Date of Award

12-31-2013

Document Type

Campus Access Thesis

Degree Name

Master of Science (MS)

Department

Biology

First Advisor

Katherine E. Gibson

Second Advisor

Michael P. Shiaris

Third Advisor

Linda Huang

Abstract

Sinorhizobium meliloti is an emerging model for the study of host-microbe interaction as it exists in two forms: free-living in the soil or symbiotically with legumes. The S. meliloti life stages are marked by a shift in cell cycle regulation where the free-living cell replicates its genome once per cell cycle while the symbiotic cell generates up to 24 copies of its genome without dividing. This massive genomic expansion, called endoreduplication, culminates in an exit from the cell cycle and initiation of nitrogen-fixation. I have found the histidine kinase, CbrA, to be important in several free-living cell cycle-related processes. Deleting cbrA causes an overproduction of the symbiotic exopolysaccharide, succinoglycan, loss of motility, cellular membrane disruption, dysregulation of DNA replication from cytokinesis, and severe morphological and symbiotic defects. In the closely related, and strictly free-living, organism Caulobacter crescentus the essential response regulator CtrA has been shown to regulate many cell cycle genes. In C. crescentus CtrA activity and stability is modulated by a complex mechanism that includes the HKs DivJ and PleC and their cognate response regulator, DivK. Polar localization is required for optimal DivK function and is reliant upon DivK's phosphory-lation state. In S. meliloti, I find loss of CbrA results in a significant decrease in DivK localization and an increase in CtrA levels. The cbrA mutant exhibits an increase in the proportion of cells with an aberrant genome complement, but I observe no drastic genome expansion when comparing the mutant to wild type, suggesting a cell division defect, rather than DNA overinitiation, is more likely to account for the filamentous morphology of mutant cells. These results allow the suggestion of a model in which CbrA functions as a DivK kinase in a regulatory network that modulates CtrA activity to elicit transcriptional changes necessary for proper cell cycle progression during both free-living and symbiotic development. Interestingly, potential extragenic suppressors of cbrA have been isolated that are observed with a reversion to several WT free-living phenotypes as well as partial suppression of symbiosis defects; the suppressors can elicit WT nodules, potentially identifying an important cell cycle component of symbiosis development.

Comments

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