Institut für Mikrobiologie, Martin-Luther-Universität Halle, Kurt-Mothes-Str. 3, D-06099 Halle, Germany1
Author for correspondence: Jan R. Andreesen. Tel: +49 345 5526350. Fax: +49 345 5527010. e-mail: j.andreesen{at}mikrobiologie.uni-halle.de
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ABSTRACT |
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Keywords: morpholine, cyclic amines, degradation, Mycobacterium, cytochrome P450
The EMBL accession number of the 16S rRNA gene of Mycobacterium sp. strain HE5 is AJ012738.
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INTRODUCTION |
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Hypothetical pathways have been proposed for the microbial biodegradation of morpholine (Swain et al., 1991 ; Mazure & Truffaut, 1994
; Combourieu et al., 1998
). Depending on the organism studied, ethanolamine and glycolate were suggested to be key intermediates in the biodegradation of morpholine (Swain et al., 1991
; Combourieu et al., 1998
). Combourieu et al. (1998)
used 1H NMR spectroscopy for a direct detection of morpholine and they were able to identify 2-(2-aminoethoxy)acetate as an intermediate of morpholine degradation, demonstrating that the initial ring cleavage occurred at the CN bond. However, little is known about the enzymes catalysing these initial steps (Knapp et al., 1982
; Cech et al., 1988
; Knapp & Brown, 1988
; Brown & Knapp, 1990
; Swain et al., 1991
; Emtiazi & Knapp, 1994
; Mazure & Truffaut, 1994
). Recently, evidence was presented for a cytochrome P450 to be involved in the initial oxidation of morpholine by an environmental Mycobacterium strain (Poupin et al., 1998
).
In this paper, we describe the isolation and characterization of a Mycobacterium strain related to Mycobacterium gilvum showing significantly increased morpholine degradation rates compared to the morpholine-utilizing organisms described so far. Evidence is presented that a cytochrome P450 is involved in morpholine, piperidine and pyrrolidine degradation by this organism.
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METHODS |
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Enrichment, isolation and growth of organisms.
Aerobic morpholine-degrading organisms were isolated from non-contaminated forest soil samples. The inocula were suspended in flasks containing 10 ml mineral salts medium and incubated at 30 °C for 14 weeks. The medium consisted of 0·09 mM CaCl2, 0·06 mM MnSO4, 0·85 mM NaCl, 2·0 mM MgSO4, 100 mM potassium phosphate (pH 7·2), 5 ml vitamin solution (Genthner et al., 1981 ), 1 ml trace element solution (Hormann & Andreesen, 1989
) and 20 mM morpholine as carbon and nitrogen source. In experiments when no nitrogen was supplied with the carbon source tested, 5·6 mM NH4Cl was added to the medium. Serial dilutions of the obtained cultures were plated on mineral agar plates (containing 2% agar) and incubated at 30 °C for 1525 d. Subsequently, single colonies were transferred again into mineral salts medium. Pure cultures of morpholine-degrading bacteria were obtained by repeating this procedure twice. The fastest growing culture, designated strain HE5, was chosen for the investigations reported in this study and deposited at the DSMZ (Braunschweig, Germany) under the accession number DSM 44238. Mass cultures were grown in baffled flasks (25 l) shaken at 130 r.p.m. Growth was followed as the increase in OD600.
The substrate range of the isolated Mycobacterium sp. and the inhibition of growth on morpholine, pyrrolidine and piperidine by metyrapone were investigated by adding the corresponding compounds to mineral salts medium.
Identification of the isolated organism.
Morphological properties of the isolated organism were determined by phase-contrast microscopy. Gram staining, acid- and alcohol-fastness, catalase and oxidase activity and pigment production were investigated according to standard procedures. The G+C content of the isolated DNA was determined as described by Tamaoko & Komagata (1984) . Analysis of fatty acids was performed as reported by Häggblom et al. (1994)
. Genomic DNA from a single colony of strain HE5 served as a template for PCR. Universal primers fD1 and rD1 were used for amplification of the 16S rDNA (Weisburg et al., 1991
). The obtained PCR product was purified and ligated into the pGEM-T vector (Promega). After transformation into Escherichia coli XL2-Blue and subsequent isolation of the plasmid vector, sequencing was carried out by using an ALF sequencer (Pharmacia) and the Cycle sequencing kit (Pharmacia). Analysis of the obtained sequence was performed by the DNASIS program and the FASTA 3 program using the EPRO database.
Determination of substrate and intermediate concentrations.
During growth of the organism, the concentration of morpholine in the mineral salts medium was determined by a colorimetric assay using naphthoquinone sulfonic acid (Knapp et al., 1982 ). Alternatively, the concentration of morpholine and other growth substrates was estimated by GC analysis using a gas chromatograph 14-B (Shimadzu), a flame-ionization detector and capillary column BGB-1701 (SCP Seitz, length 30 m, inside diameter 0·25 mm, film thickness 0·25 µm). The secondary amines were derivatized with benzenesulfonyl chloride before analysis (Hamano et al., 1981
). The conditions set at the GC were: column temperature 200 °C, injector temperature 280 °C, detector temperature 240 °C, helium carrier gas at a flow rate of 40 cm s-1 and a split of 1:20. The concentration of ammonia was estimated by a coupled enzymic assay using glutamate dehydrogenase (Boehringer).
Preparation and separation of crude extracts.
After addition of 3 µl Benzonase (Benzonuclease) per 10 ml suspension and 10 µM PMSF, the washed cells were disrupted by passing them three times through a French press at 110 MPa. The cell debris and membranes were removed by centrifugation at 33000 g for 20 min and 120000 g for 1 h, respectively. The obtained supernatant, designated crude extract, was applied to a Q Sepharose column previously equilibrated with 50 mM potassium phosphate buffer, pH 8·5 (buffer A). Unbound protein was washed off with 3 vols buffer A and subsequently the bound protein was eluted by a linear gradient from 0 to 1 M NaCl in buffer A. Protein was determined according to Bradford (1976) using bovine serum albumin as a standard.
Spectral and polarographic measurements.
The carbon monoxide difference spectra were recorded with a Uvikon 930 spectrophotometer (Kontron, Eching) using crude extract previously reduced by the addition of dithionite (2 mM). Carbon monoxide was bubbled through the cuvette and reduced extracts without carbon monoxide were used as reference. The morpholine-dependent oxygen consumption was determined polarographically using a Clark electrode (Rank Brothers).
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RESULTS |
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Growth of Mycobacterium sp. strain HE5
During investigations of the influence of pH, temperature and morpholine concentration on growth of Mycobacterium sp. strain HE5, optimal growth was obtained at a substrate concentration of 30 mM at 30 °C and pH 7·2 (Fig. 1). Under these conditions, the maximal growth rate was determined at 0·17 h-1. The growth rate halved at a morpholine concentration of 45 mM and decreased to 0·041 or 0·023 h-1, respectively, if the morpholine concentration was increased to 60 or 100 mM (Fig. 1
). However, 70% of the initial morpholine was still degraded if the strain was grown at a substrate concentration of 100 mM (data not shown). No growth occurred if morpholine was omitted from the medium. Mycobacterium sp. strain HE5 degraded 90±5% of the initial morpholine (30 mM) during the exponential growth phase, whereas the remaining substrate was utilized during the stationary phase. The yield obtained under these conditions was 0·48 g cell dry weight per g substrate utilized. If the organism was grown in the presence of 60 mM morpholine the substrate concentration decreased by 40 mM within 48 h. During this time, the turbidity of the culture increased from 0·4 to about 6·0 (Fig. 2
). The morpholine-degradation rate determined under optimal growth conditions (30 mM morpholine, 30 °C, pH 7·2) was 0·84 mmol g-1 (dry wt) h-1. No loss of morpholine was observed under the same conditions in the absence of bacterial cells. Taking all these data together, it is obvious that Mycobacterium sp. strain HE5 has a much higher morpholine-degrading capacity than the morpholine-utilizing organisms described so far, Mycobacterium aurum MO1, Mycobacterium sp. MorG and Mycobacterium sp. RP1 (Knapp et al., 1982
; Mazure & Truffaut, 1994
; Poupin et al., 1998
).
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Mycobacterium sp. strain HE5 was able to grow on the heterocycles pyrrolidine and piperidine, whereas no growth occurred in the presence of the substituted morpholine derivatives N-formyl-, N-methyl-, N-ethylmorpholine, 2-morpholinoethanol or piperazine. As shown by the succinate-grown control used in Fig. 3, the ability to grow on pyrrolidine (Fig. 3
) was delayed, as a lag phase of about 7·5 h was observed. A similar delay of growth was obtained if succinate-grown cells were transferred to a piperidine- or morpholine-containing medium (data not shown). Interestingly, no lag phase was observed if cells grown on morpholine (Fig. 3
) or piperidine (data not shown) were transferred to a pyrrolidine-containing mineral salts medium, indicating that the pathway of pyrrolidine degradation was turned on by these substrates. However, a lag phase (9 h) was observed for growth of strain HE5 on morpholine or piperidine using cells pregrown on pyrrolidine (data not shown), and also growth on piperidine led to a delayed morpholine degradation, indicating a substrate-specific step.
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DISCUSSION |
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The morpholine-degradation rate determined for Mycobacterium sp. strain HE5 was about threefold higher than previously determined for M. aurum strain MO1 and strain MorG (Knapp et al., 1982 ; Cech et al., 1988
; Mazure & Truffaut, 1994
). A doubling time of 9 h was determined for the very recently isolated strain RP1, being about twofold slower than estimated for Mycobacterium sp. strain HE5. M. aurum MO1 degrades about 11 mM morpholine within 235 h and strain RP1 catabolizes 10 mM morpholine within 50 h (Combourieu et al., 1998
; Mazure & Truffaut, 1994
). Thus Mycobacterium HE5 utilizing about 1015 mM morpholine within 10 h has by far the highest potential for morpholine degradation and might therefore be useful in biotechnological applications.
The utilization of pyrrolidine and piperidine as growth substrates seems to be a common feature of nearly all morpholine-degrading mycobacteria isolated so far (Mazure & Truffaut, 1994 ; Poupin et al., 1998
). However, none of the morpholine-degrading organisms was able to grow on piperazine although it was reported in the case of strain MorG that morpholine induces an oxidizing activity of the non-growth substrate piperazine (Knapp et al., 1982
). Investigations performed with M. aurum MO1 indicated that morpholine did not induce the degradation of pyrrolidine and piperidine in this organism (Mazure & Truffaut, 1994
). Different results were obtained for strain MorG, where morpholine seems to induce the pathway for pyrrolidine degradation (Swain et al., 1991
). For Mycobacterium HE5 the degradation pathway of pyrrolidine seems to be induced during growth on morpholine and piperidine but not vice versa.
Depending on the organism investigated, different classes of enzymes were suggested to catalyse the initial oxidation during morpholine degradation (Swain et al., 1991 ; Poupin et al., 1998
). Carbon monoxide difference spectra of extracts from morpholine-, pyrrolidine- and piperidine-grown cells of Mycobacterium sp. RP1 indicated that the organism induces a cytochrome-P450-dependent monooxygenase during growth on these substrates (Poupin et al., 1998
). This monooxygenase was proposed to be responsible for the initial hydroxylation step in morpholine degradation, leading to the formation of 2-(2-aminoethoxy)acetaldehyde (Poupin et al., 1998
). However, a morpholine-dependent enzymic activity could not be detected in Mycobacterium RP1 or strain MorG. Very recently, Poupin et al. (1999)
identified a gene encoding a cytochrome P450 involved in piperidine and pyrrolidine utilization by Mycobacterium smegmatis mc2155. An involvement of a cytochrome P450 in the initial oxidation of the three cyclic amines was supported by the data that we obtained for Mycobacterium HE5. A cytochrome P450 and a ferredoxin were isolated from Mycobacterium HE5 after growth on morpholine (to be published separately). The cytochrome P450 could only be detected by carbon monoxide difference spectra, not by an enzymic activity. However, using this assay it should be possible to investigate whether individual cytochrome P450s are expressed during growth on each cyclic amine and thus resolve the question of whether the same or different cytochrome P450s are responsible for the conversion of these substrates.
After the initial hydroxylation of morpholine to 2-hydroxymorpholine, spontaneous ring opening should occur followed by oxidation of 2-(2-aminoethoxy)acetaldehyde to the corresponding acid. As the next step, the ether bond of 2-(2-aminoethoxy)acetate has to be cleaved. This might be achieved by a second monooxygenase reaction attacking one of the carbon atoms next to the ether bond (White et al., 1996 ). However, until now nothing has been known about the enzyme catalysing this scission. The involvement of two monooxygenase reactions in morpholine degradation would be in accordance with the lag phase observed during growth of Mycobacterium sp. strain HE5 on morpholine in the case where the cells were pregrown on piperidine or pyrrolidine. Both compounds should not require a second monooxygenase in their degradation pathway. The cleavage of the ether bond was supposed to result in the formation of glycolate in the case of strains MO1, MorG and RP1 (Swain et al., 1991
; Combourieu et al., 1998
; Poupin et al., 1998
). Glycolate will only be formed if the hydroxylation occurs at the amino side of the ether bond of 2-(2-aminoethoxy)acetate, and 2-aminoacetaldehyde will be the second product. The ability of morpholine-grown Mycobacterium HE5 cells to grow on glycolate without any lag phase might indicate that the organism employs a similar pathway in morpholine degradation. Glyoxylate and ethanolamine are proposed intermediates formed if the ether is hydroxylated at the carboxyl side. The formation of ethanolamine from the proposed intermediate 2-aminoacetaldehyde (Swain et al., 1991
; Poupin et al., 1998
) has not been shown so far. An alternative pathway might be the deamination of 2-(2-aminoethoxy)acetate leading after oxidation to the formation of the symmetric ether diglycolate (2,2'-oxy-diacetate), subsequently cleaved by a second monooxygenase to glycolate and glyoxylate.
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ACKNOWLEDGEMENTS |
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Received 22 July 1999;
revised 20 December 1999;
accepted 31 January 2000.