Date of Award

December 2014

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Biological Sciences

First Advisor

Mark J. McBride

Committee Members

Charles Wimpee, Daad Saffarani, Sergei Kuchin, Sonia Bardy

Keywords

Bacteroidetes, Chitin, CTD, Flavobacterium Johnsoniae, Secretion, T9SS

Abstract

Flavobacterium johnsoniae, a member of phylum Bacteroidetes, is a gliding bacterium that digests insoluble chitin. A novel protein secretion system, the Type IX secretion system (T9SS), secretes the motility adhesins SprB and RemA and is also required for chitin utilization. In order to understand F. johnsoniae chitin utilization and the role of the T9SS, Fjoh_4555 (chiA) was targeted for analysis. Disruption of chiA resulted in cells that failed to digest chitin and complementation restored this ability. Antisera raised against ChiA were used to characterize its secretion. ChiA was secreted in soluble form by wild-type cells but remained cell-associated in T9SS mutant strains. Proteins secreted by T9SSs typically have conserved carboxy-terminal domains (CTDs) belonging to the TIGRFAM families, TIGR04131 and TIGR04183. ChiA did not exhibit strong similarity to these sequences but instead had a novel CTD. Deletion of this CTD resulted in accumulation of ChiA inside of cells. Fusion of the ChiA CTD to mCherry resulted in secretion of mCherry into the medium. These results indicate that ChiA is a soluble extracellular chitinase required for chitin utilization and that it relies on a novel CTD for its secretion by the F. johnsoniae T9SS.

Proteins involved in secretion by the T9SS include GldK, GldL, GldM, GldN, SprA, SprE, and SprT. Porphyromonas gingivalis has orthologs for each of these T9SS proteins and they are required for secretion of gingipain proteases. P. gingivalis porU and porV have also been linked to T9SS-mediated secretion and F. johnsoniae has orthologs of these. Cells of an F. johnsoniae porV deletion mutant failed to secrete ChiA and RemA, but retained the ability to secrete SprB. The porV mutant was partially deficient in attachment to glass, apparently because of the absence of RemA and other adhesins on the cell surface. The porV mutant also appeared to be deficient in secretion of numerous other proteins that have CTDs associated with targeting to the T9SS. PorU was not required for secretion of ChiA, RemA, or SprB, indicating that it does not play an essential role in the F. johnsoniae T9SS.

chiA is located downstream of a cluster of genes likely to be involved in chitin utilization. Deletion of Fjoh_4558 (cusDI) resulted in a partial defect in chitin utilization, and deletion of the region spanning Fjoh_4558 through Fjoh_4562 which includes cusDI, cusDII, cusCI and cusCII resulted in almost complete loss of ability to utilize chitin. The CusC and CusD proteins are similar in sequence to the Bacteroides thetaiotaomicron starch utilization system outer membrane proteins SusC and SusD respectively. SusC and SusD are involved in active uptake of starch oligomers across the outer membrane. The F. johnsoniae CusC and CusD proteins may perform similar functions, and cooperate with ChiA to allow efficient utilization of insoluble chitin.

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