Arch. Microbiol. 121,271-275 (1979) Archives of

Microbiolngy �9 by Springer-Verlag 1979

The Amino Acid Sequence of the Peptide Moiety of the Pseudomurein from Methanobacterium thermoautotrophicum

Helmut K6nig and Otto Kandler*

Botanisches Institut der Universit~it Mfinchen, Menzinger Strasse 67, D-8000 Mtinchen 19, Federal Republic of Germany

Abstract. The amino acid sequence of the peptide subunits of the peptide moiety of the sacculus polymer (pseudomurein) of Methanobacterium thermoauto- trophicum was elucidated by analysing overlapping peptides obtained from partial acid hydrolysates of isolated sacculi. It is suggested that the peptide subunits are attached to glycan strands via one of their glutamyl residues. Another glutamyl residue may crosslink two adjacent peptide subunits to form a dimer. The calcu- lated molar ratios of the amino acids and the per- centages of the N- or C-terminal amino acid residues of the supposed dimers are compatible with those actually found in the sacculus polymer.

Key words: Methanobacterium thermoautotrophicum - Cell wall - Pseudomurein - Amino acid sequence.

The isolated sacculi (rigid component of the cell walls; for definition see Weidl, 1964; Weidl and Pelzer, 1964) of all known species of the genus Methanobacterium contain a unique polymer consisting of either N-acetyl glucosamine or N-acetyl galactosamine and a set of L- amino acids (Glu: Lys :Ala = 2.3 : 1 : 1.2). Additional polysaccharides may or may not be present (Kandler and K6nig, 1978). It was assumed that the amino acids form covalently crosslinked peptide subunits bound to N-acetylamino-sugars. Further studies (K6nig and Kandler; paper in preparation) have revealed that the amino sugars form glycan strands of limited length, to which peptide subunits are attached by their glutamyl residues via a thus yet unknown compound X (Kandler, 1979). Therefore the sacculus polymer is not a polypep- tide substituted with single N-acetyl amino sugar resi- dues, as originally assumed (Kandler and K6nig, 1978),

* Tho whom offprint-requests should be sent.

but rather a new form of peptidoglycan which differs from the peptidoglycan of the "classical" bacterial by the lack of D-amino acids and muramic acid. In order to avoid misunderstanding, the suggestion has been made that the sacculus polymer of the "classical" bacteria be termed murein, as originally proposed by Weidl and Pelzer (1964), and that of the genus Methanobacterium pseudomurein (Kandler, 1979). Such a nomenclature expresses the functional and chemical relationship as well as the distinct differences between the two poly- mers. Each of the two forms ofpeptidoglycan may then be further subdivided into different groups and types according to their variations in the amino acid composi- tion and the types of crosslinkages (Schleifer and Kandler, 1972; Kandler and K6nig, 1978).

This paper describes the amino acid sequence of the peptide moiety of pseudomurein of Methanobacterium thermoautotrophicum as elucidated by the analysis of overlapping peptides derived from partial acid hydrolysis.

Material and Methods

Organisms and Growth Conditions

Freeze-dried cells of Methanobacterium thermoautotrophicum were provided by W. E. Balch, J. Romesser and R. S. Wolfe, University of Illinois, Urbana, Ill., U.S.A. The organisms were grown as previously described (Balch et al., 1977).

Cell Wall Preparation and Chemical Procedures

Ceil wall preparation, hydrolysis, dinitrophenylation and hydrazi- nolysis were performed as in the preceeding paper (Kandler and K6nig, 1978).

Chromatography

The following solvents were applied in paper (A, B) and thin layer (C, D, E) chromatography, respectively (v/v):

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272 Arch. Microbiol., Vol. 121 (1979)

Table 1. Amino acid and amino sugar content of the isolated sacculi ofMethanobacterium thermoautotrophicum and percentages of the N- and C- terminal residues (average of 3 determinations)

Ala Glu Lys GlcNH2 GalNH2 NH3

a 0.67 1.27 0.56 0.66 0.09 0.34 b 1.20 2.27 1.00 1.18 0.16 0.61 c - 2 1 . 0 . . . . d 13.3 4.2 9.0 - - -

a = gmol/mg dry weight; b = molar ratio; c = N-terminal in % of total amount; d = C-terminal in % of total amount

A. isopropanol:acetic acid:water = 75 : 15:10 B. ~-picoline:ammonia:water = 70:2:28 C. propanol:ammonia:water = 60:30:10 D. 1.5 M phosphate buffer pH 6 E. butanol, saturated with 2 N NH 3. The peptides were separated by repeated one-dimensional chro-

matography on Whatman paper No. 3, using first solvent A and subsequently solvent B. They were further purified by thin layer chromatography on cellulose with solvent C. The purity of the peptides was checked with an amino acid analyser without prior hydrolysis under the following conditions: 0.2 M citrate buffer pH 3.24 was used for the first 100 rain and subsequently 0.7 M citrate buffer pH 4.15; the temperature was increased from 30 - 55 ~ C during the course of the 1 h. For the separation of the pairs e- and y-Glu-Glu and c~- and 7-Glu-Ala, 0.2 M citrate buffer pH 3.0 followed by 0.7 M citrate buffer pH 3.6 after the 1 h were used at a constant temperature of 55 ~ C.

DNP-amino acids and DNP-peptides were separated in solvents B and D. The monohydrazides of glutamatic acid were identified by co-chromatography of their di-DNP-derivatives with the corre- sponding derivatives of the authentic according to Kawanishi et al. (1964) using solvents D and E.

Source of Reference Compounds

Authentic 7-Glu-Ala, ~-Glu-Glu, Lys-Glu and DNP-amino acids were obtained from Serva, Heidelberg, Germany; 7-Gtu-Glu, c~-Glu- Ala, 7-hydrazide of glutamic acid and amino acids were obtained from Sigma Chemicals, St. Louis, U.S.A. ; y-Glu-Lys was obtained from Vega-Fox-Biochemicals, Tucson, U.S.A. The ~-hydrazide of glutamic acid was prepared by hydrazinolysis of c~-Glu-Ala.

Results

The Primary Structure of the Peptide Subunit

Isolated sacculi o f Methanobacterium thermoauto- trophicum, the chemical composi t ion o f which is shown in Table 1, were subjected to partial acid hydrolysis (4 N HCI, 100 ~ C, 45 min). The hydrolysate was analysed by either paper chromatography , thin layer chromatog- raphy or the use o f an amino acid analyser. Eleven peptides were isolated in small quantitaties by repeated one-dimensional paper ch roma tog raphy using the sol- vent systems A and B. The peptides were identified by determining their amino acid composi t ion, their N- termini by dini t rophenylat ion and their C-termini by hydrazinolysis, and in some cases by cochromatog-

raphy with authentic peptides. The results are sum- marized in Table 2. The y-bonds in 7-Glu-Lys and 7- Glu-Ala were demonst ra ted by determining the sensi- tivity o f the respective 2,4-dinitrophenyl (DNP) de- rivatives to UV irradiation (Russel, 1963; Perkins, 1967). Both compounds were decarboxylated u p o n UV irradiation as indicated by the fast increase o f the extinction at 284 nm and its decrease at 348 nm. The peptide 7-Glu-Lys is also clearly distinguishable f rom ~- Glu-Lys by the RNH3 values obtained with the amino acid analyser (Table 2). Whereas authentic ~-Glu-Lys is eluted virtually together with e-(Ala)-Lys, 7-Glu-Lys is eluted shortly after glucosamine. Also the isomeric pairs ~-Glu-Ala/7-Glu-Ala and ~-Glu-Glu/7-Glu-Glu can be separated by the amino acid analyser, when buffers o f p H 3.0 and 3.6 are consecutively used at a constant temperature o f 55 ~ C as shown in Fig. 1. Co- ch roma tog raphy of a partial acid hydrolysate o f iso- lated sacculi and authentic peptides revealed the pre- sence o f only 7-Glu-Ala. Neither 7-Glu-Glu or c~-Glu- Glu could be found, a l though such peptides were to be expected, since the peptide moiety contains a high p ropor t ion o f glutamyl residues. In partial acid hy- drolysates a very small unknown peak was found almost exactly in the posit ion of ~-Glu-Glu, which, however, did no t co -ch romatograph with ~-Glu-Glu in two-dimensional paper ch romatograms using the sol- vent systems A and B. Partial alkaline hydrolysis with saturated Ca(OH)z at 100 ~ C for 24 h yielded mainly e(Ala)-Lys, 7-Glu-Ala, 7-Glu-Lys and only traces o f other peptides, but none in addit ion to those shown in Table 2.

The analysis o f the overlapping peptides indicates a pr imary structure o f the peptide subunit of the pseudomure in as depicted in Fig.2a. The exclusive occurrence o f y-glutamyl-bonds agrees with the finding that only the y-hydrazide o f glutamic acid was found in the hydrazinolysate o f the polymer.

Integration of the Peptide Subunit in the Polymer

In order to decide which of the two possibly N-terminal glutamyl residues is actually N-terminal in the polymer, the isolated sacculi were dini trophenylated and then

H. K6nig and O. Kandler: Pseudomurein from Methanobacterium thermoautotrophicum

Table 2. Analysis of peptides from the partial acid hydrolysate of isolated sacculi of M. thermoautotrophicum

273

Peptide Chromatography

RAI a e f

A B C

Molar ratio Products of Free amino hydrolysis of acids after

RNH 3 Ala Glu Lys the DNP-peptides hydrazinolysis

Structure of the peptides

I 0.98 0.57 0.46 0.19 1 1 -- DNP-Glu �9 Ala

2 0.30 0.36 0.33 0.69 - 1 1 DNP-GIu �9 a-DNP-Lys

3 0.44 0.31 0.30 1.01 - 1 1 Di-DNP-Lys O

4 0.44 0.86 0.65 1.05 1 - i DNP-Ala O c+-DNP-Lys

5 0.30 0.68 0.41 0.62 1 1 1 DNP-GIu O c~-DNP-Lys Ala

6 0.35 0.65 0.47 0.69 1 1 1 DNP-Glu O DNP-Ala Lys

7 0.30 0.23 0.21 0.64 - 2 1 DNP-Glu O a-DNP-Lys Glu

8 0.54 0.53 0.43 0.97 1 1 1 DNP-Ala �9 ~-DNP-Lys Glu

9 0.61 1.01 0.62 1.00 2 1 1 DNP-Ala Ala c~-DNP-Lys Ala, Glu

10 0.30 0.43 0.27 0.64 1 2 1 DNP-Glu O DNP-Ala Glu, Lys

11 0.25 0.79 0.49 ? 2 1 2 DNP-AIa Lys ~-DNP-Lys Glu, Lys

Glu {+Ala

Glu ~ L y s

Lys--+Glu

A l a ~ Lys

Glu

I~AlaL-{y s

Glu L~Lys

t--Ala

Glu [~Lys-+Glu

A l a ~ Lys ~ Glu

Ala~, Lys~GIu

t+Ala

Glu Lys-~ GIu

E _ Ala

Ala 7~ Lys-+Glu

L+Lys E-AIa

O - not determined A, B, C - solvents - = not present RAt a determined by paper chromatography, Rf by thin layer chromatography, enh 3 by column chromatography (amine acid analyser, standard program). DNP = 2,4 dinitrophenyl

~- Glu-ALa

~ - Gqu-Ata

a b

R

r Glu Glu

Lys ~ Glu r - - ~ Lys ~ Gtu

G,u l ~--~ Ata Glu | ~ .~L ys ~-*Gtu '

Gtu 4~ 2~ 4~ )~) 2'0 3'0 r S0 6'0 7'0 8'0 9'0 100 I i0 120 13D I~0 mm R

1 2

Fig. 1. Separation of the stereo-isomeric pairs Y- and e-Glu-Ala and Y- and a-Glu-Glu with the amino acid analyser

Fig. 2. Proposed structure of the peptide subunit and the dimer of the peptide moiety of pseudomurein. The numbers indicate the amount of the respective C-terminal residues of the mixture of incomplete dimers actually present in the polymer (see text). R = glycan moiety

274 Arch. Microbiol., Vol. 121 (1979)

Table 3. Analysis of DNP-peptides from the partial acid hydrolysate of DNP-cell walls. [(Ala)-Lys was found in the partial acid hydrolysate of both peptides]

Peptide Chromatography Products of hydrolysis Products of hydrolysis Structure (Molar ratio) after dinitrophenylation

of the DNP-peptide B D DNP-Glu Ala Glu Lys

RAI a RDNP_AI a DNP-GIu 1 1.07 1.79 1 1 1 1 DNP-Glu, DNP-ALa, t~ Lys --, Glu

Glu, Lys t Ala

Rf Rf DNP-Glu 2 0.69 0.72 1 I - 1 DNP-Glu, DNP-Ala, Lys LLys

t_ Ala

- = not present B, D = solvents DNP = 2,4-dinitrophenyl

subjected to partial acid hydrolysis. The chromato- graphic separation of the resulting peptides yielded in addition to DNP-Glu the two peptides listed in Table 3. Their presence shows that the glutamyl residue bound by its y-carboxyl group to lysine is the N-terminus of the polymer, whereas the glutamyl residue bound by its 7- carboxyl group to the alanyl residue exhibits no free amino group. The amino group may be involved in the linkage of the peptide subunit to the glycan moiety, however.

In contrast to the uniformity of the N-terminus, which is comprised solely of glutamyl residues, the C- terminus of the polymer consists of one of the three amino acid residues present. In the peptide subunit depicted in Fig.2a, however, the alanyl residue is the only one forming a C-terminus. In addition, the molar ratio of the amino acid residues of the proposed peptide subunits differs distinctly from that of the polymer in that the values for glutamic acid and alanine are too high. These discrepancies indicate that only some of the peptide subunits of the polymer may be complete, whereas the majority lacks the originally C-terminal alanyl residues or glutamyl-alanine residues. The pep- tide No. 11 (Table 2) indicates further, that the peptide subunits are crosslinked via Lys-7-Glu-Lys bridges. If it is assumed that the majority of the peptide subunits of the polymer form dimers depicted in Fig. 2b and if it is further supposed that 60 % of the originally C-terminal alanyl residues and 40 % of the adjacent glutamyl residues of the dimers are missing, the calculated molar ratios of the amino acid residues (Lys:Glu:Ala = 1" 2.30:1.20) agree almost exactly with the data ob- tained from the isolated sacculi (Table 1). Also the calculated percentages of the N-terminal glutamyl residues (21.8 %) and the C-terminal alanyl (16.3%) and glutamyl (4.35 %) residues of such dimers are close to those found in the intact sacculus polymer (Table 1).

However, the calculated percentage of C-terminal lysyl residues (20 %) of the dimer (Fig. 2b) is twice as high as that determined for the isolated sacculus (Table 1). This discrepancy may be caused by an incomplete hy- drazinolysis of the very stable peptide e(Ala)-Lys and by a partial destruction of lysine.

Discussion

The results of these studies indicate strongly that the peptide moiety of the pseudomurein contains relatively small peptide subunits composed of 4 " 6 amino acid residues. Most of them are crosslinked to form dimers but not - at least to no significant extent - higher polymers. The peptide moiety exhibits an unusually high frequency of ~- and 7-bonds which may be the cause of the distinct insensitivity of the sacculus poly- mer toward the common proteases (Kandler and K6nig, 1978). This insensitivity is also typical of the murein, but here it is mainly the result of a sequence of alternating L- and D-amino acids.

Acknowledgements. We are indebted to Dr. R. S. Wolfe, Dr. W. E. Balch and J. A. Romesser (University of Illinois, Urbana) for supplying freeze-dried cells. Excellent technical assistance by Mrs. E. Hagner and Mrs. F. Menzinger is acknowledged. This work was supported by the Deutsche Forschungsgemeinschaft and in part by National Science Foundation Grant PCM 76-02652 and U.S. Public Health Grant AI 12277 to R. S. Wolfe.

References

Balch, W. E., Magrum, L. J., Fox, G. E., Wolfe, R. S., Woese, C. R. : An ancient divergence among the bacteria. J. Mol. Evol. 9, 305 - 311 (1977)

Kandler, O.: Zellwandstrukturen bei Methan-Bakterien. Zur Evolution der Prokaryonten. Naturwissenschaften 66, 95-105 (1979)

H. K6nig and O. Kandler: Pseudomurein from Methanobacterium thermoautotrophicum 275

Kandler, O., K6nig, H. : Chemical composition of the pepdiglycan- free cell walls of methanogenic bacteria. Arch. Microbiol. 118, 141 - 152 (1978)

Kawanishi, Y., Iwai, K., Ando, T. : Determination of C-terminal arginine and asparagine of proteins by catalytic hydrazinolysis. J. Biochem. 56, 314 324 (1964)

Perkins, H. R.: The use of photolysis of dinitrophenylpeptides in structural studies on the cell-wall mucopeptide of Corynebacterium poinsettiae. Biochem. J. 102, 2 9 c - 32c (1967)

Russell, D. W.: Studies on the photochemical behaviour of 2,4- dinitrophenyl derivatives of some amino acids and peptides. Biochem. J. 87, 1 - 4 (1963)

Schleifer, K. H., Kandler, O. : Peptidoglycan types of bacterial cell walls and their taxonomic implication. Bacteriol. Rev. 36, 407 - 477 (1972)

Weidl, W. : Ein neuer Typ yon Macromolekfilen. Angew. Chem. 76, 801 - 832 (1964)

Weidl, W., Pelzer, H. : Bagshaped macromolecules : A new outlook on bacterial cell walls. Adv. Enzymol. 26, 193-232 (1964)

Received February 16, 1979

Addendum

The unknown compound X mentioned in this paper has been identified in the meantime as N-acetyltalosaminuronic acid (K6nig and Kandler, paper in preparation).