ARC Centre for Structural and Functional Microbial Genomics, Department of Microbiology, Monash University, Victoria 3800, Australia
Correspondence
Trudi L. Bannam
trudi.bannam{at}med.monash.edu.au
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ABSTRACT |
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Present address: Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
Present address: Monash Institute of Reproduction and Development, Monash University, Victoria 3800, Australia.
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INTRODUCTION |
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METHODS |
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Site-directed mutagenesis.
This was performed using the USE mutagenesis kit (Amersham Biosciences) following the manufacturer's instructions, as described previously (Kennan et al., 1997). Oligonucleotide primers were synthesized on an Applied Biosystems 392 DNA/RNA synthesizer and sequences of the mutagenic oligonucleotides are available upon request. Since the selection primer eliminated the unique AatII site within the pUC18 portion of pJIR71, mutant plasmids were initially screened by their resistance to digestion with AatII. Subsequently, the entire tetA(P) gene region was sequenced to confirm that only the required mutation was present. Sequence analysis was carried out using the PRISM Ready Reaction Dye Deoxy Terminator Cycle Sequencing Kit (Applied Biosystems) and an ABI373A automated fluorescent sequencing apparatus (Applied Biosystems).
Analysis of mutants.
The tetracycline efflux protein analysed in this study originated from C. perfringens but is active in E. coli. Our previous attempts to introduce the tetA(P) gene onto shuttle plasmids that can replicate in C. perfringens and E. coli have resulted in instability (J. Sloan & J. Rood, unpublished results). Therefore, we are currently unable to critically analyse the function of the TetA(P) protein in C. perfringens. In this study, all of the TetA(P) functional analysis was performed in E. coli DH5. For each of the tetA(P) mutants, the ability to confer tetracycline resistance was tested by determining tetracycline minimal inhibitory concentrations (MICs) as described previously (Kennan et al., 1997
). Each E. coli DH5
derivative harbouring a mutated tetA(P) gene was grown to a turbidity at 550 nm of 0·8, diluted 1 in 100 and spot-tested (10 µl) in triplicate onto 2xYT agar plates containing tetracycline at concentrations ranging from 1 to 30 µg ml-1. After incubation for 1820 h at 37 °C, the MIC was determined as the lowest concentration of tetracycline that completely inhibited growth. Immunoblot analysis of the TetA(P) protein and its derivatives was performed using inner-membrane preparations as described previously (Kennan et al., 1997
). The protein concentration of these samples was measured using the BCA Protein Assay Kit (Pierce). For each sample, 10 µg was subjected to SDS-PAGE and the separated proteins were electrotransferred onto a nitrocellulose filter. The TetA(P) protein and its derivatives were detected by probing the filter with anti-TetA(P) antibodies raised against the C-terminal 17 aa of TetA(P) conjugated to diphtheria toxoid (Kennan et al., 1997
), following the instructions of the ECL Western blotting system (Amersham Biosciences). Inner-membrane preparations of each TetA(P) derivative were isolated and immunoblotted at least three times.
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RESULTS |
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Analysis of the basic residues of Motif A
In TetA(P), the first basic residue of the extended Motif A is replaced by valine. To examine the functional significance of Val-68, it was replaced independently by leucine, threonine, alanine and, most importantly, the consensus arginine residue. These mutations had varying effects on tetracycline resistance (Table 1), although all yielded immunoreactive membrane proteins (Fig. 3
). The conservative V68L change produced wild-type tetracycline resistance, whereas the V68A and V68T mutants had an intermediate resistance. Unexpectedly, replacement of Val-68 with arginine, as in Motif A, abolished tetracycline resistance. Unlike other tetracycline efflux proteins, it is clear that at this position in TetA(P) a positively charged residue significantly disrupts efflux function.
At position 71 and 72 in TetA(P) there are the positively charged amino acids arginine and lysine, respectively. These residues form part of the Motif A consensus sequence and are highly conserved. To examine the role of Arg-71 in TetA(P), it was independently changed to lysine, glutamate and glutamine. Immunoreactive proteins were observed for all three derivatives although there was less R71E compared to wild-type (Fig. 3). The R71K, R71E and R71Q mutants were unable to confer tetracycline resistance (Table 1
), which indicated that there was a requirement for arginine at this position. Similar analysis was performed on the positively charged Lys-72 residue. Immunoreactive protein was observed for K72R, whereas K72E and K72Q had reduced levels (Fig. 3
). The K72E and K72Q derivatives were unable to confer tetracycline resistance but K72R gave an intermediate resistance level (Table 1
). These results suggest that there is a requirement for a basic amino acid at position 72 within TetA(P).
Functional significance of Glu-117
Our previous computer-based topology models of TetA(P) predicted that the variant Motif A was located within TMD3 (Bannam & Rood, 1999). After examining the well studied topology models of TetA(B) and lactose permease (Frillingos et al., 1998
; Tamura et al., 2001
), and taking into account the mutagenesis results, we considered that this region was more likely to be located within the intergenic loop region 23 (Fig. 1
). After modifying the TetA(P) model to compensate for this prediction, we observed that we had now introduced a negatively charged residue, Glu-117, into the putative TMD4 (Fig. 1
). To examine the significance of this residue, it was independently changed to glutamine, lysine and aspartate. The resultant E117Q and E117K derivatives no longer conferred tetracycline resistance (Table 1
) and no immunoreactive protein was observed for the E117K derivative (Fig. 3
). In contrast, the E117D derivative was fully functional, indicating that an acidic amino acid side chain is required at this position.
Glutamate residues in putative loop 45
To date, four functionally important glutamate residues have been identified within TMDs of the TetA(P) protein, Glu-52, Glu-59, Glu-89 and now Glu-117. Within the putative loop 45 region of TetA(P) there are five glutamate residues (Fig. 1). To examine the functional importance of this region, Glu-122, Glu-123 and Glu-131 were changed independently to glutamine. For each of these derivatives a TetA(P) protein was detected (Fig. 3
) and they still conferred resistance to tetracycline (Table 1
), indicating that these glutamate residues are not essential for TetA(P) function.
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DISCUSSION |
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A proline residue occupies the third conserved position in the TetA(P) variant Motif A region. Proline is known to have a high propensity to induce turns within proteins and to place conformational constraints on the surrounding region. Mutation of Pro-61 to residues with a smaller side-chain volume, such as alanine and glycine, abolished TetA(P) function. In addition, the non-polar bulky side chain of tryptophan at this position was also not acceptable for TetA(P) function. These results imply that side-chain spatial volume is critical at this position and that the structural constraint introduced by the presence of Pro-61 is essential for biological activity. Requirement for a proline within a similar region has also been noted for the E. coli TetA(B) protein, although a different effect was observed. When Pro-59 of TetA(B) was replaced with cysteine, resistance to tetracycline was not affected; however, this mutant demonstrated a reduced ability to transport tetracycline within everted vesicles (Kimura-Someya et al., 1998).
In TetA(B), a positively charged amino acid is found at position 67, whereas in TetA(P) it is a valine residue. When a positive charge was introduced into TetA(P) at this position, to construct the V68R derivative, resistance to tetracycline was abolished. Only non-polar residues were found to be functionally acceptable at this position, indicating that the role of this residue within TetA(P) is different to that within TetA(B). In contrast, TetA(P) does contain the two highly conserved basic residues at the end of Motif A. Mutation of Arg-71, to a negatively charged, polar or even to the basic amino acid lysine, led to loss of tetracycline resistance, suggesting a specific requirement for arginine at this position. A similar loss of function was observed with the R71C mutant (Bannam & Rood, 1999). Clearly, charge and side-chain length are important at this position in TetA(P). This result differs from that observed with TetA(B), where the corresponding residue, Arg-70, can be changed to lysine and still retain function (Yamaguchi et al., 1992
; Kimura et al., 1998
).
Mutation of the second basic residue, Lys-72, showed that proteins with either lysine or arginine at this position were able to confer tetracycline resistance. Replacement of this residue with other amino acids led to a loss of resistance. This result differed from that observed with TetA(B) as when the corresponding residue, Arg-71, was replaced with a neutral amino acid biological function was not affected (Yamaguchi et al., 1992). As this region of the TetA(P) protein has only two basic residues, rather than the usual three found in MFS transporters, it is possible that removal of one basic side chain is detrimental to the overall structure and therefore the correct steric positioning of this region. Independent replacement of each of the three positive charges within lactose permease was found not to affect lactose transport (Jessen-Marshall et al., 1995
); similarly, double mutants still retained function. However, a triple mutant had negligible levels of permease in the membrane and consequently was completely defective for transport (Pazdernik et al., 2000
). It has been suggested that for lactose permease the topology of this region is important for the ability of the protein to undergo the conformational changes required for substrate transport (Cain et al., 2000
). Similar results were observed for the eukaryotic GlutI glucose transporter (Sato & Mueckler, 1999
). The positively charged residues were required for correct structure of this protein, specifically for the correct positioning of this region with respect to the membrane (Sato & Mueckler, 1999
). It would be of interest to examine whether the mutation of the positively charged regions of TetA(P) are causing similar disruptions to the protein structure. This would require further information regarding the native membrane topology of TetA(P), which could be obtained using either alkaline phosphatase fusions or more specifically by cysteine scanning. Attempts to carry out the former have not proven to be productive (T. Bannam & J. Rood, unpublished results) and mutation of all four cysteine residues in TetA(P) significantly reduces biological activity, limiting the value of performing cysteine-scanning mutagenesis (P. Johanesen & J. Rood, unpublished results).
Previous studies have shown that Glu-52 and Glu-59 are essential for TetA(P) function (Kennan et al., 1997). In addition, this study has shown that at position 117 of TetA(P) there is a requirement for a negatively charged residue, as previously observed at position 89 (Kennan et al., 1997
). As it is known that the E. coli TetA(B) protein transports tetracycline in the form of a divalent cationtetracycline complex (Yamaguchi et al., 1990
), it would be of interest to examine whether any of these glutamate side chains are involved intimately with the transport of tetracycline. By contrast, we showed that several of the glutamate residues, which are proposed to be in loop 45, are not involved in tetracycline transport as changing these residues to the neutral glutamine had little effect on tetracycline resistance.
Finally, the conserved variant Motif A, ExPxxxxxDxxxRK, was found in putative transport proteins that have limited similarity to the TetA(P) protein. Although the solute that these proteins are capable of transporting has yet to be identified, they are unlikely to encode resistance to tetracycline. None of the host strains from which these sequences were derived is known to be tetracycline resistant. The HP1165 protein from Helicobacter pylori does not encode tetracycline resistance (Gerrits et al., 2002). In addition, the only conserved functional glutamate residue that they contain is that found within the variant Motif A region. Conservation of this motif amongst proteins that may be capable of transporting different solutes suggests that its role in transport is of a general nature. It is not likely that this region is involved in interaction with the specific target molecules to be transported. Instead, its role may be in maintaining the structural conformation required for transmembrane stability or in providing a common functional role such as energy translocation via the proton motive force.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Bannam, T. L. & Rood, J. I. (1999). Identification of structural and functional domains of the tetracycline efflux protein TetA(P) from Clostridium perfringens. Microbiology 145, 29472955.
Bao, Q., Tian, Y., Li, W. & 18 other authors (2002). A complete sequence of the T. tengcongensis genome. Genome Res 12, 689700.
Buchel, D. E., Gronenborn, B. & Muller-Hill, B. (1980). Sequence of the lactose permease gene. Nature 283, 541545.[Medline]
Cain, S. M., Matzke, E. A. & Brooker, R. J. (2000). The conserved motif in hydrophilic loop 2/3 and loop 8/9 of the lactose permease of Escherichia coli. Analysis of suppressor mutations. J Membr Biol 176, 159168.[CrossRef][Medline]
Claros, M. G. & von Heijne, G. (1994). TOPPRED II: an improved software for membrane protein structure predictions. Comput Appl Biosci 10, 685686.[Medline]
Fraser, C. M., Casjens, S. Huang, W. M. & 35 other authors (1997). Genomic sequence of a Lyme disease spirochaete, Borrelia burgdorferi. Nature 390, 580586.[CrossRef][Medline]
Frillingos, S., Sahin-Toth, M., Wu, J. & Kaback, H. R. (1998). Cys-scanning mutagenesis: a novel approach to structure function relationships in polytopic membrane proteins. FASEB J 12, 12811299.
Gerrits, M. M., de Zoete, M. R., Arents, N. L., Kuipers, E. J. & Kusters, J. G. (2002). 16S rRNA mutation-mediated tetracycline resistance in Helicobacter pylori. Antimicrob Agents Chemother 46, 29963000.
Henderson, P. J. (1990). Proton-linked sugar transport systems in bacteria. J Bioenerg Biomembr 22, 525569.[Medline]
Hirai, T., Heymann, J. A. W., Shi, D., Sarker, R., Maloney, P. C. & Subramaniam, S. (2002). Three-dimensional structure of a bacterial oxalate transporter. Nat Struct Biol 9, 597600.[Medline]
Hirai, T., Heymann, J. A. W., Maloney, P. C. & Subramaniam, S. (2003). Structural model for 12-helix transporters belonging to the major facilitator superfamily. J Bacteriol 185, 17121718.
Jessen-Marshall, A. E., Paul, N. J. & Brooker, R. J. (1995). The conserved motif, GXXX(D/E)(R/K)XG[X](R/K)(R/K), in hydrophilic loop 2/3 of the lactose permease. J Biol Chem 270, 1625116257.
Jessen-Marshall, A. E., Parker, N. J. & Brooker, R. J. (1997). Suppressor analysis of mutations in the loop 2-3 motif of lactose permease: evidence that glycine-64 is an important residue for conformational changes. J Bacteriol 179, 26162622.[Abstract]
Kennan, R. M., McMurry, L. M., Levy, S. B. & Rood, J. I. (1997). Glutamate residues located within putative transmembrane helices are essential for TetA(P)-mediated tetracycline efflux. J Bacteriol 179, 70117015.[Abstract]
Kimura, T., Nakatani, M., Kawabe, T. & Yamaguchi, A. (1998). Roles of conserved arginine residues in the metal-tetracycline/H+ antiporter of Escherichia coli. Biochemistry 37, 54755480.[CrossRef][Medline]
Kimura-Someya, T., Iwaki, S. & Yamaguchi, A. (1998). Site-directed chemical modification of cysteine-scanning mutants as to transmembrane segment II and its flanking regions of the Tn10-encoded metal-tetracycline/H+ antiporter reveals a transmembrane water-filled channel. J Biol Chem 273, 3280632811.
Lyras, D. & Rood, J. I. (1996). Genetic organization and distribution of tetracycline resistance determinants in Clostridium perfringens. Antimicrob Agents Chemother 40, 25002504.[Abstract]
Marger, M. D. & Saier, M. H., Jr (1993). A major superfamily of transmembrane facilitators that catalyse uniport, symport and antiport. Trends Biochem Sci 18, 1320.[CrossRef][Medline]
Miller, J. (1972). Experiments in Molecular Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Ng, W. V., Kennedy, S. P., Mahairas, G. G. & 40 other authors (2000). Genome sequence of Halobacterium species NRC-1. Proc Natl Acad Sci U S A 97, 1217612181.
Nguyen, T. T., Postle, K. & Bertrand, K. P. (1983). Sequence homology between the tetracycline-resistance determinants of Tn10 and pBR322. Gene 25, 8392.[CrossRef][Medline]
Nolling, J., Breton, G., Omelchenko, M. V. & 16 other authors (2001). Genome sequence and comparative analysis of the solvent-producing bacterium Clostridium acetobutylicum. J Bacteriol 183, 48234838.
Pao, S. S., Paulsen, I. T. & Saier, M. H., Jr (1998). Major facilitator superfamily. Microbiol Mol Biol Rev 62, 134.
Paulsen, I. T. & Skurray, R. A. (1993). Topology, structure and evolution of two families of proteins involved in antibiotic and antiseptic resistance in eukaryotes and prokaryotes an analysis. Gene 124, 111.[CrossRef][Medline]
Paulsen, I. T., Brown, M. H. & Skurray, R. A. (1996). Proton-dependent multidrug efflux systems. Microbiol Rev 60, 575608.[Medline]
Pazdernik, N. J., Matzke, E. A., Jessen-Marshall, A. E. & Brooker, R. J. (2000). Roles of charged residues in the conserved motif, G-X-X-X-D/E-R/K-X-G-[X]-R/K-R/K, of the lactose permease of Escherichia coli. J Membr Biol 174, 3140.[CrossRef][Medline]
Saier, M. H., Jr, Beatty, J. T., Goffeau, A. & 11 other authors (1999). The major facilitator superfamily. J Mol Microbiol Biotechnol 1, 257279.[Medline]
Sato, M. & Mueckler, M. (1999). A conserved amino acid motif (R-X-G-R-R) in the Glut1 glucose transporter is an important determinant of membrane topology. J Biol Chem 274, 2472124725.
Sloan, J., McMurry, L. M., Lyras, D., Levy, S. B. & Rood, J. I. (1994). The Clostridium perfringens Tet P determinant comprises two overlapping genes: tetA(P), which mediates active tetracycline efflux, and tetB(P), which is related to the ribosomal protection family of tetracycline-resistance determinants. Mol Microbiol 11, 403415.[Medline]
Tamura, N., Konishi, S., Iwaki, S., Kimura-Someya, T., Nada, S. & Yamaguchi, A. (2001). Complete cysteine-scanning mutagenesis and site-directed chemical modification of the Tn10-encoded metal-tetracycline/H+ antiporter. J Biol Chem 276, 2033020339.
Tomb, J.-F., White, O., Kerlavage, A. R. & 39 other authors (1997). The complete genome sequence of the gastric pathogen Helicobacter pylori. Nature 388, 539547.[CrossRef][Medline]
Yamaguchi, A., Udagawa, T. & Sawai, T. (1990). Transport of divalent cations with tetracycline as mediated by the transposon Tn10-encoded tetracycline resistance protein. J Biol Chem 265, 48094813.
Yamaguchi, A., Someya, Y. & Sawai, T. (1992). Metal-tetracycline/H+ antiporter of Escherichia coli encoded by transposon Tn10. The role of a conserved sequence motif, GXXXXRXGRR, in a putative cytoplasmic loop between helices 2 and 3. J Biol Chem 267, 1915519162.
Yoshida, K., Seki, S., Fujimura, M., Miwa, Y. & Fujita, Y. (1995). Cloning and sequencing of a 36-kb region of the Bacillus subtilis genome between the gnt and iol operons. DNA Res 2, 6169.[Medline]
Received 1 July 2003;
revised 17 October 2003;
accepted 20 October 2003.
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