Date of Award

8-31-2014

Document Type

Open Access Thesis

Degree Name

Master of Science (MS)

Department

Biology

First Advisor

Katherine E. Gibson

Second Advisor

Alexey Veraksa

Third Advisor

Michael P. Shiaris

Abstract

Sinorhizobium meliloti is a Gram-negative alphaproteobacterium and nitrogen-fixing symbiont, which undergoes a novel cell cycle modification during its' host-microbe interaction. I intend to monitor the transcriptional regulation of cell cycle-related genes during free-loving growth, in addition to monitoring their expression during symbiosis. Using genes known to be regulated by CtrA in C. crescentus or predicted to be regulated by CtrA in S. meliloti, I aim to show how certain cell cycle genes are regulated in S. meliloti. In C. crescentus, CtrA acts as a transcription factor that is active when phosphorylated and inactive when not phosphorylated. In S. meliloti, CbrA is a histidine kinase that ultimately inhibits CtrA phosphorylation. Using a ΔcbrA null mutant, which leads to increased levels of CtrA in S. meliloti, and the β-glucuronidase (GUS) reporter gene, I can monitor the expression levels of target genes that are potentially regulated by CbrA and CtrA. Promoter regions, transcription start sites, and translation start sites of target genes have been cloned into the plasmid pVO155 upstream of the GUS gene. I measured the GUS enzymatic activity using the 4-methylumelliferyl-beta-D-glucuronide (MUG) substrate. Additionally, after infecting Medicago sativa seedlings with these fusions strains, I used a different GUS substrate to test for the presence of target gene expression in root nodules. Results thus far have shown some target genes with large differences in expression coinciding with the absence of cbrA and increased CtrA levels while some target genes show only slight differences, if any at all. Tracking the expression location and patterns of target genes in root nodules has shown that some genes are expressed ubiquitously throughout the nodule while other genes are expressed in specific locations. These results are significant because no one has looked at genes regulated by CbrA or CtrA in S. meliloti, which is more applicable to host-microbe interactions than C. crescentus, especially since Agrobacterium tumefaciens and Brucella abortus both have a CbrA homologue. Additionally, I will provide critical insight into the molecular biology of the S. meliloti host-microbe interaction.

Download

Share

COinS