This project deals with the question of how bacteria grow and synthesize their cell walls. This laboratory has determined the rate and topography of cell wall peptidoglycan synthesis during the division cycle of gram- negative, rod-shaped bacteria. Prior to invagination, wall growth occurs only in the cylindrical side wall. After initiation of invagination, peptidoglycan synthesis is partitioned between the new pole and the lengthening side wall. The biochemical basis for the partitioning of peptidoglycan synthesis and the manner in which the cell initiates constrictions are unknown. Reverse-phase HPLC methods (which can resolve up to eighty different fragments in a peptidoglycan digest), combined with the membrane-elution method for cell cycle analysis, will be used to study the composition and biosynthesis of the peptidoglycan made at different times during the division cycle. The organism to be studied, Salmonella typhimurium, has been shown in this laboratory to incorporate diaminopimelic acid, a specific precursor of cell wall, 30-50 times more efficiently than any strain of Escherichia coli. The main questions to be answered are whether, when side-wall and poles are compared, there is a compositional difference, a difference in crosslinking, or a difference in maturation of the peptidoglycan. Autoradiographic analysis using the electron microscope will be used to measure the precise pattern of pole synthesis. Previous work from this laboratory has also shown that there is evidence for the insertion of an excess amount of membrane at the site of a future pole in a daughter cell. This insertion may be analogous to the periseptal annuli which are proposed to be the initial events in pole formation. The pattern of membrane synthesis during the division cycle will be measure using the membrane-elution technique. The topography of insertion of surface material during the division cycle will be analyzed using the methocel-segregation method. The stability and turnover of peptidoglycan will also be studied. Evidence from this laboratory based on the kinetics of elution of radioactivity from cells labeled with leucine and diaminopimelic acid indicates that in Salmonella typhimurium, there is no measurable turnover resulting in the release of radioactivity to the medium. In Escherichia coli, studies from other laboratories using standard techniques have revealed a great deal of turnover. Both the membrane-elution method and the standard method will be used to analyze the stability of peptidoglycan in different bacteria under identical conditions.
