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Journal of Bacteriology, September 1998, p. 4821-4827, Vol. 180, No. 18
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Topological Analysis of DcuA, an Anaerobic C4-Dicarboxylate Transporter of Escherichia coli

Paul Golby,1 David J. Kelly,2 John R. Guest,2 and Simon C. Andrews1,*

School of Animal and Microbial Sciences, University of Reading, Reading RG6 6AJ,1 and Department of Molecular Biology and Biotechnology, The Krebs Institute for Biomolecular Research, University of Sheffield, Sheffield SI0 2TN,2 United Kingdom

Received 30 April 1998/Accepted 6 July 1998

Escherichia coli possesses three independent anaerobic C4-dicarboxylate transport systems encoded by the dcuA, dcuB, and dcuC genes. The dcuA and dcuB genes encode related integral inner-membrane proteins, DcuA and DcuB (433 and 446 amino acid residues), which have 36% amino acid sequence identity. A previous amino acid sequence-based analysis predicted that DcuA and DcuB contain either 12 or 14 transmembrane helices, with the N and C termini located in the cytoplasm or periplasm (S. Six, S. C. Andrews, G. Unden, and J. R. Guest, J. Bacteriol. 176:6470-6478, 1994). These predictions were tested by constructing and analyzing 66 DcuA-BlaM fusions in which C terminally truncated forms of DcuA are fused to a beta -lactamase protein lacking the N-terminal signal peptide. The resulting topological model differs from those previously predicted. It has just 10 transmembrane helices and a central, 80-residue cytoplasmic loop between helices 5 and 6. The N and C termini are located in the periplasm and the predicted orientation is consistent with the "positive-inside rule." Two highly hydrophobic segments are not membrane spanning: one is in the cytoplasmic loop; the other is in the C-terminal periplasmic region. The topological model obtained for DcuA can be applied to DcuA homologues in other bacteria as well as to DcuB. Overproduction of DcuA to 15% of inner-membrane protein was obtained with the lacUV5-promoter-based plasmid, pYZ4.

* Corresponding author. Mailing address: School of Animal and Microbial Sciences, University of Reading, Whiteknights, P.O. Box 228, Reading RG6 6AJ, United Kingdom. Phone: 44 118-987-5123, ext. 7045. Fax: 44 118-931-0180. E-mail: S.C.Andrews{at}reading.ac.uk.

Journal of Bacteriology, September 1998, p. 4821-4827, Vol. 180, No. 18
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:

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