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Completed Project

Investigating the Elongated Cell Phenotype of Escherichia coli Overexpressing the Lysophospholipase PldB

George R. Georgiou ’18 and Teresa Garrett (Chemistry)

The overexpression of the enzyme PldB leads to increased levels of the headgroup-acylated glycerophospholipid, acyl phosphatidylglycerol (acyl PG), in the membranes of Escherichia coli. The role of acyl PG in E. coli has yet to be fully determined. Interestingly, PldB overexpression leads to an elongated phenotype, suggesting a role for acyl PG in cell length and division. To gain a better understanding of this phenotype, wildtype MG1655 and cells lacking the chromosomal pldB ( pldB) were transformed with an empty arabinose-inducible plasmid (pBAD), and a plasmid containing pldB (pPldB). These four strains (MG1655/pBAD, MG1655/pPldB,  pldB/pBAD, and  pldB/pPldB) were grown to mid-log phase and imaged via scanning electron microscopy. Quantification of cell lengths from SEM images showed a significant difference in the average length of the wild-type strain, MG1655/pBAD (1.85µm ± 0.46µm), versus MG1655/pPldB (2.69µm ± 1.12 µm) and  pldB/pPldB (3.02µm ± 2.42µm). Furthermore, many of the elongated cells appeared to contain no cleavage furrows suggesting that they failed to complete cellular division. In the future, visualization of chromosomal DNA using fluorescence microscopy will confirm if elongated cells result from incomplete binary fission. As the elongated phenotype correlates with a change in membrane lipid composition, we also attempted to separate the inner and outer membranes via isopycnic centrifugation to determine the localization of acyl PG in PldB overexpressors. Total protein and NADH oxidase activity of membrane fractions were used to assess separation. The total protein assay showed one distinct protein peak instead of two peaks (one corresponding to each membrane), and the NADH oxidase activity did not associate with the expected location of the inner membrane. More work must be done to effectively separate the membranes. Following a successful separation of the membranes, lipidomes will be assessed via mass spectroscopy.