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Effects of high hydrostatic pressure on membrane proteins of Salmonella typhimurium
International Journal of Food Microbiology 55 (2000) 115–119 www.elsevier.nl / locate / ijfoodmicro
Effects of high hydrostatic pressure on membrane proteins of Salmonella typhimurium a b b a, M. Ritz , M. Freulet , N. Orange , M. Federighi * a
´ INRA d’ Hygiene ` Alimentaire, Ecole Nationale Veterinaire ´´ Unite´ associee de Nantes, Route de Gachet, BP40706, F-44307 Nantes, France b Laboratoire de Microbiologie du Froid, Universite´ de Rouen, 43 rue Saint-Germain, F-27000 Evreux, France
Abstract Salmonella typhimurium is a leading cause of foodborne diseases. Today high hydrostatic pressure treatments are considered as alternative methods of preservation. To select optimal conditions of treatment, we have to characterize the cell targets of pressure. In this study the action of pressure on the bacterial membrane proteins is analysed. The total membrane extract is obtained by lysis of cells separated by equilibrium density gradient centrifugation. Protein content is analysed by electrophoresis SDS–PAGE and visualised by silver stain. Electrophoretic profiles reveal the presence of three major outer membrane proteins and 12 minor proteins in control bacteria outer membranes. Outer membrane protein content is drastically modified after treatments. In some cases, except for the major proteins OmpA and LamB, other outer membrane proteins seem to totally disappear. LamB is more resistant to hyperbaric exposure when the pH of the media is acidic. This behaviour could be explained by a different conformation adopted by the LamB protein depending on the extracellular pH. This work allows us to define membrane proteins as a target of high hydrostatic pressure treatments. Knowledge of the behaviour of these bacterial membrane proteins subjected to pressure under different conditions (pH, temperature, a w . . . ) could allow an increase in the efficiency of treatments. 2000 Elsevier Science B.V. All rights reserved. Keywords: High pressure; Salmonella; Membrane protein
1. Introduction High hydrostatic pressure treatment is currently considered as an attractive non-thermal process for
*Corresponding author. Tel.: 133-2-4068-7681. E-mail address: [email protected] (M. Federighi)
preserving food (Knorr, 1993). One of the primary considerations in evaluating the effectiveness of any preservation treatment is its ability to eradicate pathogenic micro-organisms and thus ensure product safety. Previous studies showed that a pressure treatment of 500 to 700 MPa readily kills vegetative cells of bacteria, yeast and mould, while bacterial spores are more resistant (Hayakawa et al., 1994; Mackey et al., 1994; Wuytack et al., 1998). Industrial equipment used to preserve foods is routinely de-
0168-1605 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0168-1605( 00 )00165-3
116
M. Ritz et al. / International Journal of Food Microbiology 55 (2000) 115 – 119
signed to generate at least 400 MPa of pressure every cycle (Zimmerman and Bergman, 1993). Resistance by micro-organisms to high pressure is variable, and reduction of microbial loads is directly related to the amount of hydrostatic pressure applied (Arroyo et al., 1997). High pressure brings about a number of changes in the morphology, cell membrane, cell wall, biochemical reactions and genetic mechanisms of micro-organisms (Hoover et al., 1989; Cheftel, 1995; Simpson and Gilmour, 1997; Wouters et al., 1998). The cell membrane represents probably one of the major targets for pressure-induced inactivation of micro-organisms. Previous studies demonstrated modifications of the fatty acid composition of membranes in relation to pressure treatment of lipid bilayers and pressure growth of micro-organisms (Macdonald, 1992; Fujii et al., 1997). Other reports demonstrated some changes in cell morphology and structure (Mackey et al., 1994), and changes in intracellular and membrane-bound enzyme activity (Wouters et al., 1998). Elucidation of the mechanistic aspects of high-pressure inactivation of microorganisms will help in food preservation. The cell envelope of Salmonella and other gramnegative enteric bacteria is a complex structure composed of three morphologically distinct layers: cytoplasmic membrane, a rigid peptidoglycan layer external to the cytoplasmic membrane, and a second membrane structure, the outer membrane or L-layer at the surface of the cell. The outer membrane contains substantial amounts of protein and phospholipid and in addition most or all of the lipopolysaccharide of the cell envelope. The aim of this work was to characterize the cell targets of pressure treatment and know if the membrane proteins of Salmonella typhimurium pressure could be this potential target. For this purpose a technique for separation of cytoplasmic membrane from outer membrane was necessary. We followed the method described by Osborn et al. (1972) with few modifications and used a technique involving a sucrose density gradient centrifugation of membranes obtained by spheroplast lysis. This communication presents general characterization of the membrane fractions obtained from Salmonella typhimurium by this technique and provides evidence that the proteins of membranes are really a target of high hydrostatic pressure treatment.
2. Materials and methods
2.1. Bacteria culture preparation S. typhimurium (ATCC 13311) was obtained from Collection de l’Institut Pasteur (Institut Pasteur, Paris, France) and stored at 2 308C in cryobeads (AES, Combourg, France). The first bacterial culture was obtained by inoculating a cryobead into brainheart infusion (BHI; Biokar, Beauvais, France) incubated at 378C for 24 h. This first culture was used to prepare a subculture by inoculating 1 ppt. (v / v) into fresh BHI to incubate for 18 h at 378C. In these conditions, the count for the initial cell population was 8 log 10 , with a standard error of 0.1.
2.2. Inoculation of the suspending medium The pressurized samples were composed of 150 ml of subculture diluted in 600 ml of buffer. Two buffers were used: a phosphate buffer (pH 7.00) composed of Na 2 HPO 4 (0.2 mol l 21 ) and NaH 2 PO 4 (0.2 mol l 21 ), and a citrate buffer (pH 5.6) composed of Na 2 HPO 4 (0.2 mol l 21 ) and C 6 H 8 O 7 (0.1 mol l 21 ) (Merck, Darmstadt, Germany). The samples were placed in a sterile bag.
2.3. Enumeration of surviving cells The number of cells was determined before and after treatment, and the efficiency of treatment was calculated by comparing these two counts in log 10 . Cells were enumerated by plating 0.1-ml volumes twice on plate count agar (PCA) (Biokar), which were then incubated for 48 h at 378C. For low numbers of viable cells, 10 ml of cell suspension were divided between two plates and poured with PCA. Thus, the pitch limit was 0.1 c / ml, corresponding to the result 0.
2.4. High-pressure treatment The sterile bags were sealed after eliminating air inside, placed in a hydrostatic pressure vessel (3 l) in a high-pressure apparatus (Alstom, Nantes, France), and treated at 208C for 10 min. High-pressure levels (350 to 600 MPa) were generated using water pressurized by a hydrostatic pump. Each treatment
M. Ritz et al. / International Journal of Food Microbiology 55 (2000) 115 – 119
117
Table 1 Cell enumeration of Salmonella typhimurium populations before and after treatment Phosphate buffer pH 7.0
Citrate phosphate buffer pH 5.6
Non-treated sample
8.04 log 10
Non-treated sample
7.99 log 10
400 MPa, 10 min 600 MPa, 10 min
,1 cell / 10 ml ,1 cell / 10 ml
350 MPa, 10 min 600 MPa, 10 min
,10 cell / 10 ml ,1 cell / 10 ml
was a combination of a pressure level, duration, and pH as shown in Table 1.
3. Results
3.1. Outer membrane fractions analysis 2.5. Isolation of cytoplasmic and outer membrane fractions The procedure was based on an equilibrium density gradient centrifugation of the total membrane extract obtained by lysis of lysozyme–EDTA spheroplasts. Cells were harvested by centrifugation at 8000 rev. / min for 15 min at 48C (Sorvall RC5B, rotor GSA). The cell pellet was rapidly resuspended in cold 0.75 M sucrose–10 mM Tris–HCl buffer, pH 7.8. Lysozyme was immediately added to a final concentration of 100 mg / ml, 2 volumes of cold 1.5 mM EDTA (Na 1 ), pH 7.5 were slowly added and the bacterial suspension was incubated at 308C for 90 min. Conversion to spheroplasts was monitored by optical microscopy. When spheroplast formation attempted about 95%, cell suspension was immersed in an ice-cold bath and sonicated four times for 30 s with Vibracell (Bioblock Scientific). Intact cells were removed by centrifugation at 10,000 rev. / min for 15 min (Sorvall RC5B, rotor SS34). Membrane extracts were collected by centrifugation at 37,000 rev. / min for 2 h (Beckman, rotor 70 Ti). Step gradients of sucrose density were prepared by layering 2 ml each of 50%, 45%, 40%, 35% and 30% sucrose solutions (w / w) containing 5 mM EDTA, pH 7.5. The membrane pellet was resuspended in 0.5 ml of cold 25% sucrose, 5 mM EDTA, pH 7.5 and applied on the top of the gradient. Centrifugation was carried out for 12 h at 37,000 rev. / min and 48C (Beckman, rotor SW 41 Ti). Fractions of 0.5 ml were collected from the bottom of the tube with a peristaltic pump. Protein content was assayed by ESL Boehringer kit and membrane extracts were analysed by electrophoresis SDS–PAGE (7% and 15% gel system). Proteins were visualised by silver stain.
After hyperbaric exposure, cells were harvested, bacterial membranes extracted and analysed by electrophoresis SDS–PAGE. Efficiency of spheroplast formation was lower (50%) for treated cells than for control bacterial suspension (95%). This suggests that the outer membranes (OM) seem to be more resistant to EDTA–lysozyme treatment after hyperbaric shock. Electrophoretic profiles revealed the presence of three major outer membrane proteins and about 12 minor proteins in the control bacteria outer membranes (Figs. 1 and 2). Major outer membrane proteins are identified as LamB (44 kDa), OmpC (37 kDa) and OmpA (35 kDa) (Nikaido and Vaara, 1985). The quantities of these proteins were found to be quite similar whatever the extra-cellular pH is, 7.0 or 5.6. After treatment at 350 MPa or 400 MPa, and more so at 600 MPa, the Salmonella typhimurium population was totally inactivated, outer membrane protein content is drastically modified whatever the extra-
Fig. 1. Outer membrane profile. Cell suspension in phosphate buffer pH 7.0. Protein content is analysed by electrophoresis SDS–PAGE and visualised by silver stain.
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M. Ritz et al. / International Journal of Food Microbiology 55 (2000) 115 – 119
Fig. 2. Outer membrane profile. Cell suspension in citrate phosphate buffer pH 5.6. Protein content is analysed by electrophoresis SDS–PAGE and visualised by silver stain.
cellular pH; 350 MPa, pH 5.6 and 400 MPa, pH 7.0 treated cells presented a different outer membrane protein pattern whereas the effect on enumeration is similar. Outer membrane extracts from treated cells in phosphate buffer at 400 MPa (Fig. 1), showed each minor and major protein except the 44 kDa protein which almost entirely disappeared. On the contrary, outer membranes obtained from cells suspended in citrate buffer at an acidic pH of 5.6 (Fig. 2), revealed only two proteins; the major outer membrane protein of 35 kDa appearing in a similar quantity as in controls, and a 44 kDa outer membrane protein less concentrated. Each other major or minor outer membrane protein seemed to disappear. Only the 35 kDa protein remained after the 600 MPa treatment whatever the pH of the suspending medium. After heating for 10 min at 1008C, the major outer membrane protein of 35 kDa is modified and migrates as a band corresponding to a relative molecular weight of 37 kDa. This heat modified behaviour is characteristic of porins (Nikaido et al., 1991). The heat modification property is conserved after hyperbaric exposure for cells in phosphate buffer but seems to be partially altered for cells suspended in citrate buffer.
3.2. Inner membrane analysis Sonication of spheroplasts from hyperbaric exposed cells needs less time than for spheroplasts from control cells. This suggests that the inner membrane seems to be made fragile by hyperbaric
Fig. 3. Internal membrane profile. Cell suspension in citrate phosphate buffer pH 5.6. Protein content is analysed by electrophoresis SDS–PAGE and visualised by silver stain.
treatment. Before treatment the inner membrane protein content is similar whatever the pH of the suspending medium. This content seems to reduce with the increase of pressure and after 600 MPa exposure (Fig. 3). The protein content in inner membrane protein is drastically diminished, but no significant difference of sensitivity was observed depending on the pH of the suspending medium.
4. Discussion Previous studies demonstrated some changes in cell structures (Mackey et al., 1994) and membranebound enzymes activity (Wouters et al., 1998). This work permits us to confirm the role of the cell membrane in the inactivation and characterizes a physical target of high hydrostatic pressure. The presence of the major outer membrane proteins identified as LamB, OmpC and OmpA for the control bacterial suspension confirm the capacity of this technique employed to characterize the content of membrane proteins. We observed lower formation of spheroplasts for treated cells than for control bacterial suspensions. This behaviour could be explained by a reorganisation of the outer membrane. The disappearance of the porins used for the macromolecule transport could explain the difficulties of lysozyme permeation. The results show that hyperbaric exposure alters drastically outer membrane proteins. However, the
M. Ritz et al. / International Journal of Food Microbiology 55 (2000) 115 – 119
major outer membrane protein OmpA seems to be more resistant to this treatment, probably due to its conformation and surrounding. This protein is well known to be involved in cell shape and integrity, which could explain its very resistant conformation to many stresses (Nikaido and Vaara, 1985). Moreover, the protein LamB displays a particular behaviour. Indeed, the protein resistance to hyperbaric shock depends on the extra-cellular pH. LamB is more resistant to hyperbaric exposure when the pH of the media is acidic. When the extra-cellular pH is close to neutrality, the porin is less resistant and 350 MPa are sufficient to cleave it. This behaviour could be explained by a different conformation adopted by the LamB depending on the extra-cellular pH.
5. Conclusion This study demonstrates the action of pressure on the membrane proteins. Even if the pressure is isostatic, the external membrane seems to be more damaged than the cytoplasmic membrane. In fact, except for the 35 kDa protein, each other major or minor outer membrane protein seems to disappear. The different destruction of membrane proteins between acidic and neutral pH could explain the greater inactivation recorded when the cells are treated in acidic suspension or food. These observations combined with the knowledge of pH of food could be integrated for the bacterial destruction models.
References Arroyo, G., Sanz, P.D., Prestamo, G., 1997. Effect of high pressure on the reduction of microbial populations in vegetables. J. Appl. Microbiol. 82, 735–742.
119
Cheftel, J.C., 1995. Review: high-pressure, microbial inactivation and food preservation. Food Sci. Technol. Int. 1, 75–90. Fujii, S., Iwahashi, H., Obuchi, K., Fujii, T., Komatsu, K., 1997. Characterization of a barotolerant mutant of the yeast Saccharomyces cerevisiae. Importance of trehalose content and membrane fluidity. In: Heremans, K. (Ed.), High Pressure Research in the Biosciences and Biotechnology, Leuven University Press, Leuven, pp. 241–244. Hayakawa, I., Kanno, T., Tomita, M., Fujio, Y., 1994. Application of high pressure for spore inactivation and protein denaturation. J. Food Sci. 59, 159–163. Hoover, D.G., Metrick, C., Papineau, A.M., Farkas, D.F., Knorr, D., 1989. Biological effects of high hydrostatic pressure on food microorganisms. Food Technol. 3, 99–107. Knorr, D., 1993. Effects of high hydrostatic pressure processes on food safety and quality. Food Technol., 156–161. Macdonald, A.G., 1992. Effects of high hydrostatic pressure on natural and artificial membranes. In: Balny, C., Hayashi, R., Heremans, K., Masson, P. (Eds.), High Pressure and Biotechnology, Colloque INSERM / John Libbey Eurotext, pp. 65– 75. ` K., Isaacs, N.S.R.S., Brooker, B., 1994. Mackey, B.M., Forestiere, The effect of high hydrostatic pressure on Salmonella thompson and Listeria monocytogenes examined by electron microscopy. Lett. Appl. Microbiol. 19, 429–432. Nikaido, H., Nikaido, K., Harayama, S., 1991. Identification and characterization of porins in Pseudomonas aeroginosa. J. Biol. Chem. 266, 770–779. Nikaido, H., Vaara, M., 1985. Molecular basis of bacterial outer membrane permeability. Microbiol. Rev. 49, 1–32. Osborn, M.J., Gander, J.E., Parisi, E., Carson, J., 1972. Mechanism of assembly of the outer membrane of Salmonella typhimurium. J. Biol. Chem. 247, 3962–3972. Simpson, R.K., Gilmour, A., 1997. The effect of high hydrostatic pressure on the activity of intracellular enzymes of Listeria monocytogenes. Lett. Appl. Microbiol. 25, 48–53. Wouters, P.C., Glaasker, E., Smelt, J.P., 1998. Effects of high pressure on inactivation kinetics and events related to proton efflux in Lactobacillus plantarum. Appl. Environ. Microbiol. 64, 509–514. Wuytack, E.Y., Boven, S., Michiels, C.W., 1998. Comparative study of pressure-induced germination of Bacillus subtilis spores at low and high pressures. Appl. Environ. Microbiol. 9, 3220–3224. Zimmerman, F., Bergman, C., 1993. Isostatic high-pressure equipment for food preservation. Food Technol. 47, 162–163.
Effects of high hydrostatic pressure on membrane proteins of Salmonella typhimurium a b b a, M. Ritz , M. Freulet , N. Orange , M. Federighi * a
´ INRA d’ Hygiene ` Alimentaire, Ecole Nationale Veterinaire ´´ Unite´ associee de Nantes, Route de Gachet, BP40706, F-44307 Nantes, France b Laboratoire de Microbiologie du Froid, Universite´ de Rouen, 43 rue Saint-Germain, F-27000 Evreux, France
Abstract Salmonella typhimurium is a leading cause of foodborne diseases. Today high hydrostatic pressure treatments are considered as alternative methods of preservation. To select optimal conditions of treatment, we have to characterize the cell targets of pressure. In this study the action of pressure on the bacterial membrane proteins is analysed. The total membrane extract is obtained by lysis of cells separated by equilibrium density gradient centrifugation. Protein content is analysed by electrophoresis SDS–PAGE and visualised by silver stain. Electrophoretic profiles reveal the presence of three major outer membrane proteins and 12 minor proteins in control bacteria outer membranes. Outer membrane protein content is drastically modified after treatments. In some cases, except for the major proteins OmpA and LamB, other outer membrane proteins seem to totally disappear. LamB is more resistant to hyperbaric exposure when the pH of the media is acidic. This behaviour could be explained by a different conformation adopted by the LamB protein depending on the extracellular pH. This work allows us to define membrane proteins as a target of high hydrostatic pressure treatments. Knowledge of the behaviour of these bacterial membrane proteins subjected to pressure under different conditions (pH, temperature, a w . . . ) could allow an increase in the efficiency of treatments. 2000 Elsevier Science B.V. All rights reserved. Keywords: High pressure; Salmonella; Membrane protein
1. Introduction High hydrostatic pressure treatment is currently considered as an attractive non-thermal process for
*Corresponding author. Tel.: 133-2-4068-7681. E-mail address: [email protected] (M. Federighi)
preserving food (Knorr, 1993). One of the primary considerations in evaluating the effectiveness of any preservation treatment is its ability to eradicate pathogenic micro-organisms and thus ensure product safety. Previous studies showed that a pressure treatment of 500 to 700 MPa readily kills vegetative cells of bacteria, yeast and mould, while bacterial spores are more resistant (Hayakawa et al., 1994; Mackey et al., 1994; Wuytack et al., 1998). Industrial equipment used to preserve foods is routinely de-
0168-1605 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0168-1605( 00 )00165-3
116
M. Ritz et al. / International Journal of Food Microbiology 55 (2000) 115 – 119
signed to generate at least 400 MPa of pressure every cycle (Zimmerman and Bergman, 1993). Resistance by micro-organisms to high pressure is variable, and reduction of microbial loads is directly related to the amount of hydrostatic pressure applied (Arroyo et al., 1997). High pressure brings about a number of changes in the morphology, cell membrane, cell wall, biochemical reactions and genetic mechanisms of micro-organisms (Hoover et al., 1989; Cheftel, 1995; Simpson and Gilmour, 1997; Wouters et al., 1998). The cell membrane represents probably one of the major targets for pressure-induced inactivation of micro-organisms. Previous studies demonstrated modifications of the fatty acid composition of membranes in relation to pressure treatment of lipid bilayers and pressure growth of micro-organisms (Macdonald, 1992; Fujii et al., 1997). Other reports demonstrated some changes in cell morphology and structure (Mackey et al., 1994), and changes in intracellular and membrane-bound enzyme activity (Wouters et al., 1998). Elucidation of the mechanistic aspects of high-pressure inactivation of microorganisms will help in food preservation. The cell envelope of Salmonella and other gramnegative enteric bacteria is a complex structure composed of three morphologically distinct layers: cytoplasmic membrane, a rigid peptidoglycan layer external to the cytoplasmic membrane, and a second membrane structure, the outer membrane or L-layer at the surface of the cell. The outer membrane contains substantial amounts of protein and phospholipid and in addition most or all of the lipopolysaccharide of the cell envelope. The aim of this work was to characterize the cell targets of pressure treatment and know if the membrane proteins of Salmonella typhimurium pressure could be this potential target. For this purpose a technique for separation of cytoplasmic membrane from outer membrane was necessary. We followed the method described by Osborn et al. (1972) with few modifications and used a technique involving a sucrose density gradient centrifugation of membranes obtained by spheroplast lysis. This communication presents general characterization of the membrane fractions obtained from Salmonella typhimurium by this technique and provides evidence that the proteins of membranes are really a target of high hydrostatic pressure treatment.
2. Materials and methods
2.1. Bacteria culture preparation S. typhimurium (ATCC 13311) was obtained from Collection de l’Institut Pasteur (Institut Pasteur, Paris, France) and stored at 2 308C in cryobeads (AES, Combourg, France). The first bacterial culture was obtained by inoculating a cryobead into brainheart infusion (BHI; Biokar, Beauvais, France) incubated at 378C for 24 h. This first culture was used to prepare a subculture by inoculating 1 ppt. (v / v) into fresh BHI to incubate for 18 h at 378C. In these conditions, the count for the initial cell population was 8 log 10 , with a standard error of 0.1.
2.2. Inoculation of the suspending medium The pressurized samples were composed of 150 ml of subculture diluted in 600 ml of buffer. Two buffers were used: a phosphate buffer (pH 7.00) composed of Na 2 HPO 4 (0.2 mol l 21 ) and NaH 2 PO 4 (0.2 mol l 21 ), and a citrate buffer (pH 5.6) composed of Na 2 HPO 4 (0.2 mol l 21 ) and C 6 H 8 O 7 (0.1 mol l 21 ) (Merck, Darmstadt, Germany). The samples were placed in a sterile bag.
2.3. Enumeration of surviving cells The number of cells was determined before and after treatment, and the efficiency of treatment was calculated by comparing these two counts in log 10 . Cells were enumerated by plating 0.1-ml volumes twice on plate count agar (PCA) (Biokar), which were then incubated for 48 h at 378C. For low numbers of viable cells, 10 ml of cell suspension were divided between two plates and poured with PCA. Thus, the pitch limit was 0.1 c / ml, corresponding to the result 0.
2.4. High-pressure treatment The sterile bags were sealed after eliminating air inside, placed in a hydrostatic pressure vessel (3 l) in a high-pressure apparatus (Alstom, Nantes, France), and treated at 208C for 10 min. High-pressure levels (350 to 600 MPa) were generated using water pressurized by a hydrostatic pump. Each treatment
M. Ritz et al. / International Journal of Food Microbiology 55 (2000) 115 – 119
117
Table 1 Cell enumeration of Salmonella typhimurium populations before and after treatment Phosphate buffer pH 7.0
Citrate phosphate buffer pH 5.6
Non-treated sample
8.04 log 10
Non-treated sample
7.99 log 10
400 MPa, 10 min 600 MPa, 10 min
,1 cell / 10 ml ,1 cell / 10 ml
350 MPa, 10 min 600 MPa, 10 min
,10 cell / 10 ml ,1 cell / 10 ml
was a combination of a pressure level, duration, and pH as shown in Table 1.
3. Results
3.1. Outer membrane fractions analysis 2.5. Isolation of cytoplasmic and outer membrane fractions The procedure was based on an equilibrium density gradient centrifugation of the total membrane extract obtained by lysis of lysozyme–EDTA spheroplasts. Cells were harvested by centrifugation at 8000 rev. / min for 15 min at 48C (Sorvall RC5B, rotor GSA). The cell pellet was rapidly resuspended in cold 0.75 M sucrose–10 mM Tris–HCl buffer, pH 7.8. Lysozyme was immediately added to a final concentration of 100 mg / ml, 2 volumes of cold 1.5 mM EDTA (Na 1 ), pH 7.5 were slowly added and the bacterial suspension was incubated at 308C for 90 min. Conversion to spheroplasts was monitored by optical microscopy. When spheroplast formation attempted about 95%, cell suspension was immersed in an ice-cold bath and sonicated four times for 30 s with Vibracell (Bioblock Scientific). Intact cells were removed by centrifugation at 10,000 rev. / min for 15 min (Sorvall RC5B, rotor SS34). Membrane extracts were collected by centrifugation at 37,000 rev. / min for 2 h (Beckman, rotor 70 Ti). Step gradients of sucrose density were prepared by layering 2 ml each of 50%, 45%, 40%, 35% and 30% sucrose solutions (w / w) containing 5 mM EDTA, pH 7.5. The membrane pellet was resuspended in 0.5 ml of cold 25% sucrose, 5 mM EDTA, pH 7.5 and applied on the top of the gradient. Centrifugation was carried out for 12 h at 37,000 rev. / min and 48C (Beckman, rotor SW 41 Ti). Fractions of 0.5 ml were collected from the bottom of the tube with a peristaltic pump. Protein content was assayed by ESL Boehringer kit and membrane extracts were analysed by electrophoresis SDS–PAGE (7% and 15% gel system). Proteins were visualised by silver stain.
After hyperbaric exposure, cells were harvested, bacterial membranes extracted and analysed by electrophoresis SDS–PAGE. Efficiency of spheroplast formation was lower (50%) for treated cells than for control bacterial suspension (95%). This suggests that the outer membranes (OM) seem to be more resistant to EDTA–lysozyme treatment after hyperbaric shock. Electrophoretic profiles revealed the presence of three major outer membrane proteins and about 12 minor proteins in the control bacteria outer membranes (Figs. 1 and 2). Major outer membrane proteins are identified as LamB (44 kDa), OmpC (37 kDa) and OmpA (35 kDa) (Nikaido and Vaara, 1985). The quantities of these proteins were found to be quite similar whatever the extra-cellular pH is, 7.0 or 5.6. After treatment at 350 MPa or 400 MPa, and more so at 600 MPa, the Salmonella typhimurium population was totally inactivated, outer membrane protein content is drastically modified whatever the extra-
Fig. 1. Outer membrane profile. Cell suspension in phosphate buffer pH 7.0. Protein content is analysed by electrophoresis SDS–PAGE and visualised by silver stain.
118
M. Ritz et al. / International Journal of Food Microbiology 55 (2000) 115 – 119
Fig. 2. Outer membrane profile. Cell suspension in citrate phosphate buffer pH 5.6. Protein content is analysed by electrophoresis SDS–PAGE and visualised by silver stain.
cellular pH; 350 MPa, pH 5.6 and 400 MPa, pH 7.0 treated cells presented a different outer membrane protein pattern whereas the effect on enumeration is similar. Outer membrane extracts from treated cells in phosphate buffer at 400 MPa (Fig. 1), showed each minor and major protein except the 44 kDa protein which almost entirely disappeared. On the contrary, outer membranes obtained from cells suspended in citrate buffer at an acidic pH of 5.6 (Fig. 2), revealed only two proteins; the major outer membrane protein of 35 kDa appearing in a similar quantity as in controls, and a 44 kDa outer membrane protein less concentrated. Each other major or minor outer membrane protein seemed to disappear. Only the 35 kDa protein remained after the 600 MPa treatment whatever the pH of the suspending medium. After heating for 10 min at 1008C, the major outer membrane protein of 35 kDa is modified and migrates as a band corresponding to a relative molecular weight of 37 kDa. This heat modified behaviour is characteristic of porins (Nikaido et al., 1991). The heat modification property is conserved after hyperbaric exposure for cells in phosphate buffer but seems to be partially altered for cells suspended in citrate buffer.
3.2. Inner membrane analysis Sonication of spheroplasts from hyperbaric exposed cells needs less time than for spheroplasts from control cells. This suggests that the inner membrane seems to be made fragile by hyperbaric
Fig. 3. Internal membrane profile. Cell suspension in citrate phosphate buffer pH 5.6. Protein content is analysed by electrophoresis SDS–PAGE and visualised by silver stain.
treatment. Before treatment the inner membrane protein content is similar whatever the pH of the suspending medium. This content seems to reduce with the increase of pressure and after 600 MPa exposure (Fig. 3). The protein content in inner membrane protein is drastically diminished, but no significant difference of sensitivity was observed depending on the pH of the suspending medium.
4. Discussion Previous studies demonstrated some changes in cell structures (Mackey et al., 1994) and membranebound enzymes activity (Wouters et al., 1998). This work permits us to confirm the role of the cell membrane in the inactivation and characterizes a physical target of high hydrostatic pressure. The presence of the major outer membrane proteins identified as LamB, OmpC and OmpA for the control bacterial suspension confirm the capacity of this technique employed to characterize the content of membrane proteins. We observed lower formation of spheroplasts for treated cells than for control bacterial suspensions. This behaviour could be explained by a reorganisation of the outer membrane. The disappearance of the porins used for the macromolecule transport could explain the difficulties of lysozyme permeation. The results show that hyperbaric exposure alters drastically outer membrane proteins. However, the
M. Ritz et al. / International Journal of Food Microbiology 55 (2000) 115 – 119
major outer membrane protein OmpA seems to be more resistant to this treatment, probably due to its conformation and surrounding. This protein is well known to be involved in cell shape and integrity, which could explain its very resistant conformation to many stresses (Nikaido and Vaara, 1985). Moreover, the protein LamB displays a particular behaviour. Indeed, the protein resistance to hyperbaric shock depends on the extra-cellular pH. LamB is more resistant to hyperbaric exposure when the pH of the media is acidic. When the extra-cellular pH is close to neutrality, the porin is less resistant and 350 MPa are sufficient to cleave it. This behaviour could be explained by a different conformation adopted by the LamB depending on the extra-cellular pH.
5. Conclusion This study demonstrates the action of pressure on the membrane proteins. Even if the pressure is isostatic, the external membrane seems to be more damaged than the cytoplasmic membrane. In fact, except for the 35 kDa protein, each other major or minor outer membrane protein seems to disappear. The different destruction of membrane proteins between acidic and neutral pH could explain the greater inactivation recorded when the cells are treated in acidic suspension or food. These observations combined with the knowledge of pH of food could be integrated for the bacterial destruction models.
References Arroyo, G., Sanz, P.D., Prestamo, G., 1997. Effect of high pressure on the reduction of microbial populations in vegetables. J. Appl. Microbiol. 82, 735–742.
119
Cheftel, J.C., 1995. Review: high-pressure, microbial inactivation and food preservation. Food Sci. Technol. Int. 1, 75–90. Fujii, S., Iwahashi, H., Obuchi, K., Fujii, T., Komatsu, K., 1997. Characterization of a barotolerant mutant of the yeast Saccharomyces cerevisiae. Importance of trehalose content and membrane fluidity. In: Heremans, K. (Ed.), High Pressure Research in the Biosciences and Biotechnology, Leuven University Press, Leuven, pp. 241–244. Hayakawa, I., Kanno, T., Tomita, M., Fujio, Y., 1994. Application of high pressure for spore inactivation and protein denaturation. J. Food Sci. 59, 159–163. Hoover, D.G., Metrick, C., Papineau, A.M., Farkas, D.F., Knorr, D., 1989. Biological effects of high hydrostatic pressure on food microorganisms. Food Technol. 3, 99–107. Knorr, D., 1993. Effects of high hydrostatic pressure processes on food safety and quality. Food Technol., 156–161. Macdonald, A.G., 1992. Effects of high hydrostatic pressure on natural and artificial membranes. In: Balny, C., Hayashi, R., Heremans, K., Masson, P. (Eds.), High Pressure and Biotechnology, Colloque INSERM / John Libbey Eurotext, pp. 65– 75. ` K., Isaacs, N.S.R.S., Brooker, B., 1994. Mackey, B.M., Forestiere, The effect of high hydrostatic pressure on Salmonella thompson and Listeria monocytogenes examined by electron microscopy. Lett. Appl. Microbiol. 19, 429–432. Nikaido, H., Nikaido, K., Harayama, S., 1991. Identification and characterization of porins in Pseudomonas aeroginosa. J. Biol. Chem. 266, 770–779. Nikaido, H., Vaara, M., 1985. Molecular basis of bacterial outer membrane permeability. Microbiol. Rev. 49, 1–32. Osborn, M.J., Gander, J.E., Parisi, E., Carson, J., 1972. Mechanism of assembly of the outer membrane of Salmonella typhimurium. J. Biol. Chem. 247, 3962–3972. Simpson, R.K., Gilmour, A., 1997. The effect of high hydrostatic pressure on the activity of intracellular enzymes of Listeria monocytogenes. Lett. Appl. Microbiol. 25, 48–53. Wouters, P.C., Glaasker, E., Smelt, J.P., 1998. Effects of high pressure on inactivation kinetics and events related to proton efflux in Lactobacillus plantarum. Appl. Environ. Microbiol. 64, 509–514. Wuytack, E.Y., Boven, S., Michiels, C.W., 1998. Comparative study of pressure-induced germination of Bacillus subtilis spores at low and high pressures. Appl. Environ. Microbiol. 9, 3220–3224. Zimmerman, F., Bergman, C., 1993. Isostatic high-pressure equipment for food preservation. Food Technol. 47, 162–163.