From the Faculty of Pharmaceutical Sciences, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan
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ABSTRACT |
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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 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.
Bacterial Strains, Plasmids, and Culture
Conditions--
E. coli DH5 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 Assay for the in Vitro Transcription of the potABCD
Genes--
The pPT104 clone was isolated from E. coli
DH5 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 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 ( 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 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.
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.
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.
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
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 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 DH5
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 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).
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 DH5
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.
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 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 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.
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 DH5
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
EXPERIMENTAL PROCEDURES
(supE44
lacU (
80
lacZ
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 DH5
, 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.
13
to +11 nucleotides (24-mer) relative to the initiation codon AUG (PE1
in Fig. 3) of the PotABCD mRNA, were end-labeled with
[
-32P]ATP using T4 polynucleotide kinase. The
32P-labeled oligonucleotides were then hybridized with
total RNA (50 µg) prepared from E. coli DH5
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.
/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.
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
[
-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
[
-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
[
-32P]TTP using the Klenow fragment. P4 DNA was
obtained by digestion of the PCR product with EcoRI and
HpaII and labeled with [
-32P]dCTP using the
Klenow fragment.
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).
-galactosidase of
E. coli DH5
carrying the fusion plasmids and
pUCpotD (or pUC-mutated potD) cultured in LB
medium containing 0.5 mM
isopropyl-
-D-thiogalactopyranoside was performed
according to the method of Miller (29).
RESULTS
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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.
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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.
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.
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.
/pPT104 was compared with
that in E. coli DH5
/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 DH5
/pPT104 was greater than that in
E. coli DH5
/pPT104 + pUCpotD (Fig.
5C). Spermidine uptake of E. coli DH5
(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
DH5 /pPT104 + pUC119 (1,
- - -
), DH5
/pPT104 + pUCpotD (2,
---
), DH5
/pUC119 (3,
---
), and
DH5
/pUCpotD (4,
- - -
) 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.
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,
-galactosidase activity in
E. coli DH5
/pACpotA279-lacZ was strongly
inhibited by 79% by PotD, but
-galactosidase activity in E. coli DH5
/pACpotA33-lacZ was inhibited by 46% by
PotD. Control
-galactosidase activity in E. coli
DH5
/pAClacZ was inhibited only by 19% by PotD. The putrescine and spermidine contents of E. coli DH5
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 DH5
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.
Effect of PotD on the expression of potA-lacZ fusion gene
cells carrying various plasmids were cultured
in LB medium containing appropriate antibiotics and 0.5 mM
IPTG until A600 = 0.4, and
-galactosidase
activity was measured as described under "Experimental Procedures."
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Fig. 6.
Effect of PotD mutants on the expression of
potA-lacZ fusion gene. E. coli DH5 cells
carrying pACpotA279-lacZ and various pUC-mutated
potD were cultured in LB medium containing appropriate
antibiotics and 0.5 mM
isopropyl-
-D-thiogalactopyranoside until
A600 = 0.4, and
-galactosidase activity was
measured as described under "Experimental Procedures." Values are
mean ± S.D. from triplicate determinations, and
-galactosidase
activity of E. coli
DH5
/pACpotA279-lacZ was 1362 Miller units
(100%).
/pUC119 and E. coli DH5
/pUCpotD was examined by Western blotting using subcellular fractions (Fig. 7).
Approximately 30-fold more of PotD existed in the periplasm of E. coli DH5
/pUCpotD than in the periplasm of the
control strain E. coli DH5
/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 DH5
/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 DH5
/pUCpotD than in
the spheroplast of E. coli DH5
/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
DH5 /pUC119 (A) and E. coli
DH5
/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
DH5
/pUC119 and E. coli DH5
/pUCpotD. The
amount of protein used for Western blotting is shown in the
figure.
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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
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.
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
DH5
/pUCpotD was estimated by comparison with the number
of RNA polymerase
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.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. K. Williams and A. J. Michael for their help in preparing this manuscript.
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FOOTNOTES |
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* 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.
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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REFERENCES |
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