The Center for Adaptation Genetics and Drug Resistance and Department of Molecular Biology and Microbiology, Tufts University School of Medicine, 136 Harrison Ave, Boston, MA 02111, USA
Correspondence
Stuart B. Levy
stuart.levy{at}tufts.edu
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ABSTRACT |
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INTRODUCTION |
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METHODS |
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Bacterial strains and plasmids.
Escherichia coli DH5 (Woodcock et al., 1989
) was the host strain used for all cloning experiments and propagation of plasmid DNA. Cell cultures were grown at 37 °C in LuriaBertani broth (Seoane et al., 1992
) supplemented with 20 µg chloramphenicol ml1 and 30 µg kanamycin ml1 as needed. Fifteen nanograms of 5
,6-anhydrotetracycline ml1 was used as a gratuitous inducer of Tet protein where applicable (Moyed et al., 1983
).
Low-copy-number plasmids pLGT2 [wild-type tetA(B), origin of replication pSC101] (Yamaguchi et al., 1990c) and its derivative pLGTS201C [Ser201Cys mutant of TetA(B)] (Kimura et al., 1997
) were kindly provided by A. Yamaguchi as were other plasmids (see text). All plasmids used during this work possess the TetB determinant comprising both TetR (repressor) and TetA (efflux pump). The TetB determinant isolated from pLGT2 by BglII/BamHI enzymic restriction was cloned into the medium-copy-number plasmid pMCL210, origin of replication p15A (Nakano et al., 1995
), to produce transitional plasmid pMCL-WT-TetB, ready to use as template for site-directed mutagenesis.
Site-directed mutagenesis and nucleotide sequencing.
Site-directed mutagenesis of tetA(B) on plasmid pMCL-WT-TetB was performed by adaptation of a PCR overlap method (Deng & Nickoloff, 1992; Wang & Malcom, 1999
). Mutagenic PCR primers were designed to incorporate a restriction endonuclease site along with the desired mutation where possible (Table 1
). Once the mutation was confirmed by restriction enzyme analysis and sequencing, the fragment was exchanged from the mutagenized plasmid into the low-copy-number unmutagenized pLGT2 plasmid (Yamaguchi et al., 1990c
). The restriction enzymes used to exchange the fragments were (i) EcoRV/EcoRI for Asp190 and Glu192 substitutions, (ii) EcoRV/BamHI for Ser201 substitutions, and (iii) EcoRV/XbaI for the Leu9Phe substitution.
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Determination of susceptibility to tetracycline and tetracycline analogues.
E. coli DH5 cells with and without plasmids bearing wild-type and tetA(B) mutated genes were grown overnight in the presence of 25 µg kanamycin ml1 on MuellerHinton agar plates. Cells were swabbed for confluent growth onto a MuellerHinton agar before the application of doxycycline, minocycline and tetracycline E-test strips (gift from AB Biodisk). The minimum inhibitory concentration (MIC) was that amount of the tetracycline showing an inhibition growth zone with the E-test after 24 h of incubation at 37 °C.
Western blot analysis.
DH5 cells expressing various plasmid-specified TetA proteins were grown in the presence of 15 ng 5
,6-anhydrotetracycline ml1 and rapidly chilled when they reached the late exponential growth phase (OD550=0·8). Cells were resuspended at an OD550 of 80 in 20 mM Tris/HCl (pH 8), 2 mM MgCl2, 1 mM EDTA and 30 µg lysozyme ml1 and disrupted by sonication (Branson Sonifier 250; Branson Ultrasonics). Total-cell lysate was used for immunological detection. Proteins were incubated in reducing sample buffer (Sambrook & Russell, 2001
) for 20 min at room temperature, then a volume of extract corresponding to 0·1 OD550 units was separated by electrophoresis in a 12 % SDS-polyacrylamide gel (Sambrook & Russell, 2001
) using a Miniprotein II gel apparatus (Bio-Rad), and the proteins were then transferred electrophoretically to a PolyScreen polyvinylidene difluoride membrane (Perkin Elmer Life Sciences) using a Trans-Blot SD Semi-Dry Transfer Cell (Bio-Rad) according to the manufacturer's recommendations. Western blot analysis was performed according to Sambrook & Russell (2001)
with blocking by 0·5 % bovine serum albumin, with the exception that the detergent Tween 80 was used instead of Tween 20. Immunological detection was carried out with polyclonal antibodies directed against the 14 carboxyl-terminal (Ct) amino acids of TetA(B) (anti-Ct antibody, kindly provided by A. Yamaguchi) (Yamaguchi et al., 1990a
). The antigenantibody complexes were detected with horseradish peroxidase coupled to anti-rabbit IgG (New England Biolabs). Blots were developed with the Western Lighting Chemiluminescence Reagent Plus kit (Perkin Elmer Life Sciences) and exposed to Kodak BioMax Light Film.
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RESULTS AND DISCUSSION |
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The pattern of MIC values for the different Glu192 substitutions was identical to that for the Asp190 replacements. In both cases, retention of negative charge was optimal, while the other changes similarly reduced, but never completely eliminated activity, with Cys giving the most drastic reduction.
Effect of substitution on tetracycline resistance at Ser201 with Thr, Ala or Cys
The plasmid bearing the Ser201Cys mutation (Kimura et al., 1997) was obtained from A. Yamaguchi. The two additional substitutions chosen were Ala (small and hydrophobic) and Thr (conservative mutation that maintains the hydroxyl group of Ser). The Ala and Thr substitutions of Ser201 each lowered the tetracycline MIC by only 1·3-fold (Table 2
). Therefore, a hydroxyl group at position 201 is not obligatory for tetracycline resistance function, nor does the residue need be hydrophilic. As expected, the Cys mutation reduced tetracycline resistance by 4·8-fold (Table 2
).
Effect of various substitutions at Asp190, Glu192 and Ser201 and a second-site Ser201Cys suppressor mutation on substrate specificity
To assay whether a region of TetA(B) is involved in tetracycline binding, one may ask if amino acid substitutions in that region change the specificity of the pump for different tetracycline analogues. For the wild-type and mutant proteins, we therefore compared the resistance to tetracycline with that to two other analogues, doxycycline and minocycline (Table 2). We noted that no resistance was offered by any of the mutants to 9-(t-butylglycylamido)-minocycline (data not shown), a glycylcycline used in previous studies (Tuckman et al., 2000
). The data are presented as the ratio of the MIC for the analogue to the MIC for tetracycline, which was then normalized to that for the wild-type.
The substitutions at Asp190 and Glu192 with Cys, Ala and Asn or Gln primarily altered tetracycline resistance without affecting analogue resistance (except Asp190Cys). The effect led to a 1·8- to 6·2-fold preference for doxycycline/minocycline over tetracycline relative to the wild-type pump (Table 2) and so altered substrate specificity. Only those substitutions that have greater than a 1·5-fold effect were considered as having altered the substrate specificity. Of note, retention of the negative charge at Asp190 and Glu192 restored tetracycline resistance, removing the preference for doxycycline and minocycline over tetracycline.
At position Ser201, substitution with Ala or Thr had no effect on specificity. However, substitution with Cys led to a 4·3- to 4·9-fold relative preference for doxycycline/minocycline. Again, this altered specificity can be seen as a loss in tetracycline resistance and not in analogue resistance, when compared to wild-type (Table 2).
Since the inactivating Ser201Cys substitution of class B is at a position similar to that of the inactivating Ser202Phe mutation of class C (Saraceni-Richards & Levy, 2000a), we examined whether the former would be suppressed by Leu9Phe, which corresponds to the Leu11Phe in TM1 which suppresses the class C mutation (Sapunaric & Levy, 2003
). The Leu9Phe replacement indeed suppressed the Ser201Cys mutation, restoring a nearly wild-type level of tetracycline resistance; the Leu9Phe replacement by itself had little effect on TetA(B) function (Table 2
). It is therefore possible that this secondary suppressor mutation modifies the structure of TM1, which in turn corrects the interdomain region that had been inactivated by the Ser201Cys mutation. The substrate specificity effect of 201Cys is suppressed by Leu9Phe, by restoring the tetracycline resistance (Table 2
).
We conclude that retention of the negative charge at residues Asp190 and Glu192 did not affect substrate specificity. If the replacement at residues 190 and 192 was uncharged, but retained the same size, the specificity was somewhat altered. If the replacement was smaller (Ala or Cys), a more dramatic specificity shift occurred. At position Ser201, substitutions of a similar size had no effect, except for Cys. At all three positions, replacement by Cys caused the most drastic shift in specificity towards the analogues. Since doxycycline and minocycline are both more lipophilic than tetracycline (Barza et al., 1975), all of the specificity-altering substitutions may somehow reduce the ability of the protein to handle hydrophilic substrates.
To further demonstrate that the substrate specificity effects attributed to the mutations at Asp190, Glu192 and Ser201 of TetA(B) were meaningful, we chose as controls' four cysteine mutants which significantly lowered activity of TetA(B) according to Tamura et al. (2001): Gly20Cys, Val339Cys, Gly366Cys and Asn184Cys. We also intentionally chose the Thr191Cys mutant for its strategical position between Asp190 and Glu192 as another control (Tamura et al., 2001
). The single cysteine mutations on a low-copy-number plasmid were provided by A. Yamaguchi and their tetracycline MICs were consistent with those reported previously (Kimura-Someya et al., 2000
; Konishi et al., 1999
; Tamura et al., 2001
). For each single cysteine mutant, the MICs and the ratio of the MIC for the two analogues to the MIC for tetracycline showed a proportional decrease in resistance to both tetracycline and to the analogues in all cases (see Table 2
). None of the five cysteine control mutations provided a change of substrate specificity towards the tetracycline analogues tested. We conclude that Asp190, Glu192 and Ser201 in the interdomain cytoplasmic loop all appear to be involved in substrate specificity and therefore are likely to interact with the substrate.
The substrate for TetA is a tetracyclinedivalent metal cation complex with a net charge of +1 (Yamaguchi et al., 1990b, 1991
). That the negative charge of Asp190 and Glu192 is not essential (Table 2
) suggests that these residues may not interact directly with the Mg2+ region of the substrate. Moreover, substitutions at the three positions cause a greater loss in activity towards tetracycline than towards the lipophilic analogues, which also utilize Mg2+.
Other negative residues located in TMs and loops of TetA(B) that are important for activity have been identified previously. The negative charge held by Asp15 (TM1) (McMurry et al., 1992; Yamaguchi et al., 1992b
), Asp66 (loop 2-3) (Yamaguchi et al., 1992b
), Asp84 (TM3) (Yamaguchi et al., 1992b
) and Asp285 (Yamaguchi et al., 1992b
), were shown to be important for transport activity. For Asp15 and Asp84, helical projections and cysteine-scanning mutagenesis demonstrated that both residues are oriented towards the putative central water-filled channel and may be a part of the translocation pathway of tetracycline (Tamura et al., 2001
). Asp66 is located in the conserved sequence motif, GXXXXRXGRR, of cytoplasmic loop 2-3, which was postulated to be a part of the entrance gate of the tetracycline divalent cation complex (Yamaguchi et al., 1992a
, 1990c
). In contrast with the previous negatively charges residues, Asp285, located in the TM9, is essential for TetA(B) activity (Yamaguchi et al., 1992b
) and cannot be replaced even with glutamate (Yamaguchi et al., 1992b
).
Other residues involved in substrate specificity in TetA(B) are Gly111Glu (TM4), Trp231Cys and Trp231Gly (TM7), Leu253Phe (TM8) and Leu308Ser (TM10) (Fig. 1) all of which increase the resistance to 9-(dimethylglycylamido)-minocycline and tigecycline GAR936 (Guay et al., 1994
; Tuckman et al., 2000
). Since these mutations lie in or near regions which probably face the putative water-filled channel (see Tamura et al., 2001
), the different substitutions may introduce a conformational change in their respective TM so as to preferentially affect the binding and/or translocation pathway of the tetracycline analogues.
In LacY, another member of major facilitator superfamily, the interdomain loop affects the insertion and stability of the protein into the membrane, but has not been implicated in substrate binding (Weinglass & Kaback, 2000). Hydrophilic residues within the interdomain loop of LacY are required to permit a temporal delay for the insertion of both domains. In TetA(B), the cytoplasmic interdomain loop is more than a linker between the
and
domains since mutations in it affect resistance (Sapunaric & Levy, 2003
; Saraceni-Richards & Levy, 2000a
). We have shown here that this loop also possesses at least three residues that appear to be involved in interacting with the tetracycline/Mg2+ substrate.
Structural analysis of the interdomain loop of tetracycline efflux pumps
The crystal structure of two transporters belonging to the major facilitator superfamily, the E. coli lactose permease (LacY) and the glycerol 3-phosphate transporter GlpT were solved at 3·5 and 3·3 Å respectively (Abramson et al., 2003; Huang et al., 2003
). In addition to their 12 TM segments, secondary structures were revealed in both interdomain loops. No function has been attributed to it, but stability of the protein appears linked to the interdomain loop of LacY (Weinglass & Kaback, 2000
). Since the interdomain loop has no homology among the related TetA proteins, it is surprising that it appears to play a part in binding to substrate. We reasoned that secondary structure might be present in the loop region of all TetA proteins.
Using PSIPred, a computational tool from University College London, that predicts secondary structure (Jones, 1999; McGuffin et al., 2000
), we analysed the secondary structure of the interdomain loop of 14 TetA efflux pump homologues. We used the 3D structure of LacY and GlpT (Abramson et al., 2003
; Huang et al., 2003
) as controls to test the validity of the prediction. The prediction from PSIPred showed two short helical segments in the interdomain cytoplasmic loop of LacY, in agreement with the resolved structure (Fig. 3a
). The degree of confidence for each of the two helices is different, with a very high score for the second structure of eight residues. The shorter one was misinterpreted by one residue. The secondary structure prediction for GlpT also proposes two short helices, one of four amino acids, 219IEEY223, and one of seven residues, 242QIFMQYV248. The actual structure of GlpT has only the second helix (Huang et al., 2003
). The predicted first short helix might be missing since it is adjacent to a disordered region from Asn232 to Leu239. Based on comparison of these predictions with the known structures, we accepted a PSIPred value of
4 as indicating a good level of confidence in predicting a secondary structure and, moreover, that a difference between two successive values equal or greater than 2 would put an end to the predicted secondary structure. As can be noted, in almost all of the tetracycline efflux pumps examined, a short
-helical segment was predicted (Fig. 3a
). The length of this secondary structure varied from four to ten residues. This new putative structured element of the interdomain loop of tetracycline efflux proteins was named I
(for interdomain
-helix). In only three tetracycline transporters [Tet(Z), Tet(31) and Tet(33)] were no helix or other secondary structures predicted in the interdomain loop. We note that Tet(Z) and Tet(33) are closely related and, unlike the other classes, come from Gram-positive organisms. For TetA(A), two different sequences are shown in Fig. 3(a)
. The first corresponds to the wild-type TetA(A) (GenBank accession no. P02982) and the second sequence, TetA(A)(2), is from a veterinary Salmonella minocycline-resistant isolate which diverges from the wild-type by three amino acid changes in the interdomain loop (Tuckman et al., 2000
). This modification is accompanied by a change in substrate specificity of the pump (Tuckman et al., 2000
). The mutations in TetA(A)(2) increase the length of the predicted I
by one residue and raise the level of confidence for the entire segment.
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We also analysed the effect of the different substitutions at Ser201 in TetA(B), since this serine is at the border of the predicted I. As can be seen in Fig. 3(b)
, each of the three substitutions increased the length of the predicted I
structure by one or two residues. Another factor is that residues 201, 203, 204, (and 211) of TetA(B), when changed to Cys, were the only residues in the interdomain loop inaccessible to a sulfhydral reagent (Tamura et al., 2001
). Thus, these four residues might be occluded, further supporting the idea of an ordered secondary or tertiary structure covering this region. In the structures of both LacY and GlpT, the longer I
is at a similar position in the interdomain loop, although no function has been attributed to it (Abramson et al., 2003
; Huang et al., 2003
). According to the structural data of LacY (Abramson et al., 2003
) and of GlpT (Huang et al., 2003
), this helix lies parallel to the membrane surface near the cytoplasmic end of TM11.
Concluding remarks
Our data suggest that residues Asp190, Glu192 and Ser201 of the interdomain loop of TetA(B) interact with substrate. The change of the substrate specificity caused by single mutations depended on the nature of the substitution itself and was associated with an increased length of a putative helical secondary structure I in the interdomain loop of TetA(A) and TetA(B).
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ACKNOWLEDGEMENTS |
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Received 28 February 2005;
accepted 28 March 2005.
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