Department of Microbiology, Monash University, Clayton, Victoria 3168, Australia1
Author for correspondence: Julian I. Rood. Tel: +61 3 9905 4825. Fax: +61 3 9905 4811. e-mail: julian.rood{at}med.monash.edu.au
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ABSTRACT |
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Keywords: tetracycline, resistance, efflux, transmembrane domains, Clostridium perfringens
Abbreviations: MFS, major facilitator superfamily; TMD, transmembrane domain
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INTRODUCTION |
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The prototype tetracycline efflux protein, TetA(B), is encoded on the transposable element Tn10 from Escherichia coli. It is an integral membrane protein which catalyses the antiport of a divalent cationtetracycline complex and a proton (Yamaguchi et al., 1990b ). It has been experimentally shown to have 12 TMDs (Allard & Bertrand, 1992
; Eckert & Beck, 1989
; Kimura et al., 1997
) and has a very high level of amino acid sequence identity to a range of tetracycline efflux proteins from other Gram-negative bacteria. Many residues that are required for TetA(B) activity have been identified (Kimura et al., 1998
; Kimura & Yamaguchi, 1996
; Yamaguchi et al., 1990a
, 1992b
, 1993a
, 1996
). In particular, three negatively charged amino acids, D15, D84 and D285, which are situated within TMDs, are essential for tetracycline transport function, probably by forming a transmembrane efflux pathway for the tetracycline complex (Yamaguchi et al., 1992a
). Other conserved negatively charged residues located within predicted loop regions of tetracycline efflux proteins from Gram-negative bacteria are also required for efficient tetracycline efflux (Yamaguchi et al., 1992c
).
Two tetracycline efflux proteins from Gram-positive organisms, TetA(K) (Mojumdar & Khan, 1988 ) and TetA(L) (McMurry et al., 1987
), differ from TetA(B). Both are larger proteins, with TetA(K) shown to consist of 14 TMDs (Ginn et al., 1997
). The TetA(K) protein contains three functional glutamate residues that are located within TMDs (Fujihara et al., 1996). These residues appear to play a similar role to the transmembrane-located aspartate residues of TetA(B). In addition, several aspartate residues that are conserved in TetA(K) and TetA(L), and are located in hydrophilic loops, are required for the function of TetA(K) (Fujihara et al., 1997). Within hydrophilic loop 23, the TetA(K) and TetA(B) proteins contain a conserved motif (GxxxxRxGRR) that is common to this family of MFS transport proteins (Henderson, 1990
; Paulsen et al., 1996
). Site-directed mutagenesis has shown that this motif plays a role in the translocation of tetracycline across the membrane (Fujihara et al., 1997; Yamaguchi et al., 1992d
). The same motif is also important for other transporters such as
-ketoglutarate permease (Seol & Shatkin, 1993
) and lactose permease (Jessen-Marshall et al., 1995
).
Tetracycline resistance in the Gram-positive anaerobe Clostridium perfringens is primarily due to the presence of the Tet(P) determinant. This novel determinant has two overlapping tetracycline-resistance genes. The tetA(P) gene encodes an integral membrane protein responsible for the active efflux of tetracycline from the cell, whereas the product of the tetB(P) gene has very strong similarity to members of the Tet(M) family of ribosomal-protection tetracycline-resistance proteins (Sloan et al., 1994 ). The efflux protein TetA(P) comprises 420 amino acids and is predicted to have 12 TMDs, but it does not have significant amino acid sequence identity to other tetracycline efflux proteins. As for most other tetracycline efflux proteins, acidic residues located within TMDs have been shown to be required for the efflux of tetracycline. Like TetA(K), TetA(P) contains two TMD-located glutamate residues that are required for function, although unlike TetA(K) these residues are both situated within putative TMD 2 (Kennan et al., 1997
).
Since TetA(P) has very little similarity to other tetracycline efflux proteins and does not contain the conserved MFS motifs, we decided to carry out additional studies on this unusual efflux protein. In this paper we report the results of random mutagenesis experiments that were successfully used to identify TetA(P) residues of structural or functional significance.
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METHODS |
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Random mutagenesis.
Random mutants of the tetA(P) gene were isolated by passaging the plasmid pJIR71 (Abraham et al., 1988 ), a pUC18 derivative which encodes the complete tetA(P) gene including the upstream regulatory region, through the DNA-repair-deficient strain XL1-Red. The mutagenesis procedure was as described in the Epicurian Coli XL1-Red Competent Cells instruction manual (Stratagene). Three independently derived samples of pJIR71 DNA were isolated after passage through XL1-Red and were used to transform competent E. coli DH5
cells. The resultant ampicillin-resistant single colonies were screened for their ability to grow in the presence of 10 µg tetracycline ml-1 and susceptible colonies were selected and retested. The tetA(P) genes from all of the tetracycline-sensitive derivatives that had a missense mutation were then completely sequenced with the PRISM Ready Reaction Dye Deoxy Terminator Cycle Sequencing Kit (Applied Biosystems) and an ABI373A automated fluorescent sequencing apparatus (Applied Biosystems). Other mutants were partially sequenced in a manner sufficient to identify the nature of the mutation.
Tetracycline MIC.
For each of the tetA(P) mutants, the tetracycline MIC was determined at 37 °C as described previously (Kennan et al., 1997 ). Each strain was tested for growth inhibition at the concentrations ranging from 1 to 30 µg tetracycline ml-1.
Immunoblot analysis.
The TetA(P) protein was detected by immunoblot analysis as described previously (Kennan et al., 1997 ), with the following modifications. E. coli cells were lysed by sonication (4x30 s) and whole cells were removed by centrifugation at 12000 g for 2 min. Samples of the resultant cell lysates were mixed with gel loading buffer at room temperature and were subjected to SDS-PAGE (Laemmli, 1970
). Samples of known protein concentrations were loaded onto the gel and, following ECL Western blotting detection (Amersham Life Sciences), the amount of each of the TetA(P)-derived bands detectable with the anti-TetA(P) antiserum (Kennan et al., 1997
) was determined using a 2022 Ultrascan laser densitometer (LKB Produkter).
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RESULTS AND DISCUSSION |
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The current model of the TetA(P) protein (Fig. 1) was derived using the TopPred II algorithm (Claros & von Heijne, 1994
), in combination with the positive-inside rule for integral membrane proteins (von Heijne, 1992
). Although the membrane structure of the TetA(P) protein is yet to be experimentally determined, previous analyses of integral membrane proteins using this or similar algorithms have shown that the computer-generated structures are usually close representations of the expected protein structure (Boyd et al., 1993
; Paulsen et al., 1995
; Roy & Isberg, 1997
). Accordingly, the predicted single amino acid changes in the mutant TetA(P) proteins were analysed in the context of the TetA(P) transmembrane model.
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The wild-type derivative DH5(pJIR71) had an MIC of 30 µg ml-1, whereas the negative control DH5
(pUC18) had an MIC of 2 µg ml-1. Each of the mutated plasmids tested had an MIC of between 2 and 15 µg ml-1 (Table 1
). Each of the truncated TetA(P) mutant proteins tested did not confer resistance to tetracycline (MIC 2 µg ml-1). The one exception was pJIR1687, which harboured a TetA(P) derivative truncated at amino acid 192, which includes the first six of the proposed 12 TMDs of TetA(P) (Fig. 1
). Strains carrying pJIR1687 had an MIC of 10 µg ml-1. It is possible that the low level tetracycline resistance observed with this mutant is due to multiple 6-TMD monomers forming a partially active TetA(P) derivative. A variation of this result was observed with the E. coli TetA(B) protein. When expressed independently, the first half of TetA(B) was not able to efflux tetracycline, but when the N-terminal and C-terminal 6 TMD regions of TetA(B) were expressed together within a cell, 40% of wild-type tetracycline efflux was observed (Yamaguchi et al., 1993b
).
Detection of mutant proteins by immunoblotting
To examine the level of expression of the mutant TetA(P) proteins, cell extracts were prepared from the 31 DH5 derivatives carrying plasmids that were predicted, from DNA sequence analysis, to produce TetA(P) proteins that contained single amino acid changes. These extracts were examined by immunoblotting with a TetA(P)-specific polyclonal antiserum that was raised by use of a synthetic peptide which consisted of the last 17 amino acids of TetA(P) (Kennan et al., 1997
). In these experiments, 18 of the 31 mutants produced detectable TetA(P) protein (Fig. 2
), with TetA(P) expression levels varying from 5 to 172% when compared to the wild-type (Table 1
). Control experiments that utilized different amounts of wild-type cell lysates showed that TetA(P) protein levels could be reliably titrated using this procedure (data not shown). Similar analysis showed that DH5
(pJIR1694), which carries the only promoter mutation detected in this study, produced slightly reduced levels of TetA(P) (P. Johanesen, D. Lyras & J. Rood, unpublished results).
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Mutants that did not produce detectable TetA(P) protein
Within the group of mutant TetA(P) proteins analysed, a wide range of protein expression levels was observed. In addition, we note that this random mutagenesis approach produced an unexpectedly high percentage (42%) of mutant TetA(P) derivatives from which no detectable TetA(P) protein was observed, each resulting in tetracycline sensitivity (MIC 2 µg ml-1). Previous studies of the TetA(B) protein, which also involved the examination of large numbers of mutant derivatives, reported that very few mutant proteins were unable to be detected with anti-TetA(B) protein antiserum (Kimura et al., 1998 ; Yamaguchi et al., 1992c
, d
).
Of the 13 mutants that did not produce detectable TetA(P) protein, six mutations involved glycine residues that were located within proposed TMDs, with each of these changes involving replacement with a charged residue, i.e. the mutants G78E, G78R, G83E, G108E, G143R and G167E. The addition of these charged residues appears to have produced unstable TetA(P) derivatives, which is consistent with the proposed location of these residues within a hydrophobic environment. It is likely that the introduction of a charged residue into a TMD has destabilized the integral transmembrane structure of the TetA(P) protein. A similar hydroxylamine-based random mutagenesis approach, when used to locate functional residues of the TetA(C) protein, also led to the identification of a high proportion of mutants containing glycine to charged amino acid changes (McNicholas et al., 1992 ).
Other pJIR71 mutants that were producing undetectable levels of TetA(P) were derivatives carrying the mutations A66V, R71C, E123G, E123K, G382D, S387P and P392S. It is not known why these changes destabilized the TetA(P) protein although it is possible that the R71 and E123 residues form salt-bridges that may be required for the correct TetA(P) structure. Note that the failure to detect the S387P and P392S proteins may reflect their inability to be recognized by the C-terminal-specific TetA(P) antibodies.
Mutants in loop 23 of TetA(P)
Of greatest interest were the mutants that harboured stably produced TetA(P) proteins with lower tetracycline MIC values. These mutants represent functional rather than purely structural changes in the protein. Most of these mutants were clustered into putative cytoplasmic or periplasmic loop regions of the TetA(P) protein. Located within or very near to the proposed loop 23 were three TetA(P) mutants, I60T, P61S and T62A (Fig. 1). Each of the mutants produced detectable TetA(P) protein and had a lower tetracycline MIC, suggesting that these residues may play a role in the function of TetA(P). In particular, the TetA(P)-I60T protein was expressed at a high level and had an intermediate resistance phenotype (Table 1
). By contrast, mutants P61S and T62A consistently produced detectable, but much lower, levels of immunoreactive TetA(P) protein. In fact, every proline mutant detected in this study produced detectable but lower amounts of the TetA(P) protein. This result was consistent with the likely structural role of proline residues within the TetA(P) protein.
The E52 and E59 residues were previously shown to be essential for tetracycline efflux (Kennan et al., 1997 ). We have now shown that the mutation of several amino acids located in close proximity to these residues also leads to loss of tetracycline resistance. These studies provide strong evidence that regions close to the proposed cytoplasmic loop 23 are important for the structure and function of TetA(P). In this region, the A66V and R71C mutations also resulted in loss of tetracycline resistance although they led to the production of either an unstable protein or a protein no longer recognized by the antiserum. Similar results were observed when the nearby residue D67 was targeted previously by site-directed mutagenesis (Kennan et al., 1997
). The equivalent loop in other tetracycline efflux proteins contains the functionally essential conserved motif (GxxxxRxGRR) (Yamaguchi et al., 1992d
). Although the TetA(P) protein does not have this exact motif it is clear that the same region of the protein is important for tetracycline efflux.
Conserved efflux protein motif
Comparison of TetA(P) with the databases only identified proteins with relatively low levels of similarity. The highest level of amino acid sequence identity observed was approximately 20%, occurring mainly with putative transport proteins whose functions are yet to be characterized. These include a hypothetical multidrug efflux transporter, BBI26, from Borrelia burgdorferi (Fraser et al., 1997 ), a hypothetical tetracycline-resistance protein, HP1165, from Helicobacter pylori (Tomb et al., 1997
) and a hypothetical 44·7 kDa protein, YxaM, from Bacillus subtilis (Yoshida et al., 1995
). Alignment of these proteins with TetA(P) identified a consensus motif, ExPxxxxxDxxxRK, where x corresponds to any amino acid residue (Fig. 3
). This motif overlaps the putative TMD 2-loop 2,3-TMD 3 region of TetA(P). It is of particular interest as the mutations which affect the function or protein stability of TetA(P) have been observed in the first four of the five conserved residues of this motif, specifically E59Q (Kennan et al., 1997
), P61S, D67N (Kennan et al., 1997
) and R71C. This observation suggests that these residues may be important for the function of each of these proteins. The DxxxRK portion of this TetA(P) motif is found in the majority of 12 TMD transport proteins, and actually forms part of the major identifying motif (Motif A) of proteins within MFS family 3 (Pao et al., 1998
; Paulsen et al., 1996
). In addition, this DxxxRK sequence is found within the consensus sequence previously identified as being conserved within tetracycline efflux proteins identified from Gram-negative species (Fig. 3
) (Yamaguchi et al., 1992d
). Functional studies have shown that the aspartate and arginine residues are required for the efflux of tetracycline by TetA(B) (Yamaguchi et al., 1992d
).
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Mutations were detected in five of the residues of loop 45. Both the G114D and A121E proteins were expressed at relatively high levels (Table 1), considering the change to charged residues, indicating that these residues are functionally important. By comparison, the A118V mutation, which would normally be considered as a relatively conservative change, led to a greater than 10-fold reduction in stable TetA(P) expression. The mutant G136R, which introduced a charged residue into putative loop 45, was producing TetA(P) protein at 20% of wild-type levels but had an MIC of only 2 and therefore also appears to be functionally important.
The proposed cytoplasmic loop 45 is of particular interest due to its high percentage of charged residues. On the basis of their low tetracycline MIC and relatively high TetA(P) levels the most interesting mutants in this region are G114D and G136R. The presence of charged residues at these positions disrupts the function of TetA(P). The role of these glycine residues is not clear but may be structural in nature. Glycine residues have previously been reported as being important for the structure of the TetA(B) protein (Yamaguchi et al., 1992d ).
TetA(P) derivatives with changes in loops 67 and 78
Each of the derivatives with alterations in the putative cytoplasmic loop 67, P188L, A190T and P191L, were stably produced at various levels. However, since with the P188L and P191L TetA(P) proteins the reduction in tetracycline MIC was related to the reduction in TetA(P) expression, it was concluded that these changes were likely to represent structural rather than functional effects. This conclusion is supported by the fact that these mutations involved proline residues, which are known to introduce conformational bends into proteins.
The next set of mutations involved the largest proposed periplasmic loop 78. E232 was the second mutagenized glutamate residue to be identified in this random screen. The E232K derivative did not confer tetracycline resistance but was expressed at 60% of wild-type TetA(P), which suggests that this residue plays an important functional role in TetA(P)-mediated tetracycline efflux. Due to the proposed location of E232 it is possible that this residue plays a gating role in the movement of tetracycline on the periplasmic membrane of the cell, in a similar way that D66 is proposed to play a gating role for tetracycline on the cytoplasmic side of TetA(B) (Yamaguchi et al., 1992c ).
Two independent variants were identified in the nearby D235 residue. Both derivatives had reduced tetracycline MIC levels but there was a large variation in the observed level of immunoreactive TetA(P), with D235N being expressed at slightly higher than wild-type levels, whereas D235G was expressed at much less than wild-type levels. Although the reduced level of resistance conferred by TetA(P)-D235G can be explained by the lower level of stability or expression, the D235N data clearly suggest that this residue is functionally important. A similar functional requirement has been observed with aspartate residues from TetA(K) (Fujihara et al., 1997).
Finally, G261 is predicted to be situated close to loop 78 but within TMD 8. The mutant identified had a charged glutamic acid residue at this position and was shown to confer only low level tetracycline resistance. The TetA(P)-G261E protein was stably produced, unlike other glycine to charged amino acid changes. Therefore, it appears that the presence of a charged residue at this position, although still allowing a stable protein to be produced, alters the structure of the protein in such a way that TetA(P) function is disrupted. Based on the hypothesis that the introduction of a charged residue into a hydrophobic region of the protein leads to instability, it is possible that this residue may be situated outside the TMD.
The TetA(P)-S360F derivative
Two mutations were identified within or very near to the proposed cytoplasmic loop 1011. Both mutants, A349T and S360F, were expressed at high levels. The latter derivative was of particular interest as the change from serine to phenylalanine resulted in a greatly reduced MIC, which suggests that this serine residue may play an important structural or functional role in TetA(P)-mediated tetracycline efflux. Although several serine residues have been identified as being part of the tetracycline translocation channel of TetA(B) (Yamaguchi et al., 1992b ), this serine residue is in a different relative position in TetA(P) and its putative role therefore remains unknown.
Functional amino acids are located throughout TetA(P)
Previous studies on TetA(P) identified three glutamic acid residues which are required for function (Kennan et al., 1997 ). These residues, E52, E59 and E89, are located within TMD 2 and close to TMD 3. We have now extended these studies by using random mutagenesis and have shown that there are potentially functional residues located over a much wider portion of the protein. In particular, we have shown that the putative cytoplasmic loops 23 and 45, and the periplasmic loop 78 are important functional domains in the efflux of tetracycline from the cell. In this study several residues of putative functional importance were identified, specifically P61, T62, A118, A121, G136, E232, D235 and S360. Analysis of the precise physical requirements at these positions using site-directed mutagenesis will aid in the further understanding of their role in tetracycline efflux by the TetA(P) protein.
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ACKNOWLEDGEMENTS |
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Received 8 March 1999;
revised 28 May 1999;
accepted 4 June 1999.