From the Center for Adaptation Genetics and Drug Resistance and the Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111
Received for publication, March 14, 2003 , and in revised form, May 22, 2003.
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ABSTRACT |
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INTRODUCTION |
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EXPERIMENTAL PROCEDURES |
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Bacterial Strains, Plasmids, and MediumTable I lists the bacterial strains and plasmids used in this study. Escherichia coli cell cultures were grown at 37 °C in Luria-Bertani (LB) broth (24) supplemented with chloramphenicol (20 µg/ml) and tetracycline (15 µg/ml) as needed. AHTc (15 ng/ml) was used as a gratuitous inducer of the Tet protein where applicable (25).
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Mutagenesis of Plasmid DNAMutagenesis in vitro was
performed with hydroxylamine, which specifically induces GC AT
transitions by deaminating the cytosine residues
(26,
27). Approximately 3 µg of
plasmid p202FCH prepared using the Qiagen Spin Mini preparation kit were
incubated in 400 mM hydroxylamine, 50 mM potassium
phosphate buffer (pH6), and 100 mM EDTA in a 100-µl volume for
40 min at 68 °C. The entire volume was drop-dialyzed against sterile water
for 1 h 30 min at room temperature on a type VS filter (0.025-µm pore size)
(Millipore, Bedford, MA). The treated DNA was ethanol precipitated and washed
with 70% ethanol before being resuspended in sterile water. Mutagenesis of
p202FCH with MNNG was performed as described previously in DH5
cells
(28).
After both mutagenic events, 40 ng of plasmid DNA were introduced by
electroporation into DH5 electro-competent cells
(24) using 0.2-cm cuvettes and
a Gene Pulser (Bio-Rad) at 200 ohms, 25 microfarads, and 25 kV/cm. To maximize
the isolation of independent mutants, the entire transformation mix (1 ml) was
divided into 10 equal portions prior to incubation and plating. Subsequently,
transformants were selected on plates containing chloramphenicol (20 µg/ml)
(for plasmid maintenance) and tetracycline (15 µg/ml). Tc-resistant mutants
appeared usually after 24 h of incubation at 37 °C. Only one single colony
per plate was chosen for analysis to ensure that the mutants isolated were
generated in separate mutagenic events. Each colony was checked for its
tetracycline susceptibility by overnight liquid growth in LB broth containing
chloramphenicol and tetracycline (15 µg/ml). The plasmids from the
Tc-resistant clones were extracted, and their copy numbers were estimated by
plasmid preparation on gels. Those plasmids whose copy numbers were not
increased were chosen to retransform DH5
to assure that the Tc
resistance observed was a plasmid-mediated mutation in the tet
gene.
Site-directed MutagenesisSite-directed mutagenesis of tet(C) on plasmid pFS1 was performed by a two-stage PCR method adapted from a PCR overlap method (29, 30). Two primers corresponding to the sense and antisense sequence of the tet(C) gene were designed to incorporate a restriction endonuclease site along with the desired mutation where possible. The following sense primers were used: primer L11F, 5'-CAATGCGCTCATCGTAATATTCGGCACCGTC-3'; primer A213T, 5'-GGGGCATGACTATCGTCACCGCACTTATGACTGTC-3'; and primer A270V, 5'-GCCTTCGTCACTGGTCCGGTCACCAAACGTTTCGGCGAG-3'. Restriction endonuclease sites (SspI and BstEII in primers L11F and A270V respectively), which were introduced to facilitate identification of the desired mutants, are underlined.
Once the mutation was confirmed by sequencing and restriction enzyme analysis, the 3.2-kb XhoI-XbaI-mutated Tet(C) determinant was exchanged by cloning into the XhoI-BglII restriction sites of the parental unmutagenized pCR2 plasmid. To facilitate the cloning of the determinants, compatible cohesive ends were produced by blunt-ended XbaI and BglII restriction sites with T4 DNA polymerase.
Nucleotide SequencingDNA sequencing was performed at the Tufts University Core Facility using a ABI3100 Genetic Analyzer.
Determination of Tetracycline SusceptibilityAG100A cells harboring plasmids bearing wild type and tet(C)-mutated genes were grown in the presence of chloramphenicol and AHTc to an A530 of 0.8. Cells were swabbed for confluent growth onto a LB agar plate containing AHTc (15 ng/ml) before the application of the tetracycline E-test strips (gift from AB Biodisk, Solna, Sweden). The minimum inhibitory concentration was that amount of tetracycline showing an inhibition growth zone with the E-test after 24 h of incubation at 37 °C.
Membrane Isolation and Western Blot AnalysisAG100A cells expressing various plasmid-specified Tet proteins were grown in the presence of AHTc (15 ng/ml) and rapidly chilled when they reached the late logarithmic growth phase (A530 = 0.8). Following centrifugation, cells were resuspended in 20 mM Tris-HCl (pH8), 2 mM MgCl2, 1 mM EDTA, and 30 µg/ml lysozyme (A530 of 100) prior to sonication (Branson Sonifier 250, Branson Ultrasonics Corporations, Danbury, CT). Membranes were collected by centrifugation at 60 000 x g for 1 h at 4 °C. Tet proteins were solubilized by incubating the membranes in 20 mM Tris-HCl (pH8), 150 mM NaCl, 10% glycerol, and 1.5% dodecylmaltoside at 4 °C for 1 h (A530 = 250). Membranes were removed by sedimentation for 30 min at 15,000 x g in 1.5-ml Eppendorf tubes, and extracts were stored at 80 °C. Before electrophoresis, extracted proteins were incubated in reducing sample buffer (24) for 20 min at room temperature. Proteins were separated by electrophoresis in a 10% SDS-polyacrylamide gel (24) using a Miniprotein II gel apparatus (Bio-Rad) and then transferred to a PolyScreen polyvinylidene difluoride transfer membrane (PerkinElmer Life Sciences) per the manufacturer's recommendations. Immunological detection was carried out with polyclonal antibodies directed against the 14 carboxyl-terminal (Ct) amino acids of Tet(B) (anti-Ct antibody, kindly provided by A. Yamaguchi) (31). The antigen-antibody complexes were detected with horseradish peroxidase coupled to the anti-rabbit IgG (New England Biolabs). Blots were developed with the Renaissance Western blot chemiluminescence reagent plus kit (PerkinElmer Life Sciences). The band intensities of each Tet(C) derivative were determined using NIH Image 1.6.2 free software (www.scioncorp.com).
Tetracycline Accumulation AssaysThe measurement of
[3H]Tc uptake by intact AG100A cells containing mutant plasmids was
adapted from previous works (1,
4). Bacteria grown to
exponential phase in LB broth containing AHTc were pelleted by centrifugation
(15,000 x g for 5 min), washed in 10 mM Tris-HCl
(pH8) buffer, and suspended in 50 mM potassium phosphate buffer (pH
6.6), 10 mM MgSO4, and 0.2% glucose
(A530 = 4). After 3 min of preincubation,
[3H]Tc was added to 270 µl of cell suspension with shaking at 30
°C in a water bath, yielding a final tetracycline concentration of 1
µM. At various intervals, 50 µl of the suspension were
removed, mixed with 10 ml of 0.1 M potassium phosphate buffer
(pH6.6) and 0.1 M LiCl at 20 °C, and filtered through a
Metricel® membrane filter (pore size, 0.45 µm). The filters were washed
with 4 ml of the same buffer and dried before the radioactivity was measured
with a liquid scintillation counter. The protonophore CCCP was added to a
final concentration of 100 µM at 18 min to deenergize the cells.
Each strain was assayed in triplicate in three separate experiments. The value
at each time point deviated 8%.
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RESULTS |
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AG100A cells devoid of the AcrAB pump were highly susceptible to tetracycline (minimum inhibitory concentration = 0.3 µg/ml) (Table II). When complemented with a low copy number plasmid bearing the wild type tet(C), the minimum inhibitory concentration in AG100A rose to 16 µg/ml. The presence of the single mutation S202F in Tet(C) decreased the tetracycline resistance by 4-fold to 4 µg/ml. Wild type Tc resistance was restored by each of the secondary mutations, A213T/S202F and A270V/S202F, and an even higher level of resistance was provided with the L11F/S202F mutation (24 µg/ml). Subsequently, each mutation was introduced by site-directed mutagenesis into Tet(C) specified by the plasmid pFS1. These mutations alone had no effect on the level of Tc resistance and could, therefore, be considered as not essential for Tet(C) activity (Table II). Thus, the secondary suppressor mutations are necessary to suppress the effect of the first mutation but are not by themselves critical for Tet protein function.
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Western Blot Analysis of Tet Protein ExpressionWestern blot analysis of the Tet protein was performed using the anti-Ct antibody. The inactivating mutation S202F slightly reduced (10%) the level of protein production (Fig. 2) but confirmed the belief that the low level of resistance was not attributable to a poor expression of the protein. The double mutant protein A270V/S202F was expressed in quantities comparable with the parental S202F mutant (Fig. 2A). On the other hand, the protein with the secondary L11F or A213T mutations showed increased amounts in the cells in which they were expressed (Fig. 2A). The introduction of each single suppressor mutation into the wild type determinant did not change protein expression or mobility in a detectable manner (Fig. 2B).
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Analysis of Tetracycline Efflux ActivityTc resistance is characterized by a reduction in the cellular accumulation of Tc brought about by a proton motive force-dependent efflux (4, 35). We measured the energy-dependent Tc efflux as the relative uptake of [3H]Tc before and after deenergization of the cells with the protonophore CCCP. AG100A, devoid of the AcrAB pump and any Tet protein, accumulated 38 pmol of [3H]Tc in 18 min (Fig. 3A). The addition of 100 µM CCCP resulted in a loss of Tc from the cell, which was attributed to the dissipation of the proton gradient across the membrane upon which Tc uptake is dependent (7). However, when AG100A expressed the wild type Tet(C), it showed a lower uptake of [3H]Tc (13.9 pmol) (Fig. 3A), which increased when cells were treated with CCCP. The strain carrying the S202F mutation accumulated nearly 21.9 pmol of [3H]Tc in 18 min but was unaffected by CCCP addition (Fig. 3A). This lack of effect of CCCP was observed previously for some low level Tet protein mutants (36).
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All three suppressor mutations restored an active Tc efflux comparable with the wild type (Fig. 3B). Modification of the wild-type Tet(C) protein with each single suppressor mutation revealed no distinguishable change in the efflux activity as compared with the wild type Tet protein (Fig. 3C).
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DISCUSSION |
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We have isolated and characterized three different types of amino acid substitution suppressor mutations arising in nine independently isolated mutants that restore the tetracycline efflux function in Tet(C) with the S202F mutation in the interdomain loop. One secondary suppressor mutation, L11F, would increase the bulkiness of the side chain at this position. Leucine 11, predicted to be relatively close to the cytoplasmic side of the membrane within TM1, is conserved among all of the classes of Gram-negative Tet proteins that have been identified to date. Its change suggests an interaction of TM1 with the interdomain loop. According to Tamura et al. (22), TM1 is a partly amphiphilic helix with several residues facing the water channel built by portions of TM2, TM4, TM5, TM7, TM8, TM10, and TM11. It is therefore possible that this secondary suppressor mutation modifies the structure of TM1 and restores a wild type tetracycline resistance phenotype by correcting the active site altered by the S202F mutation.
A second suppressor mutation, A213T in the cytoplasmic loop
(Fig. 1), slightly increases
the size of the side chain while introducing a hydrophilic residue. Prediction
deduced from hydropathy plots has shown an additional cytoplasmic
helix-(I
) located within the interdomain loop
(Fig. 1), between serine 202
and arginine 207. A similar feature of 13 amino acids has been pointed out in
the three-dimensional crystallographic structure of the efflux transporter
AcrB, and it has been postulated that the I
helix is attached to the
cytoplasmic membrane surface
(38). This feature may also be
true for Tet(C). The predicted position of A213T near the cytoplasmic surface
of the protein could improve binding of the mutated putative I
structure to the membrane. Of note, the mutation led to a 34-fold
higher amount of the protein without showing a greater efflux activity,
suggesting that the protein itself is still not completely wild type in
function. Although the reason for the increased protein is not yet clear, the
single A213T change in Tet(C) did not alter protein amount or function (Figs.
2 and
3). Thus, it appears that the
combined interdomain mutations (S202F + A213T) add stability to the protein
embedded in the membrane. Analogously, the length and nature of the residues
of the interdomain loop of LacY, a member of the major facilitator
superfamily, affected the insertion and stability of the protein in the
membrane (39). Hydrophilic
residues within the interdomain loop of LacY are required to permit a temporal
delay for the insertion of both domains.
A third suppressor mutation, A270V, predicted to be within the cytoplasmic loop between TM8 and TM9, restored an efficient tetracycline resistance phenotype and efflux activity without modifying the level of Tet production. The position on the same side of the protein suggests that the loop 8-9 interacts somehow with the interdomain loop and that the A270V mutation improves the interaction in the presence of the S202F mutation.
The loss of tetracycline efflux activity of the mutant S202F Tet(C) could
be interpreted as a modification of the conformation of the I helix
that could perturb the correct positioning of the active site involving the
interaction between
and
domains. In Tet(B), substitution of the
homologous serine 202 by a cysteine residue also led to an increase in
tetracycline susceptibility
(17). Interestingly,
cysteine-scanning mutagenesis analysis revealed no labeling of S201C with
[14C]N-ethylmaleimide. This finding suggests that the
serine, while in the water-exposed loop, is oriented into the protein interior
as a membrane-interactive residue
(17).
The introduction of the single mutations L11F, A213T, and A270V into Tet(C)
did not produce any detectable change in the wild type Tet(C) activity or
expression. This finding suggests that serine 202 plays a capital but not
essential role in the functional activity of Tet(C). In this case, any
conformational modification brought by each mutation is not sufficient to
destroy the interaction of serine 202 with other specific portion(s) of the
protein. Although the precise role of the cytoplasmic interdomain loop in
tetracycline resistance is not clear, it should be reevaluated as being more
than a simple linker between the and
domains because it affects
Tet protein-specified tetracycline resistance.
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FOOTNOTES |
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To whom correspondence should be addressed: Center for Adaptation Genetics and
Drug Resistance and Dept. of Molecular Biology and Microbiology, Tufts
University School of Medicine, 136 Harrison Ave., Boston, MA 02111. Tel.:
617-636-6764; Fax: 617-636-0458; E-mail:
stuart.levy{at}tufts.edu.
1 The abbreviations used are: TM, transmembrane domain; AHTc,
5,6-anhydrotetracycline; CCCP, carbonyl cyanide
m-chlorophenylhydrazone; MNNG,
N-methyl-N'-nitro-N-nitrosoguanidine; Tc,
tetracycline; [3H]Tc, [7-3H(N)]Tc.
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REFERENCES |
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