Membrane Topology of the Xenobiotic-exporting Subunit, MexB, of the MexA,B-OprM Extrusion Pump in Pseudomonas aeruginosa *

The MexA,B-OprM efflux pump assembly ofPseudomonas aeruginosa consists of two inner membrane proteins and one outer membrane protein. The cytoplasmic membrane protein, MexB, appears to function as the xenobiotic-exporting subunit, whereas the MexA and OprM proteins are supposed to function as the membrane fusion protein and the outer membrane channel protein, respectively. Computer-aided hydropathy analyses of MexB predicted the presence of up to 17 potential transmembrane segments. To verify the prediction, we analyzed the membrane topology of MexB using the alkaline phosphatase gene fusion method. We obtained the following unique characteristics. MexB bears 12 membrane spanning segments leaving both the amino and carboxyl termini in the cytoplasmic side of the inner membrane. Both the first and fourth periplasmic loops had very long hydrophilic domains containing 311 and 314 amino acid residues, respectively. This fact suggests that these loops may interact with other pump subunits, such as the membrane fusion protein MexA and the outer membrane protein OprM. Alignment of the amino- and the carboxyl-terminal halves of MexB showed a 30% homology and transmembrane segments 1, 2, 3, 4, 5, and 6 could be overlaid with the segments 7, 8, 9, 10, 11, and 12, respectively. This result suggested that the MexB has a 2-fold repeat that strengthen the experimentally determined topology model. This paper reports the structure of the pump subunit, MexB, of the MexA,B-OprM efflux pump assembly. This is the first time to verify the topology of the resistant-nodulation-division efflux pump protein.

Nosocomial patients with cancer, transplantation, burn, cystic fibrosis, etc. are easily infected by bacteria with low virulence. Among those opportunistic pathogens, Pseudomonas aeruginosa is particularly problematic, since the bacteria show resistance to many structurally and functionally diverse antibiotics (1). Recent studies have revealed that this type of resistance is attributable to a synergy of low outer membrane permeability and active drug extrusion (2). An increasing number of multidrug extrusion systems are being reported in both prokaryotes and eukaryotes (2)(3)(4)(5)(6)(7)(8). It is likely, therefore, that active extrusion systems play a crucial role in the cellular defense mechanism against incoming noxious compounds in many living organisms. It is of great interest and importance, therefore, to analyze the mechanism by which such universally occurring extrusion pump function.
The wild-type P. aeruginosa expresses a low level of the MexA,B-OprM drug extrusion machinery (9,10). Mutations in nalB gene cause overexpression of the mexA,B-oprM operon rendering the bacterium more resistant than the wild-type strain to a broad spectrum of antibiotics (3). Deletion of the coding region of the wild-type mexA, mexB, or oprM renders the mutant more susceptible than the wild-type strain to many antibiotics (9,11). Thus, it is apparent that the MexA,B-OprM machinery is involved in the both basal and elevated levels of intrinsic antibiotic resistance in P. aeruginosa.
The MexA,B-OprM pump consists of three subunits, MexA, MexB, and OprM, located at the inner and outer membrane, respectively (9,10). MexB consists of 1046 amino acid residues and is assumed to extrude the xenobiotics utilizing the proton motive force as the energy source (4,12). This protein belongs to the resistant/nodulation/division (RND) 1 family (13,14). MexA is an inner membrane-associated lipoprotein belonging to the membrane fusion protein family. OprM is an outer membrane protein probably forming the xenobiotics exit channel. A complex formation by these three subunit proteins has been suggested for many efflux pump assemblies in Gram-negative bacteria (13,(15)(16)(17). In fact, the functional coupling of the RND protein and the membrane fusion protein was recently demonstrated by the subunit exchange experiments using three efflux pump systems in P. aeruginosa (18 -21), while the outer membrane components could be substituted with other proteins having a similar function. However, the precise molecular mechanism of substrate recognition and efflux through these pumps remained to be clarified. For a better understanding of how this pump extrudes the xenobiotics, it is essential to elucidate the structure and membrane topology of the individual pump subunit.
The membrane topology of several RND family proteins was suggested to have 12 transmembrane domains and two large hydrophilic domains (13,22). Hydropathy analysis of MexB by the TOP-PRED II 1.1 software packages (23) suggested that it might have 12 certain and 5 putative transmembrane segments (TMS). Some other software predicted the presence of 11 TMS.
The topology of cytoplasmic membrane proteins in Gramnegative bacteria was often studied by the phoA gene fusion method. Alkaline phosphatase (AP) is enzymatically active after translocation to the periplasm, but is inactive when local-* This work was supported by grants from the Ministry of Education of Japan, Science, Sports and Culture, the Ministry of Health and Welfare of Japan, the Japan Society for Promotion of Science, and the Tokai University School of Medicine Research Project. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Recipient of the Tokai University School of Medicine Research Fellowship.
ʈ Visited Tokai University with the support of the Japan Society for Human Science.
** To whom correspondence should be addressed. ized cytoplasmically (24,25). Several tools, including TnphoA, TnTAP, pPHO7, and pBADphoA (24,26,27), have been developed to construct the phoA fusion to the target protein. Although transposon-mediated generation of gene fusion is simple, insertion of the reporter gene at a specific target site can be tedious. This difficulty is even more pronounced if a target protein has short extramembranous loops. An alternative method is cloning of PCR products to the 5Ј-end of the signal sequenceless phoA gene (28). Using these two methods, we analyzed the topology of MexB. This paper reports the twodimensional transmembrane structure of MexB.
Construction of mexB-phoA Fusions by PCR-Fusions of the truncated mexB to phoA, encoding for signal sequenceless AP, were carried out by inserting the PCR fragments of mexB into the 5Ј-end of the signal sequenceless phoA in pBADphoA. Eleven out of 15 fusions were con-structed by cloning the PCR fragments containing flanking KpnI restriction sites into pBADphoA cleaved with KpnI. The PCR primer used for the 5Ј-end of the mexB gene was cccggtaccgTCGAAGTTTTTCATT-GATAGG. The 3Ј-end primers designed for the immediately downstream of the codons Gln 34 , Glu 346 , Leu 366 , Thr 392 , Gly 440 , Thr 473 , Arg 538 , Gln 871 , Pro 897 , Ser 921 , and Cys 972 were aggatggtacCTGGTTGA-CCGGCAGACTGAG (Gln 34 ), cgcgcggtacCTCGCCGAGGGTCTTCAC-TAC (Glu 346 ), gccgcggtacCAGCGTGGCGCGGAAG (Leu 366 ), cgcggtacC-GTGTTGATCGAGAAGCC (Thr 392 ), ggcgcggtacCCCCTGGATCTGGC-CCATGGA (Gly 440 ), cgcgcggtacCGTGATGGAGAACTGCCGGTAGAT (Thr 473 ), cgcggtacCCGATGCTTGAGGATCGAC (Arg 538 ), ccggcggtacCT-GCGAGCCGGACAAGC (Gln 871 ), cgcggtacCGGAATCGACCAGCTTTC-GTACAG (Pro 897 ), cccccggtacCGACAGGCCGCGCATGGACGTCG (Ser 921 ), and ccatcggtacCGGCCGCAGACGCAT (Pro 972 ), respectively. For the fusion of phoA to codons Gly 1007 and Gln 1046 , we designed the primers allowing in-frame blunt-end ligation, because there are two KpnI sites in this part of mexB. The PCR fragments purified from agarose gel and blunted with the T4 DNA polymerase were ligated to the pBADphoA cleaved by KpnI and blunted with T4 DNA polymerase. The primer used for the 5Ј-end of the mexB gene was TCGAAGTTTT-TCATTGATAGGCCC. Two 3Ј-end primers used to generate phoA fusions downstream of the codons Gly 1007 and Gln 1046 were cGCCGATC-ACGCCGGTACCGAT (Gly 1007 ) and aTTGCCCCTTTTCGACGGACGC-CTGC (Gln 1046 ), respectively. For fusion pBAD-P9, two oligonucleotides containing the KpnI sites at the both ends were annealed and ligated directly to the pBADphoA cleaved with KpnI. The oligonucleotides for coding and noncoding strands were CGTCGAAGTTTTTCATTGATAG-GCCGGTAC and CGGCCTATCAATGAAAAACTTCGACGGTAC, respectively. The host cells used for analysis of AP activity was E. coli LMG194. For most fusion plasmids, codons for valine and proline residues were introduced at the 5Ј-and 3Ј-ends of the mexB fragments to introduce the KpnI sites.
Construction of mexB-phoA Fusions by TnTAP-To obtain in-frame fusions of TnTAP to mexB, the pMM1 containing TnTAP was transformed into the strain E. coli CC118 carrying pMEXB1, which encodes the wild-type mexB. After transposition during overnight growth, a pool of plasmid DNA was isolated and digested with NheI to destroy pMM1, but not pMEXB1 and TnTAP. The restriction digests were transformed again into strain CC118. Blue colonies on agar plates containing 40 g/ml 5-bromo-4-chloro-3-indolyl phosphate, 200 g/ml ampicillin (for pMEXB1), and 100 g/ml kanamycin were purified. Insertions into mexB were identified by PCR.
DNA Sequencing-The nucleotide sequence was determined using the ABI PRISM TM Dye Terminator Cycle Sequencing Core Kit with ampliTaq® DNA polymerase, FS. The sequencing primer used was GCAGTAATATCGCCCTGAGCAGC, reading out of the phoA gene toward the mexB gene. In addition, a primer GCGTCACACTTTGCTAT-GCC reading out of the pBADphoA vector toward the mexB gene was also used.
Assay of AP Activity-AP activity was assayed by measuring the rate of hydrolysis of p-nitrophenyl phosphate in permeabilized cells as described elsewhere (31). One unit of AP activity corresponds to the rate of p-nitrophenyl phosphate hydrolysis, 1 mol of p-nitrophenyl phosphate/min/mg of protein at 23°C.
Expression of the Hybrid Proteins-For the Western blot analysis of the hybrid proteins, the crude envelope fraction and whole cell lysate were prepared as described elsewhere (10). SDS-polyacrylamide gel electrophoresis (10%) and Western blotting were carried out as described previously (32). The monoclonal antibody raised against AP was used to probe the hybrid proteins. Boiling the MexB protein in SDS caused disappearance of the protein band from the gel.

RESULTS
Construction of the mexB-phoA Fusions-To analyze the membrane topology of the MexB protein, we took the 12-TMS model for our working hypothesis and designed the experiments accordingly (Fig. 1). We constructed 25 clones expressing COOH-terminal-truncated MexB-AP hybrids and one clone (pBAD-Q1046) expressing phoA at the COOH-terminal end of full-length MexB using pBADphoA and TnTAP (Table I). Cells harboring the pBAD-P9, pBAD-L366, pBAD-V411, pBAD-G440, pBAD-R538, pBAD-P897, pBAD-P972, and pBAD-Q1046 plasmids yielded pale blue colonies, suggesting that the AP domain is located in the cytoplasmic side of the inner membrane. The fusion pBAD-V411 was obtained accidentally in the course of pBAD-Q1046 construction. The remaining mexB-phoA fusions exhibited blue colonies on the 5-bromo-4chloro-3-indolyl phosphate plates suggesting that the AP domain is translocated to the periplasm. The fusion joints were confirmed by nucleotide sequencing and all reading frames were correct (Tables II and III). The distribution of a total of 26 fusion sites covered the entire MexB protein, and each hydrophobic segment was flanked by phoA fusions (Fig. 1).
Expression of the Hybrid Proteins-The expression of hybrid proteins derived from TnTAP and pBADphoA is under the control of lac and araBAD promotor, respectively, and therefore the cells harboring the fusions were induced in the presence of 100 M isopropyl-␤-D-thiogalactopyranoside and 100 M L-arabinose, respectively. The hybrid proteins with high and low AP activities are shown in Fig. 2, lanes 3-20 and lanes 21-27, respectively. The size of the hybrid proteins was within the range of the expected molecular mass. The protein band of the fusion at Pro 9 was undetectable, since the hybrid protein is expected to be soluble in the cytoplasm. All the bands for cytoplasmic hybrid proteins showed weaker signals than the periplasmic hybrid proteins, which probably was attributable to the proteolytic degradation of the hybrid protein as reported earlier (33,34). In addition, protein bands with a higher molecular mass than expected were seen as reported elsewhere (35). This might be explained by suggesting that the chimeric proteins maintaining the native conformation bind less SDS than fully denatured proteins in the electrophoresis buffer, because the samples were subjected to electrophoresis without heating. Fusion Gly 440 appeared only in a higher molecular weight range than expected and was barely seen. DISCUSSION Most living organisms, if not all, seem to be equipped with xenobiotics extrusion pump(s). Mammalian cells, for instance, express P-glycoproteins (8), multidrug resistance associated protein (36), and cannalicular multispecific organic anion transporter (37), which extrude anticancer drugs, bile acids, and others. Expression of the efflux proteins in bacteria renders the organisms resistant to many antibiotics, organic solvents, hydrophobic dyes, and surfactants. Structure of the efflux pump in Gram-negative bacteria is particularly complicated, since the outer membrane covers the inner membrane. An extrusion pump in P. aeruginosa consisted of three subunits, MexA, MexB and OprM. Among them, MexB is particularly important in the pump function, since this subunit primarily recognizes and extrudes the substrates. To analyze the two-dimensional membrane topology of MexB, we constructed 26 phoA fusions, which covered the entire MexB protein.
The NH 2 -terminal segment before the first hydrophobic segment consists of 9 amino acid residues containing 2 positively and 1 negatively charged residues. This segment is unlikely to cross the membrane according to the positive charge inside rule (38). The periplasmic location of the Gln 34 and Gly 1007 sites and the cytoplasmic location of the Pro 9 and Gln 1046 sites indicated that a cytoplasmic location of both the NH 2 -and COOH-terminal ends (Fig. 1). The hybrid protein Gln 34 was detected in the crude envelope fraction (Fig. 2, lane 3) indicating that TMS1 is an uncleaved signal-anchor. The periplasmic location of the Glu 346 , Thr 392 , Thr 473 , Gln 871 , Ser 921 , and Gly 1007 sites, and the cytoplasmic location of the Leu 366 , Val 411 , Gly 440 , Arg 538 , Pro 897 , and Pro 972 sites verified the transmembrane nature of the TMS 2-11 ( Fig. 1). Five weak hydrophobic segments suggested by the TOPRED II software (a-e, Fig. 1) did not function as transmembrane segments, because they were flanked by fusions with high AP activities (Fig. 1). These results supported our working model. Some of the computer programs for the topology prediction suggested the presence of 11 TMS in the FIG. 2. Western blot analysis of the MexB-AP hybrid proteins. Crude envelope fraction was prepared as described under "Experimental Procedures," subjected to polyacrylamide (10%) gel electrophoresis in SDS without heating, electroblotted to the polyvinylidene difluoride membrane and visualized with monoclonal antibody raised against alkaline phosphatase (lanes 2-20). Whole cell lysate was subjected to the electrophoresis and stained as above (lanes [21][22][23][24][25][26][27]  MexB polypeptide. Our results experimentally ruled out this possibility. We carried out computer-aided alignment analysis of the amino-terminal and carboxyl-terminal halves of the polypeptide from Met 1 to Arg 529 and Gly 530 to Gln 1046 , respectively. Fig. 3 shows 30% homology between the first and second halves. TMS 1, 2, 3, 4, 5, and 6 of the amino-terminal half could be overlaid by TMS 7,8,9,10,11, and 12 in the carboxylterminal half, respectively. This 2-fold repeat suggested that mexB is evolved from an ancestral gene encoding a protein of six TMS by an intragenic duplication as predicted earlier (13,22). This is to doubly support the transmembrane nature of the segments experimentally assigned to be the membrane-spanning domain. In addition, the distribution of positive charges in the cytoplasmic and periplasmic domains were 22 and 2, respectively, excepting the first and fourth large periplasmic domains with more than 60 amino acid residues (Fig. 1). The result was in accord with the positive inside rule (38). This is the first experimental verification of the transmembrane topology of the RND family extrusion pump protein. This experimentally verified model has the following features. (i) The MexB protein spans the membrane 12 times leaving amino and carboxyl termini at cytoplasmic side of the inner membrane as suggested for the RND family proteins (13,22). (ii) MexB has two large hydrophilic segments with 311 and 314 amino acid residues from 29 to 338 (between TMS 1 and 2) and from 558 and 871 (between TMS 7 and 8), respectively. (iii) The membrane topology of MexB appeared to have a 2-fold repeat. These big loops might interact with the periplasmic subunit, MexA, and the outer membrane subunit, OprM. It is conceivable that these large loops transmit cellular energy to the OprM channel gate. In addition, we found 5 charged amino acid residues in the transmembrane domains. These charged residues were highly conserved in the RND family efflux proteins as aligned by the Clustal W multi-alignment software (data not shown). Specific localization of the highly conserved charged residues in the TMS suggested that they might play an important role in substrate binding and proton transport. Further studies are needed to elucidate the role of these amino acid residues in the mechanism of xenobiotics extrusion.