The TetA(K) tetracycline/H(+) antiporter from Staphylococcus aureus: mutagenesis and functional analysis of motif C

Conserved motif C, identified within members of the major facilitator superfamily (MFS) of transport proteins that mediate drug export, was examined in the tetracycline resistance efflux protein TetA(K) from Staphylococcus aureus; motif C is contained within transmembrane segment 5. Using site-directed mutagenesis, the importance of the conserved glycine (G151, G155, G159, and G160) and proline (P156) residues within this motif was investigated. Over 40 individual amino acid replacements were introduced; however, only alanine and serine substitutions for glycine at G151, G155, and G160 were found to retain significant levels of tetracycline resistance and transport activity in cells expressing mutant proteins. Notably, P156 and G159 appear to be crucial, as amino acid replacements at these positions either significantly reduced or abolished tetracycline/H(+) activity. The highly conserved nature of motif C and its distribution throughout drug exporters imply that the residues of motif C play a similar role in all MFS proteins that function as antiporters.     

Mutational analysis of tetracycline resistance protein transmembrane segment insertion

The tetracycline resistance proteins (TetA) of gram-negative bacteria are secondary active transport proteins that contain buried charged amino acids that are important for tetracycline transport. Earlier studies have shown that insertion of TetA proteins into the cytoplasmic membrane is mediated by helical hairpin pairs of transmembrane (TM) segments. However, whether helical hairpins direct spontaneous insertion of TetA or are required instead for its interaction with the cellular secretion (Sec) machinery is unknown. To gain insight into how TetA proteins are inserted into the membrane, we have investigated how tolerant the class C TetA protein encoded by plasmid pBR322 is to placement of charged residues in TM segments. The results show that the great majority of charge substitutions do not interfere with insertion even when placed at locations that cannot be shielded internally within helical hairpins. The only mutations that frequently block insertion are proline substitutions, which may interfere with helical hairpin folding. The ability of TetA to broadly tolerate charge substitutions indicates that the Sec machinery assists in its insertion into the membrane. The results also demonstrate that it is feasible to engineer charged residues into the interior of TetA proteins for the purpose of structure-function analysis.     

Topology of the transposon Tn10-encoded tetracycline resistance protein within the inner membrane of Escherichia coli

The transposon Tn10-encoded tetracycline resistance protein TetA is an integral membrane protein responsible for the export of tetracycline from the cytoplasmic to the periplasmic side of the inner membrane of Gram-negative bacteria. From a plot of the average hydrophobicity along the sequence of this protein, a two-dimensional membrane topology with 12 transmembrane domains may be predicted. Using plasmid-bearing Escherichia coli maxicells we specifically radiolabeled the TetA protein. The amino terminus of this membrane protein was shown not to be processed, and its location on the inner side of the cytoplasmic membrane was demonstrated by a newly developed use of a chemical method. Spheroplasts and inside-out vesicles of the TetA protein synthesizing maxicells were subjected to limited digestion by proteases of different specificities. The TetA protein was not accessible to proteases from the periplasmic side. On the inner side of the cytoplasmic membrane, the carboxyl terminus and four sites accessible to endoproteases could be identified. The cleavage sites are proposed to be localized between amino acid residues 60-70, 110-130, 180-200, and at amino acid 327. These results allow the definition of a model for the two-dimensional topology of the TetA protein.     

Conserved cytoplasmic loops are important for both the transport and chemotaxis functions of PcaK, a protein from Pseudomonas putida with 12 membrane-spanning regions.

Chemotaxis to the aromatic acid 4-hydroxybenzoate (4-HBA) by Pseudomonas putida is mediated by PcaK, a membrane-bound protein that also functions as a 4-HBA transporter. PcaK belongs to the major facilitator superfamily (MFS) of transport proteins, none of which have so far been implicated in chemotaxis. Work with two well-studied MFS transporters, LacY (the lactose permease) and TetA (a tetracycline efflux protein), has revealed two stretches of amino acids located between the second and third (2-3 loop) and the eighth and ninth (8-9 loop) transmembrane regions that are required for substrate transport. These sequences are conserved among most MFS transporters, including PcaK. To determine if PcaK has functional requirements similar to those of other MFS transport proteins and to analyze the relationship between the transport and chemotaxis functions of PcaK, we generated strains with mutations in amino acid residues located in the 2-3 and 8-9 loops of PcaK. The mutant proteins were analyzed in 4-HBA transport and chemotaxis assays. Cells expressing mutant PcaK proteins had a range of phenotypes. Some transported at wild-type levels, while others were partially or completely defective in 4-HBA transport. An aspartate residue in the 8-9 loop that has no counterpart in LacY and TetA, but is conserved among members of the aromatic acid/H(+) symporter family of the MFS, was found to be critical for 4-HBA transport. These results indicate that conserved amino acids in the 2-3 and 8-9 loops of PcaK are required for 4-HBA transport. Amino acid changes that decreased 4-HBA transport also caused a decrease in 4-HBA chemotaxis, but the effect on chemotaxis was sometimes slightly more severe. The requirement of PcaK for both 4-HBA transport and chemotaxis demonstrates that P. putida has a chemoreceptor that differs from the classical chemoreceptors described for Escherichia coli and Salmonella typhimurium.     

TetZ, a new tetracycline resistance determinant discovered in gram-positive bacteria, shows high homology to gram-negative regulated efflux systems

The complete nucleotide sequence of the tetracycline resistance plasmid pAG1 from the gram-positive soil bacterium Corynebacterium glutamicum 22243 (formerly Corynebacterium melassecola 22243) was determined. The R-plasmid has a size of 19,751 bp and contains at least 18 complete open reading frames. The resistance determinant of pAG1 revealed homology to gram-negative tetracycline efflux and repressor systems of Tet classes A through J. The highest levels of amino acid sequence similarity were observed to the transmembrane tetracycline efflux protein TetA(A) and to the tetracycline repressor TetR(A) of transposon Tn1721 with 64 and 56% similarity, respectively. This is the first time a repressor-regulated tet gene has been found in gram-positive bacteria. A new class of tetracycline resistance and repressor proteins, termed TetA(Z) and TetR(Z), is proposed.     

Cysteine-scanning mutagenesis of transmembrane segments 4 and 5 of the Tn10-encoded metal-tetracycline/H+ antiporter reveals a permeability barrier in the middle of a transmembrane water-filled channel

Cysteine-scanning mutants as to putative transmembrane segments 4 and 5 and the flanking regions of Tn10-encoded metal-tetracycline/H(+) antiporter (TetA(B)) were constructed. All mutants were normally expressed. Among the 57 mutants (L99C to I155C), nine conserved arginine-, aspartate-, and glycine-replaced ones exhibited greatly reduced tetracycline resistance and almost no transport activity, and five conserved glycine- and proline-replaced mutants exhibited greatly reduced tetracycline transport activity in inverted membrane vesicles despite their high or moderate drug resistance. All other cysteine-scanning mutants retained normal drug resistance and normal tetracycline transport activity except for the L142C and I143C mutants. The transmembrane (TM) regions TM4 and TM5 were determined to comprise 20 amino acid residues, Leu-99 to Ile-118, and 17 amino acid residues, Ala-136 to Ala-152, respectively, on the basis of N-[(14)C]ethylmaleimide ([(14)C]NEM) reactivity. The NEM reactivity patterns of the TM4 and TM5 mutants were quite different from each other. TM4 could be divided into two halves, that is, a NEM nonreactive periplasmic half and a periodically reactive cytoplasmic half, indicating that TM4 is tilted toward a water-filled transmembrane channel and that only its cytoplasmic half faces the channel. On the other hand, NEM-reactive mutations were observed periodically (every two residues) along the whole length of TM5. A permeability barrier for a membrane-impermeable sulfhydryl reagent, 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid, was present in the middle of TM5 between Leu-142 and Gly-145, whereas all the NEM-reactive mutants as to TM4 were not accessible to 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid, indicating that the channel-facing side of TM4 is located inside the permeability barrier. Tetracycline protected the G141C mutant from the NEM binding, whereas the other mutants in TM4 and TM5 were not protected by tetracycline.     

Expression of the tetA(C) tetracycline efflux pump in Escherichia coli confers osmotic sensitivity

Expression of tetA(C) in Escherichia coli confers resistance to tetracycline as well as sensitivity to nickel and cadmium salts, lipophilic chelating agents, and aminoglycoside antibiotics. In this report we determine that high-level expression of tetA(C) also confers an osmotic sensitivity. The osmotic-sensitive phenotype is distinct from the tetracycline-resistant phenotype and can be localized to a domain contained within the first 98 amino acid residues of the TetA(C) polypeptide.     

Genetic analysis of the tetA(C) gene on plasmid pBR322.

The TetA(C) protein, encoded by the tetA(C) gene of plasmid pBR322, is a member of a family of membrane-bound proteins that mediate energy-dependent efflux of tetracycline from the bacterial cell. The tetA(C) gene was mutagenized with hydroxylamine, and missense mutations causing the loss of tetracycline resistance were identified at 30 distinct codons. Mutations that encoded substitutions within putative membrane-spanning alpha-helical regions were scattered throughout the gene. In contrast, mutations outside the alpha-helical regions were clustered in two cytoplasmic loops, between helices 2 and 3 and helices 10 and 11, suggesting that these regions play a critical role in the recognition of tetracycline and/or energy transduction. All of the missense mutations encoded a protein that retained the ability to rescue an Escherichia coli strain defective in potassium uptake, suggesting that the loss of tetracycline resistance was not due to an unstable TetA(C) protein or to the failure of the protein to be inserted in the membrane. We postulate that the mutations encode residues that are critical for the active efflux of tetracycline, except for mutations that result in the introduction of charged residues within hydrophobic regions of the TetA(C) protein.     

Purification of the Tn 10-specified tetracycline efflux antiporter TetA in a native state as a polyhistidine fusion protein

The bacterial tetracycline-resistance determinant from Tn 10 encodes a 43 kDa membrane protein, TetA, responsible for active efflux of tetracyclines. The tetA gene was cloned behind a T7 promoter/ac operator in a plasmid that provided fusion of TetA to a polyhis-tidine-carboxy terminal tail. A second plasmid provided a regulated T7 RNA polymerase. The specific activity of the TetA fusion protein was between 10–40% that of the wild-type protein as assayed by tetracycline resistance in cells and by transport in membrane vesicles. The fusion protein, overproduced approximately 3–13-fold, was purified by nickel chelation chromatography. Calculations from circular dichroism spectra of the purified protein solubilized in dodecylmaltoside gave an α-helix content of 54–64%, close to the 68% predicted from the amino acid sequence by hydropathy analysis (12 membrane-spanning helices) for the native protein in the membrane bilayer. Fluorescence studies showed binding activity of the purified protein to its substrate, the tetracycline analogue 13-(cyclopentylthio)-5-hydroxy-6-α-deoxyte-tracycline. These findings suggested that the purified protein was in a native state.     

Fe(2+)-tetracycline-mediated cleavage of the Tn10 tetracycline efflux protein TetA reveals a substrate binding site near glutamine 225 in transmembrane helix 7

TetA specified by Tn10 is a class B member of a group of related bacterial transport proteins of 12 transmembrane alpha helices that mediate resistance to the antibiotic tetracycline. A tetracycline-divalent metal cation complex is expelled from the cell in exchange for a entering proton. The site(s) where tetracycline binds to this export pump is not known. We found that, when chelated to tetracycline, Fe(2+) cleaved the backbone of TetA predominantly at a single position, glutamine 225 in transmembrane helix 7. The related class D TetA protein from plasmid RA1 was cut at exactly the same position. There was no cleavage with glycylcycline, an analog of tetracycline that does not bind to TetA. The Fe(2+)-tetracycline complex was not detectably transported by TetA. However, cleavage products of the same size as with Fe(2+) occurred with Co(2+), known to be cotransported with tetracycline. The known substrate Mg (2+)-tetracycline interfered with cleavage by Fe(2+). These findings suggest that cleavage results from binding at a substrate-specific site. Fe(2+) is known to be able to cleave amide bonds in proteins at distances up to approximately 12 A. We conclude that the alpha carbon of glutamine 225 is probably within 12 A of the position of the Fe(2+) ion in the Fe(2+)-tetracycline complex bound to the protein.     

Monoclonal antibody that binds to the central loop of the Tn10-encoded metal tetracycline/H+ antiporter of Escherichia coli

Mouse monoclonal antibodies were prepared using His-tagged Tn10-encoded metal-tetracycline/H+ antiporter [TetA(B)His] as an antigen. From them, those reacting equally with His-tagged and wild-type TetA(B) were selected and named TCL-1. Cysteine-scanning mutants were used to determine the TCL-1 binding site on the TetA(B) protein. First, 12 Cys mutants of TetA(B) in which one residue in a protruding loop region was replaced by cysteine were constructed. Western blot analysis revealed the binding of TCL-1 to all of these Cys-mutants except for R186C. Then, we constructed 13 cysteine-scanning mutants, F179C to T191C. Among them, eight mutants, F179C to T182C, N184C, and T189C to T191C, exhibited TCL-1 binding, whereas the other five, K183C, T185C, R186C, D187C, and N188C, exhibited no or lower TCL-1 binding. These results clearly indicate that the sequence recognized by TCL-1 is 183Lys-X-Thr-Arg-Asp-Asn188 in the central loop region of TetA(B). TCL-1 is the first reported antibody that binds to a region other than the C-terminus of TetA(B), and the recognized amino acid sequence was identified.     

Structural and functional characterization of nonstructural protein 2 for its role in hepatitis C virus assembly

The hepatitis C virus (HCV) is a flavivirus replicating in the cytoplasm of infected cells. The HCV genome is a single-stranded RNA encoding a polyprotein that is cleaved by cellular and viral proteases into 10 different products. While the structural proteins core protein, envelope protein 1 (E1) and E2 build up the virus particle, most nonstructural (NS) proteins are required for RNA replication. One of the least studied proteins is NS2, which is composed of a C-terminal cytosolic protease domain and a highly hydrophobic N-terminal domain. It is assumed that the latter is composed of three trans-membrane segments (TMS) that tightly attach NS2 to intracellular membranes. Taking advantage of a system to study HCV assembly in a hepatoma cell line, in this study we performed a detailed characterization of NS2 with respect to its role for virus particle assembly. In agreement with an earlier report ( Jones, C. T., Murray, C. L., Eastman, D. K., Tassello, J., and Rice, C. M. (2007) J. Virol. 81, 8374-8383 ), we demonstrate that the protease domain, but not its enzymatic activity, is required for infectious virus production. We also show that serine residue 168 in NS2, implicated in the phosphorylation and stability of this protein, is dispensable for virion formation. In addition, we determined the NMR structure of the first TMS of NS2 and show that the N-terminal segment (amino acids 3-11) forms a putative flexible helical element connected to a stable alpha-helix (amino acids 12-21) that includes an absolutely conserved helix side in genotype 1b. By using this structure as well as the amino acid conservation as a guide for a functional study, we determined the contribution of individual amino acid residues in TMS1 for HCV assembly. We identified several residues that are critical for virion formation, most notably a central glycine residue at position 10 of TMS1. Finally, we demonstrate that mutations in NS2 blocking HCV assembly can be rescued by trans-complementation.     

Mutational analysis of the major proline transporter (PrnB) of Aspergillus nidulans

PrnB, the l-proline transporter of Aspergillus nidulans, belongs to the Amino acid Polyamine Organocation (APC) transporter family conserved in prokaryotes and eukaryotes. In silico analysis and limited biochemical evidence suggest that APC transporters comprise 12 transmembrane segments (TMS) connected with relatively short hydrophilic loops (L). However, very little is known on the structure-function relationships in APC transporters. This work makes use of the A. nidulans PrnB transporter to address structure-function relationships by selecting, constructing and analysing several prnB mutations. In the sample, most isolated missense mutations affecting PrnB function map in the borders of cytoplasmic loops with transmembrane domains. These are I119N and G120W in L2-TMS3, F278V in L6-TMS7, NRT378NRTNRT and PY382PYPY in L8-TMS9 and T456N in L10-TMS11. A single mutation (G403E) causing, however, a very weak phenotype, maps in the borders of an extracellular loop (L9-TMS10). An important role of helix TMS6 for proline binding and transport is supported by mutations K245L and, especially, F248L that clearly affect PrnB uptake kinetics. The critical role of these residues in proline binding and transport is further shown by constructing and analysing isogenic strains expressing selected prnB alleles fused to the gene encoding the Green Fluorescent Protein (GFP). It is shown that, while some prnB mutations affect proper translocation of PrnB in the membrane, at least two mutants, K245E and F248L, exhibit physiological cellular expression of PrnB and, thus, the corresponding mutations can be classified as mutations directly affecting proline binding and/or transport. Finally, comparison of these results with analogous studies strengthens conclusions concerning amino acid residues critical for function in APC transporters.     

Orientation of the carboxyl terminus of the transposon Tn10-encoded tetracycline resistance protein in Escherichia coli

A site-directed antibody was generated against a synthetic polypeptide corresponding to the 14 amino acid residues of the carboxyl terminus of the Tn10 TetA protein. The antibody reacted preferentially with inside-out vesicles, rather than right-side-out vesicles, prepared from Escherichia coli cells harboring transposon Tn10. When inside-out vesicles were treated with trypsin, the TetA protein was completely digested in the vicinity of the carboxyl terminus, as judged on immunoblot analysis using the antibody. In contrast, when right-side-out vesicles were treated with trypsin, the TetA protein was hardly digested. These results indicate that the carboxyl terminus of TetA is exposed to the cytoplasmic side of the membrane.     

Affinity of Avr2 for tomato cysteine protease Rcr3 correlates with the Avr2-triggered Cf-2-mediated hypersensitive response

The Cladosporium fulvum Avr2 effector is a novel type of cysteine protease inhibitor with eight cysteine residues that are all involved in disulphide bonds. We have produced wild-type Avr2 protein in Pichia pastoris and determined its disulphide bond pattern. By site-directed mutagenesis of all eight cysteine residues, we show that three of the four disulphide bonds are required for Avr2 stability. The six C-terminal amino acid residues of Avr2 contain one disulphide bond that is not embedded in its overall structure. Avr2 is not processed by the tomato cysteine protease Rcr3 and is an uncompetitive inhibitor of Rcr3. We also produced mutant Avr2 proteins in which selected amino acid residues were individually replaced by alanine, and, in one mutant, all six C-terminal amino acid residues were deleted. We determined the inhibitory constant (K(i) ) of these mutants for Rcr3 and their ability to trigger a Cf-2-mediated hypersensitive response (HR) in tomato. We found that the two C-terminal cysteine residues and the six amino acid C-terminal tail of Avr2 are required for both Rcr3 inhibitory activity and the ability to trigger a Cf-2-mediated HR. Individual replacement of the lysine-17, lysine-20 or tyrosine-21 residue by alanine did not affect significantly the biological activity of Avr2. Overall, our data suggest that the affinity of the Avr2 mutants for Rcr3 correlates with their ability to trigger a Cf-2-mediated HR.     

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

The complete nucleotide sequence and mechanism of action of the tetracycline-resistance determinant Tet P, from Clostridium perfringens has been determined. Analysis of the 4.4 kb of sequence data revealed the presence of two open reading frames, designated as tetA(P) and tetB(P), The tetA(P) gene appears to encode a 420 amino acid protein (molecular weight 46079) with twelve transmembrane domains. This gene was shown to be responsible for the active efflux of tetracycline from resistant ceils. Although there was some amino acid sequence similarity between the putative TetA(P) protein and other tetracycline efflux proteins, analysis suggested that TetA(P) represented a different type of efflux protein. The tetB(P) gene would encode a putative 652 amino acid protein (molecular weight 72639) with significant sequence similarity to Tet(M)-like cytoplasmic proteins that specify a ribosomal-protection tetracycline-resistance mechanism. In both C. perfringens and Escherichia coli. tetB(P) encoded low-level resistance to tetracycline and minocycline whereas tetA(P) only conferred tetracycline resistance. The tetA(P) and tetB(P) genes appeared to be linked in an operon, which represented a novel genetic arrangement for tetracycline-resistance determinants. It is proposed that tetB(P) evolved from the conjugative transfer into C. perfringens of a fer (M)-like gene from another bacterium.     

Overproduction of transposon Tn10-encoded tetracycline resistance protein results in cell death and loss of membrane potential.

High-level expression of the Tn10 tetracycline resistance protein TetA in Escherichia coli caused partial collapse of the membrane potential, arrest of growth, and killing of the cells. Since alpha-methylglucoside transport was not affected, the overproduced TetA protein may cause not destruction of membrane structure but rather unrestricted translocation of protons and/or ions across the membrane.     

Membrane topology of the metal-tetracycline/H+ antiporter TetA(K) from Staphylococcus aureus.

A series of fusions to the reporter proteins alkaline phosphatase and beta-galactosidase have been constructed in the predicted periplasmic and cytoplasmic loops of TetA(K), a protein responsible for efflux-mediated tetracycline resistance in Staphylococcus aureus. The results support a topological model of 14 transmembrane segments for TetA(K).     

Membrane topology of the pBR322 tetracycline resistance protein. TetA-PhoA gene fusions and implications for the mechanism of TetA membrane insertion.

The tetracycline resistance gene of pBR322 encodes a 41-kDa inner membrane protein (TetA) that acts as a tetracycline/H+ antiporter. Based on hydrophobicity profiles, we identified 12 potential transmembrane segments in TetA. We used oligonucleotide deletion mutagenesis to fuse alkaline phosphatase (PhoA) to the C-terminal edge of each of the predicted periplasmic and cytoplasmic segments of TetA. In general, the PhoA activities of the TetA-PhoA fusions support a TetA topology model consisting of 12 transmembrane segments with the N and C termini in the cytoplasm. However, several TetA-PhoA fusions have unexpected properties. One PhoA fusion to a predicted cytoplasmic segment (C6) has high activity. However, previous protease accessibility studies on the related Tn10 TetA protein indicated that C6 is cytoplasmically localized as predicted (Eckert, B., and Beck, C. F. (1989) J. Biol. Chem. 264, 11663-11670). PhoA fusions to three predicted periplasmic segments (P1, P2, and P5) have low to intermediate activity. In each case, the preceding transmembrane segment (TM1, TM3, and TM9) contains an aspartate (Asp17, Asp86, and Asp287). We show that these aspartates act like signal sequence mutations for PhoA export: (i) Asp----Ala mutations increase the PhoA activity of fusions to P1, P2, and P5. (ii) The signal sequence mutation suppressor prlA402 increases the PhoA activity of these same fusions. We also show that the aspartates in TM1, TM3, and TM9 are critical for wild-type TetA function; they are conserved in related TetA proteins and Asp----Ala mutations reduce or eliminate tetracycline resistance. The properties of the anomalous TetA-PhoA fusions suggest that TetA sequences C-terminal to some cytoplasmic and periplasmic segments are required for the proper localization of those segments, i.e. long range interactions may be more important in determining the membrane topology of TetA than suggested in some general models.     

Substitution F569S converts UapA, a specific uric acid-xanthine transporter, into a broad specificity transporter for purine-related solutes.

UapA, a highly specific uric acid-xanthine transporter in Aspergillus nidulans, is a member of a large family of nucleobase-ascorbate transporters conserved in all domains of life. We have investigated structure-function relationships in UapA, by studying chimeric transporters and missense mutations, and showed that specific polar or charged amino acid residues (E412, E414, Q449, N450, T457) on either side of an amphipathic alpha-helical transmembrane segment (TMS10) are critical for purine binding and transport. Here, the mutant Q449E, having no uric acid-xanthine transport activity at 25 degrees C, was used to isolate second-site revertants that restore function. Seven of them were found to have acquired the capacity to transport novel substrates (hypoxanthine and adenine) in addition to uric acid and xanthine. All seven revertants were found to carry the mutation F569S within the last transmembrane segment (TMS14) of UapA. Further kinetic analysis of a selected suppressor showed that UapA-Q449E/F569S transports with high affinity (K(M) values of 4-10 microM) xanthine, hypoxanthine and uracil. Uptake competition experiments suggested that UapA-Q449E/F569S also binds guanine, 6-thioguanine, adenosine or ascorbic acid. A strain carrying mutation F569S by itself conserves high-capacity, high-affinity (K(M) values of 1.5-15 microM), transport activity for purine-uracil transport. Compared to UapA-Q449E/F569S, UapA-F569S has a distinct capacity to bind several nucleobase-related compounds and different kinetic parameters of transport. These results show that molecular determinants external to the central functional domain (L9-TMS10-L10) are critical for the uptake specificity and transport kinetics of UapA.