title: Identification and Functional Characterization of a Novel OprD-like Chitin Uptake Channel in Non-chitinolytic Bacteria.
abstract: Chitoporin from the chitinolytic marine Vibrio has been characterized as a trimeric OmpC-like channel responsible for effective chitin uptake. In this study we describe the identification and characterization of a novel OprD-like chitoporin (so-called EcChiP) from Escherichia coli The gene was identified, cloned, and functionally expressed in the Omp-deficient E. coli BL21 (Omp8) Rosetta strain. On size exclusion chromatography, EcChiP had an apparent native molecular mass of 50 kDa, as predicted by amino acid sequencing and mass analysis, confirming that the protein is a monomer. Black lipid membrane reconstitution demonstrated that EcChiP could readily form stable, monomeric channels in artificial phospholipid membranes, with an average single channel conductance of 0.55 ± 0.01 nanosiemens and a slight preference for cations. Single EcChiP channels showed strong specificity, interacting with long chain chitooligosaccharides but not with maltooligosaccharides. Liposome swelling assays indicated the bulk permeation of neutral monosaccharides and showed the size exclusion limit of EcChiP to be ∼200-300 Da for small permeants that pass through by general diffusion while allowing long chain chitooligosaccharides to pass through by a facilitated diffusion process. Taking E. coli as a model, we offer the first evidence that non-chitinolytic bacteria can activate a quiescent ChiP gene to express a functional chitoporin, enabling them to take up chitooligosaccharides for metabolism as an immediate source of energy.
authors.affiliation: From the Biochemistry-Electrochemistry Research Unit and School of Chemistry, Institute of Science and.
authors.forename: H Sasimali M
authors.initials: HS
authors.lastname: Soysa
authors.affiliation: From the Biochemistry-Electrochemistry Research Unit and School of Chemistry, Institute of Science and Center of Excellence in Advanced Functional Materials, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand wipa@sut.ac.th.
authors.forename: Wipa
authors.initials: W
authors.lastname: Suginta
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002686:Chitin / Q000235:genetics / Q000378:metabolism*; D006090:Gram-Negative Bacteria / Q000235:genetics / Q000378:metabolism*; D018272:Porins / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D018272:Porins; D002686:Chitin
keywords: Escherichia coli (E. coli); carbohydrate metabolism; chitin; chitin uptake channel; ion channel; non-chitinolytic bacteria; single channel recordings; sugar transport