aerobic and anaerobic metabolism of trimethylamine, dimethylamine

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    Journal of General Microbiology (1978), 106, 265-276. Printed in Great Britain 265

    Aerobic and Anaerobic Metabolism of Trimethylamine, Dimethylamine and Methylamine in Wyphomicrobium x

    IBy J. B. M. MEIBERG AND W. H A R D E R Department of Microbiology, Biological Centre, University of Groningen, Kerklaan 30,

    Haven (Gr.), The Netherlands

    (Received 22 December 1977)

    Hyphomicrobium strain x was grown on trimethylamine and dimethylamine as the sole sources of carbon and energy under both aerobic and anaerobic conditions and the enzymes involved in the metabolism of these compounds were investigated. During aerobic growth of the organism on trimethylamine, accumulation and subsequent utilization of dimethyl- amine was observed. When the organism was grown on trimethylamine under anaerobic conditions in the presence of nitrate, a sequential accumulation and utilization of dimethyl- amine and methylamine was found. In cell-free extracts of Hyphomicrobium x grown on trimethylamine or dimethylamine under both aerobic and anaerobic conditions the follow- ing enzyme activities were detected : trimethylamine dehydrogenase, dimethylamine dehydrogenase, y-glutamylmethylamide synthetase, N-methylglutamate dehydrogenase, methanol dehydrogenase, formaldehyde dehydrogenase, formate dehydrogenase and hydroxypyruvate reductase. Under neither growth condition were any of the following enzyme activities detected : trimethylamine mono-oxygenase, dimethylamine mono- oxygenase, trimethylamine-N-oxide aldolase (demethylase) and primary-amine dehydro- genase. Trimethylamine dehydrogenase and dimethylamine dehydrogenase were partially purified from bacteria grown on dimethylamine and the results suggest that in Hypho- microbium x a novel enzyme, namely dimethylamine dehydrogenase, participates in the oxidation of dimethylamine.

    I N T R O D U C T I O N

    Various micro-organisms can grow aerobically on trimethylamine, dimethylamine and methylamine as the sole source of carbon and energy, and the metabolism of these com- pounds has been studied extensively (Colby & Zatman, 1973; Anthony, 1975; Boulton & Large, 1977). The initial catabolism of trimethylamine in methylotrophic bacteria may proceed by one of two different pathways. The first route involves the direct demethylation to dimethylamine and formaldehyde, catalysed by trimethylamine dehydrogenase (Colby & Zatman, 1971), while in the other pathway the initial attack is an oxygenation of tri- methylamine to trimethylamine N-oxide mediated by trimethylamine mono-oxygenase (Large, Boulton & Crabbe, 1972). The N-oxide is subsequently demethylated by tri- methylamine N-oxide aldolase (demethylase) to dimethylamine and formaldehyde (Myers & Zatman, 1971; Large, 1971).

    The oxidation of dimethylamine to methylamine and formaldehyde in Pseudornonas aminovorans involves a secondary-amine mono-oxygenase (Eady & Large, 1969 ; Eady, Jarman & Large, 1971) and this enzyme was also reported to function in Bacterium 4 ~ 6 and some other organisms (Colby & Zatman, 1973). Until recently (Meiberg & Harder, 1976) no other enzyme involved in the oxidation of dimethylamine had been reported, except that

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    266 I. B. M. M E I B E R G AND W. H A R D E R the purified trimethylamine dehydrogenase from Bacterium 4 ~ 6 (Colby & Zatman, 1974) and a partially purified trimethylamine dehydrogenase from Hyphomicrobium vulgare NQ-521 (Large & McDougall, 1975) displayed some activity towards dimethylamine. Large & McDougall (1 975) also reported a dimethylamine dehydrogenase activity in an enzyme preparation obtained from H . vulgare NQ-521, but they attributed this activity to a con- taminating organism. The product of the oxidation of dimethylamine, namely methylamine, may be oxidized directly (Eady & Large, 1968, 1971) or via N-methylglutamate (Hersh, Peterson & Thompson, 1971 ; Loginova, Shishkina & Trotsenko, 1976; Bamforth & Large, 1977).

    We recently found that hyphomicrobia can be enriched and isolated by an anaerobic technique (Attwood & Harder, 1972) using trimethylamine or dimethylamine as the sub- strate and nitrate as terminal electron acceptor (J. B. M. Meiberg, unpublished results). In fact, pure cultures of several Hyphomicrobium isolates, previously obtained from enrich- ments with methanol or methylamine (Attwood & Harder, 1972), could grow on trimethylamine and dimethylamine, both aerobically and anaerobically with nitrate. Under the latter conditions it appeared to be highly improbable that dimethylamine mono-oxy- genase was involved in the catabolism of trimethylamine and dimethylamine and we therefore decided to investigate the metabolism of these compounds in one of the isolates available. Since the physiology and biochemistry of Hyphomicrobium x is well documented (Harder & Attwood, 1978), this organism was chosen for the present study which describes some aspects of its growth on trimethylamine and dimethylamine under both aerobic and anaerobic conditions and implicates a novel enzyme, namely dimethylamine dehydrogenase, in the oxidation of dimethylamine.

    A preliminary report of part of this work has been published (Meiberg & Harder, 1976).

    M E T H O D S

    Maintenance and growth of the organism. Hyphomicrobium strain x (Attwood & Harder, 1972) was grown in mineral salts medium containing (per litre of deionized water): (NHd2S04, 1.0 g; MgS04.7H20, 0.2 g ; NaH2PO4.H2O, 0.5 g; K2HP04, 1.55 g; and trace element solution (Vishniac & Santer, 1957), 0.2 ml. The pH of the medium was 7-2. After it had been heat-sterilized and cooled, filter-sterilized solutions of the various methylated amines were added to a final concentration of 10 m~ (aerobic experiments) or 20 mM (anaerobic experiments).

    The organism was maintained on the above mineral salts medium supplemented with 0.34% (w/v) methylammonium chloride and 1.5 % (w/v) Bacto-agar, and was transferred bimonthly; slopes were incubated at 30 "C for 7 d and then stored at 4 "C.

    Bacteria were grown in a 4 1 fermenter described by Harder, Visser & Kuenen (1974), filled with 3 1 of medium. During growth, the pH was maintained at 7.0 by periodic addition of sterile alkali and the culture was stirred and aerated at 30 1 h-l. The temperature was maintained at 30 "C. During anaerobic growth, KNOs was added in portions as indicated in the individual experiments. In these experiments air was replaced by nitrogen gas from which traces of oxygen had been removed by passing it through a solution of dihydroriboflavin (Strauss & Nickerson, 1961). For both aerobic and anaerobic experiments, a 200 ml culture of the organism, grown on the same substrate in 500 ml conical flasks on a shaker at 30 "C, was used as an inoculum.

    Growth was monitored by measuring the absorbance of the culture at 433 nm; one absorbance unit corresponded to 0.22 mg dry wt ml-l.

    For enzyme assays in crude cell-free extracts, bacteria from different growth phases, as indicated in the individual experiments, were harvested by centrifugation at 15000 g for 10 min at 4 "C, washed once with 50 mwpotassium phosphate buffer pH 7.0 and used immediately or stored at -20 "C. Trimethylamine and dimethylamine dehydrogenases were purified from bacteria harvested at the end of the active growth phase.

    Bacterium 5 ~ 2 was obtained from Dr L. J. Zatman, University of Reading. It was grown in the above mineral salts medium in conical flasks on a shaker at 30 "C with 0.3 % (w/v) trimethylamine as the carbon and energy source. Bacteria were harvested at the end of the exponential growth phase.

    Preparation of cell-free extracts. Bacteria (03 g wet wt) were suspended in 4 ml of 50 mwpotassium phosphate buffer pH 7.0 and 0.5 g of acid-washed glass beads (diam. 0.1 1 mm) was added to the suspension.

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    Metabolism of methylated amines 267 The bacteria were disrupted at 20 kHz in a 100 W ultrasonic disintegrator for 2.5 min at 0 "C. Whole cells, debris and glass beads were removed by centrifuging at 25000 g for 20 min. The resulting supernatant was used as the crude cell-free extract.

    Dry weight determination. Duplicate samples of 200 ml were removed from the culture, centrifuged, washed once with deionized water and dried to constant weight at 110 "C.

    Chemical estimations. Protein was measured by the method of Lowry et al. (1951), using bovine serum albumin as a standard.

    Methylatedamines were measured as follows: 10 ml samples were withdrawn from the cultures at appropri- ate intervals, and bacteria were removed by membrane filtration (pore size 0-2 pm). The concentration of the methylated amines in the filtrates was determined by gas chromatography using a Pye Unicam series 104 chromatograph with a heated dud flame ionization detector. A glass column (1.9 m x 2 mm i.d.) was used, packed with Carbopack B, coated with GP 4 % Carbowax 20 M plus 0.8 % KOH. The column oven tempera- ture was 120 "C, and the detector oven, 150 "C. Nitrogen was used as the carrier gas at a flow rate of 10 ml min-l. Before injection, 1.6 % (w/v) KOH was added to the samples to a final concentration of 0-8 % (w/v). Standards were prepared from mono-, di- and trimethylammonium chlorides dried with Mg(ClO,),.

    Nitrite was measured by a modification of the method of Egami & Taniguchi (1963) as follows: to 4 ml of appropriately diluted culture filtrate were added 0.5 ml of 1 % (w/v) sulphanilamide in 1.2


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