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Title:Elucidating the roles of the diguanylate cyclases CelR and avmA in attachment and cellulose synthesis with the response regulator divK in Agrobacterium tumefaciens
Author(s):Barnhart, David
Director of Research:Farrand, Stephen K.
Doctoral Committee Chair(s):Farrand, Stephen K.
Doctoral Committee Member(s):Tapping, Richard I.; Kuzminov, Andrei; Olsen, Gary J.
Department / Program:Microbiology
Degree Granting Institution:University of Illinois at Urbana-Champaign
cyclic diguanosine monophosphate (c-di-GMP)
Abstract:Agrobacterium tumefaciens, the causative agent of crown gall disease in plants, physically interacts with plant cells to remain anchored within the nutrient-rich rhizosphere. Part of this interaction involves the production of cellulose fibrils by the bacteria, leading to the aggregation of the cells to each other as well as to the plant surface. While this interaction between the bacteria and plants has been partially characterized, little is known concerning the regulatory system that controls cellulose synthesis within A. tumefaciens. One possible mechanism is through the secondary messenger cyclic diguanosine monophosphate (c-di-GMP), a small molecule used in numerous complex physiological processes in bacteria, including regulation of exopolysaccharide production. In this study, we attempt to characterize the effects of two diguanylate cyclases (DGC), CelR and AvmA, and other components involved in regulating cellulose production and attachment. CelR positively influences cellulose synthesis through the production of c-di-GMP. This effect occurs when the DGC is activated, likely by phosphorylation. Overexpressing celR also results in changes in cell morphology, attachment, unipolar polysaccharide (UPP) production and virulence, although deleting the gene does not affect any of these phenotypes. Overexpressing a DGC of similar structure to CelR, AvmA, also affects these processes. Deleting the gene impacts attachment, UPP deployment and virulence, suggesting that avmA is important for regulating the processes not under direct control of celR. The cellulose synthase subunit, CelA, contains a c-di-GMP-binding PilZ domain, which is necessary for the complex to produce the polymer in response to the signal molecule. Deleting divK, the first gene of a two-gene operon with celR, also reduced cellulose production. This requirement for the response regulator was alleviated by expressing a constitutively active form of CelR, suggesting that the DGC must be activated and that DivK acts upstream of this event. A constitutively-active form of DivK also results in increased cellulose production, but only in the presence of wild-type CelR. Based on bacterial two-hybrid assays, CelR forms homodimers but does not interact directly with DivK. In Caulobacter crescentus, DivK is required for polar localization of cell division components; mutants of divK form branched and elongated cells. Similarly, deleting the response regulator in A. tumefaciens results in branched and misshapen cells. The phenotype is fully corrected by complementation with divK, but not with wild-type celR or the constitutively active form of the DGC. These results support our hypothesis that the function of CelR is restricted to stimulating cellulose synthesis, and that the DivK/CelR signaling pathway in Agrobacterium separates production of the polymer from cell cycle checkpoints mediated by DivK.
Issue Date:2014-09-16
Rights Information:Copyright 2014 David Barnhart
Date Available in IDEALS:2014-09-16
Date Deposited:2014-08

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