Resistance, germination, and permeability correlates of Bacillus megaterium spores successively divested of integument layers.

A variant strain that produced spores lacking exosporium was isolated from a culture of Bacillus megaterium QM-B1551. Two additional spore morphotypes were obtained from the parent and variant strains by chemical removal of the complex of coat and outer membrane. Among the four morphotype spores, heat resistance did not correlate with total water content, wet density, refractive index, or dipicolinate or cation content, but did correlate with the volume ratio of protoplast to protoplast plus cortex. The divestment of integument layers exterior to the cortex had little influence on heat resistance. Moreover, the divestment did not change the response of either the parent or the variant spores to various germination-initiating agents, except for making the spores susceptible to germination by lysozyme. The primary permeability barrier to glucose for the intact parent and variant spores was found to be the outer membrane, whereas the barrier for the divested spores was the inner membrane.

Photometric immersion refractometry of bacterial spores.

Photometric immersion refractometry was used to determine the average apparent refractive index (n) of five types of dormant Bacillus spores representing a 600-fold range in moist-heat resistance determined as a D100 value. The n of a spore type increased as the molecular size of various immersion solutes decreased. For comparison of the spore types, the n of the entire spore and of the isolated integument was determined by use of bovine serum albumin, which is excluded from permeating into them. The n of the sporoplast (the structures bounded by the outer pericortex membrane) was determined by use of glucose, which was shown to permeate into the spore only as deeply as the pericortex membrane. Among the various spore types, an exponential increase in the heat resistance correlated with the n of the entire spore and of the sporoplast, but not of the isolated perisporoplast integument. Correlation of the n with the solids content of the entire spore provided a method of experimentally obtaining the refractive index increment (dn/dc), which was constant for the various spore types and enables the calculation of solids and water content from an n. Altogether, the results showed that the total water content is distributed unequally within the dormant spore, with less water in the sporoplast than in the perisporoplast integument, and that the sporoplast becomes more refractile and therefore more dehydrated as the heat resistance becomes greater among the various spore types.

Bacterial spore heat resistance correlated with water content, wet density, and protoplast/sporoplast volume ratio.

Five types of dormant Bacillus spores, between and within species, were selected representing a 600-fold range in moist-heat resistance determined as a D100 value. The wet and dry density and the solids and water content of the entire spore and isolated integument of each type were determined directly from gram masses of material, with correction for interstitial water. The ratio between the volume occupied by the protoplast (the structures bounded by the inner pericytoplasm membrane) and the volume occupied by the sporoplast (the structures bounded by the outer pericortex membrane) was calculated from measurements made on electron micrographs of medially thin-sectioned spores. Among the various spore types, an exponential increase in the heat resistance correlated directly with the wet density and inversely with the water content and with the protoplast/sporoplast volume ratio. Altogether with results supported a hypothesis that the extent of heat resistance is based in whole or in part on the extent of dehydration and diminution of the protoplast in the dormant spore, without implications about physiological mechanisms for attaining this state.

Synergism in the inhibition of Bacillus subtilis by combinations of lipophilic weak acids and fatty alcohols.

Combinations of two lipophilic weak acids and alcohols acted synergistically to inhibit growth of Bacillus subtilis. Alcohols contributed to both reversible and irreversible inhibitions by acids.

Effects of growth temperature on protoplast membrane properties in Bacillus megaterium.

Changes in the protoplast membrane of the KM strain of Bacillus megaterium were assessed after growth at 20, 30, or 37 degrees, C. Although the overall membrane concentrations of lipids and proteins were virtually unchanged, increased culture temperature resulted in cells with membranes that contained relatively more unbranched and long-chain fatty acids and more acidic phospholipids, as well as different proportions and numbers of individual proteins. Electrophoretic analysis revealed 23, 31, or 29 protein bands, respectively, in membranes from cells grown at the three temperatures. Protoplasts from cells grown at higher temperatures were considerably less susceptible to lysis by shearing forces. As judged by passive leakage at 30 degrees C, intact cells from cultures grown at 37 degrees C were the least permeable to erythritol. Relatively low ambient concentrations of Ca2+ or Mg2+ protected protoplasts from osmotic lysis but even much higher concentrations left erythritol leakage virtually unaffected. Thus, growth temperature affected not only membrane lipis but also membrane proteins and these changes resulted in membranes with altered mechanical properties and permeabilities.

Distribution and effects of 2,4,5-trichlorophenoxyacetic acid in cells of Bacillus megaterium.

Cell death in a resting population of an asporogenous Bacillus megaterium was accelerated by ambient concentrations of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) equal to or greater than 10 mug/ml or 5 mug/mg of cells (dry weight), but only after prolonged exposure. Conversely, populations of growing cells were not markedly influenced even at 100 mug/ml. Effects on cell respiration were not manifest until the ambient concentration reached 1,000 mug of 2,4,5-T/ml, or 500 mug/mg. Cells of B. megaterium did, however, accumulate 2,4,5-T passively to a level approximately twofold above the ambient concentration. Most of the accumulated compound was easily washed from the cells, but, of the firmly bound herbicide, about 0.5 mug/mg of cells (dry weight), nearly 60% by weight, was localized in the protoplast membrane. The foregoing results, obtained with a purified preparation of 2,4,5-T were also elicited by 2,4,5-T analytical standards. The extracted contaminants did not produce the results alone nor did they influence the results when present in combination with 2,4,5-T.

Antimicrobial actions of hexachlorophene: inhibition of respiration in Bacillus megaterium.

Hexachlorophene (HCP) inhibits both endogenous and exogenous respiration (oxygen uptake) in Bacillus megaterium, without sparing by any of several substrates. The inhibition is maximal when the cells are treated with 8 mug of HCP per mg of cells (dry weight), which corresponds to the minimal lethal dose. Levels as low as 2 mug/mg are inhibitory but not lethal. HCP also inhibits the respiration of isolated B. megaterium membranes and can act on several components of the electron transport chain in the membranes and on soluble enzymes. Although both forms of nicotinamide adenine dinucleotide, reduced form dehydrogenase and malic dehydrogenase are inhibited by HCP, they are less susceptible than is oxygen uptake. The site of maximal sensitivity is nearer the terminal electron acceptor, but the exact location depends on the cytochrome composition of the membranes. If cytochromes b(1), a, and a(3) are present, but not o, HCP inhibits electron transport on the substrate side of cytochrome b(1); if cytochromes b(1), a(3), and o are present, but not a, the inhibition occurs on the oxygen side of cytochrome b(1). Exogenous menadione, an analogue of menaquinone, reverses the inhibition in both circumstances. The primary lethal action of HCP thus appears to be respiratory inhibition at a site within the membrane-bound part of the electron transport chain.

Osmotic behavior of bacterial protoplasts: temperature effects.

Among protoplasts released from cells of Bacillus megaterium grown at 20, 30, or 37 C, osmotic swelling in NaCl solution at a given external osmotic pressure was greatest for protoplasts from cells grown at 20 C and least for protoplasts from cells grown at 37 C. Protoplasts from cells grown at lower temperaturs were also less stable to osmotic shock and lysed at higher external osmotic pressures than did protoplasts from cells grown at higher temperatures. But for cells grown at any one temperature, osmotic stabilization was itself temperature dependent so that the higher the ambient incubation temperature, the higher the osmotic pressure needed to prevent lysis of a given fraction of the input protoplast population. However, comparison of the osmotic stability of protoplasts from cells grown at different temperatures at various ambient incubation temperatures revealed that, except at 5 C where no differences were discerned, protoplasts from cells grown at lower temperatures still lysed at higher osmotic pressures than did those from cells grown at higher temperatures. The apparent internal osmolality (28 to 31 atm) did not vary significantly among whole cells from the three growth temperatures. Therefore, the observed differences in osmotic behavior could not be attributed to changes in internal osmotic pressure. Rather, it seemed likely that the differences were due to changes in membrane properties.

Location and consequences of 1,1,1-trichloro-2,2-bis(p-chlorophenyl) ethane uptake by Bacillus megaterium.

No detrimental effects of 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) were observed when cells of Bacillus megaterium were grown from small inocula in nutrient media containing up to 100 mug of DDT/ml. However, when the ratio of DDT to biomass of resting cells was held constant, levels of DDT as low as 1 mug/ml (0.5 mug/mg of cell dry weight) enhanced the rate of death in the population. The lethal action of DDT was both time- and dose-dependent so that higher doses required less time to effect the same killing than did lower doses. Intact cells bound a maximum of about 1.7 mug of DDT/mg of cell dry weight, of which about 75% was localized in the protoplast membrane. Much of the bound DDT was subsequently lost to the suspending medium and the aqueous stability of the returned DDT was enhanced, possibly by association with solubilized cell materials. A small quantity of bound DDT was converted to 1,1-dichloro-2,2-bis(p-chlorophenyl)ethane, which was released from cells somewhat faster than DDT. Apparently the lethal action of DDT was related to its binding in the membrane, but respiration was not inhibited. The atypical macroscopic appearance of membranes isolated from treated cells suggested that cell death may result from altered membrane chemistry.

Antimicrobial actions of hexachlorophene: cytological manifestations.

Hexachlorophene is a soap-compatible bisphenol that has been widely used as an antiseptic, yet its mechanism of action is undefined. The relative threshold concentration for bactericidal effect on a susceptible test organism, Bacillus megaterium, was established to be about 10 mug/mg of cell dry weight. At this or at high (>/=100 mug/mg) concentration, adsorptive uptake by cells displayed saturation kinetics. At about 30 mug/mg, the time course of adsorption occurred in three distinct stages. The triphasic pattern was interpreted to represent successive penetration of and adsorption by the cell wall, the protoplast membrane, and the cytoplasm. This interpretation was substantiated by determinations of hexachlorophene adsorption by isolated cell components. Electron microscopy disclosed cytopathology, evidenced as gaps or discontinuities, in the protoplast membrane (but not in the cell wall or cytoplasm) at > 30 mug of hexachlorophene per mg of cell dry weight. Similarly, treatment with > 30 mug/mg allowed a fluorescigenic dye (tolyl-peri acid) to penetrate into the protoplast. However, no detectable cytological manifestations were discerned at the minimum lethal concentration of 10 mug/mg. Apparently, hexachlorophene is physically disruptive at intermediate or high relative concentrations but acts in a more subtle fashion at the minimal lethal concentration.

Antimicrobial actions of hexachlorophene: release of cytoplasmic materials.

Intracellular solutes were released from growing or resting cells of Bacillus megaterium as a consequence of hexachlorophene treatment. The effect was dose dependent, with the optimum at a concentration about sevenfold greater than the minimal lethal dose. The effects of pH and temperature on the leakage process also were inconsistent with the killing effects of the drug. The types of materials released appeared to be the same with or without hexachlorophene treatment. The released materials were small molecules which apparently derived from preexisting ribonucleic acid and protein, but not from deoxyribonucleic acid. Compared to the effects of other representative surface-active agents and other bis-phenols, hexachlorophene was superior in ability to cause leakage of intracellular materials. Different microorganisms varied in their susceptibility to hexachlorophene, with Pseudomonas aeruginosa and a paracolon isolate the most resistant of the vegetative cells examined. It was concluded that the release of intracellular solutes was an effect secondary to the lethal event and presumably arose from hexachlorophene-mediated stimulation of degradative enzymes.

Antimicrobial actions of hexachlorophene: lysis and fixation of bacterial protoplasts.

Hexachlorophene was found to be both a lytic and a fixative agent for protoplasts isolated from Bacillus megaterium. Concentrations of 50 to 100 mug of drug per mg of original cell dry weight were required to lyse 4.4 x 10(9) protoplasts (2 mg of original cell dry weight). At higher drug concentrations, protoplasts became fixed against osmotic stress and reduced in sensitivity to disruption by n-butanol. Lower drug concentrations caused proportionate lysis in the protoplast population. Intact cells lost the ability to become plasmolyzed at these same hexachlorophene concentrations. Nonplasmolyzed, drug-treated cells were resistant to the action of lysozyme, whereas plasmolyzed, drug-treated cells were sensitive. But the sensitivity of isolated cell walls to lysozyme digestion was not markedly altered by hexachlorophene treatment. These effects appeared to be secondary in the killing of cells by hexachlorophene because they occurred at concentrations higher than the minimum lethal concentration.