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• W2007422151 abstract "The active accumulation of maltose and maltodextrins by Escherichia coli is dependent on the maltose transport system. Several lines of evidence suggest that the substrate specificity of the system is not only determined by the periplasmic maltose-binding protein but that a further level of substrate specificity is contributed by the inner membrane integral membrane components of the system, MalF and MalG.We have isolated and characterized an altered substrate specificity mutant that transports lactose. The mutation responsible for the altered substrate specificity results in an amber stop codon at position 99 of MalF. The mutant requires functional MalK-ATPase activity and hydrolyzes ATP constitutively. It also requires MalG. The data suggest that in this mutant the MalG protein is capable of forming a low affinity transport path for substrate. The active accumulation of maltose and maltodextrins by Escherichia coli is dependent on the maltose transport system. Several lines of evidence suggest that the substrate specificity of the system is not only determined by the periplasmic maltose-binding protein but that a further level of substrate specificity is contributed by the inner membrane integral membrane components of the system, MalF and MalG. We have isolated and characterized an altered substrate specificity mutant that transports lactose. The mutation responsible for the altered substrate specificity results in an amber stop codon at position 99 of MalF. The mutant requires functional MalK-ATPase activity and hydrolyzes ATP constitutively. It also requires MalG. The data suggest that in this mutant the MalG protein is capable of forming a low affinity transport path for substrate. The maltose transport system of the Gram-negative bacteriumEscherichia coli is responsible for the unidirectional uptake of maltooligosaccharides (α[1,4]-linkedd-glucose polymers) (1Ferenci T. Eur. J. Biochem. 1980; 108: 631-636Crossref PubMed Scopus (60) Google Scholar). This system comprises five proteins. The LamB protein (also known as the maltoporin or λ-receptor) in the outer membrane, the periplasmic maltose-binding protein (MBP), 1The abbreviation used is: MBP, maltose-binding protein.1The abbreviation used is: MBP, maltose-binding protein. and the MalF, MalG, and MalK polypeptides that together (MalFGK2) form the inner membrane complex (2Silhavy T.J. Brickman E. Bassford P.J. Casadaban M.J. Shuman H.A. Schwartz V. Guarente L. Schwartz M. Beckwith J.R. Mol. Gen. Genet. 1979; 174: 249-259Crossref PubMed Scopus (54) Google Scholar). The LamB protein, encoded by the lamBgene is responsible for the diffusion of maltooligosaccharides into the periplasm of the cell (3Szmelcman S. Schwartz M. Silhavy T.J. Boos W. Eur. J. Biochem. 1976; 65: 13-19Crossref PubMed Scopus (195) Google Scholar). MBP, a soluble periplasmic protein encoded by the malE gene, binds maltooligosaccharides with a high affinity (K d = 0.1–1 μm) (4Ferenci T. Muir M. Lee K.S. Maris D. Biochim. Biophys. Acta. 1986; 860: 44-50Crossref PubMed Scopus (43) Google Scholar, 5Kellermann O. Szmelcman S. Eur. J. Biochem. 1974; 47: 139-149Crossref PubMed Scopus (137) Google Scholar) due primarily to a slow dissociation rate (6Miller III, D.M. Olson J.S. Pflugrath J.W. Quiocho F.A. J. Biol. Chem. 1983; 258: 13665-13672Abstract Full Text PDF PubMed Google Scholar). MBP is absolutely required for the transport of maltose (7Shuman H.A. J. Biol. Chem. 1982; 257: 5455-5461Abstract Full Text PDF PubMed Google Scholar, 8Treptow N.A. Shuman H.A. J. Bacteriol. 1985; 163: 654-660Crossref PubMed Google Scholar). The three-dimensional structure of MBP has been determined in both the liganded and unliganded forms (9Spurlino J.C. Lu G.Y. Quiocho F.A. J. Biol. Chem. 1991; 266: 5202-5219Abstract Full Text PDF PubMed Google Scholar). The MalF and MalG proteins are integral membrane proteins that traverse the membrane eight and six times, respectively (10Boyd D. Manoil C. Beckwith J. Proc. Nat. Acad. Sci. U. S. A. 1987; 84: 8525-8529Crossref PubMed Scopus (202) Google Scholar, 11Froshauer S. Green G.N. Boyd D. McGovern K. Beckwith J. J. Mol. Biol. 1988; 200: 501-511Crossref PubMed Scopus (124) Google Scholar, 12Dassa E. Muir S. Mol. Microbiol. 1993; 7: 29-38Crossref PubMed Scopus (50) Google Scholar). MalK is an ATPase located on the cytoplasmic side of the inner membrane that is thought to be the energy-coupling component of the maltose transport system (3Szmelcman S. Schwartz M. Silhavy T.J. Boos W. Eur. J. Biochem. 1976; 65: 13-19Crossref PubMed Scopus (195) Google Scholar, 14Panagiotidis C.H. Reyes M. Sievertsen A. Boos W. Shuman H.A. J. Biol. Chem. 1993; 268: 23685-23696Abstract Full Text PDF PubMed Google Scholar). MalK shares strong sequence homology with other ATP-binding cassette transporter proteins (16Higgins C. Hiles I. Salmond G. Gill D. Downie J. Evans I. Nature. 1986; 323: 448-450Crossref PubMed Scopus (487) Google Scholar). The stoichiomtry of the inner membrane complex is (MalFGK2) (17Davidson A.L. Nikaidi H. J. Biol. Chem. 1990; 265: 4254-4260Abstract Full Text PDF PubMed Google Scholar). MBP has long been regarded as the prime determinant of substrate specificity for the maltose system. However, several observations suggest that the inner membrane complex plays an important role in determining substrate specificity. First, MBP-independent mutants of MalF and MalG have been isolated that transport maltose in the absence of MBP (8Treptow N.A. Shuman H.A. J. Bacteriol. 1985; 163: 654-660Crossref PubMed Google Scholar). Even in the absence of MBP, these mutants still retain substrate specificity for maltooligosaccharides, which suggests that the inner membrane complex alone is capable of maintaining the specificity of the system. These MBP-independent mutants share a similar K m for maltose transport of approximately 2 mm, but they each display a V maxthat varies from wild-type values to approximately 20-fold lower. Therefore, they all bind maltose equally well, which is suggestive of a similar substrate binding site, but they translocate with different efficiencies. The observation that the ATP-binding cassette components of two members of the ABC superfamily, the Ugp and Mal transport systems, could be exchanged without any loss of substrate specificity suggests that this component does not play a role in determining the overall specificity of the system (21Hekstra D. Tommassen J. J. Bacteriol. 1993; 175: 6546-6552Crossref PubMed Google Scholar). The Ugp transport system of E. colitransports sn-glycerol-3-phosphate. The UgpC and MalK proteins of these systems are highly homologous and both couple energy via ATP-hydrolysis (21Hekstra D. Tommassen J. J. Bacteriol. 1993; 175: 6546-6552Crossref PubMed Google Scholar). Thus, the two other components of the maltose transport system inner membrane complex, MalF and MalG, must be contributing to the specificity of the system. Mutants of MalF have been isolated that alter the range of substrates transported by the maltose transport system (22Ehrle R. Pick C. Ulrich R. Hoffman E. Ehrmann M. J. Bacteriol. 1996; 178: 2255-2262Crossref PubMed Google Scholar). The various mutations map to the malF gene and cause alterations in transmembrane domains 6, 7, and 8 of MalF. The mutants could only recognize either maltose or longer maltodextrins, but not both. The mutations cluster along the transmembrane helices, and suppressor mutations in neighboring helices of MalF suggest a physical interaction. Studies on other members of the ABC superfamily, such as P-glycoprotein and the HlyB transporter, have also suggested that the transmembrane helices play an important role in determining substrate specificity (23Raviv Y. Pollard H.B. Bruggemann E.P. Pastan I. Gottesman M.M. J. Biol. Chem. 1990; 265: 3975-3980Abstract Full Text PDF PubMed Google Scholar, 24Greenberger L.M. Yang C.P. Gindin E. Horwitz S.B. J. Biol. Chem. 1990; 265: 4394-4401Abstract Full Text PDF PubMed Google Scholar, 25Bruggemann E.P. Currier S.J. Gottesman M.M. Pastan I. J. Biol. Chem. 1992; 267: 21020-21026Abstract Full Text PDF PubMed Google Scholar, 26Zhang F. Sheps J.A. Ling V. J. Biol. Chem. 1993; 268: 19889-19895Abstract Full Text PDF PubMed Google Scholar, 27Hanna M. Brault M. Kwan T. Kast C. Gros P. Biochemistry. 1996; 35: 3625-3635Crossref PubMed Scopus (55) Google Scholar). In the present study, we describe a mutant of the maltose transport system (MalF540) that transports lactose efficiently. The MalF540 mutant we isolated carries a mutation in the malF gene that changes a glutamine codon at position 99 of MalF to an amber stop codon. This mutant suggests a novel mechanism for altering substrate specificity. The isolation and characterization of this mutant with respect to transport efficiency, maltose transport components required, and substrate specificity is described. Rich media (LB) and minimal media (M63) were prepared as described previously (30Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1972Google Scholar). Standard genetic procedures were performed by the method of Miller (30Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1972Google Scholar) unless otherwise noted. Maltose and lactose were obtained from Pfanstiehl Laboratories, Inc. All other sugars were obtained from Sigma. The following antibiotics were used at the indicated concentration unless otherwise noted: 100 μg/ml carbenicillin, 100 μg/ml ampicillin, 50 μg/ml kanamycin, 25 μg/ml chloramphenicol, 20 μg/ml tetracycline. Bacterial strains are listed in TableI. The recombination defectiverecA1 strain, GM1418, was constructed by P1vir transduction using a lysate prepared from HS3078 containing thesrl::Tn10 mutation closely linked to the recA1 allele. Tetracycline-resistant transductants of LH1375 were selected and screened for UV sensitivity.Table IBacterial strains, plasmids, and phageStrainGenotypeSourceDH5αendA1 hsdR17 supE44 thi-1 recA1 gyrA (Nalr) relA1 Δ(argF-lac)U169 (φ80dΔlacZM15)Ref. 51Hanahan D. J. Mol. Biol. 1983; 166: 557-580Crossref PubMed Scopus (8098) Google ScholarCJ236F− dut- ung- thil relA1/pCJ105Ref. 37Kunkel T.A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 488-492Crossref PubMed Scopus (4880) Google ScholarKD1087F− mutD5 argE his44 Δ(tonB trpBA) rpsERef. 32Dengen D.A. Cox E.C. J. Bacteriol. 1974; 117: 477-487Crossref PubMed Google ScholarHS3003F− araD139 Δ(araABDIC, leu)7697 Δ(lac)X74 galU galK rpsLLaboratory collectionCHP243HS3003/rpoB lacl(Q1) ΔmalB101 Δatp asn::Tn10Laboratory collectionHS3238F− araD139 Δ(argF-lac)U169 relA1 rbsR flbB5301 ptsF25 thi-1 deoC1 malTp1Tp7Laboratory collectionHS3003F− araD139 Δ(araABDIC, leu)7697 Δ(lac)X74 galU galK rpsLLaboratory collectionHS3420HS3238/ΔmalB101 rifR argHLaboratory collectionHS3239HS3238/recAl srl::Tn10Laboratory collectionHS3078F− araD139 Δ(argF-lac)U169 relA1 rbsR flbB5301 ptsF25 thi-1 deoC1 ΔmalE444 purD::Tn5 recA1 srl::Tn10Laboratory collectionHS3419HS3003/F′ KLF10 ΔmalF arg-/malE7::Tn5 recA1 ΔmalF(StuI-HpaI)Laboratory collectionHS3670F′ KLF10 malGam8/ara + Δ(argF-lac)U169 rpsL150 relA1 rbsR flbB5301 ptsF25 thi-1 deoC1 Δ(lac-pro)XIII rifR malGamV67Laboratory collectionHS3309HS3238/ΔmalE444 zjb::Tn10–729Laboratory collectionLH1616HS3003/malE7::Tn5 recA1Ref. 42Hor L.I. Genetic Studies on the Mechanism of Periplasmic Binding Protein-dependent Transport.Ph.D. dissertation. Columbia University, 1991Google ScholarLH1375F− araD139 Δ(argF-lac)U169 relA1 rbsR flbB5301 ptsF25 thi-1 deoC1 malT(c) malE7::Tn5Ref. 42Hor L.I. Genetic Studies on the Mechanism of Periplasmic Binding Protein-dependent Transport.Ph.D. dissertation. Columbia University, 1991Google ScholarGM1191HS3420/pLH22/pAB1This studyGM1305HS3239/pLH22/pAB1This studyGM1361HS3238/pAB1/pGM7This studyGM1307HS3420/pNT7/pAB1/pHS4This studyGM1308HS3420/pNT7/pAB1/pSN1This studyGM1035LH1616/pGM1This studyGM1042LH1616/pMMB207This studyGM1418LH1375/pAB1This studyGM1304GM1191/pNT7This studyGM1306GM1191/pSC101This studyGM1368F′ KLF10ΔmalF argE::Tn10/HS3238ΔmalF/pAB1/pLH22This studyGM1408F′ KLF10malGam8/HS3238 malGam8/pAB1/pLH22This studyGM1369F′ KLF10 ΔmalF argE::Tn10/HS3238ΔmalF/pAB1/pGM7This studyPlasmidGenotypeSourcepAB1pMMB207 Ptac-lacZRef.52Sadosky A.B. Wilson J.W. Steinman H.M. Shuman H.A. J. Bacteriol. 1994; 176: 3790-3799Crossref PubMed Google ScholarpSN1pACYC177 PmalB-malK(K42R)Ref. 14Panagiotidis C.H. Reyes M. Sievertsen A. Boos W. Shuman H.A. J. Biol. Chem. 1993; 268: 23685-23696Abstract Full Text PDF PubMed Google ScholarpTE18pBR322 Ptac-lacYRef. 53Teather R.M. Bramhall J. Riede I. Wright J.K. Furst M. Aichele G. Wilhelm U. Overath P. Eur. J. Biochem. 1980; 108: 223-231Crossref PubMed Scopus (187) Google ScholarpHS4pACYC177 PmalB-malKLaboratory collectionpNT7pSC101 PmalB-malERef. 8Treptow N.A. Shuman H.A. J. Bacteriol. 1985; 163: 654-660Crossref PubMed Google ScholarpLH5pBR322PmalB-malF502(G338R,A502V)-malGRef. 33Covitz K.M. Panagiotidis C.H. Hor L.I. Reyes M. Treptow N.A. Shuman H.A. EMBO J. 1994; 13: 1752-1759Crossref PubMed Scopus (65) Google ScholarpLH9pBR322 PmalB-malF-malGRef. 42Hor L.I. Genetic Studies on the Mechanism of Periplasmic Binding Protein-dependent Transport.Ph.D. dissertation. Columbia University, 1991Google ScholarpLH22pACYC177 PmalB-malK lac1 QRef. 42Hor L.I. Genetic Studies on the Mechanism of Periplasmic Binding Protein-dependent Transport.Ph.D. dissertation. Columbia University, 1991Google ScholarpMR11pACYC184 Ptrc-malKRef. 54Reyes M. Shuman H.A. J. Bacteriol. 1988; 170: 4598-4602Crossref PubMed Google ScholarpMR24pBR322 Ptrc-malGLaboratory collectionpMR28pBR322 PmalB-malFRef. 14Panagiotidis C.H. Reyes M. Sievertsen A. Boos W. Shuman H.A. J. Biol. Chem. 1993; 268: 23685-23696Abstract Full Text PDF PubMed Google ScholarpMR38pBR322 Ptrc-malFRef. 14Panagiotidis C.H. Reyes M. Sievertsen A. Boos W. Shuman H.A. J. Biol. Chem. 1993; 268: 23685-23696Abstract Full Text PDF PubMed Google ScholarpLac2pBR322PmalB-malF540(Q99(Am))-malGRef. 33Covitz K.M. Panagiotidis C.H. Hor L.I. Reyes M. Treptow N.A. Shuman H.A. EMBO J. 1994; 13: 1752-1759Crossref PubMed Scopus (65) Google ScholarpGM1pMMB207Ptac-malEThis studypGM7pACYC177PmalB-malK-malGThis studypGM8pBR322PmalB-malF541 (E39(Am))-malGThis studypGM9pBR322 PmalB-malF542(Y55(Am))-malGThis studypGM10pBR322PmalB-malF543 (E130(Am))-malGThis studypGM11pBR322 PmalB-malF544(K275(Am))-malGThis studypGM12pBR322PmalB-malF545 (Y317(Am))-malGThis studypGM13pBR322 PmalB-malF546(Q99(Am))-malGThis studypLac2F′pBR322PmalB-malF540′(BsmI)This studypLH9SMpBR322PmalB-ΔmalF(SacI-MfeI)-malGThis studypLac2SMpBR322PmalB-ΔmalF540(SacI-MfeI)-malGThis studypt-Lac2SMpBR322Ptrc-malF540(SacI-MfeI)-malGThis studyPhageGenotypeSourceλNF630′malK malE + Φ(malF-lacZ)hyb6–3lacY +Ref. 31Treptow N.A. Shuman H.A. J. Mol. Biol. 1988; 202: 809-822Crossref PubMed Scopus (66) Google Scholar Open table in a new tab The malG-amber strain, GM1408, was constructed by conjugating the strain carrying F′ KLF10 malGamV67, HS3670, with the malTp1Tp7 strain, HS3238. The mating was plated on minimal glucose plates that select only for HS3238. Recipients of the F′ were selected and screened by sensitivity to VCS-M13 phage. Independent overnight cultures of the transconjugants in LB were plated on tetrazolium maltose plates, and Mal− colonies were selected. Some of these should represent a gene conversion event between the malGamV67 on the episome and malG on the chromosome. The malTp1Tp7 allele is easily lost, so the Mal− colonies were screened for the presence ofmalTp1Tp7 by testing the ability to hydrolyze pNPG2 when lysed with chloroform. This is due to the overproduction of MalZ in response to the elevated levels of MalT. The presence of the malGamV67 was further confirmed by complementation of the Mal− phenotype with the plasmid pMR24, which carries wild-type malG, and by complementation with the φ80SupF amber suppressor phage. The pAB1 and pLH22 plasmids were introduced into the resulting strain by transformation. The malF deletion strains, GM1368 and GM1369, were constructed by conjugating the strain carrying F′ KLF10 ΔmalF argE::Tn10, HS3419, with themalTp1Tp7 strain, HS3238. F′ recipients were selected on minimal glucose plates containing tetracycline. Independent overnight cultures of the transconjugants in LB containing tetracycline were plated on tetrazolium maltose plates, and Mal− colonies were selected. Some of these should represent a gene conversion event between the ΔmalF3 on the F′ and malF on the chromosome. Mal− colonies were then screened for the presence of malTp1Tp7. The presence of theΔmalF3 was further evaluated by complementation of the Mal− phenotype with the plasmid pMR28, which carries wild-type malF. The pAB1 and either the pGM7 or pLH22 plasmids were introduced into the resulting strain by transformation. The plasmid pGM1, which carries themalE gene under control of the ptac promoter, was constructed by ligating the malE-containingEcoRI/StuI fragment from λNF630 (31Treptow N.A. Shuman H.A. J. Mol. Biol. 1988; 202: 809-822Crossref PubMed Scopus (66) Google Scholar) phage DNA, to EcoRI/SmaI-digested pMMB207 plasmid vector DNA. pGM7, which carries the malK and malG genes under the malB promoter, was constructed by ligating themalK-containing EcoRI/ScaI fragment from pHS4, to the malG-containingEcoRI/SnaBI fragment from pMR24. pLac2SM was constructed by digesting pLac2 with SacI andMfeI, thus deleting the portion of malF distal to the specificity mutation, “end-filling” and “chewing back” the respective overhangs with DNA polymerase I (Klenow) and T4 DNA-polymerase, and then ligating the resulting large DNA fragment to itself. pLH9SM was constructed in the same way except that the pLH9 plasmid was used. pLac2F′ was constructed by ligating thePstI/BsmI fragment from pLac2 containing the 5′-end of the truncated malF540 gene including the mutation, to the PstI/BsmI fragment from pBR322 that contains the origin of replication. Pt-pLac2SM was constructed by ligating the PstI/BstEII fragment from pMR38 containing the Ptrc promoter and the 5′-end of themalF gene to the PstI/BstEII fragment from pLac2SM containing the 3′-end of truncated malF540 andmalG. A plasmid carrying the malF502 MBP-independent allele, pLH5, was mutagenized in the mutator strain KD1087 (32Dengen D.A. Cox E.C. J. Bacteriol. 1974; 117: 477-487Crossref PubMed Google Scholar). This strain contains themutD5 allele, which increases the frequency of mutational events 50–100 times above that observed in a wild-type strain (mut +). KD1087 was transformed with the pLH5 plasmid, and transformants were selected on LB plates containing carbenicillin. Individual transformants were then grown in LB containing carbenicillin at 37 °C overnight. Plasmid DNA was purified from these independent cultures and used to transform the lactose indicator strain GM1305. Transformants were plated on minimal 1% lactose plates containing carbenicillin, kanamycin, chloramphenicol, and 0.2% arginine. Those transformants that grew on the indicator media after 1–3 days of incubation at 37 °C were purified by passing three times on minimal 1% lactose plates. Plasmid DNA was purified from these isolates and used to retransform the lactose indicator strain to ensure that the mutation responsible for the Lac+ phenotype is linked to the pLH5 plasmid. The nucleotide sequence of malF and malG alleles carried on pLac2 and various amber mutant plasmids (pGM8-pGM12) was determined by using double-stranded DNA templates and 13 oligonucleotide primers (33Covitz K.M. Panagiotidis C.H. Hor L.I. Reyes M. Treptow N.A. Shuman H.A. EMBO J. 1994; 13: 1752-1759Crossref PubMed Scopus (65) Google Scholar). Double-stranded DNA templates were purified, denatured, and annealed with the primers as described (34Kraft R. Tardiff J. Krauter K.S. Leinwald L.A. BioTechniques. 1988; 6: 544-546PubMed Google Scholar). The Sanger dideoxynucleotide chain termination method (35Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52250) Google Scholar) was used to determine the nucleotide sequences. Maltose and lactose transport activity was estimated by measuring the uptake of [14C]maltose (360 mCi/mmol) or [14C]lactose (57 mCi/mmol) as described previously (7Shuman H.A. J. Biol. Chem. 1982; 257: 5455-5461Abstract Full Text PDF PubMed Google Scholar). When sugars were tested for their ability to inhibit radioactive sugar uptake, cells were incubated in the presence of inhibitor sugar for 5 s prior to the addition of radioactive substrate. [14C]Maltose was obtained from Moravek Biochemicals, Inc. [14C]Lactose was obtained from Amersham International, plc. To measure the ATPase activity of the mutant maltose systems, the respective proteins must be overproduced. We used the strain HS3309, which carries the pMR11 plasmid that has the malK gene under the Ptac promoter. This strain was transformed with either pBR322 as a vector control; pNT11, which carries the malF502MBP-independent allele under Ptrc; or pt-Lac2SM, which carries the malF540 allele under Ptrc. Inside-out membrane vesicles were prepared from transformants induced with isopropyl-1-thio-β-d-galactopyranoside, and ATPase activity was measured as described (14Panagiotidis C.H. Reyes M. Sievertsen A. Boos W. Shuman H.A. J. Biol. Chem. 1993; 268: 23685-23696Abstract Full Text PDF PubMed Google Scholar, 36Reyes M. Treptow N.A. Shuman H.A. J. Bacteriol. 1986; 165: 918-922Crossref PubMed Google Scholar). Protein concentration was determined using the BCA protein assay reagent kit from Pierce. The plasmids carrying mutations in malF resulting in amber stop codons in place of amino acids Glu39, Tyr55, Glu130, Lys275, and Tyr317 were constructed by site-directed in vitro mutagenesis (37Kunkel T.A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 488-492Crossref PubMed Scopus (4880) Google Scholar). The following oligomers were used in this experiment: GM1, 5′-GGCGAACAGGTACTACCCTTGTGCG-3′, Glu39 to amber stop; GM2, 5′-CGATTGGCGAAAATCTACAGCCCCG-3′, Tyr55 to amber stop; GM3, 5′-CGCCAGTTGCCACTAATCGCCCGC-3′, Glu130 to amber stop; GM4, 5′-GGCGAGGAACGGCTACTGAATGCC-3′, Lys275 to amber stop; GM5, 5′-GCAGGACGCTAGACCGGCTTTGC-3′, Tyr317 to amber stop. The underlined bases are those that are changed in the mutants. Single-stranded plasmid DNA was made from a VCS-M13 phage lysate of thedut ung strain CJ236 carrying the pLH9 (wild-typemalF and malG) plasmid. The oligomers were phosphorylated and annealed to the template as described (38Geisselsoder J. Witney P. Yuckenburg P. BioTechniques. 1987; 5: 786-791Google Scholar). Double-stranded pDNA was then synthesized in vitro by Klenow fragment and circularized by T4 DNA ligase. An aliquot of the reaction mixtures was used to transform DH5α. Plasmid DNA was purified from individual transformants and analyzed by enzymatic digestion, and the nucleotide sequence of the region of interest was determined to verify the presence of the amber mutations. Gel electrophoresis was carried out by the method of Laemmli (39Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205516) Google Scholar). Samples were diluted in sample buffer containing 5% (v/v) 2-mercaptoethanol and heated in a boiling water bath for 5 min. Electrophoresis was carried out in 12% polyacrylamide gels. Protein was visualized by staining the gels with 0.2% Coomassie Brilliant Blue R-250 in methanol-acetic acid-water (5:1:5) and destaining in 7.5% acetic acid, 5% methanol. We examined the ability of Triton X-100 to solubilize the MalFGK proteins as described previously (14Panagiotidis C.H. Reyes M. Sievertsen A. Boos W. Shuman H.A. J. Biol. Chem. 1993; 268: 23685-23696Abstract Full Text PDF PubMed Google Scholar) with some alterations. 250 μg of protein from the inside-out membrane vesicles was incubated in 1.0 ml of 50 mmTris-HCl, pH 7.5, 1 mm dithiothreitol, 5 mmMgCl containing 2% (v/v) Triton X-100 for ½ h. The samples were centrifuged at 14,000 × g in a microcentrifuge for 20 min. The supernatant was carefully removed, and the pellet was resuspended in 50 μl of 100 mm Tris-HCl; 5 μl of this sample was loaded on gels for Coomassie staining and 15 μl for Western blots after boiling for 4 min along with 3 μl of 50% glycerol and 10 μl of sample buffer. The supernatant was added to 2.0 ml of cold ethanol and placed on dry ice for 1 h. The samples were centrifuged at 12,000 × g in a microcentrifuge for 30 min at 4 °C. The ethanol supernatant was removed, and the percipitated proteins were resuspended in 40 μl of 100 mmTris-HCl, pH 7.5. 6 μl of this sample was loaded on gels for Coomassie staining and 17 μl for Western blots after boiling for 4 min along with 3 μl of 50% glycerol and 10 μl of sample buffer. Proteins were transferred from SDS-polyacrylamide gels to sheets of nitrocellulose (BAS 0.45-μm pore size, Schleicher & Schuell) by electroblotting (20 V, overnight) as described elsewhere (40Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44643) Google Scholar). The nitrocellulose sheets were blocked with 5% (w/v) nonfat powdered milk in TBST for 1 h and then incubated with the appropriate dilution of anti-MalK or anti-MalF rabbit antibody. After extensive washing with TBST, goat anti-rabbit IgG coupled to peroxidase was added for 1 h. The resulting blot was then developed using the ECL-Western blotting kit from Amersham Life Science. The indicator strains used to detect altered substrate specificity mutants of the maltose transport system that transport lactose share some common attributes. They all have a deletion of the chromosomal lactose operon but carry the lacZ gene on a plasmid or F′-episome. Thus, these strains can metabolize intracellular lactose but are incapable of transporting it into the cell. The strains also carry themalTp1Tp7 allele (41Chapon C. EMBO J. 1982; 1: 369-374Crossref PubMed Scopus (52) Google Scholar) ensuring constitutive malgene expression in all growth media. Plasmids carrying either wild-type malF and malGor MBP-independent alleles of these genes under the control of themalB promoter on the pBR322 replicon were mutagenized in themutD5 mutator strain KD1087 (32Dengen D.A. Cox E.C. J. Bacteriol. 1974; 117: 477-487Crossref PubMed Google Scholar). The lactose indicator strains were transformed with the mutagenized plasmids, and mutants were selected for the ability to utilize lactose as a sole carbon source on minimal lactose plates. To confirm that the Lac+phenotype is linked to the plasmid, prospective mutant plasmids were retransformed into the lactose indicator strains and evaluated on minimal lactose plates. Although many plasmid-linked mutants were isolated by this technique, only one mutant displayed a strong Lac+ phenotype. This mutant allele, malF540, is carried on the plasmid pLac2. The pLac2 plasmid was derived from the mutagenized pLH5 parent plasmid (42Hor L.I. Genetic Studies on the Mechanism of Periplasmic Binding Protein-dependent Transport.Ph.D. dissertation. Columbia University, 1991Google Scholar) that carries the MBP-independent allele malF502 (8Treptow N.A. Shuman H.A. J. Bacteriol. 1985; 163: 654-660Crossref PubMed Google Scholar). It was isolated as an allele that is dominant to wild-type malF andmalG because the indicator strain used (GM1305) had all of the chromosomal maltose transport genes intact (TableII).Table IILactose phenotype in various indicator strainsPlasmidPlasmid descriptionLactose phenotype2-aLactose phenotype was scored as growth on M63 minimal plates containing 1% lactose, ampicillin, kanamycin, chloramphenicol, and 0.2% arginine after 2 days at 37 °C.GM13052-bChromosome: wild-type malF,G,K; plasmid:malK + .GM11912-cChromosome: ΔmalB101; plasmid:malK + .GM 13682-dChromosome: ΔmalF; plasmid:malK + .GM 14082-eChromosome: malGamV67; plasmid:malK + .GM13612-fChromosome: wild-type malF,G,K; plasmid:malK +, malG + .GM13692-gChromosome: ΔmalF; plasmid:malK +, malG + .GM13802-hChromosome: ΔmalF.pBR322Vector−−−−−−−pTE18lacY+++++++++++++++++++++pLH9Wild-typemalF,G−−−−−−−pLH5malF502−−−−−−−pLac2malF540,malG+++2-iPapillated and small colony morphology. Growth was found mostly around stab points.++++++++++pLH9SMWild-typemalF′,G−−ND2-jND, not determined.NDNDNDNDpLac2SMmalF540′,malG+++2-iPapillated and small colony morphology. Growth was found mostly around stab points.++++++++++pLac2FmalF540′,malG+−+−++++++2-a Lactose phenotype was scored as growth on M63 minimal plates containing 1% lactose, ampicillin, kanamycin, chloramphenicol, and 0.2% arginine after 2 days at 37 °C.2-b Chromosome: wild-type malF,G,K; plasmid:malK + .2-c Chromosome: ΔmalB101; plasmid:malK + .2-d Chromosome: ΔmalF; plasmid:malK + .2-e Chromosome: malGamV67; plasmid:malK + .2-f Chromosome: wild-type malF,G,K; plasmid:malK +, malG + .2-g Chromosome: ΔmalF; plasmid:malK +, malG + .2-h Chromosome: ΔmalF.2-i Papillated and small colony morphology. Growth was found mostly around stab points.2-j ND, not determined. Open table in a new tab The mutation responsible for the phenotype of the MalF540 mutant was mapped by performing a series of DNA fragment exchanges between the pLac2 plasmid, which carries the malF540 allele, and the pLH5 parent plasmid (Fig. 1). These exchanges suggest that the mutation is located proximal to the SacI site on the pLac2 plasmid, somewhere at the very beginning ofmalF. Not shown in Fig. 1 is the second restriction site used in these experiments, a PstI site located in the ampicillin resistance gene. The PstI-SacI DNA fragment was also used to perform exchanges between the pLac2 plasmid and the pLH9 plasmid (Fig.1). pLH9 carries the wild-type malF and malGgenes. A fragment containing the region" @default.
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• W2007422151 title "Truncation of MalF Results in Lactose Transport via the Maltose Transport System of Escherichia coli" @default.
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