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.     

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.     

Structural studies on the amino-acid-linked teichuronic acid of Bacillus subtilis AHU 1219 cell walls

Structural studies were carried out on a teichuronic acid isolated from a mild acid extract of Bacillus subtilis AHU 1219 cell walls. The teichuronic acid contained D-glucuronic acid, D-glucose, D-galactose, L-serine and L-threonine in a molar ratio of 1:1:1:0.5:0.5. Results of analyses of the polysaccharide by Smith degradation, methylation and 1H-NMR and 13C-NMR spectroscopy, in combination with data on analyses of oligosaccharides obtained by partial acid hydrolysis and alkaline hydrolysis of the polymer, led to the most likely structure for the repeating unit, ----4)(L-Ser/L-Thr)-D-GlcA(beta 1----3)-D-Glc(beta 1----4)-D-Gal(alpha 1----. In each unit, either amino acid is linked to the glucuronic acid residue through an amide bond.     

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.     

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.     

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 primary structure of teichuronic acid in Bacillus subtilis AHU 1031

Structural studies were carried out on the acidic polysaccharide fraction obtained from lysozyme digest of the cell walls of Bacillus subtilis AHU 1031. The polysaccharide fraction contained N- acetylmannosaminuronic acid ( ManNAcA ), N-acetylglucosamine (GlcNAc), glucose, glycerol and phosphorus in a molar ratio of 2:2:4:1:1, together with glycopeptide components. The results of analyses involving Smith degradation, chromium trioxide oxidation, methylation and proton magnetic resonance spectroscopy led to the conclusion that the backbone chain of the polysaccharide has the repeating unit----6)Glc(alpha 1----3/4) ManNAcA (beta 1----4)GlcNAc(beta 1----. About 50% of the N-acetylglucosamine residues in the backbone chain seem to be substituted at C-3 by the glycosidic branches, glycerol phospho-6-glucose, while the other half seem to be substituted by glucose.     

The menaquinol oxidase of Bacillus subtilis W23

The quinol oxidase appears to be mainly responsible for the oxidation of bacterial MKH₂ in Bacillus subtilis W23 growing with either glucose or succinate. The activity of the enzyme was maximum with dimethylnaphthoquinol, a water-soluble analogue of the bacterial menaquinol. Menadiol or duroquinol were less actively respired, and naphthoquinol was not oxidized at all. After fourtyfold purification the isolated enzyme contained 5.3 mol cytochrome aa ₃ per gram of protein and negligible amounts of cytochrome b and c. The turnover number based on cytochrome aa ₃ was about 10³ electrons · s^-1 at pH 7 and 37°C. The preparation consisted mainly of a M _r 57000 and a M _r 36000 polypeptide. The N-terminal amino acid sequence of the latter polypeptide differed from that predicted by the qoxA gene of B. subtilis strain 168 (Santana et al. 1992), in that asp-14 predicted by qoxA was missing in the M _r 36000 polypeptide.Abbreviations DMN 2,3-dimethyl-1,4-naphthoquinone - DMNH₂ 2,3-dimethyl-1,4-naphthoquinol - Duroquinol 2,3,5,6-tetramethyl-1,4-benzoquinol - MK menaquinone - MKH₂ menaquinol - NBH₂ 2,3-dimethoxy-5-methyl-6-(n-nonyl)-1,4-benzoquinol - TMPD N,N,N, N,-tetramethyl-1,4-phenylenediamine     

Genetic transformation and cell morphology of Bacillus subtilis grown in Mg+(+)-limited chemostat culture

The rate and frequency of genetic transformation of Bacillus subtilis grown in Mg+(+)-limited chemostat culture are dependent on the dilution rate (D) of the system and achieved maximum values at D = 0.23 h-1. Mg+(+)-limitation induced a morphological change in the cells from their normal rod shape to extended helices. Although this change in shape was a transient phenomenon, under some conditions it persisted for several days and resulted in an apparent increase in the transformation frequency.     

SP-10 bacteriophage-specific nucleic acid and enzyme synthesis in Bacillus subtilis W23.

Bacillus subtilis W23 was infected with a clear-plaque variant of SP-10 phage, namely, SP-10c. Exogenous thymidine was not incorporated into phage DNA (even in the presence of deoxyadenosine), nor was there any transfer of thymidine nucleotides from bacterial to viral DNA. The lytic program was unaffected by concentrations of 5-fluorodeoxyuridine sufficient to reduce bacterial DNA synthesis by greater than 95%. Although these data are consistent with the interpretation that thymidine nucleotides are excluded from phage DNA, formic acid digests of SP-10c DNA contained what appeared to be the four conventional bases; however, adenine and thymine were not recovered in equimolar yields. DNA-RNA hybridization and hybridization competition experiments were done. Synthesis of host RNA started to wane moments postinfection and stopped completely by 36 min. SP-10c coded for discrete classes of early and late RNA. The possibility of discrete subclasses of early RNA exists. Replication of the bacterial genome appeared to terminate 12 min postinfection. Degradation of the host DNA to acid-soluble material started at 36 min and, by the end of the latent period, greater than 90% of the host chromosome was hydrolyzed. Four apparent phage-coded enzymes have been identified. A di- and triphosphatase degraded dUTP, dUDP, dTTP, and dTDP (and, to a lesser extent, dCDP and d CTP) to the corresponding monophosphates; the enzyme had no apparent activity on dATP and dGTP. SP10c also coded for a DNA-dependent DNA polymerase, lysozyme, and a nuclease that degrades native bacterial DNA. Judging from the dependence of enzyme synthesis on the time of addition of rifampin (an inhibitor of the initiation of RNA synthesis), messengers for the di- and triphosphatase, as well as the nuclease, are transcribed from promoters that start to function 6 min postinfection. Promoters for polymerase and lysozyme did not become functional until 8 and 16 min postinfection, respectively.     

Some structural features of the teichuronic acid of Bacillus licheniformis N.C.T.C. 6346 cell walls

A teichuronic acid, containing glucuronic acid and N-acetylgalactosamine, was purified from acid extracts of Bacillus licheniformis 6346 cell walls as described by Janczura, Perkins & Rogers (1961). After reduction of the carboxyl function of glucuronic acid residues in the polysaccharide the reduced polymer contains equimolar amounts of N-acetylgalactosamine and glucose. Methylation of the reduced polysaccharide by the Hakamori (1964) technique showed the glucose residues to be substituted on C-4. A disaccharide, 3-O-glucuronosylgalactosamine, was isolated from partial acid hydrolysates of teichuronic acid. After N-acetylation the disaccharide produces chromogen readily on heating at pH7, in agreement with C-3 substitution of the reducing N-acetylamino sugar. Teichuronic acid also produces chromogen under the same conditions, with concurrent elimination of a modified polysaccharide from C-3 of reducing terminal N-acetylgalactosamine residues of the teichuronic acid chains. The number-average chain lengths of several preparations of teichuronic acid were estimated from the amounts of chromogen produced in comparison with the N-acetylated disaccharide. The values obtained are in good agreement with the weight-average molecular weight determined by ultracentrifugal analysis. The reducing terminals of teichuronic acid are shown to be exclusively N-acetylgalactosamine by reduction with sodium boro[(3)H]hydride. The number-average chain lengths of the teichuronic acid preparations were estimated by the extent of in corporation of tritium and are in agreement with values obtained by the other methods.