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A modified rapid enzymatic microtiter plate assay for the quantification of intracellular γ-aminobutyric acid and succinate semialdehyde in bacterial cells
Journal of Microbiological Methods 84 (2011) 137–139
Contents lists available at ScienceDirect
Journal of Microbiological Methods j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j m i c m e t h
Note
A modified rapid enzymatic microtiter plate assay for the quantification of intracellular γ-aminobutyric acid and succinate semialdehyde in bacterial cells C.P. O'Byrne, C. Feehily, R. Ham, K.A.G. Karatzas ⁎ Bacterial Stress Response Group, Microbiology, School of Natural Sciences, NUI Galway, Galway, Ireland
a r t i c l e
i n f o
Article history: Received 6 August 2010 Received in revised form 21 October 2010 Accepted 25 October 2010 Available online 31 October 2010
a b s t r a c t The GABase assay is widely used to rapidly and accurately quantify levels of extracellular γ-aminobutyric acid (GABA). Here we demonstrate a modification of this assay that enables quantification of intracellular GABA in bacterial cells. Cells are lysed by boiling and ethanolamine-O-sulphate, a GABA transaminase inhibitor is used to distinguish between GABA and succinate semialdehyde. © 2010 Elsevier B.V. All rights reserved.
Keywords: Listeria monocytogenes GABA Succinate semialdehyde Acid tolerance GABase
γ-aminobutyrate (GABA) is an important metabolite produced by the glutamate decarboxylase (GAD) system of various microorganisms, through the decarboxylation of glutamate in response to acidic conditions. Therefore measurements of GABA are important in research of pathogenic bacteria that use the GAD system to survive the acidic pH of the stomach (Cotter et al., 2001; Foster, 2004; Waterman and Small, 2003). This property is also important for probiotic bacteria because colonisation of the gastrointestinal tract requires survival in the stomach (Siragusa et al., 2007). Furthermore, screening of various microorganisms used in fermentations is important as they can ensure better acidification of the product, flavour and a product enriched in GABA (Komatsuzaki et al., 2005). The latter is important because GABA is beneficial for humans playing a role as a neurotransmitter in the central nervous system (Watanabe et al., 2002) and as a secretagogue of insulin in the pancreas (Adeghate and Ponery, 2002). Oral administration of GABA has also been shown to induce relaxation and enhance immunity under stress conditions (Abdou et al., 2006). Measurements of extracellular GABA (GABAe) as means of quantification of the GAD system activity can indicate the acid resistance of a microorganism (Cotter et al., 2001). These measurements are usually performed with the use of the GABase assay. However, quantification of the intracellular GABA (GABAi) is also important for the investigation of the GAD system (Karatzas et al., 2010) and has been performed in a limited number of microorganisms such as Lactobacillus paracasei (Komatsuzaki et al., 2005), Streptococcus salivarius (Yang et al., 2008)
⁎ Corresponding author. Tel.: + 353 91 495091; fax: + 353 91 494598. E-mail address: [email protected] (K.A.G. Karatzas). 0167-7012/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2010.10.017
and Saccharomyces cerevisiae (Bach et al., 2009). Until now these measurements have been performed with the use of an HPLC/amino acid analyser, which is an expensive piece of equipment while the method is relatively laborious compared to the GABase (Tsukatani et al., 2005). Furthermore, the two methods provide comparable results as it has been demonstrated by Tsukatani et al. (2005). In this manuscript we propose certain modifications of the rapid and inexpensive GABase assay described previously by Tsukatani et al. (2005) to be used for the quantification of GABAi in Listeria monocytogenes and other bacteria. The modifications include the lysis of the cells by boiling and the use of ethanolamine-O-sulphate (EOS), a GABA transaminase inhibitor to enable the distinction between GABA and succinate semialdehyde. To quantify GABAi a method to lyse the cells was required. We investigated various methods like boiling, bead beating and sonication to produce the lysates. The lysates were prepared by harvesting cells from 20 ml of culture grown overnight in BHI following centrifugation at 9000×g for 10 min. Subsequently, the supernatant was removed and the pellet was resuspended in 1 ml of sterile water and subjected to the above-mentioned lysis procedures. Lysates were centrifuged at 8000×g for 10 min to remove all debris and the supernatant was removed and placed in a sterile 1.5 ml tube. Boiling generated the highest signal when lysates were analysed with a standard GABase assay (data not shown). Subsequently, we investigated various times of boiling (10–25 min) which would be able to kill all cells and release all GABAi. Boiling for 10 min was sufficient to kill all cells as no viable cells were recovered from any sample of the lysates when plated on BHI agar. It was demonstrated experimentally that all GABAi was released as an increase in the boiling time did not result in an increase in the levels of GABAi measured (data not shown). Furthermore, the
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boiling did not lead to the degradation of GABA. This was demonstrated experimentally by the high recovery of GABA (N99%) following boiling of standards with different concentrations of GABA for different durations of time between 10 and 25 min (data not shown). Once we proved that boiling was the method of choice for cell lysis, the next step was to test the ability of the method to quantify different levels of GABA in standards prepared with boiled lysates of L. monocytogenes. Boiled lysates as well as sterile water were used to prepare standards of 0, 1, 2, and 3–10 mM GABA in triplicate and subsequently the GABase assay was performed as described previously by Tsukatani et al., 2005. Standard curves of GABA for the average values in lysate and water were constructed (Fig. 1) and statistical analysis was performed by t-test for the slope and the y-intercept resulted in P = 0.7 and 0.8 respectively. Both values were higher than 0.05 (no statistically significant difference) suggesting that L. monocytogenes does not contain any GABAi in stationary phase and that standards in water could be used instead of standards in lysate to construct the calibration curves in order to measure concentrations of GABAi. Furthermore, we determined the sensitivity of the assay which could detect as low as 0.1 mM in water. With the use of the latter value and following a series of calculations taking in account the concentration of cells and their hypothetical volume as shown previously (Karatzas et al., 2010) we could detect as low as 1.12 mM of GABAi or SSAi in the actual cell. The repeatability of the assay was also investigated by analysing in triplicate each one of the SSA standards (0.1–1 mM). The percent error of the three determinations of each standard ranged between 0.85 and 3.21% suggesting a high repeatability. The GABase (Sigma-Aldrich, Steinheim, Germany) commercial preparation comprises two enzymes: the GABA transaminase (GABA-AT) and the succinate semialdehyde dehydrogenase (SSDH) from Pseudomonas fluorescens. GABA-AT converts GABA to succinate semialdehyde (SSA) in the presence of α-ketoglutarate. Subsequently, SSA is converted to succinate in the presence of NAD(P)+ through the activity of SSDH. In the last reaction, NAD(P)+ is transformed to NAD(P)H which absorbs at 340 nm. The above reaction is stoichiometric and therefore the levels of NAD(P)H reflect the levels of GABA in the mix. Apart from being a substrate for the second reaction in the GABase assay, SSA is present in cells as a simple intermediate in the oxidative (to succinate) or reductive (to γ-hydroxybutyric acid) catabolism of GABA (Saito et al., 2009). Therefore, intracellular SSA released in the lysate would be erroneously measured as GABA by the GABase assay. This error could be corrected by the separate measurement of SSA through the use of an SSDH preparation followed by the subtraction of this value from that obtained with the use of GABase. However, no SSDH is commercially available and another alternative way to measure SSA was to inhibit the GABA-AT in the GABase
Fig. 1. Calibration curves from standards in water (◊) and lysates (♦) are similar. Water standards can be used in experiments with lysates.
mix. The GABA-AT inhibitors, taurine (Sulaiman et al., 2003) and EOS (Fowler and John, 1972) were employed for this task in concentrations of 0, 10, 20 up to 90 mM in the assay mixture. None of the taurine concentrations inhibited the P. fluorescens SSDH suggesting that concentrations higher than 90 mM might be required (data not shown). In contrast, all concentrations of EOS conferred inhibition of SSDH in water (Fig. 2A) and lysate (data not shown). Inhibition by EOS in lysate was similar to that seen in water (data not shown). More than 60 mM EOS is required to confer at least 95% inhibition of the SSDH in water and lysate (Fig. 2B). Subsequently we proceeded with experiments to demonstrate that we were able to differentiate between GABA and SSA in water or in a lysate. We prepared mixtures of GABA and SSA in water and lysate in ratios of 0:10, 1:9, 2:8 and up to 10:0 respectively. When no inhibitor was used, all mixtures in water (Fig. 3A) or lysate (data not shown) resulted in a signal similar to that of 10 mM GABA, confirming that SSA in the samples was erroneously being measured as GABA using the GABase assay. When 80 mM EOS were included in the assay mixture the GABase assay could detect accurately the amounts of SSA added, while the presence of GABA could not be detected even when this was present at 10 mM (Fig. 3A, B). These data show that it is possible to distinguish between the levels of SSA and GABA in a mixture of both in a lysate. Therefore, two GABase assays could be performed in parallel with or without EOS quantifying respectively
Fig. 2. (A) Use of 0 mM (○), 2.5 mM ( ), 5 mM ( ), 10 mM ( ), 20 mM ( ), 80 mM (●) EOS as inhibitor of the Pseudomonas fluorescens SSDH of the GABase mix in an assay against a standard of 5 mM GABA in water. (B) Inhibition expressed as percentage of the maximal signal (340 nm) during the GABase assay on 5 mM GABA in water, exerted by various concentrations of EOS. Horizontal line represents 95% inhibition and asterisk denotes the minimum concentration of EOS required to achieve this level of inhibition.
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significant under other conditions and for other organisms. We were also able to quantify the levels of GABAi in stationary phase cells of E. coli TOP10 cells grown in LB and acid challenged at pH 4 for 30 min. Following calculations taking in account the cell size of 0.65 μm3 for E. coli (Kubitschek, 1990) and the number of cells used in the assay, the estimated GABAi in the actual cell was 50.9 ± 4.15 mM. This is also the first report describing the measurement of intracellular levels of succinate semialdehyde in a bacterium. Overall, following the modifications we present here the inexpensive and accurate GABase assay method can be used to quantify accurately and rapidly the levels of GABAi and SSAi in L. monocytogenes and various other microorganisms. We expect that the method will be used in the future to investigate the GABAi, the GAD system and their role in acid resistance (Karatzas et al., 2010) and other biological processes. The work was funded by a Science Foundation Ireland (SFI) Starting Investigation Research Grant (SIRG) awarded to KimonAndreas Karatzas. Rebecca Ham was funded by a Science Foundation Ireland UREKA programme (grant 08/UR/B1350). References
Fig. 3. (A) The GABase assay without EOS detects GABA and SSA collectively (○) in mixtures prepared in lysate containing 0 and 10, 1 and 9, 2 and 8, up to 10 and 0 mM GABA and SSA respectively. The GABase assay detects only SSA (●) and not GABA in the above mixtures when 80 mM EOS is included in the GABase mixture. With the use of 80 mM EOS the GABase assay can accurately measure SSA when GABA is present in a lysate. (B) Accumulation of SSA and GABA extracellularly or intracellularly following acidification to pH 3.5 of L. monocytogenes 10403S cultures grown overnight in BHI.
SSA or GABA and SSA collectively. Subsequently, the value for GABA could be estimated by subtracting the SSA value from the collective value. This method was applied to investigate the presence of GABAi and SSAi in stationary phase cultures of L. monocytogenes grown overnight in BHI that were acidified with HCl to the non-lethal pH value of 3.5. With the use of this modified GABase assay we are able to demonstrate the presence of GABAi in L. monocytogenes and estimate its intracellular levels following the series of calculations mentioned previously (Karatzas et al., 2010; Fig. 3B). Low levels of intracellular SSAi were detected (~4.8 mM) under the conditions we tested which could be explained by the fact that L. monocytogenes possesses a gene (lmo0913) encoding a protein similar to SSDH whose substrate is thought to be SSA (Abram et al., 2008). These levels of SSAi show that the use of EOS is necessary to correct for an error of 7 to 6.6% in the estimation of the GABAi concentration. However, this error refers to a specific microorganism and conditions and it might be more or less
Abdou, A.M., Higashiguchi, S., Horie, K., Kim, M., Hatta, H., Yokogoshi, H., 2006. Relaxation and immunity enhancement effects of γ-aminobutyric acid (GABA) administration in humans. Biofactors 26, 201–208. Abram, F., Starr, E., Karatzas, K.A.G., Matlawska-Wasowska, K., Boyd, A., Wiedmann, M., et al., 2008. Identification of components of the SigB regulon in Listeria monocytogenes that contribute to acid and salt tolerance. Applied and Environmental Microbiology 74, 6848–6858. Adeghate, E., Ponery, A.S., 2002. GABA in the endocrine pancreas: cellular localization and function in normal and diabetic rats. Tissue & Cell 34, 1–6. Bach, B., Meudec, E., Lepoutre, J.-P., Rossignol, T., Blondin, B., Dequin, S., et al., 2009. New Insights into γ-aminobutyric acid catabolism: evidence for γ-hydroxybutyric acid and polyhydroxybutyrate synthesis in Saccharomyces cerevisiae. Applied and Environmental Microbiology 75, 4231–4239. Cotter, P.D., Gahan, C.G.M., Hill, C., 2001. A glutamate decarboxylase system protects Listeria monocytogenes in gastric fluid. Molecular Microbiology 40, 465–475. Foster, J.W., 2004. Escherichia coli acid resistance: tales of an amateur acidophile. Nature Reviews. Microbiology 2, 898–907. Fowler, L.J., John, R.A., 1972. Active-site-directed irreversible inhibition of rat brain 4aminobutyrate aminotransferase by ethanolamine O-sulphate in vitro and in vivo. The Biochemical Journal 130, 569–573. Karatzas, K.-A.G., Brennan, O., Heavin, S., Morrissey, J., O'Byrne, C.P., 2010. Intracellular accumulation of high levels of γ-aminobutyrate by Listeria monocytogenes 10403S in response to low pH: Uncoupling of γ-aminobutyrate synthesis from efflux in a chemically defined medium. Applied and Environmental Microbiology 76, 3529–3537. Komatsuzaki, N., Shima, J., Kawamoto, S., Momose, H., Kimura, T., 2005. Production of γaminobutyric acid (GABA) by Lactobacillus paracasei isolated from traditional fermented foods. Food Microbiology 22, 497–504. Kubitschek, H.E., 1990. Cell volume increase in Escherichia coli after shifts to richer media. Journal of Bacteriology 172, 94–101. Saito, N., Robert, M., Kochi, H., Matsuo, G., Kakazu, Y., Soga, T., et al., 2009. Metabolite profiling reveals YihU as a novel hydroxybutyrate dehydrogenase for alternative succinic semialdehyde metabolism in Escherichia coli. The Journal of Biological Chemistry 284, 16442–16451. Siragusa, S., De Angelis, M., Di Cagno, R., Rizzello, C.G., Coda, R., Gobbetti, M., 2007. Synthesis of γ-aminobutyric acid by lactic acid bacteria isolated from a variety of Italian cheeses. Applied and Environmental Microbiology 73, 7283–7290. Sulaiman, S.A.J., Suliman, F.E.O., Barghouthi, S., 2003. Kinetic studies on the inhibition of GABA-T by γ-vinyl GABA and taurine. Journal of Enzyme Inhibition and Medicinal Chemistry 18, 297–301. Tsukatani, T., Higuchi, T., Matsumoto, K., 2005. Enzyme-based microtiter plate assay for γ-aminobutyric acid: application to the screening of γ-aminobutyric acidproducing lactic acid bacteria. Analytica Chimica Acta 540, 293–297. Watanabe, M., Maemura, K., Kanbara, K., Tamayama, T., Hayasaki, H., Kwang, W.J., 2002. GABA and GABA receptors in the central nervous system and other organs. International Review of Cytology 213, 1–47. Waterman, S.R., Small, P.L.C., 2003. The glutamate-dependent acid resistance system of Escherichia coli and Shigella flexneri is inhibited in vitro by L-trans-pyrrolidine-2, 4-dicarboxylic acid. FEMS Microbiology Letters 224, 119–125. Yang, S.Y., Lü, F.X., Lu, Z.X., Bie, X.M., Jiao, Y., Sun, L.J., et al., 2008. Production of γ-aminobutyric acid by Streptococcus salivarius subsp. thermophilus Y2 under submerged fermentation. Amino Acids 34, 473–478.
Contents lists available at ScienceDirect
Journal of Microbiological Methods j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j m i c m e t h
Note
A modified rapid enzymatic microtiter plate assay for the quantification of intracellular γ-aminobutyric acid and succinate semialdehyde in bacterial cells C.P. O'Byrne, C. Feehily, R. Ham, K.A.G. Karatzas ⁎ Bacterial Stress Response Group, Microbiology, School of Natural Sciences, NUI Galway, Galway, Ireland
a r t i c l e
i n f o
Article history: Received 6 August 2010 Received in revised form 21 October 2010 Accepted 25 October 2010 Available online 31 October 2010
a b s t r a c t The GABase assay is widely used to rapidly and accurately quantify levels of extracellular γ-aminobutyric acid (GABA). Here we demonstrate a modification of this assay that enables quantification of intracellular GABA in bacterial cells. Cells are lysed by boiling and ethanolamine-O-sulphate, a GABA transaminase inhibitor is used to distinguish between GABA and succinate semialdehyde. © 2010 Elsevier B.V. All rights reserved.
Keywords: Listeria monocytogenes GABA Succinate semialdehyde Acid tolerance GABase
γ-aminobutyrate (GABA) is an important metabolite produced by the glutamate decarboxylase (GAD) system of various microorganisms, through the decarboxylation of glutamate in response to acidic conditions. Therefore measurements of GABA are important in research of pathogenic bacteria that use the GAD system to survive the acidic pH of the stomach (Cotter et al., 2001; Foster, 2004; Waterman and Small, 2003). This property is also important for probiotic bacteria because colonisation of the gastrointestinal tract requires survival in the stomach (Siragusa et al., 2007). Furthermore, screening of various microorganisms used in fermentations is important as they can ensure better acidification of the product, flavour and a product enriched in GABA (Komatsuzaki et al., 2005). The latter is important because GABA is beneficial for humans playing a role as a neurotransmitter in the central nervous system (Watanabe et al., 2002) and as a secretagogue of insulin in the pancreas (Adeghate and Ponery, 2002). Oral administration of GABA has also been shown to induce relaxation and enhance immunity under stress conditions (Abdou et al., 2006). Measurements of extracellular GABA (GABAe) as means of quantification of the GAD system activity can indicate the acid resistance of a microorganism (Cotter et al., 2001). These measurements are usually performed with the use of the GABase assay. However, quantification of the intracellular GABA (GABAi) is also important for the investigation of the GAD system (Karatzas et al., 2010) and has been performed in a limited number of microorganisms such as Lactobacillus paracasei (Komatsuzaki et al., 2005), Streptococcus salivarius (Yang et al., 2008)
⁎ Corresponding author. Tel.: + 353 91 495091; fax: + 353 91 494598. E-mail address: [email protected] (K.A.G. Karatzas). 0167-7012/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2010.10.017
and Saccharomyces cerevisiae (Bach et al., 2009). Until now these measurements have been performed with the use of an HPLC/amino acid analyser, which is an expensive piece of equipment while the method is relatively laborious compared to the GABase (Tsukatani et al., 2005). Furthermore, the two methods provide comparable results as it has been demonstrated by Tsukatani et al. (2005). In this manuscript we propose certain modifications of the rapid and inexpensive GABase assay described previously by Tsukatani et al. (2005) to be used for the quantification of GABAi in Listeria monocytogenes and other bacteria. The modifications include the lysis of the cells by boiling and the use of ethanolamine-O-sulphate (EOS), a GABA transaminase inhibitor to enable the distinction between GABA and succinate semialdehyde. To quantify GABAi a method to lyse the cells was required. We investigated various methods like boiling, bead beating and sonication to produce the lysates. The lysates were prepared by harvesting cells from 20 ml of culture grown overnight in BHI following centrifugation at 9000×g for 10 min. Subsequently, the supernatant was removed and the pellet was resuspended in 1 ml of sterile water and subjected to the above-mentioned lysis procedures. Lysates were centrifuged at 8000×g for 10 min to remove all debris and the supernatant was removed and placed in a sterile 1.5 ml tube. Boiling generated the highest signal when lysates were analysed with a standard GABase assay (data not shown). Subsequently, we investigated various times of boiling (10–25 min) which would be able to kill all cells and release all GABAi. Boiling for 10 min was sufficient to kill all cells as no viable cells were recovered from any sample of the lysates when plated on BHI agar. It was demonstrated experimentally that all GABAi was released as an increase in the boiling time did not result in an increase in the levels of GABAi measured (data not shown). Furthermore, the
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boiling did not lead to the degradation of GABA. This was demonstrated experimentally by the high recovery of GABA (N99%) following boiling of standards with different concentrations of GABA for different durations of time between 10 and 25 min (data not shown). Once we proved that boiling was the method of choice for cell lysis, the next step was to test the ability of the method to quantify different levels of GABA in standards prepared with boiled lysates of L. monocytogenes. Boiled lysates as well as sterile water were used to prepare standards of 0, 1, 2, and 3–10 mM GABA in triplicate and subsequently the GABase assay was performed as described previously by Tsukatani et al., 2005. Standard curves of GABA for the average values in lysate and water were constructed (Fig. 1) and statistical analysis was performed by t-test for the slope and the y-intercept resulted in P = 0.7 and 0.8 respectively. Both values were higher than 0.05 (no statistically significant difference) suggesting that L. monocytogenes does not contain any GABAi in stationary phase and that standards in water could be used instead of standards in lysate to construct the calibration curves in order to measure concentrations of GABAi. Furthermore, we determined the sensitivity of the assay which could detect as low as 0.1 mM in water. With the use of the latter value and following a series of calculations taking in account the concentration of cells and their hypothetical volume as shown previously (Karatzas et al., 2010) we could detect as low as 1.12 mM of GABAi or SSAi in the actual cell. The repeatability of the assay was also investigated by analysing in triplicate each one of the SSA standards (0.1–1 mM). The percent error of the three determinations of each standard ranged between 0.85 and 3.21% suggesting a high repeatability. The GABase (Sigma-Aldrich, Steinheim, Germany) commercial preparation comprises two enzymes: the GABA transaminase (GABA-AT) and the succinate semialdehyde dehydrogenase (SSDH) from Pseudomonas fluorescens. GABA-AT converts GABA to succinate semialdehyde (SSA) in the presence of α-ketoglutarate. Subsequently, SSA is converted to succinate in the presence of NAD(P)+ through the activity of SSDH. In the last reaction, NAD(P)+ is transformed to NAD(P)H which absorbs at 340 nm. The above reaction is stoichiometric and therefore the levels of NAD(P)H reflect the levels of GABA in the mix. Apart from being a substrate for the second reaction in the GABase assay, SSA is present in cells as a simple intermediate in the oxidative (to succinate) or reductive (to γ-hydroxybutyric acid) catabolism of GABA (Saito et al., 2009). Therefore, intracellular SSA released in the lysate would be erroneously measured as GABA by the GABase assay. This error could be corrected by the separate measurement of SSA through the use of an SSDH preparation followed by the subtraction of this value from that obtained with the use of GABase. However, no SSDH is commercially available and another alternative way to measure SSA was to inhibit the GABA-AT in the GABase
Fig. 1. Calibration curves from standards in water (◊) and lysates (♦) are similar. Water standards can be used in experiments with lysates.
mix. The GABA-AT inhibitors, taurine (Sulaiman et al., 2003) and EOS (Fowler and John, 1972) were employed for this task in concentrations of 0, 10, 20 up to 90 mM in the assay mixture. None of the taurine concentrations inhibited the P. fluorescens SSDH suggesting that concentrations higher than 90 mM might be required (data not shown). In contrast, all concentrations of EOS conferred inhibition of SSDH in water (Fig. 2A) and lysate (data not shown). Inhibition by EOS in lysate was similar to that seen in water (data not shown). More than 60 mM EOS is required to confer at least 95% inhibition of the SSDH in water and lysate (Fig. 2B). Subsequently we proceeded with experiments to demonstrate that we were able to differentiate between GABA and SSA in water or in a lysate. We prepared mixtures of GABA and SSA in water and lysate in ratios of 0:10, 1:9, 2:8 and up to 10:0 respectively. When no inhibitor was used, all mixtures in water (Fig. 3A) or lysate (data not shown) resulted in a signal similar to that of 10 mM GABA, confirming that SSA in the samples was erroneously being measured as GABA using the GABase assay. When 80 mM EOS were included in the assay mixture the GABase assay could detect accurately the amounts of SSA added, while the presence of GABA could not be detected even when this was present at 10 mM (Fig. 3A, B). These data show that it is possible to distinguish between the levels of SSA and GABA in a mixture of both in a lysate. Therefore, two GABase assays could be performed in parallel with or without EOS quantifying respectively
Fig. 2. (A) Use of 0 mM (○), 2.5 mM ( ), 5 mM ( ), 10 mM ( ), 20 mM ( ), 80 mM (●) EOS as inhibitor of the Pseudomonas fluorescens SSDH of the GABase mix in an assay against a standard of 5 mM GABA in water. (B) Inhibition expressed as percentage of the maximal signal (340 nm) during the GABase assay on 5 mM GABA in water, exerted by various concentrations of EOS. Horizontal line represents 95% inhibition and asterisk denotes the minimum concentration of EOS required to achieve this level of inhibition.
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significant under other conditions and for other organisms. We were also able to quantify the levels of GABAi in stationary phase cells of E. coli TOP10 cells grown in LB and acid challenged at pH 4 for 30 min. Following calculations taking in account the cell size of 0.65 μm3 for E. coli (Kubitschek, 1990) and the number of cells used in the assay, the estimated GABAi in the actual cell was 50.9 ± 4.15 mM. This is also the first report describing the measurement of intracellular levels of succinate semialdehyde in a bacterium. Overall, following the modifications we present here the inexpensive and accurate GABase assay method can be used to quantify accurately and rapidly the levels of GABAi and SSAi in L. monocytogenes and various other microorganisms. We expect that the method will be used in the future to investigate the GABAi, the GAD system and their role in acid resistance (Karatzas et al., 2010) and other biological processes. The work was funded by a Science Foundation Ireland (SFI) Starting Investigation Research Grant (SIRG) awarded to KimonAndreas Karatzas. Rebecca Ham was funded by a Science Foundation Ireland UREKA programme (grant 08/UR/B1350). References
Fig. 3. (A) The GABase assay without EOS detects GABA and SSA collectively (○) in mixtures prepared in lysate containing 0 and 10, 1 and 9, 2 and 8, up to 10 and 0 mM GABA and SSA respectively. The GABase assay detects only SSA (●) and not GABA in the above mixtures when 80 mM EOS is included in the GABase mixture. With the use of 80 mM EOS the GABase assay can accurately measure SSA when GABA is present in a lysate. (B) Accumulation of SSA and GABA extracellularly or intracellularly following acidification to pH 3.5 of L. monocytogenes 10403S cultures grown overnight in BHI.
SSA or GABA and SSA collectively. Subsequently, the value for GABA could be estimated by subtracting the SSA value from the collective value. This method was applied to investigate the presence of GABAi and SSAi in stationary phase cultures of L. monocytogenes grown overnight in BHI that were acidified with HCl to the non-lethal pH value of 3.5. With the use of this modified GABase assay we are able to demonstrate the presence of GABAi in L. monocytogenes and estimate its intracellular levels following the series of calculations mentioned previously (Karatzas et al., 2010; Fig. 3B). Low levels of intracellular SSAi were detected (~4.8 mM) under the conditions we tested which could be explained by the fact that L. monocytogenes possesses a gene (lmo0913) encoding a protein similar to SSDH whose substrate is thought to be SSA (Abram et al., 2008). These levels of SSAi show that the use of EOS is necessary to correct for an error of 7 to 6.6% in the estimation of the GABAi concentration. However, this error refers to a specific microorganism and conditions and it might be more or less
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