 39%, respectively. The results indicate that most spermidine exists as a spermidine--RNA complex, and about 40% and 50% of putrescine exists as a free form and a putrescine--RNA complex in cells, respectively. Under the conditions that the synthesis of specific proteins such as RNA replicase is stimulated by polyamines in a cell-free system, the amount of spermidine and putrescine bound to RNA was close to the value estimated in cells. Experiments to demonstrate the polyamine stimulation of MS2 RNA-directed RNA replicase synthesis in vivo were thus performed, and the results were confirmed.

The sensitivity of Escherichia coli to several aminoglycoside antibiotics was examined with E. coli DR112 transformed by the gene for polyamine-induced protein (oligopeptide-binding [OppA] protein) or polyamine transport proteins. The results clearly showed that sensitivity to aminoglycoside antibiotics (gentamicin, isepamicin, kanamycin, neomycin, paromomycin, and streptomycin) increased due to the highly expressed OppA protein. When the gene for OppA protein was deleted, sensitivity to aminoglycoside antibiotics was greatly decreased. It was also shown that isepamicin could bind to OppA protein with a binding affinity constant of 8.5 x 10(3) M-1 under the ionic conditions of 50 mM K+ and 1 mM Mg2+ at pH 7.5, and isepamicin uptake into cells was greatly stimulated by the OppA protein. These results, taken together, show that the OppA protein increases the uptake of aminoglycoside antibiotics. In addition, the OppA protein increased the transport of spermidine and an oligopeptide (Gly-Leu-Tyr). The uptake of isepamicin into cells was partially inhibited by spermidine, suggesting that the binding site for isepamicin overlaps that for spermidine on the OppA protein. Spermidine uptake activity by the OppA protein was less than 1% of that of the ordinary spermidine uptake system. Aminoglycoside antibiotics neither stimulated the synthesis of OppA protein nor increased spermidine uptake.

Excretion of putrescine from Escherichia coli was assessed by measuring its uptake into inside-out membrane vesicles. The vesicles were prepared from wild-type E. coli or E. coli transformed with plasmids containing one of the three polyamine transport systems. The results indicate that excretion of putrescine is catalyzed by the putrescine transport protein, encoded by the potE gene located at 16 min on the E. coli chromosome. Loading of ornithine (or lysine) inside the vesicles was essential for the uptake of putrescine, indicating that the protein exchanges putrescine and ornithine (or lysine) by an antiport mechanism. The Km and Vmax values for the putrescine uptake by inside-out membrane vesicles were 73 microM and 0.82 nmol/min per mg of protein, respectively. The antiport protein (potE protein) also catalyzed putrescine-putrescine and ornithine-ornithine exchange. The transport activity was not disturbed by inhibitors of energy production such as KCN and carbonyl cyanide m-chlorophenylhydrazone. When intact E. coli was used instead of the inside-out membrane vesicles, excretion of putrescine was also catalyzed by the antiport protein in the presence of ornithine in the medium.

The nucleotide sequence of one of the putrescine transport operons (pPT71), located at 16 min of the Escherichia coli chromosome, was determined. It contained the genes for an induced ornithine decarboxylase and a putrescine transport protein. The gene for the ornithine decarboxylase contained a 2,196-nucleotide open reading frame encoding a 732-amino acid protein whose calculated Mr was 82,414, and the predicted amino acid sequence from the open reading frame had 65% homology with that of a constitutive ornithine decarboxylase encoded by the gene at 64 min. The ornithine decarboxylase activity was observed in the cells carrying pPT71 cultured at pH 5.2, but not in the cells cultured at pH 7.0. The gene for the putrescine transport protein contained a 1,317-nucleotide open reading frame encoding a 439-amino acid protein whose calculated Mr was 46,494. The hydropathy profile of the putrescine transport protein revealed that it consisted of 12 putative transmembrane spanning segments linked by hydrophilic segments of variable length. The transport protein was in fact found in the membrane fraction. When the gene for the putrescine transport protein was linked to the tet promoter of the vector instead of its own promoter, the putrescine transport activity increased greatly. The results suggest that the gene expression of the operon is repressed strongly under standard conditions.

The nucleotide sequence of the gene for the spermidine and putrescine transport system that maps at 15 min on the Escherichia coli chromosome was determined. It contained four open reading frames encoding A, B, C, and D proteins. By making several subclones, we showed that expression of all the four proteins was necessary for maximal spermidine and putrescine transport activity. A single transport system was involved in the transport of both spermidine and putrescine. The A protein (Mr 43K) was found to be associated with membranes, as shown by Western blot analysis of the cell fractions. In addition, it had consensus amino acid sequences for the nucleotide binding site. B (Mr 31K) and C (Mr 29K) proteins consisted of six putative transmembrane spanning segments linked by hydrophilic segments of variable length as shown by cell localization of the proteins synthesized in maxicells and by hydropathy profiles. D protein (Mr 39K) was inferred to be a polyamine binding protein existing in a periplasmic fraction from the results of Western blot analysis of the cell fractions and from measurements of polyamine binding to the protein. These results indicate that the spermidine and putrescine transport system can be defined as a bacterial periplasmic transport system.

Escherichia coli KK313, which was deficient in spermidine transport, was isolated by treatment of E. coli MA261 with N-methyl-N'-nitro-N-nitrosoguanidine. E. coli NH1596, which was deficient in spermidine transport and has a 90% decreased putrescine transport activity, was obtained by a second treatment of E. coli KK313 with the same mutagen. Genes for polyamine transport systems were isolated by transforming E. coli NH1596 through DNA fragments from E. coli DR112 using pACYC184 as a vector. One clone for the gene of protein(s) catalyzing both putrescine and spermidine uptake (pPT104) was isolated. Two clones for the genes of protein(s) catalyzing only putrescine uptake (pPT79 and pPT71) were obtained. The genes encoded by pPT104, pPT79, and pPT71 were mapped at 15, 19, and 16 min of E. coli chromosome, respectively. Spermidine uptake by NH1596 carrying pPT104, and by MA261, was not inhibited by putrescine and several polyamine analogues, and the Kt values of these two systems were both approximately 0.1 microM. Putrescine transport by NH1596 carrying pPT104 was inhibited completely by spermidine, N,N-dimethyl-4,4'-bipyridylium (paraquat), and N1-acetyl-spermidine, and the Kt value was 1.4 microM. Putrescine uptake by NH1596 carrying pPT79 or pPT71 was not inhibited by spermidine and several polyamine analogues, and the Kt values were 0.5 and 1.8 microM, respectively. In MA261, the putrescine uptake was inhibited by 25-35% by paraquat and N1-acetyl-polyamines and showed two Kt values, 0.5 and 1.5 microM. Based on these findings, the polyamine transport systems of E. coli are discussed.

The physiological function of the polyamine-induced protein (PI protein), whose synthesis is stimulated at an early stage after the addition of putrescine to growing cells of a polyamine-requiring mutant of Escherichia coli (Mitsui, K., Igarashi, K., Kakegawa, T., and Hirose, S. (1984) Biochemistry 23, 2679-2683), has been studied. The following findings clearly show that the PI protein is a binding protein of an oligopeptide transport system. (a) PI protein was found in a periplasmic fraction. (b) When the restriction map of a clone for the PI protein gene was compared with Kohara's physical map (Kohara, Y., Akiyama, K., and Isono, K. (1987) Cell 50, 495-508), the gene was found at 27 min of the E. coli chromosome, where genes for an oligopeptide transport system were located. (c) The clone contained a 1,629-nucleotide open reading frame encoding a 543-amino acid protein whose calculated Mr was 60,901, and the predicted amino acid sequence from this open reading frame was quite similar to that of an oligopeptide binding protein of Salmonella typhimurium. (d) When the transport activity of a tripeptide, Gly-Leu-125I-Tyr, was measured in a polyamine-requiring mutant of E. coli growing both in the presence and absence of putrescine, the activity was higher in the cells growing in its presence. (e) Polyamine stimulation of cell growth was greater when an oligopeptide rather than corresponding amino acids was added to the medium. These results suggest that the polyamine stimulation of PI protein synthesis at the early stage after the addition of putrescine contributes to the polyamine stimulation of cell growth through the supply of nutrients.