Cloning and characterization of a novel, plasmid-encoded trimethoprim- resistant dihydrofolate reductase from Staphylococcus haemolyticus MUR313

Article abstract of DOI:10.1128/AAC.39.9.1920

In recent years resistance to the antibacterial agent trimethoprim (Tmp) has become more widespread, and several trimethoprim-resistant (Tmp(r)) dihydrofolate reductases (DHFRs) have been described from gram-negative bacteria. In staphylococci, only one Tmp(r) DHFR has been described, the type S1 DHFR, which is encoded by the dfrA gene found on transposon Tn4003. In order to investigate the coincidence of high-level Tmp resistance and the presence of dfrA, we analyzed the DNAs from various Tmp(r) staphylococci for the presence of dfrA sequences by PCR with primers specific for the thyE- dfrA genes from Tn4003. We found that 30 of 33 isolates highly resistant to Tmp (MICs, ≥512 μg/ml) contained dfrA sequences, whereas among the Tmp(r) (MICs, ≤256 μg/ml) and Tmp(r) isolates only the Staphylococcus epidermidis isolates (both Tmp(r) and Tmp(r)) seemed to contain the dfrA gene. Furthermore, we have cloned and characterized a novel, plasmid-encoded Tmp(r) DHFR from Staphylococcus haemolyticus MUR313. The dfrD gene of plasmid pABU17 is preceded by two putative Shine-Dalgarno sequences potentially allowing for the start of translation at two triplets separated by nine nucleotides. The predicted protein of 166 amino acids, designated S2DHFR, encoded by the longer open reading frame was overproduced in Escherichia coli, purified, and characterized. The molecular size of the recombinant S2DHFR was determined by ion spray mass spectrometry to be 19,821.2 ± 2 Da, which is in agreement with the theoretical value of 19,822 Da. In addition, the recombinant S2DHFR was shown to exhibit DHFR activity and to be highly resistant to Tmp.

Full text of DOI:10.1128/AAC.39.9.1920

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Sept. 1995, p. 1920–1924
0066-4804/95/$04.00ϩ0
Copyright ᭧ 1995, American Society for Microbiology
Vol. 39, No. 9
Cloning and Characterization of a Novel, Plasmid-Encoded
Trimethoprim-Resistant Dihydrofolate Reductase from
Staphylococcus haemolyticus MUR313
GLENN E. DALE,1† HANNO LANGEN,1 MALCOLM G. P. PAGE,2 RUDOLF L. THEN,2
1
¨
AND DIETRICH STUBER *
Departments of Gene Technologies1 and Infectious Diseases,2 Pharmaceutical Research, F. Hoffmann-La Roche Ltd.,
CH-4002 Basel, Switzerland
Received 1 February 1995/Returned for modification 17 April 1995/Accepted 27 June 1995
In recent years resistance to the antibacterial agent trimethoprim (Tmp) has become more widespread, and
several trimethoprim-resistant (Tmpr) dihydrofolate reductases (DHFRs) have been described from gram-
negative bacteria. In staphylococci, only one Tmpr DHFR has been described, the type S1 DHFR, which is
encoded by the dfrA gene found on transposon Tn4003. In order to investigate the coincidence of high-level Tmp
resistance and the presence of dfrA, we analyzed the DNAs from various Tmpr staphylococci for the presence
of dfrA sequences by PCR with primers specific for the thyE-dfrA genes from Tn4003. We found that 30 of 33
isolates highly resistant to Tmp (MICs, >512 ␮g/ml) contained dfrA sequences, whereas among the Tmpr
(MICs, <256 ␮g/ml) and Tmps isolates only the Staphylococcus epidermidis isolates (both Tmpr and Tmps)
seemed to contain the dfrA gene. Furthermore, we have cloned and characterized a novel, plasmid-encoded
Tmpr DHFR from Staphylococcus haemolyticus MUR313. The dfrD gene of plasmid pABU17 is preceded by two
putative Shine-Dalgarno sequences potentially allowing for the start of translation at two triplets separated by
nine nucleotides. The predicted protein of 166 amino acids, designated S2DHFR, encoded by the longer open
reading frame was overproduced in Escherichia coli, purified, and characterized. The molecular size of the
recombinant S2DHFR was determined by ion spray mass spectrometry to be 19,821.2 ؎ 2 Da, which is in
agreement with the theoretical value of 19,822 Da. In addition, the recombinant S2DHFR was shown to exhibit
DHFR activity and to be highly resistant to Tmp.
Antimicrobial resistance in both hospitals and the commu-
nity is a clinically important and increasing problem (10, 21).
Of particular interest is methicillin-resistant Staphylococcus
aureus (MRSA), which invariably carries genes for resistance
to other antibiotics such as erythromycin, tetracycline, strep-
tomycin, gentamicin, and sulfonamides (27). These and other
multiresistant staphylococci are a major cause of hospital-ac-
quired infections such as bacteremias, pneumonias, and surgi-
cal wound infections (10). Although trimethoprim (Tmp)
alone is not the drug of choice for the treatment of staphylo-
coccal infections, it has been used in combination with sulfa-
methoxazole to successfully treat patients infected with mul-
tiresistant staphylococci (22, 33). Tmp is a very selective potent
inhibitor of dihydrofolate reductase (DHFR) and is active in
vitro against most staphylococci, but resistance to Tmp is an
ever growing problem (9).
cus MUR313 which, when it was transformed into the Tmps
strain S. aureus 113, resulted in a high-level Tmp-resistant
strain (MIC, Ͼ1,024 ␮g/ml).
Using a PCR approach to study the molecular epidemiology
of the gene encoding the S1DHFR among staphylococci, we
isolated total DNAs from both Tmpr and Tmps strains of S.
aureus, S. epidermidis, Staphylococcus hominis, and S. haemo-
lyticus from various geographical locations and analyzed their
DNAs for sequences similar to those from the thyE-dfrA
operon. Here we report on the results of that PCR analysis of
diverse staphylococci. In addition, we report on the cloning,
expression in Escherichia coli, and purification of a novel, plas-
mid-encoded Tmpr DHFR, designated the S2DHFR, encoded
by the gene dfrD located on the plasmid pABU17 found in the
highly Tmpr strain S. haemolyticus MUR313.
The widespread resistance of staphylococci to Tmp was first
reported in the 1980s (2, 23) and was found to be due to the
plasmid-encoded production of the Tmpr type S1 DHFR
(S1DHFR) encoded by the dfrA gene located on transposon
Tn4003 (8, 11, 28). In recent studies of Tmp resistance among
coagulase-negative staphylococci (7, 17, 18), it was shown that
a specific probe for the dfrA gene hybridized with all Tmpr
Staphylococcus epidermidis isolates. However, none of the
Staphylococcus haemolyticus strains tested were shown to hy-
bridize with the S1DHFR probe (17). Recently, Burdeska (7)
isolated a small plasmid (pABU17, 3.8 kb) from S. haemolyti-
MATERIALS AND METHODS
Bacterial strains and plasmids. The staphylococcal clinical isolates used in the
present study have been described previously (7, 34). The highly Tmpr strain S.
haemolyticus MUR313 (MIC, Ͼ1,024 ␮g/ml) was isolated in Murcia, Spain, in
1987 by Martin Luengo. For the expression of DHFR in E. coli, strain
M15(pREP4) was used (32). E. coli strains were transformed as described pre-
viously (29). The cloning vector pUC18 was supplied by Boehringer Mannheim,
Mannheim, Germany. Plasmid pABU17 (7) and the expression plasmid pDS56/
RBSII,NcoI were described previously (32).
Enzymes and chemicals. Restriction enzymes, calf intestinal phosphatase, the
Klenow fragment of DNA polymerase, T4 polynucleotide kinase, and T4 DNA
ligase were obtained from Boehringer Mannheim and were used under the
conditions recommended by the manufacturer. Lysozyme was from Pharmacia,
Uppsala, Sweden; lysostaphin was from Sigma, Buchs, Switzerland. [␣-35S]dATP
was purchased from Amersham (Amersham, United Kingdom).
* Corresponding author. Phone: 0041-61-6887297. Fax: 0041-61-
6881448.
† Present address: Department of Microbiology, Biocenter, Univer-
sity of Basel, CH-4056 Basel, Switzerland.
Isolation of DNA. Plasmid DNA was isolated with the Qiagen system by
following the protocols supplied by the manufacturer (Qiagen, Basel, Switzer-
land). Isolation of plasmid DNA from S. haemolyticus MUR313 was carried out
1920

VOL. 39, 1995
Origin of straina
NOVEL DHFR FROM S. HAEMOLYTICUS
1921
TABLE 1. PCR analysis of staphylococci for the presence of dfrA sequences
No. of strains containing dfrA sequences/total no. of strains testedb
Tmp MIC, Ն 512 ␮g/ml Tmp MIC, 0.25–256 ␮g/ml
No. of strain
analyzed
A
B
C
D
A
B
C
Dc
Europe
35
18
7
5/5
2/2
3/3
0/2
5/5
4/4
7/8
1/1
1/1
0/10
0/6
6/6
4/4
3/3
South America
North America
Others
0/1
1/1
5
1/1
0/2
a Clinical isolates collected between 1983 and 1989.
b A, S. aureus; B, S. hominis; C, S. haemolyticus; D, S. epidermidis.
c For all S. epidermidis strain tested, MICs were Յ8 ␮g/ml.
by the alkaline lysis method (5) by first disrupting the cell wall with lysostaphin
(0.1 mg/ml) and lysozyme (1 mg/ml) and then by using the Qiagen system.
PCR. PCRs were performed in a thermal cycler (Biometra, Go¨ttingen, Ger-
many). The PCR profile used for determining the existence of dfrA sequences in
the total DNA extracts was an initial denaturation step for 3 min at 95ЊC; this was
followed by 25 cycles of 30 s at 95ЊC, 30 s at 55ЊC, and 30 s at 72ЊC, with 100 ng
of DNA used as the template and by using as specific primers the oligonucleo-
tides P1 (5Ј-CCCTGCTATTAAAGCACC-3Ј) and P2 (5Ј-CATGACCAGATA
ACTC-3Ј) (12). DNAs from S. aureus 157/4696 (8) and ATCC 25923 were used
as positive and negative controls, respectively. The PCR used in the subcloning
of dfrD (primers P3 and P4; see Fig. 1) followed a standard PCR amplification
protocol (19). All PCR fragments were isolated from 6% polyacrylamide gels
(29).
decrease in A340, which occurred as NADPH was oxidized and DHF was reduced
to tetrahydrofolate, by using a Kontron Uvikon 860 spectrophotometer. One unit
of DHFR activity was taken as the conversion of 1 ␮mol of dihydrofolate per
min.
Other methods. Protein samples were analyzed by sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (SDS-PAGE) in 12.5% polyacrylamide gels
and were visualized by Coomassie brilliant blue staining. Computer-aided se-
quence comparisons were performed on a VAX by using Genetics Computers
Group software of the University of Wisconsin (15).
Nucleotide sequence accession number. The EMBL Data Library accession
number of the DNA sequence for dfrD is Z50141.
RESULTS
Determination of nucleotide sequences. DNA restriction fragments were sub-
cloned in pUC18 vectors. Nucleotide sequences were determined by the dideoxy
chain termination method (30).
Dissemination of S1DHFR. On the basis of the DNA se-
quence of the S1DHFR encoded by Tn4003, we designed the
PCR primers P1 and P2 specific for the 3Ј end of the thyE gene
and the 5Ј end of the dfrA gene, respectively (12). We analyzed
by PCR 33 clinical isolates of staphylococci for which Tmp
MICs were 512 or 1,024 ␮g/ml and 32 clinical isolates for which
Tmp MICs were between 0.25 and 256 ␮g/ml for sequences
similar to dfrA encoded by Tn4003 (Table 1). The isolates were
from a variety of geographic locations. Among the highly Tmpr
clinical isolates (MICs, 512 or 1024 ␮g/ml), 30 of the 33 strains
tested produced a fragment of the expected size (262 bp).
However, among the 32 clinical isolates for which MICs were
lower, only the 13 S. epidermidis (both Tmpr and Tmps) strains
produced a fragment of the expected size (12). From the three
highly resistant strains which failed to produce a fragment of
the expected size, two were S. aureus, ATU5 and ATU6, both
of which were isolated in Colombo, Sri Lanka, in 1985; the
third strain was S. haemolyticus MUR313 (MIC, Ͼ1,024 ␮g/
ml), which was isolated in Murcia, Spain, in 1987.
Cloning and sequence analysis of the dfrD gene. S. haemo-
lyticus MUR313, which contains the 3.8-kb plasmid pABU17
(7), was negative in the PCR analysis. Since, however, pABU17
(Fig. 1) was previously shown to confer high-level Tmp resis-
tance when it was transformed into S. aureus (7), we charac-
terized the novel, plasmid-encoded Tmp resistance gene. Plas-
mid pABU17 was digested with EcoRI and BamHI, and the
restriction products were separated on a 6% polyacrylamide
gel. Four restriction fragments were isolated and subcloned
into pUC18; two EcoRI fragments of approximately 2 kb and
100 bp and two EcoRI-BamHI fragments of approximately 850
and 870 bp. By using pUC18-specific primers, each construct
was sequenced and analyzed for the presence of DHFR-re-
lated sequences. Figure 1 shows the sequence of 778 nucleo-
tides, obtained from a region encompassing part of the 2-kb
EcoRI fragment, the complete 100-bp EcoRI fragment, and
part of the 870-bp EcoRI-BamHI fragment. This DNA se-
quence contains an open reading frame of 498 nucleotides
(dfrD). The dfrD gene is preceded by two putative Shine-
Dalgarno sequences potentially allowing for the start of trans-
Expression and purification of S2DHFR. The coding region of dfrD was
amplified from pABU17 by PCR with the oligonucleotides P3 and P4 (Fig. 1).
The DNA fragment was phosphorylated at the 5Ј ends, digested with BamHI,
and then inserted into the filled-in NcoI site and the BamHI site of the expression
plasmid pDS56/RBSII,NcoI (32). In the resulting plasmid, named pS2, the
DHFR gene is under the control of the regulatable promoter-operator ele-
ment N25OPSN25OP29 and an efficient ribosomal binding site (32). Since
N25OPSN25OP29 contains two lac operators, transcription from this element is
blocked in the presence of the lac repressor, which is encoded by plasmid
pREP4, and is induced by the addition of isopropyl-␤-D-thiogalactopyranoside
(IPTG) to the culture medium. For the expression of S2DHFR, E. coli M15 cells
containing plasmids pS2 and pREP4 were grown to an A600 of 0.9 in 1 liter of
super medium (29) in the presence of 100 ␮g of ampicillin per ml and 25 ␮g of
kanamycin per ml, and then the production of the recombinant S2DHFR was
induced by the addition of IPTG (32). After 3 h, the cells (15 g from 3 liters of
culture medium) were harvested by centrifugation and were resuspended in 50
ml of buffer A (50 mM HEPES [N-2-hydroxyethylpiperazine-NЈ-2-ethanesulfonic
acid; pH 8], 20% glycerol, 0.15 M NaCl, 1 mM MgSO4, 2 mM dithiothreitol
(DTT), and 5 mM EGTA [ethylene glycol-bis (␤-aminoethyl ether)-N,N,NЈ,NЈ-
tetraacetic acid]). The suspension was passed through a French press, and the
resulting homogenate was centrifuged for 30 min at 20,000 rpm (Kontron rotor
A8.8). Essentially no DHFR activity was detected in the supernatants; therefore,
the pellet was resuspended in 25 ml of 8 M urea–40 mM Tris-HCl (pH 7.5). The
suspension was centrifuged at 54,000 ϫ g (Heraeus Sepatech HFA28.1) for 30
min. The supernatant was diluted 10-fold with 20 mM Tris-HCl–6 M urea–50
mM DTT (pH 7.5) to reduce the ionic strength. Aliquots of 10 ml were applied
to a 6-ml Resource Q column (Pharmacia) equilibrated with 20 mM Tris-HCl–6
M urea–50 mM DTT (pH 7.5). S2DHFR was eluted with a linear gradient from
0 to 1 M NaCl at a flow rate of 3 ml/min. Fractions containing the S2DHFR from
a total of 12 runs (150 ml) were combined and concentrated by a factor of 10 with
a Centriprep-10 apparatus (Amicon, Danvers, Mass.) and were applied in two
portions to a column (2.6 ϫ 60 cm) of Superdex 200 (Pharmacia) equilibrated
with 50 mM Tris-HCl (pH 8)–6 M guanidine-HCl. The fractions containing
DHFR were pooled (48 ml) and were again concentrated 10-fold with a Cen-
triprep-10 apparatus. The solution was dialyzed overnight at 4ЊC against 1 liter of
phosphate-buffered saline–5 mM DTT. Finally, the dialysate was centrifuged at
54,000 ϫ g (Heraeus Sepatech HFA28.1) for 20 min.
Ion spray mass spectrometry. Mass spectral analysis was performed on a Sciex
API-III spectrometer. Prior to the analysis, the S2DHFR was desalted on a Poros
R/H reversed-phase high-pressure liquid chromatography column (0.21 by 10
cm; Perseptive Biosystems, Boston, Mass.).
Enzyme assays. The DHFR assay has been described previously (4). Dihydro-
folate (DHF) was prepared from folic acid, and its concentration was measured
spectrophotometrically at 282 nm by using the molar extinction coefficient of 2.8
ϫ 104 MϪ1 cmϪ1 at pH 7.4 (1). The concentration of NADPH was determined
spectrophotometrically at 340 nm by using the molar extinction coefficient of 6.2
ϫ 104 MϪ1 cmϪ1 (1). The DHFR activities were determined by measuring the

1922
DALE ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
integrating its gene into the pDS expression system (32). Plas-
mid pS2 contains the S2DHFR-coding region which was am-
plified from pABU17 by PCR with primers P3 and P4 (Fig. 1).
The 3Ј PCR primer (P4) included a BamHI site following the
termination codon of dfrD; the 5Ј PCR primer (P3) included
the first codon downstream of the ATG triplet at positions 82
to 84 and is placed in pS2 just downstream of a Shine-Dalgarno
sequence; this is followed by an ATG triplet. The authenticity
of the plasmid was confirmed by DNA sequence determination
of the entire insert (both strands). E. coli M15(pREP4) cells
containing plasmid pS2 overexpressed S2DHFR (Fig. 3). The
S2DHFR could be purified in two chromatography steps to
near homogeneity (Fig. 3). Since most of the recombinant
S2DHFR was produced in inclusion bodies, 8 M urea was
necessary to solubilize the enzyme. Consequently, the ion-
exchange column and the sizing column were run in the pres-
ence of 6 M urea and 6 M guanidine-HCl, respectively. After
the sizing step about 5 mg of S2DHFR was obtained. In the
final dialysis step to refold the enzyme, more than 95% of the
protein precipitated. However, the refolded enzyme was active,
and its molecular size of 19,821.2 Ϯ 2 Da measured by ion
spray mass spectrometry was in good agreement with the cal-
culated value of 19,822.8 Da. Some kinetic parameters of the
purified S2DHFR are presented in Table 2. The Michaelis
constant (Km) values for NADPH and DHF are similar to
those determined for the other S. aureus enzymes (12),
whereas the 50% inhibitory concentration of Tmp for
S2DHFR is more than 10-fold higher than that for S1DHFR.
DISCUSSION
Since widespread Tmp resistance among staphylococci was
first reported (2, 23), a great deal of interest in understanding
the mechanism of this resistance has arisen. After the initial
observations, resistance was found to be due to the plasmid-
driven production of a Tmpr dihydrofolate reductase, the
S1DHFR, encoded by the dfrA gene located on transposon
Tn4003 (8, 11, 28). Since the dfrA gene has been found by
hybridization (7, 18) in S. aureus and S. epidermidis, a horizon-
tal transfer in nature was suggested (18). This surprisingly high
level of gene conservation, which is quite different from the
large variations observed among Tmpr DHFRs in gram-nega-
tive species, was explained by the worldwide spread of trans-
poson Tn4003 (8). However, in two independent studies an
S. aureus dfrA probe failed to hybridize to several highly Tmpr
S. haemolyticus isolates (7, 17). Additionally, Burdeska (7)
showed that a small plasmid (pABU17) from S. haemolyticus
MUR313, which was negative in the hybridization experi-
ments, could transfer high-level Tmp resistance to a recipient
strain. The results of those studies suggested that there are
other genes encoding Tmpr DHFRs in staphylococci (3, 7, 17).
We therefore used a PCR approach to determine the relation-
ship between the presence of the dfrA gene and high-level Tmp
resistance among both coagulase-positive and coagulase-neg-
ative staphylococci with the goal of identifying, cloning, and
characterizing such genes.
We analyzed a total of 65 clinical isolates from various re-
gions (7, 34) for DNA sequences homologous to those from
thyE-dfrA from Tn4003. Our results indicate that none of the
strains for which the MIC was Յ256 ␮g/ml (except S. epider-
midis [12]) contains the dfrA gene as part of transposon Tn4003
(Table 1). Consistent with that are the observations that these
strains do not seem to contain a transferable plasmid-encoded
Tmp resistance marker (7) and that at least the S. aureus
strains in this MIC category contain mutated chromosomal
DHFRs which are highly resistant to Tmp (unpublished se-
FIG. 1. (A) Diagram of the 3.8-kb plasmid pABU17 from S. haemolyticus
MUR313 displaying relevant restriction sites, the PCR primers P3 and P4, and
the location of the dfrD gene. (B) Nucleotide and deduced amino acid sequences
of the dfrD gene. The solid lines indicate the two putative Shine-Dalgarno
sequences; the putative start sites for translation are underlined. P3 and P4
indicate the oligonucleotides used to subclone the dfrD gene into an expression
plasmid; the BamHI site is underlined. Asterisks indicate the position of the stop
codon.
lation at the ATG triplet at positions 82 to 84 or at the in-frame
TTG triplet at positions 94 to 96. The predicted protein (166
amino acids) encoded by the longer open reading frame (start
of translation at the ATG triplet) was named S2DHFR. Its
amino acid sequence is most closely related to the chromo-
somal DHFR from Bacillus subtilis with approximately 50%
amino acid identity. S2DHFR is somewhat less similar to the
chromosomally encoded enzymes of S. aureus, S. epidermidis,
and E. coli K-12, with 41, 39, and 35% amino acid identities,
respectively, and it exhibits a 40% identity with the S1DHFR
encoded by Tn4003 (Fig. 2). The S2DHFR contains two struc-
tural features which are present only in those enzymes isolated
from staphylococci (8, 12, 14, 28), namely, a small hydrophobic
residue (Val in the chromosomal enzymes, Ile in the plasmid-
encoded S1DHFR) at position 35, a position occupied by a Phe
residue in the B. subtilis and E. coli chromosomal DHFRs, and
at position 99 a Phe residue, which is a small hydrophobic
residue in the chromosomal DHFRs from B. subtilis and E. coli
(Fig. 2). Therefore, S2DHFR seems to be structurally more
closely related to the staphylococcal enzymes.
Enhanced expression, purification, and analysis of S2DHFR.
The expression of S2DHFR in E. coli was accomplished by

VOL. 39, 1995
NOVEL DHFR FROM S. HAEMOLYTICUS
1923
FIG. 2. Compilation of the amino acid sequences of S2DHFR, S1DHFR (28), and the chromosomal DHFRs from S. aureus (14) S. epidermidis (12), B. subtilis (20),
and E. coli K-12 (31). The positions involved in the binding of trimethoprim (T), methotrexate (T and t), and the cofactor NADPH (n), taken from studies with the
E. coli K-12 enzyme (6, 16, 25, 26, 31), are indicated. The amino acid residues at positions 35 and 99 are marked with an asterisk (see text). The amino acid residue
marked in boldface is the tyrosine known to be the major contributor to Tmp resistance in S. epidermidis (12). The numbering of the amino acids is based on that of
S2DHFR, with the Met at its N-terminal end being at position 0.
quencing data). On the contrary, 30 of the 33 analyzed strains
for which the MIC was Ն512 ␮g/ml contained dfrA elements
(Table 1). The finding that the same gene for resistance to
Tmp is present in S. aureus, S. epidermidis, S. hominis, and S.
haemolyticus indicates that these organisms share a common
gene pool. The chromosomal DHFR gene of S. epidermidis is
almost identical (differing by only eight nucleotides) to the
thyE-dfrA region of Tn4003 (12), underscoring the importance
of S. epidermidis as a reservoir for DNA sequences that even-
tually appear in other staphylococcal species (3, 12, 24). Three
of our isolates were negative in the PCR analysis; two were S.
aureus ATU5 and ATU6, which were not further analyzed, and
the third isolate was S. haemolyticus MUR313, from which we
cloned the dfrD gene encoding the Tmpr S2DHFR.
To demonstrate that the dfrD gene encodes an active, Tmp-
resistant DHFR we decided to produce S2DHFR in E. coli, to
purify the recombinant enzyme, and to determine some of its
kinetic parameters. Since we failed to produce the recombi-
nant DHFR in a soluble and active form (with or without the
cooverproduction of the chaperones GroES and GroEL [13];
data not shown) we had to purify S2DHFR from inclusion
bodies. Upon denaturation and refolding of the enzyme, most
of the purified protein precipitated. However, after removal of
the insoluble protein sufficient amounts of soluble and active
enzyme were obtained to determine the Km for substrate and
cofactor and the 50% inhibitory concentration of Tmp (Table
TABLE 2. Enzyme kinetic and inhibition properties of purified
recombinant staphylococcal DHFRs
Km (␮M)
IC50 of Tmp
FIG. 3. Purification of S2DHFR and SDS-PAGE (10 to 20%) analysis of
protein samples obtained during purification of S2DHFR. The samples shown
correspond to the following purification steps: whole cells after solubilization
with SDS-sample buffer (lane 1), soluble extract after cell disruption and cen-
trifugation (lane 2), inclusion bodies solubilized with 6 M urea (lane 3), elution
fraction from the Resource Q column (lane 4), and peak fraction from the
Superdex 200 column prior to dialysis (lane 5). The position of S2DHFR is
indicated with an arrow. The molecular masses (in kilodaltons) of the marker
proteins (1 ␮g of each protein) are indicated on the left.
DHFR
(␮M)a
DHF
NADPH
S2DHFR
S1DHFRb
SeDHFRb
5.1
6.6
5.1
1.7
12.4
2.5
127
9.8
0.029
a IC50, 50% inhibitory concentration.
b Data for S1DHFR and SeDHFR were taken from Dale et al. (12).

1924
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ANTIMICROB. AGENTS CHEMOTHER.
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cations for catalysis. J. Biol. Chem. 257:13663–13672.
17. Froggatt, J. W., J. L. Johnston, D. W. Galetto, and G. L. Archer. 1989.
Antimicrobial resistance in nosocomial isolates of Staphylococcus haemolyti-
cus. Antimicrob. Agents Chemother. 33:460–466.
18. Galetto, D. W., J. L. Johnston, and G. L. Archer. 1987. Molecular epidemi-
ology of trimethoprim resistance among coagulase-negative staphylococci.
Antimicrob. Agents Chemother. 31:1683–1688.
2). The refolded recombinant S2DHFR is highly resistant to
Tmp and shows Km values for substrate and cofactor which are
comparable to those for the Tmp-resistant S1DHFR and the
Tmp-susceptible SeDHFR (the chromosomally encoded DHFR
of S. epidermidis ATCC 14990). Since the enzyme was isolated
from inclusion bodies, denatured, and finally refolded, the
enzyme kinetics might not be accurate. Nevertheless, the data
demonstrate that the dfrD gene encodes the novel Tmp-resis-
tant DHFR responsible for the high-level Tmp resistance of S.
haemolyticus MUR313.
Comparisons between S2DHFR and the other DHFRs show
that the active site of S2DHFR is very similar to those known
from staphylococci (8, 12, 14, 28). Like the other staphylococ-
cal enzymes, S2DHFR has a small hydrophobic amino acid
(Val) at position 35, which is occupied by a Phe residues in
chromosomal DHFRs from other organisms (Fig. 2). The va-
line residue is proposed to make strong hydrophobic contacts
with the pyrimidine ring of Tmp (26). At the far interior of the
active site and making van der Waals contacts with the pyrim-
idine ring of Tmp (26), the staphylococcal enzymes contain a
phenylalanine (residue 99), whereas this position is occupied
by a small hydrophobic amino acid in other DHFRs (Fig. 2).
Since the active sites of S2DHFR and S1DHFR seem to be
rather similar and results from mutagenesis experiments of
S1DHFR clearly implicated the presence of a Tyr residue at
position 98 as being the major determinant for Tmp resistance
(12), one could speculate that the tyrosine residue of S2DHFR
at the corresponding position (residue 105) will also be the
major determinant for resistance of this enzyme. The molecu-
lar origin of dfrD is unknown, but because of the striking
similarities between the active site of S2DHFR and those of
the other staphylococcal enzymes, it is tempting to assume that
it originates from a closely related staphylococcal species.
19. Innis, M. A., D. H. Gelfand, J. J. Sninsky, and T. J. White (ed.). 1990. PCR
protocols: a guide to methods and applications. Academic Press, Inc., San
Diego, Calif.
20. Iwakura, M., M. Kawata, K. Tsuda, and T. Tanaka. 1988. Nucleotide se-
quence of the thymidylate synthase B and dihydrofolate reductase genes
contained in one Bacillus subtilis operon. Gene 64:9–20.
21. Jacoby, G. A., and G. L. Archer. 1991. New mechanisms of bacterial resis-
tance to antimicrobial agents. N. Engl. J. Med. 324:601–612.
22. Levitz, R. E., and R. Quintiliani. 1984. Trimethoprim-sulfamethoxazole for
bacterial meningitis. Ann. Intern. Med. 100:881–890.
23. Lyon, B. R., J. W. May, and R. A. Skurray. 1983. Analysis of plasmids in
nosocomial strains of multiple antibiotic resistant Staphylococcus aureus.
Antimicrob. Agents Chemother. 23:817–826.
ACKNOWLEDGMENTS
24. Lyon, B. R., and R. Skurray. 1987. Antimicrobial resistance of Staphylococ-
cus aureus: genetic basis. Microbiol. Rev. 51:88–134.
25. Matthews, D. A., J. T. Bolin, J. M. Burridge, D. J. Filman, K. W. Volz, B. T.
Kaufman, C. R. Beddell, J. N. Champness, D. K. Stammers, and J. Kraut.
1985. Refined crystal structures of Escherichia coli and chicken liver dihy-
drofolate reductase containing bound trimethoprim. J. Biol. Chem. 260:381–
391.
We are most grateful to Sabine Demmak, Daniel Ro¨der, and Arno
Friedlein for excellent technical assistance. Special thanks go to Petra
Scheliga for consistently supplying us with DHF and to Peter Hartman
and Beat Schwaller for their advice and criticism.
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