1 Identification and Functional Characterization of a Novel OprD-Like Chitin Uptake Channel in Non-Chitonolytic Bacteria H. Sasimali M. Soysa 1 and Wipa Suginta 1,2 * 1 Biochemistry-Electrochemistry Research Unit and School of Chemistry, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima, Thailand 2 Center of Excellence in Advanced Functional Materials, Suranaree University of Technology, Nakhon Ratchasima, Thailand Running title: Identification of chitin-uptake channel in Escherichia coli Correspondence should be addressed to: Wipa Suginta, Suranaree University of Technology, Nakhon Ratchasima, Thailand 30000, E-mail: wipa@sut.ac.th Funding: 1. This work was financially supported by Suranaree University of Technology and the Office of the Higher Education Commission under the NRU project of Thailand. 2. HSMS was funded by Suranaree University of Technology through a SUT-OGRG grant. 3. WS was funded by the Thailand Research Fund and Suranaree University of Technology through a Basic Research Grant (Grant no. BRG578001) and an SUT grant (SUT1-102-57-36-18). Keywords: Black lipid membrane reconstitution, chitin uptake, chitoporin, Escherichia coli, marine Vibrios, sugar-specific channel. ABSTRACT Chitoporin from the chitinolytic marine Vibrios 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 mol. wt. 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 nS 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 about 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. Escherichia coli is a Gram-negative, heterotrophic bacterium that lives in open environments, such as soil, manure and water, but the persistence of E. coli populations depends upon the availability of carbon substrates in each natural environment. E. coli usually grows on glucose-enriched nutrients such as starch, cellulose and hemicellulose (1), but not on chitin polysaccharides, since it intrinsically lacks competent chitin-utilization machinery (2,3). The chitin-degradation http://www.jbc.org/cgi/doi/10.1074/jbc.M116.728881 The latest version is at JBC Papers in Press. Published on May 12, 2016 as Manuscript M116.728881 Copyright 2016 by The American Society for Biochemistry and Molecular Biology, Inc. by guest on May 13, 2016 http://www.jbc.org/ Downloaded from