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首页> 外文期刊>Journal of bacteriology >Chitinolytic Activity in Chromobacterium violaceum: Substrate Analysis and Regulation by Quorum Sensing
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Chitinolytic Activity in Chromobacterium violaceum: Substrate Analysis and Regulation by Quorum Sensing

机译：紫杆菌中的几丁质酶活性：群体感应的底物分析和调控。

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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 AHLN-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 ofN-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 ofC. 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.
机译：由 N -酰基高丝氨酸内酯（AHL）信号分子介导的群体感应控制已被确立为许多革兰氏阴性细菌中调节外切酶产生的关键特征。在紫杆菌 ATCC 31532中，已知许多表型特征，包括紫色素紫堇青素，氰化氢，抗生素和外蛋白酶的产生均受内源性AHL N 的调节。 -己酰基-1-高丝氨酸内酯（HHL）。在这项研究中，我们证明了 C。 violaceum 产生一组几丁质分解酶，其产生受HHL调节。在几丁质作为唯一碳源存在下生长的菌株中诱导了几丁质分解活性，并使用 N的二糖，三糖和四糖寡聚物的 p -硝基苯酚类似物对分泌蛋白进行了定量-乙酰氨基葡萄糖。通过使用与 N -乙酰氨基葡糖相同的低聚物的4-甲基伞形基类似物作为十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分离和复性的蛋白质的底物，检测到至少六种酶：对生物素酶具有高度特异性的壳二糖酶一个二聚体底物87 kDa，两个 N -乙酰氨基葡萄糖苷酶，表观分子量分别为162和133 kDa，两个内切壳多糖酶为108和67 kDa，以及壳聚糖酶为56 kDa。此外，在使用四聚体几丁质衍生物作为底物的情况下，发现了两个大于205 kDa的未识别条带。 C的多效性mini-Tn 5 突变体。还发现在HHL产生中有缺陷的紫堇（CV026）和其他群体感应调节因子均完全缺乏几丁质酶活性。该突变体在补充有 C培养上清液的几丁质基本培养基上的生长。紫胶菌野生型菌株或10μM合成的HHL可将几丁质酶的产量恢复到亲本菌株所示的水平。这些结果构成了迄今为止在革兰氏阴性细菌中通过AHL信号调节甲壳素分解活性的最完整证据。

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