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Antimicrobial Agents and Chemotherapy, March 2000, p. 647-650, Vol. 44, No. 3
Institute of Molecular and Cellular
Biosciences, University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
Received 11 August 1999/Returned for modification 9 November
1999/Accepted 16 December 1999
Together, the fomA and fomB genes in the
fosfomycin biosynthetic gene cluster of Streptomyces
wedmorensis confer high-level fosfomycin resistance on
Escherichia coli. To elucidate their functions, the
fomA and fomB genes were overexpressed in
E. coli and the gene products were characterized. The
recombinant FomA protein converted fosfomycin to fosfomycin
monophosphate, which was inactive on E. coli, in the
presence of a magnesium ion and ATP. On the other hand, the recombinant
FomB protein did not inactivate fosfomycin. However, a reaction mixture
containing FomA and FomB proteins converted fosfomycin to fosfomycin
monophosphate and fosfomycin diphosphate in the presence of ATP and a
magnesium ion, indicating that FomA and FomB catalyzed phosphorylations of fosfomycin and fosfomycin monophosphate, respectively. These results
suggest that the self-resistance mechanism of the fosfomycin-producing organism S. wedmorensis is mono- and diphosphorylation of
the phosphonate function of fosfomycin catalyzed by FomA and FomB.
Fosfomycin is a medically important
antibiotic produced by various species of Streptomyces
(4), by Pseudomonas syringae (14), and
by Pseudomonas viridiflava (9). It possesses
unique structural features, including a carbon-phosphorus bond and an epoxide. As an analog of phosphoenolpyruvate (PEP), this compound irreversibly inhibits PEP
UDP-N-acetylglucosamine-3-O-enolpyruvyltransferase (enolpyruvyltransferase), which catalyzes the first step of
peptidoglycan biosynthesis (8). Fosfomycin does not inhibit,
however, other enzymes using PEP and shows almost no toxicity in humans
(1, 7).
Several clinical isolates resistant to fosfomycin had been found to
have fosfomycin resistance genes on plasmids, such as fosA
from Serratia marcescens (12) and fosB
from Staphylococcus epidermidis (15). Both gene
products catalyzed the irreversible addition of glutathione to
fosfomycin (12). The deduced amino acid sequences of these
gene products exhibit 36.6% identity to each other. On the other hand,
to the best of our knowledge, the only precedent for self-resistance
genes in the fosfomycin-producing organisms was fosC, found
in P. syringae PB-5123 by Garcia et al. (2). They
showed that its product inactivated fosfomycin in the presence of ATP.
The inactivated products, however, were not well characterized. They
also demonstrated that this inactivation was reversed by subsequent
treatment with alkaline phosphatase.
Members of our group have cloned the fosfomycin biosynthetic gene
cluster of Streptomyces wedmorensis (11) and
identified four fosfomycin biosynthetic genes, fom1 to
fom4 (6). In addition to these genes, encoding
the enzymes necessary for fosfomycin biosynthesis, six open reading
frames (ORFs), orfA to orfF, with unknown
functions were found on a sequenced fragment including the fosfomycin
gene cluster. Among these ORFs, orfA and orfB, contained together in a 2.9-kb fragment, gave Escherichia
coli a high-level of resistance to fosfomycin, and fosfomycin was
converted to inactivated forms in the presence of ATP by a crude
extract from this transformant (10). Subsequently we
isolated the inactivated compounds and determined their structures to
be fosfomycin monophosphate and fosfomycin diphosphate. The genes
orfA and orfB were thus renamed fomA
and fomB, respectively. However, individual functions of the
fomA and fomB gene products remained to be studied.
We show here that the fomA gene confers fosfomycin
resistance on E. coli and that the fomA and
fomB gene products catalyze phosphorylations of fosfomycin
and fosfomycin monophosphate, respectively. We also describe detailed
characterization of the purified recombinant fomA and
fomB gene products overexpressed in E. coli.
Growth media.
Luria-Bertani (LB) medium containing 50 µg
of ampicillin per ml was used for pregrowth and growth of E. coli HB101 harboring pFBG1204, pFBG2204, pFBG1216, pFBG1221, or
pUC118. The pregrowth medium for E. coli K-12 strain HW8235
was nutrient broth (Eiken, Tokyo, Japan). All E. coli
strains were grown at 37°C. Strains and plasmids used are listed in
Table 1.
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Characterization of the fomA and
fomB Gene Products from Streptomyces wedmorensis,
Which Confer Fosfomycin Resistance on Escherichia
coli
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Properties of bacterial strains and plasmids
Identification of fosfomycin resistance gene(s). In order to identify the resistance gene(s), DNA fragments derived from the 12.1-kb fragment containing fosfomycin biosynthetic genes were inserted downstream of the lacZ promoter in pUC118 or pUC119 and then introduced into E. coli HB101 as described by Hanahan (3). Each transformant mixture was spread on LB agar plates containing 50 µg of ampicillin per ml and 400 µg of fosfomycin per ml and then incubated at 37°C for 24 h.
Plasmid constructions.
Deletion plasmids, pFBG1216 and
pFBG1221, derived from pFBG1204 (which includes the fomA and
fomB genes) (Fig. 1) were
constructed by using exonuclease III and mung bean nucleases as
described by Henikoff (5). These deletion plasmids were
introduced into E. coli HB101 as described by Hanahan
(3) for further experiments.
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Determination of MICs of fosfomycin. E. coli HB101 harboring one of the plasmids mentioned above (pFBG1204, pFBG2204, pFBG1216, pFBG1221, and pUC118) was grown in LB medium containing 50 µg of ampicillin per ml with or without 6.25 to 800 µg/ml of fosfomycin at 37°C. Growth of each strain was measured by optical density at 660 nm.
DNA sequencing.
DNA sequencing was carried out using a DNA
sequencer (model 4000L; Licor, Lincoln, Nebr.). Sequencing reactions
were made with a Thermo Sequenase cycle sequencing kit (U.S.
Biochemical Corporation, Cleveland, Ohio) using infrared-dye-labeled
M13 forward (
29) primer (Nisshinbo, Tokyo, Japan) or
infrared-dye-labeled M13 reverse primer (Nisshinbo).
Protein expression and purification of recombinant FomA and FomB. On the basis of the entire nucleotide sequences of fomA and fomB genes from S. wedmorensis, two pairs of oligonucleotide primers, 5'-GGGGGATCCACGCCCGATTTCTTGGCC-3' (5' of the fomA gene) plus 5'-GGGGGATCCCGCAGAAGCAGTCGTGGTG-3' (3' of the fomA gene) and 5'-GGGGGATCCCTGGAAAACCTCACGATCCGC-3' (5' of the fomB gene) plus 5'-GGGGGATCCTTCGGCAAGCTGCTTGAGCGTC-3' (3' of the fomB gene), including BamHI restriction sites (underlined), were synthesized (Amersham Pharmacia, Uppsala, Sweden). Then, these pairs of primers were used with pFBG1204 to amplify the fomA or fomB gene. By using Taq DNA polymerase (Boehringer, Mannheim, Germany) and a standard PCR protocol, an 813-bp fragment for the fomA gene or a 1,009-bp fragment for the fomB gene was amplified. These PCR fragments were cleaved with BamHI and cloned into pUC118 (Takara Shuzo, Kyoto, Japan). The sequences of the cloned DNA fragments were confirmed with a Licor DNA sequencer. The coding region of the fomA or fomB gene was cloned into the expression vector pQE30 (Qiagen, Hilden, Germany) to yield pQEFA or pQEFB, respectively.
E. coli M15 containing pREP4 (Qiagen) was used as a host for expression of FomA. E. coli (pREP4) harboring pQEFA was cultured at 37°C in 100 ml of LB medium containing 25 µg of kanamycin per ml and 200 µg of ampicillin per ml for 5 h with 2 mM isopropyl-
-D-thiogalactopyranoside. Upon reaching an
optical density at 660 nm of 0.8, cells were harvested by
centrifugation and resuspended in 100 mM Tris-HCl (pH 7.5). After 5 min
of sonication, the lysate was centrifuged at 10,000 × g for 20 min and the supernatant was collected. The supernatant
was applied to a nickel-nitrilotriacetic acid (Ni-NTA)-agarose resin
(Qiagen) column (7.5 by 10 mm). After a washing with 100 mM Tris-HCl
buffer (pH 7.5) containing 50 mM imidazole, the protein which had bound
to the Ni-NTA agarose resin was eluted with 100 mM Tris-HCl buffer (pH
7.5) containing 100 mM imidazole. The eluate was used as purified
recombinant FomA in subsequent experiments. Expression and purification
of recombinant FomB were done in a way similar to that for recombinant
FomA, with replacement of pQEFA by pQEFB.
Identification of reaction products synthesized by the fomA or fomB gene product. A reaction mixture containing the recombinant FomA protein, 5.4 mM ATP, 10 mM MgCl2, 6 mM KCl, and 5.5 mM fosfomycin was made up to 5 ml and incubated at 30°C for 3 h. One equivalent of methanol was added to the reaction mixture to stop the reaction, followed by centrifugation (15,000 × g, 10 min) to remove insoluble materials. The amount of fosfomycin remaining in the supernatant was quantitatively determined by bioassay against E. coli K-12 strain HW8235 (Meiji Seika Kaisha Ltd., Tokyo, Japan).
The supernatant (10 ml) was passed through an activated carbon column (15 by 27 mm) and, after dilution to 20 ml with H2O, subjected to Dowex 1-X8 (Cl
type, 15 by 55 mm)
chromatography with elution with 2.0% NaCl (50 ml). The eluate was
diluted twofold with water and lyophilized. After being dissolved in
H2O, the sample was analyzed by 31P nuclear
magnetic resonance (NMR) (JEOL [Tokyo, Japan] A-500 instrument).
Phosphorylation of fosfomycin monophosphate by FomB was done in a way
similar to that for phosphorylation by FomA, with replacement of
fosfomycin by 5 mM fosfomycin monophosphate.
Assay for FomA and FomB activities. FomA and FomB activities were measured by a spectrophotometric method. The reaction mixture for the fosfomycin phosphorylation by FomA contained 100 mM sodium phosphate buffer (pH 7.0), 10 mM MgCl2, 10 mM dithiothreitol, 0.5 mM NADH, 1 mM PEP, 4.2 U of pyruvate kinase, 6.6 U of lactate dehydrogenase, and several concentrations of fosfomycin and ATP. The final volume of the reaction mixture was 200 µl. The FomA enzyme assay was conducted at 37°C on a Benchmark microplate reader (Bio-Rad). The background rate of NADH oxidation was measured for the reaction mixture without fosfomycin or ATP. The reaction rate was measured for 10 min. One unit of FomA protein was defined as the amount of protein required to inactivate 1 µmol of fosfomycin per min.
The effect of temperature on the enzymatic activity was investigated over the range of 20 to 50°C. The effect of pH on the enzymatic activity was investigated over the ranges of pH 5.5 to 7.0 with morpholineethanesulfonic acid (MES)-NaOH buffer, pH 6.8 to 7.8 with sodium phosphate buffer, and pH 7.0 to 9.0 with Tris-HCl buffer. The Km for fosfomycin was determined using an enzyme assay system containing 4 mM Na2ATP2,
fosfomycin concentrations between 20 and 200 µM, and 10 µg of purified recombinant FomA. Data were analyzed on a Lineweaver-Burk plot, and the slope and x intercept were determined using a
linear-regression computer program.
The Km for Na2ATP2 was
determined using an enzyme assay system containing 4 mM fosfomycin,
Na2ATP2 concentrations between 20 and 200 µM, and 10 µg of purified recombinant FomA. Data were analyzed as
stated above for fosfomycin.
The reaction mixture for the FomB activity assay contained 100 mM
sodium phosphate buffer (pH 7.0), 10 mM MgCl2, 10 mM
dithiothreitol, 0.5 mM NADH, 1 mM PEP, 4.2 U of pyruvate kinase, 6.6 U
of lactate dehydrogenase, and several concentrations of fosfomycin
monophosphate (40 to 1,000 µM) and ATP (40 to 1,000 µM). The final
volume of the reaction mixture was 200 µl. Enzyme assays were
conducted in the same way as for FomA. The background rate of NADH
oxidation was measured for the reaction mixture without fosfomycin
monophosphate or ATP. The reaction rate was measured for 10 min. One
unit of FomB protein was defined as the amount of protein required to produce 1 µmol of fosfomycin diphosphate per min.
The Km for fosfomycin monophosphate was
determined using an enzyme assay system containing 4 mM
Na2ATP2, fosfomycin monophosphate
concentrations between 40 and 1,000 µM, and 5 µg of purified
recombinant FomB. Data were analyzed as stated above for FomA.
The Km for Na2ATP2 was
determined using an enzyme assay system containing 2 mM fosfomycin,
Na2ATP2 concentrations between 40 and 1,000 µM, and 5 µg of purified recombinant FomB. Data were analyzed as
for fosfomycin monophosphate.
Nucleotide sequence accession number. The DNA sequence of the fomA gene has been corrected (one nucleotide). The updated version is available in DDBJ/EMBL/GenBank under both the original accession number (D38561) and a new one (AB016934).
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Identification of the fosfomycin resistance gene in the biosynthetic gene cluster. Among tested plasmids, only pFBG1204 conferred fosfomycin resistance (>800 µg/ml) on E. coli. On the other hand, pFBG2204, with the same 2.9-kb BamHI-BglII fragment as pFBG1204 inserted in the opposite orientation relative to the direction of transcription of the lacZ promoter, did not provide fosfomycin resistance. Thus, fomA and/or fomB was judged to confer fosfomycin resistance on the host under the control of the lacZ promoter.
To determine the minimum region essential for fosfomycin resistance, deletion plasmids derived from pFBG1204 were constructed and then introduced into E. coli HB101. The resulting transformants were spread on LB agar plates containing various concentrations of fosfomycin. The MICs of fosfomycin for E. coli carrying pFBG1204, pFBG2204, pFBG1216, pFBG1221, and pUC118 were >800, <6.25, >800, <6.25, and <6.25 µg/ml, respectively. As can be seen from these results, a slight loss of fomA (pFBG1221) caused the complete disappearance of resistance, showing that fomA is essential for resistance. On the other hand, the loss of fomB (pFBG1216) did not influence resistance.Individual function of the fomA and fomB
gene products.
To elucidate the fomA and
fomB gene functions, we constructed plasmids for the
overexpression of the gene products followed by a one-step purification
of the recombinant enzyme by Ni-NTA-agarose resin. Based on the total
sequence of the fomA gene deposited in DDBJ/EMBL/GenBank
under accession number D38561, we first constructed a FomA
overexpression system. The overexpressed protein, however, did not
inactivate fosfomycin under the conditions described previously
(10). We then carried out resequencing of the
fomA gene around the region corresponding to the N terminus
and found an error, an omission of one base (see Materials and
Methods). We constructed the FomA overexpression system again using
primers described in Materials and Methods. The purified recombinant
FomA and FomB proteins gave homogeneous protein bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with subunit sizes of 29 and 50 kDa, respectively (Fig.
2).
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Characterization of the fomA and fomB gene
products.
The enzymatic properties of purified recombinant FomA
are summarized along with those of FomB in Table
2. The results of SDS-PAGE and gel
filtration suggested that FomA and FomB are a tetramer and a dimer,
respectively.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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We have shown in this study that the fomA and fomB gene products phosphorylated fosfomycin to fosfomycin monophosphate and fosfomycin monophosphate to fosfomycin diphosphate, respectively. These results raise two questions about the self-resistance mechanism of the fosfomycin-producing organism. (i) How does the fosfomycin-producing organism convert phosphorylated fosfomycin into active fosfomycin and export it into the fermentation medium? (ii) Why does the fomB gene product catalyze the additional phosphorylation of biologically inactive fosfomycin monophosphate?
Several clues to answer these questions may be found in the following observations: while a large amount of fosfomycin accumulated in the fermentation medium, no phosphorylated fosfomycin was detected in S. wedmorensis cells (data not shown). In addition, phosphorylation and diphosphorylation of the phosphonate function in the fosfomycin molecule had proved to be reversible (10). A simple model for the export of fosfomycin may thus be speculated as follows. Fosfomycin biosynthesized in the cytosol is rapidly converted to biologically inactive fosfomycin monophosphate by FomA to confer self-resistance on the producing organism. Next, fosfomycin monophosphate is converted by FomB to fosfomycin diphosphate, which is an energy-rich molecule. Finally, fosfomycin diphosphate is exported into the fermentation medium as its free form, fosfomycin, by utilization of free energy liberated in the hydrolysis of the two phosphate bonds in the fosfomycin diphosphate molecule. In this model, the role of the first phosphorylation by FomA is inactivation of fosfomycin for self-resistance and that of the second phosphorylation by FomB is accumulation of free energy required to export fosfomycin diphosphate outside of the cell. This reversible self-resistance mechanism together with the efflux could be reasonable for fosfomycin-producing organisms. We are now trying to identify the phosphatase involved in the hydrolysis of fosfomycin diphosphate.
It has been reported that the fosC gene product of another fosfomycin-producing organism, P. syringae PB-5123, converted fosfomycin into an inactive compound by using ATP and that the inactive compound was reactivated by treatment with alkaline phosphatase. These results suggest that like the fomA gene product, the fosC gene product converts fosfomycin into fosfomycin monophosphate. Therefore, the fosC gene product, which seems to be fosfomycin phosphotransferase, should show similarity to the fomA gene product in amino acid sequence. However, the amino acid sequence of the fomA gene product, as corrected in this study, has only 25.8% identity to the fosC gene product. This low identity may suggest that the two fosfomycin-producing organisms, taxonomically quite different from each other, independently acquired the self-resistance mechanism against fosfomycin during evolution. On the other hand, no FomB homolog has yet been found in P. syringae PB-5123.
Suarez and Mendoza had proposed that the fosfomycin resistance genes, fosA and fosB, on plasmids isolated from clinical isolates were transferred horizontally from fosfomycin-producing organisms such as Streptomyces (13). The hypothesis was based on the following two findings. (i) The GC content ratios of the fosA and fosB genes are higher than those of the other genes on the chromosome and as high as those of actinomycetes. (ii) Most antibiotic-producing organisms have self-resistance genes.
Our finding that the self-resistance mechanism of the fosfomycin-producing S. wedmorensis is quite different from those of the clinical isolates, however, strongly indicates that clinical isolates independently acquired the fosfomycin resistance mechanism. In addition, irreversible inactivation of fosfomycin by addition of glutathione is not reasonable as the self-resistance mechanism, even for fosfomycin-producing organisms with high GC content ratios other than S. wedmorensis. The resistance mechanism of the fosfomycin-producing organisms may be found in clinical isolates in the future.
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ACKNOWLEDGMENTS |
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This work was supported in part by a Grant-in-Aid for Encouragement of Young Scientists from The Ministry of Education, Science, Sports and Culture, Japan (09760114 to T.K.), and by Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists (JSPS) to S.K.
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FOOTNOTES |
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* Corresponding author. Mailing address: Institute of Molecular and Cellular Biosciences, University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan. Phone: 81-3-5841-7839. Fax: 81-3-5841-8485. E-mail: haseto{at}imcbns.iam.u-tokyo.ac.jp.
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Antimicrobial Agents and Chemotherapy, March 2000, p. 647-650, Vol. 44, No. 3
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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(2008). Crystal Structure of Fosfomycin Resistance Kinase FomA from Streptomyces wedmorensis. J. Biol. Chem.
283: 28518-28526
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