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Applied and Environmental Microbiology, June 1999, p. 2553-2557, Vol. 65, No. 6
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Chitinases from Uncultured Marine
Microorganisms
Matthew T.
Cottrell,
Jessica
A.
Moore, and
David L.
Kirchman*
College of Marine Studies, University of
Delaware, Lewes, Delaware 19958
Received 19 October 1998/Accepted 1 March 1999
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
-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.
*
Corresponding author. Mailing address: College of
Marine Studies, University of Delaware, 700 Pilottown Rd., Lewes, DE
19958. Phone: (302) 645-4375. Fax: (302) 645-4028. E-mail:
kirchman{at}udel.edu.
Applied and Environmental Microbiology, June 1999, p. 2553-2557, Vol. 65, No. 6
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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