title: Flavobacterium johnsoniae chitinase ChiA is required for chitin utilization and is secreted by the type IX secretion system.
abstract: 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-kDa 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 (LC-MS/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.
authors.affiliation: Department of Biological Sciences, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin, USA.
authors.forename: Sampada S
authors.initials: SS
authors.lastname: Kharade
authors.forename: Mark J
authors.initials: MJ
authors.lastname: McBride
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002686:Chitin / Q000378:metabolism*; D002688:Chitinases / Q000235:genetics / Q000378:metabolism*; D005417:Flavobacterium / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D009154:Mutation
chemical_list: D001426:Bacterial Proteins; D002686:Chitin; D002688:Chitinases