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After digestion of the murein-lipoprotein complex (rigid layer) of lysozyme the solubilized lipoprotein contained 2 disaccharide units of the murein per 1 lipoprotein molecule. From purely enzymatic degradation of the murein-lipoprotein complex with pronase and lysozyme the peptide GlcNAc-MurNAc-L-Ala-D-Glu-Dpm-Lys-Arg was isolated, where MurNAc = N-acetyl-muramic acid and Dpm = meso-2,6-diaminopimelic acid. The missing D-alanine in this repeating unit of the murein is replaced by the lysyl-arginine dipeptide of the lipoprotein. The lipoprotein molecules are therefore linked to the carboxyl group of the optical L-center of the diaminopimelic acid of the murein. . Therefore by the use of both enzymatic procedures one obtains one of the two macromolecules (the murein or the lipoprotein) undegraded, to which a small part of the other remains covalently bound. Previously we isolated from partial acid hydrolysates of murein-lysyl-arginine degradation products with which we could show that the lipoprotein molecules are bound to the diaminopimelic acid of nearly every tenth disaccharide unit of the murein (Dpm-Lys-Arg). It was also found that none of the degradation products bearing lysine and arginine contained D-alanine but this could have been lost by the partial acid hydrolysis. By a purely enzymatic procedure we shall show here that the disaccharide units of the murein which bear the lipoprotein molecules in fact are devoid of D-alanine and that the lipoprotein molecules actually replace the D-alanine a t the carboxyl group of the optical L-center of diaminopimelic acid. I n addition we isolated after lysozyme digestions of the mureinlipoprotein complex the lipoprotein in a soluble form and it contained two disaccharide units of the murein. MATERIALS AND METHODS Enzymatic DegradationsLysozyrne Digestion. 50 mg of the murein-lipoprotein complex prepared as described [4,7] were suspended in 100 ml 0.01 M Tris-C1 pH 7.4 and digested with 1 mg of lysozyme (Boehringer Mannheim GmbH, Mannheim) for 15 h at 34". The solution was centrifuged for 30 min a t 37 000 x g, the supernatant was either acidified to pH 3.7 or concentrated to 2 ml on a rotary evaporator for column chromatography on Sephadex G-75. The precipitate of the acidified solution was spun down, washed twice with 0.1 M acetic acid and lyophilized.Promse-Lysozyme Digestion. Incubation of the murein-lipoprotein complex with pronase P (Serva, Heidelberg) followed the procedure described [7]. After washing the resulting murein-lysyl-arginine with 4OI0 sodium dodecyIsulfate and then 4 times with water, the digestion with lysozyme followed the procedure described above. The solution of the degradation products was concentrated to about 15m1, acidified to pH 2 and run on a column of Dowex 50x2. The collected peak fractions, identified by ninhydrin reaction after alkaline hydrolysis, were tested for homogeneity by paper chromatography in the system butanol-acetic acid-water (4: 1 : 5 , upper phase) and aliquots were run on the amino acid analyzer as described [4,7]....

The rigid layer of Salmonella typhimurium LT2 and Serratia marcescens consists of murein to which depending upon the size of the cell between 75,000 and 240,000 lipoprotein molecules are covalently bound. After digestion of the rigid layer of Salmonella typhimurium with trypsin, thermolysin, papain, and pronase, only lysine and arginine remained covalently bound on the murein. By analogy to Escherichia coliit is concluded that lysine at the N-terminal end of the lipoprotein constitutes the link between the lipoprotein molecules and the murein. On average, 1 lipoprotein molecule is bound to every tenth repeating disaccharide unit of the murein from which an average distance of about 100 A between individual lipoprotein molecules along the polysaccharide chains is deduced. Some of the lipid is very tightly or even covalently bound to the protein so that the rigid layer is composed of L ittle is known about the structure of the rigid layer of the cell wall of Gram negative bacteria compared with the wealth of knowledge in Gram positive bacteria (see for the most recent review Ghuysen, 1968). This is partially due to the difficulty in isolating a pure rigid layer from the much more complex cell wall of Gram negative bacteria. It may also be due to the presence in the isolated rigid layer of additional material, roughly characterized as polysaccharide, protein, and lipid, beside the murein. Recently we were able to show for Escherichia coli (Braun and Rehn, 1969) that the protein associated with the rigid layer (Martin and Frank, 1962;Weidel and Pelzer, 1964) is in fact a lipoprotein which is covalently linked to the murein. It was found that on average 1 lipoprotein molecule is bound to every tenth * From the Max-Planck-Institut fur Biologie, 74 Tiibingen, West Germany. Receiaed July 27, 1970. This research was supported in part by the Deutsche Forschungsgemeinschaft.t To whom correspondence should be addressed. murein-protein-lipid. Lysozyme digestion of the murein-lipoprotein complex of S. typhimurium yielded the lipoprotein with 2 repeating units of the murein still attached. From the amino acid composition and gel chromatography in 2 x sodium dodecyl sulfate a molecular weight of 8000 is deduced for the lipoprotein. The lipoprotein is composed of about 60 amino acids of which about 6 5 x are polar. It does not contain glycine, proline, histidine, cysteine, and phenylalanine. In contrast to Salmonella, Serratia, and E. coli no covalently linked lipoprotein could be found on the murein of Proteus oulgaris, P. mirabilis, and Pseudomonas fluorescens. Upon treatment of the cell envelope of S. typhimurium with trypsin the turbidity decreased rapidly, and in ultrathin sections it was observed that the cytoplasmic membrane and the cell wall, otherwise attached, are separated from one another.repeating unit of the murein. The linkage consists of a peptide bond between the amino group of the N-terminal lysine of the lipoprotein and the carboxyl group of the optical L center of the diaminopimelic acid of the murein (Braun a...

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