Institut für Mikrobiologie der Westfälischen Wilhelms Universität1 and Institut für Organische Chemie der Westfälischen Wilhelms Universität2, Münster, Germany
Author for correspondence: Alexander Steinbüchel. Tel: +49 251 833 9821. Fax: +49 251 833 8388. e-mail: steinbu{at}uni-muenster.de
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ABSTRACT |
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Keywords: Rhodococcus, triacylglycerols, storage lipids
Abbreviations: ELSD, evaporative light-scattering detector; MALDI-TOF, matrix-associated laser desorption ionizationtime of flight mass spectrometry; PHA, polyhydroxyalkanoic acid; TAG, triacylglycerol
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INTRODUCTION |
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Recently, aerobic, Gram-positive bacteria belonging to the genus Rhodococcus have attracted increased interest in the biochemical and genetical characterization of their metabolic capabilities, based on their ability to transform or degrade diverse classes of mainly hydrophobic substances (Finnerty, 1992 ; Warhust & Fewson, 1994
). Rhodococcus spp. are also able to synthesize and accumulate PHAs after cultivation on different carbon sources under nitrogen-limiting conditions. The accumulation of a copolymer consisting of 3-hydroxybutyrate and 3-hydroxyvalerate from unrelated carbon sources was reported for Rhodococcus ruber (Haywood et al., 1991
). Alvarez et al. (1996
, 1997
) reported on an oleaginous hydrocarbon-degrading R. opacus strain, PD630, which is able to accumulate up to 76% of acylglycerols when grown on different carbohydrate and non-carbohydrate carbon sources under nitrogen-limiting conditions but is unable to synthesize PHAs.
The present work is concerned with the analysis of the composition and stereospecific structures of storage acylglycerols from R. opacus PD630, which have not been studied in detail previously. Stereospecific analysis of TAGs has been performed for many years to discriminate between fatty acids at the sn-1, -3 and -2 positions of TAG (Brockerhoff, 1965 ). In addition, we subjected R. opacus PD630 to chemical mutagenesis and screened for mutants defective in the accumulation of storage lipids.
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METHODS |
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Lipid extraction and TLC.
Crude oil of R. opacus PD630 was obtained from lyophilized cell material (15 g), which was stirred twice each with 500 ml chloroform/methanol (2:1, v/v) at room temperature for 3 h. The solvents were evaporated from the combined extracts, and the residual material, 5·2 g, was dissolved in light petroleum (b.p. 3040 °C) and applied to a column packed with 20 g silica gel 60 (mesh 50200 µm, Merck) equilibrated with light petroleum. The column was eluted with 500 ml light petroleum; the total eluate was collected and evaporated to dryness to obtain 4·4 g of a yellowish crude oil fraction, which consisted of 90% fatty acids in total.
To determine the identity of the lipids, TLC was carried out on silica gel 60 F254 plates (0·2 mm, Merck). For this, samples of lyophilized cell material were extracted with chloroform/methanol (2:1, v/v), and light petroleum (b.p. 3040 °C)/diethyl ether/acetic acid (70:30:1) was used as solvent. In some experiments, a double solvent system, benzene/acetic acid (85:15, v/v) followed by light petroleum (b.p. 3040 °C) applied in the same direction, was used. Preparative TLC was carried out on silica gel plates of 2 mm thickness (Merck). Plates impregnated with methanolic silver nitrate or boric acid (each 5%, w/v) were also used. Lipid fractions were visualized by heating the plates over a Bunsen flame after spraying with 40% sulfuric acid. Components on preparative plates were visualized after brief exposure to iodine vapour. Quinone compounds were detected on silica gel TLC plates by specific staining with a leukomethylene blue spray reagent (Krebs et al., 1967 ). Quinones appeared immediately as dark blue spots on a faint blue background. Osmium tetroxide vapour was used to visualize unsaturated compounds. 2-Monopalmitoylglycerol, tripalmitoylglycerol, 1,2-dipalmitoylglycerol, 1,3-dipalmitoylglycerol, cetylpalmitate, 1,2-dipalmitoyl-3-myristoyl-rac-glycerol, hexadecanol, tetradecene and oleic acid obtained from Sigma were used as reference substances.
Analysis of fatty acids.
To determine the fatty acid content of the cells and the composition of the lipids, these compounds were treated to yield fatty acid methyl esters (Brandl et al., 1988 ; Timm et al., 1990
), which were analysed by GC with a Perkin Elmer model 8420 gas chromatograph equipped with a Permaphase PEG 25Mx capillary column (25 m x 0·32 mm) and a flame ionization detector. A 2 µl portion of the organic phase was analysed after split injection (split ratio, 1:40); helium (32 ml min1) was used as carrier gas. The temperatures of the injector and detector were 230 °C and 275 °C, respectively, and a temperature programme was used for the separation of methyl esters (120 °C for 5 min; temperature increase of 8 °C min-1; 180 °C for 37 min) on the column. The fatty acids were identified by comparison of their retention times with those of standard fatty acid methyl esters.
Reversed-phase HPLC.
Reversed-phase HPLC was performed on an RP-18 Merck LiChroSphere 100 column (5 µm, 250 x 4·6 mm i.d.) using a Kontron HPLC apparatus equipped with a series 522 chromatographic pump. Elution of the compounds was monitored at 210 nm using a Kontron DAD 540 diode array detector (DAD) or an Alltech ELSD 500 evaporative light-scattering detector (ELSD) connected to a KromaSystem 2000 integrator system. The ELSD temperature was set to 55 °C. Acetonitrile/acetone (38:62, v/v) at a flow rate of 1 ml min-1 was used as solvent system. To characterize TAG mixtures further, a partition number (PN) variable was used: PN=CN-2ND, where CN and ND represent the total carbon number and the total number of double bonds in the fatty acids in the TAG molecule, respectively (Ruiz-Gutiérrez & Barron, 1995 ).
Matrix-associated laser desorption ionizationtime of flight mass spectrometry (MALDI-TOF).
MALDI-TOF was carried out using a LAZARUS III DE time of flight mass spectrometer (constructed by H. Luftmann, Institut für Organische Chemie, Münster, Germany) operated at 19 kV with delayed extraction and a path length of 2 m. A nitrogen laser was used to generate the primary beam, at 337 nm, with 3 ns pulse width. Samples were applied to the stainless steel target as 10-4 to 10-5 solutions (1 µl) mixed with an equal volume of a 0·1 M solution of 2,5-dihydroxybenzoic acid. The applied drop was allowed to dry and to crystallize before the sample was introduced into the mass spectrometer ion source.
Stereospecific analysis of TAGs.
The procedure for the stereospecific analysis of TAGs is reported in the literature (Brockerhoff, 1965 ; Yurkowski & Brockerhoff, 1966
; Christie & Moore, 1968
) and was applied to R. opacus PD630 crude oil as follows.
(i) Enzymic hydrolysis of TAGs was done with porcine pancreatic lipase (glycerol-ester hydrolase, EC 3 . 1 . 1 . 3; Merck) to obtain sn-2-monoacylglycerols.
(ii) Partial deacylation of TAGs was done by chemical hydrolysis with ethyl magnesium bromide (Alfa Aesar).
(iii) Separation of enantiomeric sn-1,2(2,3)-diacylglycerols was achieved by TLC on boric-acid-impregnated silica gel plates, followed by treatment with phosphatidyl phenol (Sigma) to obtain synthetic L- and D-phosphatidyl phenols, which were subsequently hydrolysed by stereospecific phospholipase A2 (EC 3 . 1 . 1 . 4) from rattlesnake venom (Crotalus atrox) to obtain the lysophosphatidyl phenols (Brockerhoff, 1965 ).
(iv) GC analysis of the fatty acid composition of TAGs, sn-2-monoacylglycerols and the lysophosphatidyl phenols was done as described above.
Mutagenesis.
To obtain mutants deficient in TAG accumulation, R. opacus PD630 was subjected to chemical mutagenesis using N-methyl-N'-nitro-N-nitrosoguanidine (NMG). Cells were incubated with 20 µg NMG ml-1 for 30 min (50% lethal rate). For expression of the mutations, the mutagenized cells were washed and resuspended in nitrogen-limited MSM containing 1% (w/v) sodium gluconate. Enrichment of mutants was performed by Percoll density-gradient ultracentrifugation (Guerrero et al., 1984 ). Since the buoyant density of cells of TAG-deficient mutants was higher than that of the wild-type cells, two distinct bands were obtained. The band representing the mutant cells was withdrawn from the gradient, plated on nitrogen-limited MSM agar plates containing 1% (w/v) sodium gluconate and identified by staining with ethanolic Sudan Black B and subsequent destaining with ethanol (Schlegel et al., 1970
).
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RESULTS |
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Reversed-phase HPLC
The reversed-phase HPLC analysis of purified TAG was carried out using UV detection at 210 nm and an ELSD to detect and distinguish between saturated and unsaturated TAGs. The chromatograms showed approximately 20 individual peaks with estimated PN values between 39 and 53; UV and ELSD detection gave similar peak patterns (Figs 3a and 3b
, respectively). Signals corresponding to PN values between 49 and 51 represented the most abundant peaks.
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Acylglycerol accumulation in mutants of R. opacus PD630
R. opacus PD630 was subjected to chemical mutagenesis using NMG and screened for mutants defective in the accumulation of storage lipids by staining colonies with Sudan Black B. By this method we obtained more than 100 mutants (referred to as ROM, for Rhodococcus opacus mutants) which all exhibited a significantly lower content of TAGs and 1,2-diacylglycerols in comparison with the wild-type. They all also lacked detectable 1,3-diacylglycerols. In contrast, the amounts of monoacylglycerols were slightly higher as revealed by TLC analysis (see ROM70 in Fig. 1 as an example). A recorded MALDI-TOF spectrum with crude lipid extracts of mutant ROM34 did not indicate accumulation of any TAGs (Fig. 5
). To identify the structure of the compound found at the same RF value as TAGs in the TLC analysis, it was purified by preparative TLC, and the concentrated sample was analysed by MALDI-TOF. This analysis showed that it consisted of a similar mixture of TAGs to the wild-type. For further characterization of the isolated mutants, the spectra of the fatty acids which were incorporated into the various accumulated lipids were also analysed by GC after extraction and methanolysis. All mutants possessed a similar GC profile (data not shown). The amounts of the accumulated fatty acids were drastically reduced in all mutants, to less than 1% as compared to the wild-type. Therefore, the occurrence of monoacylglycerols in the mutants did not compensate for the lower TAG content. It is therefore obvious that all mutants were still able to synthesize and accumulate trace amounts of TAGs.
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DISCUSSION |
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Stereospecific analysis of R. opacus PD630 TAGs to determine the distribution of fatty acids linked to either position of the glycerol backbone showed that fatty acids in the TAGs of R. opacus PD630 are not randomly distributed between the three possible positions. In contrast to common plant fats, where the longer and higher unsaturated fatty acids are found in position sn-2, R. opacus PD630 accumulated only the shorter and saturated fatty acids in this position. This indicates that the enzymes involved in TAG biosynthesis in R. opacus PD630 are highly specific and exhibit a substrate range different from the corresponding enzymes in plants.
In addition, we found that R. opacus PD630 synthesizes oleic acid (18:19,cis), whereas vaccenic acid (18:1
11,cis) was not detected. This provides evidence that R. opacus PD630 possesses the aerobic mechanism of fatty acid desaturation (Bloomfield & Bloch, 1960
; Fulco et al., 1964
).
All mutants defective in TAG accumulation obtained in this study still accumulated very small amounts of TAG, which were less than approximately 1% of the wild-type. The results support the two following explanations. The remaining amounts of TAG could be explained by the existence of two different pathways for the biosynthesis of TAGs in R. opacus PD630. One pathway could be only slightly active and contribute only very small amounts of TAG in both the wild-type and the mutants. The second, major, pathway could be responsible for the accumulation of the major fraction of TAGs. If only one pathway existed, completely TAG-negative mutants should have occurred among the more than 100 mutants that were isolated and screened in this study. It is implied that all the mutants isolated were most likely defective in the major pathway; mutants impaired in the minor pathway were probably not detectable by the screening method applied, using the lipophilic dye Sudan Black B, due to the putatively only faint differences in the amounts of TAGs between the wild-type and this type of mutant that were expected. On the other hand, however, it is not excluded that all the mutants isolated were substantially impaired in the regulation of TAG production. This latter possibility could also lower the rate of TAG biosynthesis and give a similar mutant phenotype.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 17 November 1999;
revised 1 February 2000;
accepted 2 February 2000.