Food Science and Nutrition
Camberg, Jodi L.
Cell and Molecular Biology
MinD; MinC; FtsZ; Oscillation; E. coli; Molecular Biology
Over two-million people in the United States are infected by antibiotic resistant bacteria each year. Of this number 23,000 die from these infections and other complications. Due to this, novel antibiotic targets are constantly being investigated. One process in prokaryotes that holds promise is cellular division. Bacterial cells grow and reproduce using a series of proteins known as the cell division machinery. This machinery enables the division of the parental cell into two identical daughter cells. The cell division machinery is similar between bacterial taxa, making it an ideal target for new classes of antibiotics. Therefore, understanding the molecular mechanisms driving bacterial cell division will provide enormous potential in the search for new antibiotic targets. In actively dividing cells, the cytoskeletal protein FtsZ drives the formation of a division septum at the cell center. FtsZ forms a large protein structure called the Z-Ring. Protein interactions between FtsZ in the Z-Ring and additional cell division proteins facilitate the cleavage of the cell. Normal division and growth require it to form at the center. Mislocalization will result in abnormal growth, which often results in cell death. Many different proteins interact with FtsZ in order to ensure correct placement. In Escherichia coli, the position of the Z-Ring is regulated by the Min system, which includes the proteins MinC, MinD, and MinE. The Min system inhibits the formation of the Z-Ring at the cell poles by oscillating between each pole. MinD utilizes cellular energy in the form of ATP (adenosine triphosphate) to bind to the membrane. MinE binds to MinD, releasing it from the membrane. MinC oscillates between the cell poles by binding to MinD and directly inhibits the formation of the Z-Ring.
The Min system is a candidate for investigation into novel antibiotic targets. To investigate Min oscillation, we tagged MinD with a fluorophore and monitored oscillation in vivo. We constructed a strain of E. coli that had the gene for green fluorescent protein fused to the beginning of the minD gene. This allows us to observe the cellular localization by fluorescence microscopy. Since specific mutations in MinC were previously reported to modify MinC oscillation, we further tested if the mutations also modify MinD oscillation. To do this, we deleted the minC gene and are in the process of inserting modified minC genes back into the chromosome (min C S16D and L194N). S16D is a Serine that has been replaced by an Aspartic Acid at the sixteenth position. L194N is a Leucine that has been replaced by an Asparagine at the one-hundred and ninety forth position. These substitutions have both previously been associated with slower oscillation rates. They have been studied in terms of MinC and we wish to now monitor oscillation of Gfp-MinD in the presence of MinC mutations. These studies will lead to a further understanding of the essential process of cell division and identify critical steps that may be susceptible to antibiotics.