When a bacterial cell dies, the cause of death is often autolysis, rather than direct damage produced by a harmful factor. Hydrolysis of peptidoglycan is a necessary stage in cell wall synthesis, and autolysis has been viewed as a result of "disbalance between peptidoglycan synthesis and hydrolysis", essentially a maladaptive mistake. This project explores the hypothesis that autolysis is not a mistake, but adaptive programmed death. Autolysis is part of the developmental process of fruiting body formation and sporulation in Myxococcus. Autolysis which is required for natural transformation in S. pneumoniae is another example of specialized adaptive programmed death. Bacteria show many features of complex organization, similar to multicellular organisms. Like in multicellular species, a bacterial population would benefit from eliminating defective cells. The aim of this project is to test the feasibility of the idea that autolysis is the mechanism of adaptive apoptosis in bacteria. Specifically, a search for regulatory components of a possible apoptotic pathway linking particular types of cell damage to activation of autolysins is being undertaken. A genetic approach is being used to identify putative apoptotic genes. E. coli will be mutagenized with mini-Tn10 transposon and selected for survival to lethal levels of a mutagen; and high temperature. Mutants that show resistance to killing (but not resistance to growth in the presence of) both factors will become candidates for being affected in genes coding for apoptotic compounds. "Random" PCR will be used to identify the flanking DNA regions, amplified DNA will be sequenced by single passage, and the genes carrying the Tn insertions will be identified using the database of the E. coli genome. The mutants will also be characterized phenotypically using a panel of different lethal factors. Strains with known mutations, such as those lacking autolysins, will also be tested for possible survival with the panel of lethal factors. This exploratory project will show whether E. coli has regulatory genes affecting autolysis and will lay the basis for future detailed studies of apoptosis in bacteria.
