Distribution of muropeptides in walls of Bacillus subtilis and a temperature-sensitive mutant.

Attempts to correlate differences in cell shape with aspects of peptidoglycan structure were investigated. The parent strain, Bacillus subtilis 168, and its temperature-sensitive tagB mutant were grown in the chemostat under different growth conditions. The composition of the peptidoglycan was similar in all samples, regardless of cellular shape and anionic polymer content. Muropeptides, released by digestion with muramidase, comprised mainly dimers and monomers with only small amounts of trimer and traces of tetramer muropeptide. Overall, cross-linking did not vary greatly and the cross-linking index was less than 38%. Reverse-phase HPLC separation showed a complex fine structure. The principal muropeptides in all samples appeared to be the tetra monomer, tetra-tetra dimer and tetra-tetra-tetra trimer. While the major components looked the same in all samples, two specific components, a monomer and a dimer, were seen exclusively in the samples that had coccal morphology.

Cell wall assembly in Staphylococcus aureus: proposed absence of secondary crosslinking reactions.

The distribution of muropeptides formed by muramidase digestion of peptidoglycan from Staphylococcus aureus H was determined by gel-filtration HPLC. The observed crosslinking pattern supports the conclusion that incorporation of peptidoglycan in S. aureus proceeds by a similar mechanism to that proposed earlier for Bacillus megaterium. In this mechanism single glycan-peptide strands are incorporated into the sacculus by crosslinking reactions that take place only between the monomer muropeptide units of the incoming glycopeptide and muropeptides present in the innermost region of wall at the wall-membrane interface: such crosslinking reactions take place only during incorporation and no other crosslinking reactions occur. This assembly process has now been termed restricted monomer addition. The present analysis shows that the distribution of muropeptides in S. aureus peptidoglycan is in excellent agreement with that predicted by this mechanism. We propose that cell wall assembly in S. aureus proceeds via restricted monomer addition without any requirement for the secondary crosslinking reactions that have been suggested to occur in this organism. The high degree of crosslinking in S. aureus, 80% in this study, may result mainly from the freedom for crosslinking provided by the pentaglycine bridge peptide.

Cell wall assembly in Bacillus megaterium: incorporation of new peptidoglycan by a monomer addition process.

The pattern of cross-linking in the peptidoglycan of Bacillus megaterium has been studied by the pulsed addition of radiolabeled diaminopimelic acid. The distribution of label in muropeptides, generated by digestion with Chalaropsis muramidase and separated by high-performance liquid chromatography, stabilized after 0.15 of a generation time. The proportion of label in the acceptor and donor positions of isolated muropeptide dimers stabilized over the same period of time. The results have led to the formulation a new model for the assembly of peptidoglycan into the cylindrical wall of B. megaterium by a monomer addition process. Single nascent glycan peptide strands form cross-linkages only with material at the inner surface of the wall. Maturation is a direct consequence of subsequent incorporation of further new glycan peptide strands, and there is no secondary cross-linking process. The initial distribution of muropeptides is constant. It follows that the final pattern of cross-linking in the wall is determined solely by, and can be forecast from, this repetitive pattern of incorporation. In a modified form, this model can also be applied to assembly of cell walls in rod-shaped gram-negative bacteria.

Cell wall composition and surface properties in Bacillus subtilis: anomalous effect of incubation temperature on the phage-binding properties of bacteria containing varied amounts of teichoic acid.

Adsorption of bacteriophage SP50 to walls and heat-killed cells of Bacillus subtilis 168 appeared to be irreversible at both 37 and 0 degree C. Few, if any, active phage were desorbed when phage-wall complexes, formed at either temperature, were suspended in fresh medium. Bacteria rich in wall teichoic acid (TA) bound phage rapidly at both 0 and 37 degrees C, binding at the higher temperature being approximately twice as fast. Bacteria containing diminished proportions of TA showed less rapid phage adsorption but the reduction in rate was greater at 37 than at 0 degree C and bacteria containing only small proportions of TA bound phage more rapidly at 0 degree C than they did at 37 degrees C. These findings show that at low phage receptor density the temperature affects some component(s) involved in the phage-bacterium interaction such that the collision efficiency is increased at the lower temperature. The possible effect of temperature on the organization of bacterial surface components is discussed.

Cell wall assembly in Bacillus subtilis: partial conservation of polar wall material and the effect of growth conditions on the pattern of incorporation of new material at the polar caps.

The use of phage SP50 as marker for cell wall containing teichoic acid in Bacillus subtilis showed clear differences in the rates at which new wall material becomes exposed at polar and cylindrical regions of the wall, though the poles were not completely conserved. Following transition from phosphate limitation to conditions that permitted synthesis of teichoic acid, old polar caps fairly rapidly incorporated enough teichoic acid to permit phage binding. Electron microscopy suggested that the new receptor material spread towards the tip of the pole from cylindrical wall so that phages bound to an increasing proportion of the pole area until only the tip lacked receptor. Eventually, receptor was present over the whole polar surface. Direct electron microscopic staining of bacteria collected during transitions between magnesium and phosphorus limitations showed that new material was incorporated at the inner surface of polar wall and later became exposed at the outer surface by removal of overlying older wall. The apparent partial conservation of the pole reflected a slower degradation of the overlying outer wall at the pole than at the cylindrical surface, the rate being graded towards the tip of the pole. The relative proportions of the new wall material incorporated into polar and cylindrical regions differed in bacteria undergoing transitions that were accompanied by upshift or downshift in growth rate. These differences can be explained on the basis that growth rate affected the rate of synthesis of cylindrical but not septal wall.

Cell wall assembly in Bacillus subtilis: visualization of old and new wall material by electron microscopic examination of samples stained selectively for teichoic acid and teichuronic acid.

Uranyl acetate staining of thin sections allowed a distinction to be made between cell wall material that contains teichoic acid and that which contains teichuronic acid. The stain was used to study the pattern of wall assembly in Bacillus subtilis undergoing transitions between growth conditions leading to incorporation of the different anionic polymers. The results showed that new material is incorporated along the inner surface of the cylindrical region of the wall confirming, by a more direct method, results obtained earlier with teichoic acid specific phages. New material appears to be evenly distributed along the inner surface and no evidence was obtained for the presence of specific zones of incorporation.

Effect of culture pH on the D-alanine ester content of lipoteichoic acid in Staphylococcus aureus.

The lipoteichoic acid in Staphylococcus aureus growing at high pH values contained very little alanine ester, showing that high overall levels of substitution were not essential for growth. The low alanine content could have resulted from a progressive loss due to base-catalyzed hydrolysis of the labile ester linkages.

Relation between wall teichoic acid content of Bacillus subtilis and efficiency of adsorption of bacteriophages SP 50 and phi 25.

Efficient adsorption of bacteriophages SP 50 and phi 25 occurred only to bacilli that contained wall teichoic acid and neither phage bound to phosphate limited bacilli that contained teichuronic acid instead of teichoic acid. Though both phages require the presence of teichoic acid, their receptors are not identical. Efficient binding of phage phi 25 required the presence of greater proportions of teichoic acid in the wall and the receptor for this phage was destroyed when bacteria or isolated walls were heated at pH 4 whereas the ability of these samples to bind phage SP 50 was unaffected by such treatment. Efficient binding of phage SP 50 was not highly dependent on the presence of glucosyl substituents on the teichoic acid. Such substituents were required for phage phi 25 binding though their anomeric configuration appeared to be unimportant since the phages bound well to both strains W23 and 168, the wall teichoic acids of which carry glucosyl substituents of opposite anomeric configuration. The differences in the nature of the receptors may be of value in the use of the phages as probes for the location and distribution of teichoic acid in the wall.

Influence of phosphate supply on teichoic acid and teichuronic acid content of Bacillus subtilis cell walls.

Bacillus subtilis 168 was grown in chemostat culture in fully defined media containing a constant concentration of magnesium and concentrations of phosphate that varied from those giving phosphate-limited growth to those in which phosphate was present in excess and magnesium was limiting. Phosphate-limited bacteria were deficient in wall teichoic acid and contained less than half as much cellular phosphate as did bacteria grown in excess of phosphate. Approximately 70% of the additional phosphate in the latter bacteria was present as wall teichoic acid, indicating that the ability of the bacteria to discontinue teichoic acid synthesis when grown under phosphate limitation permits a substantial increase in their growth yield. Since not all of the additional phosphate is present as wall teichoic acid other cellular phosphates may also be present in reduced amounts in the phosphate-limited bacteria. The content of phosphate groups in walls of magnesium-limited bacteria was similar to the content of uronic acid groups in walls of phosphate-limited bacteria, and walls of bacteria grown in media of intermediate composition contained intermediate proportions of the two anionic polymers. Phage SP50, used as a marker for the presence of teichoic acid, bound densely to nearly all of the bacteria in samples containing down to 22% of the maximum content of teichoic acid. Apparently, therefore, nearly all of these bacteria contain teichoic acid, and the population does not consist of a mixture of individuals having exclusively one kind of anionic polymer. Bacteria containing less than 22% of the maximum content of teichoic bound in a nonuniform manner, and possible explanations for this are discussed.

Cell wall turnover in phosphate and potassium limited chemostat cultures of Bacillus subtilis W23.

Turnover in phosphate and potassium limited chemostat cultures of Bacillus subtilis W23 results in the release of over 80% of the wall material present at the time of chasing equilibrium-labelled cultures. The rate at which turnover proceeds is faster in potassium limited cultures than in phosphate limited cultures but in both cases a fraction of the wall material appears to be conserved, or to undergo turnover at a lower rate. Previously we have shown that the polar wall is less active metabolically than the cylindrical wall and it is possible that the apparently conserved wall is that present in the pole.

Cell wall assembly in Bacillus subtilis: location of wall material incorporated during pulsed release of phosphate limitation, its accessibility to bacteriophages and concanavalin A, and its susceptibility to turnover.

Addition of a pulse of phosphate to a phosphate-limited chemostat culture of Bacillus subtilis W23 led to the synthesis of teichoic acid and the consequent development by the bacteria of the ability to bind phage SP50. In cultures growing at different rates, phage-binding properties became maximal approximately one generation time after addition of the pulse. Removal of the incorporated teichoic acid by turnover also reached its maximum rate after a similar interval. After pulsed release of phosphate limitation in B. subtilis NCTC 3610, the alpha-glucosyl residues of the incorporated teichoic acid, detected by their interaction with concanavalin A, became maximally exposed at the same time that phage binding was maximum. At that time the bacteria bound phage all over the cylindrical part of the surface and at about one-third of the polar caps. That fraction of the receptor material that is exposed soon after its incorporation was distributed along the cylindrical length of most of the bacteria, but few phages bound to the polar caps, except in the case of short bacteria; these bound phages in a markedly asymmetric manner at one pole and along their length. The significance of these results is discussed in relation to the mode of assembly of the cell wall.

Effect of specific growth limitations on cell wall composition of Staphylococcus aureus H.

Conditions are described for the continuous culture of a derivative of Staphylococcus aureus H in a fully defined minimal medium in which cysteine is the sole amino acid. The effects of growth under various nutrient limitations on the composition and properties of the cell wall have been studied. The proportion of ribitol teichoic acid present in the wall, and the extent to which it is substituted with N-acetylglucosamine, varies in bacteria grown under different conditions as does the composition and extent of cross-linking of the peptidoglycan. Neither the derivative nor the original strain H produced teichuronic acid when grown under phosphate limitation.

Further evidence for the structure of the teichoic acids from Bacillus stearothermophilus B65 and Bacillus subtilis var. niger WM.

Bacillus stearothermophilus B65 and Bacillus subtilis var. niger WM both contain teichoic acids in their walls composed of glycerol, phosphate and glucose. The 13C nuclear magnetic resonance spectrum of B. stearothermophilus teichoic acid showed 13C-31P coupling on the signals from the C-5 and C-6 carbon atoms of the glucose molecule and an alpha-glucosidic linkage between glucose and the C-1 atom of the glycerol moiety. These data are consistent with a poly[glucosylglycerol phosphate] as the cell-wall teichoic acid in this organism. B. subtilis var. niger WM teichoic acid was oxidized by periodate and incubated in glycine buffer at pH 10.5. This treatment did not significantly increase the phosphomonoester content (by beta-elimination of the phosphate groups) of the teichoic acid molecule (7.1 to 9.5%), which is in accordance with earlier data derived from 13C nuclear magnetic resonance spectroscopy [De Boer et al. (1976) Eur. J. Biochem. 62, 1-6], that in this organism the glucose is not an integral part of the polymer chain. Similar treatment of B. stearothermophilus B65 teichoic acid increased the phosphomonoester content of the preparation from 0.15 to 68.1%.

The linkage of sugar phosphate polymer to peptidoglycan in walls of Micrococcus sp. 2102.

1. Protein-free walls of Micrococcus sp. 2102 contain peptidoglycan, poly-(N-acetylglucosamine 1-phosphate) and small amounts of glycerol phosphate. 2. After destruction of the poly-(N-acetylglucosamine 1-phosphate) with periodate, the glycerol phosphate remains attached to the wall, but can be removed by controlled alkaline hydrolysis. The homogeneous product comprises a chain of three glycerol phosphates and an additional phosphate residue. 3. The poly-(N-acetylglucosamine 1-phosphate) is attached through its terminal phosphate to one end of the tri(glycerol phosphate). 4. The other end of the glycerol phosphate trimer is attached through its terminal phosphate to the 3-or 4-position of an N-acetylglucosamine. It is concluded that the sequence of residues in the sugar 1-phosphate polymer-peptidoglycan complex is: (N-acetylglucosamine 1-phosphate)24-(glycerol phosphate)3-N-acetylglucosamine 1-phosphate-muramic acid (in peptidoglycan). Thus in this organism the phosphorylated wall polymer is attached to the peptidoglycan of the wall through a linkage unit comprising a chain of three glycerol phosphate residues and an N-acetylglucosamine 1-phosphate, similar to or identical with the linkage unit in Staphylococcus aureus H.

Cell wall assembly in Bacillus subtilis: development of bacteriophage-binding properties as a result of the pulsed incorporation of teichoic acid.

Addition of a pulse of excess phosphate to a phosphate-limited culture of Bacillus subtilis W23 resulted in the synthesis and incorporation of wall material that contained teichoic acid. Consequently, the bacteria regained the ability to bind phage SP50 although maximum phage-binding properties did not develop until approximately half a generation time after incorporation of teichoic acid had ceased. The present findings strongly support our earlier suggestion that newly synthesized receptor material is incorporated at the inner surface of the wall and becomes exposed at the outer surface only during subsequent growth.

Bacteriophage SP50 as a marker for cell wall growth in Bacillus subtilis.

When grown under conditions of phosphate limitation, Bacillus subtilis W23 lacked wall teichoic acid and did not adsorb phage SP50. During transition from growth under conditions of phosphate limitation to those of potassium limitation, the bacteria developed an ability to adsorb phage which increased exponentially in relation to their content of wall teichoic acid. During transition in the reverse direction, the bacteria retained near-maximum phage-binding properties until their content of wall teichoic acid had fallen to a fairly low level. These observations suggest that newly incorporated wall material does not immediately appear at the cell surface in a structure to which phage can adsorb. Examination of the location of adsorbed phage particles showed that recently incorporated receptor material appeared at the cell surface first along the length of the cylindrical portion of the cell. The results are consistent with models of wall assembly in which newly synthesized wall material is intercalated at a large number of sites that are distributed along the length of the cell. This newly incorporated material may be located initially at a level underlying the surface of the cell and may become exposed at the surface only during subsequent growth. Incorporation of new material may also proceed rapidly into the developing septa, but new wall material is incorporated into existing polar caps more slowly, or perhaps not at all.