Single-channel characterization of the chitooligosaccharide transporter chitoporin (SmChiP) from the opportunistic pathogen Serratia marcescens.

Serratia marcescens is an opportunistic pathogen that can utilize chitin as a carbon source, through its ability to produce chitin-degrading enzymes to digest chitin and membrane transporters to transport the degradation products (chitooligosaccharides) into the cells. Further characterization of these proteins is important to understand details of chitin metabolism. Here, we investigate the properties and function of the S. marcescens chitoporin, namely SmChiP, a chitooligosaccharide transporter. We show that SmChiP is a monomeric porin that forms a stable channel in artificial phospholipid membranes, with an average single-channel conductance of 0.5 ± 0.02 nS in 1 M KCl electrolyte. Additionally, we demonstrated that SmChiP allowed the passage of small molecules with a size exclusion limit of <300 Da and exhibited substrate specificity toward chitooligosaccharides, both in membrane and detergent-solubilized forms. We found that SmChiP interacted strongly with chitopentaose (Kd = 23 ± 2.0 µM) and chitohexaose (Kd = 17 ± 0.6 µM) but did not recognize nonchitose oligosaccharides (maltohexaose and cellohexaose). Given that S. marcescens can use chitin as a primary energy source, SmChiP may serve as a target for further development of nutrient-based antimicrobial therapies directed against multidrug antibiotic-resistant S. marcescens infections.

Chitosugar translocation by an unexpressed monomeric protein channel.

The outer membrane protein channel EcChiP, associated with a silent gene in E. coli, is a monomeric chitoporin. In a glucose-deficient environment, E. coli can express the ChiP gene to exploit chitin degradation products. Single-channel small ion current measurements, which reveal the dynamics of single sugar molecules trapped in channel, are used here to study the exotic transport of chitosugars by E. coli. Molecules escape from the channel on multiple timescales. Voltage-dependent trapping rates observed for charged chitosan molecules, as well as model calculations, indicate that the rapid escape processes are those in which the molecule escapes back to the side of the membrane from which it originated. The probability that a sugar molecule is translocated through the membrane is thus estimated from the current data and the dependence of this translocation probability on the length of the chitosugar molecule and the applied voltage analyzed. The described method for obtaining the translocation probability and related molecular translocation current is applicable to other transport channels.

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

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.

Identification and Functional Characterization of a Novel OprD-like Chitin Uptake Channel in Non-chitinolytic Bacteria.

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.