![[Feedback]](/icons/banner/feedbackACT.gif)
![[For Subscribers]](/icons/banner/subscriberACT.gif)
![[Archive]](/icons/banner/archiveACT.gif)
![[Search]](/icons/banner/searchACT.gif)
![[Contents]](/icons/banner/tocACT.gif)
Previous Article | Next Article 
Antimicrobial Agents and Chemotherapy, December 2001, p. 3610-3612, Vol. 45, No. 12
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.12.3610-3612.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Chloramphenicol-Sensitive Escherichia
coli Strain Expressing the Chloramphenicol Acetyltransferase
(cat) Gene
Joanna
Potrykus1 and
Grzegorz
Wegrzyn1,2,*
Department of Molecular Biology, University
of Gdañsk, 80-822 Gdañsk,1 and
Marine Biology Center, Polish Academy of Sciences, 81-347 Gdynia,2 Poland
Received 18 April 2001/Returned for modification 16 July
2001/Accepted 30 August 2001
 |
ABSTRACT |
An Escherichia coli strain (strain CM2555) bearing
the chloramphenicol acetyltransferase (cat) gene was
found to be sensitive to chloramphenicol. We demonstrate that the
cat gene is efficiently expressed in strain CM2555. Our
results suggest that decreased levels of acetyl coenzyme A in
cat-expressing CM2555 cells in the presence of
chloramphenicol may cause the bacterium to be sensitive to this antibiotic.
| |
TEXT |
One of the best-characterized
bacterial antibiotic resistance mechanisms is the synthesis of
chloramphenicol acetyltransferase (CAT) (3, 9, 12, 23).
Production of this enzyme, encoded by the cat gene,
is the most common means by which bacteria become resistant to
chloramphenicol (13, 22), a small bacteriostatic antibiotic that interacts with a peptidyl transferase center
(16). CAT catalyzes transfer of the acetyl moiety from
acetyl coenzyme A (acetyl-CoA) to a chloramphenicol molecule (8,
12). Thus modified, chloramphenicol no longer binds to the
ribosomes and protein synthesis proceeds (12).
Recently, an Escherichia coli strain, strain CM2555, was
identified in which no transformants were selected when chloramphenicol was used as the selective agent during attempts to introduce plasmids carrying the cat gene (21).
cat-bearing strain CM2555 was found to be sensitive to
chloramphenicol (21). The aim of the work described here
was to investigate the mechanism of this phenomemon.
The bacterial strains and plasmids used in this work are listed
in Table 1.
For Western blotting, after sodium dodecyl sulfate (SDS)-polyacrylamide
gel electrophoresis (PAGE) with 12.5% polyacrylamide gels, or PAGE the
proteins were transferred onto a nitrocellulose membrane (Bio-Rad) with
a semidry blot apparatus (Schleicher & Schuell). The CAT protein was
visualized with a specific anti-CAT serum (Sigma Aldrich) with alkaline
phosphatase-conjugated secondary antibodies as described previously
(20). The relative amount of CAT was estimated by densitometry.
Preparation of crude cellular extracts and spectrophotometric
measurements of CAT biochemical activity were performed as described previously (11). The intracellular concentration of
acetyl-CoA was determined spectrophotometrically as described earlier
(10).
The efficiency of protein synthesis was estimated by measurement of the
level of incorporation of radioactive precursors into trichloroacetic
acid-precipitable material as described previously (18),
but 14C-labeled amino acids (UVVVR) were added to
bacterial cultures to a final concentration of 2.5 µCi/ml.
E. coli CM2555 has a genetic defect that leads to
its chloramphenicol sensitivity in the presence of
cat.
Strain CM2555 has been described as one of the
series of ilv+ dnaA mutants
(CM2555 bears the dnaA508 allele) otherwise isogenic to
strain CM732 (4). However, using a set of bacterial
strains and plasmids (Table 1), we found that the chloramphenicol
sensitivity of strain CM2555 bearing the cat gene is not
caused by the dnaA508 allele or the combination of
ilv+ and dnaA508 alleles (data
not shown). Thus, we conclude that strain CM2555 must contain another,
previously unidentified mutation(s) which makes it sensitive to
chloramphenicol, despite the presence of the cat gene.
Expression of cat gene in strain CM2555.
Densitometric analyses of electropherograms after SDS-PAGE and of
Western blots with anti-CAT serum revealed that CM2555/pBR328 produces
at least three times more CAT than the parental strain, CM732/pBR328
(data not shown). To test whether an increased level of CAT plays a
role in the mechanisms of the chloramphenicol sensitivity of CM2555, we
used plasmid pJKP1 bearing the cat gene under control of an
inducible promoter, plac, together with
another plasmid, pJMH1, harboring the lacIq
allele. Addition of
isopropyl-
-D-thiogalactopyranoside (IPTG) to a final concentration of 1 mM resulted in CAT levels (in both CM2555
and CM732 hosts) that corresponded to about 90% of that found in
CM732/pBR328 (data not shown). Contrary to the results obtained in
control experiments, the increased efficiency of cat expression correlated with the faster growth of strain
CM2555/pJKP1/pJMH1 in a chloramphenicol-containing medium only up to
IPTG concentrations of 0.1 mM, and a further increase in the efficiency
of cat expression resulted in the inhibition of bacterial
growth (Fig. 1). Therefore, we suggest
that the efficiency of expression of the cat gene may be
important for the chloramphenicol sensitivity of strain CM2555.
View larger version (25K):
[in this window]
[in a new window]
|
FIG. 1.
Effect of intracellular amount of CAT on bacterial
growth in the presence of chloramphenicol. E. coli CM732
and CM2555 harboring pJKP1 and pJMH1 were grown in Luria-Bertani
liquid medium. cat gene expression from the
plac-cat fusion was induced
overnight, and then in the course of the experiment expression was
maintained by the presence of various amounts of IPTG.
Chloramphenicol was added when the optical density at 575 nm was
0.1. Doubling times were determined by monitoring bacterial growth
(optical density at 575 nm) in the presence and absence of
chloramphenicol (34 µg/ml). Symbols: open circles, CM732/pJKP1/pJMH1
grown without chloramphenicol; closed circles, CM732/pJKP1/pJMH1 grown
in the presence of chloramphenicol; open rectangles, CM2555/pJKP1/pJMH1
grown without chloramphenicol; closed rectangles, CM2555/pJKP1/pJMH1
grown in the presence of chloramphenicol.
|
|
Activity of CAT protein in strain CM2555.
We measured the
acetylation of chloramphenicol and calculated that the total CAT
activity per 1 mg of cellular proteins in strain CM2555/pBR328 was
higher than that in analogous samples prepared from the parental
strain, CM732/pBR328 (1.20 CAT activity units in CM2555/pBR328 versus
0.73 CAT activity units in CM732/pBR328). Moreover, CAT reveals
biological activity in both CM2555/pBR328 and CM732/pBR328, as the
addition of chloramphenicol (up to 34 µg/ml) to cultures of these
strains had no significant effect on protein synthesis efficiency (data
not shown).
Sensitivity to chloramphenicol of strain CM2555 expressing
cat results from decreased levels of acetyl-CoA.
Since CAT catalyzes the transfer of the acetyl group from acetyl-CoA to
a chloramphenicol molecule, we measured the levels of acetyl-CoA in
strain CM2555 and strain CM732 bearing plasmid pBR328 in the absence
and presence of chloramphenicol. We found that the residual level of
acetyl-CoA, without the addition of the antibiotic, was slightly lower
in CM2555/pBR328 relative to that in CM732/pBR328 (the level of
acetyl-CoA in CM2555/pBR328 was about 90% of that found in
CM732/pBR328). However, after the addition of chloramphenicol, the
level of acetyl-CoA was roughly constant in CM732/pBR328, while it
gradually decreased in CM2555/pBR328 (Fig.
2A and B).
View larger version (21K):
[in this window]
[in a new window]
|
FIG. 2.
Intracellular amount of acetyl-CoA as affected by
chloramphenicol in E. coli CM732/pBR328 (A) and
CM2555/pBR328 (B) and the effect of sodium acetate on bacterial growth
in the presence of chloramphenicol (C). In the experiments whose
results are depicted in panels A and B, strains harboring pBR328 were
grown in Luria-Bertani liquid medium. Chloramphenicol (34 µg/ml) was
added when the optical density at 575 nm was 0.1 (time zero). Samples
were withdrawn every 5 min after addition of the antibiotic and were
then assayed for acetyl-CoA. For each strain, the amount of acetyl-CoA
at time zero was taken to be 1, and the relative concentration was
calculated. In the experiments whose results are depicted in panel C,
E. coli CM732 and CM2555 harboring pBR328 were grown in
Luria-Bertani liquid medium supplemented with different amounts of
sodium acetate. Chloramphenicol was added when the optical density at
575 nm was 0.1. The doubling times of the cultures were determined by
monitoring bacterial growth (optical density at 575 nm) in the presence
and absence of chloramphenicol (34 µg/ml). The values presented are
means from three experiments. In all cases the standard deviation was
below 10%. Symbols: open circles, CM732/pBR328 grown without
chloramphenicol; closed circles, CM732/pBR328 grown in the presence of
chloramphenicol; open rectangles, CM2555/pBR328 grown without
chloramphenicol; closed rectangles, CM2555/pBR328 grown in the presence
of chloramphenicol.
|
|
To test whether a decrease in the level of acetyl-CoA in strain
CM2555/pBR328 is responsible for the sensitivity of this strain to
chloramphenicol, we measured the growth rates of bacterial cultures in
the presence of sodium acetate. Sodium acetate can be used as an
alternative source of acetyl-CoA production in cells (6).
It was reported previously (12) that
cat-expressing E. coli mutants with mutations in
aceE or aceF, which cannot make acetyl-CoA
from pyruvate but which can make acetyl-CoA from sodium acetate, are
resistant to chloramphenicol only when sodium acetate is supplied.
Thus, if the hypothesis stated above were true, we should have observed
improved growth of CM2555/pBR328 in the presence of sodium acetate.
Indeed, we found that the presence of sodium acetate dramatically
improved the growth of CM2555/pBR328 in the medium containing
chloramphenicol (Fig. 2C). We also found that strain CM2555 could be
transformed by different plasmids bearing the cat gene when
selection was performed on plates containing both chloramphenicol (34 µg/ml) and 0.15% sodium acetate (data not shown). Neither the pBR328
copy number nor the amount of CAT in CM2555 cells was affected by the
presence of sodium acetate in the medium (data not shown). These
results strongly support the hypothesis that decreased levels of
acetyl-CoA in cat-expressing CM2555 cells in the presence of
chloramphenicol result in the sensitivity of the bacterium to this antibiotic.
| |
ACKNOWLEDGMENTS |
We are very grateful to S. Barañska and A. Szalewska-Palasz for assistance at the stage of preliminary
experiments and to J. Davies for discussions and support at the
beginning of this project.
This work was supported by the Polish State Committee for Scientific
Research (project 6 P04A 060 18). G.W. also acknowledges financial
support from the Foundation for Polish Science (subsidy 14/2000).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Molecular Biology, University of Gdañsk, K
adki 24, 80-822 Gdañsk, Poland. Phone: 48 (58) 346 3014. Fax: 48 (58) 301 0072. E-mail: wegrzyn{at}biotech.univ.gda.pl.
| |
REFERENCES |
| 1.
|
Bolivar, F., and K. Backman.
1979.
Plasmids of Escherichia coli as cloning vectors.
Methods Enzymol.
68:245-257[Medline].
|
| 2.
|
Chang, C. Y., and S. N. Cohen.
1978.
Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the p15A cryptic miniplasmid.
J. Bacteriol.
134:1141-1156[Abstract/Free Full Text].
|
| 3.
|
Davies, J.
1994.
Inactivation of antibiotics and the dissemination of resistance genes.
Science
264:375-382[Abstract/Free Full Text].
|
| 4.
|
Hansen, F. G., and K. von Meyenburg.
1979.
Characterization of the dnaA, gyrB, and other genes in the dnaA region of the Escherichia coli chromosome on specialized transducing phages lambda-tna.
Mol. Gen. Genet.
175:135-144[CrossRef][Medline].
|
| 5.
|
Jensen, K. F.
1993.
The Escherichia coli "wild types" W3110 and MG1655 have rph frame shift mutation that leads to pyrimidine starvation due to low pyrE expression levels.
J. Bacteriol.
175:3401-3407[Abstract/Free Full Text].
|
| 6.
|
Jones-Mortimer, M. C., and H. L. Kornberg.
1977.
The enzymic interconversion of acetate and acetyl-coenzyme A in Escherichia coli.
J. Gen. Microbiol.
102:327-336[Medline].
|
| 7.
|
Kedzierska, S.,
M. Staniszewska,
J. Potrykus, and G. Wegrzyn.
1999.
The effects of some antibiotic-resistance-conferring plasmids on the removal of heat-aggregated proteins from Escherichia coli.
FEMS Microbiol. Lett.
176:279-284[CrossRef][Medline].
|
| 8.
|
Lewendon, A., and W. V. Shaw.
1993.
Transition state stabilization by chloramphenicol acetyltransferase.
J. Biol. Chem.
268:20997-21001[Abstract/Free Full Text].
|
| 9.
|
Nikaido, H.
1994.
Prevention of drug access to bacterial targets: permeability barriers and active efflux.
Science
264:382-388[Abstract/Free Full Text].
|
| 10.
|
Prü, B. M., and A. J. Wolfe.
1994.
Regulation of acetyl phosphate synthesis and degradation, and the control of flagellar expression in Escherichia coli.
Mol. Microbiol.
12:973-984[Medline].
|
| 11.
|
Shaw, W. V.
1975.
Chloramphenicol acetyltransferase from chloramphenicol-resistant bacteria.
Methods Enzymol.
43:737-755[Medline].
|
| 12.
|
Shaw, W. V.
1983.
Chloramphenicol acetyltransferase: enzymology and molecular biology.
Crit. Rev. Biochem.
14:1-46[Medline].
|
| 13.
|
Shaw, W. V.,
L. C. Packman,
B. D. Burleigh,
A. Dell,
H. R. Morris, and B. S. Hartley.
1979.
Primary structure of a chloramphenicol acetyltransferase specified by R plasmids.
Nature
282:870-872[CrossRef][Medline].
|
| 14.
|
Silhavy, T. J.,
M. L. Berman, and L. W. Enquist.
1984.
Experiments with gene fusions.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
|
| 15.
|
Singer, M.,
T. A. Baker,
G. Schnitzler,
S. M. Deischel,
M. Goel,
W. Dove,
K. J. Jaacks,
A. D. Grossman,
J. W. Erickson, and C. A. Gross.
1989.
A collection of strains containing genetically linked alternating antibiotic resistance elements for genetic mapping of Escherichia coli.
Microbiol. Rev.
53:1-24[Abstract/Free Full Text].
|
| 16.
|
Vester, B., and R. A. Garret.
1988.
The importance of highly conserved nucleotides in the binding region of chloramphenicol at the peptidyl transfer center of Escherichia coli 23S ribosomal RNA.
EMBO J.
7:3577-3587[Medline].
|
| 17.
|
Vieira, J., and J. Messing.
1988.
The pUC plasmids, an M13mp7-derived system for insertional mutagenesis and sequencing with synthetic universal primers.
Gene
19:259-268.
|
| 18.
|
Wegrzyn, G.,
E. Kwasnik, and K. Taylor.
1991.
Replication of  plasmid in amino acid-starved strains of Escherichia coli.
Acta Biochim. Pol.
38:181-186[Medline].
|
| 19.
|
Wegrzyn, G.,
R. E. Glass, and M. S. Thomas.
1992.
Involvement of the Escherichia coli RNA polymerase  subunit in transcriptional activation by the bacteriophage CI and CII proteins.
Gene
122:1-7[CrossRef][Medline].
|
| 20.
|
Wegrzyn, A.,
G. Wegrzyn, and K. Taylor.
1995.
Protection of coliphage O initiator protein from proteolysis in the assembly of the replication complex in vivo.
Virology
207:179-184[CrossRef][Medline].
|
| 21.
|
Wegrzyn, G.,
A. Wegrzyn,
A. Pankiewicz, and K. Taylor.
1996.
Allele specificity of the Escherichia coli dnaA gene function in the replication of plasmids derived from phage .
Mol. Gen. Genet.
252:580-586[Medline].
|
| 22.
|
Zaidenzaig, Y.,
J. E. Fitton,
L. C. Packman, and W. V. Shaw.
1979.
Characterization and comparison of chloramphenicol acetyltransferase variants.
Eur. J. Biochem.
100:609-618[Medline].
|
| 23.
|
Zgurskaya, H. I., and H. Nikaido.
2000.
Multidrug resistance mechanisms: drug efflux across two membranes.
Mol. Microbiol.
37:219-225[CrossRef][Medline].
|
Antimicrobial Agents and Chemotherapy, December 2001, p. 3610-3612, Vol. 45, No. 12
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.12.3610-3612.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Top
Abstract
Text
