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(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
Chloramphenicol (Cm; Fig. 1), (
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- 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 The
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
Figure 3:
Mass
spectrum of the Cm metabolite obtained by negative ion spray. The
deduced mass of the parent ion with
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
Initiation of translation at the GTG
codon would yield a polypeptide of 178 amino acids (M
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 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 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].
Volume 270,
Number 45,
Issue of November 10, 1995 pp. 27000-27006
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A NOVEL RESISTANCE MECHANISM IN STREPTOMYCES VENEZUELAE ISP5230, A CHLORAMPHENICOL PRODUCER (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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.
)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).
C]chloramphenicol to unidentified labeled
products (Mosher et al., 1990).
Materials
Restriction endonucleases and DNA
modifying enzymes were obtained from Life Technologies, Inc., Promega,
New England Biolabs, and Pharmacia Biotech Inc. Cm and D-threo-p-nitrophenylserinol (Cm-base) were from
Sigma.Bacteria, Plasmids, and Culture Conditions
The
properties of S. lividans M252, and those of
transformants RM3 (containing pJV3) and RM4 (containing pJV4) have been
described (Mosher et al., 1990). Escherichia coli was
grown in 2 
YT broth (10 g of yeast extract, 16 g of
Bacto-tryptone and 5 g of NaCl per liter). Conditions for E. coli growth, transformation, transfection, and plasmid isolation were
essentially as described by Sambrook et al.(1989). Batch
cultures of S. lividans were grown in GNY medium (20 ml of
glycerol, 8 g of nutrient broth, 3 g of yeast extract, and 5 g of
KHPO
per liter) for 48 h at 30 °C (Malik
and Vining, 1970). Conditions for transforming Streptomyces and for plasmid isolation were as described (Hopwood et
al., 1985). Table 1lists the strains and plasmids used in
this work.
High Performance Liquid Chromatography
(HPLC)
Culture broths and extracts were analyzed for Cm and its
derivatives by HPLC on a 4.6 50-mm Phenomenex Ultracarb ODS-30
C
column (5-µm particle size). The column was
initially equilibrated with 30 mM potassium phosphate buffer,
pH 3.3. At the sample injection, a programmed methanol gradient was
started: the methanol concentration increased linearly to 25, 50, and
100% at 1, 6, and 7 min, respectively, remained at 100% for 3 min, and
then reduced to zero over 1 min. The column was re-equilibrated for 5
min before the next injection. Nitrophenyl derivatives were detected by
their absorbance at 273 nm.Isolation of 3`-Phospho-Cm from Suspensions of RM4
Mycelium
To investigate Cm metabolism by S. lividans RM3 and RM4, mycelium was grown in GNY medium supplemented with Cm
at a final concentration of 12.5 µg/ml (38.7 µM).
Cultures were routinely inoculated with spore suspensions and incubated
at 30 °C on a rotary shaker (3.7-cm eccentricity; 250 rpm).
Mycelial suspensions for converting Cm to 3`-phospho-Cm were prepared
as follows; 250 ml of RM4 cells grown for 48 h were centrifuged (35,000
g for 10 min at 4 °C), and washed aseptically with
water. They were resuspended in 250 ml of water containing Cm (200
µg/ml), and incubated in a 1-liter Erlenmeyer flask for 23 h under
the conditions used for growing cultures. The cells were then removed
by centrifugation, and the supernatant was decanted and stored at
-20 °C. The supernatant was concentrated to 1 ml at 40 °C in vacuo, and acidified with 100 µl of 1 M ammonium formate buffer, pH 2.5. The resulting solution, pH 3.4,
was clarified by filtration and applied to a C reversed-phase silica column (1 23 cm, 40-µm particle
size, Bakerbond) packed in water. After 1 ml of 0.1 M ammonium
formate buffer, pH 2.5, had been added, the products were eluted with
water. Fractions were monitored by HPLC; those that contained Cm or
3`-phospho-Cm were pooled appropriately and concentrated in
vacuo. By this procedure 3`-phospho-Cm greater than 99% pure by
HPLC and NMR analysis, and free from contamination with Cm, was
isolated in milligram quantities.Nuclear Magnetic Resonance Spectrometry
The H NMR spectra of samples were recorded in methanol at 250.1
MHz with tetramethylsilane as an internal reference; a Bruker AC-250 F
spectrometer was used. Data was accumulated using 90° pulses (9.6
µs) with delays of 1 or 2 s; the data size was 16 K zero-filled to
32 K. The H NMR spectrum for Cm contained signals at
(ppm) 3.59 (q, 1H, H-3a` J
6.1 Hz, J
6.1 Hz), 3.81 (q, 1H, H-3b`, J
7.2 Hz, J
7.2
Hz), 4.13 (ct, 1H, H-2`, J
2.6 Hz, J
2.6 Hz, J
2.6 Hz),
5.15 (d, 1H, H-1`, J
2.5 Hz), 6.22
(s, 1H, CHCl
), and 7.89 (AA` BB`, 4H, H-2, H-3, H-5, H-6, consisting of doublets at
7.62 and 8.17 ppm, J = J
= 8.9 Hz). The
H NMR spectrum of 3`-phospho-Cm
contained signals at (ppm) 3.89 (m, 1H, H-3a`), 4.07 (m,
1H, H-3b`), 4.35 (m, 1H, H-2`), 5.22 (d, 1H, H-1`, J
3.5 Hz), 6.23 (s, 1H,
CHCl
), and 7.92 (AA` BB`, 4H, H-2, H-3, H-5, H-6, consisting of doublets at 7.63 and 8.22
ppm, J 8.8 Hz, J
8.7 Hz).
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). 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.Mass Spectrometry
Mass spectra were obtained on an
API/III triple quadrupole mass spectrometer (Perkin-Elmer SCIEX
Instruments, Thornhill, Ontario) using nebulizer-assisted electrospray
ionization (ion spray). The mass spectrum of 3`-phospho-Cm was acquired
in the negative ion mode; 2 µl of a 7.5 µg/µl solution was
injected into a stream of 50% acetonitrile (flow rate 10 µl/min).
The mass spectrometer scanned over a mass range of 100-500 in
steps of 0.1 Da, with a dwell time of 2 ms/step. The spectrometer was
calibrated with a cesium nitrate standard; mass assignments were judged
to be reliable to ±0.5 Da.Antibiotic Assay
Assay disks (1.3-cm diameter)
impregnated with aqueous solutions of Cm or 3`-phospho-Cm at known
concentrations were placed on Difco nutrient agar seeded (2% v/v) with
a 48-h nutrient broth culture of Micrococcus luteus. The
relative antibiotic activities of the test samples were determined by
measuring the diameters of inhibition zones around the disks after
incubation overnight at 30 °C.Construction and Use of Shuttle Vector
pSV1.6
Standard procedures were used for manipulating DNA from Streptomyces spp. (Hopwood et al., 1985) and E.
coli (Sambrook et al., 1989). DNA fragments from
restriction digests separated by agarose gel electrophoresis were
purified with a Geneclean kit (BIO-101); E. coli plasmids were
purified with a Wizard miniprep kit (Promega). A 1.6-kbp fragment of
the 2.4-kbp insert in pJV7 was produced by digesting the plasmid with SmaI. Cloning the purified fragment in pUC19 gave pDC1.6. To
construct an E. coli/Streptomyces shuttle vector from pDC1.6,
the plasmid was digested with HindIII and ligated to pIJ6017,
also digested with HindIII. The ligation mixture was used to
transform S. lividans M252; selection for transformants
resistant to kanamycin and Cm (20 and 12.5 µg/ml, respectively)
yielded pSV1.6.Construction and Use of the ORF2/ORF3 Deletion Vector
pDC-ORF1
To test whether ORF1 alone could confer Cm resistance,
a 1-kbp fragment containing ORF2 and ORF3 was excised from pJV7. The
plasmid was first linearized with BstEII, and the 3` overhang
was filled in using the Klenow fragment of DNA polymerase I; digestion
of the product with Acc65I furnished a 1.4-kbp fragment of
insert DNA. Plasmid pJV4 was then digested with Acc65I and EcoICRI to recover the pIJ702-derived 4.7-kbp vector fragment
free of the 2.4-kbp insert. The 1.4- and 4.7-kbp fragments were
ligated, and the reaction product was used to transform S. lividans M252. From a transformant resistant to thiostrepton (20
µg/ml), plasmid pDC-ORF1 was isolated. Analysis of culture
filtrates by HPLC showed that cells containing pDC-ORF1 were not able
to convert Cm to 3`-phospho-Cm. No colonies were obtained when cells
containing pDC-ORF1 were plated on solid media containing Cm (12.5
µg/ml).Construction and Use of the Disrupted ORF2 Vector
pJV11-
The SalI site in the polylinker region
of pJV7 was removed by digesting the plasmid with HindIII and XbaI, followed by end-filling and religation. Digestion of the
resulting plasmid pJV7a with SalI and religation yielded pJV7b
in which the 363-bp SalI-SalI segment internal to
ORF2 in the plasmid insert had been deleted. The modified insert was
excised from pJV7b as a 2.0-kbp BamHI-SacI fragment,
and recloned in pHJL400 linearized by digestion with BamHI and SacI, to give pJV11-
SalSal. As a control, the intact insert
from pJV7a was recloned in pHJL400 in the same way to give pJV11.
Colonies obtained by transforming S. lividans M252 with pJV11
grew within 2 days of transfer to MYM agar containing 8 µg/ml Cm,
whereas transformants containing pJV11-Sal failed to grow within 5
days on this medium. Mycelium from the transformants grown for 48 h in
GNY medium containing 5 µg/ml thiostrepton for plasmid maintenance
was washed and resuspended in aqueous Cm (50 µg/ml). Analysis of
the suspension at intervals by HPLC showed no conversion to
3`-phospho-Cm by mycelium from pJV11-Sal transformants, whereas
conversion occurred in control suspensions of mycelium carrying pJV11.DNA Sequence and Sequence Analysis
For sequencing,
the 2.4-kbp KpnI-SstI fragment of pJV4 was subcloned
as smaller segments in the vectors M13 mp18, pTZ18R, and pBluescriptII
SK+. Sets of overlapping nested deletions were prepared with
exonuclease III and mung bean nuclease (Mosher, 1993). Single-stranded
DNA, prepared either directly or by infection of a phagemid
transformant with helper phage (VCSM13), was sequenced by the dideoxy
chain termination method (Sanger et al., 1977) using a
Sequenase version 2.0 kit (U. S. Biochemical Corp.). Both strands were
sequenced, and ambiguous regions were resolved by using synthetic
oligodeoxynucleotides (15-21-mers) derived from the generated
sequence as sequencing primers, as well as by using deaza-dGTP or dITP
in place of dGTP in the sequencing reactions.
Isolation of a Novel Cm Metabolite
Analysis by HPLC of filtrates from cultures grown in GNY
medium with Cm at the highest concentration tolerated (12.5 µg/ml)
revealed that S. lividans RM3 and RM4 rapidly converted the
antibiotic to a single major extracellular product (Fig. 2) with
an absorption maximum (273 nm) identical to that of Cm. In direct
comparisons, the retention time of the Cm product (5.9 min) differed
not only from that of Cm (6.3 min) but also from other related
reference compounds, including 1`,3`-diacetyl-Cm (6.8 min), Cm-base
(the free amine of Cm; 4.9 min), and N-acetyl-p-nitrophenylserinol (5.5 min).
Identification of the Cm Metabolite
The H NMR spectrum of the Cm metabolite was
similar to that of Cm in that it contained signals consistent with the
presence of a dichloroacetyl group and a 1,4-disubstituted
phenylpropanoid derivative. However, it differed in the spin-spin
coupling pattern of protons associated with the propanediol moiety.
Whereas the H-3a` and H-3b` resonances for Cm were
present as quartets due to coupling with each other and with H-2`, the resonances in the metabolite showed complex
spin-spin coupling, and were displaced downfield with respect to those
given by Cm. The data suggested that the propanediol protons of the
metabolite were deshielded by an electron-withdrawing functional group
probably attached at C-3`.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.
Cl
was
401.9786.
Sequence and Analysis of the pJV4 Insert
The 2.4-kbp KpnI-SstI insert of S.
venezuelae DNA cloned in pJV4 contained a 2355-bp nucleotide
sequence (Fig. 4) with 73.6% G + C content. A sequence
analysis (Fig. 5) of both strands for third position G + C
bias, and for codon usage (Wright and Bibb, 1992), indicated one
incomplete and three complete ORFs, the codon usage in which was
typical of streptomycetes (Bibb et al., 1984).
Comparison of Derived Amino Acid (aa) Sequences with Data
Base Sequences
ORF1
There are two possible ATG start sites for
ORF1 (nt 28-30 and nt 100-102; see Fig. 4); the
first gives a polypeptide 25 amino acids longer than the second. A
FASTA sequence alignment (Pearson and Lipman, 1988) comparing ORF1 with
sequences in the GenBank and EMBL data bases showed ORF1 to be 42.2%
identical over 386 amino acids to CMR, the Cm resistance protein of Rhodococcus fasciens (Desomer et al., 1992), and
39.8% identical over 372 amino acids to CmlG of S. lividans (Dittrich et al., 1991). The amino termini
of CMR and CmlG align optimally with Met-25 of ORF1 rather than with
Met-1, and since AraJ (Reeder and Schleif, 1991) and NorA (Yoshida et al., 1990), two other hydrophobic proteins with sequence
similarity to ORF1, also align at their amino termini with Met-25, the
translational start codon for ORF1 is likely to be the ATG at nt
100-102. This being so, then ORF1 is a polypeptide of 412 amino
acids (M 41,479).
-helical
segments that form a transmembrane channel (Paulsen and Skurray, 1993). ORF2
Translation of ORF2 begins at either of two
possible start codons: GTG at nt 1412-1414 or ATG at nt
1424-1426. The sequence 5`-GGTGA-3`, showing complementarity to
the 3` end of S. lividans 16 S rRNA (G =
-11.6 kcal: Tinoco et al., 1973; Bibb and Cohen, 1982)
is present 4 bp (nt 1403-1407) upstream of the GTG codon, and
also 8 bp (nt 1411-1415) upstream of the ATG codon. The 4-bp
distance is below the range (5-12 bp) commonly observed for the
separation between a ribosome-binding site and the translational start
point in streptomycetes (Strohl, 1992). Beginning 26 bp upstream of the
GTG codon, the sequence 5`-CACCGT-3` (nt 1381-1386) represents a
possible -10 hexamer recognized by E-like RNA
polymerase (Strohl, 1992). However, the region upstream of the GTG
codon contains a second such sequence (5`-TACGGT-3`; nt
1400-1405). Transcription from this hexamer should initiate at
the first nucleotide of the GTG codon. Such a concurrence of
translational and transcriptional start sites is not uncommon in
streptomycetes (Strohl, 1992).
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).
ORF3
Translation of ORF3 from the possible GTG
start codon (nt 2266-2264; Fig. 4) would generate a
polypeptide of 89 amino acids with a deduced M of
9,767. A comparison of the amino acid sequence with those in GenBank
and EMBL data bases revealed no significant similarities.Identification of the Cm Resistance Determinant in
the 2.4-kbp Insert
The striking sequence similarity between ORF1 and gene
products associated with antibiotic resistance in other bacteria
suggested a role for ORF1 in the Cm resistance of S. lividans RM4. However, the genes for CMR and CmlG confer resistance to
concentrations of Cm greater than 200 µg/ml on their hosts
(Dittrich et al., 1991; Desomer et al., 1992),
whereas the maximal resistance to Cm conferred upon S. lividans M252 by pJV4 is 12.5 µg/ml. To determine if Cm resistance is
affected by the absence of ORF2 and ORF3, a 1.4-kbp Acc651-BstEII fragment containing only ORF1 was
excised from pJV7 (E. coli vector pTZ18R carrying the 2.4-kbp KpnI-SstI insert from pJV4) and subcloned in the
residual pJV4 vector segment obtained by digesting pJV4 with Acc65I and EcoICRI to remove the 2.4-kbp insert. The
presence of the 1.4-kbp insert in the resulting plasmid (pDC-ORF1; see Fig. 6) was verified by restriction enzyme analysis.
Transformation of S. lividans M252 with pDC-ORF1 gave
Cm-sensitive colonies, a result indicating that ORF1 by itself did not
confer the Cm resistance phenotype of pJV4 transformants.
Subcloning and Expression of ORF2 in S. lividans
To determine whether ORF2 alone conferred Cm resistance in S. lividans RM4, the 1.67-kbp SmaI-SmaI
segment, lacking large segments of both ORF1 and ORF3 (see Fig. 6), was excised from pJV7 and cloned in the E. coli vector pUC19. The resultant plasmid (pDC1.6) was linearized with HindIII, and ligated to the Streptomyces vector
pIJ6017 carrying the thiostrepton-inducible promoter tip (Murakami et al., 1989). ()The E.
coli-Streptomyces shuttle vector (pSV1.6) so formed was used to
transform S. lividans M252, and colonies were selected for Cm
resistance. In bioassays these resistant transformants were inhibited
by Cm concentrations above 10 µg/ml, compared with 12.5 µg/ml
for RM4. When cultures were grown in GNY medium containing Cm, HPLC
analysis of the culture supernatant showed that the antibiotic was
converted to 3`-phospho-Cm; however, concentrations in the supernatant
were only 20% of those in RM4 cultures. We were unable to measure
3`-phospho-Cm concentrations in the cells from these cultures. Since Cm
resistance was observed without induction of the tip promoter,
the presence of a functioning promoter upstream of ORF2 seems likely.
Overall, the results indicate that phosphorylation by the ORF2 product
is mainly responsible for the Cm resistance phenotype of S.
lividans RM4, but the ORF1 product may also have a role.
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. 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.
), deletion; ORF, open reading
frame; Kan, kanamycin; Amp, ampicillin; Thio, thiostrepton; kbp,
kilobase pair; nt, nucleotide; bp, base pair(s).)
We thank Dr. D. Hooper, Chemistry Department,
Dalhousie University, for the NMR spectra and Dr. P. Thibault,
Institute for Marine Biosciences, National Research Council of Canada,
for mass spectrometry. We are also grateful to Drs. H. I. Schrempf,
Universität Osnabrück, Germany,
and M. J. Bibb, John Innes Institute, Norwich, UK, for gifts of S.
lividans M252 and plasmid pIJ6017, respectively.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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