Transcriptional Inhibition of the Operon for the Spermidine Uptake System by the Substrate-binding Protein PotD*

Fabiana Antognoni, Stefano Del Duca, Aiko Kuraishi, Eri Kawabe, Tomomi Fukuchi-Shimogori, Keiko Kashiwagi, and Kazuei IgarashiDagger

From the Faculty of Pharmaceutical Sciences, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan

    ABSTRACT
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Abstract
Introduction
References

Inhibition of spermidine uptake in Escherichia coli, which occurs in the presence of accumulated polyamines, has been studied using the spermidine uptake operon consisting of the potA, -B, -C, and -D genes. Transcription of the potABCD operon was inhibited by PotD, a spermidine-binding protein usually found in the periplasm, and the inhibitory effect of PotD was increased by spermidine. Transcription was not affected by bovine serum albumin, PotA, or PotF, suggesting that the effects of PotD are specific to the PotD protein. In the presence of 8 mM spermidine, a 50% inhibition of transcription was observed with a molar ratio of approximately 1:500 of template DNA:PotD. It was found that PotD bound to regions -258 to -209 nucleotides upstream and +66 to +135 nucleotides downstream of the ATG initiation codon of the potA gene. Binding of PotD to the downstream site was stimulated by spermidine. Overexpression of PotD in Escherichia coli DH5alpha inhibited the uptake of spermidine, the synthesis of PotABCD mRNA, and expression of a lacZ reporter gene fused downstream of a potA gene containing the PotD binding sites. In cells overexpressing PotD, a large amount of PotD existed as PotD precursor in spheroplasts. Our results indicate that PotD precursor can also inhibit spermidine transport. The amino acid residues in PotD that are involved in its interaction with the potABCD operon were determined using mutated PotD proteins. Thr-35 and Ser-85 of PotD were found to be important for this interaction. These results suggest that transcription of the spermidine transport (potABCD) operon is inhibited in vivo by PotD precursor rather than PotD through its binding to two regions close to the transcriptional initiation site of the operon.

    INTRODUCTION
Top
Abstract
Introduction
References

Polyamines (putrescine, spermidine, and spermine) are known to be necessary for cell growth (1, 2). It is thus important to understand the mechanisms by which cellular polyamines are regulated. Polyamine transport is one of the important factors that determines polyamine content. In Escherichia coli, polyamine uptake is energy-dependent, and there are separate uptake systems for putrescine and spermidine (3, 4). We have obtained and characterized three clones for E. coli polyamine transport genes (pPT104, pPT79, and pPT71) (5). The system encoded by pPT104 is a spermidine uptake system, and that encoded by pPT79 is a putrescine-specific uptake system. The characteristics of these two uptake systems, which belong to the ATP-binding cassette (ABC) superfamily (6, 7), have been systematically investigated (8-13). The third system, a putrescine transport system encoded by pPT71, is active in the excretion of putrescine from cells through a putrescine-ornithine antiporter activity (14-16).

Recently, we have begun to study regulation of the genes encoding the various polyamine transport systems. We have shown that the expression of pPT71 is positively regulated at the translational level by RNase III (17). The RNase increased the translational efficiency of mRNA derived from the E. coli gene by cutting the 5'-untranslated region of the mRNA (17). We also examined whether the spermidine uptake system, encoded by pPT104, is regulated by polyamines and found that uptake of spermidine decreased with an increase in cellular polyamine content. In this communication, we report our study on the mechanism of the decrease in spermidine uptake activity when polyamines accumulate in cells. We found that the PotD protein, a substrate-binding protein of the spermidine uptake system encoded by pPT104, as well as its precursor, inhibit transcription of the pPT104 clone by binding to two regions close to the transcriptional initiation site of the operon. The inhibitory effect of PotD (or PotD precursor) was enhanced by spermidine. Our results strongly suggest that PotD precursor is much more likely to be the primary regulator.

    EXPERIMENTAL PROCEDURES

Bacterial Strains, Plasmids, and Culture Conditions-- E. coli DH5alpha (supE44 Delta lacU (phi 80 lacZDelta M15) hsd R17 recA1 endA1 gyrA96 thi-1 relA1) was cultured in LB medium (18). A polyamine-requiring mutant, E. coli MA261 (speB speC gly leu thr thi), provided by Dr. W. K. Maas (New York University School of Medicine), was grown in medium A in the absence of polyamines as described previously (5). The resulting polyamine-depleted bacteria (A540 = 0.05) were then cultivated in either the presence or absence of putrescine (0.1 mg/ml). E. coli MM52, a temperature-sensitive secA mutant (19), was kindly supplied by Dr. H. Tokuda (University of Tokyo) and was grown in LB medium. When growth was sufficient to yield an A540 of 0.30, the cells were harvested by centrifugation at 12,000 × g for 10 min. The cells were washed once with buffer A, which contained 0.4% glucose, 62 mM potassium phosphate (pH 7.0), 1.7 mM sodium citrate, 7.6 mM (NH4)2SO4, and 0.41 mM MgSO4, centrifuged as described above, and suspended in buffer A to yield a protein concentration of 0.1 mg/ml. Protein was measured by the method of Lowry et al. (20) after trichloroacetic acid precipitation of cells. Plasmids pPT104 containing the potABCD genes and pUCpotD were prepared from pACYC184 and pUC119, respectively, as described previously (8, 10). Where indicated, E. coli DH5alpha , MA261, or MM52 containing the plasmids was grown as described above in the presence of 30 µg/ml chloramphenicol and/or 100 µg/ml ampicillin.

Assay for Spermidine Uptake-- The cell suspension (0.48 ml) was preincubated at 30 °C for 5 min, and the reaction was started by the addition of 20 µl of 250 µM [14C]spermidine (370 MBq/mmol). After incubation at 30 °C for the designated time, the cells were collected on membrane filters (cellulose acetate, 0.45 µm; Advantec Toyo), and the radioactivity on the filter was assayed with a liquid scintillation spectrometer (4).

Measurement of PotABCD mRNA by Primer Extension-- Primer extension was performed according to the method of McKnight and Kingsbury (21). Synthetic oligonucleotides, which hybridize to the -13 to +11 nucleotides (24-mer) relative to the initiation codon AUG (PE1 in Fig. 3) of the PotABCD mRNA, were end-labeled with [gamma -32P]ATP using T4 polynucleotide kinase. The 32P-labeled oligonucleotides were then hybridized with total RNA (50 µg) prepared from E. coli DH5alpha or MA261 cells by the method of Emory and Belasco (22), and the cDNA was synthesized with Moloney murine leukemia virus reverse transcriptase (U. S. Biochemical Corp.). The product of this reaction was analyzed by gel electrophoresis with 5% polyacrylamide.

Assay for the in Vitro Transcription of the potABCD Genes-- The pPT104 clone was isolated from E. coli DH5alpha /pPT104 according to the method of Sambrook et al. (23), digested with KpnI, and used as the template for an in vitro transcription assay. The transcription assay with linearized DNA template was carried out according to the method of Kajitani and Ishihama (24) with some modifications. A volume of 35 µl of a mixture containing 0.2 pmol of the template and 10-fold molar excess of E. coli RNA polymerase (Sigma) in 50 mM Tris-HCl (pH 7.8), 10 mM magnesium acetate, 100 mM KCl, 0.1 mM EDTA, 0.1 mM dithiothreitol, and 25 µg/ml nuclease-free bovine serum albumin was incubated at 37 °C for 20 min. Where indicated, spermidine, PotD, PotF, PotA, or bovine serum albumin was included in the mixture. PotD, PotF, and PotA were purified as described previously (9-11). Transcription was initiated by adding 15 µl of a prewarmed substrate-heparin mixture in the same buffer. The final concentration was 0.16 mM for ATP, GTP and CTP, 0.05 mM for [3H]UTP (74 kBq), and 200 µg/ml for heparin, respectively. RNA synthesis was carried out for 5 min and terminated by adding 50 µl of the stopping solution containing 40 mM EDTA and 300 µg/ml yeast RNA. For measurement of [3H]UTP incorporated into RNA, cold trichloroacetic acid-insoluble radioactivity was assayed with a liquid scintillation spectrometer. Where indicated, a 5.8-kb1 SmaI-KpnI fragment of pPT104 containing the potABCD genes was used as a template.

PCR Amplification-- Four kinds of probes for the mobility shift assay were made by PCR. P1 DNA was amplified using primers PRM1 (5'-ATGCGTCGACTTCCATTGGCCTTCCG-3'; position -349 to -332 of the sequence of pPT104 having SalI cut site, see Fig. 3) and PRM2 (5'-TATGGAATTCCCGCTTGCAGGGGTAAAAGT-3'; complementary sequence for the position -182 to -159 of the sequence of pPT104 with an EcoRI restriction site, see Fig. 3). It was digested with SalI and EcoRI, labeled with [alpha -32P]TTP using the Klenow fragment, and purified by polyacrylamide gel electrophoresis according to the method of Ausubel et al. (25). P2 DNA was amplified using primers PRM3 (5'-GCAAGCGGGAATATTTATCAGCATT-3'; position -170 to -146 of the sequence of pPT104, see Fig. 3) and PRM4 (5'-AGCATTTGCGAATTCCCGCCAATTG-3'; complementary sequence for the position +54 to +79 of the sequence of pPT104, see Fig. 3). It was digested with SspI and EcoRI and labeled with [alpha -32P]TTP using the Klenow fragment. Another PCR product was obtained using primers PRM5 (5'-TTCGAGCTGCTCTTTCAGCAGATGC-3'; position -378 to -354 of the sequence of pPT104, Ref. 8) and PRM6 (5'-AACACGGTCATGTGGGGGAAAAGTGC-3'; complementary sequence for the position +295 to +320 of the sequence of pPT104, Ref. 8). P3 DNA was obtained by digestion of the PCR product with FokI and Sau3AI and labeled with [alpha -32P]TTP using the Klenow fragment. P4 DNA was obtained by digestion of the PCR product with EcoRI and HpaII and labeled with [alpha -32P]dCTP using the Klenow fragment.

Electrophoretic Mobility Shift Assays-- The binding reaction was performed in a 20-µl volume containing 0.2 pmol (10,000-15,000 cpm) of DNA probe (P1, P2, P3, or P4), 2 µg of poly(dI-dC)·poly(dI-dC), 20 mM Tris-HCl (pH 7.8), 10 mM magnesium acetate, 100 mM KCl, 0.2 mM EDTA, 1 mM dithiothreitol, 5% glycerol, and various amounts of PotD protein shown in the figures in the presence or absence of 8 mM spermidine. For competition experiments, a 1000-fold molar excess of unlabeled 30-mer double-stranded oligonucleotides were added to the mixture (see Fig. 3): C1 (-368 to -339 of the sequence of pPT104), C2 (-338 to -309), C3 (-308 to -279), C4 (-278 to -249), C5 (-248 to -219), C6 (-218 to -189), C7 (-188 to -159), C8 (-15 to +15), C9 (+16 to +45), C10 (+46 to +75), C11 (+76 to +105), C12 (+106 to +135), C13 (+136 to +165), C14 (+166 to +195), and C15 (+66 to +95). The reaction mixture was incubated at 37 °C for 10 min, and the products were immediately loaded onto a pre-run 4% high ionic strength polyacrylamide gel by the method of Ausubel et al. (25). The electrophoresis was performed for 1.5-2 h at 250 V at 4 °C. After electrophoresis, gels were dried and exposed overnight at -80 °C to a Fuji x-ray film with an intensifying screen. A gel mobility supershift experiment was performed using 1-10 µl of 15 mg/ml of antiserum against PotD (8) or normal serum according to the method of Park and Katze (26).

Construction of pACpotA33-lacZ, pACpotA279-lacZ, and pAClacZ Plasmids-- Fusion plasmids containing the 5' region of the potA gene and open reading frame of the lacZ gene were prepared by overlap extension using PCR (27). Primers used for first PCR using pPT104 as template were PRM1 and 5'-AAAACGACTGCAGGTTGTTTATTCAATTTT-3' to construct pACpotA33-lacZ and PRM1 and 5'-TAAAACGACTGCAGTGTTCACATAGCGGTT-3' to construct pACpotA279-lacZ. Primers used for second PCR using pMC1871 containing lacZ (Pharmacia Biotech) as template were 5'-AAACAACCTGCAGTCGTTTTACAACGTCGT-3' and 5'-CTACGGATCCCCCCTGCCCGGTTATTA-3' (PRM7) to construct pACpotA33-lacZ and 5'-GTGAACACTGCAGTCGTTTTACAACGTCGT-3' and PRM7 to construct pACpotA279-lacZ. Then, a third PCR was performed using the first and second PCR products as templates and PRM1 and PRM7 as primers. Two PCR products thus obtained were digested with SalI and BamHI and inserted into the same restriction sites of pACYC184. Plasmid pACpotA33-lacZ contained 349 nucleotides of 5'-upstream region and 33 nucleotides of coding region of the potA gene fused to the open reading frame of the lacZ gene, and pACpotA279-lacZ contained 349 nucleotides of 5'-upstream region and 279 nucleotides of coding region of the potA gene fused to the open reading frame of the lacZ gene. To prepare pAClacZ, a 7.2-kb SalI-PstI fragment of pACpotA33-lacZ was isolated. Promoter region of lacZ was prepared by PCR using primer 5'-TAGGTCGACAGGTTTCCCGACTGGAA-3' with a SalI restriction site, sequence primer M13 (-40) 5'-GTTTTCCCAGTCACGAC-3', and pUC9 (28) as a template. After digesting the PCR product with SalI and PstI, the fragment was ligated to the above 7.2-kb SalI-PstI fragment (pAClacZ). The pUC-mutated potD plasmids were prepared as described previously (13). Assay of beta -galactosidase of E. coli DH5alpha carrying the fusion plasmids and pUCpotD (or pUC-mutated potD) cultured in LB medium containing 0.5 mM isopropyl-beta -D-thiogalactopyranoside was performed according to the method of Miller (29).

Measurement of Polyamine Contents and PotA, PotD Precursor, and PotD-- Polyamine levels in E. coli were determined by high performance liquid chromatography as described previously (30). Western blotting of PotA and PotD was performed as described previously (8). Periplasm and spheroplast of E. coli were prepared according to the method of Oliver and Beckwith (31). PotD precursor and PotD were separated by SDS gel electrophoresis with 8.0% polyacrylamide and analyzed by Western blotting.

    RESULTS

Inhibition of Spermidine Uptake by Polyamines-- We first examined whether the spermidine uptake system is regulated by polyamines using the E. coli mutant MA261, which is deficient in polyamine biosynthesis, transformed with pPT104 encoding the PotABCD uptake system. As shown in Fig. 1A, the spermidine uptake activity of E. coli MA261/pPT104 decreased about by 50% when cells were grown in the presence of 0.1 mg/ml putrescine, compared with that of the cells cultured in the absence of putrescine. The putrescine and spermidine contents of E. coli MA261/pPT104 grown in the presence and absence of putrescine were, respectively, 41.7 and <0.1 nmol/mg protein for putrescine and 5.93 and 0.78 nmol/mg protein for spermidine, similar to previous results (32, 33). When E. coli MA261/pPT104 was grown in the presence of 0.03 mg/ml spermidine, spermidine uptake activity was also decreased by about 50% (data not shown), and the putrescine and spermidine contents in these cells were 0.21 and 7.15 nmol/mg protein, respectively. The amount of PotABCD mRNA, a product of the spermidine uptake operon encoded by pPT104, was measured by primer extension (Fig. 1B). The amount of PotABCD mRNA in cells grown with putrescine was approximately half that seen in cells grown without putrescine. When E. coli MA261 (i.e. not transformed with pPT104) was grown in the presence of 0.1 mg/ml putrescine, spermidine uptake was reduced, similar to effects seen with the E. coli MA261/pPT104 cells (Fig. 1A). However, PotABCD mRNA could not be detected in E. coli MA261 cells, presumably because the native PotABCD mRNA is expressed at levels below the limit of detection in these cells. Based on the results seen with MA261/pPT104 and native MA261 cells, we hypothesized that an increase in intracellular spermidine, occurring after growth of E. coli in medium containing putrescine or spermidine, may directly inhibit transcription of the potABCD operon or may alter the stability of PotABCD mRNA.


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Fig. 1.   Spermidine uptake activity (A) and the amount of PotABCD mRNA (B) of E. coli cells cultured with or without putrescine. A, spermidine uptake activity was measured under standard conditions using E. coli MA261 (solid line) and MA261/pPT104 (dashed line) cells cultured with or without 0.1 mg/ml putrescine (PUT). Each value is the average of duplicate determinations. B, the amount of mRNA of E. coli MA261/pPT104 cultured with or without putrescine was determined using 50 µg of RNA by primer extension. PE1 shown in Fig. 3, which hybridizes with the nucleotide sequence -13 to +11 relative to the A of the translation initiation codon of potA gene, was used as the primer. C, T, A, and G represent dideoxynucleotide sequencing of M13mp19 for size comparison. Two arrows correspond to the -66 and -67 position nucleotides.

Inhibition of Transcription of the Spermidine Uptake Operon by PotD-- The effects of polyamines on transcription of the potABCD (spermidine uptake) operon were examined in an in vitro system consisting of a linearized pPT104 clone, E. coli RNA polymerase, and its appropriate substrates. By itself, spermidine did not greatly inhibit the synthesis of PotABCD mRNA when assays were carried out in the presence of 100 mM KCl and 10 mM magnesium acetate. We examined the effect of polyamine transport proteins on the synthesis of PotABCD mRNA in the presence and absence of spermidine (Fig. 2). Bovine serum albumin, PotF, a substrate-binding protein of the putrescine uptake system (9), and PotA, a protein involved in energy supply (8), did not significantly inhibit transcription. However PotD, a substrate-binding protein of the spermidine uptake system (8), inhibited transcription of the potABCD operon. Inhibition of the synthesis of PotABCD mRNA by PotD was concentration-dependent. Spermidine increased the inhibition, and 8 mM spermidine was necessary to obtain a maximal inhibition. A 50% inhibition of transcription in the presence of spermidine was observed at an approximate molar ratio of 1:500 of template DNA (0.2 pmol):PotD (100 pmol), given that the molecular mass of PotD is 36 kDa (8). When the potABCD gene by itself (a 5.8-kb SmaI-KpnI fragment of pPT104) was used as a template, similar results were obtained (data not shown). Transcription of linearized pACYC184, the parent plasmid of the pPT104 clone, was not inhibited by PotD.


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Fig. 2.   Effect of polyamine transport proteins and bovine serum albumin on the transcription of pPT104. The assay was performed as described under "Experimental Procedures" (100%: 34 pmol of [3H]UTP incorporated). Since PotA is an ATPase (11), 10-fold nucleoside triphosphates (indicated by an asterisk) were used as substrates when PotA was added to the reaction mixture. Where indicated, 8 mM spermidine (SPD) was added to the reaction mixture. Values are mean S.D. from triplicate determinations.

Identification of the Binding Site of PotD on Spermidine Uptake Operon-- Electrophoretic mobility shift assays were carried out to identify the binding site for PotD using four DNA probes (P1, P2, P3, and P4, Fig. 3B). P1 included the region upstream of the initiation site of transcription, P2 included the initiation site of transcription, P3 included the initiation site of translation and part of the open reading frame (ORF) of the potA gene, and P4 included only part of the ORF of the potA gene. As shown in Fig. 4A, there was a clear shift of the P1, P3, and P4 DNA probes induced by PotD (indicated by two stars), and the shift of P3 and P4 probes was increased about 1.4-fold by 8 mM spermidine. The shift of the P2 probe induced by PotD was faint, suggesting that PotD does not bind to the initiation site of transcription, which is encompassed by the P2 probe. Because about 7 µg of PotD caused the gel shift of P1 DNA by 30%, the binding constant of PotD for P1 DNA was estimated to be about 4 × 104 M-1. Similarly, the binding constant of PotD for P3 DNA in the presence of 8 mM spermidine was estimated to be about 1 × 104 M-1. In these experiments, PotD was stained in parallel with Coomassie Brilliant Blue R-250. The shift of PotD to the complex with the DNA probes indicated by two stars was less than 1% (data not shown). In control experiments, we found that PotA and bovine serum albumin did not cause a shift of P1, P3, or P4 DNA (data not shown).


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Fig. 3.   Nucleotide sequence of the promoter region of the spermidine uptake operon (A) and the DNA probes and competitors used for electrophoretic mobility shift assay (B). A, the deduced amino acid sequence is shown under the nucleotide sequence (GenBankTM accession number M64519). The -10 and -35 region of the promoter is boxed, and asterisks indicate the initiation site of transcription. The recognition sites of restriction enzymes (SspI, FokI, EcoRI, Sau3AI, and HpaII) are also shown. B, P1 to P4 and C1 to C15 are DNA probes and competitors used for electrophoretic mobility shift assay, respectively. PE1 is a primer used for the primer extension shown in Figs. 1 and 5. PotD protein binding sites (-258 to -209 nucleotides upstream and 66-135 nucleotides downstream from the ATG initiation codon of the potA gene) are shown as a box. The recognition sites of restriction enzymes shown in parentheses were artificially made for the preparation of P1 probe.


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Fig. 4.   Electrophoretic mobility shift of DNA probes by the PotD protein. A, the assay was performed in the presence and absence of 8 mM spermidine (SPD) as described under "Experimental Procedures." DNA probes (P1 to P4) used were shown in Fig. 3. * and ** on the left indicate DNA probe and DNA-PotD protein complex, respectively. B, gel mobility of supershift assay by adding 1-10 µl of 15 mg/ml anti-PotD antiserum or normal serum. *** and **** on the left indicate DNA-PotD-anti-PotD complex. C, competition by 1000 times higher concentrations of 30-mer oligonucleotides in electrophoretic mobility shift of DNA probe. The assay was performed with 5 µg of PotD and 8 mM spermidine. DNA probes (P1, P2, and P4) and 30-mer oligonucleotide competitors (C1 to C15) used were shown in Fig. 3.

The formation of a specific complex between P1 and P3 DNA and PotD was confirmed by the addition of antiserum against the PotD protein (Fig. 4B). The band indicated by two asterisks shifted to the position indicated by three asterisks, and a further shift indicated by four asterisks was observed by increasing the anti-PotD antiserum. Similar results were obtained in the presence of spermidine (data not shown). The antiserum against PotD by itself, or normal serum plus PotD, did not cause the super gel shift (Fig. 4B).

To pinpoint the PotD binding sites on the spermidine uptake operon, competition experiments to study the mobility shift of P1, P3, and P4 were carried out using a 1000-fold excess of 30-mer oligonucleotides, termed C1 to C15 in Fig. 3. As shown in Fig. 4C, fragment C5 was the strongest competitor among the C1 to C7 fragments for P1 DNA, but the competitive ability was weak. Fragment C11 was the strongest competitor among the C8 to C15 fragments for P3 and P4 DNA, and the competitive ability was strong compared with that seen with C5 for P1 DNA. The results indicate that PotD binds to regions -258 to -209 nucleotides upstream and +66 to +135 nucleotides downstream from the ATG initiation codon of the potA gene. The binding affinity of PotD appears to be higher in the former binding site than in the latter.

Inhibition of Spermidine Uptake and Decrease in PotABCD mRNA Levels by PotD-- To determine whether PotD reduces spermidine uptake by inhibiting transcription of the spermidine uptake operon, spermidine uptake in E. coli DH5alpha /pPT104 was compared with that in E. coli DH5alpha /pPT104 + pUCpotD, which synthesize excess PotD. As shown in Fig. 5, A and B, uptake was inhibited by PotD, and the amount of PotA was reduced in the presence of excess PotD. Under these conditions, the amount of PotABCD mRNA in E. coli DH5alpha /pPT104 was greater than that in E. coli DH5alpha /pPT104 + pUCpotD (Fig. 5C). Spermidine uptake of E. coli DH5alpha (i.e. not transformed with pPT104) was also inhibited by PotD (Fig. 5A), and the amount of PotA was found to decrease in the presence of excess PotD (Fig. 5B). The stability of PotABCD mRNA determined by addition of rifampicin to the medium (34) was not influenced by PotD. The half-life of PotABCD mRNA in the presence and absence of excess PotD was about 15 min.


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Fig. 5.   Effect of PotD on spermidine uptake activity (A), the amount of PotA (B), and of PotABCD mRNA (C). E. coli DH5alpha /pPT104 + pUC119 (1, bullet - - -bullet ), DH5alpha /pPT104 + pUCpotD (2, open circle ---open circle ), DH5alpha /pUC119 (3, bullet ---bullet ), and DH5alpha /pUCpotD (4, open circle - - -open circle ) were used for the experiments. Western blotting for PotA was performed using 1 µg (lanes 1 and 2) and 10 µg (lanes 3 and 4) protein of total cell lysate. Primer extension was performed using 50 µg of RNA.

We constructed two fusion plasmids containing the upstream region of the potABCD operon and the open reading frame of the lacZ gene. One fusion plasmid contained two PotD binding sites, the regions -258 to -209 nucleotides upstream and +66 to +135 nucleotides downstream from the ATG initiation codon of potA gene (pACpotA279-lacZ), and the other plasmid contained only the upstream PotD binding site (pACpotA33-lacZ). As shown in Table I, beta -galactosidase activity in E. coli DH5alpha /pACpotA279-lacZ was strongly inhibited by 79% by PotD, but beta -galactosidase activity in E. coli DH5alpha /pACpotA33-lacZ was inhibited by 46% by PotD. Control beta -galactosidase activity in E. coli DH5alpha /pAClacZ was inhibited only by 19% by PotD. The putrescine and spermidine contents of E. coli DH5alpha were 84.6 and 32.6 nmol/mg protein, respectively. Addition of 0.1 mg/ml putrescine or 0.03 mg/ml spermidine to the medium did not significantly influence the polyamine content or the results on lacZ expression, indicating that E. coli DH5alpha can normally synthesize enough polyamines. The results strongly suggest that PotD functions as a transcriptional inhibitor of the spermidine uptake operon rather than as a destabilizer of PotABCD mRNA.

                              
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Table I
Effect of PotD on the expression of potA-lacZ fusion gene
E. coli DH5alpha cells carrying various plasmids were cultured in LB medium containing appropriate antibiotics and 0.5 mM IPTG until A600 = 0.4, and beta -galactosidase activity was measured as described under "Experimental Procedures."

Effect of Mutated PotD Proteins on the Expression of potA-lacZ Fusion Gene-- To identify which portion of PotD functions as a negative regulator of the spermidine uptake operon, the activity of mutated PotD proteins was examined using a potA-lacZ fusion gene. As shown in Fig. 6, only the T35A2 mutation in PotD abolished its effect as a negative regulator of transcription, although the S83A mutation also greatly reduced the negative regulatory effect of PotD. Mutations at Trp-255 and Asp-257, residues that are important for spermidine binding (13), in addition to Glu-36, Ser-211 and Tyr-293 had only small effects on the negative regulation of transcription. The results suggest that the N-domain side of the central cleft of PotD, where Thr-35 and Ser-83 are located, is probably important for the interaction of PotD with the spermidine uptake operon (12, 13, 35).


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Fig. 6.   Effect of PotD mutants on the expression of potA-lacZ fusion gene. E. coli DH5alpha cells carrying pACpotA279-lacZ and various pUC-mutated potD were cultured in LB medium containing appropriate antibiotics and 0.5 mM isopropyl-beta -D-thiogalactopyranoside until A600 = 0.4, and beta -galactosidase activity was measured as described under "Experimental Procedures." Values are mean ± S.D. from triplicate determinations, and beta -galactosidase activity of E. coli DH5alpha /pACpotA279-lacZ was 1362 Miller units (100%).

Cellular Localization of PotD and PotD Precursor and Inhibition of Spermidine Uptake by PotD Precursor-- The cellular localization of the PotD protein in E. coli DH5alpha /pUC119 and E. coli DH5alpha /pUCpotD was examined by Western blotting using subcellular fractions (Fig. 7). Approximately 30-fold more of PotD existed in the periplasm of E. coli DH5alpha /pUCpotD than in the periplasm of the control strain E. coli DH5alpha /pUC119. PotD precursor was not seen in the periplasm of both E. coli strains. However, PotD located in the periplasmic space is presumably not involved in the inhibition of transcription of the potABCD gene. We therefore measured levels of PotD and of the PotD precursor protein in spheroplast. In E. coli DH5alpha /pUCpotD, a significant amount of PotD precursor was synthesized in addition to mature PotD, and levels of the PotD precursor were 100-fold higher in the spheroplast of E. coli DH5alpha /pUCpotD than in the spheroplast of E. coli DH5alpha /pUC119. Almost all the PotD precursor was located in the cytoplasmic membrane rather than in the cytoplasm (data not shown). Mature PotD was not clearly observed in the spheroplast of both E. coli strains. The results suggest that the PotD precursor may be a regulator of the transcription of spermidine uptake operon.


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Fig. 7.   Cellular localization of PotD and PotD precursor. Western blotting was performed using total cell lysate, periplasm, and spheroplast prepared from E. coli DH5alpha /pUC119 (A) and E. coli DH5alpha /pUCpotD (B). From 4 mg of total cell lysate protein, 0.5 mg of periplasmic protein and 2.9 mg of spheroplast protein were obtained. This was almost the same in E. coli DH5alpha /pUC119 and E. coli DH5alpha /pUCpotD. The amount of protein used for Western blotting is shown in the figure.

Accordingly, we tested the possibility that the PotD precursor inhibits spermidine uptake using a temperature-sensitive secA mutant MM52 (19), in which translocation of periplasmic proteins from the cytoplasm is inhibited. When E. coli MM52/pUCpotD was cultured at 37 °C, significant amounts of the PotD precursor accumulated in the spheroplast of the strain compared with E. coli MM52/pUC119. However, comparable amounts of mature PotD existed in the periplasm in both strains. Under these conditions, spermidine uptake in E. coli MM52/pUCpotD was much lower than that in E. coli MM52/pUC119 (Fig. 8). We could isolate 70% pure PotD precursor from the cytoplasmic membrane of E. coli MM52/pUCpotD. This PotD precursor inhibited the in vitro transcription of the spermidine uptake operon to almost the same degree as mature PotD (data not shown). These results suggest that PotD precursor rather than PotD acts as a negative regulator of the spermidine uptake operon.


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Fig. 8.   Amount of PotD precursor in spheroplast and inhibition of spermidine uptake activity by the precursor in a temperature sensitive secA mutant (MM52). E. coli MM52/pUC119 and E. coli MM52/pUCpotD were cultured at 37 °C until A540 = 0.3, and spermidine uptake activity was measured. Values are mean ± S.D. from triplicate determinations. From 4 mg of total cell lysate protein, 3.1 mg of spheroplast protein was obtained. The amount of protein used for Western blotting is shown in the figure.


    DISCUSSION

Our results show that PotD or more likely the PotD precursor functions as a transcriptional regulator of the spermidine uptake operon. Recently, many transcriptional regulators, such as H-NS (36, 37), HU (38, 39), Fis (40, 41), and Lrp (41, 42), have been described. These factors interact with curved DNA (36) or cause the bending of DNA (40, 42). In most cases, these factors interact with a region upstream of the initiation codon ATG. An exception is that H-NS recognizes a downstream regulation element existing in the ORF of the proU gene (36). The PotD protein recognizes two regions of the spermidine uptake operon-one on the promoter region and the other on the ORF region of the potA gene. However, there is a great difference in the affinity for DNA of H-NS and PotD. H-NS functions at concentrations of 10-100 ng/pmol DNA (36, 37), but the PotD functions at concentrations of 10-100 µg/pmol DNA; i.e. PotD (or the PotD precursor) has about a 1000-fold lower affinity for DNA than does H-NS. Intuitively, this is understandable because the major known function of H-NS is as a transcriptional regulator, whereas that of PotD is as a periplasmic polyamine-binding protein. PotD presumably inhibits transcription of the spermidine uptake operon only when excess amounts of PotD are produced. Under these conditions, large amounts of PotD precursor accumulate inside the cells, where it acts as the repressor of the spermidine uptake operon.

Because the interaction of PotD with DNA is weak, we could not precisely determine the binding site of PotD on the spermidine uptake operon by using DNase I footprinting analysis. Therefore, we determined the PotD protein binding site on the operon by using gel shift assays and competition experiments with 30-mer oligonucleotides. PotD bound to regions -258 to -209 nucleotide upstream and +66 to +135 nucleotides downstream from the ATG initiation codon of the potA gene. These two PotD binding sites on the spermidine uptake operon could possibly make a palindromic structure. The PotD binding site 1 can make stronger palindrome than the PotD binding site 2. The binding of PotD protein to site 1 was not significantly influenced by spermidine, but binding to site 2 was strengthened by spermidine. Spermidine could conceivably act to strengthen a palindromic structure.

Our results indicate that PotD precursor, but not mature PotD, accumulated in the spheroplast when excess amounts of PotD are synthesized. Using E. coli mutant that prevents protein translocation from the cytoplasm to the periplasm, we could show that PotD precursor in the spheroplast reduced the spermidine uptake. Furthermore, PotD precursor inhibited the in vitro transcription of spermidine uptake operon at almost the same degree as mature PotD. The results strongly suggest that PotD precursor is a primary regulator of the transcription of spermidine uptake operon.

To confirm that PotD precursor functions as a regulator of the spermidine uptake operon, the number of PotD precursor in spheroplast was estimated. Since it has been reported that number of RNA polymerase sigma 70 is constant during the logarithmic phase of cell growth and is estimated to be about 700 molecules/cell (43), the number of PotD precursor existed in spheroplast of E. coli DH5alpha /pUCpotD was estimated by comparison with the number of RNA polymerase sigma 70 by Western blot analysis. It was about 5 × 103 to 2.5 × 104 molecules/cell. Because a 50% inhibition of transcription was observed in an approximate molar ratio of 1:500 of template DNA:PotD, the above values can explain about 70-80% inhibition of spermidine uptake by excess PotD.

It is well known that some ribosomal proteins function as translational repressors (44). It has been reported that PutA protein also functions as membrane-bound dehydrogenase as well as the repressor of the put operon (45). In summary, PotD or more likely the PotD precursor is a new type of transcriptional regulator.

    ACKNOWLEDGEMENTS

We thank Drs. K. Williams and A. J. Michael for their help in preparing this manuscript.

    FOOTNOTES

* This work was supported by a grant-in-aid for Scientific Research from the Ministry of Education, Science, Sports and Culture, Japan, the Mochida Memorial Foundation for Medical and Pharmaceutical Research, Japan, and by the Iwaki Scholarship Foundation, Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 81-43-290-2897; Fax: 81-43-290-2900; E-mail: iga16077{at}p.chiba-u.ac.jp.

The abbreviations used are: kb, kilobase(s); PCR, polymerase chain reaction; ORF, open reading frame.

2 The mutated PotD protein T35A contains alanine instead of threonine at position 35.

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