Cycle Regulation in the Symbiont Sinorhizobium Meliloti

Gibson, Katherine

Abstract

My lab studies a regulatory network utilized by bacteria to effect an orderly progression of cell cycle events in a manner that is capable of producing differentiated cell types and is sensitive to environmental conditions. The underlying assumption of this proposal is that a mechanistic knowledge of bacterial cell cycle controls is essential to understanding how these networks evolve in response to distinct lifestyle pressures. Our broader goal is to understand how host invasion and chronic colonization impinge upon the canonical bacterial cell cycle. To this end, we study Sinorhizibium meliloti because it can colonize the soil rhizosphere as a free-living bacterium and invade the roots of leguminous plants as a symbiont to establish a chronic intracellular infection. We began our studies by examining the function of four two-component sensor histidine kinases: SmCbrA, SmCbrB, SmDivJ and SmPleC (as a group referred to as """"""""HKs""""""""). These HKs are predicted to be positioned at the top of a signal transduction pathway central to regulating S. meliloti cell cycle progression and asymmetric cell division. Preliminary data from my lab demonstrates that disruption of each HK gene produces a cell cycle defect that is reflected morphologically in aseptal filamentous growth. Our flow cytometry results further demonstrate that each HK mutant is unable to properly coordinate DNA replication initiation with cytokinesis. Thus, our preliminary data implicate HKs in cell cycle control and we will further dissect their mechanistic role in the S. meliloti cell cycle through a combination of genetic, cell biological and biochemical experiments. In order to assess gene activity and protein localization as a function of cell cycle progression, we are generating a temperature-sensitive conditional allele of DNA polymerase III holoenzyme (Pol III), encoded by SmdnaE, that will allow DNA replication elongation but block DNA replication initiation at the restrictive temperature of 37(C. We hypothesize HKs function upstream of SmDivK to regulate its phosphorylation status, which is known to impact its cellular localization. We are performing fluorescence microscopy to assess the effect HK mutations have on SmDivK-GFP localization as a measure of their potential impact on the level of SmDivK~P. We will further explore whether SmDivK is a direct target of HK enzymatic activity by performing in vitro assays and thereby examine histidine kinase autophosphorylation, as well as SmDivK phosphorylation and dephosphorylation.
We aim to determine the precise timing of HK function during cell cycle progression. We will isolate conditional cold-sensitive HK alleles in order to rapidly deplete HK function at the non-permissive temperature and thereby pinpoint its precise temporal requirement during cell cycle progression. We will quantify a variety of cell cycle events, including DNA replication initiation, SmDivK-GFP localization, formation of the cell division apparatus, and asymmetric cytokinesis. Taken together, our studies will allow us to construct a model for HK timing and mechanistic control over the cell cycle.

Public Health Relevance

The cell cycle is a fundamental process required for growth, reproduction, and developmental differentiation in all living organisms. Moreover, it is becoming clear that modification of the bacterial cell cycle occurs during chronic host colonization in a variety of species, including our model organism Sinorhizobium meliloti. Insights gained from our analysis of cell cycle regulation in S. meliloti will provide a strong basis for dissecting modes of cell cycle control in other host-associated bacteria, and can thereby be applied towards development of novel targets for antimicrobial therapeutics at a time when resistance to ?-lactams and aminoglycosides is found in a variety of potent pathogens.