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Allyssa Miller

Allyssa Miller

Advisor: Dr. Jennifer Herman

Department of Biochemisty & Biophysics

Biography:

Entered program in 2014

Research Project:

Upon nutrient starvation, Bacillus subtilis enter the developmental program of sporulation, resulting in the formation of a resting cell type called a spore. Members of the Bacillus genus are polar spore-formers; during sporulation these bacteria shift midcell division to the cell pole, where division occurs over DNA to create two asymmetrically sized compartments: the larger mother cell and the forespore. As a consequence of polar division ~30% of the forespore chromosome becomes captured inside the smaller compartment, promoting essential differential gene expression between the forespore and mother cell. Recently, we identified a DNA-binding protein, RefZ (Regulator of FtsZ) that is expressed during early sporulation. Cells lacking RefZ capture regions of the chromosome inside the forespore that are normally found inside the mother cell. RefZ binds five 20-bp, nearly palindromic sequences called RBMs (RefZ Binding Motifs). When mapped to chromosome of B. subtilis, the RBMs lie symmetric about the origin of replication with two sites at ~-40° (left arm), a single site at the origin and two sites at ~+40° (right arm). The location of the left and right arm RBMs lie adjacent to the boundaries of the chromosomal region captured by the polar septum, suggesting that they may play a role in defining where the polar septum forms during sporulation. We found that cells mutant for the five RBMs were defective in capturing the forespore chromosome similar to refZ mutants, indicating that RefZ bound to the RBMs may play a role in effectively organizing the chromosome during polar division. Bioinformatics analysis revealed that both refZ and RBMs are conserved across the Bacillus genus. Surprisingly, RBM sequences are not simply present throughout Bacillus, but their locations on the chromosome with respect to the origin of replication are also conserved in a manner that is independent of open reading frame or regulatory sequence conservation. These data suggest that the role of RefZ during sporulation is highly dependent on the arrangement of the RBMs on the chromosome. Currently, my work is concentrating on using thorough bioinformatics (skills learned in a highly recommended class taught by Dr. James Cai) to delineate RBM sequence and positional conservation from standard Bacillus phylogenic reconstruction methods, in addition to mutational analysis of refZ and RBM mutants with other known players involved in chromosome organization during sporulation.

Broader Impacts of Research Project:

Unlike their eukaryote counterparts, bacteria lack the compartmentalization.  As a consequence, bacteria like the model Gram-positive Bacillus subtilis rely heavily sub-cellular organization of the two largest structures of the cell, the cell envelope and the nucleoid, to direct and regulate major cellular processes. My work focuses on how B. subtilis uses the nucleoid as primary determinant for localizing and mediating these processes, specifically cell division during the sporulation. Division during sporulation highlights how extensively bacteria may rely on the nucleoid for positional information, as polar division occurs over a precise region of one chromosome.  We have identified short DNA sequences, or RBMs, in B. subtilis that are bound by RefZ (Regulator of FtsZ) and help ensure proper chromosome organization during polar division. We found that cells mutant for either RefZ or the RBMs were defective in capturing the forespore chromosome. Bioinformatics analysis revealed that both refZ and RBMs are conserved across the Bacillus genus. Surprisingly, RBM sequences are not simply present throughout Bacillus, but their locations on the chromosome with respect to the origin of replication are also conserved in a manner that is independent of open reading frame or regulatory sequence conservation. When we analyze the number of RBM sequences in each Bacillus species, we find that pathogenic species, including B. anthracis, B. thuringiensis, and B. cereus, have a higher abundance of sites compared to non-pathogenic Bacillus. This finding suggests a possible evolutionary role for the additional RBM sites in mediating virulence of these pathogenic species. Currently, my work is concentrating on using thorough bioinformatics to delineate RBM sequence and positional conservation from standard Bacillus phylogenic reconstruction methods. My goal is to reconstruct a phylogenic tree that helps us visualize how the RBMs have evolved in their arrangement on the chromosome with respect to their surrounding genetic sequences, and if/ how the density of RBMs correlates to the acquisition and evolution of pathogenicity. The findings of these analyses may provide unparalleled insights into how bacteria in general evolve their genomes around specific DNA sequences to ensure they remain viable positional determinants in regulating essential cellular processes.