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.     

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.     

Cell wall composition of Streptomyces roseoflavus var. roseofungini and its Nocardia-like variant

The composition of cell walls was comparatively studied in Streptomyces roseoflavus var. roseofungini 1128 and in its variant 1-68. In the logarithmic phase of growth, the content of teichoic acid in the cell wall of the parent culture was four times as high as in the cell wall of the variant. The cell walls of the parent culture contained 5 to 7 times more O-lysyl residues not only due to a higher content of teichoic acid in the walls but also owing to a lower content of lysyl groups in the teichoic acid of the variant. An additional polysaccharide comprising galactose and glucosamine was found in the cell wall of the variant but not in the parent strain. The peptidoglycan of the both cultures had a structure typical of Streptomyces spp.; its content in the cell walls of the two cultures was identical (ca. 50% of the dry cell wall biomass weight). The results are discussed in connection with the peculiarities of the variant hyphal septation.     

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.     

Genetic analysis of SCO2997, encoding a TagF homologue, indicates a role for wall teichoic acids in sporulation of Streptomyces coelicolor A3(2)

Streptomyces coelicolor contains two gene clusters putatively involved in wall teichoic acid biosynthesis. Inactivation of the tagF homologue SCO2997 or SCO2584, a component of the Streptomyces spore wall synthesizing complex, affected sporulation. The mutant phenotypes resembled those of mre mutants, suggesting a function of wall teichoic acids in the differentiation of Streptomyces.     

Structure of anionic carbohydrate-containing cell wall polymers in several representatives of the order actinomycetales

A new teichoic acid was identified in the cell walls of Streptomyces griseoviridis VKM Ac-622T, Streptomyces sp. VKM Ac-2091, and Actinoplanes campanulata VKM Ac-1319T. The polymer is poly(glycosylglycerol phosphate). The repeating units of the polymer, alpha-galactopyranosyl-(1-->3)-2-acetamido-2-deoxy-beta-galactopyran+ ++ osyl-(1-->1)-glycerols, are in phosphodiester linkage at C-3 of glycerol and C-6 of galactose. The structures of cell wall teichoic acids in the strains Streptomyces chryseus VKM Ac-200T and "Streptomyces subflavus" VKM Ac-484 similar in morphology and growth characteristics are also identical: 1,5-poly(ribitol phosphate) substituted at C-4(2) by 2-acetamido-2-deoxy-beta-glucopyranosyl residues and 1,3-poly(glycerol phosphate). The taxonomic aspects of these results are discussed.     

Peptidoglycan cross-linking and teichoic acid attachment in Streptococcus pneumoniae.

Autolysin-defective pneumococci continue to synthesize both peptidoglycan and teichoic acid polymers (Fischer and Tomasz, J. Bacteriol. 157:507-513, 1984). Most of these peptidoglycan polymers are released into the surrounding medium, and a smaller portion becomes attached to the preexisting cell wall. We report here studies on the degree of cross-linking, teichoic acid substitution, and chemical composition of these peptidoglycan polymers and compare them with normal cell walls. peptidoglycan chains released from the penicillin-treated pneumococci contained no attached teichoic acids. The released peptidoglycan was hydrolyzed by M1 muramidase; over 90% of this material adsorbed to vancomycin-Sepharose and behaved like disaccharide-peptide monomers during chromatography, indicating that the released peptidoglycan contained un-cross-linked stem peptides, most of which carried the carboxy-terminal D-alanyl-D-alanine. The N-terminal residue of the released peptidoglycan was alanine, with only a minor contribution from lysine. In addition to the usual stem peptide components of pneumococcal cell walls (alanine, lysine, and glutamic acid), chemical analysis revealed the presence of significant amounts of serine, aspartate, and glycine and a high amount of alanine and glutamate as well. We suggest that these latter amino acids and the excess alanine and glutamate are present as interpeptide bridges. Heterogeneity of these was suggested by the observation that digestion of the released peptidoglycan with the pneumococcal murein hydrolase (amidase) produced peptides that were resolved by ion-exchange chromatography into two distinct peaks; the more highly mobile of these was enriched with glycine and aspartate. The peptidoglycan chains that became attached to the preexisting cell wall in the presence of penicillin contained fewer peptide cross-links and proportionally fewer attached teichoic acids than did their normal counterparts. The normal cell wall was heavily cross-linked, and the cross-linked peptides were distributed equally between the teichoic acid-linked and teichoic acid-free fragments.     

Cell wall composition of novobiocin-resistant pleiotropic mutant staphylococci.

Physically purified cell walls were prepared from selected pleiotropic novobiocin-resistant staphylococcal strains. The quantitative amino acid, amino sugar, and phosphorus contents of these walls are reported. This pleiotype was culturally diagnosed by its inability to support the growth of typing phages, inability to release latent bacteriophage, failure to elaborate coagulase, altered sugar catabolic pattern, and resistance to novobiocin. The strains were divided into two groups on the basis of wall composition. The walls of both groups of strains appeared to possess at least two phosphorus-containing polymers. On group of strains contained elevated levels of phosphorus in the cell walls. The second group contained the novel amino sugar galactosamine in the cell walls. This amino sugar is probably associated with one of the phosphorus-containing wall polymers of this group. On the basis of the data presented, it is suggested that the pleiotropy of these strains is the result of genetic change in the control of the biosynthesis of teichoic acids.     

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.     

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.     

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.     

Characteristics of the active and inactive variants of Actinomyces sp. 26-115, a producer of actinomycin C

Two natural variants, i.e. No. 1 and No. 2, not producing actinomycin were isolated from cultures of the actinomycin C-producing organism Actinomyces sp. 26-115. Variant No. 1 differed from the active variant by the growth dynamics and colony morphology. Variant No. 2 was close to the active variant by the growth dynamics. It was shown with electron microscopy that the cells of variant No. 1 differed from those of the active variant in the number and form of the mycelial septa, more even and compact structure of the cell walls and higher sensitivity to actinomycin. Still, they were more stable to the effect of lysozyme and ultrasound. The cell walls of the inactive variant No. 1 gradually lost teichoic acid during development, while the loss of peptidoglycan was observed only on transfer to the stationary phase. The cell walls of the active variant lost teichoic acid and peptidoglycan at the same time on transfer to the stationary phase. Peptidoglycans of both variants contained diaminopimelic acid (the configuration of which was not determined) and glycine (1:1) as differentiating amino acids. The two adjacent tetrapeptides were joined with one glycine radical. The peptidoglycan peptide chains of both variants contained muramic, glutamic and diaminopimelic acids and alanine (1:1:1:2). The peptidoglycans of the inactive variant No. 1 contained in addition valine and isoleucine. However, it is hardly probable that they are contained by the peptidoglycan peptide chains.     

Analysis of autolysins in temperature-sensitive morphological mutants of Bacillus subtilis.

The content and distribution of autolysin were measured in temperature-sensitive morphological mutants of Bacillus subtilis. Strains RUB1000 and RUB1012 grew as rods at 30 C. At 45 C the mutants contained disproportionately less teichoic acid than peptidoglycan and grew as irregular spheres. The amount of enzyme that could be extracted from rods was at least 31 times the amount extracted from spheres. The rate of autolysis of cell walls was 7- to 28-fold greater in rods than in spheres. The low activity found associated with the cell walls of spheres was not compensated for by larger amounts of autolytic activity in the cytoplasm. No activity was found in the growth medium at either temperature. The failure of the mutant cells to autolyze was due to low amidase activity and relatively resistant cell walls. Revertants of RUB1012 were isolated that had 13, 23, and 55% of the normal proportions of teichoic acid when grown at the nonpermissive temperature. Cell walls from the revertants were as sensitive to added amidase as the wild-type strain. None of the revertant strains regained the wild-type ability to produce more amidase at 45 C. However, the deficiency in autolysin observed with RUB1012 was partially restored in revertants containing higher proportions of teichoic acid.     

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.     

生物矿化一蜡状芽孢杆菌聚金作用的研究

介绍了生物矿化-蜡状芽孢杆菌聚金作用原理.生物活动对矿石的风化、淋滤和沉积都有很大的影响.蜡状芽孢杆菌聚金作用主要与蜡状芽孢杆菌细胞壁的化学成分和结构功能有关.原因是其细胞壁有一层很厚的网状的肽聚糖、多糖、核酸和蛋白质结构,并且在细胞壁表面存在的磷壁酸质和糖醛酸磷壁酸质连接到网状的肽聚糖上.磷壁酸质的磷酸二脂和糖醛酸磷壁酸质的羧基使细胞壁带负电荷,具有离子交换的性质,能与溶液中带正电荷的金属离子进行交换反应.这些过程是蜡状芽孢杆菌细胞壁聚集金的主要作用机制.     

Classification of staphylococci based on chemical and biochemical properties

Summary In the present work the chemical cell wall composition and some other biochemical characteristics were studied in staphylococci with the intention of utilizing the data obtained in their classification.According to the cell wall peptidoglycans and teichoic acids, the 130 strains of staphylococci studied are divided into 10 major groups. This division of staphylococci into groups is in good agreement with their present classification only in some cases. All of the 47Staphylococcus aureus strains contain a cell wall peptidoglycan of thel-Lys-Gly_5–6 type and ribitol teichoic acid. Coagulase-negative staphylococci are more heterogeneous and are divided according to their cell wall composition into 9 major groups. 21 strains of them are classified asS. epidermidis sensu stricto. They form a natural group and are distinguished by the occurrence of thel-Lys-Gly_4–5,l-Ser_0.5–1.8 peptidoglycan type, glycerol teichoic acid and anl-lactate dehydrogenase which is activated by fructose-1,6-diphosphate. 8 strains with peptidoglycan of thel-Lys-Gly_4–5,l-Ser_0.5–1.8 type and ribitol teichoic acid are labeled asS. saprophyticus. The remaining groups have not been given species names and require further extensive comparative study.     

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.     

The interrelationship between mucopeptide and ribitol teichoic acid formation as shown by the effect of inhibitors

1. The biosynthesis of teichoic acid in cell suspensions of two strains of Staphylococcus aureus is partially inhibited by the same low concentrations of penicillin that inhibit mucopeptide synthesis by 90–100%. Further increase in the concentration of the antibiotic by several hundred-fold still fails to cause any greater inhibition of teichoic acid synthesis. 2. Other conditions, such as amino acid deficiency or the presence of cycloserine or 5-fluorouracil, that inhibit mucopeptide synthesis also inhibit teichoic acid formation. 3. The degree of inhibition of teichoic acid synthesis caused by relatively high concentrations (10μg./ml.) of benzylpenicillin depends critically on the age of the culture from which the cell suspensions have been prepared. 4. No significant amounts of soluble teichoic acid have been found in the fluid from cells incubated in the presence of penicillin. 5. A high proportion of the teichoic acid formed in the presence of penicillin can be removed from wall preparations at room temperature by 0·1n-ammonia. This is not true of the teichoic acid formed in the absence of penicillin. 6. The teichoic acid extracted with ammonia from preparations of cell walls made from cells treated with penicillin is excluded from Sephadex G-25, has a low molar ratio of glucosamine to phosphorus and contains muramic acid, alanine, glutamic acid, glycine and lysine. 7. The implications of these results for the mechanism of action of penicillin are 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.     

Streptomyces species with madurose (3-O-methyl-D-galactose) as a whole-cell sugar

Madurose, an actinomycete whole-cell sugar, was found in the strains of the genus Streptomyces: three strains of S. platensis, one strain each of S. platensis subsp. malvinus, and S. albus subsp. albus. The sugar was isolated from the hydrolysate of S. platensis IFO 14008 cells, and was identified as madurose (3-O-methyl-D-galactose) by chromatographic analyses, ¹H-NMR spectrometry, mass spectrometry as its alditol acetate, and demethylation with boron trichloride. Cell walls of the strain contained peptidoglycan and teichoic acids. ll-Diaminopimelic acid, glycine, glutamic acid, and alanine were present in the peptidoglycan fraction in molar ratios of 1.0:1.3:1.2:2.3. Madurose was detected in the teichoic acid fraction, which was composed of phosphorus, glycerol, galactose, and madurose in molar ratios of 9.3:8.5:2.9:1.0. Thus, madurose was found in the glycerol teichoic acid moiety of the cell walls of this strain.Abbreviations PC paper chromatography - TLC thin-layer chromatography - HPLC high performance liquid chromatography - GLC gas-liquid chromatography - TCA trichloroacetic acid