Chitin Oligosaccharides Research Articles

Characterization of a β-N-acetylhexosaminidase with transglycosylation activity from Metarhizium sp. A34.

In applications of chitin, one of the most abundant resources on earth, human milk oligosaccharides with many health functions were synthesized by transglycosylation of β-N-acetylhexosaminidase. Synthesis of new transfer products can be expected by other β-N-acetylhexosaminidases in nature. A total of 38 microorganisms that secrete β-N-acetylhexosaminidases with transglycosylation activity were isolated from a soil screen. Using N,N'-diacetylchitobiose as the substrate, the transfer ratio increased with a decrease in substrate degradation when it was less than 60%. Metarhizium sp. A34 β-N-acetylhexosaminidase had high transglycosylation activity and showed a maximum production of the oligosaccharides against the substrate degradation where (GlcNAc)5 and (GlcNAc)4 were produced in addition to (GlcNAc)3 . The maximum curve was attributed to a sequential reaction of transglycosylation followed by hydrolysis where oligosaccharides are an intermediate product and are hydrolyzed in a second step. The purified β-N-acetylhexosaminidase from Metarhizium sp. A34 had an optimal pH of 5 and was stable from pH 7 to 8. At pH 5, it had an optimal temperature of 40°C and was stable up to 30°C for 30min. This enzyme had high thermostability up to 55°C when bound to the cell wall. The acceptor specificity for the transglycosylation reaction was enhanced for lower molecular weight sugar alcohols in the order of glycerin (C3), erythritol (C4), and xylitol (C5). The transfer product with glycerin was identified as 1-O-β-d-N-acetylglucosaminyl glycerin, which may prove useful as a starting material for new glycolipids in food applications. PRACTICAL APPLICATION: Metarhizium sp. A34 β-N-acetylhexosaminidase produced 1-O-β-d-N-acetylglucosaminyl glycerin through the transglycosylation. Chitin oligosaccharides of the donor are obtained by hydrolysis of chitin. 1-O-β-d-N-Acetylglucosaminyl glycerin may be useful to start material for the synthesis of new glycolipids. High thermostability of this enzyme is useful to prevention of contamination in the transglycosylation reaction.

Enzymatic modification of native chitin and chitin oligosaccharides by an alkaline chitin deacetylase from Microbacterium esteraromaticum MCDA02

In this study, chitin deacetylase from Microbacterium esteraromaticum MCDA02 (MeCDA) was purified by ammonium sulfate precipitation, anion exchange chromatography, and superdex column chromatography. The molecular weight of purified MeCDA was approximately 26 kDa. The optimum pH and temperature of purified MeCDA were 8.0 and 30 °C, respectively. The enzyme activity is enhanced by metal ions K+ and Sr+ and inhibited by Co2+, Cd2+, and EDTA. The degree of deacetylation through enzymatic modification of MeCDA was removed an average of 32.75% of the acetyl groups for ɑ-chitin by acid-base titration. Meanwhile, MeCDA can catalyze the hydrolytic cleavage of the acetamido bond in GlcNAc units within chitin oligomers and polymers. Hence, the MeCDA is a potent chitin decomposer to catalyze chitin and chitin oligosaccharides deacetylation to prepare chitosan and chitosan oligosaccharide. This is a value-added utilization of chitin based biological resources.

Combinatorial pathway engineering of Bacillus subtilis for production of structurally defined and homogeneous chitooligosaccharides

Chitooligosaccharides (COSs) have a widespread range of biological functions and an incredible potential for various pharmaceutical and agricultural applications. Although several physical, chemical, and biological techniques have been reported for COSs production, it is still a challenge to obtain structurally defined COSs with defined polymerization (DP) and acetylation patterns, which hampers the specific characterization and application of COSs. Herein, we achieved the de novo production of structurally defined COSs using combinatorial pathway engineering in Bacillus subtilis. Specifically, the COSs synthase NodC from Azorhizobium caulinodans was overexpressed in B. subtilis, leading to 30 ± 0.86 mg/L of chitin oligosaccharides (CTOSs), the homo-oligomers of N-acetylglucosamine (GlcNAc) with a well-defined DP lower than 6. Then introduction of a GlcNAc synthesis module to promote the supply of the sugar acceptor GlcNAc, reduced CTOSs production, which suggested that the activity of COSs synthase NodC and the supply of sugar donor UDP-GlcNAc may be the limiting steps for CTOSs synthesis. Therefore, 6 exogenous COSs synthase candidates were examined, and the nodCM from Mesorhizobium loti yielded the highest CTOSs titer of 560 ± 16 mg/L. Finally, both the de novo pathway and the salvage pathway of UDP-GlcNAc were engineered to further promote the biosynthesis of CTOSs. The titer of CTOSs in 3-L fed-batch bioreactor reached 4.82 ± 0.11 g/L (85.6% CTOS5, 7.5% CTOS4, 5.3% CTOS3 and 1.6% CTOS2), which was the highest ever reported. This is the first report proving the feasibility of the de novo production of structurally defined CTOSs by synthetic biology, and provides a good starting point for further engineering to achieve the commercial production.

Deoxyfluorination tunes the aggregation of cellulose and chitin oligosaccharides and highlights the role of specific hydroxyl groups in the crystallization process.

Cellulose and chitin are abundant structural polysaccharides exploited by nature in a large number of applications thanks to their crystallinity. Chemical modifications are commonly employed to tune polysaccharide physical and mechanical properties, but generate heterogeneous mixtures. Thus, the effect of such modifications is not well understood at the molecular level. In this work, we examined how deoxyfluorination (site and pattern) impact the solubility and aggregation of well-defined cellulose and chitin oligomers. While deoxyfluorination increased solubility in water and lowered the crystallinity of cellulose oligomers, chitin was much less affected by the modification. The OH/F substitution also highlighted the role of specific hydroxyl groups in the crystallization process. This work provides guidelines for the design of cellulose- and chitin-based materials. A similar approach can be imagined to prepare cellulose and chitin analogues capable of withstanding enzymatic degradation.

Toward Understanding the Molecular Recognition of Fungal Chitin and Activation of the Plant Defense Mechanism in Horticultural Crops.

Large volumes of fruit and vegetable production are lost during postharvest handling due to attacks by necrotrophic fungi. One of the promising alternatives proposed for the control of postharvest diseases is the induction of natural defense responses, which can be activated by recognizing molecules present in pathogens, such as chitin. Chitin is one of the most important components of the fungal cell wall and is recognized through plant membrane receptors. These receptors belong to the receptor-like kinase (RLK) family, which possesses a transmembrane domain and/or receptor-like protein (RLP) that requires binding to another RLK receptor to recognize chitin. In addition, these receptors have extracellular LysM motifs that participate in the perception of chitin oligosaccharides. These receptors have been widely studied in Arabidopsis thaliana (A. thaliana) and Oryza sativa (O. sativa); however, it is not clear how the molecular recognition and plant defense mechanisms of chitin oligosaccharides occur in other plant species or fruits. This review includes recent findings on the molecular recognition of chitin oligosaccharides and how they activate defense mechanisms in plants. In addition, we highlight some of the current advances in chitin perception in horticultural crops.

Heterologous expression and characterization of thermostable chitinase and β-N-acetylhexosaminidase from Caldicellulosiruptor acetigenus and their synergistic action on the bioconversion of chitin into N-acetyl-d-glucosamine

The bioconversion of chitin into N-acetyl-d-glucosamine (GlcNAc) using chitinolytic enzymes is one of the important avenues for chitin valorization. However, industrial applications of chitinolytic enzymes have been limited by their poor thermostability. Therefore, it is necessary to discover thermostable chitinolytic enzymes for GlcNAc production from chitin. In this study, two chitinolytic enzyme-encoding genes CaChiT and CaHex from Caldicellulosiruptor acetigenus were identified and heterologously expressed in Escherichia coli. The purified recombinant CaChiT and CaHex showed optimal activities at 70 °C and 90 °C respectively, and exhibited good thermostability over a range of temperature below 70 °C and broad pH stability at pH range of 3.0-8.0. CaChiT and CaHex were active on colloidal chitin, pNP-(GlcNAc)2, pNP-(GlcNAc)3, and pNP-GlcNAc, pNP-(GlcNAc)2, pNP-(GlcNAc)3, pNP-Glc respectively. Besides, the chitin oligosaccharides and colloidal chitin hydrolysis profiles revealed that CaChiT degraded chitin chains through exo-mode of action. Furthermore, CaChiT and CaHex exhibited a synergistic effect in the degradation of colloidal chitin, reaching 0.60 mg/mL of GlcNAc production after 1 h incubation. These results suggested that a combination of CaChiT and CaHex have great potential for industrial applications in the enzymatic production of GlcNAc from chitin-containing biowastes.

Andrea Genre.

Andrea Genre.

Optimizing Chitin Depolymerization by Lysozyme to Long-Chain Oligosaccharides.

Chitin oligosaccharides (COs) hold high promise as organic fertilizers in the ongoing agro-ecological transition. Short- and long-chain COs can contribute to the establishment of symbiotic associations between plants and microorganisms, facilitating the uptake of soil nutrients by host plants. Long-chain COs trigger plant innate immunity. A fine investigation of these different signaling pathways requires improving the access to high-purity COs. Here, we used the response surface methodology to optimize the production of COs by enzymatic hydrolysis of water-soluble chitin (WSC) with hen egg-white lysozyme. The influence of WSC concentration, its acetylation degree, and the reaction time course were modelled using a Box–Behnken design. Under optimized conditions, water-soluble COs up to the nonasaccharide were formed in 51% yield and purified to homogeneity. This straightforward approach opens new avenues to determine the complex roles of COs in plants.

BIOREMEDIATION OF SEA FOOD WASTE IN SOLID STATE FERMENTATION ALONG WITH PRODUCTION OF BIOACTIVE AGENTS

Sea food processing generates large volumes of waste products such as skin, head, tails, shells, scales, backbones, etc. Pollution due to conventional methods of sea food waste disposal causes negative implications on environment, aquatic life, and human health. Moreover, these waste products can be used for the production of high-value products which are still untapped due to inappropriate management. Paenibacillus sp. AD, known to act on chitinolytic and proteinaceous waste was explored for its potential to degrade various types of sea food waste in solid state fermentation. Effective degradation of sea food waste, generated from variety of sources such as fish scales, crab shells, prawn shells and mixture of such wastes was observed. 30 to 40 percent degradation in terms of decrease in the mass was achieved. Along with the degradation, chitinolytic and proteolytic enzymes were produced which can have various biotechnological applications. Apart from this, value added products such as chitin oligosaccharides and peptides of various degree of polymerization were also produced which can be used for various therapeutic purposes. Results indicated that Paenibacillus sp. AD can be used for the development of a process for the infield degradation of sea food waste.

Structures and functions of carbohydrate-active enzymes of chitinolytic bacteria Paenibacillus sp. str. FPU-7.

Chitin and its derivatives have valuable potential applications in various fields that include medicine, agriculture, and food industries. Paenibacillus sp. str. FPU-7 is one of the most potent chitin-degrading bacteria identified. This review introduces the chitin degradation system of P. str. FPU-7. In addition to extracellular chitinases, P. str. FPU-7 uses a unique multimodular chitinase (ChiW) to hydrolyze chitin to oligosaccharides on the cell surface. Chitin oligosaccharides are converted to N-acetyl-d-glucosamine by β-N-acetylhexosaminidase (PsNagA) in the cytosol. The functions and structures of ChiW and PsNagA are also summarized. The genome sequence of P. str. FPU-7 provides opportunities to acquire novel enzymes. Genome mining has identified a novel alginate lyase, PsAly. The functions and structure of PsAly are reviewed. These findings will inform further improvement of the sustainable conversion of polysaccharides to functional materials.

A haustorial-expressed lytic polysaccharide monooxygenase from the cucurbit powdery mildew pathogen Podosphaera xanthii contributes to the suppression of chitin-triggered immunity.

Podosphaera xanthii is the main causal agent of cucurbit powdery mildew and a limiting factor of crop productivity. The lifestyle of this fungus is determined by the development of specialized parasitic structures inside epidermal cells, termed haustoria, that are responsible for the acquisition of nutrients and the release of effectors. A typical function of fungal effectors is the manipulation of host immunity, for example the suppression of pathogen‐associated molecular pattern (PAMP)‐triggered immunity (PTI). Chitin is a major component of fungal cell walls, and chitin oligosaccharides are well‐known PAMP elicitors. In this work, we examined the role of PHEC27213, the most highly expressed, haustorium‐specific effector candidate of P. xanthii. According to different computational predictions, the protein folding of PHEC27213 was similar to that of lytic polysaccharide monooxygenases (LPMOs) and included a conserved histidine brace; however, PHEC27213 had low sequence similarity with LPMO proteins and displayed a putative chitin‐binding domain that was different from the canonical carbohydrate‐binding module. Binding and enzymatic assays demonstrated that PHEC27213 was able to bind and catalyse colloidal chitin, as well as chitooligosaccharides, acting as an LPMO. Furthermore, RNAi silencing experiments showed the potential of this protein to prevent the activation of chitin‐triggered immunity. Moreover, proteins with similar features were found in other haustorium‐forming fungal pathogens. Our results suggest that this protein is a new fungal LPMO that catalyses chitooligosaccharides, thus contributing to the suppression of plant immunity during haustorium development. To our knowledge, this is the first mechanism identified in the haustorium to suppress chitin signalling.

Involvement of strigolactone hormone in root development, influence and interaction with mycorrhizal fungi in plant: Mini-review.

Arbuscular mycorrhizal fungi (AMF) and plant symbiosis is the old, fascinating and beneficial relation that exist on earth for the plants. In this review, we have elaborated that the strigolactones (SLs) are released from the roots and function with root parasite, seeds and symbiotic AMF as contact chemicals. They are transported through the xylem in the plants and can regulate plant architecture, seed germination, nodule formation, increase the primary root length, influence the root hairs and physiological reactions to non-living agents by regulating their metabolism. SLs first evolved in ancient plant lineages as regulators of the basic production processes and then took a new role to maintain the growing biological complexities of terrestrial plant. SLs belongs to a diversified category of butenolide-bearing plant hormones related to various processes of agricultural concern. SLs also arouses the development of spores, the divergence and enlargement of hyphae of AMF, metabolism of mitochondria, reprogramming of transcription process, and generation of chitin oligosaccharides which further stimulate the early response of symbiosis in the host plant, results from better communication in plant and ability of coexistence with these fungi. The required nutrients are transferred from the roots to the shoots, which affect the physiological, biochemical, and morphological characteristics of the plant. On the other hand, the plant provides organic carbon in the form of sugars and lipids to the fungi, which they use as a source of energy and for carried out different anabolic pathways. SLs also lead to alteration in the dynamic and structure of actin in the root region as well as changes the auxin's transporter localization in the plasma membrane. Thus, this study reveals the functions that SLs play in the growth of roots, as well as their effect and interaction with AMF that promote plant growth.

New Class of Chitosanase from Bacillus amyloliquefaciens for the Generation of Chitooligosaccharides.

Chitooligosaccharides (COS) generated from either chitin (chitin oligosaccharides) or chitosan (chitosan oligosaccharides) have a wide range of applications in agriculture, medicine, and other fields. Here, we report the characterization of a chitosanase from Bacillus amyloliquefaciens (BamCsn) and the importance of a tryptophan (Trp), W204, for BamCsn activity. BamCsn hydrolyzed the chitosan polymer by an endo mode. It also hydrolyzed chitin oligosaccharides and interestingly exhibited transglycosylation activity on chitotetraose and chitopentaose. Mutation of W204, a nonconserved amino acid in chitosanases, to W204A abolished the hydrolytic activity of BamCsn, with a change in the structure that resulted in a decreased affinity for the substrate and impaired the catalytic ability. Phylogenetic analysis revealed that BamCsn could belong to a new class of chitosanases that showed unique properties like transglycosylation, cleavage of chitin oligosaccharides, and the presence of W204 residues, which is important for activity. Chitosanases belonging to the BamCsn class showed a high potential to generate COS from chitinous substrates.

Chitin, Chitin Oligosaccharide, and Chitin Disaccharide Metabolism of Escherichia coli Revisited: Reassignment of the Roles of ChiA, ChbR, ChbF, and ChbG

Escherichia coli is unable to grow on polymeric and oligomeric chitin, but grows on chitin disaccharide (GlcNAc-GlcNAc; N,N′-diacetylchitobiose) and chitin trisaccharide (GlcNAc-GlcNAc-GlcNAc; N,N′,N′′-triacetylchitotriose) via expression of the chb operon (chbBCARFG). The phosphotransferase system (PTS) transporter ChbBCA facilitates transport of both saccharides across the inner membrane and their concomitant phosphorylation at the non-reducing end, intracellularly yielding GlcNAc 6-phosphate-GlcNAc (GlcNAc6P-GlcNAc) and GlcNAc6P-GlcNAc-GlcNAc, respectively. We revisited the intracellular catabolism of the PTS products, thereby correcting the reported functions of the 6-phospho-glycosidase ChbF, the monodeacetylase ChbG, and the transcriptional regulator ChbR. Intracellular accumulation of glucosamine 6P-GlcNAc (GlcN6P-GlcNAc) and GlcN6P-GlcNAc-GlcNAc in a chbF mutant unraveled a role for ChbG as a monodeacetylase that removes the N-acetyl group at the non-reducing end. Consequently, GlcN6P- but not GlcNAc6P-containing saccharides likely function as coactivators of ChbR. Furthermore, ChbF removed the GlcN6P from the non-reducing terminus of the former saccharides, thereby degrading the inducers of the chb operon and facilitating growth on the saccharides. Consequently, ChbF was unable to hydrolyze GlcNAc6P-residues from the non-reducing end, contrary to previous assumptions but in agreement with structural modeling data and with the unusual catalytic mechanism of the family 4 of glycosidases, to which ChbF belongs. We also refuted the assumption that ChiA is a bifunctional endochitinase/lysozyme ChiA, and show that it is unable to degrade peptidoglycans but acts as a bona fide chitinase in vitro and in vivo, enabling growth of E. coli on chitin oligosaccharides when ectopically expressed. Overall, this study revises our understanding of the chitin, chitin oligosaccharide, and chitin disaccharide metabolism of E. coli.

Structural characterization of two solute-binding proteins for N,N′-diacetylchitobiose/N,N′,N′′-triacetylchitotoriose of the gram-positive bacterium, Paenibacillus sp. str. FPU-7

The chitinolytic bacterium Paenibacillus sp. str. FPU-7 efficiently degrades chitin into oligosaccharides such as N-acetyl-D-glucosamine (GlcNAc) and disaccharides (GlcNAc)2 through multiple secretory chitinases. Transport of these oligosaccharides by P. str. FPU-7 has not yet been clarified. In this study, we identified nagB1, predicted to encode a sugar solute-binding protein (SBP), which is a component of the ABC transport system. However, the genes next to nagB1 were predicted to encode two-component regulatory system proteins rather than transmembrane domains (TMDs). We also identified nagB2, which is highly homologous to nagB1. Adjacent to nagB2, two genes were predicted to encode TMDs. Binding experiments of the recombinant NagB1 and NagB2 to several oligosaccharides using differential scanning fluorimetry and surface plasmon resonance confirmed that both proteins are SBPs of (GlcNAc)2 and (GlcNAc)3. We determined their crystal structures complexed with and without chitin oligosaccharides at a resolution of 1.2 to 2.0 Å. The structures shared typical SBP structural folds and were classified as subcluster D-I. Large domain motions were observed in the structures, suggesting that they were induced by ligand binding via the “Venus flytrap” mechanism. These structures also revealed chitin oligosaccharide recognition mechanisms. In conclusion, our study provides insight into the recognition and transport of chitin oligosaccharides in bacteria.

Chitin and Chitosan Derivatives as Biomaterial Resources for Biological and Biomedical Applications.

Chitin is a long-chain polymer of N-acetyl-glucosamine, which is regularly found in the exoskeleton of arthropods including insects, shellfish and the cell wall of fungi. It has been known that chitin can be used for biological and biomedical applications, especially as a biomaterial for tissue repairing, encapsulating drug for drug delivery. However, chitin has been postulated as an inducer of proinflammatory cytokines and certain diseases including asthma. Likewise, chitosan, a long-chain polymer of N-acetyl-glucosamine and d-glucosamine derived from chitin deacetylation, and chitosan oligosaccharide, a short chain polymer, have been known for their potential therapeutic effects, including anti-inflammatory, antioxidant, antidiarrheal, and anti-Alzheimer effects. This review summarizes potential utilization and limitation of chitin, chitosan and chitosan oligosaccharide in a variety of diseases. Furthermore, future direction of research and development of chitin, chitosan, and chitosan oligosaccharide for biomedical applications is discussed.

Unique repertoire of anti-carbohydrate antibodies in individual human serum

Humoral immunity to pathogens and other environmental challenges is paramount to maintain normal health, and individuals lacking or unable to make antibodies are at risk. Recent studies indicate that many human protective antibodies are against carbohydrate antigens; however, little is known about repertoires and individual variation of anti-carbohydrate antibodies in healthy individuals. Here we analyzed anti-carbohydrate antibody repertoires (ACARs) of 105 healthy individual adult donors, aged 20–60+ from different ethnic backgrounds to explore variations in antibodies, as defined by binding to glycan microarrays and by affinity purification. Using microarrays that contained > 1,000 glycans, including antigens from animal cells and microbes, we profiled the IgG and IgM ACARs from all donors. Each donor expressed many ACAs, but had a relatively unique ACAR, which included unanticipated antibodies to carbohydrate antigens not well studied, such as chitin oligosaccharides, Forssman-related antigens, globo-type antigens, and bacterial glycans. We also saw some expected antibodies to ABO(H) blood group and α-Gal-type antigens, although these also varied among individuals. Analysis suggests differences in ACARs are associated with ethnicity and age. Thus, each individual ACAR is relatively unique, suggesting that individualized information could be useful in precision medicine for predicting and monitoring immune health and resistance to disease.

Recognition Pattern, Functional Mechanism and Application of Chitin and Chitosan Oligosaccharides in Sustainable Agriculture.

Application of chitin attracts much attention in the past decades as the second abundant polysaccharides in the world after cellulose. Chitin oligosaccharides (CTOS) and its deacetylated derivative chitosan oligosaccharides (COS) were shown great potentiality in agriculture by enhancing plant resistance to abiotic or biotic stresses, promoting plant growth and yield, improving fruits quality and storage, etc. Those applications have already served huge economic and social benefits for many years. However, the recognition mode and functional mechanism of CTOS and COS on plants have gradually revealed just in recent years. Recognition pattern and functional mechanism of CTOS and COS in plant together with application status of COS in agricultural production will be well described in this review. By which we wish to promote further development and application of CTOS and COS-related products in the field.

Chemoenzymatic Synthesis and Function of Chitin Derivatives.

Chitin, abundant biomass found in crab shells and other marine life, has wide applications in the production of food, pharmaceuticals, and cosmetics. Our recent studies have focused on the development of new functional materials by derivatizing chitin oligosaccharides and monosaccharides. For example, we have prepared various derivatives by chemoenzymatic synthesis using N-acetylglucosamine (GlcNAc) or chitin oligosaccharide prepared from chitin as starting materials. First, we have achieved the total synthesis of two secondary metabolites (furanodictine A and B) with neuronal differentiation-inducing activity on PC12 cells by using a simple heatinduced structural transformation of GlcNAc and esterification reaction. Second, we synthesized both a novel inhibitor that has facilitated a re-examination of the reaction mechanism of hen egg-white lysozyme, and a new substrate for assaying lysozyme activity by using chitin oligosaccharides as raw materials. Thus, the development of new materials by simple derivatization of chitin mono- or oligo-saccharides is paving the way for effective use of chitin.

Regulation of Chitin-Dependent Growth and Natural Competence in Vibrio parahaemolyticus.

Vibrios can degrade chitin surfaces to soluble N-acetyl glucosamine oligosaccharides (GlcNAcn) that can be utilized as a carbon source and also induce a state of natural genetic competence. In this study, we characterized chitin-dependent growth and natural competence in Vibrio parahaemolyticus and its regulation. We found that growth on chitin was regulated through chitin sensors ChiS (sensor histidine kinase) and TfoS (transmembrane transcriptional regulator) by predominantly controlling the expression of chitinase VPA0055 (ChiA2) in a TfoX-dependent manner. The reduced growth of ΔchiA2, ΔchiS and ΔtfoS mutants highlighted the critical role played by ChiA2 in chitin breakdown. This growth defect of ΔchiA2 mutant could be recovered when chitin oligosaccharides GlcNAc2 or GlcNAc6 were supplied instead of chitin. The ΔtfoS mutant was also able to grow on GlcNAc2 but the ΔchiS mutant could not, which indicates that GlcNAc2 catabolic operon is dependent on ChiS and independent of TfoS. However, the ΔtfoS mutant was unable to utilize GlcNAc6 because the periplasmic enzymes required for the breakdown of GlcNAc6 were found to be downregulated at the mRNA level. We also showed that natural competence can be induced only by GlcNAc6, not GlcNAc2, because the expression of competence genes was significantly higher in the presence of GlcNAc6 compared to GlcNAc2. Moreover, this might be an indication that GlcNAc2 and GlcNAc6 were detected by different receptors. Therefore, we speculate that GlcNAc2-dependent activation of ChiS and GlcNAc6-dependent activation of TfoS might be crucial for the induction of natural competence in V. parahaemolyticus through the upregulation of the master competence regulator TfoX.