FEMS Microbiology Letters 28 (1985) 265-269 Published by Elsevier

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FEM 02140

Glycogen in Methanolobus and Methanococcus

(Methanolobus; Methanococcus; glycogen; nitrogen fixation; methane; metabolism)

Helmut K6nig, Elisabeth Nusser and Karl O. Stetter

Lehrstuhl far Mikrobiologie der Universitilt Regensburg, UniversitStsstrasse 31, 8400 Regensburg F.R.G.

Received 1 April 1985 Revision received 15 April 1985

Accepted 17 April 1985

1. SUMMARY

Glycogen was isolated from Methanolobus ttndarius and 4 species of Methanococcus. It was identified by the iodine reaction, determination of glucose, maltose and isomaltose in acid hydro- lysates and partial acid hydrolysates, respectively, determination of erythritol and glycerol after per- iodate oxidation and treatment with amyloglu- cosidase. Glycogen particles in the ceils are distrib- uted throughout the cytoplasm. MI. tindarius can use elemental nitrogen and cysteine as nitrogen source. The glycogen and polyphosphate content of cells of MI. tindarius increased under nitrogen limitation. During incubation without external en- ergy source, glycogen decreased and methane for- marion was correlated to the glycogen content of the cells.

2. INTRODUCTION

Glycogen is a common reserve material among eukaryotes and eubacteria [1-9] and it was also found in thermoacidophilic archaebacteria [10].

The occurrence of glycogen in the second branch of archaebacteria represented by the methanogenic and extremely halophilic bacteria has been demon- strated for Methanosarcina barkeri [11]. This paper

gives a characterization of glycogen from 2 other genera of methanogenic bacteria, Methanolobus and Methanococcus.

3. MATERIAL AND METHODS

3.1. Organisms and growth conditions MI. tindarius was grown in modified medium 3

of Balch et al. [12,13]. In nitrogen-limited media the NH4C1 concentration was decreased 100-fold (2.5 mg/1) and the serum bottles were pressurized with H~/CO 2 (80:20; 300 kPa). To test the abil- ity for nitrogen fixation NH4C1 and cysteine (re- ducing agent) were omitted and the serum bottles were gassed with N2/CO 2 (80 : 20; 300 kPa).

Methanococcus vannielii [12], Methanococcus voltae [12], Methanococcus thermolithotrophicus [14] and Methanococcusjann~chii [15] were grown as described. MI. tindarius DSM2278 and Mc. thermolithotrophicus DSM2095 were isolates from our institute, while Mc. vannielii DSM1224, Mc. voltae DSM1537 and Mc. jannaschii DSM2095 were obtained from the Deutsche Sammltmg von Mikroorganismen, GSttingen.

3.2. Electron microscopy Electron micrographs of thin sections were taken

as described [13]. The glycogen staining of thin

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sections was performed according to Lewis and Knight [16].

3.3. Chromatograph), Chromatography of sugars and corresponding

derivatives was carried out as described [10].

3.4. Analytical methods Sugars were identified by gas-liquid chromatog-

raphy according to Albersheim et al. [17]. Quanti- tative determination was performed enzymatically [18]. Methods for periodate oxidation, amyloglu- cosidase treatment, conditions for hydrolysis, polyphosphate determination and standard com- pounds were the same as described [10,13]. L-Cy- steine hydrochloride monohydrate pro analysi containing < 0.05% ammonia was purchased from Fluka.

3.5. Isolation of glycogen In the course of S-layer isolation glycogen was

always found to be the contaminating material. Therefore, the following procedure was used: cells were suspended in the basal salt solution of the culture medium and disrupted by sonification (15 s, 60 W; Branson Sonic Power Company), fol- lov~ed by incubation with DNase I (1 mg/1; Serva) and RNase A (1 mg/1; Boehringer) for 15 rain at 37°C. The suspension was spun down at 15000 rev./min for 10 min (Sorvall RC2-B, SS34 rotor, crude S-layer). The pellet was dissolved in 0.1 M Tris-HC1 buffer pH 7.5 containing 0.3% N,N-di- methyldodecylamine-N-oxide (Fluka) and solid CsC1 (0.92 g/ml; p = 1.6) was added. The centri- fugation was carried out at 40000 rev./min for 16-40 h (75 Ti rotor, 17°C, Beckman L5-65). After centrifugation the solution was removed with a Densi-Flow II (Buchler Instruments) and 25 fractions per tube were collected (Ultrorac 7000; LKB). The fractions were tested for protein [19] and carbohydrate [20] content.

4. RESULTS AND DISCUSSION

4.1. Isolation and identification of glycogen When S-layers from Methanolobus and

Methanococcus were prepared and analyzed for carbohydrate, glucose was always found in larger

amounts in the hydrolysate (Table 1). The glucose-containing polymer could be separated from the protein by CsC1 density gradient centri- fugation. It bands at a density of about 1.6 g /cm 3. With iodine the polymer showed a reddish brown colour. Maltose and isomaltose could be detected in partial acid hydrolysates by thin-layer chro- matography. The polymer could be hydrolyzed with amyloglucosidase (Table 1). Erythritol and glycerol were found after Smith degradation by gas-liquid chromatography. In Mc. thermolitho- trophicus glycerol and erythritol were found in a molar ratio of 1:8 indicating an average chain length of 9. This finding indicated that the isolated polymer should be glycogen. Glycogen is a com- mon reserve material among the archaebacteria and has been found in methanogenic bacteria [11] and in many genera of the thermoacidophiles [10]. Only in the extremely halophilic archaebacteria it could not be found so far [10].

4.2. Localization of glycogen in the cells Thin sections of MI. tindarius specifically

stained for glycogen [16] showed that glycogen is distributed in intact cells within the cytoplasm (Fig. la, b), and is not predominantly located near the cytoplasmic membrane. However, in lysed cells

Table 1

Glycogen content of the isolated S-layers

Glycogen was determined as glucose after acid hydrolysis (2 N HC1, 2 h, 100°C) of extracted S-layers. The extracted S-layers w e r e obtained from crude S-layers by Triton X-100 t r e a t m e n t

(0.5%, v/v; 60°C, 30 rain).

Species Glycogen content in % a

Mc. thermolithotrophicus 13.3 (14.3) b

Mc. jannaschii 5.7 (6.8) b

Mc. voltae 1.1 (0.7) b

Mc. vannielii 0.9 (1.1) b

MI. tindarius 2.9 (3.3) b

a Based on protein (Methanococcus), based on dry weight ( Methanolobus ).

b Determination after amyloglucosidase t r e a t m e n t .

Fig. 1. Electron micrographs of thin sections stained for glycogen. (a) Methanolobus tmdarius grown under nitrogen limitation (2.5 mg NH4C1/1); (b) Methanolobus tindarius grown under normal conditions (250 mg NH4C1/1); (c) Methanolobus tindarius grown under nitrogen limitation, but not specially stained for glycogen [13]; (d) lysed cells of Methanococcus vannielii grown under less restricted conditions of nitrogen limitation (25 mg ]',IH4C1/1 ).

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of Mc. vannielli glycogen is mainly attached to the envelope (Fig. ld).

The round holes visible in thin sections of Ml. tindarius (Fig. 1, a-c), which were found to con- tain polyphosphate granules [13], increased strongly in size under nitrogen-limiting conditions (2.5 mg NH4C1/1 instead of 250 mg NH4CI/1 ). The chemical analysis revealed a polyphosphate content of 16.9/~mol/mg protein in the nitrogen- limited culture and 0.56 #mol/mg protein in the normally grown cells. Therefore, nitrogen limi- tation leads to an increase of the energy reservoir in Ml. tindarius.

4.3. The nitrogen sources used by Methanolobus tindarius

During the growth tests under nitrogen-limiting conditions it was also found, that MI. tindarius could grow in the absence of NH4C1, when N2/CO 2 was used as gas phase. No growth was obtained, when Nz/CO: was replaced by H J C O 2 (Table 2). Cysteine could also be used as nitrogen source (Table 2). However, growth on cysteine was

Table 2

Growth of Methanolobus on different nitrogen sources.

The following concentrations of the constituents were used: • methanol (0.5%; v/v), cysteine (500 rag/l), NH4C1 (250 mg/1), H 2 / C O 2 (80:20%; 300 kPa), N2/CO2 (80:20%; 300 kPa), trimethylamine (0.5%; v/v).

Growth conditions Absorbance (578 nm)

Oh 74h 91h 115h

(a) Cysteine, methanol, H2//CO2 0.08 0.22 0.35 0.35 (b) N 2, NH4CI

(a) Methanol, H2 /CO 2 0.13 0.07 0.07 0.09 (b) N 2, NH4C1, Cysteine

(a) Methanol, N2//CO2 0.12 0.35 0.65 0.80 (b) NH4CI, Cysteine

(a) Methanol, NH4C1, H2//CO2 0.06 0.24 0.51 1.10 (b) N2, Cysteine

(a) Cysteine, H 2 / C O 2 0.03 0.03 0.02 0.02 (b) Methanol, N 2, NH4Cl

(a) Trimethylamine, H 2/CO2 (b) Methanol, N 2, Cysteine, NH4Cl OAf n.d. 0.70 0.90

(a) present; (b) omitted; n.d., not determined.

only obtained in the presence of methanol. There- fore, it could not be used as energy source. Whether or not cysteine could serve as carbon source was not tested. In addition, MI. tindarius can use am- monia and like Methanosarcina [21] methylamines (Table 2; [13]) as nitrogen source.

In archaebacteria the fixation of elemental nitrogen has recently been demonstrated in Mc. thermolithotrophicus [22] and Methanosarcina barkeri [11]. Our results indicate that another ob- ligate heterotrophic methanogen, MI. tindarius, is able to fix dinitrogen.

4.4. Glycogen production and degradation in Methanolobus tindarius

When Ml. tindarius was grown under nitrogen limiting conditions (2.5 mg NH4C1/1 ) also the glycogen content of the cells increased (Table 3, Fig. la, b). Cells subsequently centrifuged and sus- pended in medium without methanol as energy and carbon source showed a decrease of the glyco- gen content after an incubation time of 24 h. During this incubation period bacteria grown un- der nitrogen limitation produced about twice as much methane as those grown under normal con- ditions (Table 3). This indicates that methane can be formed by Ml. tindarius without exogenous substrate and that this methane production is cor-

Table 3

Production and degradation of glycogen in Methanolobus

Cells were grown under normal NH4C1 concentrations (250 mg/1; 13) and under nitrogen limitation (2.5 mg/l). Then the cells were anaerobically spun down and suspended in culture medium [13] lacking methanol as energy and carbon source and incubated for 24 h. Protein and glucose were determined before (a) and after incubation (b) and methane production only after incubation (b).

Growth conditions Content of Methane

Protein a Glucose b production c

250 mg NH4C1/I(a) 1.7 8.0 n.d. (b) 1.8 3.4 3.9

2.5 mg NHnCI/I(a) 1.9 15.2 n.d. (b) 2.0 8.8 9.3

a mg/15 ml culture. b ~mo1/15 ml culture; the glycogen content was determined as

glucose after acid hydrolysis 2 N HC1, 2 h, 100°C. c /~mol/15 ml culture.

re la ted to the g lycogen conten t of the cells. Possi- bly, me thane can be formed by degrada t ion of glycogen. However , only abou t 0 .8-1.5 /Lmol me thane pe r mol glucose were fo rmed ind ica t ing tha t abou t one ca rbon of glucose would be con- ver ted to me thane under this assumpt ion . Since the me tabo l i sm of ca rbohydra t e s was not s tudied in methanogens so far, our results may open new views on the energy and cell ca rbon me tabo l i sm of

me thanogen ic bacter ia .

A C K N O W L E D G E M E N T S

W e thank R. Semmler for excellent technical assistance. This work was suppor t ed by the Deut - sche Forschungsgemeinschaf t (SFB 43).

R E F E R E N C E S

[1] Barry, C., Gavard, R., Milhaud, G. and Aubert, J.P. (1952) Compt. Rend. 235, 1062-1064.

[2] Builder, J.E. and Walker, G.J. (1970) Carbohydrate Res. 14, 35-51.

[3] Chargaff, E. and Moore, D.H. (1944) J. Biol. Chem. 155, 493-501.

[4] DiPersio, J.R. and Deal, S.J. (1974) J. Gen. Microbiol. 83, 349-358.

[5] DiPersio, J.R., Mattingly, S.J., Higgins, M.L. and Shock- mann, G.D. (1974) Infect. Immun. 10, 597-604.

269

[6] Levine, S., Stevenson, H.J.R., Tabor, E.C., Bordner, R.H., Chambers, L.A. (1953) J. Bacteriol. 66, 664-670.

[7] Linder, J.G.E., Marcellis, J.H., DeVos, N.M. and Hoog- kamp-Korstanje, J.A.A. (1979) J. Gen. Microbiol. 111, 93-100.

[8] Weber, M. and WOber, G. (1975) Carbohydr. Res. 39, 295-302.

[9] Shil'nikova, V.K. and Signata, V.A. (1978) Izv. Akad. Nauk. SSSR Ser. Biol. 1, 52-58.

[10] Krnig, H., Skorko, R., Zillig, W. and Reiter, W.-D. (1982) Arch. Microbiol. 132, 297-303.

[11] Murray, P.A. and Zinder, S.H. (1984) Nature 312, 284-286. [12] Balch, W.E., Fox, G.E., Magrum, L.J., Woese, C.R. and

Wolfe, R.S. (1979) Microbiol. Rev. 43, 260-296. [13] KOnig, H. and Stetter, K.O. (1982) Zbl. Bakteriol. Hyg. I.

Abt. Orig. C3, 478-490. [14] Huber, H., Thomm, M., Krnig, H., Thies, G. and Stetter,

K.O. (1982) Arch. Microbiol. 132, 47-50. [15] Jones, W.J., Leigh, J.A., Mayer, F., Woese, C.R. and

Wolfe, R.S. (1983) Arch. Microbiol. 136, 254-261. [16] Lewis, P.R. and Knight, D.P. (1977) in Practical Methods

in Electron Microscopy (Glauert, A.M., Ed.), pp. 77-135. North-Holland, Amsterdam.

[17] Albersheim, P., Nevins, D.J., English, P.D. and Karr, A. (1967) Carboh. Res. 5, 340-345.

[18] Bergmeyer, H.U. (1974) Methoden der enzymatischen Analyse. Verlag Chemie, Weinheim.

[19] Peterson, G. (1977) Anal. Biochem. 83, 346-356. [20] Morris, D.L. (1948) Science 107, 254-255. [21] Hippe, H., Caspari, D., Fiebig, K. and Gottschalk, G.

(1979) Proc. Natl. Acad. Sci. USA 76, 494-498. [22] Belay, N., Spading, R. and Daniels, L. (1984) Nature 312,

286-288.