Cell wall teichoic acids from Streptomyces daghestanicus VKM Ac-1722T and Streptomyces murinus INA-00524T

The structure of cell wall teichoic acids was studied by chemical methods and NMR spectroscopy in the type strains of two actinomycete species of the "Streptomyces griseoviridis" phenetic cluster: Streptomyces daghestanicus and Streptomyces murinus. S. daghestanicus VKM Ac-1722T contained two polymers having a 1,5-poly(ribitol phosphate) structure. In one of them, the ribitol units had alpha-rhamnopyranose and 3-O-methyl-alpha-rhamnopyranose substituents; in the other, each ribitol unit was carrying 2,4-ketal-bound pyruvic acid. Such polymers were earlier found in the cell walls of Streptomyces roseolus and Nocardiopsis albus, respectively; however, their simultaneous presence in the cell wall has never been reported. The cell wall teichoic acid of Streptomyces murinus INA-00524T was is a 1,5-poly(glucosylpolyol phosphate), whose repeating unit was [-6)-beta-D-glucopyranosyl-(1 --> 2)-glycerol phosphate-(3-P-]. Such a teichoic acid was earlier found in Spirilliplanes yamanashiensis. The 13C NMR spectrum of this polymer is presented for the first time. The results of the present investigation, together with earlier published data, show that the type strains of four species of the "Streptomyces griseoviridis" phenetic cluster differ in the composition and structure of their teichoic acids; thus, teichoic acids may serve as chemotaxonomic markers of the species.     

The purification and properties of two staphylolytic enzymes from Streptomyces griseus

1. Two staphylolytic enzymes have been purified from cultures of a soil isolate of Streptomyces griseus. 2. The purified enzymes were shown to be basic proteins of low molecular weight. Each enzyme released N-acetylmuramic acid reducing groups from the cell walls of Staphylococcus aureus. 3. The enzymes lysed whole staphylococci best at higher pH values and lower ionic strengths than when the substrate was isolated cell walls or purified mucopeptide. 4. Added teichoic acid did not inhibit the enzymes, but it formed an ethanol-precipitable complex with them. 5. The possibility that teichoic acid on the surface of whole cells prevents the access of the enzymes to their mucopeptide substrate is discussed.     

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.     

Use of bacteriophage-resistant mutants to study the nature of the bacteriophage receptor site of Staphylococcus aureus

Bacteriophage-resistant strains of Staphylococcus aureus H were isolated after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. Cell walls isolated from about half of these resistant strains were incapable of inactivating phages and were shown to lack N-acetyl-d-glucosamine (GlcNAc) in their cell wall teichoic acid. Apart from the lack of GlcNAc, two of these mutant strains were deficient in cell wall phosphorus and ester-linked d-alanine. These two strains were also found to be resistant to both phage K and a host-range mutant isolated from the parent phage. These two phages could lyse the other phage-resistant mutants which lacked GlcNAc in their teichoic acid. Cell walls from the remaining phage-resistant mutant strains did inactivate phages and were found to have normal cell wall teichoic acid. Although GlcNAc in teichoic acid was required for phage inactivation, no difference in phage inactivation ability was detected with cell walls isolated from strains of S. aureus having exclusively alpha- or exclusively beta-linked GlcNAc in their cell wall teichoic acid.     

Composition of cell walls of 2 mutant strains of Streptomyces chrysomallus

The cell walls and peptidoglycans of two mutant strains, Streptomyces chrysomallus var. carotenoides and Streptomyces chrysomallus var. macrotetrolidi, were studied. The strains are organisms producing carotenes and antibiotics of the macrotetrolide group. By the qualitative composition of the peptidoglycans the mutants belong to Streptomyces and are similar. Their glycan portion consists of equimolar quantities of N-acetyl glucosamine and muramic acid. The peptide subunit is presented by glutamic acid, L, L-diaminopimelic acid, glycine and alanine. The molar ratio of alanine is 1.2-1.3. The mutant strains differ in the content of carbohydrates, total phosphorus and phosphorus belonging to teichoic acids. Teichoic acids of the cell walls of the both strains are of the ribitolhosphate nature. The cell walls of the mutants contain polysaccharides differing from teichoic acids and consisting of glucose, galactose, arabinose and fucose. The influence of the cell wall composition of the mutant strains on their morphology and metabolism and comparison of the data relative to the mutant strains with those relative to the starting strain are discussed.     

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.     

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.     

Teichoic acids from streptomyces griseus

Summary During the Schmidt-Thannhauser fractionation procedure at nucleic acid determinations of Streptomyces griseus, a phosphorous-containing compound(s) was found which could not be taken as nucleic acid or polyphosphate. The hydrolisate of the isolated substance proved to have almost the same composition as teichoic acid from Staphylococcus aureus Duncan.Teichoic acid was found both intracellular and in the isolated cell wall of Streptomyces griseus mycelia.In the cell wall only the ribitol type of teichoic acids could be detected but not the glycerol type.     

Comparative biochemical study of 2 natural inactive variants of the actinomycin C producer Actinomyces sp. 26-115 with varying sensitivity to actinomycin

Two natural variants of the actinomycin C-producing organism Actinomyces sp-26-115, i.e. H1 and H2 differ in their sensitivity to exogenic actinomycin, colony morphology, growth dynamics on the synthetic medium and stability to ultrasound and lysozyme. Both variants synthesize no actinomycin. Variant H1 is sensitive to exogenic actinomycin, while variant H2 is resistant to it. Variants H1 and H2 have some similarity in the composition of membrane proteins. Still, they differ in the protein molecular masses, which are equal to 600000--500000, 220000, 130000. The active variant A and nonactive variant H2 have the most similar compositions of membrane proteins. These variants are also close in their growth dynamics, colony morphology, sensitivity to ultrasound and lysozyme. The membranes of all the variants studied contain phosphatidyl ethanol amide as the main phospholipid component. Insignificant differences are observed only with respect to the minor components. The content of teichoic acids in the cell walls of variant H2 is very high, slightly changes during the developmental stage and insignificantly increases on addition of actinomycin to the medium. The cell wall of variant H1 contains less amounts of teichoic acids. During the developmental stage they are liberated from the wall at a higher rate than peptidoglycan. The sensitivity to actinomycin does not increase with an increase in the culture age. It is probable that teichoic acid of the cell wall is one of the factors providing resistance to actinomycin in variant H2. It may be considered as a barrier preventing transport of exogenic actinomycin into the cell.     

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.     

Regulation of bacterial cell walls: correlation between autolytic activity and cell wall turnover in Staphylococcus aureus.

Cell wall turnover was examined in parent and mutant strains of Staphylococcus aureus. Peptidoglycan and teichoic acid were observed to undergo turnover in the wild-type strain during exponential growth; however, the rate of turnover did not decrease when the growth rate slowed, as the culture entered stationary phase. Isolated native cell walls and crude soluble autolytic enzyme were prepared from cells harvested during exponential and postexponential phases of growth. Native cell walls from both phases of growth autolyzed in buffer at identical rates; similarily, crude soluble enzyme from both preparations degraded radioactive cell walls at the same rate. Therefore, the activity of the autolysin in both exponential and postexponential cells was similar. The autolysis of whole cells of a mutant tar-1 was enhanced by 1.0 M NaCl. When 1.0 M NaCl was present under growing conditions, the rate of cell wall turnover was greatly increased. The presence of chloramphenicol, which inhibits whole-cell autolysis, also inhibited turnover. Analysis of the cell wall material recovered from spent medium revealed products consistent with the known mode of action of the endogenous autolysin. It is concluded that cell wall turnover in S. aureus is independent of the stage of culture growth but is dependent instead on the activity of the autolysin.     

Association of lack of cell wall teichuronic acid with formation of cell packets of Micrococcus lysodeikticus (luteus) mutants.

Morphological mutants of Micrococcus lysodeikticus (luteus) were isolated by treatment with N-methyl-N'-nitro-N-nitrosoguanidine. They occurred on plates in large, regular cell packets, whereas the parent cells usually grew as groups of two or four cells or as short chains. The mutants required a much higher concentration of Mg2+ for growth than the parent cells. The concentrations of Mg2+ and other components of the culture medium tested did not significantly affect the morphology of either the parent or mutant strains. The mutant strains were not agglutinated by antiserum to M. lysodeikticus, which mainly interacts with teichuronic acid on the cell surface, and chemical analysis of isolated cell walls of the mutants indicated the absence of teichuronic aicd. No significant differences were detected between the parent and mutant strains in the amounts of other cell wall components, e.g., peptidoglycan, protein, and teichoic acid. They possible roles of teichuronic acid in cell separation and attachment of divalent cations are discussed.     

Cell wall teichoic acids of streptomycetes of the phenetic cluster 'Streptomyces fulvissimus'

Structures of the anionic polymers of streptomycetes Streptomyces fulvissimus VKM Ac-994(T), Streptomyces longispororuber VKM Ac-1735(T), Streptomyces aureoveticillatus VKM Ac-48(T) and Streptomyces spectabilis INA 00606 belonging to the phenetic cluster 'S. fulvissimus' were investigated by chemical and NMR spectroscopic methods. A teichoic acid from the cell wall of S. spectabilis INA 00606 was studied in more detail, and this was shown to represent 1,3-poly(glycerol phosphate) substituted with glucosamine (alpha-D-GlcNAc) and L-glutamic acid (non-stoichiometric substitution). For the first time, glutamic acid is identified as an acyl substituent in teichoic acids of streptomycetes. The polymer chain is built of the following fragments: Cell walls of other streptomycetes of the phenocluster under study contain 1,3-poly(glycerol phosphates) with glucosamine as a glycosyl substituent at O-2 of the glycerol phosphate units and L-glutamic acid and lysine as O-2 acyl substituents. Not all amino sugar residues in the polymers of these strains are N-acetylated, and the content of the glucosamine and lysine residues in the polymers of different strains is not the same. Despite certain quantitative differences in the structures of the polymers, one may consider streptomycetes of the phenocluster 'S. fulvissimus' as closely related microorganisms, the details of the structures serving as additional criteria for the determination of the species status of a strain under study.     

Function of cell wall teichoic acid in thermally injured Staphylococcus aureus.

Thermally injured cells of Staphylococcus aureus lack the ability to grow on tryptic soy agar containing 7.5% NaCl. This injury phenomenon was examined in three strains of S. aureus: MF-31; H (Str); and, isolated from H (Str), 52A5, a mutant which lacks teichoic acid in the cell wall. Temperatures for sublethal heat treatment were selected to produce maximum injury with minimum death for each strain. Examination of isolated cell walls showed that magnesium was lost from the wall during heating, and that the degree of cell injury was accentuated when magnesium ions were either removed from or made unavailable to the cell. S. aureus 52A5 was more heat sensitive than its parent strain. Cells containing higher levels of wall teichoic acid generally showed less injury than normal cells. Cells with the weaker cation-binding polymer, teichuronic acid, in the cell wall generally showed greater injury. These data suggest that cell wall teichoic acid of S. aureus aids in the survival of the cell by the maintenance of an accessible surface pool of magnesium.     

Asymmetric distribution of charge on the cell wall of Bacillus subtilis.

The cell wall of Bacillus subtilis is capable of binding different kinds of metal ions. The wall-ion complex appears to be dependent on both phosphoryl from teichoic acid and carboxylate from peptidoglycan. In the present study, cationized ferritin (CF) was used as a probe for charge distribution on the wall of B. subtilis 168. Detergent-extracted cell walls bound CF only on the outer wall face. Completed cell poles bound CF, but septa did not. When the walls were permitted to autolyze briefly, binding of CF occurred on both faces. In contrast, limited hydrolysis of the walls by egg white lysozyme resulted in the penetration of CF into the wall matrix. When walls were made teichoic acid-free, CF-binding asymmetry was preserved, suggesting that carboxyl groups were oriented toward the surface. Walls with carboxylates chemically neutralized also retained charge asymmetry. Phosphate-free and carboxyl-modified walls bound CF only poorly or not at all. These results indicate that negative charges contributed by both phosphate and carboxyl are responsible for the binding of CF and that the observed asymmetry in the distribution of the label is due to the orientation of teichoic acid and muramyl peptides toward the outside of the cell wall, above the plane of the glycan strands.     

Characteristics ofStreptomyces globisporus strain 0234A forming endospores in submerged cultures

Thermosensitive submerged endospores formed byStreptomyces globisporus 0234 and its natural variant A resembled those of thermoresistant actinomycetes not only in their morphology and ultrastructure, but also in the content of dipicolinic acid. The production of endospores containing this substance is unusual inStreptomyces while other features of the strain indicate relatedness to other streptomycetes. Chemotaxonomic analysis of variant A revealed the cell wall to be of chemotype I and fatty acid content typical ofStreptomyces. Most characteristics of surface cultures of variant A coincided with those of the original strain 0234 and its endosporeless variant B. Both the strain 0234 and its variants A and B produced identical antibiotics and pesticidal compounds.     

The properties of cell wall hydrolysates of Streptococcus group A]

Products obtained from lysis in the cell wall of group A streptococcus have been studied in different growth phases: at the end of the exponential phase and in the stationary one. Endo-beta-N-acetylmuramidase extracted from the culture liquid of Streptomyces levoris 96 has been used for lysis of streptococcus. It is stated that streptococcus cell walls isolated at different growth stages differ in the protein and polysaccharide content. High content of protein in the cell wall of a young culture makes lower the initial rate of the walls' hydrolysis by endo-beta-N-acetylmuramidase. However, with the enzyme penetration into peptidoglycan the rate of hydrolysis of cell walls gets higher and after four-hour incubation the lysis degree of walls of the 16- and 8-hour cultures reaches the equal value (63%). Studies in the protein composition of lysates of the streptococcus cell walls have shown that they contain at least 12 proteins most of which are acid and neutral ones.     

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.     

An electron microscopic study of the location of teichoic acid and its contribution to staining reactions in walls of Streptococcus faecalis 8191.

The location of the glucosylated teichoic acid in whole cells and isolated walls of Streptococcus faecalis 8191 has been investigated using ruthenium red, gold-labelled concanavalin A and concanavalin A-peroxidase-diaminobenzidine. Dense laminae were revealed in sections of osmium-fixed walls stained with ruthenium red which corresponded to similar regions stained by uranyl and lead. Such regions were not seen after teichoic acid had been extracted, suggesting that the uptake of stain was by teichoic acid. However, these regions were not labelled on exposure to gold concanavalin A or concanavalin A-peroxidase-diaminobenzidine; these stains indicated that teichoic acid was situated between the dense laminae, although the distribution of stain could have been due to the inability of the concanavalin A stains to penetrate deeply. Chemical binding studies showed that the teichoic acid was the major uranyl binding component in isolated walls, from which it might be inferred that teichoic acid was located in the densely staining regions. However, since osmification significantly increased the binding of uranyl (and lead stains) to non-teichoic acid material, such an inference was not necessarily valid. It is concluded that the presence of teichoic acid can be demonstrated in certain regions of the wall by concanavalin A, but its presence in densely staining regions has not been established. These experiments therefore suggest that teichoic acid may not be intimately associated with the mechanisms that generate contrast patterns in stained sections of cell walls of Streptococcus faecalis.     

Nature and Origins of Phosphorus Compounds in Isolated Cell Walls of Staphylococcus aureus

Preparations of purified cell walls from Staphylococcus aureus were shown to contain small amounts of phospholipid and glycerol teichoic acid. Since these are components of the cell membrane, it is probable that the wall itself contains no lipid, but does retain fragments of membrane because of physical connections between wall and membrane. In walls of S. aureus strain 52A5, which completely lacks ribitol teichoic acid, the only phosphorylated compound identified as a genuine wall component was a phosphorylated derivative of murein that gave rise to muramic acid phosphate on acid hydrolysis. Muramic acid phosphate was also identified in hydrolysates of walls from S. aureus H and strain 52A2.