Murein Hydrolases in the Envelope of Escherichia coli

Abstract

Properties of murein hydrolases, assumed to play an essential role in bacterial morphogenesis, are described. Evidence is presented that in the intact cell these enzymes are prevented from uncontrolled reaction. It is shown that the "barrier" between the enzymes and their substrate sacculus, a shape-maintaining macromolecule within the cell envelope, can be artificially broken. Assay systems to measure murein hydrolase activity in the intact cell, in isolated cell envelopes and after solubilization are described. A combination of Triton X-100 and sodium chloride is shown to liberate membrane-bound hydrolases effectively.Growth and division of a bacterial cell occur in concert with controlled modification of the cell shape. This modification involves variation of the sacculus, a shape-maintaining macromolecule which is integrated into the complex cell envelope. The sacculus consists of murein, a net-like polymer in which polysaccharide chains are intertied by short peptide bridges. I n the case of Escherichia coli these peptide bridges contain 2,6-diaminopimelic acid as an essential component (for a review see [l]). The closed murein net which completely surrounds the cell can be modified only if some of the existent covalent bonds are initially split (for a review see [ Z ] ) . This splitting has to be done by enzymes, murein hydrolases, which thus are directly involved in bacterial morphogenesis.A set of different types of murein hydrolases is found in E . coli [3,4]. This enzyme system comprises lysozyme, an N-acetylglucosaminidase [5], a transglycosidase [6,7], a muramyl-amidase [3,8], several endopeptidases [9] and a t least two carboxy-peptidases [4, lo]. The activity of a t least some hydrolases is subject to both temporal and spatial regulation; in the cellular life-cycle localized hydrolase action [ l l ] is triggered at a given stage of cell division [12]. The spatial and temporal control of hydrolase action might be correlated with the fact that most, if not all, murein hydrolases in E . coli are membrane- Eur. J. Biochem. 41 (1974) bound and thus in close contact with their substrate murein. This fact will be considered in more detail in the discussion of this paper.A study of the mechanisms by which temporal and spatial control of enzyme action is accomplished, has to start with an analysis of the properties of the complex murein hydrolase system and of its components. This paper deals with the measurement of overall murein hydrolase activity in intact cells and in isolated cell envelopes. Evidence will be presented suggesting the existence of a "barrier" between hydrolases and their substrate murein ; we speculate that it is involved in the regulation of enzyme action. I n addition, methods €or solubilization of murein hydrolases from the envelope, mentioned previously [6], are given in more detail. I n a forthcoming paper the purification and characteristics of one of the membrane-bound hydrolases, a penicillinsensitive endopeptidase, will be described. MATERIALS AND METHODS Strain, Culture Con...

Compartmentalization of murein hydrolases in the envelope of Escherichia coli

“…; 0.75 &i/pg, The Radiochemical Centre, Amersham), or 2-3H glycerol (0.6 E.tCi/l.tmol, The Radiochemical Centre, Amersham; 35 PM in medium equivalent to Penassay broth except that glucose was omitted). Radioactivity in aqueous samples was measured in a Triton-Toluene scintillator [4] , or on paper in a standard toluenebased scintillator. Enzyme tests were performed as described in the text; protein was determined [9] with tryst.…”

“…Murein hydrolases (review: 2), some of which might also function as transferases, consequently are assumed to be part of the system which produces, shapes and modifies the sacculus. E. culi contains a set of at least six different hydrolases [2, 31, some of which are membrane bound [4] and operate under topological and timed control: during cell division, hydrolase action is triggered at a given time in a defined area at the division site [5,6] .…”

“…Intimate contact between cytoplasmic membrane with the strongly bound endopeptidase [4,8], and the hydrolase substrate murein, is probably needed to permit enzyme action. How else could the reaction between a fixed enzyme and its macromolecular, nondiffusible substrate be achieved.…”

Enzymes Synthesizing and Hydrolyzing Murein in Escherichia coli

“…coli cells are known to possess a number of murein hydrolase activities (Fig. 2) within the cell envelope : transglycosylase [7], amidase [14,19], endopeptidase [12,14] and carboxypeptidase [9]. Endopeptidase and carboxypeptidase activities are found almost exclusively in the inner cytoplasmic membrane [13,15].…”

“…The undigested murein was precipitated with bovine serum albumin and trichloroacetic acid, and radioactivity in the supernatant measured as described by Hartmann et al [12].…”