Chitinolytic activity in Chromobacterium violaceum: substrate analysis and regulation by quorum sensing

Abstract

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 microM 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.

FIG. 1

Assay of chitinolytic activity on plates with SM agar medium supplemented with colloidal chitin (0.2% [wt/vol]). Clearing zones of colloidal chitin formed around the colonies of C. violaceum strains CVWT (1) and CV017 (2). Strains E. agglomerans IC1270 (3) and 40b (4) were seeded on the plate as positive and negative controls, respectively. The plate was incubated at 28°C for 4 days.

FIG. 2

(Top) Assay of chitinolytic activity on a plate with SM agar supplemented with colloidal chitin (0.2% [wt/vol]) (left) and on a plate with SM agar supplemented with colloidal chitin (0.2 [wt/vol]) plus HHL (10 μM) (right): 1 and 3, growth of mutant CV026; 2 and 4, growth of E. agglomerans IC1270 strain (positive control). Plates were incubated at 28°C for 4 days. (Bottom) Assay of chitinolytic activity of mutant CV026 grown in liquid SM medium with colloidal chitin (0.2% [wt/vol]) (left) and in the same medium supplemented with HHL (10 μM) (right). The colloidal chitin was almost completely hydrolyzed in the HHL-supplemented medium, whereas particles of nonhydrolyzed chitin are clearly visible in the unsupplemented medium. Bacteria were grown on the medium indicated at 28°C for 72 h with agitation (200 rpm).

FIG. 3

Chitinolytic activity in extracellular proteins of C. violaceum strains. Assays were performed with pNP-GlcNAc (columns 1, 3, 5, and 7) or pNP-(GlcNAc)₂ (columns 2, 4, 6, and 8). Activity shown was found in extracellular proteins of strains CVWT (columns 1 and 2), CV017 (columns 3 and 4), and CV026 (columns 5 and 6) grown in SM with chitin. Activity shown in columns 7 and 8 was found in extracellular proteins of strain CV026 grown in SM containing chitin and supplemented with HHL (10 μM). Values (mean ± standard error) were determined from 11 independently obtained preparations.

FIG. 4

Detection of chitinolytic enzymes in extracellular proteins after separation by SDS-PAGE. Chitinolytic activity was detected with the substrates 4-MU- GlcNAc (A), 4-MU-(GlcNAc)₂ (B), and 4-MU-(GlcNAc)₃ (C). Lanes 1 and 2, extracellular proteins from strains CV026 and CV017 grown on SM with chitin; lane 3, extracellular proteins from strain CV026 grown on SM with chitin supplemented with HHL (10 μM); lane 4, extracellular proteins of strain CV017 boiled for 4 min in sample buffer containing 2-mercaptoethanol prior to loading. High-range prestained SDS-PAGE standards (Bio-Rad) were used as size markers (M). The positions of protein size markers are shown on the right.