Skip to main content

The membrane topology of the Rhizobium meliloti C4-dicarboxylate permease (DctA) as derived from protein fusions with Escherichia coli K12 alkaline phosphatase (PhoA) and β-galactosidase (LacZ)

Molecular and General Genetics MGG volume 241, pages 106–114 (1993)Cite this article

Abstract

The Rhizobium meliloti dctA gene encodes the C4-dicarboxylate permease which mediates uptake of C4-dicarboxylates, both in free-living and symbiotic cells. Based on the hydrophobicity of the DctA protein, 12 putative membrane spanning regions were predicted. The membrane topology was further analysed by isolating in vivo fusions of DctA to Escherichia coli alkaline phosphatase (PhoA) and E. coli β-galactosidase (LacZ). Of 10 different fusions 7 indicated a periplasmic and 3 a cytoplasmic location of the corresponding region of the DctA protein. From these data a two-dimensional model of DctA was constructed which comprised twelve transmembrane α-helices with the amino-terminus and the carboxy-terminus located in the cytoplasm. In addition, four conserved amino acid motifs present in many eukaryotic and prokaryotic transport proteins were observed.

This is a preview of subscription content, access via your institution.

Access options

Buy single article

Instant access to the full article PDF.

43,34 €

Price includes VAT (Finland)
Tax calculation will be finalised during checkout.

References

  • Arnold W, Pühler A (1988) A family of high-copy-number plasmid vectors with single end-label sites for rapid nucleotide sequencing. Gene 70:171–179

    Google Scholar 

  • Atkins JF, Weiss RB, Gesteland RF (1990) Ribosome gymnastics — degree of difficulty 9.5, style 10.0. Cell 62:413–423

    Google Scholar 

  • Bolton E, Higgisson B, Harrington A, O'Gara F (1986) Dicarboxylic acid transport in Rhizobium meliloti: isolation of mutants and cloning of dicarboxylic acid transport genes. Arch Microbiol 144:142–146

    Google Scholar 

  • Botfield MC, Naguchi K, Tsuchiya T, Wilson TH (1992) Membrane topology of the melibiose carrier of Escherichia coli. J Biol Chem 267:1818–1822

    Google Scholar 

  • Boyd D, Beckwith J (1990) The role of charged amino acids in the localization of secreted and membrane proteins. Cell 62:1031–1033

    Google Scholar 

  • Brickman E, Beckwith J (1975) Analysis of the regulation of Escherichia coli alkaline phosphatase synthesis using deletions and ϕ80 transducing phages. J Mol Biol 96:307–316

    Google Scholar 

  • Chen EY, Seeburg PH (1986) Supercoil sequencing: a fast and simple method for sequencing plasmid DNA. DNA 4:165–170

    Google Scholar 

  • Chou PY, Fasman GD (1978) Prediction of the secondary structure of proteins from their amino acid sequence. Adv Enzymol 47:45–148

    Google Scholar 

  • Eisenberg D, Schwarz E, Komaromy M, Wall R (1984) Analysis of membrane and surface protein sequences with the hydrophobic moment plot. J Mot Biol 179:125–142

    Google Scholar 

  • El-Din AKYG (1992) A Succinate transport mutant of Bradyrhizobium japonicum forms ineffective nodules on soybeans. Can J Microbiol 38:230–234

    Google Scholar 

  • Engelke T, Jagadish MN, Pühler A (1987) Biochemical and genetical analysis of Rhizobium meliloti mutants defective in C4-dicarboxylate transport. J Gen Microbiol 133:3019–3029

    Google Scholar 

  • Engelke T, Jording D, Kapp D, Pühler A (1989) Identification and sequence analysis of the Rhizobium meliloti dctA gene encoding the C4-dicarboxylate carrier. J Bacteriol 171:5551–5560

    Google Scholar 

  • Finan TM, Wood JM, Jordan DC (1981) Succinate transport in Rhizobium leguminosarum. J Bacteriol 148:193–202

    Google Scholar 

  • Finan TM, Wood JM, Jordan DC (1983) Symbiotic properties of C4 dicarboxylic acid transport mutants of Rhizobium leguminosarum. J Bacteriol 154:1403–1413

    Google Scholar 

  • Foster DL, Boublik M, Kaback HR (1983) Structure of the lac carrier protein of Escherichia coli. J Biol Chem 258:31–34

    Google Scholar 

  • Gött P, Boos W (1988) The transmembrane topology of the sn-glycerol-3-phosphate permease of Escherichia coli analysed by phoA and IacZ protein fusions. Mol Microbiol 2:655–663

    Google Scholar 

  • Henderson PJF (1990) The homologous glucose transport proteins of prokaryotes and eukaryotes. Res Microbiol 141:316–327

    Google Scholar 

  • Henderson PJF, Maiden MCI (1990) Homologous sugar transport proteins in Escherichia coli, and their relatives in both prokaryotes and eukaryotes. Phil Trans R Soc London B 326:391–410

    Google Scholar 

  • Herrero M, de Lorenzo V, Neilands JB (1988) Nucleotide sequence of the iucD gene of the pCo1V-K30 aerobactin operon and topology of its product studied with phoA and lacZ gene fusions. J Bacteriol 170:56–64

    Google Scholar 

  • Holmes DS, Quigley M (1981) A rapid boiling method for the preparation of bacterial plasmid. Anal Biochem 114:193–197

    Google Scholar 

  • Jagadish MN, Szalay AA (1984) Directed transposon Tn5 mutagenesis and complementation in slow-growing, broad host range cowpea Rhizobium. Mol Gen Genet 196:290–300

    Google Scholar 

  • Jennings ML (1989) Topography of membrane proteins. Annu Rev Biochem 58:999–1027

    Google Scholar 

  • Jiang J, Gu B, Albright LM, Nixon BT (1989) Conservation between coding and regulatory elements of Rhizobium meliloti and Rhizobium leguminosarum dct genes. J Bacteriol 171:5244–5253

    Google Scholar 

  • Klipp W, Masepohl B, Pühler A (1988) Identification and mapping of nitrogen fixation genes of Rhodobacter capsulatus: duplication of a nifA-nifB region. J Bacteriol 170:693–699

    Google Scholar 

  • Kyte J, Doolittle RF (1982) A simple method for displaying the hydropathic character of a protein. J Mol Biol 157:105–132

    Google Scholar 

  • Lee C, Li P, Inouye H, Brickman ER, Beckwith J (1989) Genetic studies on the inability of β-galactosidase to be translocated across the Escherichia coli cytoplasmic membrane. J Bacteriol 171:4609–4616

    Google Scholar 

  • Lewis MJ, Chang JA, Simoni RD (1990) A topological analysis of subunit a from Escherichia coli F1F0-ATP synthase predicts eight transmembrane segments. J Biol Chem 265:10541–10550

    Google Scholar 

  • Lloyd AD, Kadner RJ (1990) Topology of the Escherichia coli uhpT sugar-phosphate transporter analyzed by using TnphoA fusions. J Bacteriol 172:1688–1693

    Google Scholar 

  • Maloney PC (1990) A consensus structure for membrane transport. Res Microbiol 141:374–396

    Google Scholar 

  • Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York

    Google Scholar 

  • Manoil C (1990) Analysis of protein localization by use of gene fusions with complementary properties. J Bacteriol 172:1035–1042

    Google Scholar 

  • Manoil C, Beckwith J (1985) TnphoA: a transposon probe for protein export signals. Proc Natl Acad Sci USA 82:8129–8133

    Google Scholar 

  • Manoil C, Mekalanos JJ, Beckwith J (1990) Alkaline phosphatase fusions: sensors of subcellular location. J Bacteriol 172:515–518

    Google Scholar 

  • McRae DG, Miller RW, Berndt WB, Joy K (1989) Transport of C4 dicarboxylates and amino acids by Rhizobium meliloti bacteroids. Mol Plant-Microbe Interact 2:273–278

    Google Scholar 

  • Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York

    Google Scholar 

  • Pourcher T, Bassilana M, Sarkar HK, Kaback HR, Leblanc G (1990) The melibiose/Na+ symporter of Escherichia coli: kinetic and molecular properties. Phil Trans R Soc Lond B 326:411–423

    Google Scholar 

  • Rao JKM, Argos P (1986) A conformational preference parameter to predict helices in integral membrane proteins. Biochem Biophys Acta 869:197–214

    Google Scholar 

  • Ronson CW, Lyttleton P, Robertson JG (1981) C4-dicarboxylate transport mutants of Rhizobium trifolii form ineffective nodules on Trifolium repens. Proc Natl Acad Sci USA 78:4284–4288

    Google Scholar 

  • Ronson CW, Astwood PM, Nixon BT, Ausubel FM (1987) Deduced products of C4-dicarboxylate transport regulatory genes of Rhizobium leguminosarum are homologous to nitrogen regulatory gene products. Nucleic Acids Res 15:7921–7934

    Google Scholar 

  • Saier MH Jr (1989) Evolution of permease diversity and energycoupling mechanisms: an introduction. Res Microbiol 141:281–286

    Google Scholar 

  • San Francisco MID, Jacobson GR (1985) Uptake of Succinate and malate in cultured cells and bacteroids of two slow-growing species of Rhizobium. J Gen Microbiol 131:765–773

    Google Scholar 

  • Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467

    Google Scholar 

  • Silhavy TJ, Beckwith JR (1985) Uses of lac fusions for the study of biological problems. Microbiol Rev 49:398–418

    Google Scholar 

  • Simon R, Priefer U, Puhler A (1983) A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram-negative bacteria. BioTechnology 1:784–791

    Google Scholar 

  • Simon R, O'Connell M, Labes M, Puhler A (1986) Plasmid vectors for the genetic analysis and manipulation of Rhizobia and other Gram-negative bacteria. Methods Enzymol 118:640–659

    Google Scholar 

  • Staden R (1986) The current status and portability of our sequence handling software. Nucleic Acids Res 14:217–231

    Google Scholar 

  • Tolner B, Poolman B, Wallace B, Konings WN (1992) Revised nucleotide sequence of the gltP gene, which encodes the protonglutamate-aspartate transport protein of Escherichia coli K-12. J Bacteriol 174:2391–2393

    Google Scholar 

  • van Slooten JC, Bhuvanasvari TV, Bardin S, Stanley J (1992) Two C4-dicarboxylate transport systems in Rhizobium sp. NGR234: rhizobial dicarboxylate transport is essential for nitrogen fixation in tropical legume symbioses. Mol Plant-Microbe Interact 5:179–186

    Google Scholar 

  • Vieira J, Messing J (1982) The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19:159–268

    Google Scholar 

  • von Heijne G (1992) Membrane protein structure prediction. Hydrophobicity analysis and the positive-inside rule. J Mol Biol 225:487–494

    Google Scholar 

  • Wang Y-P, Birkenhead K, Boesten B, Martian S, O'Gara F (1989) Genetic analysis and regulation of the Rhizobium meliloti genes controlling C4-dicarboxylic acid transport. Gene 85:135–144

    Google Scholar 

  • Watson RJ (1990) Analysis of the C4-dicarboxylate transport genes of Rhizobium meliloti: nucleotide sequence and deduced products of dctA, dctB, and dctD. Mol Plant-Microbe Interact 3:174–181

    Google Scholar 

  • Watson RJ, Chan Y-K, Wheatcroft R, Yang A-F, Han S (1988) Rhizobium meliloti genes required for C4-dicarboxylate transport and symbiotic nitrogen fixation are located on a megaplasmid. J Bacteriol 170:927–934

    Google Scholar 

  • Wu J, Tisa LS, Rosen BP (1992) Membrane topology of the ArsB protein, the membrane subunit of an anion-translocating ATPase. J Biol Chem 267:12570–12576

    Google Scholar 

  • Yarosh OK, Charles TC, Finan TM (1989) Analysis of C4-dicarboxylate transport genes in Rhizobium meliloti. Mol Microbiol 3:813–823

    Google Scholar 

Download references

Author information

Affiliations

  1. Lehrstuhl fur Genetik, Fakultät für Biologie, Universität Bielefeld, D-33501, Bielefeld, Germany

    Doris Jording & Alfred Pühler

Authors
  1. Doris Jording
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Alfred Pühler
    View author publications

    You can also search for this author in PubMed Google Scholar

Additional information

Communicated by J. Lengeler

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Jording, D., Pühler, A. The membrane topology of the Rhizobium meliloti C4-dicarboxylate permease (DctA) as derived from protein fusions with Escherichia coli K12 alkaline phosphatase (PhoA) and β-galactosidase (LacZ). Molec. Gen. Genet. 241, 106–114 (1993). https://doi.org/10.1007/BF00280207

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00280207

Key words

  • Rhizobium meliloti
  • C4-dicarboxylate transport system
  • Protein topology
  • Monitor protein fusions
  • Conserved motifs