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Journal of Bacteriology, August 2003, p. 4693-4698, Vol. 185, No. 16
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.16.4693-4698.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Detachment of Actinobacillus actinomycetemcomitans Biofilm Cells by an Endogenous ß-Hexosaminidase Activity

Jeffrey B. Kaplan,* Chandran Ragunath, Narayanan Ramasubbu, and Daniel H. Fine

Department of Oral Biology, New Jersey Dental School, Newark, New Jersey 07103

Received 7 March 2003/ Accepted 5 May 2003

When cultured in broth, fresh clinical isolates of the gram-negative periodontal pathogen Actinobacillus actinomycetemcomitans form tenaciously adherent biofilm colonies on surfaces such as plastic and glass. These biofilm colonies release adherent cells into the medium, and the released cells can attach to the surface of the culture vessel and form new colonies, enabling the biofilm to spread. We mutagenized A. actinomycetemcomitans clinical strain CU1000 with transposon IS903{phi}kan and isolated a transposon insertion mutant that formed biofilm colonies which were tightly adherent to surfaces but which lacked the ability to release cells into the medium and disperse. The transposon insertion in the mutant strain mapped to a gene, designated dspB, that was predicted to encode a secreted protein homologous to the catalytic domain of the family 20 glycosyl hydrolases. A plasmid carrying a wild-type dspB gene restored the ability of biofilm colonies of the mutant strain to disperse. We expressed A. actinomycetemcomitans DspB protein engineered to contain a hexahistidine metal-binding site at its C terminus in Escherichia coli and purified the protein by using Ni affinity chromatography. Substrate specificity studies performed with monosaccharides labeled with 4-nitrophenyl groups showed that DspB hydrolyzed the 1->4 glycosidic bond of ß-substituted N-acetylglucosamine, which is consistent with the known functions of other family 20 glycosyl hydrolases. When added to culture medium, purified DspB protein, but not heat-inactivated DspB, restored the ability of the mutant strain to release cells and disperse. DspB protein also caused the detachment of cells from preformed biofilm colonies of strain CU1000 grown attached to plastic and the disaggregation of highly autoaggregated clumps of CU1000 cells in solution. We concluded that dspB encodes a soluble ß-N-acetylglucosaminidase that causes detachment and dispersion of A. actinomycetemcomitans biofilm cells.

* Corresponding author. Mailing address: Medical Science Building, Room C-636, 185 S. Orange Ave., Newark, NJ 07103-2714. Phone: (973) 972-5051. Fax: (973) 972-0045. E-mail: kaplanjb{at}umdnj.edu.

Journal of Bacteriology, August 2003, p. 4693-4698, Vol. 185, No. 16
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.16.4693-4698.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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