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Journal of Bacteriology, October 1998, p. 5484-5488, Vol. 180, No. 20
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Relationship between Spontaneous Aminoglycoside
Resistance in Escherichia coli and a Decrease in
Oligopeptide Binding Protein
Keiko
Kashiwagi,1
Maria Heloisa
Tsuhako,1
Kaori
Sakata,1
Tomoko
Saisho,1
Atsuko
Igarashi,2
Sérgio Olavo
Pinto da Costa,3 and
Kazuei
Igarashi1,*
Faculty of Pharmaceutical Sciences, Chiba
University, Inage-ku, Chiba 263-8522,1 and
Niigata University School of Dentistry, 2-5274 Gakkocho-dori,
Niigata 951-8514,2 Japan, and
Universidade de São Paulo, Instituto de
Ciéncias Biomédicas, Departmento de Microbiologia,
São Paulo 05508-900, Brazil3
Received 15 September 1997/Accepted 15 August 1998
 |
ABSTRACT |
Changes in the amount of oligopeptide binding protein (OppA) in
spontaneous kanamycin-resistant mutants of Escherichia coli were investigated. Among 20 colonies obtained from 108
cells cultured in the presence of 20 µg 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.
| |
TEXT |
Uptake of aminoglycoside antibiotics
is a complex process that is still a matter of controversy (7,
21). Streptomycin is thought to be taken up by Escherichia
coli by a process that may be subdivided into three consecutive
phases: first, a rapid electrostatic binding to the cell; second, a
slow rate of accumulation; and third, a much-enhanced rate of
accumulation. Since there are several reports that aminoglycoside
antibiotics are accumulated in E. coli by an active
transport system (1, 2, 6), the second phase (a slow rate of
accumulation) may involve the active transport system. The third phase,
a much-enhanced rate of accumulation, may be explained by membrane
permeabilization caused by the insertion of mistranslated proteins into
the cytoplasmic membrane (3, 8). Another possibility has
also been suggested for the third phase: the enhanced streptomycin
uptake may involve the induction of a polyamine transport system by
streptomycin, which can be utilized by streptomycin itself
(10).
We isolated three clones carrying polyamine transport genes (pPT104,
pPT79, and pPT71) and characterized them (9, 13, 16, 23).
Using these three clones, we showed that aminoglycoside antibiotics do
not up-regulate the polyamine transport system (15). We also
proposed that the oligopeptide transport system is a candidate for the
second phase, a slow rate of accumulation. This was based on the
finding with E. coli DR112 (18) that sensitivity to aminoglycoside antibiotics increased due to the highly expressed oligopeptide binding protein (OppA), a component of the oligopeptide transport system, and decreased in cells lacking the oppA
gene (15). To clarify whether the oligopeptide transport
system is involved in the active transport of aminoglycoside
antibiotics, we isolated spontaneous kanamycin-resistant mutants.
Complete loss of or decrease in OppA was observed in 8 of 20 of these
mutants. These results indicate that the oligopeptide transport system is involved in the uptake of aminoglycoside antibiotics, and that the
system is down-regulated in some of the spontaneous kanamycin-resistant mutants.
Bacterial strains, plasmid, and culture conditions.
E.
coli J53 (met pro thi) (5) was grown in
Luria-Bertani medium (medium A) or M9 minimal medium (24)
containing 100-µg/ml concentrations each of methionine and proline
and 10 µg of thiamine/ml (medium B). Most of the experiments were
performed with medium B. Spontaneous kanamycin-resistant mutants were
isolated on 1.5% agar plates of Luria-Bertani medium containing 20 µg of kanamycin/ml by incubating the plates overnight at 37°C. The
plasmid pPI5, containing the oppA gene located at 27 min of
the E. coli chromosome, was prepared as described previously
(17). E. coli cells containing pPI5 were cultured
in the presence of 30 µg of chloramphenicol/ml to maintain the
plasmid in E. coli cells. Cell growth was monitored by
measuring the A540.
Determination of the nucleotide sequence of the oppA
gene and measurement of the levels of OppA mRNA and OppA protein.
The oppA gene was amplified by PCR with
5'-GGGGAATTCGCCACATCATAATCC-3' (sequence for positions 
570
to 553 of the oppA gene, containing the EcoRI
site) as 5'-end primer and
5'-GGGGTCGACACTCCTGCCCCACG-3' (complementary sequence
for positions 1657 to 1641 of the oppA gene, containing the
SalI site) as 3'-end primer. The nucleotide sequence of the
2.2-kb oppA gene was determined by the dideoxy method of
Sanger et al. (25). Determination of the transcription initiation site, dot blot analysis of OppA mRNA, and Western blot analysis of OppA were performed as described previously (12, 17) with E. coli cells harvested at an
A540 of 0.3.
Measurement of OppA synthesis by an immunoprecipitation method.
E. coli cells were grown in medium B in which the methionine
content was decreased from 100 to 10 µg/ml. When the
A540 reached 0.2, [35S]methionine
(1 MBq) was added to each 5-ml aliquot and cells were allowed to grow
for 20 min. The amount of OppA synthesized was determined as described
previously (12) by using 1,000,000 cpm of
[35S]methionine-labeled protein and antibody against
OppA. Radioactivity of labeled OppA was quantified with a Fujix Bas
2000 II imaging analyzer.
Measurement of isepamicin uptake and polyphenylalanine
synthesis.
Determination of isepamicin uptake by intact cells was
performed with 30 µM [14C]isepamicin (300 MBq/mmol) as
described previously (15). Poly(U)-directed polyphenylalanine synthetic activity of ribosomes was measured in
accordance with a previously published protocol (26).
Measurement of polyamine and amino acid contents.
Polyamine
and amino acid contents were determined with a Toyo Soda
high-performance liquid chromatography system (11) and a
Hitachi 835-10 amino acid analyzer (14), using the
trichloroacetic acid supernatant of E. coli. Protein levels
were determined by the method of Lowry et al. (19).
Properties of spontaneous kanamycin-resistant mutants of E. coli.
Twenty spontaneous kanamycin-resistant colonies were
isolated by culturing 108 E. coli J53 cells on a
1.5% agar plate containing 20 µg of kanamycin/ml. The amount of OppA
was then determined by Western blot analysis using these 20 colonies.
Colonies were classified into three groups: colonies having a normal
amount of OppA (n = 12), colonies having 60 to 70%
less OppA than the parent strain (n = 7), and colonies having no detectable OppA (n = 1). Since we were
interested in the relationship between kanamycin resistance and its
transport, the properties of mutants in the second and third groups
were examined. The mutants from the second and third groups were termed m1 and m2, respectively.
The structure of the oppA gene in the m1 and m2 mutants
was determined from their nucleotide sequences (Fig.
1A). The synthesis of OppA mRNA in
E. coli J53, m1, and m2 started at 266 nucleotides upstream from the initiation codon AUG. The sequence in the m1 mutant
was the same as that in the parent strain, J53. Two mutations were
observed in the nucleotide sequence of the oppA gene in the m2 mutant. Those were at positions 368 (A to G) and 460 (A insertion) (Fig. 1A). Thus, a termination codon appeared at amino acid position 166 of OppA.
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FIG. 1.
Gene structure of oppA gene (A), OppA mRNA
level (B), OppA protein level (C), and the rate of OppA synthesis (D)
in E. coli J53, m1, and m2. The gene structure shown in
panel A was constructed from the nucleotide sequences of
oppA. TI, transcriptional initiation site. Shading in the
open reading frame of the m2 mutant indicates encoded amino acids
different from those of parent strain J53 due to the "A" insertion
at position 460. Experiments were performed as described in the text.
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|
The level of OppA mRNA was next measured by dot blot analysis. As shown
in Fig. 1B, the level of OppA mRNA in the m1 mutant was the same as
that of the parent strain, but only a small amount of OppA mRNA existed
in the m2 mutant. The amount of OppA was measured by Western blot
analysis. As shown in Fig. 1C, the amount of OppA in the m1 mutant was
about one-third of that in the parent strain. OppA was not found in the
m2 mutant. The amount of OppA was proportional to OppA synthesis (Fig.
1D).
The degree of kanamycin resistance was determined by adding various
concentrations of kanamycin to medium B. Cell growth was
followed by
measuring the
A540. As shown in Fig.
2, the growth
of the parent strain was
inhibited strongly by 1 µg of kanamycin/ml,
while the growth of the
m1 and m2 mutants was resistant to 10-
and 100-µg/ml concentrations
of kanamycin, respectively (Fig.
2A, C, and E). The mutants transformed
with the
oppA gene exhibited
greatly increased sensitivity,
but the parent strain transformed
with the
oppA gene showed
only slightly increased sensitivity
(Fig.
2). The growth of the m1 and
m2 mutants was completely inhibited
by 10- and 100-µg/ml
concentrations of kanamycin, respectively.
These results indicate that
OppA, a component of the active transport
system of oligopeptides, is
partly involved in the sensitivity
to kanamycin.
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FIG. 2.
Effect of kanamycin on growth of E. coli J53,
m1, and m2. E. coli cells were transformed with either
pACYC184 vector (A, C, E) or pPI5 containing the oppA gene
(B, D, F). The concentrations of kanamycin (Km) used are shown in the
figure. Each value is the average of duplicate determinations.
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|
The sensitivity of the m1 and m2 mutants to other aminoglycoside
antibiotics was next examined. As shown in Fig.
3, the m1
and m2 mutants were resistant
to streptomycin, neomycin, and isepamicin.
The degree of resistance was
much greater in the m2 mutant than
in the m1 mutant, similar to the
degree of resistance seen with
kanamycin. It has been reported that
triornithine is taken up
by the oligopeptide transport system and cell
growth is inhibited
due to its accumulation (
20). Cell
growth of the m1 and m2 mutants
was resistant to triornithine by two-
to threefold (data not shown).
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FIG. 3.
Effect of aminoglycoside antibiotics on growth of
E. coli J53 (A), m1 (B), and m2 (C).  , no antibiotic;
 , streptomycin (10 µg/ml);  , streptomycin (30 µg/ml);  ,
neomycin (3 µg/ml);  , neomycin (10 µg/ml); ×, neomycin (30 µg/ml);  , isepamicin (3 µg/ml);  , isepamicin (10 µg/ml); +,
isepamicin (30 µg/ml). Each value is the average of duplicate
determinations.
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|
The aminoglycoside transport activity of the m1 and m2 mutants was
measured with [
14C]isepamicin. As shown in Fig.
4A, the activity of the two mutants
was
much lower than that of the parent strain. Protein synthetic
activity
of ribosomes from the mutants was then measured, since
ribosomal
mutation is also involved in the resistance to aminoglycoside
antibiotics (
4,
22). As shown in Fig.
4B, protein synthetic
activity of ribosomes from the m2 mutant was more resistant to
kanamycin than that of ribosomes from the parent strain and the
m1
mutant. These results suggest that the m2 mutant is a double
mutant of
the
oppA gene and a gene for ribosomal protein, which
explains the strong resistance to aminoglycoside antibiotics.
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FIG. 4.
Isepamicin (ISP) uptake activity (A) and
polyphenylalanine synthetic activity of ribosomes (B) of E. coli J53, m1, and m2. Experiments were performed as described in
the text with E. coli cells harvested at
A540 = 0.3. Each value is the average of
duplicate determinations.
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|
We next investigated why the amount of OppA is smaller in the m1 mutant
than in the parent strain. The amount of OppA mRNA
in the m1 mutant was
almost equal to that in the parent strain
(Fig.
1B), suggesting that
OppA synthesis is negatively regulated
at the posttranscriptional level
in the m1 mutant. Synthesis of
OppA was measured by immunoprecipitation
of [
35S]methionine-labeled protein with antibody against
OppA (
12).
The rate of OppA synthesis in the m1 mutant was
about one-third
of that in the parent strain (Fig.
1D), suggesting that
decrease
in OppA in the m1 mutant is due to a decreased rate of OppA
synthesis.
It has been reported that OppA synthesis is greatly stimulated by
polyamines at the translational level (
12,
17). Thus,
the
amounts of polyamines and amino acids were measured. As shown
in Fig.
5A, the level of putrescine was lower in
the m1 mutant
than in the parent strain, J53. The levels of most amino
acids
were very similar in J53 and the m1 mutant, but the content of
ornithine was much lower in the m1 mutant than in J53 (Fig.
5B).
The
results suggest that decrease in polyamines due to the change
of
ornithine metabolism may be one of the reasons for the decrease
in OppA
synthesis.
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FIG. 5.
Polyamine (A) and amino acid (B) contents in E. coli J53 and m1. Experiments were performed as described in the
text with E. coli cells harvested at
A540 = 0.3. Each value is the average of
duplicate determinations.
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|
The results, taken together, indicate that the oligopeptide transport
system is important for the uptake of aminoglycoside
antibiotics, since
a decrease in OppA was observed in about 40%
of spontaneous kanamycin
(aminoglycoside)-resistant mutants. However,
it remains to be clarified
which gene is mutated in the m1 mutant.
| |
ACKNOWLEDGMENTS |
We thank A. J. Michael and K. Williams for their kind
suggestions and help in preparing the manuscript. Thanks are also due to F. Ikegami and S. Yamaji for the operation of the amino acid analyzer and for kindly supplying isepamicin.
This work was supported in part by a grant-in-aid for Scientific
Research from the Ministry of Education, Science, Sports and Culture,
Japan.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Faculty of
Pharmaceutical Sciences, Chiba University, 1-33 Yayoi-cho, Inage-ku,
Chiba 263-8522, Japan. Phone: 81-43-290-2897. Fax: 81-43-290-2900. E-mail: iga16077{at}p.chiba-u.ac.jp.
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Journal of Bacteriology, October 1998, p. 5484-5488, Vol. 180, No. 20
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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