The Chitin Catabolic Cascade in the Marine Bacterium Vibrio Cholerae: Characterization of a Unique Chitin Oligosaccharide Deacetylase

Department of Biology, The Johns Hopkins University, Baltimore, MD 21218, USA.
Glycobiology (Impact Factor: 3.15). 01/2008; 17(12):1377-87. DOI: 10.1093/glycob/cwm096
Source: PubMed


Chitin, one of the most abundant organic substances in nature, is consumed by marine bacteria, such as Vibrio cholerae, via a multitude of tightly regulated genes (Li and Roseman 2004, Proc Natl Acad Sci USA. 101:627–631). One such gene, cod, is reported here. It encodes a chitin oligosaccharide deacetylase (COD), when cells are induced by chitobiose, (GlcNH2)2, or crude crab shells. COD was molecularly cloned (COD-6His), overproduced, and purified to apparent homogeneity. COD is
secreted at all stages of growth by induced V. cholerae. The gene sequence predicts a 26 N-terminal amino acid signal peptide not found in the isolated protein. COD is very active
with chitin oligosaccharides, is virtually inactive with GlcNAc, and slightly active with colloidal ([3H]-N-acetyl)-chitin. The oligosaccharides are converted almost quantitatively to products lacking one acetyl group. The latter
were characterized by mass spectrometry (ESI-MS), and treatment with nitrous acid. COD catalyzes the following reactions (n = 2–6): (GlcNAc)n→ GlcNAc-GlcNH2-(GlcNAc)n−2 + Ac−. That is, COD hydrolyzes the N-acetyl groups attached to the penultimate GlcNAc residue. The gene bank sequence data show that cod is highly conserved in Vibrios and Photobacteria. One such gene encodes a deacetylase isolated from V. alginolytics (Ohishi et al. 1997, Biosci Biotech Biochem. 61:1113–1117; Ohishi et al. 2000, J Biosci Bioeng. 90:561–563), that is specific for (GlcNAc)2, but inactive with higher oligosaccharides. The COD enzymatic products, GlcNAc-GlcNH2-(GlcNAc)n, closely resemble those obtained by hydrolysis of the chitooligosaccharides with Nod B: GlcNH2-(GlcNAc)3-4. The latter are key intermediates in the biosynthesis of Nod factors, critically important in communications between the
symbiotic nitrogen fixing bacteria and plants. Conceivably, the COD products play equally important roles in cellular communications
that remain to be defined.

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    • "Hence, COD is key enzyme for producing this signal disaccharide. Untill now, CE-4 CODs have been isolated from only following four species of Vibrio besides V. parahaemolyticus KN1699: Vibrio alginolyticus H-8 [4], Vibrio cholerae EI Tor N16961 [5], Vibrio sp. SN184 [6], and Vibrio harveyi ATCC BAA-1116 [7]. "
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    ABSTRACT: The X-ray crystal structure of chitin oligosaccharide deacetylase from Vibrio parahaemolyticus (Vp-COD) was determined at an 1.35 Å resolution. The amino acid sequence and structure of Vp-COD show that the enzyme comprises one polysaccharide deacetylase domain (PDD) and two carbohydrate-binding domains (CBDs). On the basis of a chitin-binding assay with Vp-COD and its CBDs-deleted mutant, it was confirmed that CBDs can adhere to chitin. The catalytic activity of the CBDs-deleted mutant was only mildly depressed compared with that of Vp-COD, indicating that CBDs are unlikely to affect the configuration of the active center residues in active site of PDD. Copyright © 2014. Published by Elsevier B.V.
    Full-text · Article · Dec 2014 · FEBS Letters
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    • "Chitinases are a wide spread group of enzymatic systems that breakdown chitin an abundant insoluble linear polymer of β-1, 4-linked N- Acetyl glucosamine (Lee et al., 2007). Chitinase enzymes are found in many organisms and their properties to be closely related to their biological functions (Li et al., 2007). "
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    ABSTRACT: ARTICLE INFO ABSTRACT An extracellular chitinase producing bacterium Bacillus subtilis TS22 was isolated from shrimp shell waste. It was identified by as gram positive rod shaped bacteria, Indole, Citrate, Oxidase, Catalase, Nitrate positive and Methyl Red, Vogeus Proskauer negative. The carbohydrate fermentation occurs by producing acid but not gas in Glucose, Maltose and Sucrose. The genomic size of Bacillus subtilis TS22 was 870bp confirmed with the help of 16s rRNA primer viz forward primer F5-AGAGTTTGATCMTGGCTCAG-3 | and reverse primer 5 | AAGGAGGTGWTCCARCC-3 | .The obtained gene sequence were submitted and deposited to NCBI under accession number JQ 727436. When plotted for phylogenetic tree showed 98% similarities with Bacillus species. The Bacillus subtilis TS22 produced the extracellular chitinase having 127U/ml of chitinolytic activity. The molecular weight of chitinase was 50KDa in 12% sodium dodecyl sulphate polyacrylamide gel electrophoresis. The Bacillus subtilis TS22 showed antifungal activity against three fungal pathogens such as Phytophthora parasitica, Alternaria solani and Pythium aphanidermatum.
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    • "These data demonstrated that the host upregulates the expression of one chitinase gene and one chitin synthase gene just before dawn. Simultaneously, the resident bacterial symbionts increase the transcription of genes that have been reported to be important for chitin recognition and breakdown in Vibrio cholerae (Meibom et al., 2004; Li et al., 2007). In addition, V. fischeri is genetically and physiologically capable of chitin breakdown and utilization (Ruby et al., 2005; Sugita and Ito, 2006). "
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    ABSTRACT: The light-organ symbiosis of Euprymna scolopes, the Hawaiian bobtail squid, is a useful model for the study of animal-microbe interactions. Recent analyses have demonstrated that chitin breakdown products play a role in communication between E. scolopes and its bacterial symbiont Vibrio fischeri. In this study, we sought to determine the source of chitin in the symbiotic organ. We used a commercially available chitin-binding protein (CBP) conjugated to fluorescein to label the polymeric chitin in host tissues. Confocal microscopy revealed that the only cells in contact with the symbionts that labeled with the probe were the macrophage-like hemocytes, which traffic into the light-organ crypts where the bacteria reside. Labeling of extracted hemocytes by CBP was markedly decreased following treatment with purified chitinase, providing further evidence that the labeled molecule is polymeric chitin. Further, CBP-positive areas co-localized with both a halide peroxidase antibody and Lysotracker, a lysosomal marker, suggesting that the chitin-like biomolecule occurs in the lysosome or acidic vacuoles. Reverse transcriptase polymerase chain reaction (PCR) of hemocytes revealed mRNA coding for a chitin synthase, suggesting that the hemocytes synthesize chitin de novo. Finally, upon surveying blood cells from other invertebrate species, we observed CBP-positive regions in all granular blood cells examined, suggesting that this feature is a shared character among the invertebrates; the vertebrate blood cells that we sampled did not label with CBP. Although the function of the chitin-like material remains undetermined, its presence and subcellular location in invertebrate hemocytes suggests a conserved role for this polysaccharide in the immune system of diverse animals.
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