Date of Award

12-31-2017

Document Type

Campus Access Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology/Molecular, Cellular, and Organismal Biology

First Advisor

Katherine E. Gibson

Second Advisor

Alexey Veraksa

Third Advisor

Adán Colón-Carmona

Abstract

Sinorhizobium meliloti grows either as a free-living bacterium in the soil, or as an intracellular symbiont within its legume host for the purpose of nitrogen fixation. During host colonization, novel changes occur to its cell cycle. In order to understand whether these cell cycle outcomes are required for host colonization, we must first understand how the free-living cell cycle is regulated. The goal of this research is to determine the role of the histidine kinase SMc04212, which we named MorA, in S. meliloti cell cycle regulation as bioinformatics predicts it functions within the CtrA pathway. In order to test this hypothesis, deletion strains ΔmorA and ΔcbrA ΔmorA were created and cell cycle phenotypes were analyzed. The ΔmorA mutation causes cells to have a significant motility defect and thus the protein has been named MorA for Motility regulator A. Phenotypic assays show that the ΔmorA single mutant does not present with obvious cell cycle defects, but it does display additive defects in cell cycle progression when combined with ΔcbrA. Additionally, constitutive ectopic expression of morA is able to suppress several ΔcbrA cell cycle defects and partially suppresses the symbiosis defect of a ΔcbrA mutant. This data indicates that CbrA and MorA possess a similar function, which was found to be cognate DivK kinase activity through an in vitro phosphotransfer assay. In order to better understand MorA function, a MorA pull-down assay followed by mass spectrometry was performed to identify potential interaction partners. Through this assay, it was found that MorA may interact with numerous proteins predicted to function in stress response pathways. While ΔmorA and ΔcbrA mutants individually did not exhibit growth defects in various stress conditions, the ΔcbrA ΔmorA double mutant exhibited synthetic growth defects when grown in temperature, osmotic, and pH stress. When levels of CtrA were examined in ΔmorA at low and high temperatures, it was found that CtrA levels were significantly lower than in wild type. This data supports the hypothesis that MorA may have a primary function in cell cycle regulation during environmental stresses in addition to its role in regulating motility under standard growth conditions.

Comments

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