Characterization of the arcD Arginine:Ornithine Exchanger of Pseudomonas aeruginosa LOCALIZATION IN THE CYTOPLASMIC MEMBRANE AND A TOPOLOGICAL MODEL*

The arcDABC operon of Pseudomonas aeruginosa encodes the enzymes of the arginine deiminase pathway and is induced by oxygen limitation. The arcD gene specifies a 53-kDa protein with arginine: ornithine exchange activity. The ArcD protein of P. aeruginosa, like the LysI lysine transporter of Coryne- bacterium glutamicum, has 13 hydrophobic regions which could span the cytoplasmic membrane. Fusion of a Caa (colicin A) epitope to the N-terminal part of ArcD permitted the localization, by immunoblotting, of the hybrid protein in the inner membrane of P . aeruginosa. Fusion of PhoA (alkaline phosphatase) to the very C terminus of ArcD produced another hybrid protein, which exhibited PhoA activity. Both ArcD hybrid proteins retained arginine transport activity and served to support a topological model which pro- poses that the N terminus is oriented toward the cytoplasm and the C terminus faces

enzymes of the arginine deiminase pathway are encoded by the arcDABC operon (1-3) and coordinately induced by oxygen limitation (2, 4,5). Induction requires the transcriptional activator protein ANR, which recognizes a sequence motif in the -40 region of the arc promoter (5-7). The arcABC genes encode the soluble enzymes arginine deiminase, catabolic ornithine carbamoyltransferase, and carbamate kinase, respectively (1,2,8,9). The arcD gene specifies a highly hydrophobic protein, which can be detected in the total membrane fraction of Escherichia coli maxicells after overexpression of the cloned arcD gene (3). A hydrophobicity plot of the deduced ArcD amino acid sequence shows 13 potential transmembrane segments (3). Point mutations in arcD render P. aeruginosa unable to utilize extracellular arginine for anaerobic growth although in these arcD mutants the enzymes of the arginine deiminase pathway remain active and inducible (1,3). Under aerobic conditions, P. aeruginosa utilizes arginine as a carbon and nitrogen source, primarily via the W-succinylarginine pathway (10). The arcD mutants are not impaired in aerobic arginine catabolism (1). Taken together, these findings suggest that the ArcD protein is involved in a specific uptake system that delivers arginine to the arginine deiminase pathway during conditions of oxygen limitation.
The arginine deiminase pathway allows the utilization of arginine as an energy source in a variety of bacteria other than P. aeruginosa, e.g. in Lactococcus species. In lactococci, a specific membrane transport system catalyzes the stoichiometric, electroneutral exchange between arginine and ornithine, which is produced in the cytoplasm by the arginine deiminase pathway (11)(12)(13). The driving force for arginine uptake and ornithine excretion is provided by the concentration gradients of these amino acids. The arginine-ornithine exchanger of Lactococcus lactis has been extracted and functionally reconstituted in proteoliposomes (13). This has permitted an analysis of the kinetic mechanism; the exchanger appears to have a single substrate binding site which may be exposed alternately to the inner and outer surface of the cytoplasmic membrane (14). The exchange activity in proteoliposomes is not affected by the electrical potential across the membrane or the protonmotive force (13, 14). Thus, the energetic costs for arginine uptake and ornithine release are minimized in the arginine deiminase pathway. The gene encoding the exchanger, however, has not yet been identified in lactococci.
Recent experiments have shown that the ArcD protein of P. aeruginosa has arginine:ornithine antiporter activity which is similar to that found in lactococci. An E. coli strain lacking arginine permease has been transformed with a recombinant 5417 of P. aeruginosa plasmid expressing the arcD gene of P. aeruginosa. Membrane vesicles from this recombinant strain exchange intracellularly accumulated ornithine for external arginine. The ArcD protein can be solubilized from membrane vesicles and reconstituted into liposomes in a functionally active state (15). In addition to the exchange reaction, ArcD also catalyzes arginine and ornithine uptake driven by the protonmotive force (15). In this study, we report the construction of gene fusions of arcD to phoA (alkaline phosphatase) or caa (colicin A).
The properties of the resulting hybrid proteins lead us to propose a model for the topology of the ArcD protein in the cytoplasmic membrane.
Growth Conditions-Media, anaerobic growth in the Gas-Pak jar, conditions of good or limiting aeration, and selective antibiotic concentrations have been described (1,2,7,25,26). The pJRD253 derivative pME3608 was selected in P. aeruginosa on minimal medium containing streptomycin (75 pglml). DNA Manipulations-The conditions for plasmid isolation, restriction, BAL31 digestion, ligation, and DNA sequencing by the chain termination method have all been described (2,7,25,27).
Construction of 'phoA Fusions-The target plasmid for TnphoA insertions was constructed by inserting the 1.95-kb' HindIII-BamHI fragment encompassing the arc promoter, arcD, and part of arcA ( Fig.  L4) into pBLS. In the resulting plasmid pME3750, the arc genes are expressed from the lac promoter on the vector. E. coli CC202 was transformed with pME3750. Transposition of TnphoA was obtained by selection for high level kanamycin resistance on nutrient agar containing kanamycin (1000 pg/ml), ampicillin (100 pg/ml), XP (40 rg/ml), and isopropyl thiogalactoside (100 p~; added to induce the lac promoter). 20 blue or white colonies were purified 3 had a TnphoA insertion in arcD (Table I). From the latter plasmids, the segments carrying the arc promoter and the arcD'-'phoA open reading frame were excised by HindIII + XhoI digestion and inserted into pKT240 (23) cut by the same enzymes. The kanamycin promoter of pKT240, which might have interfered with phoA expression, was eliminated by a 1.6-kb XhoI-HpaI deletion, giving pME3761, pME3762, and pME3765 (Table I). In uitro phoA fusions were constructed in E. coli CC118. The AatII site in arcD (position 910) carried by pME3750 and the PstI site upstream of phoA on pCH39 were both rendered blunt by T4 DNA polymerase (27) to allow in-frame joining; subcloning into pKT240 gave pME3763 (Table I). The PstI site in arcD pME3764 (Table I) L4) was derived from pVLl (161, from which a 0.5-kb NcoI-AuaI fragment was removed and replaced by a multiple cloning site. The NcoI site spans codons 29-31 of the caa gene (Fig. 1B). For the construction of pME3801, the proximal part of the arcD gene on pME183 (Ref. 2) was deleted by BAL31 digestion to position 1220 in arcD and ligated to a HindIII linker, as described previously (3). A 19-base pair fragment from pBLS was joined to the HindIII site. This created a new Sal1 site upstream of arcD. The 0.75-kb SalI-BamHI fragment carrying 'arcD-arcA' was then inserted into the corresponding sites in pEPlink, giving pME3801 (Fig. 1, A and B ) . The construction of pME3802 involved cloning of the 1.7-kb MaeI-BamHI fragment containing 'arcD-arcA' into the EcoRV and BamHI sites of pEPlink (Fig. 1, A and B ) . Plasmid pME3808 was derived from pME3802 as follows: the caa promoter was removed by a 0.25-kb BAL31 deletion initiated from the unique PuuII site; after insertion of a phosphorylated BamHI linker (Pharmacia LKB Biotechnology Inc.), the promoterless 1.8-kb caa'-'arcD arcA fragment was inserted into pJRD253 such that the T7a10 promoter reads into caa'-'arcD ( Fig. L4).
Induction of the caa Promoter-E. coli cells carrying pEPlink derivatives were grown to OD, , . , z 0.5 in LB medium (27) and then induced with mitomycin C (0.3 pg/ml; Sigma (16)) or with an analog of nalidixic acid, norfloxacin (4 pg/ml; Merck Sharp & Dohme), for 2 to 4 h (28). Alternatively, induction was achieved by UV irradiation (20 J/mZ (28)). strain PA0 were prepared by the method of Hancock and Nikaido Membrane Preparations of P. aeruginosa-Total membranes of (29) and fractionated on two discontinuous sucrose gradients (29,30) in the presence of 1 mM phenylmethylsulfonyl fluoride (PMSF).
Enzyme and Protein Assays-Alkaline phosphatase was determined in P. aeruginosa cells permeabilized with 0.005% SDS and 5% (v/v) chloroform in 1 M Tris-HCI, pH 8; the substrate was p-nitrophenyl phosphate (34). NADH oxidase was assayed by the method of Osborn et al. (35). One enzyme unit is defined as 1 nmol of product formed per min. Anaerobic arginine consumption by whole cells of P. aeruginosa was measured by a modified Sakaguchi reaction (36). Protein concentrations were estimated by the method of Bradford (37) using a commercial reagent (Bio-Rad) and bovine serum albumin as a standard.

Construction of Caa'-'ArcD Fusion Proteins and Their
Expression in E. coli-We wished to detect the ArcD protein of P. aeruginosa specifically in membrane fractions. As no antibodies against ArcD are available, we tagged ArcD with an epitope which is derived from the colicin A (Caa) protein and which is recognized by a specific monoclonal antibody (16,33). The epitope consists of amino acids 13 to 26 of the Caa protein.' The construction of Cas'-' ArcD hybrid proteins was facilitated by the expression vector pEPlink ( Fig. 1 A ) .   Table I. Horizontal arrows designate promoters of the arc operon (Pam), the caa gene (Pcm), or the T7@10 sequence (Pel,,). Numbers in brackets below restriction sites refer to the complete nucleotide sequence of the arcDABC operon (3,8,9). Restriction sites are abbreviated B, BamHI; Bg, BglII; E, EcoRI, EV, EcoRV; H, HindIII, M, MaeI; Ps, PstI; Pu, PuuII; S, SalI; Sm, SmaI; X , XhoI. MCS, multiple cloning site (see B). B, the 31 N-terminal amino acid residues (boldface) of the caa gene (38,39) are connected to the MCS in pEPlink. All restriction sites (NcoI to BamHI) in the MCS are unique. The monoclonal antibody IC11 (33) recog nizes amino acid residues 13 to 26.' In pME3801, the first arcD amino acid residues Ala-Leu-Trp-Leu-Thr-Asn are in the putative transmembrane helix IX (Fig. 6). In pME3802, the first arcD residues Leu-Gly-Ala-Leu-Thr are in the putative transmembrane helix I (Fig. 6).  codons + 472 'arcD codons). The molecular mass of the deduced C a a ' -' A r~D~7~ hybrid polypeptide was calculated to be 54 kDa. Note that the first putative transmembrane segment in the wild type ArcD protein is believed to start at Leu-11 (3; cf. Fig. 6). As the Caa epitope replaces the first 10 amino acid residues of ArcD, the c a a ' -' A r~D~~~ hybrid should have an intact transmembrane segment 1. A second hybrid caul-'arcD gene was constructed on pME3801 (Fig. 1A). This derivative of pEPlink also expresses the caa N terminus under the control of the caa promoter. The 31 N-terminal codons of caa were fused, via a 17-codon linker fragment, to the 149 C-terminal codons of arcD. Details of construction are explained under "Materials and Methods.'' The resulting fusion protein, designated C~~' -' A~C D~~~, has a calculated molecular mass of 21 kDa. Its probable topology will be discussed later (Fig. 6).
In E. coli W3110(Nalr) carrying pME3801 or pME3802, these Caa'-'ArcD fusion proteins were expressed after SOS induction with mitomycin C, UV irradiation, or norfloxacin, and detected by Western blotting using the specific monoclonal antibody directed against the Caa epitope. Without SOS induction, no proteins were revealed in Western blots (data not shown), demonstrating the tight LexA control of the vector promoter on pEPlink.
The Cas'-'ArcD proteins could be localized in the cytoplasmic membrane of E. coli. Cells of strain W3110(Nal') harboring pME3801 or pME3802 were induced with mitomycin C and disrupted by sonication. The total membrane fraction purified by sucrose block gradient centrifugation (40) was extracted with sodium lauryl sarcosinate (SLS), a detergent known to solubilize selectively inner membrane proteins of E. coli (41). The total membrane fractions and the SLS extracts were subjected to SDS-PAGE. A Western blot of an SLS extract revealed the c a a ' -' A r~D~~~ protein as a single band corresponding to an apparent molecular mass of 40 kDa (Fig. 2, lune 1 ). In the total membrane preparation, this protein was also visible, along with some supposed degradation products (Fig. 2, lane 3 ) . It appears that SLS extraction resulted in a purification of the Caa'-'ArcD4,* protein, removing the contaminating degradation polypeptides (Fig. 2, lune  I). T h e c a a ' -' A r~D~~~ protein lacks more than two-thirds of ArcD; this hybrid was nevertheless found in the total membrane fraction (Fig. 2, lane 4 ) . Extraction with SLS gave a SLS. E. coli W3110(Nalr) carrying pME3801 or pME3802 was induced with mitomycin C. Cells of a 50-ml culture were resuspended in 2 ml of 35 mM Tris-HC1, pH 7.8, 20% (w/v) sucrose, 0.2 mM PMSF, 10 pg of DNase/ml and broken by sonification (3 pulses of 30 s). EDTA was added to 5 mM final concentration. Total membranes were purified by sucrose block gradient centrifugation (40,43). Part of this preparation was extracted with 0.5% (w/v) SLS (final concentration) in 500 pl of 100 mM Tris-HC1, pH 7.9, at room temperature for 20 min (41). The reaction was stopped by 20-fold dilution in 10 mM Tris-HC1, pH 7.9, 5 mM EDTA, 0.2 mM PMSF. After ultracentrifugation at 100,000 X g and 4 "C for 100 min, the supernatant with added carrier protein (bovine serum albumin, 50 pg/ml) was precipitated with 0.5 volume of 50% (w/v) trichloroacetic acid. The precipitate was dissolved in loading buffer (see "Materials and Methods"). The Caa epitope was visualized with l C l l antibody by Western blotting of an SDS gel. Numbers indicate size standards (kDa). 1, Caa'-'ArcD17:! encoded by pME3802 after SLS extraction; 2, Cas" ' A~C D~~~ encoded by pME3801 after SLS extraction; 3, total membrane fraction containing Caa'-'ArcD,,:!; 4, total membrane fraction containing CBB'-'A~CD~,~. single 18-kDa band (Fig. 2, lane 2), indicating that this protein was located in the cytoplasmic membrane as well. The membrane fraction contained the 18-kDa c a a ' -' A r~D~~~ protein together with an aggregated form, perhaps representing a dimer (Fig. 2A, lane 4 ) . The experimental M, values for Caa'-' A~c D~~~ and Caa'-'ArcDllg are both smaller than the values calculated from sequence data. This discrepancy was observed previously for the ArcD protein (3) and is typical of integral membrane proteins. These hydrophobic proteins bind excessive amounts of SDS, resulting in an anomalously high electrophoretic mobility (42).
The orientation of the Caa epitope in the inner membrane was probed by two methods. First, right-side-out membrane vesicles of strain W3110(Nal') carrying pME3801 or pME3802 were prepared from osmotically lysed spheroplasts (35). Inner membrane vesicles were treated with proteinase K (25 pg/ml) on ice for 15 min. This treatment did not affect the integrity of t h e c a a ' -' A r~D ]~~ a n d c a a ' -' A r~D~~~ proteins (Fig. 3, lunes 1 and 3 ) . Apparently, the periplasmic loops of the ArcD recombinant proteins were not accessible to proteinase K. After lysis of the membrane vesicles with Triton X-100, however, the Caa epitope was proteolytically degraded (Fig. 3, lanes 2 and 4 ) . We infer from this result that in both fusion proteins the Caa epitope faces the cytoplasm.
This finding was confirmed with E. coli cells whose outer membrane had been permeabilized with EDTA in Tris-HC1 buffer (44). This procedure allows access of trypsin to the periplasm (43,44). When the Caa epitope is exposed to the periplasm, as is the case in a Caa'-'TolQ hybrid protein, 3. Accessibility of Cas'-'ArcD hybrid proteins to proteinase K in right-side-out membrane vesicles. E. coli W3110(Nalr) carrying pME3801 or pME3802 was induced with mitomycin C. Cells from a 500-ml culture were converted to spheroplasts and osmotically lysed (35). After centrifugation at 1,200 X g for 20 min (to remove whole cells) and ultracentrifugation at 360,000 X g for 1 h, the membrane pellet was resuspended in 2 ml of 10 mM Tris, 5 mM EDTA, pH 7.8, 25% (w/v) sucrose, and purified by sucrose block (60 to 30% (w/v)) gradient centrifugation (40) in a Beckman SW41 rotor at 250,000 X g for 18 h. Fractions containing inner membrane vesicles were pooled. Proteinase K (25 pg/ml, final concentration) was added to this preparation (100 pg of protein/ml) at 0°C; Triton X-100 (0.5% (v/v), final concentration) was further included in two samples. After incubation at 0 "C for 15 min, the reaction was stopped by addition of PMSF (10 mM, final concentration). The entire trichloroacetic acid-precipitated samples were immediately submitted to SDS-PAGE and analyzed by Western blotting using l C l l antibody. Numbers indicate the molecular masses of size standards (kDa). I , Caa'-'ArcDllg specified by pME3801, without Triton X-100; 2, same as 1 but with Triton X-100; 3, Caa'-'ArcD472 specified by pME3802, without Triton X-100; 4, same as 3 but with Triton X-100. ..

FIG. 4.
Distribution of the Caa'-'ArcD4,* fusion protein in membrane fractions of P. aeruginosa. A, P. aeruginosa PA0 harboringpME3808 and pJRD7pol was grown in 1 liter of LB medium supplemented with tetracycline (50 pg/ml) and streptomycin (75 pg/ ml) at 30 "C. When the cell density had reached ODsoo,, = 0.5, the culture was shifted to 42 "C for 30 min and then the incubation was continued in the presence of rifampicin (200 pg/ml) at 30 "C for 100 min. Cells were disrupted in a French Press, and total membranes were fractionated by two sucrose gradients (29,30). Fractions (400 pl) of the second sucrose gradient were collected from the bottom of the tube and tested for protein concentration. Peak fractions were assayed for NADH oxidase activity; fractions 9 (OM, outer membrane) and 22 (CM, cytoplasmic membrane) contained 11 units/ml and 166 units/ml, respectively. B, from each even-numbered fraction, 100 pg of protein was precipitated with trichloroacetic acid and then subjected to SDS-PAGE. Immunoblot analysis with antibody l C l l revealed the Caa'-'ArcD17* protein.
trypsin/EDTA treatment leads to rapid digestion of the epitope (43). In contrast, we could not detect any damage of the Caa epitope by trypsin/EDTA in c a a ' -' A r~D~~~ or Caa'-' A~c D~~~, suggesting that indeed the epitope in these fusion proteins was not directed toward the periplasmic side of the inner membrane (data not shown).
Expression of Caul-'ArcD Fusion Proteins in P. aeruginosa-The caa promoter-operator region and the caa'-'arcD coding sequences of the E. coli plasmids pME3801 and pME3802 were subcloned into the broad-host-range vector pKT240 (cleaved with HpaI and BamHI). The resulting recombinant plasmids pME3803 and pME3804 allowed expression of Caa'-'ArcD14g and c a a ' -' A r~D~~~, respectively, in E. coli after induction with mitomycin C, norfloxacin, or UV irradiation (data not shown). After conjugative transfer of these plasmids to P. aeruginosa PAO1, we attempted to obtain expression of the fusion proteins by exposure to mitomycin C (1.5 pg/ml), UV irradiation (20 J/m2), or norfloxacin (4 pg/ ml). It is known that such DNA damaging agents induce the RecA protein and pyocins in P. aeruginosa (28,45,46). However, the PAOl/pME3803 or pME3804 transconjugants did not express the Cas'-'ArcD proteins under these conditions, suggesting that the caa promoter of E. coli is not inducible in P. aeruginosa. The reasons for the lack of induction are not clear; P. aeruginosa contains a LexA-like repressor protein (47).
To determine the cellular location of the c a a ' -' A r~D~~~ protein in P. aeruginosa, we therefore decided to use the T7@10 promoter on the broad-host-range vector pJRD253 (22) as a substitute for the caa promoter. We constructed the recombinant plasmid pME3808 (Fig. LA)    (argP) carrying pME3802 was induced with norfloxacin in LB medium for 4 h. Membrane vesicles were prepared as described (15) and loaded with 500 p~ L-ornithine in 50 mM potassium phosphate, pH 6.0, at 0 "C overnight, the protein concentration being 0.5 mg/ml. The membrane vesicles were exposed to 37 "C for 5 min, concentrated by centrifugation at 120,000 X g and 4 "C, and stored on ice. The exchange reaction was started by diluting the membrane vesicles (approximately 6 to 7 mg of protein per ml) 50-fold in 50 mM potassium phosphate, pH 6.0, containing 0.375 p~ L-["Clarginine (11 GBq/mmol). The reaction was stopped at intervals with 0.1 M LiCl. Radioactivity was determined in vesicles collected on a nitrocellulose filter (13). E. coli JC182-5/pME3802 vesicles loaded ( . ) or nonloaded (0) with ornithine. B, E. coli JC182-5/pME3806 was grown in LB medium, and membrane vesicles were prepared as described (15). Reaction conditions are as in panel A. E. coli JC182-5 /pME3806 vesicles loaded (0) or nonloaded (0) with ornithine. the caa ribosome binding site, was removed by BAL31 deletion. Strain PA01 was transformed with pME3808 and pJRD7pol (helper plasmid coding for the T7 RNA polymerase under the control of the thermoinducible X P L promoter (22)). After a temperature shift from 30 "C to 42 "C, the Caa'-' A~c D~~~ protein could be detected by Western blotting. Moreover, this protein was found to be associated mostly with the cytoplasmic membrane, as judged by Western blot analysis of a sucrose gradient that was used to separate the inner and outer membrane fractions (Fig. 4). Thus, it appears that the C~B ' -' A~C D~~~ protein is localized in the cytoplasmic membrane in P. aeruginosa as well as in E. coli.
ArginincOrnithine Exchanger Activity of the C~~' -' A~C D~,~ Protein in E. coli-To demonstrate transport activity of the Caa'-'Ar~D~7~ fusion protein, we introduced pME3802 into the arginine permease-negative E. coli strain JC182-5. After induction with norfloxacin (4 pg/ml) for 4 h, membrane vesicles were prepared from osmotically lysed spheroplasts.
(Induction with mitomycin C or UV interfered with the isolation of membrane vesicles.) The vesicles were loaded with unlabeled ornithine at 0 "C overnight. Incubation in the presence of [I4C]arginine at 25 "C resulted in arginine uptake ( Fig.  5A; W), whereas arginine uptake was low in vesicles not loaded with ornithine (Fig. 5A, 0). In a control experiment, membrane vesicles from strain JC182-5 without plasmid did not take up arginine (data not shown). We conclude that the 10 N-terminal amino acids of ArcD are not essential for transport activity in that they can be replaced by the Caa epitope. The vector was pKT240. Care was taken to avoid read-through from vector promoters into the arcD'-'phoA region. The exception is pME3806 where the promoter of the kanamycin resistance gene of pKT240 lies 0.8 kb upstream of the arc promoter, resulting in moderate phosphatase activity even when the arc promoter is not induced.
Numbers and dots shown above the sequence indicate the last arcD nucleotides (3). Horizontal lines above the sequence represent linkers; 0 stands for a 16-codon sequence at the end of TnphoA (18): GAC TCT TAT ACA CAA GTA GCG TCC TGG ACG GAA CCT TTC CCG TTT TGC Asp Ser Tyr Thr Glu Val Ala Ser Trp Thr Glu Pro Phe Pro Phe Cys. * refers to the first nucleotide in mature alkaline phosphatase (50). Amino acid sequences represent ArcD (boldface), linkers, and PhoA (italics), in this order. Numbers designate the amino acid residue in wild type PhoA (50).
Alkaline phosphatase levels are expressed in nmol per min and ml of culture at ODmo ",,, = 1.0. Cells were grown in YEA medium with good aeration (+02) or oxygen limitation (-02). 'The linker sequence consists of a BglII linker spliced to a BamHI-PstI fragment of pVBC18 (Ref. 24) and the PstI site of pCH40.
constructed by two approaches. First, TnphoA insertions (18) were obtained in the multicopy E. coli plasmid pME3750 carrying arcD (see "Materials and Methods"). Most TnphoA insertions occurred in the ampicillin resistance gene of the vector and only three different arcD::TnphoA fusions were isolated, which gave blue colonies on X P plates. To test the arcD'-'phoA fusions in P. aeruginosa, we subcloned the fragments containing the arc promoter and the urd'-'phoA open reading frame into the broad-host-range vector pKT240. The resulting plasmids pME3761 (Table I and Fig. lA), pME3762, and pME3765 (Table I) could be transferred to P. aeruginosa.
It turned out that the arcD::TnphoA insertions of pME3762 and pME3765 gave very low alkaline phosphatase activities in P. aeruginosa (Table I). Hence, the corresponding precursor plasmids in E. coli had indicated a false positive PhoA' phenotype.
In the second approach, a 'phoA fragment (21) was fused, in uitro, to the arcD gene at three different sites, the vector being again pKT240 (Table I) Table I). All arcD'-'phoA fusions were constructed in-frame and checked by DNA sequencing.
The phoA constructs were introduced into P. aeruginosa by transformation. Since all arcD'-'phoA hybrid genes are under the control of the arc promoter, they should be expressed during oxygen limitation. This was found to be the case. P.
aeruginosa carrying pME3761, pME3764, or pME3806 had high alkaline phosphatase activities, which were inducible by lack of aeration (Table I). On X P plates, these strains formed blue colonies (Table I) rule (55, 56). Arrows mark the fusion sites of Caa' or 'PhoA hybrid proteins; numbers indicate the plasmid constructs shown in Fig. L4 and Table I. +, high PhoA activity; -, low PhoA activity.
plasmids pME3762, pME3763, or pME3765 yielded very low alkaline phosphatase activities and produced pale blue or white colonies on X P plates ( Table I), suggesting that the PhoA part of the hybrid proteins faces the cytoplasm or has no access to the periplasm. The topological model presented below (Fig. 6) incorporates these findings.
Plasmid pME3806 codes for a hybrid protein that has all ArcD amino acid residues and, therefore, could retain arginine:ornithine antiporter activity. This was tested by complementation. A recombination-deficient arcD mutant of P. aeruginosa, PT06138, was transformed with pME3806. On yeast extract-arginine (YEA) medium, the transformant grew anaerobically in a Gas-Pak jar, whereas the arcD mutant could not grow. Moreover, in liquid medium containing 25 mM arginine, the transformant PT06138/pME3806 consumed 98% of the arginine initially present within 24 h of incubation under oxygen limitation. Arginine consumption by the wild type PA01 was similar (3), whereas the arcD mutant PT06138 used only 26% of the arginine in the medium. T o establish the transport activity of the ArcD-'PhoA fusion protein directly, we performed arginine uptake experiments with membrane vesicles from E. coli JC182-5 transformed with pME3806. Membrane vesicles prepared from these cells rapidly accumulated [14C]arginine after loading with unlabeled ornithine (Fig. 5B, 0 ) . Uptake of arginine was low when vesicles were not loaded with ornithine (Fig. 5B, 0). These results indicate that the ArcD moiety specified by pME3806 remains active for arginine uptake.

DISCUSSION
The ArcD protein of P. aeruginosa transports arginine, ornithine, and lysine and catalyzes their energy-independent exchange. The exchange reaction is much faster than the uptake driven by the protonmotive force (15). The physiological role of ArcD in the arginine deiminase pathway is to take up arginine from the medium in exchange for ornithine, which is formed from arginine in the cytoplasm (1,3). The ArcD protein has 43% sequence identity with the LysI protein of Corynebacterium glutamicum (51). LysI mediates lysine uptake and exchanges lysine for alanine, valine, or isoleucine (52). The hydrophobicity profiles (53) of ArcD and LysI are very similar; both proteins have 13 hydrophobic regions (3,

VI VI1 Vlll IX
x XI XI1 Xlll 51). Eleven hydrophobic segments of ArcD each have about 21 amino acids and are presumed to traverse the cytoplasmic membrane. The slightly shorter hydrophobic segments XI and XI1 might be embedded in the membrane or just span it (Fig. 6). It is of particular interest that ArcD appears to have 13 membrane-spanning segments; usually 12 membranespanning segments are found in bacterial secondary transport proteins (54). When we apply the "positive inside-rule'' (56), the N terminus of ArcD should lie in the cytoplasm whereas the C terminus would be exposed to the periplasm (Fig. 6). According to this topology, there are 15.6% positively charged (arginine, lysine) residues in the cytoplasmic loops of ArcD uersus 6.7% positive charges in the periplasmic loops (Fig. 6), which is in agreement with von Heijne's postulates (55, 56). In the present study, we have located the ArcD protein in the inner membrane and we have examined the plausibility of the topological model by Caa' and 'PhoA fusions.
The 'PhoA fusion to full-length ArcD (encoded by pME3806) gave high alkaline phosphatase activity (Table I), and a substantial amount of the hybrid protein was associated with the cytoplasmic membrane (data not shown). This result is consistent with our model (Fig. 6). The Caa epitope fused to the 11th ArcD residue in c a a ' -' A r~D ,~~ was protected from protease attack and hence is likely to face the cytoplasm (Fig.   3). The Caa N terminus contains 1 lysine and 1 arginine residue, as does the N-terminal fragment of ArcD which has been replaced by the Caa epitope. The Caa N terminus consists essentially of hydrophilic amino acids and should not influence the targeting of following hydrophobic segments. For these reasons we expect that the c a a ' -' A r~D~~~ hybrid should behave like native ArcD protein with respect to insertion into the membrane.
If the assumption is correct, the topology proposed by our model (Fig. 6) is again supported. The 'PhoA and the Caa' fusion proteins of pME3806 and pME3802 both retained ArcD transport activity (Fig. 5, A and  B ) . The 'PhoA and Caa' extensions may therefore provide useful handles for affinity purification of the ArcD protein.
The construction and testing of arcD'-'phoA fusions required certain precautions. Since some false positive results were observed in an E. coli background, all fusions were tested in P. aeruginosa. Direct TnphoA insertions into an arc+ broadhost-range plasmid of the IncQ group were avoided because Tn5 insertions in IncQ plasmids are notoriously unstable in Pseudomonas species (57, 58). Therefore, the fusions of arcD to 'phoA or TnphoA fragments were subcloned, in uitro, into pKT240 derivatives (Table I), and this procedure limited the number of constructs that could be analyzed. The PhoA activities of four internal ArcD'-'PhoA constructs (pME3761 to pME3764; Table I) were compatible with the model presented in Fig. 6. The PhoA-phenotype of a further fusion protein (encoded by pME3765) suggests that perhaps the fusion site lies closer to the cytoplasm than predicted by the hydrophobicity plot (Fig. 6). This seems possible because the periplasmic loop between segments VI1 and VI11 contains several hydrophobic amino acids (VVGHWGA) that might be embedded in the membrane (Fig. 6). Clearly, further fusions would be necessary to confirm the proposed topology of the inner part of ArcD. However, an important implication of our present model, the uneven number of membrane-spanning segments in ArcD, ought not to be affected by the properties of additional 'PhoA fusions.
The C~~' -' A~C D ,~~ hybrid was recovered from the inner membrane ( Figs. 2 and 3) and appeared to be stable, indicating that the five C-terminal hydrophobic segments are sufficient to direct the truncatedprotein into the membrane. C-terminal fragments of the Lacy lactose carrier can also be incorporated into the cytoplasmic membrane but they need the presence of a complementary N-terminal fragment for stabilization (59,60). Protease accessibility experiments suggest that the Caa epitope in the c a a ' -' A r~D , ,~ hybrid protein is oriented toward the cytoplasm, as expected from the model (Fig. 6).
The usefulness of PhoA fusions in topological studies of membrane proteins has been amply documented (48,49). Similarly, fusions of the Caa epitope (33) to membrane proteins can be exploited to determine the orientation of a protein in bacterial membranes. Previous applications of the Caa tag system have shown that the Caa epitope can either be located in the cytoplasm (as in the case of the immunity protein to colicin A (16)) or be translocated to the periplasm (as in the case of the TolQ protein (43)). Furthermore, it is also possible to insert the Caa epitope at an internal site of a polypeptide chain. For example, a hybrid PhoE-Caa outer membrane protein has been constructed in P. putidu. In this hybrid, the epitope is exposed to the cell surface, allowing immunochemical detection of the recombinant bacteria.' The present study on the ArcD inner membrane protein confirms the utility of the Caa tagging method.