Localization of the Outer Membrane Subunit OprM of Resistance-Nodulation-Cell Division Family Multicomponent Efflux Pump in Pseudomonas aeruginosa*

The outer membrane subunit OprM of the multicomponent efflux pump of Pseudomonas aeruginosa has been assumed to form a transmembrane xenobiotic exit channel across the outer membrane. We challenged this hypothesis to clarify the underlying ambiguity by manipulating the amino-terminal signal sequence of the OprM protein of the MexAB-OprM efflux pump in P. aeruginosa. [3H]Palmitate uptake experiments revealed that OprM is a lipoprotein. The following lines of evidence unequivocally established that the OprM protein functioned at the periplasmic space. (i) The OprM protein, in which a signal sequence including Cys-18 was replaced with that of periplasmic azurin, appeared in the periplasmic space but not in the outer membrane fraction, and the protein fully functioned as the pump subunit. (ii) The hybrid OprM containing the N-terminal transmembrane segment of the inner membrane protein, MexF, appeared exclusively in the inner membrane fraction. The hybrid protein containing 186 or 331 amino acid residues of MexF was fully active for the antibiotic extrusion, but a 42-residue protein was totally inactive. (iii) The mutant OprM, in which the N-terminal cysteine residue was replaced with another amino acid, appeared unmodified with fatty acid and was fractionated in both the periplasmic space and the inner membrane fraction but not in the outer membrane fraction. The Cys-18-modified OprM functioned for the antibiotic extrusion indistinguishably from that in the wild-type strain. We concluded, based on these results, that the OprM protein was anchored in the outer membrane via fatty acid(s) attached to the N-terminal cysteine residue and that the entire polypeptide moiety was exposed to the periplasmic space.

Pseudomonas aeruginosa shows natural resistance to structurally and functionally diverse antibiotics including ␤-lactams, fluoroquinolones, tetracycline, and chloramphenicol, a phenomenon that is largely attributable to an interplay between tight outer membrane permeability and low level expression of the xenobiotic efflux pumps, namely MexAB-OprM (1)(2)(3) and MexXY (4). P. aeruginosa achieves an elevated level of multiantibiotic resistance upon chromosomal mutation of nalB (5) or MexR (6, 7) due to a higher level of expression of the MexAB-OprM efflux pump compared with the level in the wild-type strain (8,9).
The MexAB-OprM pump consists of three subunit proteins including inner membrane-spanning MexB, inner membraneassociated periplasmic MexA, and the outer membrane subunit, OprM (8,10). MexA and MexB belong to the hydrophobe/ amphiphile efflux-1 (HAE-1) pump family and membrane fusion protein family, respectively, of the resistance-nodulation-cell division (RND) 1 superfamily (11,12). OprM belongs to the RND pump-associated outer membrane protein family (12). MexB constitutes the proton gradient-energized efflux pump, which traverses the inner membrane 12 times and has two large periplasmic domains (13). It has been assumed that MexA links the inner and outer membranes, bypassing the periplasmic space for the xenobiotic exit (14,15). OprM seems to be lipoprotein in nature (8) and has been thought to form the xenobiotic exit channel across the outer membrane (1)(2)(3)8).
The lipoproteins of prokaryotes were synthesized and translocated across the inner membrane, and the N-terminal cysteine residue in the mature protein formed a thioether with diglyceride. This was followed by cleavage of the signal peptide by the lipoprotein signal peptidase and then aminoacylation of the cysteine residue (17). The resulting lipoprotein is localized to either the inner or the outer membrane depending on the amino acid residue next to the lipid-modified cysteine (18,19). Most lipoproteins localizing in the outer membrane are transported by the lipoprotein-specific transporter (19) and are anchored in the membrane via the fatty acid residue (17). If P. aeruginosa had the lipoprotein transporter similar to the Lol system in Escherichia coli (19), OprM may be transported to and anchored in the outer membrane via the fatty acid moiety. On one hand the membrane-spanning outer membrane proteins fold the ␤-barrel structure without exception (20 -26), which is significantly different from the secondary structure of OprM. This is the underlying ambiguity in the membrane topology and function of OprM of the MexAB-OprM pump. We investigated this problem and report that the polypeptide moiety of OprM protrudes to the periplasmic space and is anchored to the outer membrane via N-terminal cysteine linked to the fatty acid moiety and functions in situ.

EXPERIMENTAL PROCEDURES
Bacterial Strains, Plasmids, and Growth Conditions-The bacterial strains and plasmids used are listed in Table I. Cells were grown aerobically in L-broth containing 10 g of tryptone, 5 g of yeast extract, and 5 g of NaCl per liter (pH 7.2) at 37°C.

Incorporation of [ 3 H]Palmitic
Acid-This was carried out by the procedure reported earlier (15) except that OprM was immunoprecipitated with anti-OprM antibody.
Manipulation of the DNA-Preparation of the plasmid DNA, the restriction enzyme treatment, ligation, and transformation were carried out essentially as described by Sambrook et al. (27). All the PCR products were subjected to nucleotide sequencing. Genetic constructions were carried out on E. coli hosts DH5␣ or XL10-Gold, and the constructed plasmids were transformed to P. aeruginosa.
Cloning of the oprM Gene and Attachment of the Polyhistidine Tag-The chromosomal oprM was amplified by PCR using forward and reverse primers, AGCGAGCTCAGCAGGCGTCCGTCGAAAAGG and CAGAAGCTTGCATGGCGCGGAAG, respectively. The product was treated with SacI and HindIII, cloned on pMMB67EH, and treated with SacI and HindIII yielding pOprM. Next, the oprM fragment on pNOT322 (28) was amplified by PCR using forward and reverse primers, ATCCTCGAGGCCGAGCAC and CGGAAGCTTGGCGCGGAAGG-CGATCAGTGGTGATGGTGGTGATGAGCCTGGGGATCTTCCTTCT-TCG, respectively. After the T4 polymerase treatment, the product was ligated to pHSG398 treated with SmaI. The XhoI-HindIII fragment derived from this newly constructed plasmid was ligated to pOprM1 (29) treated with XhoI and HindIII yielding pOprM-His. The histidinetagged OprM was used to purify and determine amino acid sequence of the protein.
Construction of the azurin(signal)-oprM Fusion Gene-We constructed the oprM gene by replacing the signal sequence including Cys-18 with the signal sequence of azurin, a periplasmic protein (15). We first amplified oprM by inserting XhoI and EcoRI sites using forward and reverse primers, AGGCTCGAGCCTGATCCCCGACTACCAG and GCAGAATTCTTGCATGGCGCGGAAGG, respectively. The PCR product was treated with XhoI and EcoRI and ligated to pHSG398 carrying the Azu signal sequence pretreated with SalI and EcoRI. The HindIII-EcoRI fragment of azu(signal)-oprM was subcloned to pMMB67HE and pretreated with HindIII and EcoRI.
Construction of OprM Containing a Transmembrane Segment of MexF-We constructed OprM containing an N-terminal transmembrane segment of MexF and a polyhistidine 6 tag at the C-terminal end. Three different lengths of the mexF fragment were amplified by PCR using the forward primer, GCGGAGCTCCGCGGCGATAGGAAGAAC-CG, and one of three reverse primers, GTGCTGCAGCACTTCCGGGT-ATTCGCTGAT, CAGCTGCAGGTCGCCGAGGCCGAACAACTG, or Cells were grown in the presence of [ 3 H]palmitate and disrupted in the presence of octyl ␤-glucoside. OprM was collected by immunoprecipitation using anti-OprM antibody as described earlier (15). The precipitate was subjected to SDS-PAGE, and a dried gel was analyzed by a Fluoroimage analyzer. Plasmid-borne OprM was expressed in the host strain TNP072 lacking chromosomal oprM. pMMB67EH is the plasmid not carrying oprM. The faint bands that appeared in pAzu-OprM are probably due to uptake of the degradative product of [ 3 H]palmitate.

FIG. 2. SDS-PAGE of periplasmic materials from genetically engineered OprM.
The periplasmic materials were extracted by the cold shock method as described under "Experimental Procedures." The extract containing 15 g of protein was subjected to SDS-PAGE, and the protein was visualized with anti-OprM antibody. PAO4290 is the wild-type strain expressing chromosomal OprM. The plasmid-borne OprM was expressed in the host cell, TNP072, lacking chromosomal oprM. Only a portion of the gel profile is shown. Protein bands that appear below the mature OprM are proteolytically cleaved OprM as confirmed by the N-terminal amino acid sequencing. CTGCTGCAGGACGATGGAGTAGTCCATGCC. These three fragments encode N-terminal 42, 186, or 331 amino acid residues of the MexF protein (30). Next, the oprM fragment lacking the signal sequence including Cys-18 was amplified by PCR using forward and reverse primers AGCCTGCAGTCGCTGATCCCCGACTAC and CAGAAGCTTGCA-TGGCGCGGAAGGCGATCA, respectively. The mexF fragments digested with SacI and PstI and the oprM fragment digested with PstI and HindIII were ligated to pHSG397 digested with SacI and HindIII. The resulting mexF-oprM on pHSG397 was treated with SacI and BamHI and ligated to pOprM or pOprM-His digested with SacI and BamHI to yield pMexF-OprM and pMexF-OprM-His, respectively.
Site-directed Mutagenesis of Cysteine 18 Residue-We replaced cysteine 18 with glycine, phenylalanine, or tryptophan by site-directed mutagenesis by the splicing-overlapping extension method (31) using the oprM gene cloned on pHSG398 as a template. The primers used were forward (A), TACGCAAGGCGACAAGGTGCTGATG (based on the sequence of pHSG398) and (B) CTTCAGCGTCAGGTAGGCG (based on oprM). The forward primers (C) used to insert C18G, C18F, and C18W were CTGTCCGGCGGCTCGCTGAT, GTCCGGCTTCTCGCTGAT, and GTCCGGCTGGTCGCTGAT, respectively. The reverse primers (D) for C18G, C18F, and C18W were CGGGGATCAGCGAGCCGCCG, CGG-GGATCAGCGAGAAGCCG, and CGGGGATCAGCGACCAGCCG, respectively. The first PCR was carried out using primers A and D and the second PCR was carried out using primers C and B. The third PCR was carried out using the first and the second PCR products as the templates and A and B primers. The third PCR product was treated with SalI and SacI and ligated to pOprM or pOprM-His treated with SalI and SacI yielding, for example, pOprM(C18G) and pOprM(C18G)-His for C18G and C18G-His-tag, respectively.
Cold Shock Experiment-A preculture grown overnight in 40 ml of L-broth was diluted into 400 ml of fresh prewarmed L-broth and incubated at 37°C until A 600 units reached about 1.0. Isopropyl-1-thio-␤-Dgalactopyranoside up to 2 mM was added as needed. Cells were harvested by centrifugation and suspended in 50 mM Tris-HCl, pH 7.3, containing 0.2 M MgCl 2 in the cell to buffer ratio of 1:5 (w/w). The cell suspension was placed in a 37°C water bath for 10 min and in an icy water bath for 15 min for two cycles. Cells were centrifuged at 12,000 rpm for 15 min at 10°C (32). The supernatant was centrifuged at 45,000 rpm for 60 min at 10°C. This centrifuged supernatant was saved as the periplasmic material.
Other Methods-Cells were fractionated by the EDTA-lysozyme method as described previously (33). SDS-PAGE was carried out as described by Laemmli (34). Protein concentration was quantified by the method of Lowry et al. (35).

[ 3 H]Palmitate
Labeling of OprM-The N-terminal signal sequence of OprM bears a lipid modification consensus sequence, LSGC (8). To ascertain whether OprM contains fatty acid, we carried out a [ 3 H]palmitic acid incorporation experiment. Fluorography of the materials collected by the anti-OprM antibody from cells expressing the wild-type OprM showed a radioactive 52-kDa protein corresponding to the size of the wild-type OprM (Fig. 1, lane pOprM). In contrast, OprM was undetectable in the similarly treated materials from the OprM deletion mutant, TNP072 (lane TNP072). These results assured that OprM was a lipoprotein.
Substitution of the OprM Signal Sequence with That of Periplasmic Azurin-We designed an experiment to replace the OprM signal sequence including Cys-18 with a signal sequence of azurin. We found that the N-terminal amino acid sequence of OprM encoded by the azu-oprM fusion gene was AESSLIP-DYQ, which is identical to the sequence designed for Azu(signal)-OprM. The cellular fractionation experiment revealed that the majority of OprM was located in the periplasmic space   (Fig. 2, lane pAzu-OprM). The protein band below the mature OprM was probably proteolytically cleaved OprM. A considerable amount of OprM was found at the inner membrane fraction but not in the outer membrane (Fig. 3). When this mutant was subjected to the [ 3 H]palmitate uptake experiment, we found a trace of radioactive OprM, but the amount was far less than in the wild-type OprM (Fig. 1, lane pAzu-OprM). A trace of labeling was probably due to the uptake of degraded radioactive materials. When this mutant protein was expressed in the host lacking OprM, the cell exhibited 16-fold higher MICs of erythromycin, ofloxacin, and aztreonam, and an 8-fold higher MIC of chloramphenicol than the MICs in the cell lacking OprM. These values were indistinguishable from that in the wild-type strain demonstrating that the mutant OprM was fully functional without anchoring the outer membrane (Table II). Note that the amount of the plasmid-borne OprM was severalfold higher than that encoded by chromosomal oprM (Fig. 2).
Construction of the MexF-OprM Hybrid Protein-Next, we attached the transmembrane segment of MexF to the N-terminal end of OprM. MexF is the inner membrane subunit of the P. aeruginosa MexEF-OprN pump, which is not expressed under normal growth conditions (36). Because this protein shows high homology to MexB, it was assumed that the protein would span the membrane several times. We fused mexF encoding N-terminal consisting of a 42-, 186-, or 331-amino acid residue with oprM encoding signal sequence-less OprM tagged with polyhistidine. We first investigated localization of the hybrid proteins and found that protein bands with a molecular mass of about 56, 72, and 86 kDa corresponding to OprMs containing 42, 186, and 331 residues of MexF polypeptide, respectively, were located in the inner membrane (Fig. 4). None of these hybrid proteins was detectable in the outer membrane (Fig. 4) or in the periplasmic fraction (data not shown). We confirmed that all these hybrid proteins contained an N-terminal sequence identical to MexF. The [ 3 H]palmitate-labeling experiment showed that the proteins were delipidated (Fig. 5).
The MICs of aztreonam in cells expressing OprM with 42-, 186-, and 331-amino acid extensions were 0.39, 3.13 and 3.13 mg/liter, respectively (Table II). These results indicated that the MexF-OprM hybrid protein having 186 and 331 residues of MexF fully functioned as the MexAB-OprM efflux pump sub-unit whereas the hybrid protein with 42 residues was inactive (Table II).
Site-directed Mutation of the Cysteine 18 Residue-To identify the fatty acid modification site, we constructed a mutant bearing the OprM containing a glycine substitution for an 18-residue cysteine, a unique cysteine in OprM. Similarly, we also substituted Cys-18 with phenylalanine or tryptophan. To test whether these mutant OprMs retained the fatty acid, [ 3 H]palmitate-labeling experiments were carried out. The results clearly demonstrated that radiolabeled OprM was undetectable in cells expressing Cys-18-modified OprM (Fig. 1). This result demonstrated that fatty acid modification in OprM occurred at the N-terminal cysteine residues.
When the Cys-18-modified OprM was expressed in the strain lacking chromosomal oprM, MICs of antibiotics were fully restored to the level of the wild-type strain (Table II), demonstrating that fatty acid modification is not essential for pump function. A cellular fractionation experiment showed that the Cys-18-modified OprM was located in the periplasmic space ( Fig. 2) but not in the outer membrane fraction (Fig. 6). A considerable amount of the OprM was seen in the inner membrane fraction (Fig. 6). The N-terminal amino acid sequencing confirmed that two bands appearing in the membrane fraction (Fig. 6) were mature and premature OprMs.
We tested whether inner membrane localization of OprM was a consequence of the interaction of OprM with MexA and MexB subunits by expressing the wild-type OprM in a host with or without the MexA and MexB subunits. We found only a small amount of the inner membrane-associated OprM in the host lacking MexA and MexB, whereas a significant quantity of OprM was seen in the wild-type strain (data not shown). DISCUSSION We reported in this paper that the delipidated OprM is located in the periplasmic space and functions in situ. Furthermore, the inner membrane-anchored OprM via a transmembrane segment from MexF also had a full function despite the fact that the hybrid protein was exclusively located in the inner membrane. One may argue that a fraction of the mutant OprM is localized at the outer membrane. This is unlikely, however, because the lipoprotein transporter only recognizes and transports an acylated protein (19). The results reported in this paper provide novel information for improved understanding of the transport mechanism by the RND family efflux pump in P. aeruginosa. The question now arises about whether the MexAB-OprM pump assembly alone can translocate the substrates across the outer membrane without the assistance of another auxiliary protein(s) such as TolC in the E. coli outer membrane. Although the classical view was that the outer membrane subunit forms a transmembrane channel across the outer membrane (1)(2)(3)8), in fact, all the transmembrane outer membrane proteins so far analyzed contain a ␤-barrel structure (20 -26). Therefore, the OprM is distinct from a family of these membrane-spanning outer membrane proteins. Because membrane anchoring and topology of OprM is different from that of most other transmembrane outer membrane proteins, we propose designating the OprM protein as MexM and accordingly the oprM gene as mexM.
A recent paper reported that MexM bears ␤-sheet-based 16-transmembrane segments across the outer membrane (37). This conclusion is unlikely for the following reasons. (i) Circular dichroism spectroscopy of homogeneously purified MexM revealed that MexM had 53, 0, and 47% contents of ␣-helix, ␤-sheet, and random coils, respectively. 2 This result clearly ruled out the possibility that MexM forms ␤-strand-based 16transmembrane segments because the outer membrane proteins with ␤-barrel structure had 60 -70% contents of the ␤-sheet (38 -40). (ii) The lipoproteins of Gram-negative bacteria destined to reach the outer membrane are transported by the lipoprotein-specific Lol family transporter (19) and are anchored in the outer membrane via fatty acids exposing most of the polypeptide moiety to the periplasmic space (17).
On the basis of the present results, we propose a new model for the structure of RND multicomponent efflux pump assembly (Fig. 7). The inner membrane pump subunit, MexB, crosses the membrane 12 times, having two large periplasmic domains at TMS-1 to TMS-2 and TMS-7 to TMS-8 (13). Membrane fusion protein, MexA, is anchored in the inner membrane via fatty acid exposing most of the polypeptide to the periplasmic space (14,15). As we reported here, outer membrane lipoprotein, MexM, anchored the periplasmic side of the outer membrane and protruded the entire polypeptide portion into the periplasmic space. The MexA and MexM homologues of the pump assembly may interact with large periplasmic domains of the MexB homologues as illustrated in Fig. 7. It is possible that unidentified outer membrane protein(s) analogous to TolC of E. coli is involved in the xenobiotic export in P. aeruginosa. This newly proposed model prompted us to reconsider the role of membrane-associated periplasmic proteins, such as MexM and MexA, in the xenobiotic transport by the RND efflux pump assembly.
Addendum-During preparation of this manuscript, the authors learned that the three-dimensional structure of TolC, an E. coli outer membrane protein, was solved (16). This protein spans the outer membrane by ␤-barrel structure formed with a trimeric aggregate. This protein is distinct from MexM because TolC is unacylated.
Note Added in Proof-The revised nucleotide sequence of MexM (OprM) has been deposited in the DDBJ/EMBL/GenBank™ nucleotide data bases with accession number AB011381.