From the Biozentrum, University of Basel, Department of Microbiology, Klingelbergstrasse 70, CH-4056 Basel, Switzerland
Received for publication, August 23, 2000, and in revised form, October 23, 2000
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ABSTRACT |
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Pip is a
pristinamycin-induced transcriptional regulator
protein detected in many Streptomyces species
by its ability to specifically bind sequence motifs within the promoter
of a Streptomyces pristinaespiralis multidrug resistance
gene (ptr). To investigate the possible role of Pip in
regulating multidrug resistance, it was purified from a genetically
characterized species, Streptomyces coelicolor, utilizing
an affinity matrix of the ptr promoter conjugated to magnetic beads. Reverse genetics identified the corresponding locus and
confirmed that it encoded Pip, a protein belonging to the TetR family
of procaryotic transcriptional repressors. Pip binding motifs were
located upstream of the adjacent gene pep, encoding a major
facilitator antiporter homologous to ptr. In vivo analysis
of antibiotic susceptibility profiles demonstrated that pep conferred
elevated levels of resistance only to pristinamycin I (PI), a
streptogramin B antibiotic having clinical importance. Purified
recombinant Pip was a dimer (in the presence or absence of PI) and
displayed a high affinity for its palindromic binding motifs within the
ptr promoter and the upstream region of pep. The Pip/ptr promoter complex was dissociated by PI but not
by any of the other nonstreptogramin antibiotics that were described previously as transcriptional inducers. These procaryotic
regulatory elements served as the basis for the development of systems
allowing repression or induction of cloned genes in mammalian and plant cells in response to streptogramin antibiotics (including
pristinamycin, virginiamycin, and Synercid®).
Streptomyces are Gram-positive mycelial soil bacteria
that undergo a complex developmental program involving morphological differentiation coordinated with biosynthesis of a vast array of
structurally diverse secondary metabolites. Although most are of
unknown function, many have antimicrobial activity, making this genus
the most abundant known source of antibiotics. Streptomyces require extensive collections of resistance genes and corresponding regulatory genes to protect themselves from these endogenous
metabolites as well as those produced by competing species. These genes
are believed to be the progenitors of resistance determinants acquired by pathogenic bacteria (1, 2) that are progressively eroding the
efficacy of antibiotics.
The genetic control and mechanism of tetracycline resistance has been
well characterized. The expression of tetA, encoding an integral membrane protein of the major facilitator superfamily (MFS)1 that exports
tetracycline, is under the control of the TetR repressor. In the
absence of tetracycline, transcription of tetA and the divergent tetR (3) is repressed by TetR. tetA is
efficiently expressed only when TetR is released from its operator
sites by its association with tetracycline or its analogs. Based on
these characteristics, the laboratories of Hillen and Bujard have
engineered systems for tetracycline-regulated gene expression in
eucaryotic cells (4). An advantage of such systems is that the
regulatory protein, along with its DNA recognition motif and ligand,
are procaryotic and thus minimize pleiotropic effects within host regulatory circuits.
Pairs of genes homologous to tetR/tetA found within
Streptomycete biosynthetic gene clusters serve to respond to
and export the cognate antibiotics. For example, ActII-orf1 and TcmR
repress promoters controlling divergent structural genes encoding
proteins that export actinorhodin (actII-orf2-3) (5)
or tetracenomycin (tcmA) (6). However, QacR, a TetR-like
repressor of a multidrug resistance gene in Staphylococcus
aureus, can bind heterogeneous compounds (7).
Resistance and corresponding regulatory genes present in organisms
producing several antibiotics have potentially interesting multidrug
recognition capabilities. Streptomyces pristinaespiralis produces the human oral streptogramin antibiotic Pyostacin® (8). Like
other streptogramins, it is a mixture of two structurally dissimilar
molecules, the streptogramin A component pristinamycin II (PII), a
polyunsaturated macrolactone, and the streptogramin B component
pristinamycin I (PI), a cyclic hexadepsipeptide (8). The water-soluble
form of pristinamycin, Synercid®, recently approved in the United
States and Europe for use against most multiple drug-resistant
Gram-positive bacteria, is composed of dalfopristin, a 26-sulfonyl
derivative of PII, and quinupristin, which is derived from PI by
synthetic addition of a
(5 A S. pristinaespiralis pristinamycin
resistance gene, ptr, encoding a MFS antiporter
was cloned in Streptomyces lividans where it provided an
increase in resistance to not only PI and PII but also rifampicin (10).
Interestingly, the ptr gene was not situated within the
pristinamycin biosynthetic cluster (11, 12).
It was surprising to find that many Streptomyces hosts, most
not known to produce streptogramin antibiotics, nevertheless had
systems to control the ptr promoter (13). Studies carried out in S. lividans and Streptomyces coelicolor
showed that the ptr promoter (Pptr) was activated
not only by PI and PII but also by a wide range of heterogeneous
compounds (14). Gel retardation using a DNA fragment encoding the
Pptr indicated the activity of a
pristinamycin-induced DNA binding
protein (Pip). Analysis of mutations and DNA protection
assays identified the binding sites as a series of three similar motifs
having dyad symmetry (GTACRSYGTAY) within the Pptr region
(13).
Here we report the purification of Pip, identification of the
corresponding locus in S. coelicolor, and definition of its ligand specificity. Based on these results, Pip has been adapted as a
central element in the design of novel systems for regulated gene
expression in mammalian and plant cells utilizing clinically approved
streptogramin antibiotics as controlling agents
(15).2
Bacterial Strains and Plasmids--
S. lividans 1326 and S. coelicolor J1501 (hisA1 uraA1 strA1 pgl
SCP1 Isolation of the Pptr and Ppep Promoter Fragment--
Plasmids
were isolated from E. coli by alkaline lysis and PEG 8000 precipitation (19). Fragments were precipitated in isopropanol and
purified on a 1% TBE (90 mM Tris, 90 mM
borate, and 2 mM EDTA, pH 8.0) agarose gel.
The Pptr promoter was isolated from a genomic DNA clone by
subcloning it on a 219-bp Sau3A fragment into pUC19
(pIP1911, 14). The Ppep promoter was isolated from genomic
DNA (clone pRM515) by subcloning it on a 228-bp
BamHI/HinfI fragment into pK18. Both Pptr and Ppep fragments were purified after
digestion with EcoRI and HindIII.
Gel Retardation Assays--
The promoter fragments were labeled
using T4 polymerase to fill in the HindIII end with
[ Preparation of Magnetic Beads Containing the Pptr Promoter
Fragment--
The Pptr fragment (100 µg) was biotinylated
by filling in the HindIII end with biotin-labeled dATP using
T4 polymerase (Roche Molecular Biochemicals). Unincorporated
dATP-biotin was removed using a Quick spin desalting column (Roche
Molecular Biochemicals). The efficiency of the labeling reaction was
monitored by testing the ability of streptavidin (Sigma) to retard
migration of the biotin-labeled fragment during electrophoresis in a
1% agarose gel. Fully biotinylated Pptr fragment was then
mixed with streptavidin-coated magnetic beads.
Purification of Pip--
S. coelicolor J1501 was
cultured in 350 ml of YEME liquid medium (17) in a baffled
1-liter Erlenmeyer flask at 30 °C for 48 h on a rotary
shaker. Aliquots of this "preculture" (40 ml) were used to
inoculate ten 5-liter baffled Erlenmeyer flasks, each containing 2 liters of YEME. These cultures were then incubated for 20 h at
30 °C. Pip synthesis was induced for 3 h by adding 20 µg/ml
PI (13). Pip activity was monitored throughout using the gel
retardation assay with radiolabeled Pptr fragment.
Cells were harvested from YEME (20 liters) by centrifugation (30 g of
wet weight), washed in lysis buffer (200 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, 5% (v/v)
glycerol, 50 mM Tris, pH 8.0), resuspended in 150 ml of the
same buffer, and sonicated (Branson Sonifier 250). After cell
disruption, the extract was diluted with an equal volume of TGED
containing 2 mM phenylmethanesulfonyl fluoride, 1 mM pepstatin, and 1 mM benzamidine. The cell
debris was separated from the supernatant by two centrifugation steps: 1) 10,000 rpm for 15 min in a Sorvall GSA rotor, and 2)
100,000 × g for 30 min in a TFT 45/94 Kontron rotor.
Salt was added to the extract bringing its conductivity up to the
equivalent of 100 mM NaCl. This extract was loaded at a
rate of 3 ml/min onto a 100-ml DEAE column (XK26/50; Amersham Pharmacia
Biotech) equilibrated with TGED + 100 mM NaCl. The matrix
was washed with five column volumes of the same buffer and monitored by
adsorption at 280 nm. The protein was eluted from the column with 10 volumes of buffer providing a linear gradient of NaCl (100-500
mM). Pip activity was observed in fractions containing
~200 mM NaCl. These fractions were dialyzed against TGED + 75 mM NaCl to bring the conductivity equal to that of 100 mM NaCl. The dialysate was then loaded at a rate of 1.5 ml/min onto a 25-ml Sp-Sepharose column (XK16/20; Amersham Pharmacia
Biotech) equilibrated with TGED + 100 mM NaCl. The column
was washed until a stable adsorption base line was attained, and the
protein was eluted in a linear gradient of TGED + 500 mM
NaCl (seven column volumes). Pip activity was observed in the eluent
having conductivity corresponding to 230-300 mM NaCl. The
active fractions were pooled and incubated in a suspension (3 ml of
TGED + 100 mM NaCl, 300 µg of poly(dI-dC)·poly(dI-dC)) of the Pptr fragment coupled to magnetic particles. The mix
was incubated at room temperature for 1 h while keeping the
particles suspended. The particles were collected using a magnet,
washed with 2 ml of TGED + 100 mM NaCl containing 100 µg
of poly(dI-dC)·poly(dI-dC) and 2 ml TGED + 250 mM NaCl.
Pip was eluted in a step (1 ml) gradient ranging from 300 mM to 2.5 M NaCl (in TGED).
Column Chromatography--
Apparent molecular weight was
determined by column sizing chromatography (Superdex 75 SMART System)
in comparison with protein molecular weight standards. Protein bound
with putative ligand were separated on Superdex75 and further analyzed
by HPLC reverse phase chromatography using a C18 column in a gradient
of H2O/acetonitrile/trifluoroacetic acid 0.1%.
Peptide and N-terminal Analysis--
After SDS-PAGE,
proteins were stained with Coomassie blue in 40% methanol/1% acetic
acid. Regions of the gel containing the protein bands were cut out,
rinsed in water, and digested with porcine trypsin (Sigma). Peptides
were separated by HPLC (DEAE-C18) in a gradient of
acetonitrile/trifluoroacetic acid (0.1%) and then analyzed by Edmann degradation.
Isolation of the pip Gene--
PCR reactions were performed in a
thermal cycler (Biometra, Göttingen, Germany); reaction products
were purified on 5% polyacrylamide TBE gels for analysis and cloning
in pCR2 (Stratagene). Amplification of the Pip gene was achieved using
degenerate primers corresponding to the two peptides (pep1
(DXVWLGEGR); gt(g/c)tggct(g/c)(a/t)(g/c)(g/c)gg(g/c)gaggg; pep2 (HPDPDAGLD; reverse strand):
ccagtc(g/c)ag(g/c)cc(g/c)gcgtc(g/c)gggt). The reaction was carried out
in 25 cycles including 30-s stages of annealing (58 °C in 10%
Me2SO), denaturation (94 °C), and extension using
Taq polymerase (Roche Molecular Biochemicals; 72 °C).
In Vivo Functional Analysis of pep--
The pep gene
was amplified from the genomic clone using the primers P1 and P2 (P1:
5'-TCTAGAACTACTTCATCGGTGGTGGC-3'; P2:
5'-AAGCTTCCGCCGGTCGGCGAAGCG-3'), tagged with a
XbaI and HindIII recognition sequences (bold
type), using the high fidelity Expand PCR system (Roche Molecular
Biochemicals) under the conditions recommended by the manufacturer (but
with the addition of 5% Me2SO). The fragment was cloned
into pGemT (Promega), and its sequence was confirmed. The cloned
fragment was excised with XbaI/HindIII and cloned
into pIJ486 (pRM516).
Expression and Purification of Recombinant Pip--
The coding
region of pip was amplified by PCR using the
oligonucleotides P3 and P4 (P3,
5'-gggaattccatATGagtcgaggagaggtgcgcatg-3'; P4,
5'-gatcaagcttggcggacgactagggcctgtc-3'), which were designed to
incorporate a NdeI site overlapping the start codon and a
HindIII site following the stop codon and direct repeats.
This fragment was cloned into the PCR cloning vector pGEMT, and a clone
was verified by sequencing (pRPM4). A
NdeI/HindIII fragment was subsequently subcloned
into the expression plasmid pDS56/RBSII, generating pRPM10.
Overproduction, in a 25-liter fermentor, was accomplished using
E. coli M15 (pREP4) cells harboring pRPM10 in the presence of 100 mg/liter ampicillin and 25 mg/liter kanamycin. Expression was
induced by the addition of
isopropyl- Drug Resistance Measurements--
Antibiotic resistance was
measured on NE medium (11) using either a disc (discs provided
by Pasteur Diagnostics) diffusion assay (zones of inhibition were
measured after 48 h at 30 °C) or as the concentration of
antibiotic lethal for colonial growth (scored after 1 week at
30 °C).
Ultracentrifugation--
Sedimentation velocity and
sedimentation equilibrium centrifugations were carried out at 20 °C
using a solution of 1 mg/ml protein in a TA buffer (10 mM
Tris, pH 7.5, 10 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol, 0.1% Triton, 10% glycerol) in
a XLA Beckman analytical ultracentrifuge equipped with adsorption
optics. The sedimentation velocity run was made in a 12-mm double
sector cell at 54,000 rpm and scanned at 277 nm. The sedimentation
equilibrium run was carried out with the same cell at a rotor speed of
18,000 rpm. Both sectors, one with the protein solution and one with the reference, were filled to a height of 2.5 mm on top of the Fc-43
bottom fluid. The molecular masses were evaluated using a floating
base-line computer program that adjusts the base-line absorbance to
obtain the best linear fit of lnA versus
r2 (A = absorbance;
r = radial distance). A partial specific volume (v) of 0.73 was assumed. The solution density (1.034 g/cm3) and viscosity (1.30 centipoise) were taken from the
CRC Handbook.
Purification of Pip from S. coelicolor--
Purification of Pip
from PI-induced S. coelicolor cultures was performed using
various combinations of standard column chromatography. Initial studies
established a convenient protocol using DEAE/SP-Sepharose chromatography to enrich Pip. Pip activity was detected by gel mobility
shifts resulting from Pip binding to the three operator sites in the
ptr promoter (details found under "Experimental
Procedures").
The final purification step, which allowed Pip to be convincingly
identified and sequenced, was based on a Pptr DNA affinity matrix (Fig. 1A). The
Pptr affinity matrix was prepared from biotin end-labeled
Pptr fragment conjugated to streptavidin-coated magnetic beads ("Experimental Procedures"). The Pip-enriched fractions from
SP Sepharose were incubated with the affinity beads. The beads were
then sedimented with a magnet and washed repeatedly in low salt buffer.
Proteins were stepwise eluted in buffers containing increasing salt
concentration (Fig. 1B). Only minor amounts of Pip activity
eluted in a buffer containing poly(dI-dC)·poly(dI-dC) (Fig. 1B,
lane 3); many proteins were observed by SDS-PAGE in this
fraction (Fig. 1A, lane 3). Most Pip activity
eluted in fractions of higher salt concentration (Fig. 1B,
lanes 4 (300-400 mM), and 5 (600 mM-2.5 M NaCl)). A 28-kDa protein, the major
band detected in these high salt washes, coeluted with Pip activity.
The Pptr affinity matrix allowed final purification of 10 µg of Pip from ~100 mg of crude protein extract.
Cloning of the S. coelicolor pip Gene--
The 28-kDa protein was
eluted from an SDS-PAGE gel and digested with trypsin to generate
peptide fragments that were isolated by HPLC and subjected to
N-terminal analysis by Edmann degradation. Four peptide
sequences were obtained: pep1, DxVWLGEGR; pep2, HPDPDAGLD; pep3, PWSSR;
and pep4, VAEMLDR. The longest peptides, pep1 and pep2, were also
identified in tryptic digests of Pip similarly purified from S. lividans (data not shown). A 300-bp PCR fragment, amplified using
the degenerate oligonucleotides P1 and P2, was used to probe Southern
blots of BamHI-digested S. coelicolor genomic DNA. A hybridizing fragment having a convenient size (~5.5 kilobase pairs) was isolated from the gel and cloned into the BamHI
site of pUC18 (pIP515). Crude extracts prepared from pRM515
transformants had Pip activity in the gel retardation assay (data not shown).
Nucleotide Sequence Analysis of the pip Locus--
Nucleotide
sequence (accession number AF193856) within the 5.5-kilobase pair
BamHI fragment provided further proof that it encoded Pip,
the ptr transcriptional regulator. The pip gene sequence predicted a protein with a pI (5.8), mass (28 kDa; 259 amino
acids), and peptide sequences (N-terminal as well as pep1-4) indistinguishable from Pip purified from crude extracts.
The Pip protein sequence was most closely related to RifQ, a putative
transcriptional repressor in the rifamycin biosynthetic cluster of
Amycolatopsis mediterranei (106/243, 43% identical amino
acids) (20), and ActII-orf1, a repressor in the actinorhodin biosynthetic pathway (38 of 122, 31% identical amino acids) (5). The
N-terminal region of these proteins contained a helix-turn-helix motif
that fitted the consensus of the tetracycline resistance repressor
family (TetR) (3).
The nucleotide sequence upstream of the pip gene predicted a
transcriptionally coupled open reading frame (Fig. 3; pep)
whose amino acid sequence had significant similarity to the MFS
14-spanner drug antiporters (21). The best matches were to
streptogramin resistance genes: the S. pristinaespiralis
pristinamycin resistance gene (encoding Ptr, 73% amino acid identity)
(10) and the Streptomyces virginiaensis virginiamycin
resistance gene (encoding VarS, 67% amino acid identity) (22).
Nucleotide homology between the pep and ptr loci
extended for at least 200 bp upstream of their coding sequences (Fig.
2). Pep had strong similarity (65%
identical amino acids) to RifP, encoded by an open reading frame
immediately upstream of rifQ (20). Many other proteins
having 25-40% identity with Pep were involved either with antibiotic
export in Streptomyces that produce various antibiotics or
with multidrug resistance in diverse bacteria (21).
Regulatory Pip binding motifs identified within the Pptr
(13) were also found in the corresponding region upstream of
pep (Fig. 2). Only two of the three pip operator
sites in the Pptr promoter were conserved in the putative
region of S. coelicolor pep promoter (Ppep). The
regions of the Ppep which aligned with the Pptr
promoter hexamers (Fig. 2) were well conserved (6 of 6 in the In Vivo Functional Analysis of pep--
The pep gene
along with 205 bp of upstream sequence was cloned into pIJ486 (pRM516)
and introduced into S. lividans. Transformants were assayed
by disc diffusion tests for antibiotic susceptibility against a
spectrum of 60 structurally and functionally dissimilar antibiotics
including bacitracin, chloramphenicol, clindamycin, erythromycin,
fusidic acid, lincomycin, PI, PII, rifampicin, spiramycin, streptomycin, and tetracycline. The pep gene conferred a
detectable elevated resistance only to PI. Subsequent determination of
the minimal inhibitory concentration (10 µg/ml) on solid medium
showed a 4-fold increase in resistance to PI for strains harboring
pRM516 (40 µg/ml).
Pip Sequence-specific Interactions--
Recombinant Pip purified
from E. coli extract had chromatographic characteristics (on
DEAE and heparin) similar to the native protein purified from S. coelicolor. Moreover, both bound specifically to repeated sequence
motifs present within Pptr and Ppep having the
consensus RTACRSYGTAY. The gel retardation pattern generated by Pip
corresponded to the degree of site occupancy, a total of three discrete
retarded bands for the Pptr fragment (Fig.
3A, points A-H) as
previously documented (13), and two for the Ppep fragment
(Fig. 3B, points A-H). These titration profiles
on each promoter can be interpreted as a noncooperative binding to each of the operator sites. The binding curve determines an approximate Kd of 10 Ligand Recognition Specificity of Pip--
The ligand specificity
of Pip was established using gel retardation to test the ability of
purified recombinant Pip to bind either Ppep fragment (Fig.
4) or Pptr fragment (data not
shown) in the presence of various antibiotics. Pip was released from the Ppep by equimolar amounts of PI. Quinupristin, a
chemically modified PI derivative, apparently had 3 orders of magnitude
less affinity for Pip. In contrast, a series of other antibiotics that induce the Pptr in vivo, including bacitracin,
chloramphenicol, clindamycin, erythromycin, fusidic acid, lincomycin,
PI, PII, rifampicin, spiramycin, streptomycin, and tetracycline, did
not significantly affect the Pip operator binding (representative results are shown in Fig. 4). These data showed that the PI component of the streptogramin antibiotic complex was the preferred Pip ligand. A
binding curve (Fig. 5) representing the
inhibition of shift as a function of increasing PI approximates a
Kd of 10
Pip (PI-treated or untreated) eluted from a sizing column with an
apparent molecular mass of ~50 kDa, indicating a dimeric nonaggregated form under native conditions (Fig.
6). Fractions from the sizing column
containing PI-treated or untreated Pep were then analyzed by reverse
phase chromatography (Fig. 6) under conditions expected to denature Pep
and release the ligand. Indeed, a compound that comigrated with PI was
detected only in the PI-treated Superdex fraction. Approximate
quantification of the HPLC peaks suggested an equimolar binding
stoichiometry between the native protein monomer and PI. This proved
that the interaction was of a noncovalent nature allowing a reversible
association of the antibiotic ligand with this regulatory protein.
Ultracentrifugation--
Sedimentation equilibrium data predicted
Pip to have a molecular mass of 64 kDa (Fig.
7A). This was not
significantly different (the significant difference of these
measurements is ~5%) from the predicted mass of a Pip dimer (57,032 Da). The addition of PI (Fig. 7B) did not change its
measured mass (60 kDa).
Sedimentation velocity analyses (Fig. 7) showed that recombinant Pip
purified from E. coli was >90% soluble, presumably in its
native conformation. Its sedimentation coefficient in the absence of PI
(s20, w = 3.4) was not significantly altered by
the addition of PI (s20, w = 3.5).
Thus, Pip was dimeric, and PI did not generate detectable changes in
multimerization (dimer) or shape (Pip f/f0 = 1.70; Pip+PI f/f0 = 1.58). Although PI was
presumably complexed to Pip, in the absence of dramatic shape changes,
the additional mass (853 Da) would not be expected to have a detectable
effect on its sedimentation velocity or sedimentation
equilibrium-measured molecular weight.
Pip proved to be a protein with homology and function similar to
the TetR transcriptional repressor that recognizes a streptogramin B
antibiotic rather than tetracycline. The affinity of Pip for PI
measured in vitro (Kd = 10 Multidrug resistance can be dependent on global regulators of general
stress responses such as sigma factors or transcriptional activators.
Mutations in sigma factors that mediate stress responses can decrease
antibiotic resistance in S. aureus (24) and E. coli (25). In E. coli, batteries of efflux systems
having overlapping specificity provide resistance to hydrophobic
compounds (26). Several of these loci encode an efflux protein and
divergently transcribed TetR homolog (26). In at least one well
characterized system, acr, transcription of the transporter
gene involves the AraC-like transcriptional activator proteins
marA, soxS, and rob (27, 28). In
Neisseria gonorrhoeae, a comparable multidrug resistance
locus including a TetR-like protein (AcrR), is also activated by an
AraC-like protein (29).
Pip may play a subordinate role as a secondary modulator in a similar
multidrug responsive regulatory system. Two Pip homologs, AcrR and
MtrR, are not required for initial transcriptional activation during
the multidrug resistance response. Instead, they act primarily as
repressors, apparently limiting growth inhibitory effects resulting from overexpression of their corresponding MFS pumps (30). Although compounds that release AcrR and MtrR repressors from their binding sites are unknown, we have identified a specific antibiotic (PI) as a
Pip ligand.
Many antibiotic resistance genes are linked to antibiotic biosynthetic
clusters in Streptomyces (31). This does not appear to be
the case for pep. Streptogramins (PI-like) have not been detected in S. coelicolor. In addition, its adjacent
sequence (4 kilobase pairs downstream of pep; S. coelicolor cosmid D13)3
did not indicate linkage to known antibiotic biosynthetic genes. Although Pep may be an independent drug resistance element that responds to and provides resistance specifically to PI, its genetic organization was unlike homologous antibiotic resistance loci of
Gram-negative organisms.
Hillen and Berens (3) have shown that the characteristic divergent
polarity of TetR regulators and MFP genes is important for inducibility
to toxic compounds. The arrangement of operator sites having different
TetR affinities between the promoters provides for differential
expression of the two genes. This apparently allows transcription of
tetR that is sufficient to effectively repress
tetA under noninducing conditions. The second characteristic feature of this arrangement is that the tetA promoter is
fully activated in response to a narrow range of tetracycline
concentration (3). In contrast, the coupled transcriptional
organization of pep and pip does not provide for
such an amplified response of the resistance gene relative to its repressor.
Similar linkage of antibiotic export genes to those encoding
Pip/TetR-like regulators has been observed in antibiotic biosynthetic gene clusters including: tetracenomycin (tcmR/tcmA) (32),
actinorhodin (actII-orf1/actII-orf2 and
orf3) (33), rifamycin (rifP/rifQ) (20), and landomycin
(lanJ/lanK) (16). Thus, antibiotic-producing Streptomyces species are a rich source of similar regulatory
elements responding to a wide variety of structurally diverse secondary metabolic compounds. These elements may have important future applications.
Studies reported here have lead to the design of a family of novel
systems for regulated expression of cloned genes in mammalian (15) and
plant cells.2 These systems are based on binding of Pip, or
Pip fused to eucaryotic transcriptional activators or repressors, to
its operators engineered into eucaryotic promoter sequences. Such
constructions have been used to achieve streptogramin-regulated
induction or repression of various therapeutic proteins in diverse cell
lines. The streptogramin-based expression technology was functionally
compatible with a tetracycline-regulated system, thus enabling the
selective use of different antibiotics to independently control two
different gene activities in the same cell. These may serve as
important tools in controlling the timing and levels of gene expression
in plant cells, mammalian cells, transgenic animals, and perhaps future
human gene therapies.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
R)-[(3S)-quinuclidinyl] thiomethyl
group (9).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
SCP2
) were used in these studies (17).
Systems for cloning in Escherichia coli included pBluescript
(Stratagene)/XL1 Blue (Invitrogen), pGemT (Promega)/XL1 Blue and
M15(pRep4)/pDS56/RBSII (18) (provided by Dr. D. Stüber).
-32P]dATP and then purified on a Quick spin column
(Roche Molecular Biochemicals). Crude or semi-purified proteins (5 µg) in 30 µl of TGED (50 mM Tris, pH 8.0, 1 mM dithiothreitol, 1 mM EDTA, 5% (v/v)
glycerol) + 100 mM NaCl were incubated with radiolabeled DNA fragments (2-5 ng) for 30 min at 30 °C in the presence of 1 µg of poly(dI-dC)·poly(dI-dC) (Amersham Pharmacia Biotech). The
reaction mixtures were then resolved on a nondenaturing 5% polyacrylamide gel in TBE buffer run at room temperature and constant voltage (7 V cm
1). After migration, gels were dried, and
bands were visualized by autoradiography. The ability of Pip to retard
migration of fragments containing its operator motif (13) was defined
as "Pip activity."
-D-thiogalactopyranoside to a final
concentration of 0.5 mM. Purification of the protein was
done according to the procedure above omitting the DNA affinity step.
The purified protein retained full activity for more than a year
when stored a
80 °C in 10% glycerol.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Purification of Pip using a Pptr
affinity matrix. A, SDS-PAGE analysis of proteins
binding to the Pptr matrix. Pip activity, partially purified
by conventional column chromatography (DEAE/SP-Sepharose
chromatography) was loaded on a Pptr affinity matrix
prepared by binding biotin-labeled Pptr fragment to
streptavidin-coated magnetic beads. Proteins were eluted in 1-ml
fractions using increasing concentrations of NaCl. After precipitation
in acetone, 20% of the material was loaded on the gel. Lane
1, molecular mass standards. Lane 2, nonbinding
proteins present in the flowthrough. Fractions were eluted in TGED
buffer containing 100 µg of competitor DNA poly(dI-dC) (lane
3), pooled stepwise fractions 300-400 mM NaCl
(lane 4), or 600 mM to 2.5 M NaCl
(lane 5). B, gel retardation assay of
Pip/Pptr binding activity during purification. Lane
1, radiolabeled Pptr in the absence of protein.
Lanes 2-5, in the presence of 1 µl aliquots corresponding
to eluents described in A. The cartoon on the right
represents Pip binding randomly to one, two or three motifs
(A, B, or C) within the ptr
promoter generating the three characteristic retarded bands (13).
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Fig. 2.
Locus and promoter structure of
pep and ptr loci. A,
Ptr (S. pristinaespiralis) and Pep (S. coelicolor) are homologous MFS proteins conferring resistance to
pristinamycin I. pep is followed by its cognate
tetR-like regulatory gene, whereas the sequence downstream
of ptr is unknown. Homologous sequence is present upstream
(B, identical nucleotides are boxed) including
functional Pip binding motifs (inverted repeats shown in
gray). The arrow indicates the transcriptional
start point of Pptr. Presumed RNAP recognition hexamers
( 10 and
35) are indicated.
35
hexamer and 5 of 6 in the
10 hexamer). No obvious promoter sequences
or ribosome binding sites were detected within the nucleotides
separating the end of the pep open reading frame and the
pip start codon (37 bp). These data suggested that Pip was
located in a position that would allow it to both control pep and to autoregulate its own expression.
9 M.
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Fig. 3.
The affinity of Pip to ptr
and pep promoters. Gel retardation
quantification of Pip binding to radiolabeled fragments (~1
pM) encoding Pptr (A) or
Ppep (B) (insets). Promoters were
titrated with a range of purified Pip: lane A, 0.028 nM; lane B, 0.14 nM; lane
C, 0.28 nM; lane D, 1.4 nM;
lane E, 2.8 nM; lane F, 7 nM; lane G, 14 nM; lane
H, 28 nM. Shifted fragments represent all possible
permutations of site occupancy (three in Pptr and two in
Ppep) (13). The percentage of bound fragment was measured
using a densitometer by dividing the complexed signal by total signal
per lane.
6 M.
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Fig. 4.
Pip ligand recognition specificity. Gel
retardation assay showing binding of Pip to radiolabeled
Ppep fragment (~1 pM) in the absence and
presence of various antibiotics. Lane A, Ppep in
the absence of Pip. Lane B, all of the binding sites are
saturated with relatively large amounts of purified Pip (70 nM). Lane C, 7 nM Pip, nonsaturated
site occupancy used to test ligand specificity. Inhibition of shift
with titration (50, 0.5, and 0.005 µM) of
representative antibiotics. Tet, tetracycline;
Rif, rifampicin; QP, quinupristin. Note that
diffusion from lane containing the highest concentration of PI (50 µM) affected the shift in the lane containing 5 nM Tet.
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Fig. 5.
Affinity of Pip/PI binding estimated by
dissociation of the Pip/Pptr complex. Pip was
titrated against radiolabeled Pptr fragment (~1
pM; lane J) to obtain full saturation of the
three operator sites (lane I; 14 nM). This
complex was exposed to various concentrations of pristinamycin I:
lane A, 0.5 nM; lane B, 5 nM; lane C, 50 nM; lane
D, 250 nM; lane E, 0.5 µM;
lane F, 2.5 µM; lane G, 5 µM; lane H, 25 µM. Pip was
completely released from the Pptr fragment by excess molar
concentrations of PI. The percentage of bound fragment was
measured using a densitometer by dividing the complexed signal by total
signal per lane and plotted against PI concentration.
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Fig. 6.
HPLC chromatogram showing noncovalent
interaction of PI and Pip. Pip (50 µg) was incubated with a
molar excess of PI (20 µg) and applied to a Superdex 75 sizing
column. Pip or Pip exposed to PI eluted with the same apparent
molecular mass. The specific, reversible binding of PI to Pip was
confirmed by analysis on a HPLC reverse phase C18 column that resolved
two peaks comigrating with Pip and PI run separately. The integrated
adsorption (215 nm) of the peaks was compared with standards of PI or
Pip (defined by the Bradford assay) to estimate approximately equimolar
amounts of Pip and PI released from the complex.
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Fig. 7.
Analysis of Pip multimerization by
ultracentrifugation. Sedimentation equilibrium of Pip
(A) or a Pip/PI complex (B) was done for 92 min
at 18,000 rpm at 20 °C and scanned at 277 nm. Sedimentation velocity
of Pip (C) or a Pip/PI complex (D) was carried
out at 54,000 rpm at 20 °C and scanned at 277 nm.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
6
M) was similar to that of TetR for tetracycline
(Kd = 10
6 M). However,
in vivo, tetracycline is likely to be complexed with
divalent cations such as Mg2+ that increase the
affinity of tetracycline for TetR more than 1000-fold (23). This lower
affinity of Pip for PI in vitro may reflect the absence of a
cofactor or imply the existence of an alternative natural ligand
in vivo. However, Pip had the same drug recognition
specificity for PI as its associated antibiotic resistance gene, Pep.
These observations proved that Pip did not mediate multidrug activation
of Pptr transcription through direct interactions with
structurally heterogeneous ligands.
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ACKNOWLEDGEMENTS |
---|
We thank Ariel Lustig for carrying out the ultracentrifugation studies and Martin Fussenegger for enthusiastic support.
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FOOTNOTES |
---|
* This work was funded by Swiss Priority Program in Biotechnology Grant 3100-039669.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF193856.
Present address: Morphochem, Schwarzwaldallee 215, Basel, BS 4058, Switzerland.
§ Present address: Infectious Disease Group, Aventis-Hoechst Marion Roussel, 102 route de Noisy F-93235 Romainville Cedex, France.
¶ Present address: Dept. of Developmental Biology, 279 Campus Dr., Beckman Center B-343, Stanford University School of Medicine, Stanford, CA 94305-5329.
To whom correspondence should be addressed. Tel.:
41-61-267-2116; Fax: 41-61-267-2118;
Charles-J.Thompson{at}unibas.ch.
Published, JBC Papers in Press, October 24, 2000, DOI 10.1074/jbc.M007690200
2 A. D. Frey, M. Rimann, J. E. Bailey, P. T. Kallio, C. J. Thompson, and M. Fussenegger,submitted for publication.
3 R. P. Morris and C. J. Thompson, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are: MFS, major facilitator superfamily; HPLC, high pressure liquid chromatography; PAGE, polyacrylamide gel electrophoresis; PI, pristinamycin I; PII, pristinamycin II; bp, base pair(s); PCR, polymerase chain reaction; pep, pristinamycin resistance gene from S. coelicolor; ptr, pristinamycin resisance gene from S. pristinaespiralis; tetA, tetracycline resistance gene; tetR, repressor of the tetracycline resistance gene.
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