Chitin Catabolism in the Marine Bacterium Vibrio furnissii

Abstract

Chitin catabolism by the marine bacterium Vibrio furnissii involves many genes and proteins, including two unique periplasmic hydrolases, a chitodextrinase and a ␤-N-acetylglucosaminidase (Keyhani, N. O., and Roseman, S. (1996) J. Biol. Chem. 271, 33414 -33424 and 33425-33432). A specific chitoporin in the outer membrane may be required for these glycosidases to be accessible to extracellular chitooligosaccharides, (GlcNAc) n , that are produced by chitinases. We report here the identification and molecular cloning of such a porin. An outer membrane protein, OMP (apparent molecular mass 40 kDa) was expressed when V. furnissii was induced by (GlcNAc) n , n ‫؍‬ 2-6, but not by GlcNAc or other sugars. Based on the N-terminal sequence of OMP, oligonucleotides were synthesized and used to clone the gene, chiP. The deduced amino acid sequence of ChiP is similar to several bacterial porins; OMP is a processed form of ChiP. In Escherichia coli, two recombinant proteins were observed, corresponding to processed and unprocessed forms of ChiP. A null mutant of chiP was constructed in V. furnissii. In contrast to the parental strain, the mutant did not grow on (GlcNAc) 3 and transported a nonmetabolizable analogue of (GlcNAc) 2 at a reduced rate. These results imply that ChiP is a specific chitoporin.We have previously reported that the chitin catabolic cascade and signal transduction systems expressed by the marine bacterium Vibrio furnissii comprise a large number of genes and proteins, only some of which have been identified (6 -15). 1Two of these proteins are unique periplasmic glycosidases. One is a chitodextrinase (11), and the other is a ␤-N-acetylglucosaminidase (12). The concerted action of these two enzymes yields GlcNAc and (GlcNAc) 2 from the higher oligosaccharides, which are then taken up by specific cytoplasmic membrane transporters (9, 10, 13).But how do the higher oligosaccharides penetrate the outer membrane/cell wall complex so that they can be hydrolyzed in the periplasm? There is an extensive literature on outer membrane proteins or porins that are thought to mediate this process (16 -18). The nonspecific, constitutive porins of Escherichia coli permit the diffusion of solutes that range up to about 600 Da (19), depending on shape, or roughly the size of a trisaccharide. A few sugar-specific porins, or glycoporins, have been reported. These include, most notably, the LamB protein or phage receptor protein, whose crystal structure has been resolved (20) and which permits the diffusion of malto-oligosaccharides (21,22), and ScrY, involved in sucrose transport (23). The RafY protein is involved in raffinose uptake (24) and may not have a specific binding site for the trisaccharide but functions because it is wider than the constitutive E. coli porins (25).The present report presents evidence for another glycoporin designated chitoporin. The porin is expressed by V. furnissii in its outer membrane and is induced by chitin oligosaccharides but not by GlcNAc nor by other sugars. The structural gene for the po...

The Chitin Disaccharide,N,N′-Diacetylchitobiose, Is Catabolized byEscherichia coli and Is Transported/Phosphorylated by the Phosphoenolpyruvate:Glycose Phosphotransferase System

“…Similarly, both organisms have the same array of non-PTS sugars: Gal, Mal, glycerol, Rib, and Glc-6-P (20). 4 Furthermore, the structural genes of three of the sugar-specific Enzyme II (Glc, GlcNAc, Man) complexes of V. furnissii have been cloned and sequenced and show 30 -67% identity to the corresponding genes of E. coli (22,23). These V. furnissii proteins efficiently substitute for the corresponding E. coli proteins in vivo.…”

The Vibrio cholerae chitin utilization program

“…VC0972, which encodes a protein that is moderately homologous (40% identity, 66% similarity) to a previously characterized chitin-specific porin of V. furnissi (10), was found to be powerfully induced by crab shell-attached bacteria and by chitin oligosaccharides in a ChiS-dependent manner. To determine whether VC0972 also encodes a chitoporin, it was disrupted and the mutant found to grow poorly in a liquid medium containing colloidal chitin.…”

Comparative Genomics and Experimental Characterization of N-Acetylglucosamine Utilization Pathway of Shewanella oneidensis

“…Homologous genes are marked by matching colors. In Vibrionales, chito-oligosaccharides are known to enter the periplasmic space via a specific chitoporin encoded by the (GlcNAc) 2 -induced gene chiP (11,41), a predicted member of the NagC regulon. Based on the genome context analysis, we predicted the involvement of two other types of outer membrane proteins, ChiP-II and ChiP-III, in the uptake of GlcNAc and/or its oligosaccharides.…”