Identification and Characterization of the Gene Cluster Involved in Chitin Degradation in a Marine Bacterium, Alteromonas sp. Strain O-7

Abstract

Alteromonas sp. strain O-7 secretes chitinase A (ChiA), chitinase B (ChiB), and chitinase C (ChiC) in the presence of chitin. A gene cluster involved in the chitinolytic system of the strain was cloned and sequenced upstream of and including the chiA gene. The gene cluster consisted of three different open reading frames organized in the order chiD, cbp1, and chiA. The chiD, cbp1, and chiA genes were closely linked and transcribed in the same direction. Sequence analysis indicated that Cbp1 (475 amino acids) was a chitin-binding protein composed of two discrete functional regions. ChiD (1,037 amino acids) showed sequence similarity to bacterial chitinases classified into family 18 of glycosyl hydrolases. The cbp1 and chiD genes were expressed in Escherichia coli, and the recombinant proteins were purified to homogeneity. The highest binding activities of Cbp1 and ChiD were observed when ␣-chitin was used as a substrate. Cbp1 and ChiD possessed a chitin-binding domain (ChtBD) belonging to ChtBD type 3. ChiD rapidly hydrolyzed chitin oligosaccharides in sizes from trimers to hexamers, but not chitin. However, after prolonged incubation with large amounts of ChiD, the enzyme produced a small amount of (GlcNAc) 2 from chitin. The optimum temperature and pH of ChiD were 50°C and 7.0, respectively.Chitin, an insoluble linear ␤-1,4-linked polymer of N-acetylglucosamine (GlcNAc), is the second most abundant polymer in nature. This polysaccharide is particularly an important nutrient source for maintaining the ecosystem in the marine environment (7). Chitinolytic marine bacteria play a critical role in the process of recycling chitinous materials such as the exoskeletons of crustaceans and insects. If the insoluble form of chitin could not be returned to the ecosystem in a biologically usable form, the marine environment would be completely depleted of a carbon and nitrogen source in a relatively short time (39). To degrade chitin, chitinolytic microorganisms produce two classes of enzymes: chitinases (EC 3.2.1.14) and ␤-N-acetylglucosaminidases (GlcNAcases; EC 3.2.1.30). Chitinases hydrolyze chitin to soluble oligosaccharides, mainly chitobiose, which are further hydrolyzed to GlcNAc by GlcNAcases. Finally, the degradation products, mainly GlcNAc, are then taken up by the cells as a carbon and nitrogen source.Alteromonas sp. strain O-7 is a gram-negative, flagellated, motile, and aerobic rod-shaped bacterium of marine origin (30). This strain was isolated from a sediment sample at Sagami Bay in Japan as an efficient producer of chitinolytic enzymes (30). We have been studying the chitin degradation system of the strain as a model for defining the various components involved in chitin utilization. It was made clear that the chitinolytic system of the strain consists of at least four chitinases (Chi85, ChiA, ChiB, and ChiC) and three ␤-Nacetylglucosaminidases (GlcNAcaseA, GlcNAcaseB, and GlcNAcaseC). The genes for these enzymes have been cloned and characterized previously to clarify the role of individual enzymes in the ...

Nutrient regime regulates complex transcriptional start site usage within a Pseudoalteromonas chitinase gene cluster

“…O-7 chiD, cbp1 and chiA (Genbank accession no. AB063629; Tsujibo et al, 2002) genes are also arranged in this order in a cluster, and the domains identified within each gene are similarly located in S91 chiABC ( Figure 1 and Table 5; Techkarnjanaruk and Goodman, 1999). The GC content of each gene cluster is similar (46% for S91 and 45% for O-7).…”

Purification, Cloning, and Sequence Analysis of β-N-Acetylglucosaminidase from the Chitinolytic BacteriumAeromonas hydrophilaStrain SUWA-9

“…13,[24][25][26] On the other hand, NAGs have been reported to be located in periplasmic space or in cytoplasm in the case of marine bacteria. 22,23,27) This intracellular localization of enzymes appears to be advantageous for an efficient hydrolysis of chitin in sea water, since hydrolyzed products can easily be taken into cells. The existence of a signal peptide in a precursor peptide of NAG20A suggests that NAG20A can be exported across the cellular membrane.…”

“…A chitinolytic enzyme system has not been clarified so far for bacteria living in fresh water, although a detailed characterization has been reported for marine bacteria, such as Altermonas sp. O-7 22) and All sequences corresponded to the domain III regions (residues 336-818) of S. marcescens chitobiase, which contains catalytic center residues. Numerals show the number of amino acid residues initiated from the N-terminus of the total ORF.…”

Isolation and characterization of mutants of Pseudomonas maltophila PM-4 altered in chitinolytic activity and antagonistic activity against root rot pathogens of clusterbean (Cyamopsis tetragonoloba)

“…Chitinases hydrolyze chitin to soluble oligosaccharides, mainly chitobiose, which are further hydrolyzed to GlcNAc by GlcNAcases which is then taken up by the cells as a carbon and nitrogen source 5 . Chitinases are produced by a number of microorganisms such as Bacillus, Pseudomonas, Serratia, Stenotrophomonas, Streptomyces and Vibrio; and is one of the mechanism that has been implicated in biocontrol of fungal diseases 6 .…”