A decrease of the absorbance a t 578 nm of a cell wall suspension of mid log phase E . coli occurs when the suspension is incubated with trypsin. The reaction is so rapid that 55O/, of the total decrease is obtained within the first 2 min [ratio of enzyme to total cell wall protein = I : 50 (w/w), room temperature]. The rate of the reaction is specific for trypsin when compared with other proteases, different lipases, lysozyme and other glycosidases. A peptide bond especially sensitive to trypsin could be localized within the complex cell wall by the demonstration that the decrease of the absorbance is paralleled by the splitting of the protein from the murein.This protein could be shown to be a lipoprotein with a part of the lipid probably covalently bound to the protein. It is called murein-lipoprotein. The link between the lipoprotein and the murein is lysine. After trypsin digestion lysine is the only additional amino acid remaining a t the murein. The ratio of the amount of lysine to the known constituents of the murein demonsstrates that on the average one lipoprotein molecule is covalently bound to every tenth repeating unit of the murein (N-acetylglucosamine-N-acetylmuramic acid-L-alanine-D-glutamic acidmeso-diaminopimelic acid-D-alanine). After 3 min incubation with trypsin, the isolated lipoprotein molecules have a lysine to arginine ratio of 4:4 as compared with 5 : 4 in the native molecule. The lipoprotein has an unusual amino acid composition since it contains about 65O/, polar amino acids and no glycine, cysteine, proline, phenylalanine, and histidine could be found. On a weight basis the lipoprotein accounts for more than 40°/, of the rigid layer. Since the murein is held together exclusively by covalent bonds one can get a fairly accurate idea of the distribution of the lipoprotein molecules in the cell wall. About lo5 lipoprotein molecules per cell should be distributed 103 A apart along the glycosidic chains of the murein.The lipoprotein has an important function in stabilizing the total structure of the cell wall.It seems that cleavage of only one peptide bond adjacent to the lysine link between the lipoprotein and the murein causes the rapid decrease of the absorbance and as shown by electron microscopic examination of ultrathin sections of trypsin treated cell walls, two separated membrane structures appear which otherwise are closely adjacent to one another.There are several lines of evidence suggesting that cell walls are more than mere cell envelopes protecting the essential cell functions which are supposed to take place in the cytoplasm. For example cell walls of E. coli not only contain the systems of active transport but interact with colicins (bacteriocins) in such a way that suggests that the whole complex memUnwnutl Abbreviation. Dansyl-, l-dimethylaminonaphthalene-5-sulfonyl-.Enzymes. Trypsin (EC 3.4.4.4); chymotrypsin (EC 3.4.4.5) ; papain (EC 3.4.4.10) ; pronase (EC 3.4.4) ; lysozyme (EC 3

A mutant of Escherichia coli K12 is described which, depending on temperature and Mg2+ concentration, can grow as a rod or sphere. The mutational defect was traced to an alteration in the cytoplasmic membrane. Rod-shaped cells and spherical cells did not differ measurably in the chemistry of their murein.With the aim of obtaining some insight concerning steps and control mechanisms involved in the assembly of the Escherichia coli cell envelope we have isolated a number of mutants with conditionally defective envelopes. Such mutants will grow, more or less normally, a t 30 "C and will lyse, virtually completely, a t 42 "C. Among them one was found which does not lyse a t 42 "C when Mg2+ is present at concentrations of 2 mM or greater. It was seen that in the presence of Mg2+ a t 42 "C this mutant grows as a sphere and a t 30 "C or below, with or without Mg2+, as a rod; on lowering the temperature from 42 "C to 30 "C spheres become rods again.The mutant was studied in more detail because it appeared to offer an opportunity to attack experimentally the question of the unknown basis of E . coli's normal morphology. Furthermore, it obviously was tempting to determine whether the morphological transitions could be used to learn a t least something about the biochemistry of bacterial morphogenesis, which as yet is rather mysterious.I n this first communication we present evidence that the altered component of the mutant is a component of the cytoplasmic membrane.Nomenclature and Abbreviations. Murein (synonyms : peptidoglycan, mucopeptide, glycosaminopeptide) is the chemical name of the bacterial polymer; sacculus, its morphological designation [21] ; a sacculus thus consists of murein ; dap, his, leu, lys, thi, thr, thy, trp, indicate inability to synthesize diaminopimelate, histidine, leucine, lysine, thiamine, threonine, thymine, and tryptophan, respectively; gal, lac, indicate inability to use galactose or lactose as sole carbon source; rod, loss of the ability to grow in rod shape.Enzymes. Lysozyme (EC 3.2.1.17); pronase (EC 3.4.4.-). _ _ _~ MATERIALS AND METHODS Media and ReagentsCells were grown with aeration in a complete Mutant IsolationStrain W945T3282, the parent of the mutant under study, was obtained from strain W945T1 (F-, thi, thr, leu, trp, lac, gal [I], in the following way. W945T1 was made his (diethylsulfate mutagenesis, penicillin selection), Zys (same procedure), and thy [2]. Penicillin selection for lysine requirement automatically selects for diaminopimelate-decarboxylasenegative mutants, as earlier blocks in the lysine biosynthetic pathway yield diaminopimelate requirers which will not survive starvation. 15 different lys derivatives were tested for "leakiness" by growth in the presence of [3H]diaminopimelate [3]. Only two were found not to incorporate any label into lysine. One of these was then made dap: among

Rod-shaped "ghosts" that are free of murein have been isolated from E. coli. The shape of these "ghosts" is maintained by a unit membrane soluble in sodium dodecyl sulfate. Ghosts consist of about 20-30% phospholipid (almost exclusively phosphatidylethanolamine) and 50-60% protein; a large fraction of the remaining material is lipopolysaccharide. Sodium -dodecyl sulfate-gel electrophoresis reveals 4-5 different bands corresponding to molecular weights between 10,000 and 40,000. Treatment of ghosts with Pronase reduces this number to 3, and the rod shape still is not lost. Results of treatment of ghosts with a crude extract from Dictyostelium discoideum have supplied tentative evidence that at least one of these proteins is involved in the maintenance of rod shape. It does not appear too unlikely that these polypeptide chains are the final products of the genetic information specifying cellular shape.E. coli is a rod-shaped organism and, as for all other bacteria, it is not known how this shape is determined, i.e., how genetic information is translated into cellular morphology. The expression of specificity for a certain morphology must, of course, involve assembly of the cell envelope. The E. coli cell envelope consists of three layers: cytoplasmic membrane, outer membrane or cell wall, and in between the two, the murein or peptidoglycan (1-3). The murein can be isolated intact and the isolated layer, the "sacculus," retains the shape and dimensions of the cell from which it was made (4).However, the specificity we are looking for cannot reside solely in the sacculus or in its precursors. This covalently closed net is not a self-assembly system. Thus, the information required to produce sacculi of a certain shape can only involve the relevant biosynthetic enzymes in connection with a matrix onto which murein is laid down.In this communication, we report purification and some properties of a membrane from E. coli K12 that is a good candidate for such a matrix. MATERIALS AND METHODSCells, Media, and Growth Conditions. Strain W945-T3282 (subsequently called 3282), which among other markers (5) carries diaminopimelate and lysine auxotrophies, was grown at 300 in Antibiotic Medium no. 3 (Difco) under aerobic conditions. The medium was supplemented with diaminopimelate (20 ,ug/ml) and thymine (50 ,ug/ml).Isolation of Ghosts. All operations were performed at 20-250, and all solutions contained 8 mM MgSO4.Procedure I: Cells grown to 5 to 8 X 108/ml were collected by centrifugation, washed once with 0.85% NaCl, and suspended, per g wet weight, in 10 ml of 20 mM Tris HCl (pH 7.5) containing 40% sucrose. Lysozyme was added (150 2033 Ag/ml) and was allowed to act until >90% of the cells lysed upon dilution into water (about 2 hr). About 0.1 volume of chloroform was added; the suspension was shaken vigorously and then left overnight. Viscosity was broken by forcing the suspension through a hypodermic needle. The cells were centrifuged (10 min, 16,000 X g) and resuspended in the same volume of 20 mM Tris HCl (pH 9). C...

The surface area per repeating murein unit (i.e. per molecule of diaminopimelate) has been determined for the cell envelopes of the Escherichia coli strains K-12 and W. This area was constantly found to be 1.3 nm 2 . Using this value and other previously determined properties of E. coli murein, a three-dimensional model of murein is proposed. The model specifies a monomolecular layer in which disaccharide units are each 1.03 nm long, and the polysaccharide chains, all parallel, are 1.25 nm apart. The cross-linking peptide side-chains have the same atomic coordinates and are arranged above or below the polysaccharide chains.

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...