Structural and Functional Characterization of a Novel Broad-Spectrum Oligosaccharide Uptake Channel OmpV25 in Vibrio campbellii.

Vibrio campbellii (formerly Vibrio harveyi) is a marine pathogen that causes vibriosis, a major disease affecting global aquaculture. This bacterium inhabits chitin-rich environments and uses chito-oligosaccharides as a main nutrient source. Here, we identify the novel outer membrane protein OmpV25 as a porin mediating broad-spectrum oligosaccharide uptake in V. campbellii. OmpV25 was heterologously expressed in Escherichia coli BL21 (DE3) Omp8, purified by nickel affinity and size exclusion chromatography, and crystallized. The resulting 2.70 Å crystal structure revealed the 25-kDa OmpV25 to be a monomeric β-barrel composed of 12 β-strands connected by six extracellular loops and five periplasmic turns. Single-channel electrophysiology demonstrated that OmpV25 forms stable monomeric pores in artificial lipid bilayers characterized by a conductance of 0.19 ± 0.003 nS in 1 M KCl. The channel facilitated binding of mono-, di-, tri- and tetragalacturonic acids, as well as chitotetraose and maltotetraose. Fluorescence spectroscopy revealed moderate-affinity binding to tetragalacturonic acid (Kd = 3.04 ± 0.42 μM), chitotetraose (Kd = 6.79 ± 0.27 μM), and maltotetraose (Kd = 5.21 ± 0.78 μM). A liposome swelling assay confirmed that OmpV25 enables translocation of a broad range of oligosaccharides beyond oligogalacturonic acids. These findings define the physiological role of OmpV25 as a broad-specificity sugar channel that may support environmental resilience of Vibrio species in nutrient-variable marine habitats.