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Chapter 3 : Effects of High Pressure on Bacterial Spores

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Abstract:

High pressures (50 to 800 MPa) were found to inactivate spores many years ago. The killing of spores by pressure is unusual in that very high pressures are often less effective than are lower pressures. The initial germination may be essential for efficient spore killing by pressure, since germinated spores are much more sensitive to stress factors, in particular, heat, than are dormant spores. Consequently, in order to fully understand the effects of high pressures on spores, it is essential to first understand (i) the structure of dormant spores, (ii) some of the factors that contribute to spore resistance, and (iii) the normal mechanism(s) for germination of bacterial spores, as high-pressure germination uses some components of normal spore germination pathways. Spores of and species have a structure very different from that of growing cells, with a number of layers unique to spores and many spore-specific macromolecules. The nutrients that trigger germination vary in a species- and strain-specific manner, but common ones are L-amino acids, D-sugars, and purine nucleosides. spores have three functional germinant receptors. The GerA receptor responds to L-alanine, while the GerB and GerK receptors together trigger germination with a mixture of L-asparagine (or L-alanine), D-glucose, D-fructose, and K ions. Dipicolinic acid (DPA) and other ions that are released early in germination comprise ~25% of the spore core’s dry weight. Germination of spores of some strains, in particular, some strains, is triggered by inorganic salts such as KBr.

Citation: Setlow P. 2008. Effects of High Pressure on Bacterial Spores, p 35-52. In Michiels C, Bartlett D, Aersten A (ed), High-Pressure Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555815646.ch3

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Acyl Coenzyme A
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Fatty Acid Desaturase
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Integral Membrane Proteins
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Figures

Image of Figure 1.
Figure 1.

Dormant-spore structure. The various layers of the dormant spore are not drawn to scale. Note that the exosporium is not present in spores of many species.

Citation: Setlow P. 2008. Effects of High Pressure on Bacterial Spores, p 35-52. In Michiels C, Bartlett D, Aersten A (ed), High-Pressure Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555815646.ch3
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Image of Figure 2.
Figure 2.

Structure of DPA.

Citation: Setlow P. 2008. Effects of High Pressure on Bacterial Spores, p 35-52. In Michiels C, Bartlett D, Aersten A (ed), High-Pressure Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555815646.ch3
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Image of Figure 3.
Figure 3.

Events in various stages of spore germination. This figure is adapted from Fig. 3 in reference .

Citation: Setlow P. 2008. Effects of High Pressure on Bacterial Spores, p 35-52. In Michiels C, Bartlett D, Aersten A (ed), High-Pressure Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555815646.ch3
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Image of Figure 4.
Figure 4.

Rates of DPA release from spores upon treatment with mHP and uHP at various temperatures. Spores of PS533, a derivative of strain 168 carrying plasmid pUB110 ( ), were incubated at an optical density at 600 nm of 2 in 50 mM Tris-HCl (pH 7.5) and treated for various times and at various temperatures with either 150 MPa (0) or 500 MPa (●). Spore germination was assessed in duplicate samples by monitoring DPA release by measuring the optical density at 270 nm of the supernatant fluid from 1-ml samples centrifuged in a microcentrifuge. The release of DPA in this experiment was linear with time (data not shown). The variation in values for the duplicate samples was ±7% or less. The data for this experiment are from reference .

Citation: Setlow P. 2008. Effects of High Pressure on Bacterial Spores, p 35-52. In Michiels C, Bartlett D, Aersten A (ed), High-Pressure Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555815646.ch3
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Image of Figure 5.
Figure 5.

Effects of changes in nutrient receptors on mHP germination. Spores of various isogenic strains of at an optical density at 600 nm of 1 were treated with 150 MPa of pressure at 37°C in 50 mM Tris-HCl (pH 7.5). The germination of duplicate samples of the treated spores was assessed by flow cytometry after staining the spores with the nucleic acid stain Syto 16 (Molecular Probes, Eugene, OR) as described previously ( , 67). Dormant-spore nucleic acids are not stained by Syto 16, which only stains fully germinated spores. Symbols represent the various strains of spores used as follows: ○, PS533 (wild type [ ]); ●, FB72 ( ) (lacks all three functional germinant receptors); □, PS3301 (lacks the only lipoprotein diacylglycerol transferase [ ]); ▲, FB68 (lacks GerD [ ]); and □, PS3476 ( ) (contains 20-fold-higher levels of the GerA germinant receptor, the major germinant receptor responding to mHP). The variation in values for the duplicate samples was ±5% or less. Data are from references and .

Citation: Setlow P. 2008. Effects of High Pressure on Bacterial Spores, p 35-52. In Michiels C, Bartlett D, Aersten A (ed), High-Pressure Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555815646.ch3
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Image of Figure 6.
Figure 6.

Effects of sporulation temperature and loss of DPA on mHP germination. Spores of strain PS533 (wild type [ ]) were prepared at various temperatures, and spores of strain FB122 (DPA-less [ ]) were prepared at 37°C. Spores were treated with 150 MPa of pressure at 37°C, and spore germination in duplicate samples was assessed as described in the legend to Fig. 5. The symbols for the spores of the strains used are as follows: ○, PS533 spores prepared at 23°C; ●, PS533 spores prepared at 30°C;Δ, PS533 spores prepared at 37°C;□, PS533 spores prepared at 44°C; ▲, FB122 (DPA-less) ( ) spores prepared at 37°C. The variation in values for the duplicate samples was ±8% or less. Data are from reference .

Citation: Setlow P. 2008. Effects of High Pressure on Bacterial Spores, p 35-52. In Michiels C, Bartlett D, Aersten A (ed), High-Pressure Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555815646.ch3
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Image of Figure 7.
Figure 7.

Effect of loss of germinant receptor function or DPA on uHP germination. Spores of various strains at an optical density at 600 nm of 1 were treated with 500 MPa of pressure at 50°C, and spore germination in duplicate samples was assessed as described in the legend to Fig. 5. The symbols for the spores of the strains used are as follows: ○, PS533 (wild type [ ]); ●, FB72 (lacks all germinant receptors [ ]);Δ, PS3301 (lacks the lipoprotein diacylglycerol transferase [ ]); ▲, FB68 (lacks GerD [ ]); and □, FB122 (DPA-less [ ]). The variation in values for the duplicate samples was :7% or less. Data are from reference .

Citation: Setlow P. 2008. Effects of High Pressure on Bacterial Spores, p 35-52. In Michiels C, Bartlett D, Aersten A (ed), High-Pressure Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555815646.ch3
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Image of Figure 8.
Figure 8.

Effect of inner membrane fatty acid composition and sporulation temperature on uHP spore germination. Spores of various strains prepared at different temperatures were treated at an optical density at 600 nm of 1 with 500 MPa of pressure at 50°C, and spore germination in duplicate samples was assessed as described in the legend to Fig. 5. The symbols for the spores of the strains used are as follows: ○, PS533 (wild-type [ ]) sporulated at 23°C; ●, PS533 sporulated at 37°C; ▲, PS533 sporulated at 44°C; Δ, PS3628 (lacking fatty acid desaturase and containing no unsaturated fatty acids in the spore’s inner membrane [ ]) prepared at 37°C; and □, PS3624 (has elevated levels of fatty acid desaturase and >10-fold-higher levels of unsaturated fatty acids in the spore’s inner membrane compared to PS533 spores [ ]) prepared at 37°C. The variation in values for the duplicate samples was ±7% or less. Data are from reference .

Citation: Setlow P. 2008. Effects of High Pressure on Bacterial Spores, p 35-52. In Michiels C, Bartlett D, Aersten A (ed), High-Pressure Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555815646.ch3
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