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

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.

由N-酰基高丝氨酸内酯（N-acyl homoserine lactone, AHL）信号分子介导的群体感应（quorum sensing）调控，已被证实是诸多革兰氏阴性菌胞外酶产生调控的关键特征。在紫色色杆菌ATCC 31532（Chromobacterium violaceum ATCC 31532）中，已知其多项表型特征——包括紫色色素紫菌素（violacein）、氰化氢、抗生素以及胞外蛋白酶的产生——均由内源N-己酰基-L-高丝氨酸内酯（N-hexanoyl-L-homoserine lactone, HHL）所调控。本研究证实，紫色色杆菌可产生一组受HHL调控的几丁质酶（chitinolytic enzymes）。几丁质酶活性在以几丁质作为唯一碳源培养的菌株中被诱导产生，并通过使用N-乙酰葡糖胺（N-acetylglucosamine）二糖、三糖及四糖寡聚体的对硝基苯酚（p-nitrophenol）类似物，对分泌蛋白中的几丁质酶活性进行定量检测。采用相同N-乙酰葡糖胺寡聚体的4-甲基伞形酮基（4-methylumbelliferyl）类似物作为底物，对经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE）分离并复性的蛋白进行检测，共发现至少6种酶：一种对二聚体底物具有高度特异性的87 kDa壳二糖酶（chitobiase）、两种表观分子量分别为162 kDa和133 kDa的N-乙酰葡糖胺酶（N-acetylglucosaminidases）、两种分子量分别为108 kDa和67 kDa的内切几丁质酶（endochitinases），以及一种56 kDa的壳二糖苷酶（chitobiosidase）。此外，当以四聚体几丁质衍生物作为底物时，还检测到两条分子量大于205 kDa的未鉴定条带。一株缺陷于HHL合成及其他群体感应调控因子的紫色色杆菌多效性mini-Tn5突变株CV026，其几丁质酶活性完全缺失。将该突变株接种于添加了野生型紫色色杆菌培养上清液或10 μM合成HHL的几丁质基本培养基中培养，其几丁质酶产量可恢复至亲本菌株水平。本研究结果为革兰氏阴性菌中AHL信号调控几丁质酶活性提供了迄今为止最为完整的实验证据。