An in vitro system for the biosynthesis of spore cortex peptidoglycan

Abstract

A simple crude system for the incorporation of tritiated diaminopimelic acid (3H-DAP) into a polymer with characteristics of spore cortical peptidoglycan has been obtained from cells of Bacillus cereus var. alesti, harvested and disrupted in a French pressure cell, at the time of late cortex formation (sporulation stage 5). The small fraction of whole cells remaining in the homogenate were not responsible for the observed incorporation. The radioactive product was sensitive to digestion by lysozyme as is cortex formed in vivo. Preparations obtained at the same time of sporulation, from a mutant unable to form cortex, were unable to incorporate 3H-DAP into peptidoglycan. However, homogenates prepared at the time of germ cell wall formation (early stage 4) from both parent and cortexless mutant produced radioactive peptidoglycan which was more resistant to lysozyme as is germ cell wall produced in vivo in this species. Bacitracin and vancomycin inhibited incorporation of 3H-DAP into peptidoglycan by over 90% in both cell wall and cortical preparations. Methicillin caused a striking inhibition of 3H-DAP incorporation into peptidoglycan by the cortical system in contrast to its almost total lack of inhibition of the cell wall or germ cell wall systems. Over 90% of the DAP incorporated was the expected meso-isomer. 3H-DAP-labelled lipid intermediate was produced by the system. Cross-linking of newly synthesized material was observed. This in vitro system appears satisfactory for the further study of some aspects of the biosynthesis of cortical peptidoglycan.