FEMS Microbiology Letters 61 (1989) 323-328
Published by Elsevier
323
FEMSLE 03717
Isolation of a nucleotide activated disaccharide pentapeptide
precursor from Methanobacterium thermoautotrophicum
Evamarie H a r t m a n n
1, H e l m u t
K~Snig a, Otto Kandler
2, Walter
Hammes 3
t Abteilungf~r Angewandte Mikrobiologie, Universitiit Ulm, Ulm, 2 Botanisches lnstitut der Universitdt, Mi~nchen,
and 3 lnstitut fiir Lebensmitteltechnologie, Universtitiit Hohenheim, Stuttgart, F.R.G.
Received 8 May 1989
Revision received 2 June 1989
Accepted 5 June 1989
Key words: Methanogens; Archaebacteria; M e t h a n o b a c t e r i u m t h e r m o a u t o t r o p h i c u m ; Cell walls;
Peptidoglycan; Murein; Pseudomurein; Biosynthesis
This compound is supposed to be a putative precursor of the pseudomurein.
1. S U M M A R Y
A uridine diphosphate activated disaccharide
pentapeptide was isolated from trichloroacetic acid
extracts of Methanobacterium thermoautotrophicum:
UDP--GlcNAc--NAcTalNUA
~ Glu ~ Ala
Lys ~ Ala.
Glu
Correspondence to: Prof. Dr. Helmut KiSnig, Abteilung Angewandte Mikrobiologie, Universit~t Ulm, Oberer Eseilsberg
M23, 7900 Ulm, F.R.G.
Abbreviations: N%AcLys, N%acetyllysine, N'-AcLys, N'acetyllysine; GlcNAc, N-acetylglucosamine; GlcNitol, glucosaminitoi; GalNAc, N-acetylgalactosamine; NAcTalNUA, Nacetyltalosaminuronic acid; UDP, uridine diphosphate;
MurNAc, N-acetylmuramic acid; Glu, glutamic acid, Ala,
alanine, Lys, lysine; TCA, trichloroacetic acid; DNP, dinitrophenyl-.
Standards: Glu-Lys (Serva), Lys-Ala (Serva), y-Glu-Ala
(Serva), N'-Ala-Lys (isolated from intact pseudomurein sacculi), N"-AcLys (Serva), N'-AcLys (Aldrich).
2. I N T R O D U C T I O N
A m o n g the archaebacteria only the Methanobacteriales have developed a cell wall polymer
(pseudomurein), which resembles structurally and
chemically the eubacterial murein. However, the
two peptidoglycan types have also remarkable differences [1]: Muramic acid is replaced by talosaminuronic acid and the peptide subunits show
not only a different amino acid sequence, but do
not contain D-amino acids. While the biosynthesis
of the murein has been studied in detail [2], virtually nothing is known about the biosynthesis of
the pseudomurein [1]. During the biosynthesis of
murein a U D P and an undecaprenyl pyrophosphate activated MurNAc-pentapeptide are subsequently formed. At the lipid stage G l c N A C is
added to form a lipid activated disaccharide pentapeptide.
In contrast, from cell extracts of the pseudomurein containing M e t h a n o b a c t e r i u m t h e r r n o a u t o -
0378-1097/89/$03.50 © 1989 Federation of European Microbiological Societies
324
trophicum [3], a UDP-activated disaccharide composed of GIcNAc and NAcTalNUA was isolated
as a main intermediate. No monomeric derivative
of NAcTalNUA was found, while the UDPactivated amino sugars GIcNAc and GalNAc were
present in cell extracts [3].
In this paper we describe the isolation and
characterization of a further putative intermediate
of the pseudomurein.
Silica gel (glass plates; Merck)
f. chloroform : methanol : acetic acid = 95 : 5 : 1
(running distance: 12 cm).
Bands containing nucleotides were detected in
the UV light (254 nm).
3.4. Column chromatography
Sephadex gel filtration was performed as previously described [3,6]. Ion-exchange chromatography was carried out on a Fractogel TSK DEAE650 (S) column (15 cm × 2 cm; Merck) using a
3. MATERIALS A N D M E T H O D S
3.1. Organisms and growth conditions
A stock culture of Methanobacterium thermoautotrophicum strain Marburg was purchased from
the Deutsche Sammlung von Mikroorganismen
(DSM 2133, Braunschweig). The organism was
grown in medium 1 [4] in a 10-1 fermenter at
64 ° C. The cells were harvested in the early stationary phase by centrifugation. The pellet was
washed with phosphate buffer (0.1 mol/1; p H 7)
and kept at - 20 ° C.
3.2. Extraction
The preparation of the cell extracts was performed as previously described [3,5].
3.3. Thin layer chromatography
Thin layer chromatography was performed
using the following solvents (v/v) and thin layer
plates:
RP 18 (glass plates; Merck)
a. 0.01 M triethyalamine/formate buffer, p H
8.0, containing 35% methanol (running distance:
10 cm).
PEI-cellulose (Schleicher and Schtill)
b. 0.3 M triethylamine/formate buffer, p H 8.0
(running distance: 10 cm).
Cellulose F (aluminium sheets; Merck)
c. isobutyric acid : ammonia (25%) : water =
198 : 6 : 99 (running distance: 14 cm).
d. a-picoline : a m m o n i a (25%) : water =
70 : 2 : 28 (running distance: 18 cm).
Cellulose (Polygram CEL 400; Macherey and
Nagel)
e. 1.5 M phosphate buffer, p H 6.0, (running
distance: 12 cm).
Table 1
Analysis of the isolated compound
DEAE-column
Peak No.
buffer (M)
0.17-0.25
TLC (Rr)
solvent a
solvent b
solvent c
0.85
0.13
0.37
Absorption
260 : 280
250 : 280
0.38
0.76
Composition
(molar ratio)
Uracil
Phosphate
Glu
Ala
Lys
GIcNAc
NAcTalNUA
1.0
1.9
1.9
2.1
1.0
1.1
1.2 ~
N-terminus
C-terminus
Glu
Ala
Proposed
Structure
UDP-G-T
1
Glu
Ala
3'
Lys '-- Glu
Ala
G = GlcNAc; T = NAcTalNUA. a The TalNUA content was
calculated by substracting the GlcN content determined by the
amino acid analyzer from values for total amino sugars obtained by the Morgan-Elson test after hydrolysis (4 N HCI, 20
rain) and N-acetylation.
325
linear gradient of 0.1-0.5 M t r i e t h y l a m i n e /
formate buffer, p H 8.0, with a flow rate of 1
m l / m i n . 60 fractions of 3 ml were collected. The
elution profile was determined at 254 nm.
[14]. The linkage of the carbohydrates was determined by the direct M o r g a n - E l s o n test [15] and
by alkaline treatment [16].
3.6. Hydrolytic conditions
(a) Total hydrolysis: 4 N HC1, 16 h, 100 o C.
(b) Partial hydrolysis: 4 N HC1, 15 min, 100 ° C.
3.5. Analytical methods
A m i n o acids, aminuronic acids and amino
sugars were determined with an amino acid
analyzer (Biotronik L C 5000).
Uracil was identified as previously described
[3,7]. Quatitative determination of phosphate was
p e r f o r m e d b y the m o l y b d a t e m e t h o d [8].
A m i n u r o n i c acids and amino sugars were also
quantitatively determined by the E l s o n - M o r g a n
test [9]. N - and C-terminal amino acids were determined by "dinitrophenylation [10,11] and hydrazinolysis [12], respectively. Talosaminuronic
acid was identified by thin layer c h r o m a t o g r a p h y
(solvent d) and with the amino acid analyzer [13].
The peptide anlaysis was performed as described
4. R E S U L T S
4.1. Fractionation of the TCA extract
The elution profile of the T C A extracts separated on c o m b i n e d Sephadex G 50 fine and
Sephadex G 25 fine columns showed four distinct
peaks [3]. The chemical analysis of the c o m p o u n d s
of peaks I I - I V containing a U D P - a c t i v a t e d disaccharide c o m p o s e d of G l c N A c and N A c T a l N U A ,
the a m i n o sugar derivatives U D P - G l c N A c , U D P G a l N A c and the free nucleotide U D P has been
Table 2
Dipeptides isolated from the nucleotide activated compound (Table 1)
No.
Chromatography a
Chemical composition
of the hydrolysate of
Rr
g
f
the dinitrophenylated
Structure of the
original compound
or dipeptide
compounds
1
2
3
4
0.03
0.14
0.30
0.00
0.35
0.00
0.00
0.60
DNP-Ala, a-DNP-Lys
DNP-GIu, ~-DNP-Lys
a, c-di-DNP-Lys, Ala
DNP-GIu, Ala
c-Ala-Lys
Lys ~ Glu b
Lys-Ala
y-Glu-Ala b
Standard compounds
1
0.32
2
0.05
3
0.00
4
0.07
5
0.02
6
0.21
7
0.00
8
0.00
9
0.03
10
0.28
0.00
0.33
0.60
0.00
0.66
0.05
0.62
0.50
0.60
0.51
a, c-di-DNP-Lys, Ala
a-DNP-Lys, DNP-AIa
DNP-Glu, Ala
c-DNP-Lys, DNP-Glu,
DNP-Glu, Ala
a, ~-di-DNP-Lys
a-DNP-Lys
c-DNP-Lys
DNP-Glu
DNP-Ala
Lys-Ala
~-Ala-Lys
7-Glu-Ala
Glu-Lys
a-Glu-Ala
Lys
N~-AcLys
N~-AcLys
Glu
Ala
a = solvent system f and g (running distance: 10 cm).
b = running distance: 18 cm.
For the isolation of the dipeptides the nucleotide activated compound was partially hydrolyzed and the peptides in the hydrolysate
were dinitrophenylated. The dinitrophenylated peptides were then purified by TLC in solvents f and g. The dinitrophenylated
standards were prepared by dinitriphenylation of the corresponding amino acids or dipeptides. In the case of Na-AcLys and
N'-AcLys the acetyl residues were split off after dinitrophenylation by acid (4 N HC1, 16 h, 100 o C).
326
reported recently [3]. Here we describe the chemical characterization of a compound of peak I [3].
Three peaks were obtained, when peak I was
fractionated on a TSK-DEAE column: peak 1
(tubes 12-22; buffer molarity: 0.17-0.25 M), peak
2 (tubes 23-34; buffer molarity: 0.25-0.32 M) and
peak 3 (tubes 35-50; buffer molarity: 0.32-0.45
M). The compounds of each peak were separated
on RP-18 plates (solvent a). The UV absorbing
bands were eluted and subsequently run on PEIcellulose plates (solvent b) and Alugram CEL 300
plates (solvent c).
4.2. Characterization of a precursor isolated from
peak 1
A nucleotide-activated precursor could be
purified (Table 1). It was composed of Glu, Ala,
Lys, GlcNAc, NAcTalNUA, uracil and phosphate. In partial acid hydrolysates the dipeptide
"¢-Glu-Ala was identified with the amino acid
analyzer [14]. Half the alanine residues were Cterminal and half the glutamic acid residues were
found to have a free amino group. After dinitrophenylation DNP-Glu was identified by thin
layer chromatography (Rt = 0.62, solvent e). The
position of the N-terminal glutamic acid residue
was revealed by the isolation of a dinitrophenylated dipeptide composed of lysine and DNPglutamic acid (Re=0.33, solvent e; Rf=0.06,
solvent f) after partial acid hydrolysis of the dinitrophenylated compound (Table 1). Lysine is
linked to the y-carboxylic group of glutamic acid,
since the dipeptide is sensitive against UV irradiation [17,18]. When the dinitrophenylated dipeptide
obtained by partial acid hydrolysis was again subjected to dinitrophenylation and total acid hydrolysis c-DNP-Lys and DNP-Glu were found. This
indicates that lysine is linked via its a-amino
group to the 3,-carboxylic group of glutamic acid.
When UDP was split off by mild acid treatment (0.01 N HC1, 10 rain, 100 ° C), GlcN could
be completely reduced to GlcNitol by NaBH4 as
revealed by the amino acid analyzer [3]. The complete destruction of glucosamine under alkaline
conditions and a positive Morgan-Elson test (120%
color development, based on the N-acetylglucosamine content determined by the amino acid
analyzer) indicates a 1.3 linkage, which is also true
for the intact glycan strand [9,10]. N,N'-diacetyl
chitobiose (no color development) and chondrosine (150% color development) were used as standards for comparison. The molar ratio (Table 1)
of the components, the structure of the additionally characterized dipeptides (Table 2) and the
other data indicate, that the isolated compound is
a UDP-activated disaccharide pentapeptide with a
similar peptide sequence found in the intact pseudomurein [1], with the exception that one alanine
residue is bound via its amino group to the
carboxylic group of lysine. The structure of the
peptide-free disaccharide has been recently described [3].
5. DISCUSSION
We suppose that the isolated soluble compound
is a pseudomurein precursor. Pseudomurein biosynthesis may start with the formation of the
UDP-activated amino sugars UDP-GlcNAc and
UDP-GalNAc. Thereafter a UDP-activated disaccharide composed of GlcNAc and NAcTalNUA is
synthesized. Since NAcTalNUA is not found as a
monomeric derivative [3], it is suggested that
NAcTalNUA is formed at the disaccharide level
by epimerisation of UDP-GalNAc. UDP-GalNAc
occurs in relatively high amounts in the cell extracts of Methanobacterium thermoautotrophicum
strain AH [3] and may serve as precursor of the
above mentioned disaccharide, whereas it is only
found in trace amounts in the glycan strands of
the intact pseudomurein sacculi of this organism
[6]. Parallel to the formation of the UDP-activated
disaccharide di-, tri-, and pentapeptide intermediates are formed (Hartmann and K~nig,
unpublished results). Finally, the pentapeptide intermediate may be transferred to the UDPactivated disaccharide thus forming a UDPactivated disaccharide pentapeptide.
The formation of a nucleotide activated disaccharide [3], and a nucleotide activated disaccharide pentapeptide indicates that the biosynthesis of
the pseudomurein and the murein follow different
pathways.
327
ACKNOWLEDGEMENTS
This work was supported by a grant of the
Deutsche Forschungsgemeinschaft (Ko 785/1-1).
REFERENCES
[1] Kandler, O. and Ki3nig, H. (1985) in The Bacteria. Vol. 8.
(Woese, C.R. and Wolfe, R.S., eds.), pp. 413-457,
Academic Press, New York.
[2] Tipper, D.J., Wright, A. (1979) in The Bacteria, Vol. 7,
(Gunsalus, C., Sokatch, J.R. and Ornston, L.N., eds.), pp.
291-426, Academic Press, New York.
[3] K~Snig, H., Kandler, O., Hammes, W. (1989) Can. J.
Microbiol. 35, 176-181.
[4] Balch, W.E., Fox, G.E., Magrum, L.J., Woese, C.R., Wolfe,
R.S. (1979) Microbiol. Rev. 43, 260-296.
[5] Stickgold, R.A., Neuhaus, F.C. (1967) J. Biol. Chem. 242,
1331-1337.
[6] K/3nig, H., Kralik, R., Kandler, O. (1982) Zentralbl.
Bakteriol. Microbiol. Hyg. I. Abt. Orig. C 3, 179-191.
[7] Dunn, D.B., Hall, R.H. (1975) in Handbook of Biochemistry, 3rd ed., (Fasman, G.D. and Sober, H.A., eds.), pp.
65-215, CRC-Press, Cleveland.
[8] Chen, P.S., Toribara, T.Y., Warner, H. (1956) Anal. Chem.
28, 1756-1758.
[9] Johnson, A.R. (1971) Anal. Biochem. 44, 628-635.
[10] Takebe, I. (1965) Biochim. Biophys. Acta 101, 124-126.
[11] Rao, K.R., Sober, H.A. (1954) J. Am. Chem. Soc. 76,
1328-1331.
[12] Ghuysen, J.M., Tipper, D.J., Strominger, J.L. (1966) in
Methods in Enzymology. Vol. 8 (Colowick, S.P. and
Kaplan, N.O. eds.), pp. 685-699, Academic Press, New
York.
[13] K/3nig, H., Kandler, O. (1979) Arch. Microbiol. 123,
295-299.
[14] KOnig, H., Kandler, O. (1979) Arch. Microbiol. 121,
271-275.
[15] Tipper, D.J. (1968) Biochem. 7, 1441-1449.
[16] Horton, D. (1969) in The amino sugars. Vol. AI (Jeanloz,
R.W., ed.), pp. 1-211, Academic Press, New York.
[17] Perkins, H.R. (1967) Biochem. J. 102, 29c-32c.
[18] Russell, D.W. (1963) Biochem. J. 81, 1-4.