Departamento de Bioquímica, Facultad de Ciencias Naturales, Universidad Nacional de la Patagonia San Juan Bosco, CC 1078, Km 4, 9000 Comodoro Rivadavia, Chubut, Argentina1
Institut für Organische Chemie der Westfälischen Wilhelms-Universität Münster, Corrensstraße 40, D-48149 Münster, Germany2
Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad Nacional de Buenos Aires, Buenos Aires, Argentina3
Institut für Mikrobiologie der Westfälischen Wilhelms-Universität Münster, Corrensstraße 3, D-48149 Münster, Germany4
Author for correspondence: Héctor M. Alvarez. Tel: +54 297 4550 339. Fax: +54 297 4550 339. e-mail: halvarez{at}unpata.edu.ar
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ABSTRACT |
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Keywords: Rhodococcus opacus PD630, phenyldecane, triacylglycerol, wax ester, phenyldecylphenyldecanoate
Abbreviations: ESI-MS, electron spray ionization mass spectrometry; TAG, triacylglycerol
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INTRODUCTION |
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Phenyldecane-grown cells of Rhodococcus opacus PD630, which were also able to catabolize phenylacetic acid, intracellularly accumulated oxidation products of the substrate, with phenyldecanoic acid as the major compound and also lesser amounts of phenyloctanoic, phenylhexanoic, phenylnonanoic acids and diacylglycerols (Alvarez et al., 1996 ). Members of the genus Rhodococcus are widely distributed in soil environments. These micro-organisms show a broad capacity and metabolic spectrum for the biodegradation of different kinds of pollutants, such as hydrocarbons, herbicides or other xenobiotic compounds (Finnerty, 1992
; Warhust & Fewson, 1994
). In addition, recent studies demonstrated that these bacteria are able to accumulate triacylglycerols (TAGs) from different carbon sources, including hydrocarbons, during cultivation under nitrogen-starvation conditions (Alvarez et al., 1996
, 1997
, 2000
). In this context, the biodegradation and production of cellular lipids by Nocardia globerula 432 from the recalcitrant branched hydrocarbon pristane under conditions restricting growth has been reported recently (Alvarez et al., 2001
). These results suggest that these Gram-positive bacteria may be employed to eliminate pollutants from the environment under a broad range of metabolic and environmental conditions. Several reports considered the potential of these bacteria for in situ bioremediation of contaminated environments (Yakimov et al., 1999
; Wagner-Dobler et al., 1998
; White et al., 1998
).
On account of the ability of R. opacus PD630 to accumulate metabolites and lipids under unbalanced growth conditions, we investigated in this study the catabolism and assimilation of derivatives of phenyldecane in this strain.
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METHODS |
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Growth was monitored by noting the occurrence of visible growth (turbidity) in the culture medium, using a KlettSummerson photometer and a filter at a wavelength of 436 nm.
Induction procedure.
Induction studies based on measuring the time-dependent release of CO2 due to the mineralization of the carbon source were carried out as described by Frederikson et al. (1991) . For this purpose, the cells were grown at 25 °C on mineral salts medium agar plates containing 0·3 ml of the respective carbon source on a filter-paper disc in the lid or containing 1% (w/v) sodium gluconate in the agar. After 48 h incubation, the cells were harvested from the plates, washed twice with sterile NaCl solution (0·85%, w/v) and subsequently resuspended to an optical density at 436 nm of 2·5 in 500 ml flasks containing 50 ml mineral salts medium plus the hydrocarbon as sole carbon source. Chloramphenicol (200 µg ml-1) was also added to prevent further protein synthesis. Controls without chloramphenicol were done under identical conditions. Each flask was equipped with a vial containing 2 ml 1 M NaOH to absorb CO2 produced by the cells; these vials were exchanged every 24 h with new vials containing fresh NaOH solution during the time course of the experiment. The flasks were tightly sealed with wrapped rubber stoppers and incubated for 24 h at 25 °C on a rotary shaker at 120 r.p.m. CO2 production was monitored by titration with 1 M HCl.
TLC of extracted aromatic compounds and lipids.
Aromatic compounds were extracted from the supernatants with methanol and were subjected to TLC on silica-gel 60F254 plates (Merck), applying chloroform/acetic acid (9:1, v/v) as the solvent system. Compounds were visualized under UV light and identified by comparison of their RF values with those of phenylacetic acid, phenylpropionic acid, homogentisic acid, 4-hydroxyphenylpropionic acid, DL-mandelic acid (Sigma) and 4-hydroxybenzoic acid (Merck), which were used as reference substances.
For identification of lipids, whole cells were extracted with a mixture of chloroform and methanol (2:1, v/v), and the extracts were separated by TLC, which was performed on silica-gel 60F254 plates (Merck) using a mixture of hexane, diethyl ether and acetic acid (80:20:1, by vol.) as the solvent system. Lipid fractions were visualized after brief exposure to iodine vapour. Palmitic acid, stearic acid, dipalmitoylglycerol, tripalmitoylglycerol and cetylpalmitate (Merck) were used as reference substances.
Qualitative determination of intermediates in supernatants.
Culture supernatants obtained by centrifugation were analysed for excreted intermediates by liquid chromatography, using HPLC apparatus (Knauer). Separation was achieved by reversed-phase chromatography on Nucleosil-100 C18 (5 µm particle size, 250 mmx4·0 mm column) with a gradient of 0·1% (v/v) formic acid (eluent A) and acetonitrile (eluent B) in a range of 20100% (v/v) eluent B and at a flow rate of 1 ml min-1. The compounds were identified by their retention times and the corresponding spectra were obtained with a diode array detector (WellChrom Diodenarray-Detektor K-2150; Knauer) (Priefert et al., 1997 ).
Analysis of neutral lipids and fatty acids.
To determine the fatty acid content of the cells and the composition of lipids, 35 mg lyophilized whole cells or the triacylglycerol fraction obtained from preparative TLC were subjected to methanolysis in the presence of 15% (v/v) sulfuric acid. The resulting fatty acid methyl esters were analysed by GC on a Konik HRGC3000 gas chromatograph equipped with an Innowax capillary column (30 mx0·53 mm) and a flame-ionization detector (Brandl et al., 1988 ; Alvarez et al., 1996
). A 2 µl portion of the organic phase was analysed after split injection (split ratio 1:20), and nitrogen was used as the carrier gas at a flow rate of 50 ml min-1. The temperature of the injector and the detector was 260 °C, whereas a temperature programme was used for efficient separation of the methyl esters on the column (150 °C for 1 min, temperature increases of 5 °C min-1, 240 °C for 10 min).
Electron spray ionization mass spectrometry and tandem electron spray ionization mass spectrometry.
Electron spray ionization mass spectrometry (ESI-MS) and tandem ESI-MS/MS experiments were conducted with a Micromass type Quattro LCZ (Beverly) quadrupole mass spectrometer. In MS/MS mode, fragmentation was achieved by introducing argon in the reaction chamber in front of the second quadrupole. The lipid samples were directly transferred from TLC plates with a mixture of chloroform and methanol (5:1, v/v; flow rate 100 µl min-1) into the ESI source using a TLC plate elution probe (DGMS 2001, P31, constructed by H. Luftmann, Institut für Organische Chemie, Westfälische Wilhelms-Universität, Münster, Germany).
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RESULTS |
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Analysis of intracellular metabolites accumulated by R. opacus during growth on phenyldecane
Cells of strain PD630 accumulated neutral lipids during cultivation on phenyldecane as sole carbon source under nitrogen-limiting conditions. These lipids were separated by TLC analysis and identified by comparision of their RF values with those of reference substances and by additional chemical analysis. After 4 d cultivation of the cells on phenyldecane, four spots of lipids were detected on the TLC plates (data not shown). The spot exhibiting an RF value of 0·13 contained the diacylglycerol fraction, as revealed by comparison with the respective RF value of a dipalmitoylglycerol standard. The occurrence of diacylglycerols in phenyldecane-grown cells of strain PD630 has been reported previously (Alvarez et al., 1996 ). In addition, detailed analysis of the spots occurring in TLC in a previous study confirmed the occurrence of diacylglycerols in cells of R. opacus PD630 (Wältermann et al., 2000
). The other compounds were identified by ESI-MS and ESI-MS/MS after separation on TLC plates. The spot exhibiting an RF value of 0·43 contained a mixture of TAGs with pseudomolecular ions between m/z [M+Na]+ 850 and 950. The mass spectrum obtained was very similar to that of TAGs accumulated by strain PD630 during cultivation on gluconate (Wältermann et al., 2000
). These TAGs contained odd- and even-numbered aliphatic fatty acids with carbon chain lengths ranging from 13 to 19 carbon atoms, palmitic acid, margaric acid, cis
9-heptadecenoic acid and oleic acid being major components (Alvarez et al., 1996
; Wältermann et al., 2000
). An ESI mass spectrum of the purified TLC fraction exhibiting an RF value 0·54 revealed pseudomolecular ions in the range, for m/z [M+Na]+, 800 to 880 (Fig. 1a
). The main ions represented a mixture of TAGs in which one acyl group was replaced by a phenyldecanoic acid residue. The occurrence of fatty acids with a phenyl group in the TAG of this TLC fraction was confirmed by ESI-MS/MS analysis. One ESI-MS/MS spectrum and the fragmentation pattern of the pseudomolecular ion with an m/z [M+Na]+ of 847 are presented in Fig. 1(b)
as an example. The spectrum showed the occurrence of odd-numbered fatty acids among the TAGs, since the mass differences between ionic groups frequently amounted to 14. Similar results were previously reported by Wältermann et al. (2000)
for TAGs accumulated by gluconate-grown cells of R. opacus PD630.
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DISCUSSION |
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However, phenyldecanoic acid is directed not only to catabolic pathways (- and ß-oxidation) in R. opacus PD630 but also towards anabolic routes, such as the biosynthesis of a novel wax ester and novel TAGs. In contrast, it is very interesting that (though it remains unclear why) wax esters composed of regular fatty acids and the corresponding alcohols were not synthesized when R. opacus PD630 was grown on gluconate under nitrogen starvation (Wältermann et al., 2000
). To our knowledge, this is the first report on the formation of waxes and TAGs containing aromatic constituents in bacteria. In this context, the accumulation of unusual polyhydroxyalkanoates bearing a phenyl group by Pseudomonas species has to be mentioned (Fritzsche et al., 1990
; García et al., 1999
). The synthesis of such polyhydroxyalkanoates required the occurrence of a polyhydroxyalkanoate synthase with broad substrate specificity. In contrast, R. opacus PD630 is unable to store 3-hydroxyalkanoic acids as polyhydroxyalkanoates but accumulates fatty acids as neutral lipids under the culture conditions in this study (Alvarez et al., 1996
).
Whether or not the formation of waxes and TAGs with aromatic constituents in R. opacus is physiologically relevant remains to be evaluated. As result of its broad metabolic capacity, unusual fatty acids such as phenyldecanoic acid may be generated from phenylalkanes, which may disturb the membrane fluidity if they are incorporated into phospholipids. Therefore, their incorporation into acylglycerols or wax esters may provide a defence strategy to regulate the fatty acid composition of the membrane phospholipids. Recently, Dahlqvist et al. (2000) identified an acyl-CoA-independent enzyme in plants, which is responsible for the biosynthesis of TAGs using phospholipids as acyl donor. This enzyme probably contributes only slightly to the accumulation of TAGs, but its role may be important for maintaining the functional fluidity of cellular membranes. If a similar enzyme occurs in strain PD630, it may be responsible for the biosynthesis of TAGs containing aromatic fatty acids. Assimilation of phenyldecane by strain PD630 was not complete under the cultivation conditions used in this study, since intermediates of phenyldecane oxidation were accumulated intracellularly, and others were excreted into the medium. These surplus intermediates may be further oxidized when an external carbon source becomes limiting.
In conclusion, this study provides a physiological and biochemical approach for investigating the catabolism and assimilation of phenyldecane by R. opacus strain PD630 under conditions of restricted growth. Such conditions normally predominate in natural environments. The understanding of the metabolic response of bacteria to the substances investigated in this study could be important not only for bioremediation processes but also for obtaining novel compounds, such as TAGs and wax esters containing aromatic compounds, by using biotechnological processes.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Alvarez, H. M., Kalscheuer, R. & Steinbüchel, A. (1997). Accumulation of storage lipids in species of Rhodococcus and Nocardia and effect of inhibitors and polyethylene glycol. Fett/Lipid 9, 239-246.
Alvarez, H. M., Kalscheuer, R. & Steinbüchel, A. (2000). Accumulation and mobilization of storage lipids by Rhodococcus opacus PD630 and Rhodococcus ruber NCIMB 40126. Appl Microbiol Biotechnol 54, 218-223.[Medline]
Alvarez, H. M., Souto, M. F., Viale, A. & Pucci, O. H. (2001). Biosynthesis of fatty acids and triacylglycerols by 2,6,10,14-tetramethyl pentadecane-grown cells of Nocardia globerula 432. FEMS Microbiol Lett 200, 195-200.[Medline]
Brandl, H., Gross, R. A., Lenz, R. W. & Fuller, R. C. (1988). Pseudomonas oleovorans as a source of poly(hydroxyalkanoates) for potential applications as biodegradable polyesters. Appl Environ Microbiol 54, 1977-1982.
Dahlqvist, A., Stähl, U., Lanman, M., Banas, A., Lee, M., Sandager, L., Ronne, H. & Stymne, S. (2000). Phosholipid:diacylglycerol acyltransferase: an enzyme that catalyzes the acyl-CoA-independent formation of triacylglycerol in yeast and plants. Proc Natl Acad Sci USA 12, 6487-6492.
Finnerty, W. R. (1992). The biology and genetics of the genus Rhodococcus. Annu Rev Microbiol 46, 193-218.[Medline]
Frederickson, J. K., Brockman, F. J., Workman, D. J., Li, S. W. & Stevens, T. O. (1991). Isolation and characterization of subsurface bacterium capable of growth on toluene, naphthalene, and other aromatic compounds. Appl Environ Microbiol 57, 796-803.
Fritzsche, K., Lenz, R. W. & Fuller, R. C. (1990). An unusual bacterial polyester with a phenyl pendant group. Macromol Chem 191, 1957-1965.
García, B., Olivera, E. R., Minabres, B., Fernándes-Valverde, M., Canedo, L. M., Prieto, M. A., García, J. L., Martínez, M. & Luengo, J. M. (1999). Novel biodegradable aromatic plastics from a bacterial source. J Biol Chem 274, 29228-29241.
Gibson, D. T. & Subramanian, V. (1984). Microbial degradation of aromatic hydrocarbons. In Microbial Degradation of Aromatic Hydrocarbons , pp. 181-242. Edited by D. T. Gibson. New York:Dekker.
Olivera, E. R., Minambres, B., García, B., Muniz, C., Morena, M. A., Ferrández, A., Díaz, E., García, J. L. & Luengo, J. M. (1998). Molecular characterization of the phenylacetic acid catabolic pathway in Pseudomonas putida U: the phenylacetyl-CoA catabolon. Proc Natl Acad Sci USA 95, 6419-6424.
Priefert, H., Rabenhorst, J. & Steinbüchel, A. (1997). Molecular characterization of genes of Pseudomonas sp. strain HR199 involved in bioconversion of vanillin to protocatechuate. J Bacteriol 179, 2595-2607.[Abstract]
Sariaslani, F. S., Harper, D. B. & Higgins, I. J. (1974). Microbial degradation of hydrocarbons. Catabolism of 1-phenylalkanes by Nocardia salmonicolor. Biochem J 140, 31-45.[Medline]
Schlegel, H. G., Kaltwasser, H. & Gottschalk, H. (1961). Ein Submersverfahren zur Kultur wasserstoffoxydierender Bakterien: Wachstumsphysiologische Untersuchungen. Arch Mikrobiol 38, 209-222.
Sikkema, J., De Bont, J. A. M. & Poolman, B. (1995). Mechanisms of membrane toxicity of hydrocarbons. Microbiol Rev 59, 201-222.
Wagner-Dobler, I., Bennasar, A., Vancanneyt, M., Strompl, C., Brummer, I., Eichner, C., Grammel, I. & Moore, E. R. (1998). Microcosm enrichment of biphenyl-degrading microbial communities from soils and sediments. Appl Environ Microbiol 64, 3014-3022.
Wältermann, M., Luftmann, H., Baumeister, D., Kalscheuer, R. & Steinbüchel, A. (2000). Rhodococcus opacus strain PD630 as a new source of high-value single cell oil? Isolation and characterization of triacylglycerols and other storage lipids. Microbiology 146, 1143-1149.
Warhust, A. M. & Fewson, C. A. (1994). Biotransformation catalyzed by the genus Rhodococcus. Crit Rev Biotechnol 14, 29-73.[Medline]
White, L. G., Hawari, J., Zhou, E., Bourbonnière, L., Innis, W. E. & Greer, H. W. (1998). Biodegradation of variable-chain-length alkanes at low temperatures by a psychotrophic Rhodococcus sp. Appl Environ Microbiol 64, 2578-2584.
Yakimov, M. M., Giuliano, L., Bruni, V., Scarfi, S. & Golyshin, P. N. (1999). Characterization of antarctic hydrocarbon-degrading bacteria capable of producing bioemulsifiers. New Microbiol 22, 249-256.[Medline]
Received 28 August 2001;
revised 11 December 2001;
accepted 11 January 2002.
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