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Alkaline phosphatase activity of Flexibacter chinensis under starvation stress in water microcosms
Enzyme and Microbial Technology 40 (2006) 13–16
Alkaline phosphatase activity of Flexibacter chinensis under starvation stress in water microcosms Jamshid Raheb a,∗ , Shamim Naghdi a , Ken P. Flint b a
National Institute for Genetic Engineering and Biotechnology (NIGEB), P.O. Box 14155-6343, Tehran, Iran b Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK Received 18 March 2005; received in revised form 19 December 2005; accepted 21 December 2005
Abstract Alkaline phosphatase activity was assayed in Flexibacter chinensis to determine the effects of long term starvation stress and the effect of different nutrient amendments to starvation medium on the reactivation of this enzyme. Alkaline phosphatase activity increased at all temperature in starvation medium showing that reactivation could occur in cells previously grown in a high phosphate medium even under conditions where cell viability was declining, suggesting that in Flexibacter, de novo synthesis of enzyme occurs under starvation conditions. Addition of glucose and urea led to reactivation of alkaline phosphatase while the addition of inorganic phosphate had no effect on alkaline phosphatase activity. © 2006 Elsevier Inc. All rights reserved. Keywords: Flexibacter chinensis; Alkaline phosphatase activity; Starvation stress
1. Introduction The Flexibacter genus belongs to the flexibacter–flavobacter–cytophaga group of organisms. The genus is Gramnegative, chemoorganotroph, non-photosynthetic and facultative anaerobes. Some of this genus species have pathogenic importance (in aquatic animals) and some of them have pharmacologic importance [1]. Alkaline phosphatase (ALP, EC 3.1.3.1) is an extracellular enzyme enabling utilization of phosphomonoesters as the source of inorganic phosphate (Pi) required for the maintenance of cellular metabolism. This enzyme exact biological function is not yet known, however it has been suggested that it may be associated with the transport of inorganic phosphate into the cell. It is an adaptive enzyme whose biosynthesis is controlled by the concentration of Pi in the medium [2]. ALP is induced under phosphate limitation [3]. The ability of cells to grow in the presence of sufficient carbon, nitrogen and the other essential elements allows to utilize organic phosphate compounds as a phosphate source by synthesize of ALP [4]. Addition of carbon and nitrogen sources to the starvation medium resulted in reactivation of ALP activity [5]. Similarly,
the addition of glucose, or glucose and ammonium nitrate to soil samples has led to an increase in ALP activity [6]. There is little known about the activity of ALP when cells enter the viable but non-culturable state. In this paper, we report the role of ALP in the survival of Flexibacter chinensis under long term starvation. Also, the effects of different nutrient additions to the starvation medium and the effects of different incubation temperature on ALP activity were examined. Our data showed that ALP reactivated under the above condition in starved medium. 2. Materials and methods 2.1. Bacterial strain The bacterial strain used in this study was F. chinensis obtained from Dr. Flint (Warwick University, UK).
2.2. Bacterial growth media and conditions The bacterial strain was routinely grown in Luria Broth (10 g/l bacto tryptone, 5 g/l yeast extract, 5 g/l NaCl, pH 7.2) or on Luria agar (10 g/l bacto tryptone, 5 g/l yeast extract, 5 g/l NaCl and 15 g/l agar). Plates were incubated at 30 ◦ C.
2.3. Starvation experiments ∗
Corresponding author. Fax: +98 21 44580399. E-mail address: [email protected] (J. Raheb).
0141-0229/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2005.12.024
Elgastat water (100 ml) was dispensed into 250 ml sterile glass Erlenmeyer flasks and autoclaved at 121 ◦ C for 15 min; the flasks were amended with carbon,
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J. Raheb et al. / Enzyme and Microbial Technology 40 (2006) 13–16
nitrogen and phosphorus sources, prior to autoclaving as required. Autoclaved flasks stored at 4 ◦ C until required [1].
2.4. Nutrient source amendments Carbon and KH2 PO4 (as a phosphorus source) were used with 0.5 g/l and 100 mg/ml as final concentration, respectively. In addition, urea, amino acids and ammonium sulphate were used at a maximum final concentration of 100 mg/l as nitrogen sources [1].
2.5. Viable cell counts The viable cell counts (cfu/ml) were determined using the surface spread plate technique [1]. Samples (1 ml) were taken from the flasks and serial dilutions prepared as a 10-fold serial dilution in quarter-strength Ringers solution (2.25 g/l NaCl, 0.12 g/l CaCl2 , 0.05 g/l NaHPO4 and 0.105 g/l KCl in 1 l distilled water). One hundred microliters of the diluted samples was spread on duplicate LBA plates. The plates were incubated at 30 ◦ C for least 48 h. Plates were counted by using an illuminated colony counter.
2.6. Total counts The total count was determined using a Coulter counter ZM (Coulter Euro Diagnostics GMBH) with a 30 m orifice probe. The data were analyzed using Coulter channelyzer software to estimate the size distribution. The samples were diluted in an isotonic buffer containing 0.4% (v/v) glutaraldehyde to fix the cells. Total count is expressed as total particles/ml. The software also determines mean cell size and volume [1].
2.7. Alkaline phosphatase assay F. chinensis was grown in the minimal media with 2 mg Pi/ml for 24 h at 30 ◦ C. The cells were harvested by centrifugation and washed twice in sterile distilled water. One ml samples of the resuspended cells were inoculated into 100 ml of sterile water in a 250 ml flask to give an initial viable cell count of approximately 107 cfu/l. The flasks were incubated in the dark without shaking at the desired temperatures. The ALP activity of the culture was determined by taking 1 ml from the flask, mixing with 2 ml of 0.1 M Tris buffer (pH 9.0) and adding 0.5 ml, 5% (w/v, in sterile distilled water) para-nitrophenyl disodium orthophosphate (pNPP). This reaction mixture was incubated at 30 ◦ C in a water bath for 30–240 min. The reaction was stopped by the addition of 0.2 ml, 10 M NaOH and the absorbance was measured at 420 nm. Controls containing no substrate and containing no cells were included to correct the absorbance changes due to cell density and spontaneous hydrolysis of the pNPP [5].
3. Results The effect of different temperatures on the alkaline phosphatase activity and survival of F. chinensis was investigated in a starvation medium. Over a 90-day starvation period, there was an increase in ALP activity at all incubation temperatures (Fig. 1a and b). The greatest increase in activity was found at 30 and 25 ◦ C. At 37 ◦ C, ALP activity increased less rapidly than at the other temperatures. However, after 90 days starvation, the enzyme activity at 37 ◦ C was close to that the other temperatures. There was no decrease at any of the temperatures used which suggests that a very stable ALP enzyme exists in F. chinensis. Except at 37 and 30 ◦ C, the viable cell counts at all temperatures were constant until after 25 days starvation when there was small decline in viable cell count at 4 and 15 ◦ C. The viable cell count at 37 ◦ C declined below the detection limits within 2 days of the onset of starvation but ALP activity continued to increase over the starvation period.
Fig. 1. The effect of different temperatures on alkaline phosphatase activity and the viable cell count of F. chinensis in starvation medium. The alkaline phosphatase activity (a) and viable counts (b) were assayed at 30 ◦ C. The enzyme activity was expressed as M/l PNP released ml−1 h−1 .
Addition of d-glucose to starvation medium as an amendment showed an increase in ALP activity in comparison to the control over a period of 25 days (Fig. 2). The viable cell counts of F. chinensis initially increased slightly in these samples which
Fig. 2. The effect of d-glucose on alkaline phosphatase activity and the viable cell count of F. chinensis at 15 ◦ C in a starvation medium.
J. Raheb et al. / Enzyme and Microbial Technology 40 (2006) 13–16
15
Fig. 3. The effect of urea on alkaline phosphatase activity and the viable cell count of F. chinensis at 15 ◦ C in a starvation medium.
suggest that cell growth was possible under these conditions. Reactivation of ALP in the presence of a carbon source is related to the absence of phosphate in this medium which therefore resembles a phosphate-limited medium. Addition of urea to starvation medium as an amendment showed that ALP activity doubled over the 40-day starvation period in both the unamended controls and in the nitrogenamended test flasks (Fig. 3). The viable cell count declined less in the flasks amended with the nitrogen source compared with the control. Addition of phosphate to starvation medium as an amendment showed that there was a much larger increase in ALP activity over the 40 days period of starvation in the control flasks with no phosphate amendment than in the flask amended with inorganic phosphate (Fig. 4). Initially, there was a small increase in enzyme activity in the amended flask over the first 5 days of starvation; the enzyme activity then remained relatively constant until the end of the 40 days experimental period. The viable cell count in the unamended control and the amended flask declined to the same extent. In the case of the unamended control, this decline in viable cell count occurred while ALP activity was still increasing. Fig. 5 shows that ALP activity doubled during starvation in the flasks amended with glucose and urea but there was only a small increase in the flask amended with inorganic phosphate.
Fig. 4. The effect of inorganic phosphate on the viable cell count and alkaline phosphatase activity of F. chinensis at 15 ◦ C in a starvation medium.
Fig. 5. The effects of different nutrient amendments on total alkaline phosphatase activity in F. chinensis incubated at 15 ◦ C in a starvation medium.
There was also close to a doubling of activity in the unamended control flask. All the flasks which show an increase in activity could be considered as phosphate limited whereas the flask which had inorganic phosphate added would not be phosphate limited. 4. Discussion In this study, ALP activity was assayed to determine the effects of long term starvation stress and the effects of different nutrient amendments to starvation medium on the reactivation of this enzyme. The effects of different temperatures at which the starvation microcosms were incubated on the ALP activity of F. chinensis were also examined. In the starvation experiments, ALP activity was measured over a period of up to 90 days at a range of incubation temperatures from 4 to 37 ◦ C. At all temperatures, activity continued to increase even as the viable cell count declined. The continued increase in ALP activity even during the period when the viable cell count was declining suggests that the ALP in F. chinensis is produced under starvation conditions. ALP activity has also been shown to increase in Escherichia coli starved in nutrient-free sea water [7] and in the same organisms starved for 70 days at temperatures between 4 and 37 ◦ C in lake water [8]. Enzyme activity was still observed to increase at 30 and 37 ◦ C after the cells began to lose their culturability and is believed to enter the viable but non-culturable state. After 60 days starvation, these bacteria could be recovered in a minimal medium or in diluted nutrient broth. After 90 days, the cells at 37 ◦ C were no longer recoverable but ALP activity was still increasing and activity was still measurable. It is possible that, at this time, the activity is being released from dead or lysing cells and the increase in activity is an artifact due to the reduction in diffusion barriers as cells lyse. However, there was no decrease in total cell count observed and it is believed to be unlikely that cell lysis was a significant factor in the increase in ALP
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J. Raheb et al. / Enzyme and Microbial Technology 40 (2006) 13–16
activity. Similar results were obtained from ref. [8] who also showed that ALP activity still increased and remained detectable in starved cultures of E. coli in which it was not possible to detect viable bacteria. The effect of different nutrient amendments on ALP activity any effect on the survival of the bacteria and on the reactivation or inactivation of ALP under starvation conditions. ALP activity increased over that of the unamended control after the addition of d-glucose to the starvation medium. Lim [9] reported a similar increase in ALP activity of Aeromonas hydrophila in sterile amended lake water microcosms. He also suggested that this increase was dependent upon the length of the starvation period and on the temperature at which starvation experiments were conducted. There was a greater increase in enzyme activity and in cells number at higher temperatures as the extra nutrient sources, such as carbon or nitrogen, were primarily needed for enzyme synthesis. Here, experiments were only conducted at 15 ◦ C as this was the optimal growth temperature for F. chinensis and is a temperature which has some environmental significance. Ozkanca and Flint [5] also showed that reactivation of ALP activity in E. coli occurred after the addition of carbon sources (particularly glucose and succinate) to the starvation medium. They suggested that the increase in enzyme activity could be a reflection of the fact that the addition of carbon or nitrogen sources to the starvation medium makes it more likely that phosphate-limited conditions occur which would lead to reactivation of enzyme synthesis if there was a carbon source to meet the energy requirement of the cell. Lim [9] showed that the greatest reactivation of ALP activity in A. hydrophila occurred after the addition of d-glucose, which is why this was the only carbon source tried in these series of experiments. The addition of urea to the microcosms also reactivated the synthesis of the enzyme. The addition of a nitrogen source (both organic nitrogen, such as amino acids or urea, and inorganic source such as ammonium ions) increased the survival time for E. coli without increasing the ALP activity [10]. The increase in phosphatase activity in A. hydrophila in nitrogen-amended microcosms was much more marked than with E. coli [8]. It is probable that the ability to synthesize the enzyme under these conditions is related to the ability of the cell to utilize the trace amounts of carbon which will be in the microcosms as energy sources.
When inorganic phosphate at a concentration of 100 mg PO4 /l was added to the starvation medium, ALP activity remained relatively constant over a 40 days starvation period. This suggests that these microcosms were not phosphate limited and that the increase in ALP activity seen in the other experiments is a direct response to phosphate limitation in the starvation medium. In all these experiments, the total counts remained constant, and ALP activity increased although cell viability decreased, in some cases to a non-detectable level. This suggests that the de novo synthesis of ALP seen here is a response of the cell to survival in an impoverished environment (certainly survival in phosphate-limited environment) rather than a facet of bacterial growth. It seems unlikely that the bacteria have to be able to grow to reactive ALP as there was no increase in cell numbers seen in any of these experiments and activity increased even though the viable cell count declined. References [1] Raheb J. The molecular and physiological effects of starvation and other stresses on Flexibacter chinensis, PhD thesis. University of Warwick: UK; 1998. [2] Orhanovic S, Pavela-Vrancic M. Alkaline phosphatase activity in seawater: influence of reaction conditions on the kinetic parameters of ALP. Croat Chem Acta 2000;73:819–30. [3] Nyc JF, Kadner RJ, Croken BJ. A repressible alkaline phosphatase in Neurospora crassa. J Biol Chem 1966;241:1468–72. [4] Filloux A, Bally M, Socia C, Murgier M, Lazdunski A. Phosphate regulation in Pseudomonas aeruginosa: cloning of the alkaline phosphatase gene and identification of PhoB and PhoR-like genes. Mol Gen Genet 1988;212:510–3. [5] Ozkanca R, Flint KP. Alkaline phosphatase activity of Escherichia coli starved in sterile lake water microcosms. J Appl Bacteriol 1996;80:252–85. [6] Sardans J, Penuelas J. Drought decrease soil enzyme activity in a Mediterranean Quercus ilex L. forest. Soil Biol Biochem 2005;37: 455–61. [7] Gauthier MJ, Flatau GN, Clement RL. Influence of phosphate ions and alkaline phosphatase activity of cells on survival of Escherichia coli in sea water. Microb Ecol 1990;20:245–51. [8] Ozkanca R. Survival and physiological status of Escherichia coli in lake water under different nutrient condition. PhD thesis. University of Warwick: UK; 1993. [9] Lim CH. The effect of environmental factors on the physiology of Aeromonas hydrophila in lake water, PhD thesis. University of Warwick: UK; 1995. [10] Lim CH, Flint KP. The effect of nutrients on the survival of Escherichia coli in lake water. J Appl Bacteriol 1989;66:5576–6569.
Alkaline phosphatase activity of Flexibacter chinensis under starvation stress in water microcosms Jamshid Raheb a,∗ , Shamim Naghdi a , Ken P. Flint b a
National Institute for Genetic Engineering and Biotechnology (NIGEB), P.O. Box 14155-6343, Tehran, Iran b Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK Received 18 March 2005; received in revised form 19 December 2005; accepted 21 December 2005
Abstract Alkaline phosphatase activity was assayed in Flexibacter chinensis to determine the effects of long term starvation stress and the effect of different nutrient amendments to starvation medium on the reactivation of this enzyme. Alkaline phosphatase activity increased at all temperature in starvation medium showing that reactivation could occur in cells previously grown in a high phosphate medium even under conditions where cell viability was declining, suggesting that in Flexibacter, de novo synthesis of enzyme occurs under starvation conditions. Addition of glucose and urea led to reactivation of alkaline phosphatase while the addition of inorganic phosphate had no effect on alkaline phosphatase activity. © 2006 Elsevier Inc. All rights reserved. Keywords: Flexibacter chinensis; Alkaline phosphatase activity; Starvation stress
1. Introduction The Flexibacter genus belongs to the flexibacter–flavobacter–cytophaga group of organisms. The genus is Gramnegative, chemoorganotroph, non-photosynthetic and facultative anaerobes. Some of this genus species have pathogenic importance (in aquatic animals) and some of them have pharmacologic importance [1]. Alkaline phosphatase (ALP, EC 3.1.3.1) is an extracellular enzyme enabling utilization of phosphomonoesters as the source of inorganic phosphate (Pi) required for the maintenance of cellular metabolism. This enzyme exact biological function is not yet known, however it has been suggested that it may be associated with the transport of inorganic phosphate into the cell. It is an adaptive enzyme whose biosynthesis is controlled by the concentration of Pi in the medium [2]. ALP is induced under phosphate limitation [3]. The ability of cells to grow in the presence of sufficient carbon, nitrogen and the other essential elements allows to utilize organic phosphate compounds as a phosphate source by synthesize of ALP [4]. Addition of carbon and nitrogen sources to the starvation medium resulted in reactivation of ALP activity [5]. Similarly,
the addition of glucose, or glucose and ammonium nitrate to soil samples has led to an increase in ALP activity [6]. There is little known about the activity of ALP when cells enter the viable but non-culturable state. In this paper, we report the role of ALP in the survival of Flexibacter chinensis under long term starvation. Also, the effects of different nutrient additions to the starvation medium and the effects of different incubation temperature on ALP activity were examined. Our data showed that ALP reactivated under the above condition in starved medium. 2. Materials and methods 2.1. Bacterial strain The bacterial strain used in this study was F. chinensis obtained from Dr. Flint (Warwick University, UK).
2.2. Bacterial growth media and conditions The bacterial strain was routinely grown in Luria Broth (10 g/l bacto tryptone, 5 g/l yeast extract, 5 g/l NaCl, pH 7.2) or on Luria agar (10 g/l bacto tryptone, 5 g/l yeast extract, 5 g/l NaCl and 15 g/l agar). Plates were incubated at 30 ◦ C.
2.3. Starvation experiments ∗
Corresponding author. Fax: +98 21 44580399. E-mail address: [email protected] (J. Raheb).
0141-0229/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2005.12.024
Elgastat water (100 ml) was dispensed into 250 ml sterile glass Erlenmeyer flasks and autoclaved at 121 ◦ C for 15 min; the flasks were amended with carbon,
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J. Raheb et al. / Enzyme and Microbial Technology 40 (2006) 13–16
nitrogen and phosphorus sources, prior to autoclaving as required. Autoclaved flasks stored at 4 ◦ C until required [1].
2.4. Nutrient source amendments Carbon and KH2 PO4 (as a phosphorus source) were used with 0.5 g/l and 100 mg/ml as final concentration, respectively. In addition, urea, amino acids and ammonium sulphate were used at a maximum final concentration of 100 mg/l as nitrogen sources [1].
2.5. Viable cell counts The viable cell counts (cfu/ml) were determined using the surface spread plate technique [1]. Samples (1 ml) were taken from the flasks and serial dilutions prepared as a 10-fold serial dilution in quarter-strength Ringers solution (2.25 g/l NaCl, 0.12 g/l CaCl2 , 0.05 g/l NaHPO4 and 0.105 g/l KCl in 1 l distilled water). One hundred microliters of the diluted samples was spread on duplicate LBA plates. The plates were incubated at 30 ◦ C for least 48 h. Plates were counted by using an illuminated colony counter.
2.6. Total counts The total count was determined using a Coulter counter ZM (Coulter Euro Diagnostics GMBH) with a 30 m orifice probe. The data were analyzed using Coulter channelyzer software to estimate the size distribution. The samples were diluted in an isotonic buffer containing 0.4% (v/v) glutaraldehyde to fix the cells. Total count is expressed as total particles/ml. The software also determines mean cell size and volume [1].
2.7. Alkaline phosphatase assay F. chinensis was grown in the minimal media with 2 mg Pi/ml for 24 h at 30 ◦ C. The cells were harvested by centrifugation and washed twice in sterile distilled water. One ml samples of the resuspended cells were inoculated into 100 ml of sterile water in a 250 ml flask to give an initial viable cell count of approximately 107 cfu/l. The flasks were incubated in the dark without shaking at the desired temperatures. The ALP activity of the culture was determined by taking 1 ml from the flask, mixing with 2 ml of 0.1 M Tris buffer (pH 9.0) and adding 0.5 ml, 5% (w/v, in sterile distilled water) para-nitrophenyl disodium orthophosphate (pNPP). This reaction mixture was incubated at 30 ◦ C in a water bath for 30–240 min. The reaction was stopped by the addition of 0.2 ml, 10 M NaOH and the absorbance was measured at 420 nm. Controls containing no substrate and containing no cells were included to correct the absorbance changes due to cell density and spontaneous hydrolysis of the pNPP [5].
3. Results The effect of different temperatures on the alkaline phosphatase activity and survival of F. chinensis was investigated in a starvation medium. Over a 90-day starvation period, there was an increase in ALP activity at all incubation temperatures (Fig. 1a and b). The greatest increase in activity was found at 30 and 25 ◦ C. At 37 ◦ C, ALP activity increased less rapidly than at the other temperatures. However, after 90 days starvation, the enzyme activity at 37 ◦ C was close to that the other temperatures. There was no decrease at any of the temperatures used which suggests that a very stable ALP enzyme exists in F. chinensis. Except at 37 and 30 ◦ C, the viable cell counts at all temperatures were constant until after 25 days starvation when there was small decline in viable cell count at 4 and 15 ◦ C. The viable cell count at 37 ◦ C declined below the detection limits within 2 days of the onset of starvation but ALP activity continued to increase over the starvation period.
Fig. 1. The effect of different temperatures on alkaline phosphatase activity and the viable cell count of F. chinensis in starvation medium. The alkaline phosphatase activity (a) and viable counts (b) were assayed at 30 ◦ C. The enzyme activity was expressed as M/l PNP released ml−1 h−1 .
Addition of d-glucose to starvation medium as an amendment showed an increase in ALP activity in comparison to the control over a period of 25 days (Fig. 2). The viable cell counts of F. chinensis initially increased slightly in these samples which
Fig. 2. The effect of d-glucose on alkaline phosphatase activity and the viable cell count of F. chinensis at 15 ◦ C in a starvation medium.
J. Raheb et al. / Enzyme and Microbial Technology 40 (2006) 13–16
15
Fig. 3. The effect of urea on alkaline phosphatase activity and the viable cell count of F. chinensis at 15 ◦ C in a starvation medium.
suggest that cell growth was possible under these conditions. Reactivation of ALP in the presence of a carbon source is related to the absence of phosphate in this medium which therefore resembles a phosphate-limited medium. Addition of urea to starvation medium as an amendment showed that ALP activity doubled over the 40-day starvation period in both the unamended controls and in the nitrogenamended test flasks (Fig. 3). The viable cell count declined less in the flasks amended with the nitrogen source compared with the control. Addition of phosphate to starvation medium as an amendment showed that there was a much larger increase in ALP activity over the 40 days period of starvation in the control flasks with no phosphate amendment than in the flask amended with inorganic phosphate (Fig. 4). Initially, there was a small increase in enzyme activity in the amended flask over the first 5 days of starvation; the enzyme activity then remained relatively constant until the end of the 40 days experimental period. The viable cell count in the unamended control and the amended flask declined to the same extent. In the case of the unamended control, this decline in viable cell count occurred while ALP activity was still increasing. Fig. 5 shows that ALP activity doubled during starvation in the flasks amended with glucose and urea but there was only a small increase in the flask amended with inorganic phosphate.
Fig. 4. The effect of inorganic phosphate on the viable cell count and alkaline phosphatase activity of F. chinensis at 15 ◦ C in a starvation medium.
Fig. 5. The effects of different nutrient amendments on total alkaline phosphatase activity in F. chinensis incubated at 15 ◦ C in a starvation medium.
There was also close to a doubling of activity in the unamended control flask. All the flasks which show an increase in activity could be considered as phosphate limited whereas the flask which had inorganic phosphate added would not be phosphate limited. 4. Discussion In this study, ALP activity was assayed to determine the effects of long term starvation stress and the effects of different nutrient amendments to starvation medium on the reactivation of this enzyme. The effects of different temperatures at which the starvation microcosms were incubated on the ALP activity of F. chinensis were also examined. In the starvation experiments, ALP activity was measured over a period of up to 90 days at a range of incubation temperatures from 4 to 37 ◦ C. At all temperatures, activity continued to increase even as the viable cell count declined. The continued increase in ALP activity even during the period when the viable cell count was declining suggests that the ALP in F. chinensis is produced under starvation conditions. ALP activity has also been shown to increase in Escherichia coli starved in nutrient-free sea water [7] and in the same organisms starved for 70 days at temperatures between 4 and 37 ◦ C in lake water [8]. Enzyme activity was still observed to increase at 30 and 37 ◦ C after the cells began to lose their culturability and is believed to enter the viable but non-culturable state. After 60 days starvation, these bacteria could be recovered in a minimal medium or in diluted nutrient broth. After 90 days, the cells at 37 ◦ C were no longer recoverable but ALP activity was still increasing and activity was still measurable. It is possible that, at this time, the activity is being released from dead or lysing cells and the increase in activity is an artifact due to the reduction in diffusion barriers as cells lyse. However, there was no decrease in total cell count observed and it is believed to be unlikely that cell lysis was a significant factor in the increase in ALP
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J. Raheb et al. / Enzyme and Microbial Technology 40 (2006) 13–16
activity. Similar results were obtained from ref. [8] who also showed that ALP activity still increased and remained detectable in starved cultures of E. coli in which it was not possible to detect viable bacteria. The effect of different nutrient amendments on ALP activity any effect on the survival of the bacteria and on the reactivation or inactivation of ALP under starvation conditions. ALP activity increased over that of the unamended control after the addition of d-glucose to the starvation medium. Lim [9] reported a similar increase in ALP activity of Aeromonas hydrophila in sterile amended lake water microcosms. He also suggested that this increase was dependent upon the length of the starvation period and on the temperature at which starvation experiments were conducted. There was a greater increase in enzyme activity and in cells number at higher temperatures as the extra nutrient sources, such as carbon or nitrogen, were primarily needed for enzyme synthesis. Here, experiments were only conducted at 15 ◦ C as this was the optimal growth temperature for F. chinensis and is a temperature which has some environmental significance. Ozkanca and Flint [5] also showed that reactivation of ALP activity in E. coli occurred after the addition of carbon sources (particularly glucose and succinate) to the starvation medium. They suggested that the increase in enzyme activity could be a reflection of the fact that the addition of carbon or nitrogen sources to the starvation medium makes it more likely that phosphate-limited conditions occur which would lead to reactivation of enzyme synthesis if there was a carbon source to meet the energy requirement of the cell. Lim [9] showed that the greatest reactivation of ALP activity in A. hydrophila occurred after the addition of d-glucose, which is why this was the only carbon source tried in these series of experiments. The addition of urea to the microcosms also reactivated the synthesis of the enzyme. The addition of a nitrogen source (both organic nitrogen, such as amino acids or urea, and inorganic source such as ammonium ions) increased the survival time for E. coli without increasing the ALP activity [10]. The increase in phosphatase activity in A. hydrophila in nitrogen-amended microcosms was much more marked than with E. coli [8]. It is probable that the ability to synthesize the enzyme under these conditions is related to the ability of the cell to utilize the trace amounts of carbon which will be in the microcosms as energy sources.
When inorganic phosphate at a concentration of 100 mg PO4 /l was added to the starvation medium, ALP activity remained relatively constant over a 40 days starvation period. This suggests that these microcosms were not phosphate limited and that the increase in ALP activity seen in the other experiments is a direct response to phosphate limitation in the starvation medium. In all these experiments, the total counts remained constant, and ALP activity increased although cell viability decreased, in some cases to a non-detectable level. This suggests that the de novo synthesis of ALP seen here is a response of the cell to survival in an impoverished environment (certainly survival in phosphate-limited environment) rather than a facet of bacterial growth. It seems unlikely that the bacteria have to be able to grow to reactive ALP as there was no increase in cell numbers seen in any of these experiments and activity increased even though the viable cell count declined. References [1] Raheb J. The molecular and physiological effects of starvation and other stresses on Flexibacter chinensis, PhD thesis. University of Warwick: UK; 1998. [2] Orhanovic S, Pavela-Vrancic M. Alkaline phosphatase activity in seawater: influence of reaction conditions on the kinetic parameters of ALP. Croat Chem Acta 2000;73:819–30. [3] Nyc JF, Kadner RJ, Croken BJ. A repressible alkaline phosphatase in Neurospora crassa. J Biol Chem 1966;241:1468–72. [4] Filloux A, Bally M, Socia C, Murgier M, Lazdunski A. Phosphate regulation in Pseudomonas aeruginosa: cloning of the alkaline phosphatase gene and identification of PhoB and PhoR-like genes. Mol Gen Genet 1988;212:510–3. [5] Ozkanca R, Flint KP. Alkaline phosphatase activity of Escherichia coli starved in sterile lake water microcosms. J Appl Bacteriol 1996;80:252–85. [6] Sardans J, Penuelas J. Drought decrease soil enzyme activity in a Mediterranean Quercus ilex L. forest. Soil Biol Biochem 2005;37: 455–61. [7] Gauthier MJ, Flatau GN, Clement RL. Influence of phosphate ions and alkaline phosphatase activity of cells on survival of Escherichia coli in sea water. Microb Ecol 1990;20:245–51. [8] Ozkanca R. Survival and physiological status of Escherichia coli in lake water under different nutrient condition. PhD thesis. University of Warwick: UK; 1993. [9] Lim CH. The effect of environmental factors on the physiology of Aeromonas hydrophila in lake water, PhD thesis. University of Warwick: UK; 1995. [10] Lim CH, Flint KP. The effect of nutrients on the survival of Escherichia coli in lake water. J Appl Bacteriol 1989;66:5576–6569.