S

Crq hydrophobic interactions. Experiments are now in progress to clarify the properties of the transmembrane proteins of polyamine-uptake systems.

The uptake of polyamines decreases following their accumulation in E. coli. One of the reasons for this is that spermidine inhibits the ATPase activity of PotA. Overexpression of PotD in E. coli also inhibited the uptake of spermidine and the synthesis of PotABCD messenger RNA (mRNA) (32). A 50% inhibition of the transcription was observed with a molar ratio of approx 1:500 of template DNA/PotD in the presence of spermidine. PotD bound to regions -258 to -209 nucleotides upstream and +66 to +135 nucleotides downstream of the ATG initiation codon of the potA gene (Fig. 4). Binding of PotD to the downstream site was stimulated by spermidine. PotD exists as the PotD precursor in spheroplasts. Thus, the transcription of the potABCD operon is inhibited in vivo by the PotD precursor through its binding to two regions close to the transcriptional initiation site of the operon. To confirm that the PotD precursor functions as a regulator of the spermidine preferential uptake system operon, the number of PotD precursors in the spheroplast was estimated. Because the number of molecules of the major a subunit of RNA polymerase (a70) is constant during the exponential phase of cell growth (about 700 molecules/cell) (33), the number of PotD precursor was estimated by comparison with the number of RNA polymerase a70 molecules. It was estimated to be 5000 to 25,000 PotD precursor molecules/cell in the spheroplast of E. coli-overexpressing PotD. These values can account for a 70-80% inhibition of spermidine uptake by excess PotD (32). It appears that PotD, or more likely the PotD precursor, is a new type of transcriptional regulator.

PotE encoded by pPT71 and CadB belong to another class of polyamine transporter 34-38). They consist of 12 transmembrane segments linked by hydrophilic segments of variable length with the NH2- and the COOH-termini located in the cytoplasm. PotE and CadB can catalyze both the uptake at neutral pH and excretion at acidic pH of putrescine and cadaverine, respectively. Uptake of putrescine and cadaverine by PotE and CadB was dependent on the membrane potential, and the Km value for putrescine and cadaverine was 1.8 and 20.8 |iM Excretion of putrescine and cadaverine was catalyzed by putrescine/ornithine and cadaverine/lysine antiporter activities of PotE and CadB. The Km value for putrescine and cadaverine for excretion was 73 and 303 |iM The exchange ratio between putrescine (cadaverine) and ornithine (lysine) was 1:1. Excretion was increased by carbonyl cyanide m-chlorophenylhydrazone, which inhibits the membrane potential-dependent reuptake of putrescine and cadaverine. As for PotE protein, amino acid residues which are involved in both activities (Cys62, Glu77, Tyr92, Trp201, Glu207, Cys210, Cys285, Cys286, Trp292, Tyr425, and Glu433) were located at the cytoplasmic surface

Fig. 3. Structure of PotD (A) and schematic drawing of PotD and PotF (B). (A) The conserved sequence motif observed in maltodextrin-binding protein and PotD is shown in the circle, and the binding site for the spermidine molecule is marked by the side chains of the four acidic residues. (B) The polar and hydrophobic residues are drawn as ellipses and rectangles, respectively. The most crucial residues are shown in white ellipses and rectangles, and the other residues are shown is thick and thin shaded ellipses and rectangles. White circles represent water molecule. (Reproduced with permission from refs. 28 and 31.)

Pota Spermidine Binding

Fig. 4. (A) Inhibition of spermidine uptake by overexpression of PotD and PotD-binding sites on the potABCD operon. (B) The level of PotABCD mRNA was determined by primer extension. (C) PotD binding sites (-258 to -209 nucleotides upstream and 66 to 135 nucleotides downstream from the ATG initiation codon of the potA gene), which were determined by elec-trophoretic mobility shift assay, are shown as a box. (Reproduced with permission from ref. 32.)

Fig. 4. (A) Inhibition of spermidine uptake by overexpression of PotD and PotD-binding sites on the potABCD operon. (B) The level of PotABCD mRNA was determined by primer extension. (C) PotD binding sites (-258 to -209 nucleotides upstream and 66 to 135 nucleotides downstream from the ATG initiation codon of the potA gene), which were determined by elec-trophoretic mobility shift assay, are shown as a box. (Reproduced with permission from ref. 32.)

and the vestibule of the pore consisting of 12 transmembrane segments (36,37). These results suggest that the active site of PotE is located at the cytoplasmic side.

The gene for potE (cadB) together with the speF (cadA) gene for inducible ornithine decarboxylase (ODC) (lysine decarboxylase) constitutes an operon. Cell growth of a polyamine-requiring mutant was stimulated slightly at neutral pH by the uptake activity and greatly at acidic pH by the antiporter activity. At acidic pH, these two operons were induced in the presence of ornithine and cadaverine, respectively. The induction of the operon caused neutralization of the extracellular medium and made possible the production of CO2 and polyamines (Fig. 5). CO2 produced by ornithine (lysine) decar-boxylase contributes to the various metabolic pathways including nucleotide biosynthesis. Ornithine (lysine) decarboxylase also generates a pH gradient by consumption of a cytoplasmic proton. This process causes the increase in the level of ATP in cells.

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Fig. 5. Physiological functions of PotE and CadB in Escherichia coli. PotE, putrescine transporter; CadB, cadaverine transporter; iODC, inducible ornithine decarboxylase; CadA, lysine decarboxylase. iODC and CadA generate a pH gradient by consumption of a cytoplasmic proton in acidic conditions.

Because the speF-potE operon encoding inducible ODC and PotE is inducible at acidic pH, a protein involved in the enhancement of expression of the operon was sought. Using a fused gene containing the upstream sequence of the speF-potE operon and the open reading frame of P-galactosidase as a reporter gene, a clone that caused an increase of P-galactosidase activity at acidic pH was isolated. The clone was identified as a gene encoding RNase III (39). Our results suggest that the initiation codon AUG and Shine-Dalgarno sequence are exposed on the surface of the mRNA by the RNase III processing of the 5' untranslated region of mRNA. Although this is the first example of an RNase III-stimulated mRNA processing derived from E. coli gene, stimulation of the synthesis of ^N protein and T7 0.3 gene protein by RNase III has been reported (40,41). It is noteworthy that expression of the speF-potE operon is positively regulated at the posttranscriptional level.

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