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Exocellular phosphatidylethanolamine production and multiple-metal tolerance in Pseudomonas fluorescens
ELSEVIER
FEMS Microbiology
Letters 131 (1995) 53-56
Exocellular phosphatidylethanolamine production and multiple-metal tolerance in Pseudomonas fluorescens Vasu D. Appanna
*,
Hugh Finn, Micheal St. Pierre
Department of Chemistry and Biochemistry, Laurentian Uniuersity, Ramsey Lake Road, Sudbury, Ont. P3E 2C6, Canada Received 22 May 1995; accepted
12 June 1995
Abstract Pseudomonas fltwrescens appeared to circumvent the challenge imposed by millimolar amounts of metals (5 mM A13+, 5 mM Fe3+, 2 mM Cazf, 1 mM Ga3+ and 3 mM Zn*+) by the formation of phosphatidylethanolamine. This lipid moiety constituted an important organic component of an insoluble gelatinous residue in which most of the test metals were immobilized at stationary phase of growth. Ultracentrifugation and dialysis experiments showed that the metals were associated with phosphatidylethanolamine from early stages of growth. Transmission electron microscopy revealed metal rich bodies in the cytoplasm prior to their secretion in the spent fluid. These results demonstrate a role of phosphatidylethanolamine in multiple-metal homeostasis. Keywor&
Phosphatidylethanolamine;
Multiple-metal
tolerance;
Insolubilization;
1. Introduction Metals constitute
an essential
nutritional
require-
ment for all living organisms due to their participation in a wide variety of biological activities. Thus in order to ensure normal cellular activities, the metal content of each cell is intricately regulated. Storage proteins, transport proteins and modified metabolic pathways are known to play an instrumental role in the attainment of metal homeostasis [l]. For instance siderophore production is initiated to circumvent a dearth of iron, while bacterioferritin helps regulate cellular iron concentration when the level of this trivalent element is excessive [2,3]. Owing to industrial pollution and acid rain, living organisms are
Corresponding author. Tel.: +l (705) 675 1151 ext. 2112; Fax: + 1 (705) 675 4844; e-mail: [email protected]. l
Federation of European Microbiological sSDIO378-1097(95)00234-O
Societies
Pseudomonas fluorescens
constantly being challenged by metal stress. Although higher organisms are known to succumb easily to these varying inorganic fluxes in their environment, microbes on the other hand, have evolved diverse strategies to adapt to the presence of elevated levels of metals in their ecological niches. Modification in the transport systems, accumulation, insolubilization, and volatization of the metals are some of the stratagems employed to combat the toxic effects of metals [4]. Even though numerous reports on the interaction of cellular systems with separate individual metals exist, studies on the impact of multiple metals, a condition prevalent in nature, are quite uncommon. In this report we have examined the impact of multiple-metal stress on the soil bacterium Pseudomonas jluorescens. It appears that tolerance to millimolar quantities of aluminum, iron, calcium, zinc and gallium is mediated by the elaboration of exocellular phosphatidylethanolamine.
54
V.D. Appanna et al. / FEMS Microbiology Letters 131 (1995) 53-56
2. Materials and methods
3. Results and discussion
The bacterial strain Pseudomonas j7uorescens ATCC 13525 was from American type culture collection (Rockville, Maryland, USA). It was maintained and grown in a mineral medium as described in [5]. The test metals aluminum chloride (5 mM), iron (III), chloride (5 mM), calcium chloride (2 mM), zinc chloride (3 mM) and gallium nitrate (1 mM) were complexed to citrate, the sole carbon source prior to sterilization. The media were dispensed in 200 ml amounts in 500-ml Erlenmeyer flasks and inoculated with 1 ml of stationary phase bacterial cells grown in a control medium. Cell yield was recorded by monitoring solubilized bacterial protein by the methods of Lowry et al. and Bradford [6,7]. Exocellular carbohydrate was measured colorimetrically [8]. While aluminum in the spent fluid, pellet and bacterial fractions were analysed by the aluminon assay [9], the other four test metals were analysed by atomic absorption spectroscopy. The five metals were further studied by x-ray fluorescence spectroscopy as described in [lo]. At various incubation times, aliquots of 20 ml of spent fluid devoid of bacteria were centrifuged at 159000 x g for 3.5 h. The pellet was extracted with a mixture of CH,OH:CHCl,:H,O (2:1:0.8). The extracted lipids were placed as spots on thin layer silica gel plates (Whatman, Germany) and resolved ascending chromatography using by CHCl,:CHsOH:NH,OH (65:25:5 v/v>. The lipids were visualized with I, vapor, ninhydrin and ammonium molybdate reagents [ 111. Phospholipids in these samples were quantitated with the aid of the dye Victoria blue R [12] and with ammonium molybdate [ll] following the liberation of phosphate by acidic hydrolysis. The metal content of this pellet isolated by ultracentrifugation was analysed by x-ray fluorescence spectroscopy, calorimetric assay by atomic absorption spectroscopy and x-ray diffraction spectroscopy. At timed intervals the supematant fluid was dialysed in membranes with molecular mass cut-off of 3.5 kDa. Bacterial cells harvested at various incubation periods were processed according to standard methods and visualized by a Zeiss 902 transmission electron microscope equipped with an electron energy loss spectroscopic (EELS) device.
When cultured in a medium unamended with test metals, Pseudomonas j7uorescens attained a cellular yield of 465 pg ml-’ of soluble protein at stationary phase of growth. Although a decrease in growth rate and a 22% diminution in cellular yield was recorded in the metal supplemented cultures, citrate the sole carbon source was rapidly utilized. There was no significant variation in the concentration of exocellular protein and carbohydrates in the control and test media. In both media the pH increased to 8.0-8.8. As bacterial multiplication progressed, an insoluble gelatinous residue formed. Most of the test metals were immobilized in this precipitate. X-ray fluorescence analyses indicated that the metal-rich insoluble residue was initially formed at 35 h and continued until 55 h when most of the aluminum, iron, zinc, calcium and gallium were insolubilized (Fig. 1). The pellet was apparently devoid of carbohydrates and proteins but contained CHCl,/CH,OH extractable component(s). Examination of this component by thin layer chromatography revealed a major band with a R, value of 0.42 that responded positively with ninhydrin and ammonium molybdate sprays. This spot was found to corn&rate with standard phosphatidylethanolamine. Ultracentrifugation and dialysis experiments revealed that the nature of the test metals was changing from early stages of growth. At 22 h of incubation a pellet rich in test metals was initially isolated by ultracentrifugation (Fig. 2). This residue also 500
125
t
Fig. 1. Microbial insolubilization of multiple metals. (0, % of metals in supematant; n , % of metals in pellet; A, bacterial growth).
55
V.D. Appanna et al. / FEMS Microbiology Letters 131 (I 995) 53-56
Incubation
Time
(hrs)
Fig. 2. Aluminum, iron and phosphatidylethanolamine in pellet isolated from spent fluid by ultracentrifugation. (Aluminum, 0; iron, W ; phosphatidylethanolamine, A 1.
comprised of phosphatidylethanolamine. However, as growth continued, a decrease in ultracentrifugable pellet was concomitant with the formation of the gelatinous residue. The test metals were also found to be retained in dialysis membranes (molecular mass cut-off of 3.5 kDa). Phosphatidylethanolamine was also detected in the dialysate (Fig. 3). Transmission electron microscopy and EELS helped in the identification of metal rich inclusions in the cytoplasm of the bacterial cells. These metallic bodies were absent in cells harvested after pellet formation (Fig. 4).
Fig. 4. Transmission electron micrograph of metal inclusions gelatinous residue (Bar represents 0.52 pm).
Fig. 3. Aluminum, iron and phosphatidyl ethanolamine in dialysate from membrane with molecular mass cut-off of 3.5 kDa. (0, aluminum; n , iron; A, phosphatidyl ethanolamine).
The foregoing data indicate that Pseudomonas fluorescens survived a medium supplemented with millimolar amounts of metals by the elaboration of a gelatinous residue. Phosphatidylethanolamine appeared to be an important organic constituent of the pellet. When present individually in the medium, the test metals elicited disparate responses tailored for each metal. While calcium homeostasis was attained via the formation of calcite, gallium was immobilized as soluble organic conjugate localized in the spent fluid [5,13]. Zinc triggered the synthesis of exocellular proteins and aluminum evoked the production of a polycarboxylated metabolite [14]. Iron
in bacterial
cells. No inclusions
were observed
following
the formation
of
56
V.D. Appanna et al. /FEMS Microbiology Letters 131 (1995) 53-56
detoxification was mediated by phosphatidylethanolamine [15]. In this instance it appears that when the metals were presented simultaneously, the microbe utilized phosphatidylethanolamine in the insolubilization of all the five metals. Ultracentrifugation and dialysis studies revealed that the metals were associated with phosphatidylethanolamine from early stages of growth, and it was only after a critical concentration was reached that spontaneous formation of a gelatinous residue was observed. The metal inclusions within the cytoplasm and their eventual elimination would indicate that the metals were processed within the bacterial cells. The phospholipid moieties of the inner membrane and/or the inner leaflet of the outer membrane may provide the nucleating sites for the metals or the metal-organic conjugate prior to their secretion. As the concentration of the phosphatidylethanolamine containing residues increased, precipitation occurred. Magnetosome membrane that has similar composition as cell membranes appears to be the site of magnetite formation in magnetotactic bacteria [16]. The involvement of phospholipid vesicles in metal deposition has also been shown in vitro [17]. Furthermore the absence of lipopolysaccharide in the residue or in the spent fluid would indicate that the metals are not being fortuitously bound to the outer membrane and that their subsequent elimination is indeed mediated by phosphatidylethanolamine. The initial sequestration of the metals within the bacterial cells and their elimination would point to such a possibility. Although further studies are needed to unravel the mechanism(s) involved in multiplemetal tolerance, it is tempting to propose that phosphatidylethanolamine is instrumental in this process. Furthermore it is important to note that instead of detoxifying the metals individually by disparate processes, the organism eliminates the metals predominantly as an insolwith phosresidue uble associated phatidylethanolamine. This strategy may be ensuring a more efficient utilization of energy under this stressed situation. Acknowledgements This investigation was supported by grants from the Ontario Ministry of Northern Development and
Mines and the National Science and Engineering Council of Canada. We would also like to thank Mr. A. Fook Yan (Agriculture Canada, Ottawa) for TEM analyses. References [II Frausto
da Silva, J.J.R. and Williams, R.J.P. (1993) The Biological Chemistry of the Elements, Clarendon Press, Oxford. L21Blakemore, R.P. and Blakemore, N.A. (1990) Metabolic magnetogens. In: Iron Biominerals (Frankel, R.B. and Blakemore, R.P., Eds.), pp. 51-68. Plenum Press, New York, NY. 131Appanna, V.D. and Viswanatha, T. (1986) Effects of some substrate analogs on aerobactin synthetase from Aerobacter aerogenes 62-1. FEBS I&t. 202, 107-110. [41 Silver, S., Misra, J.K. and Laddaga, R.A. (1989) Bacterial resistance to toxicity of heavy metals. In: Metal Ions and Bacteria (Beveridge, T.J. and Doyle, R.J., Eds.), pp. 121-140. John Wiley and Sons, New York, NY. 151Anderson, S., Appanna, V.D., Huang, J. and Viswanatha, T. (1992) A novel role for calcite in calcium homeostasis. FEBS L&t. 308, 94-96. [61Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. [71 Bradford, M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein-dye binding. Analyt. Biochem. 72, 248254. [81 Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A. and Smith, F. (1956) C&orimetric method for determination of sugar and related substances. Analyt. Chem. 28, 350-356. [91 Hsu, P.S. (1963) Effect of pH, phosphate and sillicate on the determination of aluminum with aluminon. Soil Sci. 96, 230-238. [lOI Anderson, S. and Appanna, V.D. (1993) Indium detoxification in Pseudomontas@orescens. Environ. Pollut. 82,33-37. [Ill Kates, M. (1988) Techniques in Lipidology, Isolation, Analysis, and Identification. 2nd edn. Elsevier, New York, NY. t121 Eryomin, A.E. and Poznyakon, S.P. (1989) Quantitative determination of phospholipids using dyes Victoria Blue R and B. Anal. B&hem. 180, 186-191. [131 Al-Aoukaty, A., Appanna, V.D. and Falter, H. (1992) Gallium toxicity and adaptation in Pseudomonas fluorescens. FEMS Microbial. Lett. 92, 265-272. 1141Appanna, V.D. and St. Pierre, M. (1994) Influence of phosphate on aluminum tolerance in Pseudomonas fluorescens. FEMS Microbial. L&t. 124, 327-332. WI Appanna, V.D. and Finn, H. (1995) Biometals Vol. 8. Ml Gorby, Y.A., Beveridge, T.J. and Blakemore, R.P. (1988) Characterization of the bacterial magnetosome membrane. J. Bacterial. 154, 708-718. [171 Archibald, D.D. and Mann, S. (1993) Template mineralization of self assembled anisotropic lipid microstructures. Nature 364, 430-433.
FEMS Microbiology
Letters 131 (1995) 53-56
Exocellular phosphatidylethanolamine production and multiple-metal tolerance in Pseudomonas fluorescens Vasu D. Appanna
*,
Hugh Finn, Micheal St. Pierre
Department of Chemistry and Biochemistry, Laurentian Uniuersity, Ramsey Lake Road, Sudbury, Ont. P3E 2C6, Canada Received 22 May 1995; accepted
12 June 1995
Abstract Pseudomonas fltwrescens appeared to circumvent the challenge imposed by millimolar amounts of metals (5 mM A13+, 5 mM Fe3+, 2 mM Cazf, 1 mM Ga3+ and 3 mM Zn*+) by the formation of phosphatidylethanolamine. This lipid moiety constituted an important organic component of an insoluble gelatinous residue in which most of the test metals were immobilized at stationary phase of growth. Ultracentrifugation and dialysis experiments showed that the metals were associated with phosphatidylethanolamine from early stages of growth. Transmission electron microscopy revealed metal rich bodies in the cytoplasm prior to their secretion in the spent fluid. These results demonstrate a role of phosphatidylethanolamine in multiple-metal homeostasis. Keywor&
Phosphatidylethanolamine;
Multiple-metal
tolerance;
Insolubilization;
1. Introduction Metals constitute
an essential
nutritional
require-
ment for all living organisms due to their participation in a wide variety of biological activities. Thus in order to ensure normal cellular activities, the metal content of each cell is intricately regulated. Storage proteins, transport proteins and modified metabolic pathways are known to play an instrumental role in the attainment of metal homeostasis [l]. For instance siderophore production is initiated to circumvent a dearth of iron, while bacterioferritin helps regulate cellular iron concentration when the level of this trivalent element is excessive [2,3]. Owing to industrial pollution and acid rain, living organisms are
Corresponding author. Tel.: +l (705) 675 1151 ext. 2112; Fax: + 1 (705) 675 4844; e-mail: [email protected]. l
Federation of European Microbiological sSDIO378-1097(95)00234-O
Societies
Pseudomonas fluorescens
constantly being challenged by metal stress. Although higher organisms are known to succumb easily to these varying inorganic fluxes in their environment, microbes on the other hand, have evolved diverse strategies to adapt to the presence of elevated levels of metals in their ecological niches. Modification in the transport systems, accumulation, insolubilization, and volatization of the metals are some of the stratagems employed to combat the toxic effects of metals [4]. Even though numerous reports on the interaction of cellular systems with separate individual metals exist, studies on the impact of multiple metals, a condition prevalent in nature, are quite uncommon. In this report we have examined the impact of multiple-metal stress on the soil bacterium Pseudomonas jluorescens. It appears that tolerance to millimolar quantities of aluminum, iron, calcium, zinc and gallium is mediated by the elaboration of exocellular phosphatidylethanolamine.
54
V.D. Appanna et al. / FEMS Microbiology Letters 131 (1995) 53-56
2. Materials and methods
3. Results and discussion
The bacterial strain Pseudomonas j7uorescens ATCC 13525 was from American type culture collection (Rockville, Maryland, USA). It was maintained and grown in a mineral medium as described in [5]. The test metals aluminum chloride (5 mM), iron (III), chloride (5 mM), calcium chloride (2 mM), zinc chloride (3 mM) and gallium nitrate (1 mM) were complexed to citrate, the sole carbon source prior to sterilization. The media were dispensed in 200 ml amounts in 500-ml Erlenmeyer flasks and inoculated with 1 ml of stationary phase bacterial cells grown in a control medium. Cell yield was recorded by monitoring solubilized bacterial protein by the methods of Lowry et al. and Bradford [6,7]. Exocellular carbohydrate was measured colorimetrically [8]. While aluminum in the spent fluid, pellet and bacterial fractions were analysed by the aluminon assay [9], the other four test metals were analysed by atomic absorption spectroscopy. The five metals were further studied by x-ray fluorescence spectroscopy as described in [lo]. At various incubation times, aliquots of 20 ml of spent fluid devoid of bacteria were centrifuged at 159000 x g for 3.5 h. The pellet was extracted with a mixture of CH,OH:CHCl,:H,O (2:1:0.8). The extracted lipids were placed as spots on thin layer silica gel plates (Whatman, Germany) and resolved ascending chromatography using by CHCl,:CHsOH:NH,OH (65:25:5 v/v>. The lipids were visualized with I, vapor, ninhydrin and ammonium molybdate reagents [ 111. Phospholipids in these samples were quantitated with the aid of the dye Victoria blue R [12] and with ammonium molybdate [ll] following the liberation of phosphate by acidic hydrolysis. The metal content of this pellet isolated by ultracentrifugation was analysed by x-ray fluorescence spectroscopy, calorimetric assay by atomic absorption spectroscopy and x-ray diffraction spectroscopy. At timed intervals the supematant fluid was dialysed in membranes with molecular mass cut-off of 3.5 kDa. Bacterial cells harvested at various incubation periods were processed according to standard methods and visualized by a Zeiss 902 transmission electron microscope equipped with an electron energy loss spectroscopic (EELS) device.
When cultured in a medium unamended with test metals, Pseudomonas j7uorescens attained a cellular yield of 465 pg ml-’ of soluble protein at stationary phase of growth. Although a decrease in growth rate and a 22% diminution in cellular yield was recorded in the metal supplemented cultures, citrate the sole carbon source was rapidly utilized. There was no significant variation in the concentration of exocellular protein and carbohydrates in the control and test media. In both media the pH increased to 8.0-8.8. As bacterial multiplication progressed, an insoluble gelatinous residue formed. Most of the test metals were immobilized in this precipitate. X-ray fluorescence analyses indicated that the metal-rich insoluble residue was initially formed at 35 h and continued until 55 h when most of the aluminum, iron, zinc, calcium and gallium were insolubilized (Fig. 1). The pellet was apparently devoid of carbohydrates and proteins but contained CHCl,/CH,OH extractable component(s). Examination of this component by thin layer chromatography revealed a major band with a R, value of 0.42 that responded positively with ninhydrin and ammonium molybdate sprays. This spot was found to corn&rate with standard phosphatidylethanolamine. Ultracentrifugation and dialysis experiments revealed that the nature of the test metals was changing from early stages of growth. At 22 h of incubation a pellet rich in test metals was initially isolated by ultracentrifugation (Fig. 2). This residue also 500
125
t
Fig. 1. Microbial insolubilization of multiple metals. (0, % of metals in supematant; n , % of metals in pellet; A, bacterial growth).
55
V.D. Appanna et al. / FEMS Microbiology Letters 131 (I 995) 53-56
Incubation
Time
(hrs)
Fig. 2. Aluminum, iron and phosphatidylethanolamine in pellet isolated from spent fluid by ultracentrifugation. (Aluminum, 0; iron, W ; phosphatidylethanolamine, A 1.
comprised of phosphatidylethanolamine. However, as growth continued, a decrease in ultracentrifugable pellet was concomitant with the formation of the gelatinous residue. The test metals were also found to be retained in dialysis membranes (molecular mass cut-off of 3.5 kDa). Phosphatidylethanolamine was also detected in the dialysate (Fig. 3). Transmission electron microscopy and EELS helped in the identification of metal rich inclusions in the cytoplasm of the bacterial cells. These metallic bodies were absent in cells harvested after pellet formation (Fig. 4).
Fig. 4. Transmission electron micrograph of metal inclusions gelatinous residue (Bar represents 0.52 pm).
Fig. 3. Aluminum, iron and phosphatidyl ethanolamine in dialysate from membrane with molecular mass cut-off of 3.5 kDa. (0, aluminum; n , iron; A, phosphatidyl ethanolamine).
The foregoing data indicate that Pseudomonas fluorescens survived a medium supplemented with millimolar amounts of metals by the elaboration of a gelatinous residue. Phosphatidylethanolamine appeared to be an important organic constituent of the pellet. When present individually in the medium, the test metals elicited disparate responses tailored for each metal. While calcium homeostasis was attained via the formation of calcite, gallium was immobilized as soluble organic conjugate localized in the spent fluid [5,13]. Zinc triggered the synthesis of exocellular proteins and aluminum evoked the production of a polycarboxylated metabolite [14]. Iron
in bacterial
cells. No inclusions
were observed
following
the formation
of
56
V.D. Appanna et al. /FEMS Microbiology Letters 131 (1995) 53-56
detoxification was mediated by phosphatidylethanolamine [15]. In this instance it appears that when the metals were presented simultaneously, the microbe utilized phosphatidylethanolamine in the insolubilization of all the five metals. Ultracentrifugation and dialysis studies revealed that the metals were associated with phosphatidylethanolamine from early stages of growth, and it was only after a critical concentration was reached that spontaneous formation of a gelatinous residue was observed. The metal inclusions within the cytoplasm and their eventual elimination would indicate that the metals were processed within the bacterial cells. The phospholipid moieties of the inner membrane and/or the inner leaflet of the outer membrane may provide the nucleating sites for the metals or the metal-organic conjugate prior to their secretion. As the concentration of the phosphatidylethanolamine containing residues increased, precipitation occurred. Magnetosome membrane that has similar composition as cell membranes appears to be the site of magnetite formation in magnetotactic bacteria [16]. The involvement of phospholipid vesicles in metal deposition has also been shown in vitro [17]. Furthermore the absence of lipopolysaccharide in the residue or in the spent fluid would indicate that the metals are not being fortuitously bound to the outer membrane and that their subsequent elimination is indeed mediated by phosphatidylethanolamine. The initial sequestration of the metals within the bacterial cells and their elimination would point to such a possibility. Although further studies are needed to unravel the mechanism(s) involved in multiplemetal tolerance, it is tempting to propose that phosphatidylethanolamine is instrumental in this process. Furthermore it is important to note that instead of detoxifying the metals individually by disparate processes, the organism eliminates the metals predominantly as an insolwith phosresidue uble associated phatidylethanolamine. This strategy may be ensuring a more efficient utilization of energy under this stressed situation. Acknowledgements This investigation was supported by grants from the Ontario Ministry of Northern Development and
Mines and the National Science and Engineering Council of Canada. We would also like to thank Mr. A. Fook Yan (Agriculture Canada, Ottawa) for TEM analyses. References [II Frausto
da Silva, J.J.R. and Williams, R.J.P. (1993) The Biological Chemistry of the Elements, Clarendon Press, Oxford. L21Blakemore, R.P. and Blakemore, N.A. (1990) Metabolic magnetogens. In: Iron Biominerals (Frankel, R.B. and Blakemore, R.P., Eds.), pp. 51-68. Plenum Press, New York, NY. 131Appanna, V.D. and Viswanatha, T. (1986) Effects of some substrate analogs on aerobactin synthetase from Aerobacter aerogenes 62-1. FEBS I&t. 202, 107-110. [41 Silver, S., Misra, J.K. and Laddaga, R.A. (1989) Bacterial resistance to toxicity of heavy metals. In: Metal Ions and Bacteria (Beveridge, T.J. and Doyle, R.J., Eds.), pp. 121-140. John Wiley and Sons, New York, NY. 151Anderson, S., Appanna, V.D., Huang, J. and Viswanatha, T. (1992) A novel role for calcite in calcium homeostasis. FEBS L&t. 308, 94-96. [61Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. [71 Bradford, M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein-dye binding. Analyt. Biochem. 72, 248254. [81 Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A. and Smith, F. (1956) C&orimetric method for determination of sugar and related substances. Analyt. Chem. 28, 350-356. [91 Hsu, P.S. (1963) Effect of pH, phosphate and sillicate on the determination of aluminum with aluminon. Soil Sci. 96, 230-238. [lOI Anderson, S. and Appanna, V.D. (1993) Indium detoxification in Pseudomontas@orescens. Environ. Pollut. 82,33-37. [Ill Kates, M. (1988) Techniques in Lipidology, Isolation, Analysis, and Identification. 2nd edn. Elsevier, New York, NY. t121 Eryomin, A.E. and Poznyakon, S.P. (1989) Quantitative determination of phospholipids using dyes Victoria Blue R and B. Anal. B&hem. 180, 186-191. [131 Al-Aoukaty, A., Appanna, V.D. and Falter, H. (1992) Gallium toxicity and adaptation in Pseudomonas fluorescens. FEMS Microbial. Lett. 92, 265-272. 1141Appanna, V.D. and St. Pierre, M. (1994) Influence of phosphate on aluminum tolerance in Pseudomonas fluorescens. FEMS Microbial. L&t. 124, 327-332. WI Appanna, V.D. and Finn, H. (1995) Biometals Vol. 8. Ml Gorby, Y.A., Beveridge, T.J. and Blakemore, R.P. (1988) Characterization of the bacterial magnetosome membrane. J. Bacterial. 154, 708-718. [171 Archibald, D.D. and Mann, S. (1993) Template mineralization of self assembled anisotropic lipid microstructures. Nature 364, 430-433.