Characterization of the fomA andfomB Gene Products from Streptomyces wedmorensis, Which Confer Fosfomycin Resistance on Escherichia 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. colistrains were grown at 37°C. Strains and plasmids used are listed in Table 1.

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 andfomB 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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Fig. 1.

Fosfomycin biosynthetic gene cluster and deletion plasmids used to determine the ORFs necessary to confer fosfomycin resistance on E. coli HB101. Abbreviations for restriction sites: Ba, BamHI; Bg, BglII; E, EcoRI; P, PstI; S, SphI. Arrows indicate the direction of transcription from the lac promoter of pUC118 or pUC119 with respect to the inserted fragments.

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 offomA and fomB genes from S. wedmorensis, two pairs of oligonucleotide primers, 5′-GGGGGATCCACGCCCGATTTCTTGGCC-3′ (5′ of thefomA gene) plus 5′-GGGGGATCCCGCAGAAGCAGTCGTGGTG-3′ (3′ of thefomA gene) and 5′-GGGGGATCCCTGGAAAACCTCACGATCCGC-3′ (5′ of thefomB gene) plus 5′-GGGGGATCCTTCGGCAAGCTGCTTGAGCGTC-3′ (3′ of thefomB gene), including BamHI restriction sites (underlined), were synthesized (Amersham Pharmacia, Uppsala, Sweden). Then, these pairs of primers were used with pFBG1204 to amplify thefomA 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 orfomB 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-β-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 thefomA 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 K_m 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 K_m 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 K_m 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 K_m 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).