Role of autolytic enzymes in the growth and morphogenesis of bacterial cell walls

J. theoret. Biol. (1971) 33, 63-75

Role of Autolytic Enzymesin the Growth and Morphogenesisof Bacterial Cell Walls J. S. THOMPSON Department of Biological Chemistry, University of Manchester, Manchester Ml3 9PL, England (Received 11 December 1970)

A model for bacterial cell wall synthesis is proposed based on recent biochemical and cytological data. Unlihe.~pmvious theories, cleavage of pre-existing mucop#&le to ‘provide &ceptor sites for mucopeptide synthesis is not a prerequisite for growth and expansion. On the contrary, it is proposed that polymerization occurs on a membrane-bound lipid precursor. The strip of septum produced extends away from the membrane. Autolytic enzyme(s) SQlit it into-two halves which grow towards opposite poles of the cell. Two factors are considered to be important in determining cell shape. First, the site of orientation of consecutive division planes. Second, the shape of the end of the:celi formed..du@rg cell division. The relative rates of wall synthesis and autolytic activity at the septum could control the latter feature ‘by analogy with the operation of a lathe, Jnterference with the balance between synthesis and autolysis is discussed with particular reference to, penicillin,,actibn. It is suggested that this balance determines whether elongation or septum formation occur. An explanation for the two qualitatively different effects of penicillin is proposed. At low penicillin concentrations, filaments are formed; higher concentrations result in classical lysis. 1. The Basic Model According to a recent model of cell. wall growth, synthesis occurs in the region nearest the hub of an annularly closing cross wall (Fig. 1; Higgins & Shockman, 1970). Incorporation of precursors at this Point results in eventual formation of both cross-wall and Peripheral wall. Conversion of crosswall to peripheral wall occurs by splitting the cross-wall in two by autolytic action. The region of autolytic activity extends well down into the septum in advance of the actual split (Higgins, Pooley & Shockman, 1970). It has been suggested that autolytic enzyme action is important not only for initiation of new sites of wall synthesis in old walls but also for cell separation throughout 63

/Wall band

(a)

Crass-wall

(d)

FIG. 1. Diagrammatic representation of wall division model for Streptococcus ficulis (Hiins & Shockman, 1970).This simulated time lapse sequencewas reconstructedfrom a large number of electron micrographs.At the top (a), a crosswall has nearly completely separateda dip1ococcusinto two daughter cells.A wall band rings the equator of the forming cell. All of the wall surrounding this coccushas been synthesizedin the preceding generation and is caged old wall (stippled).New wall growth beginsat the coccalequator. Between(a) and (b), two new wall bands were formed, and between them the external wall was notched or split. Limited centripetal cross wag growth occurred along with some extension of peripheral wall between the two wall bands. Both peripheral and crosswall extensionare thought to be due to synthetic activity in the area of the tip of the septum. However, between stages(b) and (c) centripetal penetration of the crosswall appears to remain relatively constant white the wall bands which mark the separation of new wall from old move apart. When the wag bands are at or near the equators of the two new daughter cells, at stage (c), centri#etal penetration of the cross wall resumes,to finally separatethe two daughter cells[stage(d)]. Before completion of the crosswall at stage(d), initiation of a new area of wall growth at the subequatorialportion is often observed.This is shown by the forming daughter coccuson the left at stage(c). The cyclets then repeated. Note that the cocci on the right at stages(a) and (c) are identical exceptfor the designation of wall age and that only the poles of the daughter cellscontain old wall. , old wall; q , new wall.

AUTOLYSIS

AND

BACTERIAL

65

MORPHOGENESIS

the division cycle (Higgins & Shockman, 1970). The model described in Fig. 2 takes this argument a step further by questioning whether, in fact, the provision of acceptor sites by autolytic activity is necessary at all. If the polysaccharide chain is spun out continuously from a lipid-bound carrier (Robbins, Bray, Dankert & Wright, 1967; Nikaido, 1968), then localized autolytic Cleavage

I

plane

,

Peripheral wall A

I

Region ‘of maturation

Region of outolytic enzyme action. Ingrowing septum

Region of tronspeptidation reaction. ‘-\ Nascent mucoaeatide chain linked td lipid carrier in close association with polymerase.

Mesosomal membrane extending both sides of the septum.

FIG. 2. Auto&tic enzymeaction in symmetricalcell wall replication. Thin linesrepresent the polysaccharideportion of mucopeptide; thick lines representthe peptide moiety. The diagram is intended to illustrate a concept and not representthe actual detailed vt of the polymers. The polysaccharidechains may be intertwined and the peptide units ‘ly regularly arranged. Although the peplinking the polysaccharidechainsare not necesmrr tide portion traversesthe cleavageplane in the diagram, this does not imply that the autolytic enzymebreakspeptide bonds. Separationinto two halvescould equally well occur by giycosidaseactivity at the sites indicated by the heavy arrows. The regions of transpeptidation and autolytic enxyme action may well overlap. Maturation may involve a loosen& of the network so that someof the chainscan stretchout and becomecross-linked to other chainswhich weresynthesizedbefore them. The site of cell wall thickening depends on the species(seetext). T.B. 5

66

I.

S. THOMPSON

breakdown to separate the cross-wall into two parts may be the only type of degradative activity required during normal growth, except perhaps to a very minor extent in the early stages of initiation of new division sites. Growth of mucopeptide chains outwards from a lipid membrane such as the mesosome would fit in nicely with Robbins’ model for the biosynthesis of complex oligosaccharides whereby addition of new units occurs at the reducing-terminal end of the chain (Robbins et al., 1967). All polymer synthesis occurs in or on the membrane and two carrier lipid molecules side by side, with polymerase molecules close by, can theoretically “spin out” a polysaccharide chain of indefinite length (Nikaido, 1968). The direction of growth of the mucopeptide chain has not yet been reported. Growth from the reducing end would accord with Nikaido’s model (1968) and Fig. 2; growth from the opposite end would require extracellular polymerases which would gradually work their way out from the membrane. Growth would presumably become less and less efficient as the precursors would have further to diffuse from the membrane, the further the chain extended away from it. There would also be a continuing need to start new chains at the membrane. Primers could presumably be supplied by autolysis, though the acceptor groups released by these enzymes are often inappropriate for existing models of biosynthesis (Landman, Ryter & Frthel, 1968; Tipper, 1969). Provision of acceptor sites by autolytic enzyme action would involve the production of localized weakness. This would have to be very closely controlled because of the considerable stress from the internal osmotic pressure of the cell. Shockman (1965) has written: “It is difficult to envision a mechanism for increasing the surface area of the bacterial cell wall that does not involve structural weakness at the areas of active wall synthesis.”

Weidel & Pelzer (1964) stated: “It is certain a priori that growth of a bag-shaped macromolecule with nothing but covalent bonds to keep it together requires continuous breakage of some of these bonds. This must happen wherever additional subunits, suitably activated, are to be inserted into the macromolecular structure.”

The present model avoids production of these weakened areas of cell wall, at least under conditions of balanced growth, by forming the new bonds before autolytic action, not after. The product of autolysin action is not therefore a weakened product, but represents the structure of the final wall, except, perhaps, for some further maturation by completion of cross-linking and thickening. It is proposed that production of a continuous ribbon of mucopeptide by addition at the reducing terminal end (Robbins et al., 1967; Nikaido, 1968) with subsequent addition of teichoic acid and other wall

AUTOLYSIS

AND

BACTERIAL

MORPHOGENESIS

67

polysaccharides, or covalently-linked lipoprotein (Braun & Wolff, 1970), is followed by closely controlled autolytic septum cleavage (Higgins & Shockman, 1970). This seems to provide a relatively simple and elegant model for wall growth on the experimental evidence at present available. It may be necessary to modify this model to allow for some degree of prefabrication in the mesosome, though apart from the similarity of some of the granular material in walls and mesosomes under the electron microscope (Ellar, Lundgren & Slepecky, 1967), there is little direct evidence for this; the mesosome-associated material could even represent degradation products (Higgins et al., 1970). In addition to septum formation and cell elongation, mucopeptide synthesis occurs during wall thickening (Shockman, 1965). There is no a priori reason why this should involve autolytic enzymes, provided sufficient acceptor groups are available for the addition of new mucopeptide units. Thickening occurs in cells which have entered the post-exponential phase of growth by restriction of protein synthesis; at this time, elongation and division are minimal. Under these conditions, autolytic activity is much less than in exponential phase cells (Shockman, 1965); it starts to increase again only after recovery from amino acid starvation has proceeded for about 30 to 70 minutes (Pooley & Shockman, 1969). Thickening is not confined to the postexponential phase, however, but seems to be a minor but normal feature of mid-exponential growth, occurring away from the sites of active autolytic enzyme in Streptococcus faecalis (Pooley & Shockman, 1970). The present model is entirely compatible with subsequent maturation and thickening of the wall. 2. Symmetry of Bacterial Growth

The models discussed so far (Figs 1 and 2) have assumed that extension of a bacterium is symmetrical on either side of a division plane. Early evidence for asymmetric growth at the poles of bacteria was obtained by relatively unsophisticated techniques and has tended to be discounted in more recent publications (Cole, 1965). A recent careful study suggests that growth may be unidirectional in small cells growing in minimal medium, bidirectional in the longer cells which predominate in rich medium (Donachie & Begg, 1970). If the mechanism of symmetrical cell wall synthesis proposed in Fig. 2 is to be of value, it should be able to provide a model for asymmetrical growth with a minimum of adaptation. Fig. 3(a) and (b) represents the asymmetrical and symmetrical mechanisms described in Fig. 6 of Donachie & Begg’s paper (1970). Figure 3(c) is a modification of Fig. 3(b). Figure 3(b) is incompatible with the growth models in Figs 1 and 2 because the growth sites are not

(a)

Plane of penicillin- sensilive sites ond ccl I divisions

TimeImin)

’

sites and ceil divisions (cl

3

(b)

4

2

sites a-d ceil divisions

I

Units of cell length

Plane of penicillin-sensitive

120

I IO

100

90

80

‘70

Time(min)

0

Plone of penicillin -sensitive

t

Units of cell length

FIG. 3. (a) Asymmetrical cell growth (after Donachie & Begg, 1970). (b) Symmetrical cell growth (after Donachie & Begg, 1970). (c) Symmetrical cell growth (present model). --f = direction of movement of newly synthesized wall material; the vertical line at the head or tail of the arrow indicates the growth site. In the imaginary experiment depicted, a unit cell is grown for one cell cycle in a medium where the mass doubling time is 60 min [3(a)]. At 60 mm, the daughter cell on the left is transferred to a richer medium where the mass doubling time is 30 min [3(b) and (c)]. The normal cell cycle for cells growing continuously in these two media is shown by the groups of cells connected by the arrow to the left of them. During a shift up of the kind shown here, there will be a 60 min interval between transfer to the new medium and the next cell division. Consequently, the length of cells at this division will be twice the length of the dividing cells in the old medium. For simplicity, the increase in cell volume after the shift up is assumed to result from increase in length without increase in diameter. Each growth site gives rise to two new sites of opposite polarities when the cell reaches a length of two unit cells.

60

50

Units of cell length 2 I Time (min) 1 I

AUTOLYSIS

AND

BACTERIAL

MORPHOGENESIS

69

identical with the sites of incipient cell division; this is rectified in Fig. 3(c) which seems to be equally consistent with Donachie & Begg’s data. Figure 3(a) and (c) is compatible with the more detailed mechanisms shown in Figs 4 and 2 respectively. h-existing wall.

Region of limited outolysis Involving the loosening ond exponsion of the existing wall, ond onostomosis between old ond new wall.

Region of tronseptidation reaction

Mesosomal membrone extending to the right hand side of the septum Nascent mucopeptide linked to lipid carrier

chain

FIO. 4. Autolytic enzyme action in asymmetrical cell wall replication. Thin lines and thick lines: as Fig. 2.

Asymmetrical wall growth may perhaps indicate that an active mesosome is located only on the side of the growing point which is actually extending. Conversion to symmetrical growth may involve expansion of the mesosome until it extends across the plane of growth; this may represent completion of a round of replication and separation of the two chromosomes by movement of the two attachment points to opposite sides of the potential division plane. It is interesting to note that Donachie & Begg (1970) observed asymmetrical growth only in minimal medium where cells with a single replicating chromosome are most likely to exist. 3. Explanation of Penicillin Action in Terms of the Model

The present model provides a plausible explanation for the two qualitatively different effects of penicillin on Escherichiu cdi in terms of the balance between autolytic activity and mucopeptide synthesis. Many other physio-

70

J.

S.

THOMPSON

logical treatments which produce localized weakness, particularly in the plane of incipient cell division probably involve similar effects on this balance (Shockman, 1965; Matheson & Donaldson, 1968; Higgins & Shockman, 1970). At low penicillin concentrations, a sharp drop in mucopeptide synthesis occurred over the concentration range (10 to 50 units of penicillin G/ml) in which cell division was specifically inhibited without affecting longitudinal growth (Schwarz, Asmus & Frank, 1969). This may indicate that the autolytic enzyme cleaves the newly synthesized wall so fast relative to the rate of mucopeptide synthesis that the septum never achieves net centripetal penetration and remains a vestigial structure associated with the suboptimally active growth site. They also observed a nick in the mucopeptide at the centre of the division plane analogous to that reported by Higgins & Shockman (1970); this suggests a basic similarity between Gram-positive and Gram-negative cell wall growth. At higher penicillin concentrations, longitudinal growth was inhibited and autolysis occurred (Schwarz et al., 1969). In this case, greater inhibition of mucopeptide synthesis would allow autolytic activity to overtake mucopeptide synthesis and kill the cells by cleavage in the plane where septum formation should have occurred. Thus when the penicillin effects are considered in terms of the balance of mucopeptide synthesis and autolysis, the two different actions may perhaps be explainable as quantitative rather than qualitative variants of the same basic process. Two factors are involved in the disturbance of this balance. Addition of penicillin G to Staphylococcus aureus H both inhibited mucopeptide synthesis and increased the sensitivity to autolysis. Mucopeptide synthesized in the presence of penicillin tends to be more susceptible to enzymic degradation, perhaps because it is less highly cross-linked and requires cleavage of fewer bonds to produce solubilization. In addition, there is a faster rate of attack in terms of bonds broken in penicillin-synthesized mucopeptide, presumably because decreased cross-linking lowers steric hinderance (Takebe, Singer, Wise & Park, 1970). Despite clear evidence that cell division is more penicillin-sensitive than cell elongation, the biphasic nature of the relationships between osmotic stability, mucopeptide synthesis and penicillin concentration is hard to correlate with the presence of two or more separate penicillin-sensitive processes in the growing cell. The difficulty in interpretation arises because measurements of mucopeptide synthesis were estimated from the incorporation of labelled diaminopimelic acid (Schwarz et al., 1969). Recoveries of mucopeptide are likely to be lower than the amount actually synthesized, since in the presence of penicillin G, the proportion of soluble mucopeptide polymer synthesized by an E. coli cell free system is considerably increased (Izaki, Matsuhashi & Strominger, 1966). In a cell free S. aureu.s system, mucopeptide synthesized in

AUTOLYSIS

AND

BACTERIAL

MORPHOGENESIS

71

the presence of penicillin was preferentially lost when isolation was carried out by the Park and Hancock technique (Takebe et al., 1970). This was attributed at least in part to degradation at 0°C during preparation. Nevertheless, walls and whole cells of highly autolytic preparations of Bacillus licheniformis N.C.T.C. 6346 which have not been penicillin-treated exhibit appreciable autolysis at 0°C (J. S. Thompson, unpublished work). It is therefore highly unlikely that Schwarz et al. (1969) achieved quantitative isolation of mucopeptide from penicillin-treated E. coli cells by their technique; this involved manipulations at 2°C over a period of at least an hour, followed by hot detergent treatment. 4. Polymer Orientation

and Autolysis as Determinants of Cell Shape

The model for bacterial cell wall synthesis outlined in Fig. 2 assumes that a mucopeptide ribbon is formed on the mesosome with the polysaccharide chains parallel to the plane of the septum. This provides a simple and aesthetically pleasing model of vectorial biosynthesis. An alternative model, based on the organization of the structurally related polymers, cellulose and chitin, implies that the polysaccharide chains run in opposite directions (Kelemen & Rogers, 1969); if such a structure is synthesized from one end only, the complexity of the process is increased, as in the biosynthesis of double-stranded DNA. Testing of these models by physical methods for the determination of polymer orientation is urgently required. Although some information has been obtained on the topography of the bacterial cell wall (Glauert & Thornley, 1969), the correlation of visible structures with chemical components is still at an early stage, particularly in Gram-positive bacteria. Physical methods such as X-ray diffraction and electron microscopy have so far provided no clue as to the macromolecular structure and organization of mucopeptide. Evidence exists suggesting that mucopeptide is anisotropic and speculations can be made about its properties in three mutually perpendicular directions. It is possible that the wall is stronger along the circumference of the cylinder than along the axis since photographs of tears in walls from mechanically broken cells usually show them occurring perpendicular to this axis. This could be due partially to weakening by limited autolysis at incipient division planes during preparation. However it would certainly be advantageous to the cell if the walls were stronger in the direction perpendicular to the longitudinal axis, since the shearing force from internal pressure in a cylinder is greater in a radial than in a longitudinal direction (Rogers, 1965). During autolysis of S. fuecalis, separation of the wall into bands parallel to the division plane occurs (Shockman & Martin, 1968). If the strands of wall material emerge from the septum and bend round as shown in Fig. 2, then this

72

J.

S.

THOMPSON

separation into bands away from the septal region bears a different and rather paradoxical relationship (cleavage perpendicular to the strands) to the long axis of the polymer strands compared with lysis in the plane of the septum (cleavage parallel to the strands). It should be remembered that the cleavage plane seen by electron microscopy need not correspond to the cleavage plane of the individual covalent bonds (Fig. 2). Whatever model of wall growth is eventually found to be correct, the existence of these bands, and the concentric bands in thickened walls of Bacillus subtilis mutants (Rogers, McConnell & Burdett, 1970), provide support for a non-amorphous mucopeptide structure and a stimulus to investigate its anisotropic nature. From Higgins & Shockman’s data (1970), it can be deduced that the outer surface of the cell wall has been produced by splitting down the centre of the developing septum with, in the case of S. faecalis, an autolytic muramidase. The outer surface of the wall should therefore contain reducing-terminal muramic acid residues. Figure 2 shows polysaccharide chains (thin lines) growing outwards parallel to the cleavage plane, then bending over at right angles; this may well be an oversimplification and represent only the general direction of movement during growth. Fibre nets occur in plant and fungal cell walls (Rogers, 1962). Mucopeptide chains could be intertwined to form a woven type of structure. By analogy with the movement of the warp during the manufacture of patterned textiles, this could involve displacement of the lipid carriers bearing the nascent chains by a form of membrane flow or oscillation. Consequently, polysaccharide chains may cross and recross the presumptive cleavage plane. It will be interesting to discover, in organisms containing more than one autolytic enzyme, how many enzymes are involved in cell separation. Investigation of the effects of phenotypic and genotypic alterations in the relative amounts of each enzyme on the biogenesis of structure and orientation in the septal region will be of vital importance to the understanding of the mechanism of cell division. Despite differences in mucopeptide composition and autolytic enzymes involved, cell division in Bacillus megaterium and S.faecalis probably exhibits an overall similarity, though the spatial and temporal organization of septum-splitting is different. Thickening of the cell wall and production of capsular material occurs at the septum in advance of the progress inwards of a constriction to separate the daughter Bacilli. Mesosomes appear to remain associated with this area until thickening is complete (Ellar et al., 1967). This contrasts with S. faecalis, where wall thickening occurs on newly synthesized wall just after separation (Higgins & Shockman, 1970). It remains to be seen whether these cytological variations in cell division can be related to taxonomic groupings in the same way as gross mucopeptide composition (Ghuysen, 1968).

AUTOLYSIS

AND

BACTERIAL

MORPHOGENESIS

73

Although synthesis is localized at regular spatial intervals in Grampositive walls, this may not be the case in Gram-negative bacteria (Tubergen & Setlow, 1961; Rogers, 1965; Rogers & Perkins, 1968). The latter group may contain a larger number of synthetic sites giving the impression of randomness, but certainly at least some zonal growth occurs in E. coli (Schwarz et al., 1969). Most of the evidence to the contrary has been derived from fluorescent antibody studies, which may sometimes be difficult to interpret. Mesosomes are readily seen in Gram-positive bacteria, and detailed evidence suggesting a role in cell division has been obtained (Ellar et al., 1967). They are much less prominent in Gram-negative bacteria, but under appropriate conditions, transverse septal divisions composed of membrane with a thin mucopeptide core have been observed (F&z-James, 1965; Rogers, 1970); this suggests that the basic division mechanism may not differ very much from that of Gram-positive bacteria. Rogers has discussed some of the geometrical implications concerned in the production of cell shape, particularly in Cocci. There has been much more discussion of this sort of problem in connection with plant cells, probably because in this case, the fibre directions can be determined, and a more fruitful interplay of theory and experiment is possible (Rogers, 1965). The peripheral wall is, apart from the ends of cells, a hollow cylinder, long in filaments, shorter in Bacilli, Bacilli and Cocci both possess rounded ends, and it may be profitable to seek biochemical unity by thinking of Cocci as Bacilli with almost infinitesimally short cylindrical sections. In both cases, once division has reached the stage at which the septum starts to constrict, the same problem of sealing off the hole between the daughter cells must be overcome. No longer can the cells continue to turn out a cylinder by simple elongation of strands at a fixed number of points as described above. There must be some method of reducing the number of fibres as the cross-sectional area decreases from the cylinder to the axial point at which sealing off occurs. This could be achieved by maintaining a fairly constant number of synthetic sites per unit length of the septal internal circumference. Final sealing of the pore must involve cross-linking between the few remaining fibres in the centre, perhaps closely followed by coating this area internally by the advancing wave of wall thickening. The cylindrical portion of the Streptococcal wall is synthesized by an annular area of slightly smaller diameter than the finished wall. The product is probably sufficiently flexible and elastic to be stretched to its final diameter by internal osmotic pressure as it grows away from the septum. Ou & Marquis (1970) have described the wall as a flexible polymer which probably owes its flexibility to the peptide portion; the glycan is likely to be fairly rigid, by analogy with chitin and cellulose. The wall has many of the proper-

74

J. S. THOMPSON

ties of polyelectrolyte gels, which change in volume as a result of pH and ionic strength affecting their ionic interactions. The tonus of the elastic structure is maintained by the balance of electrostatic interactions and the mechanical force of cell turgor pressure (Ou & Marquis, 1970). In the absence of this turgor pressure, bacterial walls collapse, but still retain the general shape of the cell. Apart from the length of the cylindrical section determining the shape of Bacillary forms, there must be other controls involved. The site and orientation of consecutive division planes determine the differences between Streptococci, Staphylococci and Sarcinae. Malfunction in the formation of division planes can result in abnormal cellular morphology. These changes may be phenotypic or genotypic; the exact steps involved are as yet imperfectly understood (Rogers, McConnell & Burdett, 1968). There must also be some control of the shape of the more or less rounded ends produced when bacteria divide. This shape is species-specific. Although hemispherical ends are common, they are not universal. Tapered ends occur in fusiform Bacilli and are presumably genetically controlled. As a corollary to the present model for cell growth, it is proposed that the relative rates of two processes contribute to the shape of the end: (i) longitudinal wall formation; (ii) constriction of the diameter of the mucopeptide septum by inward movement of the annulus of coordinated autolytic activity and wall synthesis. This is analogous to the determination of the shape of the end of a metal rod by the relative rates of longitudinal and inward movements of the cutting edge during the operation of a lathe. Complex shapes can be obtained when these two rates of movement are expressed by non-linear functions. There seems to be no simple relationship between the fine structure of mucopeptide and bacterial shape (Rogers, 1970). Perhaps fine structure, such as cross-linking, determines the strength, flexibility and extensibility of the wall, and the coarser features of cellular morphology are determined by a process akin to the lathe model. I would like to thank Professor G. R. Barker and Dr S. R. Ayad for helpful criticism during the preparation of this paper. REFERENCES BRAUN, V. & WOLFF, H. (1970). Eur. J. Biochem. 14, 387. COLE,R. M. (1965). Butt. Rev. 29, 326. DONACHE, W. D. & BEGG, K. J. (1970). Nature, Lond. 227, 1220. ELLAR,D. J., LUNDGREN, D. G. & SLEPECKY, R. A. (1967). J. Bact. 94, 1189. FITZ-JAMES, P. C. (1965). Symp. Sot. gen. Microbial. 15, 369. GHUYSEN, J. M. (1968). Bat. Rev. 32, 425. GLAUERT, A. M. & THORNLEY, M. J. (1969). A. Rev. Microbial. 23, 159. HIGGINS, M. L. & SHOCKMAN, G. D. (1970). J. Bat. 101, 643. HIGGINS,M. L., POOLEY, H. M. & SHOCKMAN, G. D. (1970). J. Bat. 103, 504.

AUTOLYSIS

AND

BACTERIAL

MORPHOGENESIS

75

IZAKI, K., MATSUHASHI, M. & STROMINGER, J. L. (1966). Proc. nutn. Acad. Sci. U.S.A. 55, 656. KBLBMEN, M. V. & ROGERS, H. J. (1969). J. gen. Microbial. 57, xiii. LANDMAN, 0. E., RYTER, A. & FRI?HEL, C. MATHI?SON, A. & DONALDSON, D. M. (1968). NIKAIDO, H. (1968). Adv. Enzymol. 31, 77. Ou, L-T, & MARQUIS, R. E. (1970). J. Bact. POOLEY, H. M. & SHOCKMAN, G. D. (1969). POOLEY, H. M. & SHOCKMAN, G. D. (1970). ROBBINS, P. W., BRAY, D., DANKERT, M. &

(1968). J. Butt. %, J. Butt. 95, 1892.

2154.

101, 92. J. Butt. J. Bat. WRIGHT,

100, 617. 103, 457.

A. (1967). Science, N. Y. 158, 15 36. ROGERS, H. J. (1962). Biochem. Sot. Symp. 22, 55. ROGERS, H. J. (1965). Symp. Sot. gen. Microbial. 15, 186. ROGERS, H. J. (1970). Bact. Rev. 34, 194. ROGERS, H. J. & PERKINS, H. R. (1968). Celf Walls and Membrunes. London: E. & F. N. Spon, Ltd. ROGERS, H. J., MCCONNELL, M. & BURDEN, I. D. J. (1968). Nature, Land. 219, 285. ROGERS, H. J., MCCONNELL, M. & BURDEN, I. D. J. (1970). J. gen. Microbial. 61, 155. SCHWARZ, U., Amus, A. 8c FRANK, H. (1969). J. molec. Biol. 41, 419. SHOCKMAN, G. D. (1965). Bact. Rev. 29, 345. SHOCKMAN, G. D. & MARTIN, J. T. (1968). J. Bat. 96, 1803. TAKEBE, I., SINGER, H. J., WISE, E. M. & PARK, J. T. (1970). J. Butt. 102, 14. TIPPER, D. J. (1969). J. Bat. 97, 837. TUBERGEN, R. P. VAN, & SETLOW, R. B. (1961). Biophys. J. 1, 589. WEIDEL, W. & PELZER, H. (1964). Adv. Enzymof. 26, 193.