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Journal of Bacteriology, September 1998, p. 4435-4441, Vol. 180, No. 17
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Chitinolytic Activity in Chromobacterium violaceum: Substrate Analysis and Regulation by Quorum Sensing

Leonid S. Chernin,^1,* Michael K. Winson,^2,3, Jacquelyn M. Thompson,^2,3 Shoshan Haran,¹ Barrie W. Bycroft,³ Ilan Chet,¹ Paul Williams,³ and Gordon S. A. B. Stewart³

The Otto Warburg Center for Biotechnology in Agriculture and the Department of Plant Pathology and Microbiology, Faculty of Agricultural, Food, and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot 76100, Israel,¹ and School of Biology, University of Nottingham, Leicestershire LE12 5RD,² and School of Pharmaceutical Sciences, University of Nottingham, Nottingham NG7 2RD,³ United Kingdom

Received 25 March 1998/Accepted 18 June 1998

Quorum sensing control mediated by N-acyl homoserine lactone (AHL) signaling molecules has been established as a key feature of the regulation of exoenzyme production in many gram-negative bacteria. In Chromobacterium violaceum ATCC 31532 a number of phenotypic characteristics, including production of the purple pigment violacein, hydrogen cyanide, antibiotics, and exoproteases are known to be regulated by the endogenous AHL N-hexanoyl-L-homoserine lactone (HHL). In this study we show that C. violaceum produces a set of chitinolytic enzymes whose production is regulated by HHL. The chitinolytic activity was induced in strains grown in the presence of chitin as the sole carbon source and quantitated in the secreted proteins by using p-nitrophenol analogs of disaccharide, trisaccharide, and tetrasaccharide oligomers of N-acetylglucosamine. By using 4-methylumbelliferyl analogs of the same oligomers of N-acetylglucosamine as substrates for proteins separated and renatured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, at least six enzymes were detected: a chitobiase with high specificity to a dimeric substrate of 87 kDa, two N-acetylglucosaminidases with apparent molecular masses of 162 and 133 kDa, two endochitinases of 108 and 67 kDa, and a chitobiosidase of 56 kDa. In addition, two unidentified bands of >205 kDa were found where a tetrameric chitin derivative was used as a substrate. A pleiotropic mini-Tn5 mutant of C. violaceum (CV026) that is defective in HHL production and other quorum-sensing-regulated factors was also found to be completely deficient in chitinolytic activity. Growth of this mutant on minimal medium with chitin supplemented with culture supernatant from the C. violaceum wild-type strain or 10 µM synthetic HHL restored chitinase production to the level shown by the parental strain. These results constitute the most complete evidence so far for regulation of chitinolytic activity by AHL signaling in a gram-negative bacterium.

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^* Corresponding author. Mailing address: The Hebrew University, Otto Warburg Center for Biotechnology in Agriculture, Faculty of Agriculture, P.O. Box 12, Rehovot 76100, Israel. Phone: 972-8-9481128. Fax: 972-8-9468785. E-mail: chernin{at}agri.huji.ac.il.

Present address: Institute of Biological Sciences, University of Wales, Aberystwyth, Ceredigion SY23 3DD, United Kingdom.

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Journal of Bacteriology, September 1998, p. 4435-4441, Vol. 180, No. 17
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

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