Mechanisms of the Killing and Development of Spores of Bacillus Species

Abstract

Abstract : Notable findings in the final year of support include the following. 1) The proteins that comprise the mechanosensitive channels were fused to Green Fluorescent Protein (GFP) and localized in Bacillus subtilis cells. All of these proteins were in the plasma membrane, and at least two may be in a helical array. 2) The phospholipid composition of B. subtilis spores' inner membrane could be altered drastically without large effects on spore resistance or germination. 3) Spores of a cotE gerE mutant lacked most coat layers but retained a thin layer of insoluble coat proteins. 4) A major reason for the effort to prepare and characterize the cotE gerE spores was that it was hoped that these spores would be: a) generally stained less well by hydrophobic stains that often bind non-specifically to the spore's outer layer(s), presumably by adsorbing to spore coats; stains in this category include nucleic acid stains such as DAPI and acridine orange, as well as various membrane potential-sensitive dyes; and b) less autofluorescent. 5) Dormant B. subtilis spores give a distinctive X-ray scattering pattern. We used spores of mutants lacking the DNA protective alpha/beta-type small, acid-soluble spore proteins, as well as cotE, gerE and cotE gerE spores to show that one or more coat proteins under the direct or indirect control of the GerE transcription factor is responsible for the X-ray scattering. 6) We studied the physical state of water in B. subtilis spores. This work led to three major conclusions: a) the water in the spore core is not in a glass-like state; b) proteins in the spore core are rotationally immobilized; and c) the rate of permeation of water across the spore's inner membrane is at least several orders of magnitude slower than rates of permeation across a growing cell membrane.