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.     

The teichuronic acid of cell walls of Bacillus subtilis W23 grown in a chemostat under phosphate limitation.

Cell walls of Bacillus subtilis W23 contain teichuronic acid when grown in a chemostat under phosphate limitation at a low dilution rate, but teichoic acid at a higher dilution rate. The teichuronic acid was purified and shown to be a polymer of glucuronic acid and N-acetylgalactosamine.     

Cell wall metabolism in Bacillus subtilis subsp. niger: effects of changes in phosphate supply to the culture

Chemostat cultures of Bacillus subtilis subsp. niger WM were exposed to changes in the availability of phosphorus by means of a resuspension technique. Responses in wall metabolism were recorded by measuring the amounts of peptidoglycan and anionic polymers (teichoic or teichuronic acid) in the wall and extracellular fluid fractions. With respect to the wall composition, the effect of a change in orthophosphate supply was a complete shift in the nature of the anionic polymer fraction, the polymer originally present in the walls ("old" polymer) being replaced by the alternative ("new") anionic polymer. The peptidoglycan content of the walls remained constant. It was concluded that the incorporation of old polymer was completely blocked from the moment the orthophosphate supply was changed. However, from a measurement of the total amount of polymer in the whole culture during the course of the experiments, it was evident that synthesis of old polymer continued, but it was secreted. Synthesis of the new polymer started immediately, and it was incorporated exclusively into the wall. During adaption of the cells to the new environment, wall turnover continued in an identical fashion to that extant in steady-state cultures. It was concluded that the primary adaptive response to a change in orthophosphate supply occurred through a mechanism interacting with polymer incorporation and thus at the level of wall assembly at the membrane.     

Control of teichoic acid and teichuronic acid biosynthesis in chemostat cultures of Bacillus subtilis var. niger

1. Quantitative determination of the anionic polymers present in the walls of Bacillus subtilis var. niger organisms undergoing transition, in a chemostat culture, from either Mg(2+)-limitation to PO(4) (3-)-limitation or K(+)-limitation to PO(4) (3-)-limitation showed that teichuronic acid synthesis started immediately the culture became PO(4) (3-)-limited and proceeded at a rate substantially faster than the rate of biomass synthesis. 2. Simultaneously, the cell-wall teichoic acid content diminished at a rate greater than that due to dilution by newly synthesized wall material, and fragments of teichoic acid and mucopeptide accumulated in the culture extracellular fluid. 3. Equally rapid reverse changes occurred when a PO(4) (3-)-limited B. subtilis var. niger culture was returned to being Mg(2+)-limited. 4. It is concluded that in this organism both teichoic acid and teichuronic acid syntheses are expressions of a single genotype, and a mechanism for the control of synthesis of both polymers is suggested. 5. These results are discussed with reference to the constantly changing environmental conditions that obtain in a batch culture and the variation in bacterial cell-wall composition that is reported to occur throughout the growth cycle.     

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.     

Lipoteichoic acid from Bacillus subtilis subsp. niger WM: isolation and effects on cell wall autolysis and turnover.

Lipoteichoic acid (LTA) was extracted by means of hot aqueous phenol from Bacillus subtilis subsp. niger WM cells grown under various conditions in chemostat culture. The extracts were partially purified by nuclease treatment and gel permeation chromatography. Chemical analyses revealed a composition consistent with a polyglycerol phosphate polymer. The influence on autolysis of the LTAs thus obtained was studied with both whole cells and autolysin-containing native walls of B. subtilis subsp. niger WM. Lysis rates of phosphate-limited cells could be reduced to about 40% of the control rate by the addition of LTA, whereas lysis of cells grown under phosphate-sufficient conditions was affected to a much lesser extent. The lysis of native walls prepared from variously grown cells proved to be fairly insensitive to the addition of LTA. The effect of LTA on wall turnover was studied by following the release of radioactively labeled wall material during exponential growth. The most obvious effect of LTA was a lowered first-order rate of release of labeled wall material; calculations according to the model for cell wall turnover in Bacillus spp. formulated by De Boer et al. (W. R. De Boer, F. J. Kruyssen, and J. T. M. Wouters, J. Bacteriol. 145:50-60, 1981) revealed changes in wall geometry and not in turnover rate in the presence of LTA.     

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.     

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%.     

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.     

Role of teichuronic acid in Bacillus licheniformis: defective autolysis due to deficiency of teichuronic acid in a novobiocin-resistant mutant.

nov-12, a novobiocin-resistant mutant of Bacillus licheniformis ATCC 9945, grows as long chains of cells, a characteristic of autolytic-deficient (Lyt-) mutants. Isolated walls from nov-12 autolyzed at a rate equal to 5% of that displayed by wild-type walls, thus confirming the Lyt- phenotype. Protein-free nov-12 walls displayed marked resistance to, and also failure to bind, added autolysin solubilized from wild-type walls. Comparison of isolated cell walls revealed a deficiency in teichuronic acid in the mutant. Lesser differences were observed in walls of this strain, including a reduction in galactose, an increase in the proportion of peptidoglycan, and small quantitative differences in peptidoglycan composition though the proportions of protein and teichoic acid were similar in walls of both strains. Autolytic sensitivity was studied in walls in which protein, teichoic acid, and teichuronic acid were removed successively by selective extraction procedures. Autolysis of wild-type walls was unaffected by removal or protein or teichoic acid, but teichuronic acid removal rendered wild-type walls as insensitive to autolysis as mutant walls had been throughout. Therefore, in this mutant, deficiency in teichuronic acid alone leads to the Lyt- phenotype and, hence, activity and binding of autolysin(s) are dependent upon teichuronic acid but not teichoic acid. Also, the potential rate of autolysis of cell walls in this organism was correlated with the proportion of teichuronic acid in the wall. The possible significance of these findings with respect to control of autolysis and cell separation is discussed.     

The cell wall of Bacillus licheniformis N.C.T.C. 6346. Linkage between the teichuronic acid and mucopeptide components

1. After extraction of teichoic acid from cell walls of Bacillus licheniformis with dilute alkali, the insoluble residue contains the teichuronic acid and mucopeptide components and a small amount of residual phosphorus. 2. A complex of teichuronic acid and a part of the mucopeptide was isolated from the soluble fraction obtained by lysozyme treatment of alkali extracted walls. 3. Small-molecular-weight mucopeptide fragments, not containing teichuronic acid, are obtained from the soluble fraction in yields similar to those obtained after treatment of whole walls or acid-extracted walls with lysozyme. 4. The covalent linkages between teichuronic acid and mucopeptide are broken by treatment with dilute acid. The release of teichuronic acid chains is accompanied by the hydrolysis of N-acetylgalactosaminide linkages and the exposed N-acetylgalactosamine residues form chromogen under very mild conditions, indicating that they are substituted on C-3. 5. The initial rate of formation of reactive N-acetylgalactosamine residues during mild acid hydrolysis is parallel to the rate of extraction under the same conditions of teichuronic acid from alkali-treated insoluble walls, and to the rate of acid hydrolysis of glucose 1-phosphate. 6. The results suggest that the teichuronic acid chains are attached through reducing terminals of N-acetylgalactosamine residues to phosphate groups in the mucopeptide. 7. Muramic acid phosphate was isolated from the insoluble mucopeptide remaining after extraction of walls with dilute alkali followed by dilute acid.     

Major sites of metal binding in Bacillus licheniformis walls.

Isolated and purified walls of Bacillus licheniformis NCTC 6346 his contained peptidoglycan, teichoic acid, and teichuronic acid (0.36 mumol of diaminopimelic acid, 0.85 mumol of organic phosphorus, and 0.43 mumol of glucuronic acid per mg [dry weight] of walls, respectively). The walls also contained a total of 0.208 mumol of metal per mg. When these walls were subjected to metal-binding conditions (T. J. Beveridge and R. G. E. Murray, J. Bacteriol. 127:1502-1518, 1976) for nine metals, the amount of bound metal above background ranged from 0.910 mumol of Na to 0.031 mumol of Au per mg of walls. Most were in the 0.500-mumol mg-1 range. Electron-scattering profiles from unstained thin sections indicated that the metal was dispersed throughout the wall fabric. Mild alkali treatment extracted teichoic acid from the walls (97% based on phosphorus) but left the peptidoglycan and teichuronic acid intact. This treatment reduced their capacity for all metals but Au. Thin sections revealed that the wall thickness had been reduced by one-third, but metal was still dispersed throughout the wall fabric. Trichloroacetic acid treatment of the teichoic acid-less walls removed 95% of the teichuronic acid (based on glucuronic acid) but left the peptidoglycan intact (based on sedimentable diaminopimelic acid). The thickness of these walls was not further reduced, but little binding capacity remained (usually less than 10% of the original binding). The staining of these walls with Au produced a 14.4-nm repeat frequency within the peptidoglycan fabric. Sedimentation velocity experiments with the extracted teichuronic acid in the presence of metal confirmed it to be a potent metal-complexing polymer. These results indicated that teichoic and teichuronic acids are the prime sites of metal binding in B. licheniformis walls.     

The isolation of structural components present in the cell wall of Bacillus licheniformis N.C.T.C. 6346

1. Four of the known components of wall preparations of vegative cells of Bacillus licheniformis N.C.T.C. 6346 have been isolated free of each other after successive treatments of the walls with trichloroacetic acid and lysozyme: (a) a mucopeptide consisting of glucosamine, muramic acid, alphain-diaminopimelic acid, glutamic acid and alanine in the molar proportions 1.0:0.8:1.0:1.2:1.7; (b) an insoluble protein; (c) teichoic acid containing phosphorus and glucose in equimolar amounts; (d) teichuronic acid containing equimolar amounts of N-acetylgalactosamine and glucuronic acid, as found by Janczura, Perkins & Rogers (1961). 2. Evidence has been obtained for the presence in the soluble fraction obtained by lysozyme treatment of whole walls of a stable covalent complex of the teichoic acid and the mucopeptide components. 3. The molar ratio of phosphorus to glucose in the teichoic acid present in intact walls or the soluble fractions obtained by extraction of the walls with lysozyme or trichloroacetic acid is 1.0:0.25, in contrast with values of about unity obtained for the purified teichoic acid. 4. Intact walls have been shown to contain polyribitol phosphate chains bearing different amounts of glucose substituents. 5. Trichloroacetic acid extracts of walls also contain polyribitol phosphate compounds of different chain lengths. Dialysis of trichloroacetic acid extracts removes the short chains of polyribitol phosphate that have been found to carry only very low amounts of glucose side chains. By contrast, the longer chains present in the non-diffusible fraction contain phosphorus and glucose in almost equimolar amounts.     

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.     

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.     

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.     

Activation and inactivation of synthesis of secondary wall polymers in Bacillus subtilis W23

The progress of activation and inactivation of synthesis of the wall polymers, teichoic acid and teichuronic acid, in response to changes in the phosphate content of the growth medium has been examined using toluenised cells of B. subtilis W23. Activation of teichoic acid synthesis from nucleotide precursors was independent of protein synthesis, but chloramphenicol prevented activation when DL-glycerol 3-phosphate and CTP replaced CDP-glycerol as one of the substrates of the reaction. Activation of teichuronic acid synthesis was dependent on synthesis of protein. Inactivation of synthesis of both polymers was slowed, but not prevented, by inhibition of protein synthesis. Evidence was obtained that a protein synthesised during phosphate starvation retards the activation of teichoic acid synthesis.