Date of Award

6-1-2015

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

Adan Colon-Carmona

Abstract

Sinorhizobium meliloti is an Alphaproteobacteria that grows as a free-living organism in soil or as a symbiont within legumes. As a free-living organism, S. meliloti undergoes an asymmetrical cell division with once-and-only-once DNA replication per cell cycle; however, inside the plant, the bacterium differentiates into a bacteroid through a novel cell cycle pathway of endoreduplication without cell division. The goal of my research was to understand how a DivK-dependent two-component signaling (TCS) pathway regulates these distinct cell cycle outcomes. Based on the Caulobacter crescentus model, I hypothesized that this TCS pathway requires the histidine kinases (HKs) PleC and DivJ, as well as a novel histidine kinase CbrA, to directly regulate phosphorylation of the response regulator DivK and the activity of its downstream target CtrA. CbrA is required for symbiosis and cell cycle progression and I first discovered that a cbrA null mutant is unable to properly regulate CtrA resulting in increased levels. My focus was to understand the mechanisms that contribute to regulation of CtrA activity. CtrA depletion and overexpression showed that CtrA is required for proper cell morphology, motility, and cell wall integrity. While many of the known C. crescentus regulators of CtrA phosphorylation and proteolysis are phylogenetically conserved within S. meliloti, the latter lacks the regulator PopA that is required for CtrA degradation. Therefore, I studied CtrA stability in vivo and found that CtrA is unstable during exponential growth and that its stability is regulated in a CbrA-dependent manner. CtrA degradation in C. crescentus requires that CpdR direct CtrA to the ClpXP protease. I therefore investigated the contribution of the S. meliloti ortholog CpdR1 to CtrA regulation. Constitutive ectopic expression of a CpdR1 variant that cannot be phosphorylated restores CtrA instability to the cbrA mutant at wild type levels and also suppresses all known cbrA mutant phenotypes. These observations demonstrate that CtrA is important to the S. meliloti cell cycle and its stability is regulated by CbrA and CpdR1. By characterizing free-living cell cycle regulation via CbrA, CtrA, and CpdR1 I addressed how host symbiosis modifies the canonical free-living cell cycle program.

Comments

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