Tetsuaki Tsuchido

Effect of preincubation temperature on the heat resistance of Escherichia coli having different fatty acid compositions.

The heat resistance of Escherichia coli depends on the temperature of incubation prior to heat treatment at 50°C. This dependence is affected by the fatty acid composition in bacterial membranes. The results are discussed in relation to the change in fluidity of membrane lipids.

Interaction of the Fluorescent Dye 1-N-Phenylnaphthylamine with Escherichia coli Cells during Heat Stress and Recovery from Heat Stress

The fluorescent dye 1-N-phenylnaphthylamine permeated Escherichia coli cells after exposure to a heat stress at 55 degrees C in Tris/Mg2+ buffer, pH 8.0. The rate of dye permeation increased with time during heat treatment and decreased gradually during subsequent incubation at 37 degrees C in a minimal medium. The initial level of rapid adsorption of the dye also increased with heating time, although it remained roughly constant during post-heating incubation. The results obtained suggest that the permeability barrier to the dye in the outer membrane was damaged by heat stress and was repaired after sublethal heating. RNA, protein and lipid syntheses, as well as an energy-yielding process, appeared to be necessary for the repair of impermeability to the dye.

Cell death of Bacillus subtilis caused by surfactants at low concentrations results from induced cell autolysis

Abstract A cationic surfactant, palmityltrimethylammonium iodide at 6 to 40 μM caused the death and lysis of Bacillus subtilis 168 cells growing logarithmically in a chemically defined medium. The relationship between the induced death and lysis was investigated by using an autolytic enzyme-defective mutant and by exposure of cells to several treatments. While pretreatment with erythromycin rendered cells resistant to the surfactant, pretreatment with cerulenin caused the sensitization of cells. Addition of cardiolipin neutralized the lytic and killing action of the surfactant. The results obtained suggest that the cell death caused by the surfactant at low concentrations results from the induction of autolysis. At higher concentrations of the surfactant, however, no substantial cell lysis occurred, although much cell death was observed. Similar situations were observed with other surfactants tested.

Autolysis of Bacillus subtilis induced by monovalent cations

SummaryMonovalent cations including K+, Na+, NH4+, Cs+, Rb+, or Li+ at 100 mM concentration caused cellular lysis of Bacillus subtilis 168 in the log phase. The lysis caused by 100 mM K+ was inhibited by divalent cations at 100 mM including Mg++, Ca++, Mn++, Co++, or Ni++. These in vivo phenomena agreed well with the effect of cations on in vitro autolysin activity. No cellular lysis occurred, even in the presence of monovalent cations, when both carbon and nitrogen sources were added. An uncoupler of oxidative phosphorylation, respiratory inhibitors, inhibitors of cell wall synthesis, or lauric acid caused rapid lysis in the presence of 100 mM K+, glucose, and NH4+, but not in the absence of monovalent cations or in the presence of 100 mM Mg++. These results suggest that autolysis of B. subtilis is evoked by a two-step mechanism: the first might be the blocking of energy metabolism or disturbance of the cell surface structure, and the second is the activation of autolysin by a high concentration of monovalent cations.

Isolation and characteristics of a Bacillus subtilis mutant tolerant to the lytic action of sucrose esters of long-chain fatty acids

Abstract A spontaneous mutant of Bacillus subtilis 168, SR3, tolerant to the autolysis-inducing action of sucrose monoesters of long-chain fatty acids, was isolated. It was shown that its susceptibility to the lytic action of sucrose esters and glycerol esters of short-chain fatty acids, fatty acids themselves, some surfactants, uncouplers of oxidative phosphorylation, and β-lactam antibiotics against the mutant strain was similar to that of the wild-type strain. In the absence of sucrose monoesters, no substantial differences were observed between the mutant and the wild-type strains in cell autolysis and autolysin activity. It was found that in the mutant the cellular uptake of the molecules of sucrose ester of palmitic acid was suppressed. Also, a protein having a molecular weight of 41 kDa was richer in the membrane of strain SR3 than that of 168. The tolerance of the mutant to the lytic action of the ester is suggested to be derived from a decrease in the amount of ester molecules transferred into the membrane, where the activity of autolytic enzymes may be controlled.

Photodynamic inactivation of bacteria on immobilized α-terthienyl film

SummaryThe photodynamic inactivation by near UV and α-terthienyl (αT) of bacteria immobilized in poly(methyl methacrylate) (PMMA) was observed. Killing curves for Escherichia coli strains exhibited multi-hit kinetics, whereas curves for Pseudomonas aeruginosa and Staphylococcus aureus exhibited single-hit kinetics. Cell inactivation by irradiated αT film at an immobilized concentration of 20 μg/cm2 was substantially caused by generated singlet oxygen (1O2), but not by that from free αT molecules released from the film after irradiation with near UV. The inactivation rate was found to depend on the surface density of αT in the film. A linear relationship between the concentration of 1O2 generated on the film surface and the apparent rate of cell death was obtained.

A modified assessment of growth inhibition from growth-delay time in a cell population exposed to an environmental stress

Abstract An assay method for the evaluation of a microbial growth-inhibition caused by an inhibitory substance was proposed by modification of the original growth-delay method, which has been reported previously [J. Ferment. Technol., 60, 189, 1982]. The use of an incubator equipped with an automatic OD-detection apparatus and connected to a computer-assisted-data-analysis system made it easy to calculate the growth delay time and also, to evaluate the inhibition.

Autolysis of Bacillus subtilis induced by low temperature

Abstract By exposure to a temperature below the membrane phase transition point, Bacillus subtilis 168 lost their permeability control followed by the leakage of intracellular K + and incapability of glucose uptake, resulting in cellular lysis in the following incubation at 37°C in the presence of a high concentration (∼ 100 mM) of monovalent cations. The result confirmed that the concomitance of energy deprivation and the presence of monovalent cations were the factors that caused the lysis observed after low temperature exposure.

Dependence of autolysis of Bacillus subtilis cells on macromolecule synthesis under nutrient limitation

Abstract Autolysis of Bacillus subtilis 168 cells in buffer solution was induced by high concentrations of monovalent cations, which activate N- acetylmuramoyl- l -alanine amidase, a major autolysin in this bacterium. However, cells did not lyse in Spizizen salt medium containing high K+ ion concentration. Therefore, autolysis of washed cells of B. subtilis in the log phase was studied in various nutrient solutions in the presence of 100 mM K+. Glucose inhibited autolysis in 100 mM KCl solution, but ammonium ions together with glucose were required to inhibit lysis in potassium phosphate buffer (KPB) solution. Penicillin G or vancomycin caused cell lysis in KPB + glucose + ammonium ion (KPB non-lytic solution), but not in KCl + glucose (KCl non-lytic solution). A tracer experiment showed that synthesis of both protein and peptidoglycan was active in KPB non-lytic solution but not in KCl non-lytic solution. Chloramphenicol-treated cells did not lyse in KPB non-lytic solution upon addition of penicillin G. Autolysis observed during active protein synthesis without peptidoglycan synthesis in KPB + glucose solution was suppressed by chloramphenicol. From these results, we concluded that autolysis occurred when protein synthesis was active but peptidoglycan synthesis was incomplete.