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Polyphosphate production by strains of Acinetobacter
FEMS MicrobiologyLetters 70 (1990) 37-40 Pub~lis.hedby Elsevier
37
FEMSLE 04039
Polyphosphate production by strains of Acinetobacter G. Vasiliadis ~, A. D u n c a n 2, R.C. Bayly ~ a n d J.W. M a y 1 t Department of Microbiology, Monash University, Clayton, Victoria and 2 CSIRO Division of Chemicals and Polymers, Clayton, Victoria, Australia
Received25 January 1990 Accepted 5 March 1990 Key words: Acinetobacter; Biological phosphate removal; Sewage
1. S U M M A R Y Of four strains of Acinetobacter isolated from a pilot plant exhibiting enhanced biological phosphate removal from sewage, two strains (RA3116 and RA3117) accumulated more than 10 times the amount of polyphosphate accumulated by the other two strains (RA3114 and RA3123). Variants isolated from RA3116 and RA3117 showed polyphosphate levels similar to RA3114 and RA 3123. N o correlation was found between the polyphos.. phate content of the strains and levels of several enzymes that have been implicated in polyphosphate formation.
activity of the enzyme(s) responsible for its formation. Polyphosphate kinase has frequently been implicated in the formation of polyphosphate [1-3] but there is some evidence that this may not be the relevant enzyme in Acinetobacter [4]. We have examined several strains of Acinetobacter for their ability to accumulate polyphosphate and the activities of several enzymes implicated in polyphosphate formation in Acinetobacter and in some other bacteria.
3. MATERIALS A N D M E T H O D S 3.1. Microorganisms Acinetobacter strains
2. I N T R O D U C T I O N Biological removal of phosphate from sewage in activated-sludge plants is generally attributed to accumulation of intracellular polyphosphate by species of Acinetobacter. If this is so, then in such Acinetobacter there should be a correlation between the ability to form polyphosphate and the
Correspondence to: G. Vasiliadis. Department of Microbiology.
Monash University,Clayton, Victoria 3168, Australia
RA3114, RA3117 and RA3123 were obtained from a pilot-plant (5 m 3 capacity): details of the plant and of the isolation and identification of the strains have been described previously [5]. Variant strains RA3739 and RA3197 were isolated from strains RA3116 and RA3117 respectively. Strain RA3116 or RA3117 were grown in a fill-draw continuous-culture system programmed to operate alternately under aerobic and anaerobic conditions (3 h each). Culture was withdrawn at the end of the aerobic phase and fresh medium (ADM) was added just after the start of the anaerobic phase to give an overall dilution rate of 0.2/d.
0378-1097/90/$03.50 © 1990 Federation of European MicrobiologicalSocieties
Samples were withdrawn from the system, diluted, and 0.1 ml plated on to triethanolaminebuffered ADM and incubated for two to three days at 30 °C. Colonies with a slightly different appearance from the parent strain were subcultured and studied further. The variants were confirmed as Acinetobacter using the transformation system of Juni [6] and the genospecies was ascertained by the percentage positive matrix described by Bouvet and Grimont [7].
3.2. Media and growth conditions The chemically defined medium contained: 5.6 g CH3-COONa. 3H20, 1.0 g NH4CI, 0.5 g KCl, 0.5 g MgC! z • 6H20, 0.05 g CaCI z • 2H20, 0.1 g Na2SO4, 0.5 g NaH2PO 4 • 2HzO, 12.1 g Tris(hydroxymethyl) aminomethane and 1 ml of a trace element solution [8] per litre of deionized water. This medium (designated ADM) in which acetate was the sole carbon and energy source had a final pH of 7.4. Triethanolamine-buffered ADM contained 18.6 g . l -~ triethanolamine instead of Tris(hydroxymethyl) aminomethane. Cultures were grown at 30 o C on a rotary shaker (New Brunswick Scientific, Model G-52) at 150 rpm in 2 fitre flasks which contained 500 ml medium. Cells were harvested in mid-exponential growth phase.
3.3. Analytical methods Phosphate and polyphosphate: Cells were extracted by the method of Clark et al. [9] to produce an acid-soluble and two acid-insoluble fractions but removal of proteins by phenol/ chloroform extraction and separation of polyphosphate from nucleic acid was not undertaken. The acid-insoluble fractions and part of the acid-soluble fraction were each hydrolysed separately to orthophosphate by boiling in 1 M HCI for 10 rain [10] and then diluted with distilled water as necessary. A volume (0.3 ml) of the diluted phosphate solution was further diluted to 1 mi with a mixture of 10% ascorbic acid and 0.425 (w/v) ammonium molybdate (1:6) in 0.5 M H2SO4. The assay was then as described by Ames [11]. The other portion of the acid-soluble fraction
was analyzed for the presence of orthophosphate at pH 4 as described by Panusz et al. [12]. Enzyme assays: Enzyme activities were determined in cell-free extracts prepared by disruption of washed cell suspensions in a French pressure cell (Aminco, Silver Springs, MD, USA) and removal of cell debris by centrifugation at 25 000 x g for 30 min at 4°C. Extracts were kept on ice and all assays were completed within 6 h of preparation of the extracts. Polyphosphate-AMP phosphotransferase and polyphosphate-dependent N A D + kinase were determined by the methods of van Groenestijn et al. [13]. Polyphosphate glucokinase (EC 2.7.1.63) and 3-phosphoglycerate kinase (EC 2.7.4.17) were assayed as described by Wood and Goss [14]. Polyphosphate kinase (EC 2.7.4.1) was determined according to the method described by Robinson et al. [15]. Protein determination: The method of Lowry et al. [16] was used. Protein was extracted from whole cells by boiling in 4.6 M NaOH for 5 rain. Metachromatic staining: The Neisser stain [17] was used.
4. RESULTS AND DISCUSSION Table 1 shows the metachromatic staining and the content of phosphates for the 6 strains used in this study. Compared with RA3114 and RA3123, strains RA3116 and RA3117 showed markedly stronger metachromatic staining and higher levels of phosphates, particularly the first acid-insoluble fraction. Not all of the phosphate in the acid-insoluble fractions would have been derived from polyphosphate, since some would have been produced by hydrolysis of extracted nucleic acids. However, assuming an equal contribution of nucleic acids to the post-hydrolysis phosphate in both pairs of strains, the much higher values in the first acid-insohible fraction of RA3116 and RA3117 can reasonably be attributed to polyphosphate. The two variants, RA3739 (from RA3116) and RA3197 (from RA3117) showed levels of acid-insoluble phosphates very similar to those in the natural isolates RA3114 and RA3123, although they failed to show any metachromatic
39 staining. Overall, when the two acid-insoluble fractions of each strain were summed, the values for RA3116 and RA3117 were from 10 to 20 times higher than those for the other four strains. In all of the strains, the acid-soluble fraction contained only orthophosphate and no detectable polyphosphate. Activities of polyphosphate glucokinase and polyphosphate-dependent N A D + kinase were not detected in either RA3116 and RA3117 and were not therefore assayed in the remaining strains. The levels of another three enzymes that have been implicated in polyphosphate formation are shown in Table 2. For each enzyme, the level in all six strains was generally very similar. The levels of polyphosphate A M P phosphotransferase were very low. This contrasts with the finding of van Groenestijn et al. [18] who detected quite high levels of this enzyme in the polyphosphate-accumulating Acinetobacter strain 210A. Levels of polyphosphate kinase were on average about 7 times higher than those of the former enzyme. Of the various enzymes implicated, polyphosphate kinase has been claimed to be the major enzyme responsible for polyphosphate formation in most microorganisms [19] and
Table l Content of phosphatesin Acinetobacter strainsgrown in acetate defined medium Values are the means of duplicate experiments. Strain
Metacl~omatic staining
Phosphatesa in: Acid-soluble Acid-insoluble fractionb fractions
RA3114 RA3123 RA3116 RA3117 RA3197 RA3739
+ + +++ +++ -
30 27 52 i19 28 17
1
2
21 24 336 476 25 10
8 13 39 26 6 6
a Content of phosphates expressedas /tg of phosphate-phosphorus (mr protein)-I. (Note: 1 mg dry weight of cells contained about 0.13 mg protein.) b Valuesobtained by assayingat pH 4 withoutprior hydrolysis (i.e. orthophosphate),no significantincreasebeing observed followinghydrolysisof this fraction from any of the strains.
Table 2 Specificactivitiesa of some polyphosphate-formingenzymesin Acinetobacter strains grown on acetate defined medium Strain
RA3114 RA3123 RA3116 RA3ll7 RA3197 RA3739
Polyphosphate AMP-phosphotransferase 2.2 ND b 6.5 5.5 3.3 7.5
Polyphosphate kinase 20 36 20 18 32 43
3-phosphoglycerate kinase 55 62 71 57 92 68
a Meansof duplicateexperiments,expressedas nmol substrate utilized or product formed rain- J (mg protein)- i. b ND, no activity detected.
in Propionibacterium shermanii is claimed to be the only enzyme involved [3]. Nevertheless, the level of polyphosphate kinase in RA3116 and RA3117 was never greater than the levels in the poor polyphosphate-accumulating strains. The levels of 3-phosphoglycerate kinase were significantly higher than those for polyphosphate kinase. These relatively high levels of the 3-phosphoglycerate kinase were probably due to the cells having been grown on acetate and the consequent need for gluconeogenesis. Since no strain showed any association between its content of polyphosphate and the level of a particular enzyme, it is probable that polyphosphate formation in Acinetobacter RA3116 and RA3117 is due to an enzyme different from any of those that were measured in this study. Work is continuing on identifying the enzyme and further elucidating the mechanisms of polyphosphate accumulation by A cinetobacter.
ACKNOWLEDGEMENTS This work was supported by grants from the Australian Water Research Advisory Council, the Urban Water Research Association of Australia and the Monash University Special Research Grant. We thank Mr. W. Raper for useful discussions.
REFERENCES [1] Harold, F.M. (1966) Bact. Rev. 30, 772-789. [2] Li, H. and Brown, G.G. (1973) Biochem. Biophys. Res. Commun. 53, 875-881. [3] Wood, H.G., Robinson, N.A., Pepin, C.A. and Clark, J.E. (1987) in Phosphate Metabolism and Cellular Regulation in Microorganisms (Torriani-Gorini, A., Rothman, F.G., Silver, S., Wright, A. and Yagil, E., eds.), pp. 225-232, American Society for Microbiology, Washington D.C. [4] T'Seyn, J., Malnou, D., Block, J.C. and Paup, G. (1985) Wet. Sci. Tech. 17, 43-56. [5] Duncan, A., Vasiliadis, G.E., Bayly, R.C. and May, J.W. (1988) Biotechnol. Lett. 10, 831-836. [6] Jani, E. (1972) J. BacterioL 112, 917-931. [7] Bouvet, P.J.M. and Grimont, P.A.D. (1987) Ann. Inst. Pasteur/Microbiol. 138, 569-578. [8] Mitchison, J.M. (1970) Methods Cell Physiol. 4,131-165. [9l Clark, J.E., Beegen, H. and Wood, H.G. (1986)J. Bacteriol. 168,1212-1219. [10] Poindexter, J.S. and Eley, L.F. (1983) J. Microbiol. Methods 1,1-17.
[II] Ames, B.N. (1966) in Methods in Enzymology (Neufeld, E.F. and Ginsberg, V., eds.), 8, pp. 115-118, Academic Press, New York, london. [12] Panusz, H.T., Graczyk, G., Wilmanska* D. and Skarzynski, J. (1970) Anal. Biochem. 35, 494-504. [13] van Groenestijn, J.W., Deinema, M.H. and Zehnder, A.J.B. (1987) Arch. Microbioi. 148,14-19. [14] Wood, H.G. and Goss, N.H. (1985) Prof. Natl. Acad. Sci. U.S.A. 82, 312-315. [15] Robinson, N.A., Clark, J.E. and Wood, H.G. (1987) J. Biol. Chem. 262, 5216-5222. [16] Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. [17] Cowan, S.T. (1974) in Cowan and Steel's Manual for the Identification of Medical Bacteria, 2nd Edn., p. 162, Cambridge U~iversity Press, london. [181 van Groenestijn, J.W., lkntvelsen, M.M.A., Deinema, M.H. and Zehnder, AJ.B. (1989) Appl. Environ. Microbiol. 55, 219-223. [191 Kulaev, I.S. (1979) 3"he Biochemistry of Inorganic Polyphosphates. John Wiley and Sons Ltd., Chichester, New York, Brisbane, Toronto.
37
FEMSLE 04039
Polyphosphate production by strains of Acinetobacter G. Vasiliadis ~, A. D u n c a n 2, R.C. Bayly ~ a n d J.W. M a y 1 t Department of Microbiology, Monash University, Clayton, Victoria and 2 CSIRO Division of Chemicals and Polymers, Clayton, Victoria, Australia
Received25 January 1990 Accepted 5 March 1990 Key words: Acinetobacter; Biological phosphate removal; Sewage
1. S U M M A R Y Of four strains of Acinetobacter isolated from a pilot plant exhibiting enhanced biological phosphate removal from sewage, two strains (RA3116 and RA3117) accumulated more than 10 times the amount of polyphosphate accumulated by the other two strains (RA3114 and RA3123). Variants isolated from RA3116 and RA3117 showed polyphosphate levels similar to RA3114 and RA 3123. N o correlation was found between the polyphos.. phate content of the strains and levels of several enzymes that have been implicated in polyphosphate formation.
activity of the enzyme(s) responsible for its formation. Polyphosphate kinase has frequently been implicated in the formation of polyphosphate [1-3] but there is some evidence that this may not be the relevant enzyme in Acinetobacter [4]. We have examined several strains of Acinetobacter for their ability to accumulate polyphosphate and the activities of several enzymes implicated in polyphosphate formation in Acinetobacter and in some other bacteria.
3. MATERIALS A N D M E T H O D S 3.1. Microorganisms Acinetobacter strains
2. I N T R O D U C T I O N Biological removal of phosphate from sewage in activated-sludge plants is generally attributed to accumulation of intracellular polyphosphate by species of Acinetobacter. If this is so, then in such Acinetobacter there should be a correlation between the ability to form polyphosphate and the
Correspondence to: G. Vasiliadis. Department of Microbiology.
Monash University,Clayton, Victoria 3168, Australia
RA3114, RA3117 and RA3123 were obtained from a pilot-plant (5 m 3 capacity): details of the plant and of the isolation and identification of the strains have been described previously [5]. Variant strains RA3739 and RA3197 were isolated from strains RA3116 and RA3117 respectively. Strain RA3116 or RA3117 were grown in a fill-draw continuous-culture system programmed to operate alternately under aerobic and anaerobic conditions (3 h each). Culture was withdrawn at the end of the aerobic phase and fresh medium (ADM) was added just after the start of the anaerobic phase to give an overall dilution rate of 0.2/d.
0378-1097/90/$03.50 © 1990 Federation of European MicrobiologicalSocieties
Samples were withdrawn from the system, diluted, and 0.1 ml plated on to triethanolaminebuffered ADM and incubated for two to three days at 30 °C. Colonies with a slightly different appearance from the parent strain were subcultured and studied further. The variants were confirmed as Acinetobacter using the transformation system of Juni [6] and the genospecies was ascertained by the percentage positive matrix described by Bouvet and Grimont [7].
3.2. Media and growth conditions The chemically defined medium contained: 5.6 g CH3-COONa. 3H20, 1.0 g NH4CI, 0.5 g KCl, 0.5 g MgC! z • 6H20, 0.05 g CaCI z • 2H20, 0.1 g Na2SO4, 0.5 g NaH2PO 4 • 2HzO, 12.1 g Tris(hydroxymethyl) aminomethane and 1 ml of a trace element solution [8] per litre of deionized water. This medium (designated ADM) in which acetate was the sole carbon and energy source had a final pH of 7.4. Triethanolamine-buffered ADM contained 18.6 g . l -~ triethanolamine instead of Tris(hydroxymethyl) aminomethane. Cultures were grown at 30 o C on a rotary shaker (New Brunswick Scientific, Model G-52) at 150 rpm in 2 fitre flasks which contained 500 ml medium. Cells were harvested in mid-exponential growth phase.
3.3. Analytical methods Phosphate and polyphosphate: Cells were extracted by the method of Clark et al. [9] to produce an acid-soluble and two acid-insoluble fractions but removal of proteins by phenol/ chloroform extraction and separation of polyphosphate from nucleic acid was not undertaken. The acid-insoluble fractions and part of the acid-soluble fraction were each hydrolysed separately to orthophosphate by boiling in 1 M HCI for 10 rain [10] and then diluted with distilled water as necessary. A volume (0.3 ml) of the diluted phosphate solution was further diluted to 1 mi with a mixture of 10% ascorbic acid and 0.425 (w/v) ammonium molybdate (1:6) in 0.5 M H2SO4. The assay was then as described by Ames [11]. The other portion of the acid-soluble fraction
was analyzed for the presence of orthophosphate at pH 4 as described by Panusz et al. [12]. Enzyme assays: Enzyme activities were determined in cell-free extracts prepared by disruption of washed cell suspensions in a French pressure cell (Aminco, Silver Springs, MD, USA) and removal of cell debris by centrifugation at 25 000 x g for 30 min at 4°C. Extracts were kept on ice and all assays were completed within 6 h of preparation of the extracts. Polyphosphate-AMP phosphotransferase and polyphosphate-dependent N A D + kinase were determined by the methods of van Groenestijn et al. [13]. Polyphosphate glucokinase (EC 2.7.1.63) and 3-phosphoglycerate kinase (EC 2.7.4.17) were assayed as described by Wood and Goss [14]. Polyphosphate kinase (EC 2.7.4.1) was determined according to the method described by Robinson et al. [15]. Protein determination: The method of Lowry et al. [16] was used. Protein was extracted from whole cells by boiling in 4.6 M NaOH for 5 rain. Metachromatic staining: The Neisser stain [17] was used.
4. RESULTS AND DISCUSSION Table 1 shows the metachromatic staining and the content of phosphates for the 6 strains used in this study. Compared with RA3114 and RA3123, strains RA3116 and RA3117 showed markedly stronger metachromatic staining and higher levels of phosphates, particularly the first acid-insoluble fraction. Not all of the phosphate in the acid-insoluble fractions would have been derived from polyphosphate, since some would have been produced by hydrolysis of extracted nucleic acids. However, assuming an equal contribution of nucleic acids to the post-hydrolysis phosphate in both pairs of strains, the much higher values in the first acid-insohible fraction of RA3116 and RA3117 can reasonably be attributed to polyphosphate. The two variants, RA3739 (from RA3116) and RA3197 (from RA3117) showed levels of acid-insoluble phosphates very similar to those in the natural isolates RA3114 and RA3123, although they failed to show any metachromatic
39 staining. Overall, when the two acid-insoluble fractions of each strain were summed, the values for RA3116 and RA3117 were from 10 to 20 times higher than those for the other four strains. In all of the strains, the acid-soluble fraction contained only orthophosphate and no detectable polyphosphate. Activities of polyphosphate glucokinase and polyphosphate-dependent N A D + kinase were not detected in either RA3116 and RA3117 and were not therefore assayed in the remaining strains. The levels of another three enzymes that have been implicated in polyphosphate formation are shown in Table 2. For each enzyme, the level in all six strains was generally very similar. The levels of polyphosphate A M P phosphotransferase were very low. This contrasts with the finding of van Groenestijn et al. [18] who detected quite high levels of this enzyme in the polyphosphate-accumulating Acinetobacter strain 210A. Levels of polyphosphate kinase were on average about 7 times higher than those of the former enzyme. Of the various enzymes implicated, polyphosphate kinase has been claimed to be the major enzyme responsible for polyphosphate formation in most microorganisms [19] and
Table l Content of phosphatesin Acinetobacter strainsgrown in acetate defined medium Values are the means of duplicate experiments. Strain
Metacl~omatic staining
Phosphatesa in: Acid-soluble Acid-insoluble fractionb fractions
RA3114 RA3123 RA3116 RA3117 RA3197 RA3739
+ + +++ +++ -
30 27 52 i19 28 17
1
2
21 24 336 476 25 10
8 13 39 26 6 6
a Content of phosphates expressedas /tg of phosphate-phosphorus (mr protein)-I. (Note: 1 mg dry weight of cells contained about 0.13 mg protein.) b Valuesobtained by assayingat pH 4 withoutprior hydrolysis (i.e. orthophosphate),no significantincreasebeing observed followinghydrolysisof this fraction from any of the strains.
Table 2 Specificactivitiesa of some polyphosphate-formingenzymesin Acinetobacter strains grown on acetate defined medium Strain
RA3114 RA3123 RA3116 RA3ll7 RA3197 RA3739
Polyphosphate AMP-phosphotransferase 2.2 ND b 6.5 5.5 3.3 7.5
Polyphosphate kinase 20 36 20 18 32 43
3-phosphoglycerate kinase 55 62 71 57 92 68
a Meansof duplicateexperiments,expressedas nmol substrate utilized or product formed rain- J (mg protein)- i. b ND, no activity detected.
in Propionibacterium shermanii is claimed to be the only enzyme involved [3]. Nevertheless, the level of polyphosphate kinase in RA3116 and RA3117 was never greater than the levels in the poor polyphosphate-accumulating strains. The levels of 3-phosphoglycerate kinase were significantly higher than those for polyphosphate kinase. These relatively high levels of the 3-phosphoglycerate kinase were probably due to the cells having been grown on acetate and the consequent need for gluconeogenesis. Since no strain showed any association between its content of polyphosphate and the level of a particular enzyme, it is probable that polyphosphate formation in Acinetobacter RA3116 and RA3117 is due to an enzyme different from any of those that were measured in this study. Work is continuing on identifying the enzyme and further elucidating the mechanisms of polyphosphate accumulation by A cinetobacter.
ACKNOWLEDGEMENTS This work was supported by grants from the Australian Water Research Advisory Council, the Urban Water Research Association of Australia and the Monash University Special Research Grant. We thank Mr. W. Raper for useful discussions.
REFERENCES [1] Harold, F.M. (1966) Bact. Rev. 30, 772-789. [2] Li, H. and Brown, G.G. (1973) Biochem. Biophys. Res. Commun. 53, 875-881. [3] Wood, H.G., Robinson, N.A., Pepin, C.A. and Clark, J.E. (1987) in Phosphate Metabolism and Cellular Regulation in Microorganisms (Torriani-Gorini, A., Rothman, F.G., Silver, S., Wright, A. and Yagil, E., eds.), pp. 225-232, American Society for Microbiology, Washington D.C. [4] T'Seyn, J., Malnou, D., Block, J.C. and Paup, G. (1985) Wet. Sci. Tech. 17, 43-56. [5] Duncan, A., Vasiliadis, G.E., Bayly, R.C. and May, J.W. (1988) Biotechnol. Lett. 10, 831-836. [6] Jani, E. (1972) J. BacterioL 112, 917-931. [7] Bouvet, P.J.M. and Grimont, P.A.D. (1987) Ann. Inst. Pasteur/Microbiol. 138, 569-578. [8] Mitchison, J.M. (1970) Methods Cell Physiol. 4,131-165. [9l Clark, J.E., Beegen, H. and Wood, H.G. (1986)J. Bacteriol. 168,1212-1219. [10] Poindexter, J.S. and Eley, L.F. (1983) J. Microbiol. Methods 1,1-17.
[II] Ames, B.N. (1966) in Methods in Enzymology (Neufeld, E.F. and Ginsberg, V., eds.), 8, pp. 115-118, Academic Press, New York, london. [12] Panusz, H.T., Graczyk, G., Wilmanska* D. and Skarzynski, J. (1970) Anal. Biochem. 35, 494-504. [13] van Groenestijn, J.W., Deinema, M.H. and Zehnder, A.J.B. (1987) Arch. Microbioi. 148,14-19. [14] Wood, H.G. and Goss, N.H. (1985) Prof. Natl. Acad. Sci. U.S.A. 82, 312-315. [15] Robinson, N.A., Clark, J.E. and Wood, H.G. (1987) J. Biol. Chem. 262, 5216-5222. [16] Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. [17] Cowan, S.T. (1974) in Cowan and Steel's Manual for the Identification of Medical Bacteria, 2nd Edn., p. 162, Cambridge U~iversity Press, london. [181 van Groenestijn, J.W., lkntvelsen, M.M.A., Deinema, M.H. and Zehnder, AJ.B. (1989) Appl. Environ. Microbiol. 55, 219-223. [191 Kulaev, I.S. (1979) 3"he Biochemistry of Inorganic Polyphosphates. John Wiley and Sons Ltd., Chichester, New York, Brisbane, Toronto.