Phenotypic Characterization of psd and pss Mutant Strainsof Escherichia coli
Oluwadamilola Oke, Vassar College ’17 and Prof. Teresa A. GarrettEscherichia coli contains a variety of glycerophospholipids (GPLs). Phosphatidylethanolamine (PE) is particularly important as it is the only zwitterionic phospholipid and makes up 70-80% of total GPLs. Phosphatidylserine synthase (Pss) condenses CDP-diacylglycerol and serine to form phosphatidylserine (PS) and releases cytidine monophosphate. PS is a short-lived intermediate that is rapidly decarboxylated to PE by the phosphatidylserine decarboxylase (Psd) enzyme. This study investigates the impact of altering head group acylated GPLs has on E. coli that are deficient in Pss and Psd using three genes of interest: pldB and pagP, which converts phosphatidylglycerol to acyl phosphatidylglycerol (acyl PG) and At1g78690, a lyso GPL acyltransferase that, when overexpressed leads to the accumulation of acyl PG. We have obtained a strain of RA2000 E. coli with the pssA21 mutation (strain RA2021), a lesion that causes the Pss enzyme to be temperature sensitive. We have also obtained a strain of ES4 E. coli with the psd mutation (strain EH150), a null mutation that causes the Psd enzyme to be temperature sensitive. Both mutations lead to decreased levels of PE in the cell membrane. To investigate the role of acylated GPLs we have transformed all four strains with plasmids containing pldB, pagP or At1g78690. A variety of cellular phenotypes, such as temperature sensitivity, cell morphology, antibiotic resistance, and lipid composition, were determined. Expression of At1g78690 promotes reversion of the temperature sensitive phenotype of the pssA21 mutant. Expression of pagP results in longer cells. Future experiments will use fluorescently-tagged proteins involved in cell division to determine if the increased cell length is the result of cell division defects promoted by accumulation of head group acylated GPLs.