Globin Coupled Sensor Signaling in P. carotovorum: A Model of Biofilm Regulation through Oligomerization of Diguanylate Cyclases Open Access

Joynt, Shawnna Christin (2013)

Permanent URL: https://etd.library.emory.edu/concern/etds/4b29b630z?locale=en
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Abstract

Bacterial infestation of plants by Pectobacterium carotovorum ssp carotovorum (P. carotovorum), commonly called soft rot, creates world-wide devastation of many crops. Studies have shown that P. carotovorum grows rapidly in a more hypoxic state and becomes less virulent in aerobic environments. These studies, however, do not consider how oxygen may be involved in regulating virulence by switching the bacteria from an actively growing state to a sessile biofilm and whether that switch is due to oxygen sensitive globin coupled sensors (GCS). Diatomic gases can bind to the heme domain within a GCS to activate or turn-off the protein's catalytic domain. GCSs with diguanylate cyclase activity studied to date produce the secondary signaling molecule bis-(3'-5')-cyclic diguanosine monophosphate (c-di-GMP), which is positively associated with biofilm growth. Therefore, we hypothesize that a globin coupled sensor within P. carotovorum (PccGCS) has a direct role in the regulation of biofilm formation. This study characterizes the basic biochemical properties and mechanism of PccGCS regulation. We show that PccGCS is an active response regulator that may use its globin domain to induce dimerization and subsequent activation of its diguanylate cyclase domain.

Table of Contents

Table of Contents

Introduction…………………………………………………………………………….. 1

P. carotovorum Infection……………………………………………….………. 1

Diguanylate Cyclase Domains……………………………………….…………. 2

Heme-Binding Signal Transducers……………………………….………..… 3

Globin Coupled Sensors………………...……….……………………..……….. 4

Results and Discussion……………………………………………………………… 8

Discovery and Activity of PccGCS………………………………………..... 8

Sequence Alignment…………………………………………………………...…… 8

Initial Characterization…………………………………………………….………. 9

Initial Activity…………………………………………………………………......… 11

Optimized Expression………………………………………………………...…… 12

Enzyme Coupled Assay…………………………………………………..….…… 13

Oligomerization……………………………………………………………..…………. 19

Native PAGE Gel Analysis………………………………………………...……… 19

Distributions of Oxygen Bound and Unligated Heme States….… 20

Role of the His Tag……………………………………………………………....… 23

Distributions of R364A BpeGreg…………………………………….………... 24

Conclusion and Future Directions……………………………………………. 27

Experimental Methods……………………………………………………………... 31

References………………………………..……………………………………………… 40

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