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. 1999 Jun;65(6):2553-7.
doi: 10.1128/AEM.65.6.2553-2557.1999.

Chitinases from uncultured marine microorganisms

M T Cottrell  1 , J A Moore, D L Kirchman
Affiliations

Chitinases from uncultured marine microorganisms

M T Cottrell et al. Appl Environ Microbiol. 1999 Jun.

Abstract

Our understanding of the degradation of organic matter will benefit from a greater appreciation for the genes encoding enzymes involved in the hydrolysis of biopolymers such as chitin, one of the most abundant polymers in nature. To isolate representative and abundant chitinase genes from uncultivated marine bacteria, we constructed libraries of genomic DNA isolated from coastal and estuarine waters. The libraries were screened for genes encoding proteins that hydrolyze a fluorogenic analogue of chitin, 4-methylumbelliferyl beta-D-N,N'-diacetylchitobioside (MUF-diNAG). The abundance of clones capable of MUF-diNAG hydrolysis was higher in the library constructed with DNA from the estuary than in that constructed with DNA from coastal waters, although the abundance of positive clones was also dependent on the method used to screen the library. Plaque assays revealed nine MUF-diNAG-positive clones of 75,000 screened for the estuarine sample and two clones of 750,000 for the coastal sample. A microtiter plate assay revealed approximately 1 positive clone for every 500 clones screened in the coastal library. The number of clones detected with the plaque assay was consistent with estimates of the portion of culturable bacteria that degrade chitin. Our results suggest that culture-dependent methods do not greatly underestimate the portion of marine bacterial communities capable of chitin degradation.

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Figures

FIG. 1
FIG. 1
Restriction patterns (XbaI with KpnI) of clones that hydrolyze MUF-diNAG. (A) Clone identified by screening the library by plaque assay. Lanes: 1, pG1; M, molecular weight marker. (B) Clones identified by screening the library in microtiter plates. Lanes: 1, p2F9; 2, p3A6; 3, p4H11; 4, p5C7; 5, p5F2; 6, p6D5; 7, p6E11; 8, p7C8; 9, p7D9; 10, p7F6; 11, p9D5; 12, p10D3; 13, p14E11; M, molecular weight marker.
FIG. 2
FIG. 2
Calcofluor-stained glycol chitin gel of proteins extracted from E. coli bearing the plasmids pBluescript KS(−) (lane 1), p2F9 (lane 2), p3A6 (lane 3), p4H11 (lane 4), and p5C7 (lane 5). Regions of the gel with hydrolyzed chitin were distinguishable by their lack of fluorescence (dark bands).
See this image and copyright information in PMC

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