Isolation, Identification, and Characterization of a Lipoate-Degrading ...

JOURNAL OF BACTERIOLOGY, Dec 1972, p. 1043-1051 Copyright O 1972 American Society for Microbiology

Vol. 112, No. 3 Printed in U.S.A.

Isolation, Identification, and Characterization of a Lipoate-Degrading Pseudomonad and of a Lipoate Catabolite JASON C. H. SHIH, LEMUEL D. WRIGHT, AND DONALD B. McCORMICK

Graduate School of Nutrition and Section of Biochemistry and Molecular Biology, Cornell University, Ithaca, New York 14850 Received for publication 26 July 1972

A strain of bacteria that can degrade lipoic acid was isolated from soil. The bacterium, adapted to use 0.4% dl-lipoate as the sole organic substrate to supply carbon, sulfur, and energy, was identified morphologically and physiologically as a strain of Pseudomonas putida. Degradation of 1, 6- 14C-lipoic acid, synthesized from 1,6-1 4C-adipic acid, was evidenced by: (i) loss of approximately 50% of the total radioactivity from the medium after bacterial growth; (ii) appearance of 14C-degradation products upon paper and thin-layer chromatography of the culture medium; and (iii) oxygraphically measured utilization of 02 by cells in the presence of lipoate or other oxidizable substrates. Analyses of the benzene extract of culture medium by infrared, nuclear magnetic resonance, and mass spectrometry, and by gas-liquid chromatography after desulfuration, have characterized bisnorlipoic acid, or 4, 6-dithiohexanoic acid, as the major catabolite present in the medium. (-Oxidation of the side chain is thus proven to be a pathway employed by the pseudomonad to degrade lipoic acid. from Amersham/Searle Corp. The methylating reagent BF3/methanol (14%, w/v) was obtained from Applied Science Laboratories. Deuterated chloroform was from International Chemical and Nuclear Corp. Sodium hippurate and all media used in the identification of the bacterium were from Difco. Four authentic strains of Pseudomonas putida were obtained from the American Type Culture Collection; the strain numbers were 17484, 17390, 12633, and 4359. Preparation of lipoate media and culture conditions. Stock solutions of lipoate were prepared by suspending lipoic acid at a concentration of 2 g per 100 ml in water, adjusting the pH to 8.0 with KOH, and sterilizing by filtration. The sterile solutions were then stored in brown bottles at 5 C. The composition of the basal medium is shown in Table 1. To avoid precipitation of magnesium phosphate during autoclaving of the medium, magnesium chloride solutions were autoclaved separately and added aseptically to autoclaved media. Similarly, because of the MATERIALS AND METHODS relative instability of lipoic acid, lipoate from Materials. dl-Lipoic, caprylic (octanoic), caproic aseptic stock solutions was also added to the basal (hexanoic), and butyric (butanoic) acids were pur- medium after autoclaving. A 0.4% lipoate medium is chased from Sigma Chemical Co. Adipic acid, mono- now used routinely for the growth of the bacterium. methyl adipate, and dimethyl adipate were from In the early stages of isolation of the bacterium from Aldrich Chemical Co. Benzylamine and creatine soil, and in the process of adaptation (see Results), were from Eastman Kodak Co. Mannitol and trigothe media contained 0.2% lipoate and 0.05% glucose nelline were purchased from Nutritional Biochemi- (lipoate-glucose medium) or 0.2% lipoate and 0.05% cals Corp. The 1, 6- 14C-adipic acid was obtained potassium acetate (lipoate-acetate medium). 1043

Lipoic acid was first isolated in 1951 (17), and its structure was soon established as 6,8dithioctanoic acid (3). Lipoic acid is an essential coenzyme for all systems of a-keto acid dehydrogenase complexes that have been investigated. Although considerable information is available concerning the mechanism of lipoic acid action (14, 15), little is known concerning the metabolism of this compound (12, 19). In this laboratory, a strain of bacteria that grows on lipoate as the sole organic substrate to supply carbon, sulfur, and energy has been isolated from soil. In this paper, the isolation, adaptation, and identification of the bacterium will be described, and, in addition, evidence will be presented supporting d-oxidation as the initial step in the degradative pathway.

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SHIH, WRIGHT, AND McCORMICK TABLE 1. Composition of basal medium

Ingredient

Amt

NH4NO3 ......................... MgCl2 *6H20 ..................... CaCl 2 -2H2 O......... .... FeCl2 *4H20 ...................... Trace element solution .............. Molybdate solution ... .............. KH 2PO 4/K 2HPO,4 (pH 6.8.) ......... Distilled water to make up 1 liter ...

2.0 g 0.3 g 3.0 mg 3.0 mg 0.1 ml 0.1 ml 0.04 mole

Hoagland et al. (5). "Composed of 27.7 mg of Na2MoO4 2H20 in 1 liter of distilled water. a

Cells were cultured in 125-ml Erlenmeyer flasks containing 25 ml of medium. Growth was at 28 to 30 C with aeration by rotation of the flasks at 140 rev/min. To avoid photolysis and photoactivated polymerization of lipoate, the cultures were grown in the dark. The growth of the cells, indicated by turbidity, was measured by optical density at 650 nm with a Bausch & Lomb spectrophotometer (Spectronic 20). Optical density of the bacterium was found to be correlated linearly with the dry weight of bacterial cells. Authentic P. putida strains, which were obtained in the form of lyophilized powders, were first revived in nutrient broth and then transferred to lipoate medium to test for their ability to grow on lipoate. Isolation and identification of the bacterium. Soil samples were added to lipoate-glucose medium and incubated as described. Transfer was made from incubated medium to fresh medium every 5 days. After about 2 months of transferring and incubating, an increase in turbidity after each transfer indicated bacterial growth. Streaking onto lipoate-glucose agar plates was then done, and a pure culture was obtained from a single colony. The bacterium was characterized by morphology and a number of physiological tests. Chemical synthesis of 1,6- 4C-lipoic acid. 1,6'4C-dl-lipoic acid was synthesized from 1,6- 4C adipic acid after the method described by Pritchard et al. (13) as modified from the method of Acker and Wayne (1). The infrared spectrum of the synthesized product is identical to that of a commercial, nonradioactive sample, and the product is homogeneous on the basis of paper and thin-layer chromatography. Measurement of radioactivity. Radioactivity in the culture medium was determined on 0.2-ml samples, which were acidified with one drop of 1 N HCl, evaporated to dryness under reduced pressure at room temperature, redissolved in 0.2 ml of water and 10 ml of Bray's solution (2), and counted in a Packard liquid scintillation counter (model 3375). Radioactivity on paper chromatograms was determined with a Nuclear-Chicago Actigraph III. Preparation of benzene extracts of the culture medium. Whole cultures were centrifuged at 8,000 x g for 10 min to remove bacterial cells. The clear supernatant solution was acidified with 1 N HCl to pH 1 to 2 and then extracted in a separatory funnel

J. BACTERIOL.

twice with half-volume portions of benzene. After drying over anhydrous Na2SO4, the benzene extracts were evaporated to a small volume under reduced pressure at 30 to 40 C. The concentrated extracts were then subjected to different analyses (see Fig. 3). Analytical techniques. On paper chromatograms, lipoate and derivaties containing disulfide bonds were detected by the reddish color developed after spraying with a solution of 2% sodium nitroferricyanide and 5% sodium cyanide after the method of Reed and DeBusk (16). Thin-layer chromatograms on MN silica gel N-HR precoated plastic sheets, purchased from Brinkman Instrument Co., were developed in ascending chloroform-methanol-formic acid (8: 1: 1, v/v). Organic compounds were detected by the brown spots formed when the plates were exposed to iodine vapor in a closed chamber. Radioactivity was detected by counting, in a liquid scintillation counter, samples prepared by scraping silica gel areas into Bray's solution. Infrared spectra were obtained on NaCl plates in a Perkin-Elmer infrared spectrophotometer. Nuclear magnetic resonance spectra were obtained with a Varian A60A (60 MHz) NMR spectrometer. To prepare the sample, the benzene extract was evaporated to dryness and redissolved in a small volume of deuterated chloroform. Mass spectrum analyses were carried out by the Cornell High Resolution Mass Spectral Facility, Department of Chemistry, Cornell University. Desulfurization of lipoic acid and of the benzene extracts of the medium. Reductive desulfurization of lipoic acid was carried out by refiuxing, for 5 hr, 0.2 g of lipoic acid and 3.0 g of Raney nickel catalyst (11) in 15 ml of absolute ethanol and 1.2 ml of water. After the reaction, the nickel catalyst was separated from the reaction mixture by centrifugation and washed four times with 0.1 N NaOH. The reaction mixture and washings were combined and evaporated to dryness under reduced pressure; the residue was taken up in water. The aqueous solution was then acidified and extracted with two portions of ether. The ether extract, which was dried with anhydrous Na 2SO4, was evaporated under reduced pressure. The product, in a yield of 95%, gave an infrared spectrum identical to that obtained with a reference sample of octanoic acid. Similar reductive desulfurization procedures were carried out on samples of the benzene extracts of the culture media after various periods of bacterial growth. The desulfurized derivatives thus obtained were then characterized by gas-liquid chromatography. Gas-liquid chromatography. The products of the desulfuration of the benzene extracts obtained from cultures after various periods of incubation, as well as authentic reference samples of octanoic, hexanoic, and butanoic acids, were methylated with the BFJ/ methanol reagent after the method described by Metcalfe and Schmitz (10). The methyl esters, extracted in petroleum ether, were injected into the gas-liquid chromatograph for analysis on the basis of retention time. An F & M gas chromatograph (model 400) with flame ionization detector was used. The column was glass tubing, 4 ft (122 cm) in length and 3 mm in inside diameter, packed with silicone-XE-

VOL. 112, 1972

BACTERIAL DEGRADATION OF LIPOATE

60, 5% on Gas-Chrom Q (Applied Science Laboratories, Inc.). Because of the high volatility of the methyl esters of short-chain fatty acids, a low column temperature, as well as a low flow rate of carrier gas, was used. Operating conditions were as follows: column temperature, 90 C; injection point temperature, 140 C; detector block temperature, 160 C; carrier gas (N2) flow rate, 20 ml per min; hydrogen flow rate, 25 ml per min; air flow rate, 100 ml per min.

Substrate oxidation by intact cells. Bacterial cells grown in 1 liter of 0.2% lipoate medium for 3 days were collected and washed three times with basal medium. The washed cells were suspended in 50 ml of basal medium and aerated by vigorous shaking at 30 C for 1 hr to oxidize the endogenous oxidizable substrates. After refrigeration overnight, cells were transferred to fresh basal medium and aerated for another hour at 30 C. The cells that had been repeatedly aerated were then centrifuged. About 0.5 g of packed cells resulted. The cells were then resuspended in 40 ml of basal medium and were used for the substrate oxidation experiments. The polarographic "oxygen electrode" technique (4) was used to measure the utilization of dissolved oxygen by the action of the bacterial cells on different substrates. A Gilson Medical Electronics, Inc., oxygraph was used to carry out the experiment. First, 0.5 ml of cell suspension was incubated with 1.0 ml of basal medium at 30 C; then 10 pliters of concentrated substrate solution was added to start the reaction, and the rate of oxygen utilization was recorded automatically. The substrates that have been tested are glucose, a-glycerol phosphate, succinate, acetate, butyrate, octanoate, and lipoate.

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first isolated from, and grown on, lipoate-glucose medium were transferred at frequent intervals in the lipoate-glucose medium, except that the level of glucose was gradually reduced. Viability could not be maintained under these conditions. On the other hand, the same process was also carried out with the isolated bacterium, except that potassium acetate, instead of glucose, was used as a supplement to the lipoate medium. Through the process of continuous transfer and culture in the media with gradually decreasing acetate, the bacterium finally grew on repeated transfer in the lipoate that was free from acetate. The growth of the bacterium in the 0.4% '4C-lipoate medium, along with the loss of radioactivity from (0.2%)-Glucose (0.05 %) 060pLipoote As O. D.

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RESULTS

Isolation and adaptation of the bacterium to lipoate medium. A pure culture of the lipoate-degrading organism as first isolated by use of the lipoate (0.2%)-glucose (0.05%) medium. A growth curve and evidence for the degradation of lipoic acid by loss of '4C from labeled lipoate when the bacterium was grown on the lipoate-glucose medium are given in Fig. 1. Radioactivity disappeared from the culture concomitant with the growth of the bacterium. At the stationary phase of growth, approximately 50% of the radioactivity, originally present as 1,6- '4C-lipoate, had disappeared from the medium. After cessation of growth, the addition of either 0.025% glucose or 0.1% "4C-lipoate to the medium permitted the resumption of growth. A greater stimulation of growth resulted with glucose than with lipoate. With the addition of lipoate, again approximately 50% of that added as 1,6- '4C-lipoate disappeared from the medium. Attention was then turned to adapting the bacterium to growing on lipoate as the sole organic substrate. As shown in Table 2, cells

0.04 0

2

3

4

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DAYS FIG. 1. Growth of the bacterium and loss of ra-

dioactivity from the 0.2% 1,6- '4C-lipoate-0.05% glumedium, and the effect of further additions of glucose (0.025%) or 1,6-'4C-lipoate (0.1%) during the stationary phase of the growth cycle. cose

TABLE 2. Adaptation of the pseudomonad to the glucose- or acetate-free lipoate medium Growth

Glucose or acetate ( %)

Lipoate-glucose

0.10

+++

0.05 0.04 0.03 0.02 0.01 0

++ ++

+±+ + 4

_

Lipoate-acetate +++ +++ ++

++ ++ +

aThe cell culture, first grown on lipoate (0.2%)glucose (0.05%) or lipoate (0.2%)-acetate (0.05%), was subsequently transferred to a corresponding medium which had decreased glucose or acetate.

1046

SHIH, WRIGHT, AND McCORMICK

the 1,6- 14C-lipoate, is described by the data of Fig. 2. Even though the inoculum used was from an actively growing culture, a more-orless lag phase was always observed prior to the log phase of growth. In the log phase of growth, the cell-doubling time was about 6 hr. The loss of '4C from the medium again reached a plateau of about 50%. The optimal temperature for growth is in the range of 25 to 30 C. No growth of the bacterium was observed at temperatures either higher than 40 C or lower than 7 C. Optimal pH values ranged from 6.5 to 8.0 with little difference in the rate or amount of growth within this range. A pH of 6.8 was selected for routine studies. None of the four authentic strains of P. putida grew on 0.4% lipoate medium after incubation for 1 week. Strain 17390 was selected to try for adaptation through decreasing acetate from lipoate-acetate medium as mentioned above. Unlike the isolated bacterium, its viability could not be maintained when acetate was totally absent. Characterization and identification of the bacterium. The bacteria are short, motile rods bearing more than one flagellum at one end. The presence of flagella was confirmed by examination under an electron microscope, by use of the method of negative stain. No spores were formed, and the bacterium was gram negative. After the process of Gram staining, cells from the lipoate medium were spheroidal and separate, whereas the cells from the nutrient broth were rods, with some in filamentous arrangement. When streaked on nutrient agar plates, growth was abundant, spreading, opaque, and viscid. Colonies were circular, 0.60 Lipoate (0.4%)

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