Studies on membrane synthesis in Bacillus megaterium KM

Abstract

Radioactive palmitic acid was used to label specifically the cell membrane of exponentially growing Bacillus megaterium KM. The purified membranes contained approximately 80% of the total incorporated radioactivity. Extraction of the membrane lipids with 90 % aqueous acetone and 2:1 chloroform-methanol, and subsequent separation by column and thin-layer chromatography, demonstrated that the incorporated label was primarily in membrane phospholipid. Turnover and metabolism of membrane lipid was found to be small with respect to lipid synthesis in logarithmically growing cells. The site of lipid incorporation into the membrane was studied by two radioautographic techniques. In the first, the distribution of silver grains in whole cells and in half cells was determined for both pulse-labeled and fully-labeled cells. The experimental data were compared with theoretical distributions. The half-cell distribution was symmetric for fully-labeled cells but was highly asymmetric for pulse-labeled cells. Both whole cell distributions approximated Poisson distributions. The asymmetric pulse-label distribution was further examined by serial section radioautography. Analysis of the silver-grain distribution in the first four cell sections from the end of the cell suggests a site of lipid incorporation with the first 0.2μ of the cell end (the limit of resolution of the experiment). The distribution of membrane in cell division was studied by growth of fully-labeled cells on agar-coated microscope slides followed by radioautography. Membrane growth was found to be primarily end-localized, confirming the pulse-label experiment. The membrane lipid was also found, in general, to be conserved in cell growth and division.