Molecular Uptake of Chitooligosaccharides through Chitoporin from the Marine Bacterium Vibrio harveyi

Abstract

BackgroundChitin is the most abundant biopolymer in marine ecosystems. However, there is no accumulation of chitin in the ocean-floor sediments, since marine bacteria Vibrios are mainly responsible for a rapid turnover of chitin biomaterials. The catabolic pathway of chitin by Vibrios is a multi-step process that involves chitin attachment and degradation, followed by chitooligosaccharide uptake across the bacterial membranes, and catabolism of the transport products to fructose-6-phosphate, acetate and NH3.Principal FindingsThis study reports the isolation of the gene corresponding to an outer membrane chitoporin from the genome of Vibrio harveyi. This porin, expressed in E. coli, (so called VhChiP) was found to be a SDS-resistant, heat-sensitive trimer. Immunoblotting using anti-ChiP polyclonal antibody confirmed the expression of the recombinant ChiP, as well as endogenous expression of the native protein in the V. harveyi cells. The specific function of VhChiP was investigated using planar lipid membrane reconstitution technique. VhChiP nicely inserted into artificial membranes and formed stable, trimeric channels with average single conductance of 1.8±0.13 nS. Single channel recordings at microsecond-time resolution resolved translocation of chitooligosaccharides, with the greatest rate being observed for chitohexaose. Liposome swelling assays showed no permeation of other oligosaccharides, including maltose, sucrose, maltopentaose, maltohexaose and raffinose, indicating that VhChiP is a highly-specific channel for chitooligosaccharides.Conclusion/SignificanceWe provide the first evidence that chitoporin from V. harveyi is a chitooligosaccharide specific channel. The results obtained from this study help to establish the fundamental role of VhChiP in the chitin catabolic cascade as the molecular gateway that Vibrios employ for chitooligosaccharide uptake for energy production.

Chitoporin from Vibrio harveyi, a Channel with Exceptional Sugar Specificity

“…To resolve the uncertainty surrounding the function of chitoporin, we previously performed liposome swelling assays to demonstrate possible translocation of chitohexaose, but not other non-chitooligosaccharides, through VhChiP (11). To quantify translocation, we employed the BLM reconstitution technique based on channel occlusion for ion current.…”

“…However, the specific function of chitoporin as a sugar-specific channel had never been clearly elucidated. We recently employed black lipid membrane (BLM) 4 reconstitution and liposome swelling assays to demonstrate that chitoporin from V. harveyi (VhChiP) is a pore-forming channel that performs highly specific translocation of chitooligosaccharides (11). In the present study, we focused on the kinetic evaluation of single chitooligosaccharide translocation through a single VhChiP.…”

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

“…In contrast to the parental strain, a mutant strain lacking VfChiP did not grow on GlcNAc 3 , implying that VfChiP was selective for chitooligosaccharides (6). We recently identified and characterized the chitin uptake channel (so-called VhChiP) from the bioluminescent marine bacterium Vibrio harveyi (7,8). VhChiP is a trimeric OmpC-like porin located in the outer membrane and responsible for the molecular uptake of chitin breakdown products that are generated by the action of secreted chitinases (4, 9 -11).…”

Chitoporin from the Marine Bacterium Vibrio harveyi

“…We recently identified chitoporin from V. harveyi (known as VhChiP) as a pore-forming channel that performs highly specific translocation of chitooligosaccharides (9,10). Single channel recordings using a black lipid membrane (BLM) 4 reconstitution technique showed that VhChiP inserted into the artificial bilayer membranes and formed a trimeric channel that remained steadily open under applied potentials of up to Ϯ150 mV.…”