Abstract
The function of CymA, 1 of the 10 gene products involved in cyclodextrin uptake and metabolism by Klebsiella oxytoca, was characterized. CymA is essential for growth on cyclodextrins, but it can also complement the deficiency of a lamB (maltoporin) mutant of Escherichia coli for growth on linear maltodextrins, indicating that both cyclic and linear oligosaccharides are accepted as substrates. CymA was overproduced in E. coli and purified to apparent homogeneity. CymA is a component of the outer membrane, is processed from a signal peptide-containing precursor, and possesses a high content of antiparallel beta-sheet. Incorporation of CymA into lipid bilayers and conductance measurements revealed that it forms ion-permeable channels, which exhibit a substantial current noise. CymA-induced membrane conductance decreased considerably upon addition of alpha-cyclodextrin. Titration experiments allowed the calculation of a half-saturation constant, K(S), of 28 microM for its binding to CymA. CymA assembled in vitro to two-dimensionally crystalline tubular membranes, which, on electron microscopy, are characterized by a p1-related two-sided plane group. The crystallographic unit cell contains four monomeric CymA molecules showing a central pore. The lattice parameters are a = 16.1 nm, b = 3.8 nm, gamma = 93 degrees. CymA does not form trimeric complexes in lipid membranes and shows no tendency to trimerize in solution. CymA thus is an atypical porin with novel properties specialized to transfer cyclodextrins across the outer membrane.
Highlights
The function of CymA, 1 of the 10 gene products involved in cyclodextrin uptake and metabolism by Klebsiella oxytoca, was characterized
CymA is essential for growth on cyclodextrins, but it can complement the deficiency of a lamB mutant of Escherichia coli for growth on linear maltodextrins, indicating that both cyclic and linear oligosaccharides are accepted as substrates
We show that ␣-CDs cross the outer membrane of K. oxytoca and of recombinant Escherichia coli cells via a specific porin that has been identified as the product of the cymA gene
Summary
Media and growth conditions were as described previously [3, 4]. Construction of Plasmid pCYMA2—Plasmid pCYMA2 is a pSU2719 derivative [12] which carries a K. oxytoca DNA fragment starting 216 base pairs 5Ј of cymA and extending to base pair 175 of cymB. PSU2719 was restricted with SacI and XbaI, dephosphorylated, and ligated with the insert, which was prepared by analogous restriction of plasmid pCYMA [3]. Cloning of the lamB Gene of K. oxytoca M5a1—The lamB gene of K. oxytoca was cloned via PCR using phosphorylated oligonucleotides derived from the nucleotide sequence of the homologous gene from Klebsiella pneumoniae [13].
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