Functional analysis of an unusual porin-like channel that imports chitin for alternative carbon metabolism in Escherichia coli

Abstract

Escherichia coli have the genetic potential to use chitin as a carbon source in the absence of glucose, importing it via the chitin-uptake channel EcChiP for processing by the glucosamine catabolic pathway. The chip gene is usually not expressed when E. coli are grown on glucose-enriched nutrients, providing a general regulatory mechanism for the pathway. EcChiP is unusual in that it is homologous to porins and monomeric instead of trimeric, the typical form of sugar-specific channels, making it unclear how this channel operates. We recently reported that EcChiP could form a stable channel in lipid membranes and that the channel is specific for chitooligosaccharides. This report describes the biophysical nature of sugar-channel interactions and the kinetics of sugar association and dissociation. Titrating EcChiP with chitohexaose resulted in protein fluorescence enhancement in a concentration-dependent manner, yielding a binding constant of 2.9*105 M-1, consistent with the value of 2.5*105 M-1 obtained from isothermal titration microcalorimetry. Analysis of the integrated heat change suggested that the binding process was endothermic and driven by entropy. Single-channel recordings confirmed the voltage dependence of the penetration of chitohexaose molecules into and their release from EcChiP. Once inside the pore, the sugar release rate (koff) from the affinity site increased with elevated voltage, regardless of the side of sugar addition. Our findings revealed distinct thermodynamic and kinetic features of the activity of sugar-specific EcChiP and advance our knowledge of the physiological possibility of chitin utilization by non-chitinolytic bacteria.