Journal of Environmental Science and Health, Part B (2015) 50, 146–150 Copyright © Taylor & Francis Group, LLC ISSN: 0360-1234 (Print); 1532-4109 (Online) DOI: 10.1080/03601234.2015.975626
Listeria monocytogenes batch culture growth response to metabolic inhibitors S. R. MILILLO1,2, B. LUNGU1, C. A. O’BRYAN1, S. E. DOWD3, A. MUTHAIYAN1, M. G. JOHNSON1 and S. C. RICKE1 1
Department of Food Science and Center for Food Safety, University of Arkansas, Fayetteville, Arkansas, USA Department of Food Science, Pennsylvania State University, University Park, Pennsylvania, USA 3 USDA-ARS-LIRU, Lubbock, Texas, USA 2
In certain environments nutrient and energy sources available to microorganisms can be limited. Foodborne pathogens must efficiently adapt in order to be successfully transmitted through the food chain to their hosts. For the intracellular foodborne pathogen Listeria monocytogenes, little is known regarding its response to nutrient/energy-limiting conditions. The alternative stress responsive sigma factor s B has been reported to contribute to survival under specific stresses. Therefore, the effects of several metabolic inhibitors on growth of L. monocytogenes wild-type and a DsigB mutant were examined. In the absence of inhibitors, both strains reached stationary phase after 18 h at 23 C and 10 h at 37 C. All of the metabolic inhibitors slowed growth of either strain, with few differences observed among the different inhibitors. Keywords: Listeria monocytogenes, metabolic inhibitors, potassium cyanide, iodoacetate, sodium arsenate.
Introduction Perhaps because of the varied environments they encounter during transmission, foodborne pathogens can be talented adaptors. The intracellular foodborne pathogen, Listeria monocytogenes is a facultative anaerobe and one member of a genus containing mostly saprophytes.[1] L. monocytogenes can persist in numerous adverse environmental conditions such as low temperature, low pH, high salt concentration, and nutrient starvation.[2–4] Compared to model organisms such as E. coli and Salmonella, the specifics of L. monocytogenes’ metabolic pathways and mechanisms it employs during adaptation to new environments are not well described. It has been shown that glucose can be fermented by the Embden–Meyerhoff pathway to pyruvate and lactate (with 6 mol ATP generated per mol of glucose), but in anaerobic conditions glucose is fermented primarily to Present address for B. Lungu: Aviagen Veterinary Laboratory, Elkmont, AL 35620, USA. Present address for S. E. Dowd: MR DNA (Molecular Research), 503 Clovis Road, Shallowater, TX 79363, USA. Address correspondence to S. C. Ricke, 2650 N. Young Ave., Department of Food Science, University of Arkansas, Fayetteville, AR 72704, USA; E-mail: [email protected] Received July 9, 2014.
lactic acid.[1] Reportedly, L. monocytogenes can also ferment several other carbohydrates, including fructose, mannose, galactose, cellobiose, trehalose, and rhamnose.[1,5] It apparently has a discontinuous TCA cycle as no evidence for the conversion of a-ketoglutarate to succinate has been observed (i.e. no functional a-ketoglutarate dehydrogenase).[6] Therefore, in comparison to a traditional TCA cycle, L. monocytogenes’ metabolism of pyruvate results in less energy production by one unit of NADH and one GTP molecule. In addition, L. monocytogenes appears to lack complex IV in the electron transport chain, the cytochrome C oxidase.[1,5] Without the cytochrome C oxidase, L. monocytogenes’ power to generate the proton motive force (PMF) is reduced relative to other microorganisms possessing all the electron transport chain components. The PMF generates energy via the transfer of ions across a membrane.[6] As in the better-characterized Bacillus subtilis, the alternative sigma factor, s B, is a general stress response regulator contributing to L. monocytogenes’ ability to rapidly adapt to and survive under conditions of in vitro environmental stress.[7–9] However, Chaturongakul and Boor [10] noted that the pathway for Rsb-dependent regulation of s B activity differs between L. monocytogenes and B. subtilis. RsbV contributes to sigB activation under both environmental and energy stresses.[11] For L. monocytogenes,
Response of L. monocytogenes to metabolic inhibitors
147
RsbT in addition to RsbV were both shown to contribute to s B-dependent survival under environmental and energy stress conditions. Through use of animal models and microarray analyses, a role for s B in facilitating L. monocytogenes’ transition from saprophyte to pathogen is increasingly being recognized.[12,13] The basic response of L. monocytogenes to sudden changes in nutrient/energy availability is unclear and this is an important question to address because during its transmission L. monocytogenes routinely faces multiple stresses simultaneously, such as those encountered in food-processing plants or host cells. Thus, in the present study metabolic inhibitors were utilized to provide insight into L. monocytogenes mechanisms of growth and survival under conditions of nutrient and energy stress following interference with two important energy generating and metabolic pathways, oxidative phosphorylation, and the proton motive force and substrate-level phosphorylation and the TCA cycle. In addition, since s B has been shown to be activated under stress conditions, an L. monocytogenes strain containing a sigB null mutation was also studied.[7,10,14]
measured every 2 h until the control cultures (those without metabolic inhibitor added) reached stationary phase. The mean OD600 values from three separate experiments were plotted over time. Statistical analyses Correlation analyses were performed using multivariate analyses functions of JMP 6.0 (SAS Institute).
Results and discussion Transmission through the food chain requires L. monocytogenes to balance multiple stresses simultaneously, ranging from those associated with food-processing plants to those encountered in hosts. The effects of metabolic inhibitors interfering with oxidative and substrate-level phosphorylation on L. monocytogenes were investigated to gain insight into L. monocytogenes’ response to nutrient and
Materials and methods Bacterial strains and metabolic inhibitors Stock cultures of L. monocytogenes strains wild-type (WT; 10403S) and a nonpolar isogenic null mutant DsigB (FSL A1-254)[15] were stored at ¡80 C in Brain Heart Infusion (BHI; TEKnova, Hollister, CA, USA) broth with 20% glycerol. Prior to each experiment, frozen stocks were individually grown in BHI for 18 h at 37 C with aeration (200 rpm), then subcultured in fresh BHI for another 18 h (also at 37 C with aeration). The metabolic inhibitors potassium cyanide (CY), iodoacetate (IA), sodium fluoride (SF), sodium arsenite (SAs), sodium arsenate (SA), and 2,4-dinitrophenol (DNP; all from Sigma-Aldrich Inc, St Louis, MO, USA) were selected for their ability to inhibit substrate-level phosphorylation or oxidative phosphorylation.[16] Stock solutions were prepared and filter sterilized prior to use. The metabolic inhibitors were added aseptically to growth media to obtain the desired final concentration Growth studies L. monocytogenes WT and DsigB strains were grown individually in 10 mL volumes of BHI with aeration (200 rpm) at 23 C and 37 C to an absorbance at 600 nm (OD600) of 0.2 to 0.25, reflecting early log phase. In addition to 37 C, 23 C was selected to approximate one of the temperatures foods might experience during processing and handling, particularly if temperature abuse were to occur. The metabolic inhibitors CY, IA, SF, SAs, SA, and DNP were added to these early log phase cultures to a final concentration of 5 mM. The absorbance was subsequently
Fig. 1. Growth of L. monocytogenes WT strain at 23 C in the presence of various metabolic inhibitors. In preparation for each growth experiment, frozen stocks of the L. monocytogenes WT strain were grown in BHI for 18 h at 37 C with aeration (200 rpm), then subcultured in fresh BHI for another 18 h (also at 37 C with aeration). For the growth study, cells were grown individually in 10 mL volumes of BHI with aeration (200 rpm) at 23 C to an absorbance at 600 nm (OD600) of approximately 0.2 at which time the metabolic inhibitors potassium cyanide (CY), iodoacetate (IA), sodium fluoride (SF), sodium arsenite (SAs), sodium arsenate (SA), and 2,4-dinitrophenol (DNP) were added to a final concentration of 5 mM. Measurements were made every 2 h until the control cultures (those without added metabolic inhibitor) reached stationary phase. Data points reflect the mean OD600 values from three independent experiments, error bars indicate standard deviation from the mean.
148
Fig. 2. Growth of L. monocytogenes DsigB strain at 23 C in the presence of various metabolic inhibitors. In preparation for each growth experiment, frozen stocks of the L. monocytogenes DsigB strain were grown in BHI for 18 h at 37 C with aeration (200 rpm), then subcultured in fresh BHI for another 18 h (also at 37 C with aeration). For the growth study, cells were grown individually in 10 mL volumes of BHI with aeration (200 rpm) at 23 C to an absorbance at 600 nm (OD600) of approximately 0.2 at which time the metabolic inhibitors potassium cyanide (CY), iodoacetate (IA), sodium fluoride (SF), sodium arsenite (SAs), sodium arsenate (SA), and 2,4-dinitrophenol (DNP) were added (as indicated by the arrows) to a final concentration of 5 mM. Measurements were made every 2 h until the control cultures (those without added metabolic inhibitor) reached stationary phase. Data points reflect the mean OD600 values from three independent experiments, error bars indicate standard deviation from the mean.
energy stress, including the potential ability of the alternative stress responsive sigma factor s B to over-ride these inhibitors. Our results show that L. monocytogenes growth was arrested following exposure to six different metabolic inhibitors at 5 mM concentration. Phenotypic characterization of L. monocytogenes WT and DsigB strains exposed to six metabolic inhibitors reveals dramatic but few unique effects on growth. The effects on actively growing L. monocytogenes WT and DsigB strains of metabolic inhibitors interfering with substrate-level (IA, SF, and SAs) and oxidative (CY SA, and DNP) phosphorylation[16] were assessed at 23 C and 37 C. In the absence of inhibitors, the strains reached stationary phase after 18 h at 23 C (Figs. 1 and 2) and 10 h at 37 C (Figs. 3 and 4). Addition of any of the metabolic inhibitors to the culture medium drastically slowed growth of either L. monocytogenes strain, but few differences were observed among the different inhibitors. At either temperature, exposure to IA (an inhibitor of substrate-level
Milillo et al.
Fig. 3. Growth of L. monocytogenes WT strain at 37 C in the presence of various metabolic inhibitors. In preparation for each growth experiment, frozen stocks of the L. monocytogenes WT strain were individually grown in BHI for 18 h at 37 C with aeration (200 rpm), then subcultured in fresh BHI for another 18 h (also at 37 C with aeration). For the growth study, cells were grown individually in 10 mL volumes of BHI with aeration (200 rpm) at 37 C to an absorbance at 600 nm (OD600) of approximately 0.2 at which time the metabolic inhibitors potassium cyanide (CY), iodoacetate (IA), sodium fluoride (SF), sodium arsenite (SAs), sodium arsenate (SA), and 2,4-dinitrophenol (DNP) were added (as indicated by the arrows) to a final concentration of 5 mM. Measurements were made every 2 h until the control cultures (those without added metabolic inhibitor) reached stationary phase. Data points reflect the mean OD600 values from three independent experiments, error bars indicate standard deviations from the mean.
phosphorylation) had the most severe result, essentially arresting growth. Interestingly, the effect of SF appeared to be temperature dependent, as growth at 37 C was less affected than growth at 23 C; it is possible that this perceived effect is a result of the differing growth rates at these temperatures and further research should be done to elucidate this. Although the metabolic inhibitors’ concentration was selected to be sublethal, it was nonetheless surprising to see little difference between the effects on cell viability of inhibitors with disparate modes of action. The nutrient rich environment provided by the BHI culture medium may be an explanation for this observation as it has been shown by a recent study that the presence of exogenous organic material supports L. monocytogenes survival during times of stress.[16] Literature searches failed to uncover any prior research conducted to characterize the relative importance of substrate-level phosphorylation to L. monocytogenes metabolism and energy generation. However, it has been previously shown that the use of bacteriocin PMF dissipaters, like nisin and pediocin JD, have detrimental effects on L. monocytogenes.[17–19] Due to their respective interference with two important avenues for energy generation,
149
Response of L. monocytogenes to metabolic inhibitors
04637) as well as the Department of Food Science at the University of Arkansas.
References
Fig. 4. Growth of L. monocytogenes DsigB strain at 37 C in the presence of various metabolic inhibitors. In preparation for each growth experiment, frozen stocks of the L. monocytogenes DsigB strain were grown in BHI for 18 h at 37 C with aeration (200 rpm), then subcultured in fresh BHI for another 18 h (also at 37 C with aeration). For the growth study, cells were grown individually in 10 mL volumes of BHI with aeration (200 rpm) at 37 C to an absorbance at 600 nm (OD600) of approximately 0.2 at which time the metabolic inhibitors potassium cyanide (CY), iodoacetate (IA), sodium fluoride (SF), sodium arsenite (SAs), sodium arsenate (SA), and 2,4-dinitrophenol (DNP) were added (as indicated by the arrows) to a final concentration of 5 mM. Measurements were made every 2 h until the control cultures (those without added metabolic inhibitor) reached stationary phase. Data points reflect the mean OD600 values from three independent experiments, error bars indicate standard deviations from the mean.
oxidative and substrate-level phosphorylation, the PMF dissipater DNP[20,21] and SAs, an inhibitor of the glycolytic enzyme pyruvate dehydrogenase that catalyzes production of acetyl CoA for the TCA cycle, were utilized in the subsequent gene expression analyses. In conclusion, the environments experienced by foodborne pathogens during passage through the food chain or during infection involve conditions of severe nutrient and energy stress. This study investigated the effect(s) on L. monocytogenes’ growth of interference with two important energy generating pathways: oxidative phosphorylation and substrate-level phosphorylation. All of the metabolic inhibitors we studied slowed growth of both L. monocytogenes WT and DsigB strains, but there was little difference observed in effects of the different inhibitors.
Funding This study was supported in part by grants from the USDA-Food Safety Consortium and the USDA/ CSREES NRI funding program (award no. 2008-35201-
[1] Benedict, R.C. Listeria monocytogenes: Physiology and metabolism. In Foodborne Listeriosis, Miller, A.J., Smith, J.L., Somkuti, G.A., Ed.; Elsevier: New York, NY, 1990, p. 13–24. [2] Becker, L.A.; Evans, S.N.; Hutkins, R.W.; Benson, A.K. Role of sigma (B) in adaptation of Listeria monocytogenes to growth at low temperature. J. Bacteriol. 2000, 182, 7083–7087. [3] Ferreira, A.; Sue, D.; O’Byrne, C.P. K.J. Boor, K.J. Role of Listeria monocytogenes sigma(B) in survival of lethal acidic conditions and in the acquired acid tolerance response. Appl. Environ. Microbiol. 2003, 69, 2692–2698. [4] Lungu, B.; Ricke, S.C.; Johnson, M.G. Resistance of nutrientdeprived Listeria monocytogenes 10403S and a delta sigB mutant to chemical stresses in the presence or absence of oxygen. J. Food Sci. 2008, 73, 339–345. [5] Seeliger, H.P.R.; Jones, D. Genus Listeria Pirie 1940. In Bergey’s Manual of Systematic Bacteriology, Sneath, P.H.A., Mair, N.S., Sharpe, M.E., Holt, J.G. Ed., Williams and Wilkins: Baltimore, MD, 1986, Vol. 2, p. 1235–1245. [6] Trivett, T.L.; Meyer, E.A. Citrate cycle and related metabolism of Listeria monocytogenes. J. Bacteriol. 1971, 107, 770–779. [7] Ferreira, A.; O’Byrne, C.P.; Boor, K.J. Role of sigma (B) in heat, ethanol, acid, and oxidative stress resistance and during carbon starvation in Listeria monocytogenes. Appl. Environ. Microbiol. 2001, 67, 4454–4457. [8] Hoper, D.; Volker, U.; Hecker, M. Comprehensive characterization of the contribution of individual SigB-dependent general stress genes to stress resistance of Bacillus subtilis. J. Bacteriol. 2005, 187, 2810–2826. [9] Horsburgh, M.J.; Aish, J.L.; White, I.J.; Shaw, L.; Lithgow, J.K.; Foster, S.J. s B modulates virulence determinant expression and stress resistance: Characterization of a functional rsbU strain derived from Staphylococcus aureus 8325–4. J. Bacteriol. 2002, 184, 5457–5467. [10] Chaturongakul, S.; Boor, K.J. RsbT and RsbV contribute to sigma B-dependent survival under environmental, energy, and intracellular stress conditions in Listeria monocytogenes. Appl. Environ. Microbiol. 2004, 70, 5349–5356. [11] Ferreira, A., Gray, M.; Wiedmann, M.; Boor, K.J. Comparative genomic analysis of the SigB operon in Listeria monocytogenes and in other gram-positive bacteria. Curr. Microbiol. 2004, 48, 39–46. [12] Garner, M., Njaa, B.; Wiedmann, M.; Boor, K. Sigma B contributes to Listeria monocytogenes gastrointestinal infection but not to systemic spread in the guinea pig infection model. Infect. Immun. 2006, 74, 876–886. [13] Toledo-Arana, A.; Dussurget, O.; Nikitas, G.; Sesto, N.; GuetRevillet, H.; Balestrino, D.; Loh, E.; Gripenland, J.; Tiensuu, T.; Vaitkevicius, K.; Barthelemy, M.; Vergassola, M.; Nahori,, M.; Soubigou, G.; Regnault, B.; Coppee, J.; Lecuit, M.; Johansson, J.; Cossart, P. The Listeria transcriptional landscape from saprophytism to virulence. Nature 2009, 459, 950–956. [14] Chaturongakul, S.; Boor, K.J. Sigma B activation under environmental and energy stress conditions in Listeria monocytogenes. Appl. Environ. Microbiol. 2006, 72, 5197–5203. [15] Wiedmann, M.; Arvik, T.; Hurley, R.; Boor, K. General stress transcription factor Sigma B and its role in acid tolerance and virulence of Listeria monocytogenes. J. Bacteriol. 1998, 180, 3650–3656. [16] Lungu, B.; Saldivar, J.C.; Story, R.; Ricke, S.C.; Johnson, M. G. The combination of energy-dependent internal adaptation
150 mechanisms and external factors enables Listeria monocytogenes to express a strong starvation survival response during multiple-nutrient starvation. Foodborne Pathogens Dis. 2010, 7, 499–505. [17] Christensen, D.P.; Hutkins, R.W. Glucose uptake by Listeria monocytogenes Scott A and inhibition by pediocin JD. Appl. Environ. Microbiol. 1994, 60, 3870–3873. [18] Lungu, B.; Johnson, M.G. Fate of Listeria monocytogenes inoculated onto the surface of model turkey frankfurter pieces treated with zein coatings containing nisin, sodium diacetate,
Milillo et al. and sodium lactate at 4 degrees C. J. Food Prot. 2005, 68, 855–859. [19] Winkowski, K., Bruno, M.E.; Montville, T.J. Correlation of bioenergetic parameters with cell death in Listeria monocytogenes cells exposed to nisin. Appl. Environ. Microbiol. 1994, 60, 4186–4188. [20] Loomis, W.F.; Lipman, F. Reversible inhibition of the coupling between phosphorylation and oxidation. J. Biol. Chem. 1948, 173, 807–808. [21] Shioi, J.; Taylor, B.L. Oxygen taxis and proton motive force in Salmonella Typhimurium. J. Biol. Chem. 1984, 259, 10983–10988.
Copyright of Journal of Environmental Science & Health, Part B -- Pesticides, Food Contaminants, & Agricultural Wastes is the property of Taylor & Francis Ltd and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.
Listeria monocytogenes batch culture growth response to metabolic inhibitors S. R. MILILLO1,2, B. LUNGU1, C. A. O’BRYAN1, S. E. DOWD3, A. MUTHAIYAN1, M. G. JOHNSON1 and S. C. RICKE1 1
Department of Food Science and Center for Food Safety, University of Arkansas, Fayetteville, Arkansas, USA Department of Food Science, Pennsylvania State University, University Park, Pennsylvania, USA 3 USDA-ARS-LIRU, Lubbock, Texas, USA 2
In certain environments nutrient and energy sources available to microorganisms can be limited. Foodborne pathogens must efficiently adapt in order to be successfully transmitted through the food chain to their hosts. For the intracellular foodborne pathogen Listeria monocytogenes, little is known regarding its response to nutrient/energy-limiting conditions. The alternative stress responsive sigma factor s B has been reported to contribute to survival under specific stresses. Therefore, the effects of several metabolic inhibitors on growth of L. monocytogenes wild-type and a DsigB mutant were examined. In the absence of inhibitors, both strains reached stationary phase after 18 h at 23 C and 10 h at 37 C. All of the metabolic inhibitors slowed growth of either strain, with few differences observed among the different inhibitors. Keywords: Listeria monocytogenes, metabolic inhibitors, potassium cyanide, iodoacetate, sodium arsenate.
Introduction Perhaps because of the varied environments they encounter during transmission, foodborne pathogens can be talented adaptors. The intracellular foodborne pathogen, Listeria monocytogenes is a facultative anaerobe and one member of a genus containing mostly saprophytes.[1] L. monocytogenes can persist in numerous adverse environmental conditions such as low temperature, low pH, high salt concentration, and nutrient starvation.[2–4] Compared to model organisms such as E. coli and Salmonella, the specifics of L. monocytogenes’ metabolic pathways and mechanisms it employs during adaptation to new environments are not well described. It has been shown that glucose can be fermented by the Embden–Meyerhoff pathway to pyruvate and lactate (with 6 mol ATP generated per mol of glucose), but in anaerobic conditions glucose is fermented primarily to Present address for B. Lungu: Aviagen Veterinary Laboratory, Elkmont, AL 35620, USA. Present address for S. E. Dowd: MR DNA (Molecular Research), 503 Clovis Road, Shallowater, TX 79363, USA. Address correspondence to S. C. Ricke, 2650 N. Young Ave., Department of Food Science, University of Arkansas, Fayetteville, AR 72704, USA; E-mail: [email protected] Received July 9, 2014.
lactic acid.[1] Reportedly, L. monocytogenes can also ferment several other carbohydrates, including fructose, mannose, galactose, cellobiose, trehalose, and rhamnose.[1,5] It apparently has a discontinuous TCA cycle as no evidence for the conversion of a-ketoglutarate to succinate has been observed (i.e. no functional a-ketoglutarate dehydrogenase).[6] Therefore, in comparison to a traditional TCA cycle, L. monocytogenes’ metabolism of pyruvate results in less energy production by one unit of NADH and one GTP molecule. In addition, L. monocytogenes appears to lack complex IV in the electron transport chain, the cytochrome C oxidase.[1,5] Without the cytochrome C oxidase, L. monocytogenes’ power to generate the proton motive force (PMF) is reduced relative to other microorganisms possessing all the electron transport chain components. The PMF generates energy via the transfer of ions across a membrane.[6] As in the better-characterized Bacillus subtilis, the alternative sigma factor, s B, is a general stress response regulator contributing to L. monocytogenes’ ability to rapidly adapt to and survive under conditions of in vitro environmental stress.[7–9] However, Chaturongakul and Boor [10] noted that the pathway for Rsb-dependent regulation of s B activity differs between L. monocytogenes and B. subtilis. RsbV contributes to sigB activation under both environmental and energy stresses.[11] For L. monocytogenes,
Response of L. monocytogenes to metabolic inhibitors
147
RsbT in addition to RsbV were both shown to contribute to s B-dependent survival under environmental and energy stress conditions. Through use of animal models and microarray analyses, a role for s B in facilitating L. monocytogenes’ transition from saprophyte to pathogen is increasingly being recognized.[12,13] The basic response of L. monocytogenes to sudden changes in nutrient/energy availability is unclear and this is an important question to address because during its transmission L. monocytogenes routinely faces multiple stresses simultaneously, such as those encountered in food-processing plants or host cells. Thus, in the present study metabolic inhibitors were utilized to provide insight into L. monocytogenes mechanisms of growth and survival under conditions of nutrient and energy stress following interference with two important energy generating and metabolic pathways, oxidative phosphorylation, and the proton motive force and substrate-level phosphorylation and the TCA cycle. In addition, since s B has been shown to be activated under stress conditions, an L. monocytogenes strain containing a sigB null mutation was also studied.[7,10,14]
measured every 2 h until the control cultures (those without metabolic inhibitor added) reached stationary phase. The mean OD600 values from three separate experiments were plotted over time. Statistical analyses Correlation analyses were performed using multivariate analyses functions of JMP 6.0 (SAS Institute).
Results and discussion Transmission through the food chain requires L. monocytogenes to balance multiple stresses simultaneously, ranging from those associated with food-processing plants to those encountered in hosts. The effects of metabolic inhibitors interfering with oxidative and substrate-level phosphorylation on L. monocytogenes were investigated to gain insight into L. monocytogenes’ response to nutrient and
Materials and methods Bacterial strains and metabolic inhibitors Stock cultures of L. monocytogenes strains wild-type (WT; 10403S) and a nonpolar isogenic null mutant DsigB (FSL A1-254)[15] were stored at ¡80 C in Brain Heart Infusion (BHI; TEKnova, Hollister, CA, USA) broth with 20% glycerol. Prior to each experiment, frozen stocks were individually grown in BHI for 18 h at 37 C with aeration (200 rpm), then subcultured in fresh BHI for another 18 h (also at 37 C with aeration). The metabolic inhibitors potassium cyanide (CY), iodoacetate (IA), sodium fluoride (SF), sodium arsenite (SAs), sodium arsenate (SA), and 2,4-dinitrophenol (DNP; all from Sigma-Aldrich Inc, St Louis, MO, USA) were selected for their ability to inhibit substrate-level phosphorylation or oxidative phosphorylation.[16] Stock solutions were prepared and filter sterilized prior to use. The metabolic inhibitors were added aseptically to growth media to obtain the desired final concentration Growth studies L. monocytogenes WT and DsigB strains were grown individually in 10 mL volumes of BHI with aeration (200 rpm) at 23 C and 37 C to an absorbance at 600 nm (OD600) of 0.2 to 0.25, reflecting early log phase. In addition to 37 C, 23 C was selected to approximate one of the temperatures foods might experience during processing and handling, particularly if temperature abuse were to occur. The metabolic inhibitors CY, IA, SF, SAs, SA, and DNP were added to these early log phase cultures to a final concentration of 5 mM. The absorbance was subsequently
Fig. 1. Growth of L. monocytogenes WT strain at 23 C in the presence of various metabolic inhibitors. In preparation for each growth experiment, frozen stocks of the L. monocytogenes WT strain were grown in BHI for 18 h at 37 C with aeration (200 rpm), then subcultured in fresh BHI for another 18 h (also at 37 C with aeration). For the growth study, cells were grown individually in 10 mL volumes of BHI with aeration (200 rpm) at 23 C to an absorbance at 600 nm (OD600) of approximately 0.2 at which time the metabolic inhibitors potassium cyanide (CY), iodoacetate (IA), sodium fluoride (SF), sodium arsenite (SAs), sodium arsenate (SA), and 2,4-dinitrophenol (DNP) were added to a final concentration of 5 mM. Measurements were made every 2 h until the control cultures (those without added metabolic inhibitor) reached stationary phase. Data points reflect the mean OD600 values from three independent experiments, error bars indicate standard deviation from the mean.
148
Fig. 2. Growth of L. monocytogenes DsigB strain at 23 C in the presence of various metabolic inhibitors. In preparation for each growth experiment, frozen stocks of the L. monocytogenes DsigB strain were grown in BHI for 18 h at 37 C with aeration (200 rpm), then subcultured in fresh BHI for another 18 h (also at 37 C with aeration). For the growth study, cells were grown individually in 10 mL volumes of BHI with aeration (200 rpm) at 23 C to an absorbance at 600 nm (OD600) of approximately 0.2 at which time the metabolic inhibitors potassium cyanide (CY), iodoacetate (IA), sodium fluoride (SF), sodium arsenite (SAs), sodium arsenate (SA), and 2,4-dinitrophenol (DNP) were added (as indicated by the arrows) to a final concentration of 5 mM. Measurements were made every 2 h until the control cultures (those without added metabolic inhibitor) reached stationary phase. Data points reflect the mean OD600 values from three independent experiments, error bars indicate standard deviation from the mean.
energy stress, including the potential ability of the alternative stress responsive sigma factor s B to over-ride these inhibitors. Our results show that L. monocytogenes growth was arrested following exposure to six different metabolic inhibitors at 5 mM concentration. Phenotypic characterization of L. monocytogenes WT and DsigB strains exposed to six metabolic inhibitors reveals dramatic but few unique effects on growth. The effects on actively growing L. monocytogenes WT and DsigB strains of metabolic inhibitors interfering with substrate-level (IA, SF, and SAs) and oxidative (CY SA, and DNP) phosphorylation[16] were assessed at 23 C and 37 C. In the absence of inhibitors, the strains reached stationary phase after 18 h at 23 C (Figs. 1 and 2) and 10 h at 37 C (Figs. 3 and 4). Addition of any of the metabolic inhibitors to the culture medium drastically slowed growth of either L. monocytogenes strain, but few differences were observed among the different inhibitors. At either temperature, exposure to IA (an inhibitor of substrate-level
Milillo et al.
Fig. 3. Growth of L. monocytogenes WT strain at 37 C in the presence of various metabolic inhibitors. In preparation for each growth experiment, frozen stocks of the L. monocytogenes WT strain were individually grown in BHI for 18 h at 37 C with aeration (200 rpm), then subcultured in fresh BHI for another 18 h (also at 37 C with aeration). For the growth study, cells were grown individually in 10 mL volumes of BHI with aeration (200 rpm) at 37 C to an absorbance at 600 nm (OD600) of approximately 0.2 at which time the metabolic inhibitors potassium cyanide (CY), iodoacetate (IA), sodium fluoride (SF), sodium arsenite (SAs), sodium arsenate (SA), and 2,4-dinitrophenol (DNP) were added (as indicated by the arrows) to a final concentration of 5 mM. Measurements were made every 2 h until the control cultures (those without added metabolic inhibitor) reached stationary phase. Data points reflect the mean OD600 values from three independent experiments, error bars indicate standard deviations from the mean.
phosphorylation) had the most severe result, essentially arresting growth. Interestingly, the effect of SF appeared to be temperature dependent, as growth at 37 C was less affected than growth at 23 C; it is possible that this perceived effect is a result of the differing growth rates at these temperatures and further research should be done to elucidate this. Although the metabolic inhibitors’ concentration was selected to be sublethal, it was nonetheless surprising to see little difference between the effects on cell viability of inhibitors with disparate modes of action. The nutrient rich environment provided by the BHI culture medium may be an explanation for this observation as it has been shown by a recent study that the presence of exogenous organic material supports L. monocytogenes survival during times of stress.[16] Literature searches failed to uncover any prior research conducted to characterize the relative importance of substrate-level phosphorylation to L. monocytogenes metabolism and energy generation. However, it has been previously shown that the use of bacteriocin PMF dissipaters, like nisin and pediocin JD, have detrimental effects on L. monocytogenes.[17–19] Due to their respective interference with two important avenues for energy generation,
149
Response of L. monocytogenes to metabolic inhibitors
04637) as well as the Department of Food Science at the University of Arkansas.
References
Fig. 4. Growth of L. monocytogenes DsigB strain at 37 C in the presence of various metabolic inhibitors. In preparation for each growth experiment, frozen stocks of the L. monocytogenes DsigB strain were grown in BHI for 18 h at 37 C with aeration (200 rpm), then subcultured in fresh BHI for another 18 h (also at 37 C with aeration). For the growth study, cells were grown individually in 10 mL volumes of BHI with aeration (200 rpm) at 37 C to an absorbance at 600 nm (OD600) of approximately 0.2 at which time the metabolic inhibitors potassium cyanide (CY), iodoacetate (IA), sodium fluoride (SF), sodium arsenite (SAs), sodium arsenate (SA), and 2,4-dinitrophenol (DNP) were added (as indicated by the arrows) to a final concentration of 5 mM. Measurements were made every 2 h until the control cultures (those without added metabolic inhibitor) reached stationary phase. Data points reflect the mean OD600 values from three independent experiments, error bars indicate standard deviations from the mean.
oxidative and substrate-level phosphorylation, the PMF dissipater DNP[20,21] and SAs, an inhibitor of the glycolytic enzyme pyruvate dehydrogenase that catalyzes production of acetyl CoA for the TCA cycle, were utilized in the subsequent gene expression analyses. In conclusion, the environments experienced by foodborne pathogens during passage through the food chain or during infection involve conditions of severe nutrient and energy stress. This study investigated the effect(s) on L. monocytogenes’ growth of interference with two important energy generating pathways: oxidative phosphorylation and substrate-level phosphorylation. All of the metabolic inhibitors we studied slowed growth of both L. monocytogenes WT and DsigB strains, but there was little difference observed in effects of the different inhibitors.
Funding This study was supported in part by grants from the USDA-Food Safety Consortium and the USDA/ CSREES NRI funding program (award no. 2008-35201-
[1] Benedict, R.C. Listeria monocytogenes: Physiology and metabolism. In Foodborne Listeriosis, Miller, A.J., Smith, J.L., Somkuti, G.A., Ed.; Elsevier: New York, NY, 1990, p. 13–24. [2] Becker, L.A.; Evans, S.N.; Hutkins, R.W.; Benson, A.K. Role of sigma (B) in adaptation of Listeria monocytogenes to growth at low temperature. J. Bacteriol. 2000, 182, 7083–7087. [3] Ferreira, A.; Sue, D.; O’Byrne, C.P. K.J. Boor, K.J. Role of Listeria monocytogenes sigma(B) in survival of lethal acidic conditions and in the acquired acid tolerance response. Appl. Environ. Microbiol. 2003, 69, 2692–2698. [4] Lungu, B.; Ricke, S.C.; Johnson, M.G. Resistance of nutrientdeprived Listeria monocytogenes 10403S and a delta sigB mutant to chemical stresses in the presence or absence of oxygen. J. Food Sci. 2008, 73, 339–345. [5] Seeliger, H.P.R.; Jones, D. Genus Listeria Pirie 1940. In Bergey’s Manual of Systematic Bacteriology, Sneath, P.H.A., Mair, N.S., Sharpe, M.E., Holt, J.G. Ed., Williams and Wilkins: Baltimore, MD, 1986, Vol. 2, p. 1235–1245. [6] Trivett, T.L.; Meyer, E.A. Citrate cycle and related metabolism of Listeria monocytogenes. J. Bacteriol. 1971, 107, 770–779. [7] Ferreira, A.; O’Byrne, C.P.; Boor, K.J. Role of sigma (B) in heat, ethanol, acid, and oxidative stress resistance and during carbon starvation in Listeria monocytogenes. Appl. Environ. Microbiol. 2001, 67, 4454–4457. [8] Hoper, D.; Volker, U.; Hecker, M. Comprehensive characterization of the contribution of individual SigB-dependent general stress genes to stress resistance of Bacillus subtilis. J. Bacteriol. 2005, 187, 2810–2826. [9] Horsburgh, M.J.; Aish, J.L.; White, I.J.; Shaw, L.; Lithgow, J.K.; Foster, S.J. s B modulates virulence determinant expression and stress resistance: Characterization of a functional rsbU strain derived from Staphylococcus aureus 8325–4. J. Bacteriol. 2002, 184, 5457–5467. [10] Chaturongakul, S.; Boor, K.J. RsbT and RsbV contribute to sigma B-dependent survival under environmental, energy, and intracellular stress conditions in Listeria monocytogenes. Appl. Environ. Microbiol. 2004, 70, 5349–5356. [11] Ferreira, A., Gray, M.; Wiedmann, M.; Boor, K.J. Comparative genomic analysis of the SigB operon in Listeria monocytogenes and in other gram-positive bacteria. Curr. Microbiol. 2004, 48, 39–46. [12] Garner, M., Njaa, B.; Wiedmann, M.; Boor, K. Sigma B contributes to Listeria monocytogenes gastrointestinal infection but not to systemic spread in the guinea pig infection model. Infect. Immun. 2006, 74, 876–886. [13] Toledo-Arana, A.; Dussurget, O.; Nikitas, G.; Sesto, N.; GuetRevillet, H.; Balestrino, D.; Loh, E.; Gripenland, J.; Tiensuu, T.; Vaitkevicius, K.; Barthelemy, M.; Vergassola, M.; Nahori,, M.; Soubigou, G.; Regnault, B.; Coppee, J.; Lecuit, M.; Johansson, J.; Cossart, P. The Listeria transcriptional landscape from saprophytism to virulence. Nature 2009, 459, 950–956. [14] Chaturongakul, S.; Boor, K.J. Sigma B activation under environmental and energy stress conditions in Listeria monocytogenes. Appl. Environ. Microbiol. 2006, 72, 5197–5203. [15] Wiedmann, M.; Arvik, T.; Hurley, R.; Boor, K. General stress transcription factor Sigma B and its role in acid tolerance and virulence of Listeria monocytogenes. J. Bacteriol. 1998, 180, 3650–3656. [16] Lungu, B.; Saldivar, J.C.; Story, R.; Ricke, S.C.; Johnson, M. G. The combination of energy-dependent internal adaptation
150 mechanisms and external factors enables Listeria monocytogenes to express a strong starvation survival response during multiple-nutrient starvation. Foodborne Pathogens Dis. 2010, 7, 499–505. [17] Christensen, D.P.; Hutkins, R.W. Glucose uptake by Listeria monocytogenes Scott A and inhibition by pediocin JD. Appl. Environ. Microbiol. 1994, 60, 3870–3873. [18] Lungu, B.; Johnson, M.G. Fate of Listeria monocytogenes inoculated onto the surface of model turkey frankfurter pieces treated with zein coatings containing nisin, sodium diacetate,
Milillo et al. and sodium lactate at 4 degrees C. J. Food Prot. 2005, 68, 855–859. [19] Winkowski, K., Bruno, M.E.; Montville, T.J. Correlation of bioenergetic parameters with cell death in Listeria monocytogenes cells exposed to nisin. Appl. Environ. Microbiol. 1994, 60, 4186–4188. [20] Loomis, W.F.; Lipman, F. Reversible inhibition of the coupling between phosphorylation and oxidation. J. Biol. Chem. 1948, 173, 807–808. [21] Shioi, J.; Taylor, B.L. Oxygen taxis and proton motive force in Salmonella Typhimurium. J. Biol. Chem. 1984, 259, 10983–10988.
Copyright of Journal of Environmental Science & Health, Part B -- Pesticides, Food Contaminants, & Agricultural Wastes is the property of Taylor & Francis Ltd and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.
