Cell Lysis and Consumption during Cannibalistic Growth of Bacillus subtilis 168

Abstract

Bacillus subtilis is a Gram-positive organism that forms long-lasting endospores under unfavorable environmental conditions, such as nutrient limitation. As sporulation is a time and energy-consuming developmental process, B. subtilis seeks to delay entry into sporulation until absolutely necessary. In a survival strategy called cannibalism a subpopulation of cells, the “cannibals”, secrete two proteinaceous toxins, SKF and SDP, to attack sibling cells. These toxins induce cell death and lysis in the prey cell, which involves endogenous, cell wall lytic enzymes (i.e. autolysins). Ultimately, the cannibals feed on the nutrients released by the decay of prey cells and sporulation of B. subtilis is delayed. Thus, a cell population is sacrificed for the sake of survival of the species. The first aim of this study was to investigate the role of autolysins in cannibalism and to identify which autolysins are required for prey lysis. The focus was set on the major autolysins of B. subtilis LytC, LytD, LytE, and LytF as it was reported that a mutant lacking all four autolysins fails to lyse during sporulation. Cannibalistic behavior was monitored with agar diffusion experiments using toxin-expressing and autolysin-defective cells as well as isolated SDP toxin, along with fluorescence microscopy of labeled strains. The role of specific autolysins was shown to be highly dependent on preculturing conditions, and no particular autolysin appeared to be absolutely essential. The second aim was to investigate the flow of nutrients from lysed prey cells to the cannibals, with an emphasis on the salvage of cell wall fragments. Therefore, a cell wall labeling strain was generated in this study, which can serve as a tool to study cell wall decay and consumption during cannibalism. B. subtilis degrades and recycles its own peptidoglycan cell wall also during vegetative growth, when the cell wall undergoes a steady remodeling and turnover. Known to be important in this process is the N-acetyl¬muramic acid (MurNAc) recycling operon, which encodes the genes required for the salvage of the cell wall sugar MurNAc. In this operon next to the exo-N-acetylgluco¬saminidase nagZ and the N-acetylmuramyl-L-alanine amidase amiE, a gene of previously unknown function ybbC (namZ) is located. We identified ybbC as the coding gene for a novel peptidoglycan recycling enzyme that functions as unique exo-lytic β-N-acetyl¬muramidase (named NamZ). The enzyme was characterized by HPLC-MS analyses of culture supernatants of B. subtilis mutants, showing differences in the cell wall fragment releases as well as fragment release upon peptidoglycan digestions using combinations of purified NagZ, AmiE and NamZ enzymes. A biochemical characterization using the chromogenic substrate pNP-MurNAc as well as the purified minimal natural substrate MurNAc-GlcNAc, reveled that NamZ is identical to an enzyme reported in earlier studies ((Del Rio and Berkeley 1976); DOI: 10.1111/j.1432-1033.1976.tb10382.x) that was not linked to a coding gene. NamZ is the founding member of a novel family of glycosyl hydrolases, now classified as GH171 within the “carbohydrate-active enzymes” CAZy-database (www.cazy.GH171.html) and involved in the sequential hydrolysis of peptidoglycan from the non-reducing end, in concert with the exo-lytic N-acetylglucosaminidase NagZ and the exo-acting amidase AmiE. The role of NamZ and the entire MurNAc recycling cluster during sporulation still remains enigmatic.

Die Dissertation ist gesperrt bis zum 06. Mai 2026 !