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Chapter 4 : Inactivation of by High Pressure

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Abstract:

This chapter examines the effects of lethal pressures above 100 MPa on cellular structure and function of and attempts to distinguish between events that may be associated with loss of viability and those that are not. This is summarized in a provisional model of the mechanisms of inactivation in based on our current understanding. The confirmatory tests for identifying events as being lethal may be carried out with cells which show different degrees of resistance to pressure, e.g., in different physiological states, treated in particular media, or subjected to different pressure intensities. Researchers investigated the response of stationary-phase cells of a pressure-resistant strain of O157:H7, EC-88, to a sublethal pressure of 100 MPa for 15 min. More than 100 genes were up-regulated or downregulated following pressure treatment, but in only 36 of these was the change regarded as significant. The major functional categories affected were (i) stress responses, (ii) thioldisulfide redox system, (iii) iron-sulfur cluster assembly, (iv) spontaneous mutation, and (v) several miscellaneous genes. An important conclusion from this study was that high pressure adversely affects the cell’s redox homeostasis. The role of protein denaturation, including cytoplasmic and membrane proteins, in cell death remains to be determined, as does the role of chaperones in protecting against pressure damage. The interrelationships between the various manifestations of pressure-induced damage, both direct and indirect, and cell death will be a fascinating topic of study for the future.

Citation: Mackey B, Mañas P. 2008. Inactivation of by High Pressure, p 53-85. In Michiels C, Bartlett D, Aersten A (ed), High-Pressure Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555815646.ch4

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Environmental Microbiology
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Outer Membrane Proteins
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Elongation Factor Tu
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Reactive Oxygen Species
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Figures

Image of Figure 1.
Figure 1.

Scheme of experimental approach. Events labeled A to E represent independent measurements of changes in the cell structures or functions caused by pressure. Events that happen before loss of colony-forming ability (A and B) or after it (D and E) are most unlikely to be the cause of cell death, whereas events occurring coincidentally with loss of colony-forming ability (C) may be responsible for death.

Citation: Mackey B, Mañas P. 2008. Inactivation of by High Pressure, p 53-85. In Michiels C, Bartlett D, Aersten A (ed), High-Pressure Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555815646.ch4
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Image of Figure 2.
Figure 2.

Effect of growth temperature on pressure resistance of exponential-phase (□) or stationary-phase (■) cells of NCTC 8164. Pressure resistance is expressed as the pressure of onset of cell death (± standard error). Reproduced from ( ) with permission.

Citation: Mackey B, Mañas P. 2008. Inactivation of by High Pressure, p 53-85. In Michiels C, Bartlett D, Aersten A (ed), High-Pressure Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555815646.ch4
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Image of Figure 3.
Figure 3.

Pressure resistance of J1 in exponential (○) and stationary (●) phases: percentage of survivors after 8 min of treatment at different pressures. Each point corresponds to the mean of at least three independent experiments. The initial inoculum levels were 5 X 10 cells/ml for stationary-phase cells and 10 cells/ ml for exponential-phase cells. Reproduced from ( ) with permission.

Citation: Mackey B, Mañas P. 2008. Inactivation of by High Pressure, p 53-85. In Michiels C, Bartlett D, Aersten A (ed), High-Pressure Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555815646.ch4
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Image of Figure 4.
Figure 4.

Effect of pressure on general appearance of J1: phase-contrast photomicrographs of exponential- and stationary-phase cells. (A) Exponential-phase cells, untreated (100% viable cells); (B) stationary-phase cells, untreated (100% viable cells); (C) exponential-phase cells treated with 300 MPa for 8 min (0.002% viable cells);(D) stationary-phase cells treated with 600 MPa for 8 min (0.01% viable cells). Bar marker, 2 fm. Reproduced from ( ) with permission.

Citation: Mackey B, Mañas P. 2008. Inactivation of by High Pressure, p 53-85. In Michiels C, Bartlett D, Aersten A (ed), High-Pressure Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555815646.ch4
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Image of Figure 5.
Figure 5.

Pressure-induced condensation of the nucleoid and aggregation of cellular protein in J1. Phase-contrast/fluorescence photomicrographs of exponential-phase cells, left untreated (100% viable) and stained with DAPI (A); exponential-phase cells treated with 400 MPa for 8 min (<0.0001% viable cells) and stained with DAPI (B); stationary-phase cells treated with 400 MPa for 8 min (61% viable cells) and stained with DAPI (C); and stationary-phase cells treated with 300 MPa for 8 min (73% viable) and stained with FITC (D). Bar marker, 2 fm. Reproduced from ( ) with permission.

Citation: Mackey B, Mañas P. 2008. Inactivation of by High Pressure, p 53-85. In Michiels C, Bartlett D, Aersten A (ed), High-Pressure Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555815646.ch4
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Image of Figure 6.
Figure 6.

Morphological changes in caused by high pressure. Shown are transmission electron micrographs of thin sections of untreated exponential-phase cells (A), exponential-phase cells pressure treated at 200 MPa for 2 min (30% survival) (B), untreated stationary-phase cells (C), and stationary-phase cells pressure treated at 400 MPa for 4 min (<1% survival) (D). Bar marker, 0.25 fm.

Citation: Mackey B, Mañas P. 2008. Inactivation of by High Pressure, p 53-85. In Michiels C, Bartlett D, Aersten A (ed), High-Pressure Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555815646.ch4
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Image of Figure 7.
Figure 7.

Effect of pressure on the osmotic response of J1: phase-contrast photomicrographs of exponential- and stationary-phase cells placed on agar containing 0.75 M NaCl. (A) Exponential-phase cells, untreated (100% viable cells); (B) exponential-phase cells treated with 200 MPa for 8 min (0.02% viable cells); (C) stationary-phase cells, untreated (100% viable cells); (D) stationary-phase cells treated with 200 MPa for 8 min (79% viable cells); (E) stationary-phase cells treated with 500 MPa for 8 min (43% viable cells); (F) stationary-phase cells treated with 600 MPa for 8 min (0.01% viable cells). Bar marker, 2 µm. Reproduced from ( ) with permission.

Citation: Mackey B, Mañas P. 2008. Inactivation of by High Pressure, p 53-85. In Michiels C, Bartlett D, Aersten A (ed), High-Pressure Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555815646.ch4
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Image of Figure 8.
Figure 8.

Effect of pressure on membrane integrity of exponential-phase cells of J1: fluorescence photomicrographs of cells stained with lipophilic membrane probe FM 4-64. (A) Untreated cells (100% viable cells); (B) cells pressurized at 300 MPa for 8 min (0.002% viable cells). Bar marker, 2 fm. Arrows show internal and external vesicles. Reproduced from ( ) with permission.

Citation: Mackey B, Mañas P. 2008. Inactivation of by High Pressure, p 53-85. In Michiels C, Bartlett D, Aersten A (ed), High-Pressure Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555815646.ch4
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Image of Figure 9.
Figure 9.

Relationship between transient membrane permeability and loss of viability in stationary-phase J1. The relative extent of transient membrane permeability is expressed as a percentage of the maximum fluorescence reading of cells pressurized in the presence of PI (bars). Viability was determined by viable counts of cells given the same treatment (♦).

Citation: Mackey B, Mañas P. 2008. Inactivation of by High Pressure, p 53-85. In Michiels C, Bartlett D, Aersten A (ed), High-Pressure Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555815646.ch4
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Image of Figure 10.
Figure 10.

Effect of membrane fatty acid composition on pressure resistance. K1060, a fatty acid auxotroph, was grown at 37°C in medium supplemented with linoleic (C [■]), oleic (C [♦]), or elaidic ( C [Δ]) acid to exponential phase (A) or stationary phase (B), and pressure resistance was determined at 200 or 300 MPa, respectively. Reproduced from ( ) with the kind permission of Springer Science and Business Media.

Citation: Mackey B, Mañas P. 2008. Inactivation of by High Pressure, p 53-85. In Michiels C, Bartlett D, Aersten A (ed), High-Pressure Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555815646.ch4
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Image of Figure 11.
Figure 11.

Multiplication of stationary-phase cells containing protein aggregates stained with FITC.

Citation: Mackey B, Mañas P. 2008. Inactivation of by High Pressure, p 53-85. In Michiels C, Bartlett D, Aersten A (ed), High-Pressure Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555815646.ch4
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Image of Figure 12.
Figure 12.

Model for the mechanisms of inactivation of by high hydrostatic pressure.

Citation: Mackey B, Mañas P. 2008. Inactivation of by High Pressure, p 53-85. In Michiels C, Bartlett D, Aersten A (ed), High-Pressure Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555815646.ch4
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Tables

Generic image for table
TABLE 1.

Mutations causing a decrease in the resistance of to lethal pressures

Citation: Mackey B, Mañas P. 2008. Inactivation of by High Pressure, p 53-85. In Michiels C, Bartlett D, Aersten A (ed), High-Pressure Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555815646.ch4
Generic image for table
TABLE 2.

Mutations causing an increase in resistance of to lethal pressures

Citation: Mackey B, Mañas P. 2008. Inactivation of by High Pressure, p 53-85. In Michiels C, Bartlett D, Aersten A (ed), High-Pressure Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555815646.ch4
Generic image for table
TABLE 3.

Genes whose transcription is affected by sublethal pressure but which cause no significant change in the pressure resistance of

Citation: Mackey B, Mañas P. 2008. Inactivation of by High Pressure, p 53-85. In Michiels C, Bartlett D, Aersten A (ed), High-Pressure Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555815646.ch4

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