Flavobacterium johnsoniae Chitinase ChiA Is Required for Chitin Utilization and Is Secreted by the Type IX Secretion System
Flavobacterium johnsoniae Chitinase ChiA Is Required for Chitin Utilization and Is Secreted by the Type IX Secretion System
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DOI：
10.1128/jb.01170-13
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发表时间：
2014-03-01
影响因子：
3.2
通讯作者：
McBride, Mark J.
中科院分区：
文献类型：
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作者：
Kharade, Sampada S.;McBride, Mark J.
约氏黄杆菌（Flavobacteriumjohnsoniae）是拟杆菌门（Bacteriodetes）的一种游动细菌，能降解不溶性甲壳素等多糖。一种新的蛋白质分泌系统，IX型分泌系统（T9SS），需要滑行运动和几丁质利用。通过基因组分析鉴定了5种潜在的几丁质酶。Fjoh_4555（ChiA）是一种具有两个糖苷水解酶家族18（GH18）结构域的168.9-kDa蛋白，被靶向用于分析。通过插入突变破坏chiA导致细胞不能消化几丁质，并且在质粒上与野生型chiA互补恢复几丁质利用。用抗重组ChiA的抗血清检测该蛋白，并分析其分泌特性。约翰逊氏菌。ChiA由野生型细胞以可溶性形式分泌，但在携带T9SS基因、gldK、gldL、gldM、gldNO、sprA、sprE和sprT中的任一个突变的菌株中保持细胞缔合。蛋白质印迹和液相色谱-串联质谱（LCMS/MS）分析表明，ChiA被蛋白水解加工成两个含GH18结构域的蛋白质。由T9SS分泌的蛋白质通常具有属于TIGRFAM家族TIGR04131和TIGR04183的保守羧基末端结构域（CTD）。ChiA与这些序列没有很强的相似性，而是具有新的CTD。该CTD的缺失导致细胞内ChiA的积累。ChiA CTD与重组mCherry的融合导致mCherry分泌到培养基中。结果表明，ChiA是一种可溶性的胞外几丁质酶，它依赖于一种新的CTD来分泌几丁质。约翰逊氏菌T9SS.
Flavobacterium johnsoniae, a member of phylum Bacteriodetes, is a gliding bacterium that digests insoluble chitin and many other polysaccharides. A novel protein secretion system, the type IX secretion system (T9SS), is required for gliding motility and for chitin utilization. Five potential chitinases were identified by genome analysis. Fjoh_4555 (ChiA), a 168.9-kappa Da protein with two glycoside hydrolase family 18 (GH18) domains, was targeted for analysis. Disruption of chiA by insertional mutagenesis resulted in cells that failed to digest chitin, and complementation with wild-type chiA on a plasmid restored chitin utilization. Antiserum raised against recombinant ChiA was used to detect the protein and to characterize its secretion by F. johnsoniae. ChiA was secreted in soluble form by wild-type cells but remained cell associated in strains carrying mutations in any of the T9SS genes, gldK, gldL, gldM, gldNO, sprA, sprE, and sprT. Western blot and liquid chromatography-tandem mass spectrometry (LCMS/MS) analyses suggested that ChiA was proteolytically processed into two GH18 domain-containing proteins. Proteins secreted by T9SSs typically have conserved carboxy-terminal domains (CTDs) belonging to the TIGRFAM families TIGR04131 and TIGR04183. ChiA does not exhibit strong similarity to these sequences and instead has a novel CTD. Deletion of this CTD resulted in accumulation of ChiA inside cells. Fusion of the ChiA CTD to recombinant mCherry resulted in secretion of mCherry into the medium. The results indicate that ChiA is a soluble extracellular chitinase required for chitin utilization and that it relies on a novel CTD for secretion by the F. johnsoniae T9SS.