Effects of carbon source and growth rate on cell wall composition of Bacillus subtilis subsp. niger.

A study was made to determine whether factors other than the availability of phosphorus were involved in the regulation of synthesis of teichoic and teichuronic acids in Bacillus subtilis subsp. niger WM. First, the nature of the carbon source was varied while the dilution rate was maintained at about 0.3 h-1. Irrespective of whether the carbon source was glucose, glycerol, galactose, or malate, teichoic acid was the main anionic wall polymer whenever phosphorus was present in excess of the growth requirement, and teichuronic acid predominated in the walls of phosphate-limited cells. The effect of growth rate was studied by varying the dilution rate. However, only under phosphate limitation did the wall composition change with the growth rate: walls prepared from cells grown at dilution rates above 0.5 h-1 contained teichoic as well as teichuronic acid, despite the culture still being phosphate limited. The wall content of the cells did not vary with the nature of the growth limitation, but a correlation was observed between the growth rate and wall content. No indications were obtained that the composition of the peptidoglycan of B. subtilis subsp. niger WM was phenotypically variable.     

Influence of growth condition on the concentration of potassium in Bacillus subtilis var. niger and its possible relationship to cellular ribonucleic acid, teichoic acid and teichuronic acid

1. Mg(2+)-limited Bacillus subtilis var. niger, growing in a chemostat in a simple salts medium, contained considerably more potassium and phosphorus than Mg(2+)-limited Aerobacter aerogenes growing in a similar medium at corresponding dilution rates. 2. Growth of the bacillus in a K(+)-limited environment did not lower the cellular potassium and phosphorus contents, the molar proportions of cell-bound magnesium, potassium, RNA (as nucleotide) and phosphorus being approximately constant at 1:13:5:13 (compared with 1:4:5:8 in Mg(2+)-limited or K(+)-limited A. aerogenes). 3. Growth of B. subtilis in a phosphate-limited environment caused the cellular phosphorus content to be lowered to a value similar to that of Mg(2+)-limited A. aerogenes, but the potassium content was not correspondingly lowered; the molar potassium:magnesium ratio varied from 14 to 17 with changes in dilution rate from 0.4 to 0.1hr.(-1). 4. Whereas over 70% of the cell-bound phosphorus of Mg(2+)-limited or K(+)-limited A. aerogenes was contained in the nucleic acids, these polymers accounted for less than 50% of the phosphorus present in similarly limited B. subtilis; much phosphorus was present in the walls of the bacilli, bound in a teichoic acid-type compound composed of glycerol phosphate and glucose (but no alanine). 5. Phosphate-limited B. subtilis cell walls (from organisms grown at a dilution rate of 0.2hr.(-1)) contained little phosphorus and no detectable amounts of teichoic acid, but 40% of the cell-wall dry weight could be accounted for by a teichuronic acid-type compound; this contained a glucuronic acid and galactosamine, neither of which could be detected in the walls of Mg(2+)-limited B. subtilis grown at a corresponding rate. 6. It is suggested that the high concentration of potassium in growing B. subtilis (compared with A. aerogenes) results from the presence of large amounts of anionic polymer (teichoic acid or teichuronic acid) in the bacillus cell walls.     

Teichoic acid is an essential polymer in Bacillus subtilis that is functionally distinct from teichuronic acid

Wall teichoic acids are anionic, phosphate-rich polymers linked to the peptidoglycan of gram-positive bacteria. In Bacillus subtilis, the predominant wall teichoic acid types are poly(glycerol phosphate) in strain 168 and poly(ribitol phosphate) in strain W23, and they are synthesized by the tag and tar gene products, respectively. Growing evidence suggests that wall teichoic acids are essential in B. subtilis; however, it is widely believed that teichoic acids are dispensable under phosphate-limiting conditions. In the work reported here, we carefully studied the dispensability of teichoic acid under phosphate-limiting conditions by constructing three new mutants. These strains, having precise deletions in tagB, tagF, and tarD, were dependent on xylose-inducible complementation from a distal locus (amyE) for growth. The tarD deletion interrupted poly(ribitol phosphate) synthesis in B. subtilis and represents a unique deletion of a tar gene. When teichoic acid biosynthetic proteins were depleted, the mutants showed a coccoid morphology and cell wall thickening. The new wall teichoic acid biogenesis mutants generated in this work and a previously reported tagD mutant were not viable under phosphate-limiting conditions in the absence of complementation. Cell wall analysis of B. subtilis grown under phosphate-limited conditions showed that teichoic acid contributed approximately one-third of the wall anionic content. These data suggest that wall teichoic acid has an essential function in B. subtilis that cannot be replaced by teichuronic acid.     

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.     

Cell wall teichoic acid as a reserve phosphate source in Bacillus subtilis.

Although exponential growth of Bacillus subtilis 168 in a phosphate-limited medium halted with the exhaustion of inorganic phosphate, the bacteria continued to grow at a slower rate for a further 3 to 4 h at 37 degrees C. This postexponential growth in the absence of an exogenous phosphate supply was accompanied by a loss of teichoic acid from the cell walls of the bacteria. Quantitative analysis of walls and culture fluids showed that the phosphate loss from the walls could not be accounted for by an increase in phosphate-containing compounds in the medium, which implied that the cells were using their own wall teichoic acids to supply phosphate necessary for growth. Addition of exogenous teichoic acid to phosphate-starved cultures resulted in stimulation of growth and in the simultaneous disappearance of teichoic acid phosphate from the medium. It is proposed that teichoic acids, which can contain more than 30% of the total phosphorus of exponential-phase cells, can be used as a reserve phosphate source when the bacteria are starved for inorganic phosphate.     

Effect of Temperature on the Growth and Cell Wall Chemistry of a Facultative Thermophilic Bacillus

The morphology and cell wall composition of Bacillus coagulans, a facultative thermophile, were examined as a function of growth temperature. The morphology of the organism varied when it was grown at different temperatures; at 37 C the organism grew as individual cells which increased in length with increasing growth temperature. At 55 C it grew in long chains of cells. Cell wall prepared from cells grown at 37 C contained 44% teichoic acid by weight, whereas cells grown at 55 C contained 29% teichoic acid. Teichoic acid from these cells was a polymer of glycerol phosphate containing galactose and ester alanine. The ratio of ester alanine to phosphate was significantly higher in cell walls and teichoic acid from 37 C-grown cells compared with those from 55 C-grown cells. Other differences observed were that cells grown at 55 C contained a lower level of autolytic ability, produced cell walls which bound more Mg(2+), and contained less peptide cross-bridging in its peptidoglycan layer than cells grown at 37 C.     

Cell wall metabolism in Bacillus subtilis subsp. niger: accumulation of wall polymers in the supernatant of chemostat cultures

Cell wall polymers were measured both in the cells and in the cell-free medium of samples from steady-state chemostat cultures of Bacillus subtilis, growing at various rates under magnesium or phosphate limitation. The presence of both peptidoglycan and anionic wall polymers in the culture supernatant showed the occurrence of wall turnover in these cultures. Variable proportions of the total peptidoglycan present in the culture samples were found outside the cells in duplicate cultures, indicating that the rate of peptidoglycan turnover is variable in B. subtilis. Besides peptidoglycan, anionic wall polymers were detected in the culture supernatant: teichoic acid in magnesium-limited cultures and teichuronic acid in phosphate-limited cultures. In several samples, the ratio between the peptidoglycan and the anionic polymer concentrations was significantly lower in the extracellular fluid than in the walls. This divergency was attributed to the occurrence of direct secretion of anionic polymers after their synthesis.     

Control of teichoicand teichuronic acid biosynthesis in Bacillus subitlis 168trp−: Evidence for repression of enzyme synthesis and inhibition of enzyme activity

Phosphate starvatiion induced teichuronic acid synthesis in cells of Bacillus subtilis 168trp^? which had previously been grown with excess phophate. This induction was prevented when protein synthesis was inhibited immediately prior to phosphate starvation and under these conditions cells continued to form teichoic acid. The converse was true when phosphate was added to cells previously grown in phosphate-limited chemostat. The increase in teichoic acid synthesis normally following phosphate addition was prevented by chlorampehnicol or amino acid starvation and cells continued to make teichuronic acid. The suggestion that repression of enzyme synthesis is involved in controlling the type of wall polymer made was supported by the low levels of UDP-glucose dehydrogenase found in cells grown with excess phosphate and of CDP-glycerol pyrophosphorylase in phophate-limited cells. The greater amounts of teichoic acid made under phosphate limitation and of teichuronic acid with excess phosphate when protein synthesis was also inhibited indicated that modulation of enzyme activity occurs. Glycerol starvation of a glycerol-requiring mutant did not derepress teichuronic acid synthesis, indicating that glycerol-containing intermediates do not act as repressors.     

Binding of magnesium ions to cell walls of Bacillus subtilis W23 containing teichoic acid or teichuronic acid.

When grown in a chemostat under various nutritional conditions, cells of Bacillus subtilis W23 produce walls containing teichoic acid or teichuronic acid. The binding of Mg2+ to these walls and to the isolated anionic polymers in solution was measured by equilibrium dialysis. In solution the ribitol teichoic acid bound Mg2+ in the molar ratio Mg2+/P=1:1 with an apparent association constant (Kassoc.) of 0.61 X 10(3)M-1, and the teichuronic acid bound Mg2+ in the ratio Mg2+/CO2-=1.1, Kassoc.=0.3 X 10(3)M-1. Cell walls containing teichuronic acid exhibited closely similar binding properties to those containing teichoic acid; in both cases Mg2+ was bound in the ratio Mg/P or Mg/CO2- of 0.5:1 and with a greater affinity than displayed by the isolated polymers in solution. It was concluded that Mg2+ ions are bound bivalently between anionic centres in the walls and that the incorporation of teichoic acid or teichuronic acid into the walls gives rise to similar ion-binding and charged properties. The results are discussed in relation to the possible functions of anionic polymers in cell walls.     

Morphology and anionic polymer content in the cell wall of a glycerol-requiring mutant ofBacillus subtilis

A glycerol-requiring mutant ofBacillus subtilis formed irregular spheres and showed disturbed septum formation, when subjected to growth limitation by the supply of glycerol. Under phosphate limitation the cells were also round and developed asymmetric septa. In magnesium-limited cultures the cells contained a thickened wall, as compared with that of the parent strain grown under the same conditions. Chemical analysis revealed the presence of teichoic acid as the major anionic polymer in the wall of the glycerol-, as well as the magnesium-limited cells of the glycerol-requiringB. subtilis mutant.Under phosphate limitation teichuronic acid was the only anionic polymer present in the wall. Thus, in this respect, there were no apparent differences between mutant organisms and the parent strain when grown under magnesium and phosphate limitation, respectively and the observed morphological deviations could not be correlated with an altered anionic polymer content of the wall.     

The wall content and composition of Bacillus subtilis var. niger grown in a chemostat

Bacillus subtilis var. niger was grown in a chemostat with various growth limitations and at various growth rates. The wall content and composition of the organism grown under these conditions were determined. The wall content, expressed as a percentage of the dry weight of organisms, varied with the growth rate. Analysis of wall samples showed that their composition also varied, particularly with respect to the phosphorus content. Wall samples extracted with trichloroacetic acid under carefully controlled conditions were found to contain various amounts of phosphorus, this being present as a glycerol phosphate polymer containing hexose (glucose and in some cases galactose), i.e. a teichoic aid. Teichoic acids were present in the walls of organisms grown under all conditions except when phosphorus limited growth. Then a different anionic polymer, composed of glucuronic acid and N-acetylgalactosamine (a teichuronic acid), was present. Under the specific growth conditions at pH7.0 and 35 degrees C in a chemostat, teichoic acid and teichuronic acid appeared to be mutually exclusive.     

Control of teichoic acid synthesis in Bacillus licheniformis ATCC 9945

Analysis of cell walls of Bacillus licheniformis ATCC 9945 grown under phosphate limitation showed that teichoic acid could be replaced by teichuronic acid under these conditions. Teichuronic acid, however, was always present in the walls to some extent irrespective of the growth conditions. The enzymes involved in teichoic acid synthesis were investigated and the synthesis of these was shown to be repressed when the intracellular Pi level fell. CDP-glycerol pyrophosphorylase was studied in some detail and evidence is presented to show that the enzyme is inactivated under phosphate-limited conditions. The mechanism of inactivation is unknown but it has been shown that it does not require protein synthesis de novo.     

The interaction of magnesium ions with teichoic acid.

The binding of Mg2+ to the wall teichoic acid of Lactobacillus buchneri N.C.I.B. 8007 was measured by equilibrium dialysis at controlled ionic concentration and pH. In an aqueous solution containing 10mM-NaCl at pH 5.0 one Mg2+ ion was bound for every two phosphate groups of the teichoic acid, with an apparent association constant, Kassoc. = 2.7 x 10(3) M-1. On lowering the pH below the pKa of the phosphate groups the amount of bound Mg2+ decreased concomitantly with decreasing ionization of the phosphate groups. Both the amount of Mg2+ bound to the teichoic acid and the apparent association constants were similar in the presence of 10 mM concentrations of NaCl or KCl but decreased markedly in the presence of 10 mM-CaCl2 because of competition between Ca2+ and Mg2+ for the binding sites. A similar effect was found when the concentration of NaCl was increased from 0 to 50 mM. The results are discussed in relation to the function of teichoic acid in the walls of Gram-positive bacteria.     

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

Efficient adsorption of bacteriophages SP 50 and 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 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 dependant on the presence of glucosyl substituents on the teichoic acid. Such substituents were required for phage 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 differeneces 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.Non-Standard Abbreviation PAE Phage adsorption efficiency, as defined by Archibald and Coapes (1976)     

Cell Wall Composition and Associated Properties of Methicillin-Resistant Staphylococcus aureus Strains

Methicillin-resistant (MR) Staphylococcus aureus strains have previously been reported to be deficient in surface negative charge; this has been correlated with methicillin resistance and ascribed to a deficiency of teichoic acid at the cell surface (A. W. Hill and A. M. James, Microbios 6:157-167, 1972). Teichoic acid was present in walls of MR organisms as revealed by appreciable phosphate levels and detection of ribitol residues. Phosphate levels in walls from five MR strains (0.54 to 0.77 mumol/mg of wall) were lower than in three unrelated methicillin-sensitive (MS) strains (0.86 to 1.0 mumol/mg of wall). However, two MS strains derived from two of the MR strains had wall phosphate levels very similar to those of the MR strains. No evidence for unusual wall polymers was found. Simple deficiency of wall teichoic acid does not result in methicillin resistance since an independently isolated teichoic acid-deficient strain (0.1 mumol of phosphate per mg of wall) was not methicillin resistant. In studies of biological properties possibly related to wall teichoic acid, it was discovered that walls isolated from MR organisms grown in the presence of methicillin autolyzed more rapidly than those isolated from organisms grown in the absence of the drug. Since methicillin resistance is enhanced by NaCl and suppressed by ethylenediaminetetraacetate, the effects of these compounds on autolysis of isolated walls were studied. NaCl (1.0 M) and ethylenediaminetetraacetate (1.0 mM) inhibited the autolysis of walls isolated from MR and MS strains. An MR strain bound phage 47, 52A, and 3A only slightly less well than their respective propagating strains.     

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.     

The function of teichoic acids in cation control in bacterial membranes

1. The effects of teichoic acids on the Mg(2+)-requirement of some membrane-bound enzymes in cell preparations from Bacillus licheniformis A.T.C.C. 9945 were examined. 2. The biosynthesis of the wall polymers poly(glycerol phosphate glucose) and poly(glycerol phosphate) by membrane-bound enzymes is strongly dependent on Mg(2+), showing maximum activity at 10-15mm-Mg(2+). 3. When the membrane is in close contact with the cell wall and membrane teichoic acid, the enzyme systems are insensitive to added Mg(2+). The membrane appears to interact preferentially with the constant concentration of Mg(2+) that is bound to the phosphate groups of teichoic acid in the wall and on the membrane. When the wall is removed by the action of lysozyme the enzymes again become dependent on an external supply of Mg(2+). 4. A membrane preparation that retained its membrane teichoic acid was still dependent on Mg(2+) in solution, but the dependence was damped so that the enzymes exhibited near-maximal activity over a much greater range of concentrations of added Mg(2+); this preparation contained Mg(2+) bound to the membrane teichoic acid. The behaviour of this preparation could be reproduced by binding membrane teichoic acid to membranes in the presence of Mg(2+). Addition of membrane teichoic acid to reaction mixtures also had a damping effect on the Mg(2+) requirement of the enzymes, since the added polymer interacted rapidly with the membrane. 5. Other phosphate polymers behaved in a qualitatively similar way to membrane teichoic acid on addition to reaction mixtures. 6. It is concluded that in whole cells the ordered array of anionic wall and membrane teichoic acids provides a constant reservoir of bound bivalent cations with which the membrane preferentially interacts. The membrane teichoic acid is the component of the system which mediates the interaction of bound cations with the membrane. The anionic polymers in the wall scavenge cations from the medium and maintain a constant environment for the membrane teichoic acid. Thus a function of wall and membrane teichoic acids is to maintain the correct ionic environment for cation-dependent membrane systems.     

Alterations in the composition and bacteriophage-binding properties of walls of Staphylococcus aureus H grown in continuous culture in simplified defined media.

A nutritional mutant of Staphylococcus aureus H has been isolated and grown in media in which the only amino acids are arginine, cysteine, glutamic acid and proline. Walls of the bacteria grown in such media in continuous culture under potassium limitation differ in composition from walls of the bacteria grown in batch culture in rich nutrient broth in that they contain less glycine, the peptidoglycan component is less highly cross-linked and the teichoic acid component contains a reduced proportion of N-acetylglucosaminyl substituents. Walls of the potassium-limited bacteria retain the ability to bind bacteriophage 52a but are more susceptible to the action of lytic peptidases than are wall samples in which the peptidoglycan is more highly cross-linked. Teichoic acid was present in walls of the bacteria grown under phosphate limitation in the defined medium and these walls were also able to absorb bacteriophage 52a.     

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.     

Ratio of Teichoic Acid and Peptidoglycan in Cell Walls of Bacillus subtilis Following Spore Germination and During Vegetative Growth

Cell walls were isolated from cells of Bacillus subtilis strain Marburg during synchronous outgrowth of spores, during the two synchronous cell divisions which followed, and at various times during exponential and early stationary growth. The amounts of teichoic acid and peptidoglycan components were determined in each cell wall preparation. The peptidoglycan is composed of hexosamine, alanine, diaminopimelic acid, and glutamic acid. The ratio of these was relatively constant in the cell walls at each stage of growth. The teichoic acid is composed of glycerol, phosphate, glucose, and ester-linked alanine. With the exception of glucose and ester-linked alanine, the ratios of these components were relatively constant throughout the growth cycle. There was a slight increase in the glucose content of the teichoic acid as the cells aged. There was no correlation between the amount of ester-linked alanine and the stage of growth. The ratio of teichoic acid (based upon phosphate content) to peptidoglycan (based upon diaminopimelic acid content) remained at nearly a constant level throughout the growth cycle. The conclusion is presented that these two cell wall polymers are coordinately synthesized during spore outgrowth and throughout the vegetative growth cycle.