Repetitive Sequences in the Murein-Lipoprotein of the Cell Wall of Escherichia Coli (Covalent Lipid/Peptidoglyean/Mucopeptide) V
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2354 Corrections: Proc. Nat. Acad. Sci. USA 69 (1972) Correction. In the article "A Structure of Pyridine Correction. In the article "Insulin Receptors in Human Nucleotides in Solution," by Oppenheimer, N. J., Arnold, Circulating Cells and Fibroblasts," by Gavin, J. R., III, L. J. & Kaplan, N. O., which appeared in the December Roth, J., Jen, P. & Freychet, P., which appeared in the 1971 issue of Proc. Nat. Acad. Sci. USA 68, 3200-3205, March 1972 issue of Proc. Nat. Acad. Sci. USA 69, 747- the column heads for Table 1, p. 3202, should read: 751, the fifth line from the bottom of the right-hand column on p. 747 should read "Tris buffer [25 mM Tris- PC4HA PC4HB J5-4A J5-4B 10 mM dextrose-1 mM EDTA-1.4 mM sodium acetate- 5.0 mM KCI-120 mM NaCl-2.4 mM MgSO4-1% bovine serum albumin (pH 7.4)]." Correction. In the article "Effect of Dibutyryladenosine 3': 5'-Cyclic Monophosphate on Growth and Differen- Correction. In the article "Repetitive Sequences in the tiation in Caulobacter crescentus," by Shapiro, L., Agabian- Murein-Lipoprotein of the Cell Wall of Escherichia coli," Keshishian, N., Hirsch, A. & Rosen, 0. M., which ap- by Braun, V. & Bosch, V., which appeared in the April peared in the May 1972 issue of Proc. Nat. Acad. Sci. 1972 issue of Proc. Nat. Acad. Sci. USA 69, 970-974, USA 69, 1225-1229, the bacteria-Escherichia coli B- lines 48/49, page 972, should read: ...treated with di- shown in Table 3, p. 1228, should read: Escherichia coli isopropylfluorophosphate, and not (... treated with dini- Crookes. trofluorobenzene). Downloaded by guest on September 28, 2021 Proc. Nat. Acad. Sci. USA Vol. 69, No. 4, pp. 970-974, April 1972 Repetitive Sequences in the Murein-Lipoprotein of the Cell Wall of Escherichia coli (covalent lipid/peptidoglyean/mucopeptide) V. BRAUN* AND V. BOSCH* Max-Planck-Institut fXr Biologie, Tabingen, West Germany Communicated by Eugene P. Kennedy, February 3, 1972 ABSTRACT The amino-acid sequence of the murein- lipoprotein further. Of special interest were the attachment lipoprotein of the Escherichia coli cell wall is presented. sites of the covalently linked lipid and the murein to the This protein is covalently bound to a lipid component as well as to the murein (peptidoglycan, mucopeptide). The polypeptide chain. We report here the complete amino-acid sequence is also highly repetitive. At the N-terminal por- sequence of the protein, the attachment site of the lipid, tion, there are three adjacent almost identical sequences, and the mode of binding of the lipoprotein on the murein. indicating repeated duplication of a gene coding originally Experimental details and the complete list of sequenced for 15 amino acids. The C-terminal part of the poly- peptide chain is more variable but still shows striking peptides will be published elsewhere. homology when certain sequence gaps are introduced. The lipid is bound to the N-terminal serine of the di- METHODS peptide (Ser-Ser) that extends from the repetitive se- Experimental procedure quence. At the C-terminal end where the murein is bound, a tripeptide extends from the repetitive portion. Here there The following preparations and techniques have been de- are several basic amino acids and the only aromatic amino scribed (2-4): isolation of murein-lipoprotein complex from acid in the lipoprotein. The sequence is Lys-Tyr-Arg- E. coli B, grown to 5 X 108 cells/ml in glucose-minimal Lys. The linkage to the murein is formed between the -amino group of the C-terminal lysine and the carboxyl medium (6) or Difco M3; digestion of murein-lipoprotein group of the optical L-center of meso-diaminopimelic with hen egg white lysozyme (EC 3.2.1.17) and isolation acid. The polypeptide chain is composed of 57 amino of lipoprotein either by precipitation at pH 3.7 or chroma- acids and lacks glycine, proline, cysteine, phenylalanine, tography on Sephadex G-75; preparation of murein from a histidine, and tryptophan. 63% of the amino acids are hy- with Pronase drophilic, but because of the covalently linked lipid this digest of murein-lipoprotein complex (EC structural membrane protein has very hydrophobic prop- 3.4.4), preparation of a lipopeptide (7) from such a digest, erties. partial acid hydrolysis of murein, isolation of a murein repeating unit or a portion of the murein peptide side chain The cell wall of Escherichia coli is a favorable system for containing in addition 1 mol of lysine and arginine (3, 4); obtaining insight into the molecular organization of biological amino-acid and amino-sugar analysis on a Unichrom amino- membranes by chemical means. It contains the so-called acid analyzer (Beckman, Munich). rigid layer, which is linked together by covalent bonds. The structure of this layer is therefore preserved even under Preparation of peptides rigorous isolation conditions during which other membranes The peptides used for determination of the amino-acid are dissociated into their individual components with ir- sequence were obtained from digests of the murein-lipo- reversible loss of their in vivo arrangement. protein complex with Pronase, thermolysin, subtilisin The basic structure of the rigid layer is the murein (peptido- (EC 3.4.4.16), or trypsin (EC 3.4.4.4). For example, glycan, mucopeptide), which consists of polysaccharide 3.2 g murein-lipoprotein complex in 300 ml of 0.01 M Tris- chains crosslinked by short peptide bridges (1). It forms a HCl buffer were digested for 2 hr at 60° with Pronase (pH net that encloses the whole cell. From our previous studies 7.5) or thermolysin (pH 8). Insoluble murein was removed (2-5) we concluded that, depending upon the size of the by a 2-hr centrifugation at 37,000 X g, and then lipopeptide cell, about 105 lipoprotein molecules are covalently linked to was precipitated by adjusting the pH oA the clear supernatant the murein of one cell. We further hypothesized that these to 3.2. The soluble peptides were chromatographed. lipoprotein molecules may represent the covalently anchored Special treatments were necessary to cleave the insoluble core of subunits of which the cell wall is built. The other com- lipoprotein with trypsin. Lipoprotein still bound to the ponents of the cell wall could be organized around the lipo- murein, lysozyme-released lipoprotein dispersed by soni- protein so that the pattern of lipoprotein molecules on the fication, lipoprotein treated with maleic anhydride (8), or murein would reflect the overall organization of the cell lipoprotein dispersed in 8 M urea and then diluted to 0.1 M wall. To support this model, we studied the structure of the urea were incubated with trypsin [protein: enzyme ratio 50:1 (w/w) I for 15 hr at 370 at pH 8-9. Hydrolysis with Abbreviations: Dpm, meso-2.6-diaminopimelic acid; MurNAc, trypsin was incomplete but fortunately yielded different N-acetylmuramic acid. peptide spectra. The soluble peptides were separated into * Present address: Max-Planck-Institut ffir Molekulare Genetik, groups by column chromatography on Dowex 50 X 2 with 1000 Berlin 33, Ihnestrasse 63-73, Germany. the following logarithmic gradient: starting with 0.1 M 970 Proc. Nat. Acad. Sci. USA 69 (1972) Structure of Murein-Lipoprotein 971 pyridine adjusted to pH 2 with formic acid, the molarity and as shown in Fig. 1. The sequence is presented to show maximal pH of the pyridine were raised to final values of 2 M and homology between peptides within the polypeptide chain. pH 5.0. The peptides were further purified on phospho- Such a degree of repetition has not previously been ob- cellulose with a gradient as used on Dowex 50 X 2, or on served (see, e.g., discussion in refs. 18 and 19). Starting from Dowex 1 X 2 (9) with an N-ethylmorpholine-pyridine- the third amino acid, a sequence of 14 amino acids is nearly acetate gradient. The purity of the peptides was checked by identically repeated by the next sequence of 15 amino acids paper chromatography with butanol-acetic acid-water followed again by 7 amino acids. The structural gene probably (4:1:5, upper phase), or, for basic peptides, methanol- coded originally for 15 amino acids; it doubled and the water-pyridine-12 N HCl (80:17.5:10:2.5). When only triplet coding for lysine or arginine at the C-terminal end a single spot was observed, the purity was evaluated by was deleted. Then, only half of the original gene was fused determination of the amino-acid composition with the amino- with the doubled gene. Now a more variable part of the acid analyzer. Peptides were usually hydrolyzed in 5.7 N protein starts whose descendence from the original gene is HCl for 20 hr at 1050. However, peptides containing amino obvious when all the following peptides are aligned so that sugars of the murein were hydrolyzed for 15 hr in 4 N HCl they end either with lysine or arginine. The observed ho- at 1050. The amino-acid sequence of peptides was mostly mologies are still striking, but several amino-acid deletions determined by the subtractive Edman procedure (10, 11), (17) have to be introduced. The repetitive N-terminal but the sequences of some were, in addition, directly deter- portion of the polypeptide chain, which has remained essen- mined from the cleaved phenylthiohydantoin derivatives tially constant, may reflect a high selection pressure during of the N-terminal amino acids by chromatography on starch- evolution that allowed only few, and then very conservative, paper (12) or on silica gel thin-layer plates (13). In addition, amino acid replacements (Ile/Val; Gln/Asn/Ser; Ala/Ser; sequential digestions of peptides by carboxypeptidases A and Ala/Thr; Lys/Arg).