(Received for publication, April 25, 1995; and in revised form, September 6, 1995)
From the
Plasmid pJV4, containing a 2.4-kilobase pair insert of genomic
DNA from the chloramphenicol (Cm) producer Streptomyces venezuelae ISP5230, confers resistance when introduced by transformation into
the Cm-sensitive host Streptomyces lividans M252 (Mosher, R.
H. Ranade, N. P., Schrempf, H., and Vining, L. C.(1990) J. Gen.
Microbiol. 136, 293-301). Transformants rapidly metabolized
Cm to one major product, which was isolated and purified by reversed
phase chromatography. The metabolite was identified by nuclear magnetic
resonance spectroscopy and mass spectrometry as
3`-O-phospho-Cm, and was shown to have negligible inhibitory
activity against Cm-sensitive Micrococcus luteus. The
nucleotide sequence of the S. venezuelae DNA insert in pJV4
contains an open reading frame (ORF) that encodes a polypeptide (19
kDa) with a consensus motif at its NH terminus
corresponding to a nucleotide-binding amino acid sequence (motif A or
P-loop; Walker, J. E., Saraste, M., Runswick, M. J., and Gay, N.
J.(1982) EMBO J. 1, 945-951). When a recombinant vector
containing this ORF as a 1.6-kilobase pair SmaI-SmaI
fragment was used to transform S. lividans M252, uniformly
Cm-resistant transformants were obtained. A strain of S. lividans transformed by a vector in which the ORF had been disrupted by an
internal deletion yielded clones that were unable to phosphorylate Cm,
and exhibited normal susceptibility to the antibiotic. The results
implicate the product of the ORF from S. venezuelae as an
enzymic effector of Cm resistance in the producing organism by
3`-O-phosphorylation. We suggest the trivial name
chloramphenicol 3`-O-phosphotransferase for the enzyme.
Chloramphenicol (Cm; Fig. 1), ()a
broad-spectrum antibiotic that inhibits protein biosynthesis by binding
reversibly to the peptidyl transferase center of 50 S ribosomal
subunits (Pongs, 1979), is produced by Streptomyces venezuelae and some related species (Vining and Westlake, 1984). During
growth under conditions where Cm is not produced, S. venezuelae is relatively sensitive to the antibiotic, but high-level
resistance is induced by exposure to Cm; in cultures grown under
Cm-producing conditions, resistance increases concurrently with Cm
synthesis. However, ribosomes extracted from producing or nonproducing
mycelium are equally sensitive to the antibiotic (Malik and Vining,
1970, 1972). Resistance to Cm is mediated in most eubacteria by
chloramphenicol acetyltransferase (CAT; EC 2.3.1.28: reviewed by
Shaw(1983) and Shaw and Leslie(1991)); this enzyme modifies Cm by
acetylation, yielding 3`-O-acetyl-Cm, which is only very
weakly bound by ribosomes and thus is not an antibiotic. Since S.
venezuelae lacks CAT activity (Shaw and Hopwood, 1976; Nakano et al., 1977), alternative mechanisms have been sought to
explain the ability of the producing organism to avoid inhibition by
one of its own products (Vining and Westlake, 1984).
Figure 1: Structure of chloramphenicol and its derivatives.
By cloning
genomic S. venezuelae DNA in the streptomycete vector pIJ702
and transforming the Cm-hypersensitive Streptomyces lividans M252 to resistance, Mosher and co-workers(1990) obtained a
transformant (RM3) harboring a recombinant plasmid (pJV3) with a
6.5-kbp insert. Deletion from pJV3 of a 5.2-kbp segment (encompassing
4.1-kbp from the insert and 1.1-kbp from the pIJ702 vector) yielded
pJV4, from which a region implicated in Cm resistance was subcloned as
a 2.4-kbp KpnI-SstI fragment. Cultures of S.
lividans RM3 rapidly metabolized
[U-C]chloramphenicol to unidentified labeled
products (Mosher et al., 1990).
We report here the isolation and characterization of the major product of Cm metabolism by S. lividans RM3 and RM4 (M252 transformed with pJV4). Identification of this metabolite as the 3`-phospho ester of Cm implicates a mechanism of Cm resistance for the producing organism that has not hitherto been encountered in streptomycetes or other microbial systems.
The C NMR spectrum for Cm was recorded in acetone at
62.5 MHz using solvent
C as a reference. Data were
accumulated using 90° pulses (6 µs) with delays of 1 or 2 s.
The data size was 32 K. The spectrum contained signals at
(ppm)
58.5 (d, C-2`), 62.2 (t, C-3`), 67.4 (d, CHCl
), 71.3 (d,
C-1`), 124.2 (d, C-2, C-6), 128.3 (d, C-3, C-5), 148.6 (s, C-1), 151.6
(s, C-4), and 166.6 (s, CO). The
C NMR spectrum of
3`-phospho-Cm was recorded in D
O, locked to solvent
deuterium; signals were present at
(ppm) 58.3 (d, C-2`), 66.6 (t,
C-3`), 68.7 (d, CHCl
), 73.3 (d, C-1`), 126.4 (d, C-2, C-6),
129.9 (d, C-3, C-5), 149.9 (s, C-1), 151.0 (s, C-4), and 169.6 (s, CO).
The P NMR spectrum of 3`-phospho-Cm was recorded at
101.2 MHz in D
O with 85% phosphoric acid as an external
reference. Data were accumulated using 90° pulses (6 µs) with
delays of 1 or 2 s. The data size was 32 K.
Figure 2: Chromatographic analysis by HPLC of an aqueous Cm solution incubated 12 h with mycelium of S. lividans RM4.
Mycelium grown in a variety of media was active in converting Cm to its major metabolite; activity persisted in mycelium harvested after the end of the growth phase, or resuspended in water. Complete conversion was achieved by adding Cm at the concentration tolerated (12.5 µg/ml) to cultures of S. lividans RM4 growing in GNY medium. Furthermore, complete conversion was obtained by incubating washed mycelium with 200 µg/ml of Cm in water for 48 h. The use of aqueous cell suspensions simplified isolation of the metabolite, and facilitated its purification by reversed phase chromatography. The major chromatographic product was collected as a single sharp peak, well separated from other substances absorbing at 273 nm, and was evaporated to dryness at 40 °C in vacuo.
The C NMR spectrum of the
metabolite was similar to that of Cm, with most of the signals slightly
downfield of corresponding Cm signals. However, the signal assigned to
C-3` showed a larger shift, from 62.2 to 66.6 ppm, consistent with the
presence of an electron-withdrawing substituent on this carbon. That
the latter is an orthophosphate group is supported by the
P NMR spectrum of the product. The chemical shift (
P,
= 6.474 ppm) of the single strong signal
was in the region of the spectrum predicted for an organophosphate
ester (data not shown). Examination of the sample by low resolution,
negative ion-spray mass spectrometry gave a group of molecular ions in
the relative proportions expected for a substance containing two
chlorine atoms (Fig. 3). The deduced M
of
the compound is 401.97, the value predicted for a monophosphate ester
of chloramphenicol. From the combined evidence, the most probable
structure of the metabolite is, therefore, 3`-phospho-Cm.
Figure 3:
Mass
spectrum of the Cm metabolite obtained by negative ion spray. The
deduced mass of the parent ion with Cl
was
401.9786.
To determine whether 3`-phospho-Cm exhibited antibiotic activity, the purified compound was compared with Cm in a disk-diffusion assay using M. luteus as the test organism. Inhibition zones were observed only with relatively large samples of 3`-phospho-Cm (>350 µg/disk), whereas 0.15 µg of Cm gave a measurable zone; in a standard assay, the apparent specific activity of 3`-phospho-Cm was 0.04% that of Cm. We conclude from this result that phosphorylation of the 3`-OH position of Cm reduces its antibiotic activity significantly.
Figure 4: The nucleotide and deduced amino acid sequences of the 2.4-kbp KpnI-SstI fragment of S. venezuelae ISP5230 DNA cloned in pJV4. Potential translational start and stop codons are overlined; plausible ribosome-binding sites are underlined, and inverted repeats have underlying facing arrows. The amino acid motifs for nucleotide binding are double underlined.
Figure 5: A, analysis of %G + C in the 2355-bp sequence of S. venezuelae DNA using the FRAME programme of Bibb et al.(1984) adapted for the Macintosh Plus microcomputer (Uchiyama and Weisblum, 1985; Doran et al., 1990). B, restriction enzyme sites on the cloned S. venezuelae DNA fragment (thick bar, same scale as A); the arrows immediately below represent the ORFs deduced from the FRAME analysis, and the location of translational start and stop codons. The short arrows show the sequencing strategy using both the exonuclease derived deletions and primers deduced from sequenced DNA.
ORF1 shows greater than 60%
sequence similarity over 300 amino acids to a number of antibiotic
resistance proteins presumed to be integral membrane components and
capable of promoting active and specific antibiotic efflux (Levy,
1992). Examples include the quinolone resistance determinant (NorA) of Staphylococcus aureus noted above, the methylenomycin
resistance protein (Mmr) of the methylenomycin producer Streptomyces coelicolor (Neal and Chater, 1987), and the
tetracycline resistance protein (TetL) of Bacillus
stearothermophilus (McMurry et al., 1987). All such
proteins probably contain 12-14 membrane-spanning -helical
segments that form a transmembrane channel (Paulsen and Skurray, 1993).
Initiation of translation at the GTG
codon would yield a polypeptide of 178 amino acids (M 18,804), whereas a polypeptide initiated at the ATG codon would
contain only 174 amino acids (M
18,315). Although
a FASTA comparison of the amino acid sequence with the GenBank and EMBL
data bases revealed no significant similarities, use of the alignment
program MPsrch (Sturrock and Collins, 1993) showed resemblance between
the NH
-terminal region of the ORF2 product and predicted
nucleotide-binding sites in such ATP-requiring proteins as pantothenate
kinase of E. coli (Song and Jackowski, 1992) and GlnQ of B. stearothermophilus (Wu and Welker, 1991).
Examination of ORF2 using the Motifs program (Genetics Computer Group
Inc., version 7.3), showed the presence of the ``P-loop''
phosphate-binding motif found in many classes of adenine and guanine
nucleotide-binding proteins (Saraste et al., 1990). The ORF2
sequence GGSSAGKS (aa 10-17; see Fig. 4) fits the P-loop
motif consensus (A/G)XXXXGK(S/T) for an ATP/GTP binding site
(Walker et al., 1982; Saraste et al., 1990), and
implicates ORF2 in a process such as phosphoryl transfer. Also present
in the ORF2 polypeptide is the sequence DADG (aa 57-60; see Fig. 4), which corresponds to a proposed consensus element
(DXXG) for GTP/GDP-binding sites (Dever et al.,
1987). The linear separation between the aspartic acid of
DXXG, which interacts with nucleotide-bound
Mg
, and the P-loop lysine, thought to interact with
the
- and
-phosphates of the bound nucleotide, conforms to
the observed separation (40-80 residues) between these amino
acids (Saraste et al., 1990; Mimura et al., 1991).
Figure 6: A schematic diagram of the vectors described in the text. Numbers refer to the nucleotide sequence of restriction enzyme sites in the 2.4-kbp KpnI-SstI segment of pJV4 DNA.
Evidence that resistance depended on the presence of an intact ORF2 in pJV4 was obtained by deleting a segment of DNA between two SalI sites within ORF2. The deletion plasmid was constructed in the Streptomyces-E. coli shuttle vector pHJL400. Transformation of S. lividans M252 with the vector carrying the 2.4-kbp S. venezuelae DNA insert from which the SalI-SalI fragment had been deleted yielded Cm-sensitive colonies, in contrast to the Cm-resistant transformants obtained with a control plasmid in which ORF2 was intact. Transformants with the deletion in ORF2 also differed from those with ORF2 intact in that they failed to convert Cm to 3`-phospho-CM.
In a previous report (Mosher et al., 1990), the
presence of small quantities of metabolites derived from Cm-base in
cultures of the Cm-resistant S. lividans strain RM3
supplemented with Cm was offered as evidence that a chloramphenicol
amide hydrolase mediated Cm resistance in S. venezuelae. In
the present study, a more exhaustive analysis of the culture broths of S. lividans RM3 and RM4 revealed a hitherto unidentified polar
metabolite as the major product of Cm metabolism. Enough of the
metabolite was obtained by incubating Cm with resting RM4 cells to
allow its isolation, purification by C reverse-phase
column chromatography, and identification as the
3`-O-phosphoryl ester of Cm. That ORF2 has a role in the Cm
resistance conferred by pJV4 was established by the concurrent loss of
Cm kinase activity and resistance when an internal segment was deleted.
We conclude that ORF2 encodes the enzyme that catalyses the
phosphorylation of Cm, for which we have proposed the trivial name
chloramphenicol 3`-O-phosphotransferase.
Much early work on the relationship between the structure of Cm and its antibiotic activity (summarized by Pongs, 1979) emphasized the importance of an unmodified primary alcohol at C-3`. Consistent with this, bacterial resistance to Cm is commonly mediated by enzymic acetylation of the C-3` hydroxyl group (Shaw and Leslie, 1991). The discovery that a Cm-producing streptomycete modifies the same functional group by an alternative mechanism suggests that this transformation is responsible for self-resistance. Phosphorylation is widely used to confer resistance to antibiotics, both in producing and in susceptible nonproducing organisms (Cundliffe, 1992). Nonetheless, in the more than four decades during which Cm has been used to treat infections in humans and animals, phosphorylation has not hitherto been reported as a mechanism for inactivating this antibiotic. Its presence in a Cm-producing streptomycete implies that, here at least, the origin of a resistance mechanism cannot easily be attributed to horizontal transfer of a gene conferring protection on an antibiotic producer.
Although
the deduced primary structure of chloramphenicol
3`-O-phosphotransferase does not show end-to-end similarity to
proteins in current data bases, it does contain localized regions of
sequence similarity to a number of proteins requiring nucleotide
cofactors. The presence near the NH terminus of a consensus
P-loop sequence similar to known ATP/GTP-binding sites is compatible
with the role of chloramphenicol 3`-O-phosphotransferase in
phosphoryl transfer. The P-loop motif occurs in a wide variety of
proteins that, although diverse in biochemical function, have both a
common nucleotide binding sequence and similar structures (Saraste et al., 1990; Schulz, 1992). Among these are the human
Ha-ras p21 protein, E. coli adenylate kinase, EF-Tu
and dethiobiotin synthetase (Jurnak, 1985; Pai et al., 1989,
1990; Muller and Schulz, 1992; Huang et al., 1994; Alexeev et al., 1994). The ATP-binding site is also present in the
transport protein superfamily that includes the cystic fibrosis gene
product (Hyde et al., 1990; Mimura et al., 1991). In
many proteins of this class the polypeptide is folded into a barrel of
parallel
-sheets surrounded by
-helices to give a ``core
structure'' (Milner-White et al., 1991). The glycine-rich
region of the loop contributes to forming a giant anion hole
accommodating the nucleotide triphosphate oxygen atoms.
Although three intact ORFs are present in the 2.4-kbp KpnI-SstI DNA fragment cloned in pJV4 from S. venezuelae, large segments of ORF1 and ORF3 can be deleted with only a 10-20% loss of the Cm resistance phenotype conferred on S. lividans M252 transformants by the plasmid. Cm resistance and the ability to phosphorylate Cm are retained only when ORF2 remains intact; selective disruption of ORF2 eliminates both capabilities. The conclusion that cpt is involved in the resistance conferred by pJV4 is consistent with chemical characterization of the Cm inactivation product as 3`-phospho-Cm, and sequence analysis of the deduced gene product indicated that the primary structure contains a nucleotide-binding motif. Although ORF1 contributes to the Cm resistance of S. lividans RM4, it does not have a predominant role. This is unexpected given its similarity to proteins associated with resistance to greater than 200 µg/ml Cm in other bacteria. Its lack of activity in pJV4 could be attributed to the absence of upstream regulatory sequences, but the comparable Cm resistance conferred by pJV3, in which 4.1 kbp of upstream sequence from S. venezuelae remains intact, renders this explanation unsatisfactory. A possible alternative is that ORF1 encodes an efflux protein for 3-phospho-Cm, rather than for Cm itself, and that it functions in the presence of chloramphenicol 3`-O-phosphotransferase to facilitate the export of inactivated antibiotic.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U09991[GenBank].