Defense Date

2011

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Integrative Life Sciences

First Advisor

Marconi Richard

Abstract

Treponema denticola levels in the gingival crevice become elevated as periodontal disease develops. Oral treponemes may account for as much as 40% of the total bacterial population in the periodontal pocket. The stimuli that trigger enhanced growth of T. denticola and the mechanisms associated with the transmission of these signals remain to be defined. A hypothesis was set that the T. denticola ORFs tde1970 (histidine kinase) and tde1969 (response regulator) constitute a functional two component regulatory system that regulates, at least in part, responses to the changing environmental conditions associated with the development of periodontal disease. The results presented demonstrate that tde1970 and tde1969 are conserved, universal among T. denticola isolates and transcribed as part of a 7 gene operon in a growth phase dependent manner. Tde1970 undergoes autophosphorylation and transfers phosphate to Tde1969. Henceforth the proteins encoded by these ORFs are designated as Hpk2 and Rrp2 respectively. Hpk2 autophosphorylation kinetics was influenced by environmental conditions and by the presence or absence of a Per Arnt Sim (PAS) domain. It can be concluded that Hpk2 and Rrp2 constitute a functional two-component system that contributes to environmental sensing. This study also sought to determine the molecular basis of Hpk2 function in response to environmental stimuli. Hpk2 was shown to bind hemin via a putative heme-binding domain within the PAS domain. Hemin binding to Hpk2 positively regulated its autokinase ability under anaerobic conditions, suggesting that Hpk2 activation may play a role in the migration of T. denticola away from the aerobic zone deeper into developing periodontal pockets. In this study we have generated point mutations of conserved amino acid residues in the sensor PAS domain of Hpk2 and assessed their role in kinase activation under both aerobic and anaerobic conditions depending on their oligomeric state, hence providing a strong basis to correlate ligand binding, kinase activity and oligomeric states of the protein that may provide stability of these complex interactions. Ultimately this study provides a comparative linkage between the responses of PAS domain to sensory inputs controlling access to its kinase domain within which is contained the dimerization domain, which ultimately leads to fine-tuned control of interactions between Hpk2 dimerization and catalytic domain.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

September 2011

Included in

Life Sciences Commons

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