Screen for Conditional Alleles of the Cell Cycle Histidine Kinase CbrA of Sinorhizobium meliloti

Sinorhizobium meliloti is a Gram-negative alpha-proteobacterium which normally grows as a free-living organism. However, while growing on nitrogen limiting soils, S. meliloti can also form a symbiotic relationship with certain genera of leguminous plants, which results in a plant organ: the nodule. During free-living growth, S. meliloti undergoes asymmetrical cell division with no greater than a 2N complement of its genome, which implies that the bacteria initiate DNA replication only once per cell cycle. However, S. meliloti undergoes a novel cell cycle while inside the plant host. The organism replicates its DNA multiple times but does not divide. Unfortunately, little is known about the S. meliloti cell cycle. Our laboratory found that the loss of one histidine kinase, CbrA, interrupts the normal coordination between DNA replication initiation and cytokinesis during free-living growth. Thus, I am interested in determining the precise role that CbrA plays in S. meliloti cell cycle coordination. My hypothesis is that conditional alleles of cbrA can be isolated and these mutations will illuminate the phase of the cell cycle that requires cbrA function.

Currently, I have screened 487 colonies and identified eight mutants. I am now testing their phenotype to determine whether they are conditional alleles. Once I confirm the phenotypes of my two calcofluor mutants, the cbrA allele will be sequenced to identify the point mutation responsible for the conditional phenotype. Ultimately, I will then be able to perform temperature-shift experiments and understand the role CbrA plays in the cell cycle.