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Functional and Biochemical Characterization of Escherichia coli Sugar Efflux Transporters

1999; Elsevier BV; Volume: 274; Issue: 33 Linguagem: Inglês

10.1074/jbc.274.33.22977

1083-351X

Jia Yeu Liu, Paul Miller, Jennifer Willard, Eric R. Olson,

Amino Acid Enzymes and Metabolism

A family of bacterial transporters, the SET (sugar efflux transporter) family, has been recently reported (Liu, J. Y., Miller, P. F., Gosink, M., and Olson, E. R. (1999) Mol. Microbiol. 31, 1845–1851). In this study, the biochemical and cell biological properties of the three Escherichia coli members (SetA, SetB, and SetC) of the family are characterized. We show that both SetA and SetB can transport lactose and glucose. In addition, SetA has broad substrate specificity, with preferences for glucosides or galactosides with alkyl or aryl substituents. Consistent with the observed in vitro substrate specificities, strains that hyperexpress SetA or SetB are desensitized to lactose analogues as measured by induction of the lac operon. In addition, strains that hyperexpress SetA are resistant to the growth inhibitory sugar analogueo -nitrophenyl-β-d-thiogalactoside. Strains disrupted for any one or all of the set genes are viable and show no defects in lactose utilization nor increased sensitivity to inducers of the lac operon and nonmetabolizable sugar analogues. The data suggest that the set genes are either poorly expressed under normal laboratory growth conditions or are redundant with other cellular gene products. A family of bacterial transporters, the SET (sugar efflux transporter) family, has been recently reported (Liu, J. Y., Miller, P. F., Gosink, M., and Olson, E. R. (1999) Mol. Microbiol. 31, 1845–1851). In this study, the biochemical and cell biological properties of the three Escherichia coli members (SetA, SetB, and SetC) of the family are characterized. We show that both SetA and SetB can transport lactose and glucose. In addition, SetA has broad substrate specificity, with preferences for glucosides or galactosides with alkyl or aryl substituents. Consistent with the observed in vitro substrate specificities, strains that hyperexpress SetA or SetB are desensitized to lactose analogues as measured by induction of the lac operon. In addition, strains that hyperexpress SetA are resistant to the growth inhibitory sugar analogueo -nitrophenyl-β-d-thiogalactoside. Strains disrupted for any one or all of the set genes are viable and show no defects in lactose utilization nor increased sensitivity to inducers of the lac operon and nonmetabolizable sugar analogues. The data suggest that the set genes are either poorly expressed under normal laboratory growth conditions or are redundant with other cellular gene products. methyl-α-glucoside isopropyl-β-d-thiogalactoside o-nitrophenyl-β-d-thiogalactoside carbonyl cyanidem -chlorophenylhydrazone 4-morpholinepropanesulfonic acid The recently described bacterial SET (sugarefflux transporter) family of efflux pumps shares amino acid sequence similarity with the Major Facilitator Superfamily of transport proteins (1Liu J.Y. Miller P.F. Gosink M. Olson E.R. Mol. Microbiol. 1998; 31: 1845-1851Crossref Scopus (41) Google Scholar, 2Pao S.S. Paulsen I.T. Saier Jr., M.H. Microbiol. Mol. Biol. Rev. 1998; 62: 1-34Crossref PubMed Google Scholar, 3Paulsen I.T. Brown M.H. Skurray R.A. Microbiol. Rev. 1996; 60: 575-608Crossref PubMed Google Scholar). Members of the Major Facilitator Superfamily perform diverse functions including the uptake of nutrients such as sugars and the secretion of noxious agents, including antibiotics (2Pao S.S. Paulsen I.T. Saier Jr., M.H. Microbiol. Mol. Biol. Rev. 1998; 62: 1-34Crossref PubMed Google Scholar, 3Paulsen I.T. Brown M.H. Skurray R.A. Microbiol. Rev. 1996; 60: 575-608Crossref PubMed Google Scholar). The SET proteins were first identified inEscherichia coli , which encodes three members (SetA, SetB, and SetC) that share a high degree (at least 70%) of amino acid sequence similarity (1Liu J.Y. Miller P.F. Gosink M. Olson E.R. Mol. Microbiol. 1998; 31: 1845-1851Crossref Scopus (41) Google Scholar). Prior to this work, setA, setB , andsetC were given the generic names yabM, yeiO , andyicK , respectively, for E. coli open reading frames of unknown function. It was shown that two of the proteins (SetA and SetB) could catalyze the secretion of lactose (1Liu J.Y. Miller P.F. Gosink M. Olson E.R. Mol. Microbiol. 1998; 31: 1845-1851Crossref Scopus (41) Google Scholar). Two additional members were identified as open reading frames in Deinococcus radiodurans and Yersinia pestis . A recent examination of the sequenced microbial genomes data base yielded six additional, more distantly related, proteins. 1M. Saier, manuscript in preparation. At present, the transport properties of these new family members have not been characterized. The SET proteins are not ubiquitously present in bacteria, suggesting an ecologically specialized role for this family of pumps. Sugar efflux has been reported in many bacterial species (4Reizer J. Novotny M.J. Panos C. Saier Jr., M.H. J. Bacteriol. 1983; 156: 354-361Crossref PubMed Google Scholar, 5Reizer J. Saier Jr., M.H. J. Bacteriol. 1983; 156: 236-242Crossref PubMed Google Scholar), including E. coli (6Huber R.E. Pisko Dubienski R. Hurlburt K.L. Biochem. Biophys. Res. Commun. 1980; 96: 656-661Crossref PubMed Scopus (16) Google Scholar, 7Huber R.E. Hurlburt K.L. Can. J. Microbiol. 1984; 30: 411-415Crossref PubMed Scopus (8) Google Scholar, 8Wilson T.H. Kashket E.R. Biochim. Biophys. Acta. 1969; 173: 501-508Crossref PubMed Scopus (21) Google Scholar, 9Winkler H.H. J. Bacteriol. 1971; 106: 362-368Crossref PubMed Google Scholar). It was shown that sugar efflux is an integral part of the metabolism of lactose in E. col i (7Huber R.E. Hurlburt K.L. Can. J. Microbiol. 1984; 30: 411-415Crossref PubMed Scopus (8) Google Scholar). In a strain constitutive for the lac operon, the addition of lactose led to the immediate and stoichiometeric appearance of the products (glucose, galactose, and allolactose) of β-galactosidase action in the medium (6Huber R.E. Pisko Dubienski R. Hurlburt K.L. Biochem. Biophys. Res. Commun. 1980; 96: 656-661Crossref PubMed Scopus (16) Google Scholar). Consistent with this hypothesis, mutants defective in the uptake of glucose and galactose grow poorly on lactose as the sole carbon source (7Huber R.E. Hurlburt K.L. Can. J. Microbiol. 1984; 30: 411-415Crossref PubMed Scopus (8) Google Scholar). Physiological evidence supports the hypothesis that efflux systems are involved in the detoxification of nonmetabolizable sugars in E. coli (8Wilson T.H. Kashket E.R. Biochim. Biophys. Acta. 1969; 173: 501-508Crossref PubMed Scopus (21) Google Scholar, 9Winkler H.H. J. Bacteriol. 1971; 106: 362-368Crossref PubMed Google Scholar, 10Andrews K.J. Lin E.C. J. Bacteriol. 1976; 128: 510-513Crossref PubMed Google Scholar, 11Hoffee P. Englesberg E. Lamy F. Biochim. Biophys. Acta. 1964; 79: 337-350Crossref PubMed Scopus (36) Google Scholar). Methyl-α-glucoside (MG),2 a competitive inhibitor of glucose utilization (12Hagihira H. Wilson T.H. Lin E.C.C. Biochim. Biophys. Acta. 1963; 78: 505-515Crossref PubMed Scopus (27) Google Scholar, 13Stock J.B. Waygood E.B. Meadow N.D. Postma P.W. Roseman S. J. Biol. Chem. 1982; 257: 14543-14552Abstract Full Text PDF PubMed Google Scholar), enters the cell mainly by the transporter for glucose and mannose, the products of the genesptsG and pstM , respectively, and accumulates to high levels as both the phosphorylated and the unmodified forms (9Winkler H.H. J. Bacteriol. 1971; 106: 362-368Crossref PubMed Google Scholar,13Stock J.B. Waygood E.B. Meadow N.D. Postma P.W. Roseman S. J. Biol. Chem. 1982; 257: 14543-14552Abstract Full Text PDF PubMed Google Scholar). When glucose is the sole carbon source, growth inhibition by MG is due to both decreased uptake of glucose and interference with the utilization of intracellular glucose-6-phosphate, the latter because of the accumulation of MG and MG-6-phosphate (9Winkler H.H. J. Bacteriol. 1971; 106: 362-368Crossref PubMed Google Scholar, 12Hagihira H. Wilson T.H. Lin E.C.C. Biochim. Biophys. Acta. 1963; 78: 505-515Crossref PubMed Scopus (27) Google Scholar). It was shown that both MG and MG-6-phosphate are secreted from the cell by an uncharacterized mechanism, the latter being first dephosphorylated before secretion (9Winkler H.H. J. Bacteriol. 1971; 106: 362-368Crossref PubMed Google Scholar, 11Hoffee P. Englesberg E. Lamy F. Biochim. Biophys. Acta. 1964; 79: 337-350Crossref PubMed Scopus (36) Google Scholar, 12Hagihira H. Wilson T.H. Lin E.C.C. Biochim. Biophys. Acta. 1963; 78: 505-515Crossref PubMed Scopus (27) Google Scholar). Many nonmetabolizable lactose analogues such as isopropyl-β-d-thiogalactoside (IPTG) and methyl-β-d-thiogalactoside are growth inhibitory when lactose is the only carbon source (10Andrews K.J. Lin E.C. J. Bacteriol. 1976; 128: 510-513Crossref PubMed Google Scholar). These compounds enter the cell through the lactose permease, the product of the lacY gene (14Herzengerg L. Biochim. Biophys. Acta. 1959; 31: 525-538Crossref PubMed Scopus (72) Google Scholar). To prevent the accumulation of IPTG and methyl-β-d-thiogalactoside, these sugar analogues are first acetylated by the LacA transacetylase and then secreted from the cell by an unknown transporter (8Wilson T.H. Kashket E.R. Biochim. Biophys. Acta. 1969; 173: 501-508Crossref PubMed Scopus (21) Google Scholar, 10Andrews K.J. Lin E.C. J. Bacteriol. 1976; 128: 510-513Crossref PubMed Google Scholar). Re-entry into the cell is prevented because the acetylated sugar analogues are not substrates for the permease (8Wilson T.H. Kashket E.R. Biochim. Biophys. Acta. 1969; 173: 501-508Crossref PubMed Scopus (21) Google Scholar). In this report, a role for the SET proteins (SetA, SetB, and SetC) in the metabolism of lactose or the detoxification of nonmetabolizable sugar analogues is investigated. As we show in this study, the range of sugars that are efflux substrates for the E. coli SET proteins include selective monosaccharides and disaccharides, in addition to glycoside analogues such as IPTG. Because lactose and IPTG are both substrates for Set protein-catalyzed efflux, we also generated null mutations in setA , setB , and setC and used these to help define the role of the Set proteins in E. coli sugar metabolism. The bacterial strains and plasmids used in this study are listed in Table 1. Cultures were grown in L broth (10 g tryptone/liter, 5 g yeast extract/liter, 5 g NaCl/liter) at 37 °C unless otherwise indicated. Plates contained 15 g agar/liter and top agar contained 7.5 g agar/liter. Antibiotics and arabinose, purchased from Sigma, were added to the following concentrations: 50 μg/ml ampicillin, 10 μg/ml tetracycline, 12.5 μg/ml chloramphenicol, 15 μg/ml streptomycin, 35 μg/ml kanamycin, 15 mm arabinose. M63 medium contained 3.0 g KH2PO4/liter, 7.0 g of K2HPO4/liter, 2.0 g (NH4)2SO4/liter, 0.5 mg FeSO4/liter, 1 mm MgSO4/liter, 0.1 mg thiamine/liter, and 2.0 g glycerol/liter. Sugars, sugar analogues, hexokinase, glucose-6-phosphate dehydrogenase, CCCP, and NADP+ were purchased from Sigma. Ciprofloxacin was obtained from the Parke-Davis chemical collection. Radiolabeled lactose (55.0 mCi/mmol) was purchased from Amersham Pharmacia Biotech, and radiolabeled glucose (8.36 mCi/mmol) was purchased from NEN Life Science Products.Table IE. coli strains and plasmidsStrainsRelevant genotype or phenotypePlasmidSource or referenceW3110F-λ-IN1 rph-1 thyA36 deoC2noneK. BertrandMN102W3110 Δ acrABnoneK. BertrandMC4100F-λ-araD139 Δ(argF-lac )U169rpsL 150 relA 1 flb B5301noneT. SilhavyML308lacInoneATCC 15224JL172ML 308 setA::Ω Smr setB ::ΩCmr setC ::ΩTetrzib-207::Tn10noneThis studyJL186W3110 setA ::ΩSmr setB ::ΩCmrnoneThis studyB359AB1157recD ::Tn10noneLab stockPlasmidRelevant propertiesSource or referencepBAD 18Ampr33Guzman L.M. Belin D. Carson M.J. Beckwith J. J. Bacteriol. 1995; 177: 4121-4130Crossref PubMed Scopus (4031) Google ScholarpBR322Ampr, Tetr32Bolivar F. Rodriguez R.L. Greene P.J. Betlach M.C. Heyneker H.L. Boyer H.W. Gene (Amst.). 1977; 2: 95-113Crossref PubMed Scopus (3627) Google ScholarpBR-SetA, -SetB, or -SetCAmpr set genes cloned into Nhe I-Bam HI site of pBR322This studypBAD-SetA, -SetB, or -SetCAmpr,set genes cloned into Sac I-Hin dIII site of pBAD18This study Open table in a new tab Standard DNA manipulation techniques were used (15Sambrook J. Fritsh E.F. Maniatis T. Molecular Cloning: A Laboratory Manual.2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). DNA corresponding to each of theset genes was obtained by the polymerase chain reaction. All polymerase chain reaction products were sequenced, and except for three silent mutations in yabM , the products were identical to the sequences reported in the EBI/GenBankTM data base. Construction of the plasmids pBAD-SetA, pBAD-SetB, and pBAD-SetC, previously referred to as pBAD-YabM, pBAD-YeiO, and pBAD-YicK (1Liu J.Y. Miller P.F. Gosink M. Olson E.R. Mol. Microbiol. 1998; 31: 1845-1851Crossref Scopus (41) Google Scholar), respectively, were described earlier (1Liu J.Y. Miller P.F. Gosink M. Olson E.R. Mol. Microbiol. 1998; 31: 1845-1851Crossref Scopus (41) Google Scholar). Plasmids pBR-SetA, pBR-SetB, and pBR-SetC are derivatives of plasmid pBR-LacY (1Liu J.Y. Miller P.F. Gosink M. Olson E.R. Mol. Microbiol. 1998; 31: 1845-1851Crossref Scopus (41) Google Scholar) where thelacY gene was replaced with the gene for eithersetA , setB , or setC , respectively. Overnight cultures of strains ML308/pBAD18, ML308/pBAD-SetA, and ML308/pBAD-SetB grown in L broth containing ampicillin were diluted 100-fold into the same medium or medium containing 15 mm arabinose. After 1.6 h of growth, ONPTG, dissolved at 20 times the final concentration in 60% dimethyl sulfoxide, or solvent only, was added to the indicated concentrations. The cultures were grown in a 96-well plate, and growth was followed with a SpectraMax 96-well plate reader (Molecular Devices Corp, Sunnyvale, CA). Cells were grown in L broth with arabinose to an A 600of 0.6–1.0 and harvested for inside-out vesicles, essentially as described previously (16Yamaguchi A. Udagawa T. Sawai T. J. Biol. Chem. 1990; 265: 4809-4813Abstract Full Text PDF PubMed Google Scholar). In the experiments described in Fig. 1(A and C ), the culture was grown in shake flasks. In the experiments described in Fig. 1 (B and D ), the culture was grown in a fermentor with L broth containing ampicillin to an A 600 of 0.6–1.0. Subsequently, arabinose was added to 15 mm, and the culture was harvested 2 h later. Cell pellets were washed once with ice-cold lysis buffer (50 mm MOPS-KOH, pH 6.6, 180 mm NaCl, 10 mm EDTA), resuspended in lysis buffer to anA 600 of 40–80, and lysed in a French Press at 5000 p.s.i. The lysate was centrifuged at 27,000 ×g for 10 min to remove unlysed cells and debris. The supernatant was centrifuged for 1 h at 100,000 ×g to pellet total membranes. Protein concentrations were determined by the Bradford protein assay kit (Bio-Rad) using bovine serum albumin as the standard. The transport assay was performed at 21 °C. A 1.5-μl solution of 50 mm ATP, pH 7.0, 50 mm MgSO4 was added to 13.5 μl of a suspension of membrane vesicles (14.0 μg protein/ml) followed by incubation at 21 °C for 30 s. Transport was initiated by the addition of 60 μl of Buffer A (50 mm MOPS, pH 7.5, 10 mmMgSO4, 192 mm NaCl) containing the labeled sugar ([14C]lactose: 0.181 mm, 55.0 mCi/mmol, or [14C]glucose: 0.57 mm, 8.36 mCi/mmol). At the specified times, the entire solution was mixed with 3 ml of ice-cold stop buffer (50 mm MOPS-KOH, pH 7.5, 180 mm LiCl) followed by filtration through Whatman glass fiber filters. The filters were washed twice with 3 ml of the same buffer, and radioactivity was measured in a liquid scintillation counter. For the CCCP-treated samples, CCCP was added to 80 μm at the indicated time after the initiation of transport and allowed to incubate further before stopping the assay with stop buffer. For the kinetic studies of SetA catalyzed [14C]lactose transport, the stock [14C]lactose solution was diluted with unlabeled lactose so that the final concentrations in the assay were 0.33, 0.5, 1.0, 2.0, and 5.0 mm. In the assay for inhibitors of SetA catalyzed transport of [14C]lactose, Buffer A contained the tested inhibitor at 31.25 mm and [14C]lactose at 0.181 mm (55.0 mCi/mmol). The final concentrations of the test compound and lactose were 25 and 0.145 mm, respectively. Log phase cultures (A 600 = 0.2–0.4) were grown at the indicated concentration of IPTG or lactose for 1 h. Subsequently, β-galactosidase activity in the culture was determined as described (17Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1972: 352-355Google Scholar). To follow the kinetics of IPTG induction of the lac operon, IPTG was added to early log phase cultures (A 600 = 0.1) to final concentrations of 2.5 and 5.0 μm. At the indicated times, samples of the culture were removed and assayed for β-galactosidase activity. Gradient plates were prepared by the method described previously (18Gambino L. Gracheck S.J. Miller P.F. J. Bacteriol. 1993; 175: 2888-2894Crossref PubMed Google Scholar). Antibiotics were used at the following concentrations: 15 μg/ml kanamycin, 15 μg/ml neomycin, 0.008 μg/ml ciprofloxacin. The quantitative assay for glucose is essentially that described previously (6Huber R.E. Pisko Dubienski R. Hurlburt K.L. Biochem. Biophys. Res. Commun. 1980; 96: 656-661Crossref PubMed Scopus (16) Google Scholar). Log phase cells growing in M63 with 0.2% glycerol (6Huber R.E. Pisko Dubienski R. Hurlburt K.L. Biochem. Biophys. Res. Commun. 1980; 96: 656-661Crossref PubMed Scopus (16) Google Scholar) were pelleted and washed once with buffer containing 0.1 mNaPO4, 1.0 mm MgSO4, pH 7.6. Cells were resuspended in assay buffer (0.1 m NaPO4, 7.0 mm MgSO4, 6.0 mm ATP, 0.3 units/ml hexokinase, 2.5 μg/ml glucose-6-phosphate dehydrogenase, 0.5 mg/ml NADP+, pH 7.6) to an A 600 of 0.25. The assay was initiated by the addition of lactose to 1.0 mm, and the absorbance at 340 nm was followed. To inhibit lactose uptake by LacY, cells were preincubated in assay buffer with CCCP at 60 μm for 3.0 min before the addition of lactose. The glucose efflux activity was normalized to the β-galactosidase activity (17Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1972: 352-355Google Scholar) in the solution. The disruption markers (19Fellay R. Frey J. Krisch H. Gene (Amst.). 1987; 52: 147-154Crossref PubMed Scopus (583) Google Scholar), referred to as interposons (denoted by the symbol Ω), which conferred resistance to streptomycin, chloramphenicol, or tetracycline, were inserted into setA (at Msc I), setB (atBsg I), and setC (at Bsg I), respectively, which were cloned in pBAD18. The disrupted alleles were individually integrated into the chromosome by one of two independent methods: 1) homologous recombination following transformation with linearized DNA into a recD strain (20Russell C.B. Thaler D.S. Dahlquist F.W. J. Bacteriol. 1989; 171: 2609-2613Crossref PubMed Google Scholar) and 2) cloning of the disrupted alleles into the temperature sensitive replication plasmid pMAK705 (21Hamilton C.M. Aldea M. Washburn B.K. Babitzke P. Kushner S.R. J. Bacteriol. 1989; 171: 4617-4622Crossref PubMed Google Scholar) to allow use of a two-step method (21Hamilton C.M. Aldea M. Washburn B.K. Babitzke P. Kushner S.R. J. Bacteriol. 1989; 171: 4617-4622Crossref PubMed Google Scholar) to obtain double cross-over recombinants. The disrupted alleles were confirmed both by polymerase chain reaction analysis and by P1 mapping studies. The triple mutant was assembled in both the W3110 and ML308 strain backgrounds by P1 transduction (22Silhavy T.J. Berman M.L. Enquist L.W. Experiments with Gene Fusions. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1984: 107-112Google Scholar). A study of the transport properties of the SET proteins was undertaken as a first step toward elucidating the physiological function of this newly identified family of efflux proteins. BothsetA , setB , and setC were cloned downstream from the arabinose-inducible promoter, generating the plasmids pBAD-SetA, pBAD-SetB, and pBAD-SetC, respectively, and the resulting plasmids transformed into E. coli strain MC4100. Inside-out membrane vesicles were prepared from cells that hyperexpressed the plasmid-encoded Set proteins. (In this configuration, an efflux pump would be expected to transport a radiolabeled substrate into the inside of the vesicle, which can be monitored as the accumulation of radioactivity in the vesicle interior.) It was shown previously and confirmed here that both SetA and SetB transport [14C]lactose (Fig.1, A and B ). Transport was sensitive to the addition of the protonophore CCCP, which caused the release of the accumulated radioactivity to near basal levels. The accumulation of the labeled lactose was also dependent on the presence of either ATP or NADH, which gave equivalent rates of labeled lactose transport (data not shown) and was not observed in vesicles prepared from cells that harbored the control plasmid pBAD18. Transport activity in the SetB-containing vesicles was higher than that of SetA. The SetB and more recent preparations of SetA vesicles were prepared from cells grown with increased aeration. The transport activity of recent preparations of SetA vesicles is comparable with SetB and is likely due to increased SetA protein expression (data not shown). The ability of SetA and SetB to transport [14C]glucose was also tested. In this assay, a higher concentration of [14C]glucose, which was of a lower specific activity than that of the [14C]lactose, was used. Both SetA and SetB promoted the transport of [14C]glucose, which was accumulated in the vesicle interior (Fig. 1, C andD ). The accumulation of the radiolabeled glucose was also dependent on the presence of ATP, was sensitive to CCCP, and was not observed in vesicles prepared from cells that harbored the control plasmid pBAD18. We were unable to observe the transport of [14C]galactose by SetA or SetB (data not shown). In addition, vesicles prepared from the strain that harbored thesetC expression plasmid was unable to transport any of the three sugars tested; however, the level of protein expressed was not determined. Because our data from the SetA and SetB overexpression strains indicated that SetA has a broader substrate specificity than SetB (see below), an assay was developed to define the range of sugars and sugar analogues that could serve as SetA substrates. To facilitate the design of the assay, which was based on the inhibition of transport of radiolabeled lactose into inside-out vesicles, a study of the kinetic properties of lactose transport by SetA was conducted. Fig.2 A shows the kinetics of lactose transport at varying substrate concentrations. The rate of transport appears to be saturable (Fig. 2 B ) with an apparentK m of 1.9 mm andV max of 0.12 pmol lactose/μg protein/s (Fig.2 C ). In the assay for possible substrates or inhibitors of lactose transport, the accumulation of radiolabeled lactose into inside-out vesicles was performed in the presence of unlabeled test compounds at 25 mm and [14C]lactose at 0.14 mm. (Note that this assay cannot distinguish between substrates that are effluxed by SetA and inhibitors that can compete with lactose for binding but that themselves are not transported.) The results of the competition assay are shown in Fig.3 and are briefly summarized here. Of the tested sugars, trioses, tetroses, pentoses, and heptoses are poor inhibitors, whereas selective hexoses and disaccharides, the best beingd-glucose (72% inhibition; Fig. 3 B ) and cellobiose (88% inhibition; Fig. 3 C ), respectively, show inhibitory activity. Glucosides and galactosides with large alkyl or aryl aglycone substituents are the most potent inhibitors of lactose transport (91–100% inhibition; Fig. 3 D ). When the alkyl substituent is a small methyl group, such as that found in the methyl-galactosides, there is a significant reduction in the inhibitory activity (methyl-α-galactoside 0%; methyl-β-galactoside 50%) in addition to a pronounced preference for a β-linkage of the aglycone to the sugar (Fig. 3 D ). Interestingly, α- and β-d-glucose-1-phosphate are both inactive inhibitors. The inactivity is likely due to the presence of the negative charge and not a steric effect of the phosphate group (Fig. 3 D ). The first position can be replaced with the larger and uncharged phenyl substituent found in phenyl-α and phenyl-β-d-glucosides, which are both potent inhibitors (Fig. 3 D ).Figure 3Inhibition of SetA catalyzed [14C]lactose transport into inside-out vesicles. The assay measured the amount of SetA-dependent [14C]lactose taken up into inside-out vesicles in 30 s in the presence of the indicated inhibitor at 25 mm. The amount of [14C]lactose transported in the presence of 25 mm NaCl is taken to be 100. The classes of compounds tested for inhibitory activity were as follows: A , nonsugars, 3, 4, and 5 carbon monosaccharides. B , 6 and 7 carbon monosaccharides. C , disaccharides and trisaccharides.D , glucosides and galactosides. Each data point represents the mean of triplicate determinations ± S.D.View Large Image Figure ViewerDownload Hi-res image Download (PPT) A series of cell-based assays were designed to test whether SetA and SetB transport the substrates identified by the in vitro transport assays. A set of pBR322 derived plasmids were constructed that constitutively expressed each of the set genes by cloning them downstream from the promoter for the tetracycline resistance gene. The plasmids were transformed individually intoE. coli strain W3110, which is wild type at thelac locus, and the resulting strains were each expected to hyperexpress one of the Set proteins. These strains were tested for the ability of the individual Set proteins to transport either IPTG or lactose. Because IPTG and lactose are both inducers of thelac operon, the level of β-galactosidase activity should reflect the intracellular concentration of the inducer. If the inducer is effluxed from the cell by a Set protein, the level of β-galactosidase activity would be expected to be lower. Strains harboring individual set plasmids were titrated with either lactose or IPTG, and the level of β-galactosidase activity was measured. Fig. 4 A shows that only SetA can efflux IPTG, because the level of β-galactosidase activity in the setA -containing plasmid was equal to that of the background at 0.1 mm IPTG. The level of β-galactosidase activity increased as the concentrations of IPTG rose from 1.0 to 100 mm, where it plateaued. This is the expected result if SetA is simply an efflux pump for the inducer. At high extracellular concentrations of the inducer, the rate of entry of the inducer exceeds the rate of efflux by SetA. In contrast, strains with either the setB or the setC plasmid, as well as the strain harboring the control plasmid pBR322, all showed high levels of β-galactosidase activity at 0.1 mm IPTG. The plateau level of β-galactosidase activity in the strain with thesetB plasmid was lower than that of the control strain with pBR322, possibly reflecting the slow growth phenotype of this strain. Fig. 4 B shows that both SetA and SetB can efflux lactose. The β-galactosidase activity of the strain with the setA plasmid remained at background levels when titrated with lactose at up to 10 mm. Growth of this strain was not affected by the addition of lactose at the concentrations used. The β-galactosidase activity of the strain with the setB plasmid was equal to that of the background level at 0.1 mm lactose and was 22% of the level present in the control strain with pBR322 at 1 and 10 mm lactose. In contrast, the level of β-galactosidase activity in the strain with the setC plasmid was similar to that of the control strain with pBR322. Comparison of the lactose and IPTG titration curves indicates that IPTG is a better inducer of thelac operon, which is due to the fact that lactose is not active as an inducer until it is rearranged to allolactose by the action of β-galactosidase (23Burstein C. Cohn M. Kepes A. Monod J. Biochim. Biophys. Acta. 1965; 95: 634-639Crossref PubMed Scopus (56) Google Scholar). Because the in vitro transport studies indicated that aryl-glycosides may possibly serve as substrates for SetA, a cell-based assay was therefore designed to test this. It was previously reported that the intracellular accumulation of the toxic β-galactoside analogue, ONPTG, inhibits growth (24Muller Hill B. Crapo L. Gilbert W. Proc. Natl. Acad. Sci. U. S. A. 1968; 59: 1259-1264Crossref PubMed Scopus (279) Google Scholar). Growth inhibition is dependent on the expression of lacY , because ONPTG is a substrate for the LacY permease (25Flagg J.L. Wilson T.H. J. Bacteriol. 1976; 128: 701-707Crossref PubMed Google Scholar). If ONPTG is also a substrate for SetA efflux, it would be expected that strains that hyperexpress SetA would be more resistant to the sugar analogue. A strain constitutive for thelac operon (ML308) was transformed with plasmids pBAD-SetA, pBAD-SetB or the control plasmid pBAD18. Transformants were tested for resistance to ONPTG by following the growth curves of cultures in the absence or presence of arabinose; the latter condition is expected to induce expression of SetA or SetB in the corresponding transformants. In the pBAD18 transformant, the presence of ONPTG caused a decrease in the growth rate (Fig. 5 A ). The severity of the growth inhibition was dependent upon the concentration of ONPTG in the medium. The presence of arabinose slightly decreased the growth yield (compare Fig. 5, A and B without ONPTG) but did not change