Surface electrical properties of <Emphasis Type="Italic">bacillus subtilis</Emphasis> cells and the effect of interaction with silicon dioxide particles

Depending on the amount of phosphate in the growth medium, the surface layer of the Bacillus subtilis cell wall may be composed mainly of either teichoic or teichuronic acids. Only in the former case the negative charge of the cell surface increases upon addition of silicon dioxide particles into the cell suspension.     

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.     

Effect of ristomycin on the electrokinetic properties of Staphylococcus aureus cells

The electrophoretic mobility of the cells of Staph. aureus cultivated on the medium with ristomycin is markedly decreased at pH 3.0-5.0. This indicates that ristomycin added to the cultivation medium lowers the number of the phosphate groups in teichoic acids of the staphylococcal cell wall. The effect of ristomycin on the cells of the staphylococci was studied in vitro during the process of their incubation. It was shown that almost within the first minutes of the incubation ristomycin interacts immediately with the ionogenic groups of the cell wall and first of all with the phosphate groups of teichoic acids.     

Structural differentiation of the Bacillus subtilis 168 cell wall.

Exponential-growth-phase cultures of Bacillus subtilis 168 were probed with polycationized ferritin (PCF) or concanavalin A (localized by the addition of horseradish peroxidase conjugated to colloidal gold) to distinguish surface anionic sites and teichoic acid polymers, respectively. Isolated cell walls, lysozyme-digested cell walls, and cell walls treated with mild alkali to remove teichoic acid were also treated with PCF. After labelling, whole cells and walls were processed for electron microscopy by freeze-substitution. Thin sections of untreated cells showed a triphasic, fibrous wall extending more than 30 nm beyond the cytoplasmic membrane. Measurements of wall thickness indicated that the wall was thicker at locations adjacent to septa and at pole-cylinder junctions (P < 0.001). Labelling studies showed that at saturating concentrations the PCF probe labelled the outermost limit of the cell wall, completely surrounding individual cells. However, at limiting PCF concentrations, labelling was observed at only discrete cell surface locations adjacent to or overlying septa and at the junction between pole and cylinder. Labelling was rarely observed along the cell cylinder or directly over the poles. Cells did not label along the cylindrical wall until there was visible evidence of a developing septum. Identical labelling patterns were observed by using concanavalin A-horseradish peroxidase-colloidal gold. Neither probe appeared to penetrate between the fibers of the wall. We suggest that the fibrous appearance of the wall seen in freeze-substituted cells reflects turnover of the wall matrix, that the specificity of labelling to discrete sites on the cell surface is indicative of regions of extreme hydrolytic activity in which alpha-glucose residues of the wall teichoic acids and electronegative sites (contributed by phosphate and carboxyl groups of the teichoic acids and carboxyl groups of the peptidoglycan polymers) are more readily accessible to our probes, and that the wall of exponentially growing B. subtilis cells contains regions of structural differentiation.     

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.     

Choline-containing bacteriophage receptors in Streptococcus pneumoniae.

Choline-containing teichoic acid seems to be essential for the adsorption of bacteriophage Dp-1 to pneumococci. This conclusion is based on the following observations: In contrast to pneumococci grown in choline-containing medium, cells grown in medium containing ethanolamine or other submethylated aminoalcohols instead of choline were found to be resistant to infection by Dp-1. Live choline-grown bacteria and heat- or UV-inactivated cells and purified cell walls prepared from these cells were capable of adsorbing phage Dp-1; ethanolamine-grown pneumococci or cell wall preparations were unable to do so. Adsorption of Dp-1 to choline-containing cell walls was competitively inhibited by phosphorylcholine and by several choline-containing soluble cell surface components, such as the Forssman antigen and the teichoic acid-glycan complexes formed by autolytic cell wall degradation. Cell walls prepared from pneumococci grown in ethanolamine or phosphorylethanolamine were inactive. Electron microscopic studies with pneumococci that had segments of choline-containing cell wall material amid ethanolamine-containing regions indicated that the Dp-1 phage particles adsorbed exclusively to the choline-containing surface areas. We suggest that the choline residues of the pneumococcal teichoic acid are essential components of the Dp-1 phage receptors in this bacterium.     

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.     

Inactivation of the dlt operon in Staphylococcus aureus confers sensitivity to defensins, protegrins, and other antimicrobial peptides

Positively charged antimicrobial peptides with membrane-damaging activity are produced by animals and humans as components of their innate immunity against bacterial infections and also by many bacteria to inhibit competing microorganisms. Staphylococcus aureus and Staphylococcus xylosus, which tolerate high concentrations of several antimicrobial peptides, were mutagenized to identify genes responsible for this insensitivity. Several mutants with increased sensitivity were obtained, which exhibited an altered structure of teichoic acids, major components of the Gram-positive cell wall. The mutant teichoic acids lacked D-alanine, as a result of which the cells carried an increased negative surface charge. The mutant cells bound fewer anionic, but more positively charged proteins. They were sensitive to human defensin HNP1-3, animal-derived protegrins, tachyplesins, and magainin II, and to the bacteria-derived peptides gallidermin and nisin. The mutated genes shared sequence similarity with the dlt genes involved in the transfer of D-alanine into teichoic acids from other Gram-positive bacteria. Wild-type strains bearing additional copies of the dlt operon produced teichoic acids with higher amounts of D-alanine esters, bound cationic proteins less effectively and were less sensitive to antimicrobial peptides. We propose a role of the D-alanine-esterified teichoic acids which occur in many pathogenic bacteria in the protection against human and animal defense systems.     

Colony spreading in Staphylococcus aureus

Wild-type Staphylococcus aureus rapidly expands on the surface of soft agar plates. The rates of expansion and the shapes of the resultant giant colonies were distinct for different strains of laboratory stocks and clinical isolates. The colony spreading abilities did not correlate with the biofilm-forming abilities in these strains. Insertional disruption of the dltABCD operon, which functions at the step of D-alanine addition to teichoic acids, and of the tagO gene, which is responsible for the synthesis of wall teichoic acids, decreased the colony spreading ability. The results indicate that wall teichoic acids and D-alanylation of teichoic acids are required for colony spreading.     

Ultrastructural localization of cell wall teichoic acids in Streptococcus faecalis by means of concanavalin A

Some teichoic acids are known to be partially substituted by α-D-glucopyranosyl residues such as the teichoic acids of Streptococcus faecalis NCIB 8191. They will, therefore, bind specifically the phytohemagglutinin concanavalin A. Concanavalin A labelled with mercury or colloidal gold coated with concanavalin A has been used to mark isolated cell walls in order to localize the teichoic acids at the ultrastructural level. Besides these two direct marking techniques, the indirect concanavalin A-peroxidase technique (localization of peroxidase by the diaminobenzidine method followed by postosmication) has been applied to thin sections of premarked cells. All three methods gave almost identical results, namely, a dense and homogeneous distribution of the cell wall teichoic acids. In control experiments total inhibition was achieved in the presence of methyl-α-D-mannopyranoside. After trichloroacetic acid or alkali extraction of the teichoic acids from isolated walls no marking could be detected.     

Synthesis of teichoic acids. VII. Synthesis of teichoic acids during spore germination

The synthesis of teichoic acids has been examined during germination in Bacillus licheniformis ATCC 9945 and in B. subtilis W-23. Teichoic acids are absent from the spores of both organisms. B. licheniformis spores lack the enzymes responsible for teichoic acid synthesis. The appearance of these enzymes during germination is correlated with the appearance of teichoic acids in the cell. The appearance of teichoic acid-synthesizing enzymes and of teichoic acids in the cell are inhibited by the addition of chloramphenicol to the germination medium. In B. subtilis W-23 the situation is similar for the synthesis of polyribitolphosphate. The synthesis of glucosyl polyribitolphosphate is only partially inhibited by chloramphenicol, puromycin, and penicillin, and uridine diphosphate-d-glucose polyribitol-phosphate glucosyl transferase can be demonstrated in spores. The possible implications of some of these observations are discussed.     

Isolation of the Teichoic Acid of Bacillus Subtilis 168 by Affinity Chromatography

A column of insoluble concanavalin A was prepared by coupling the protein to cyanogen bromide-activated Sepharose. When autolysates of Bacillus subtilis 168 cell walls were passed over the column, the alpha glucosylated teichoic acid component of the cell wall was retained. The teichoic acid could be eluted with dilute alpha-methylglucopyranose. The teichoic acid prepared by affinity chromatography from cell wall autolysates had a higher sedimentation rate than teichoic acids obtained by conventional methods.

Several authors have shown that concanavalin A (con A) forms complexes with alpha-glucosylated teichoic acids^1–3. Doyle and Birdsell¹ found that the teichoic acid of Bacillus subtilis 168 (trp C2) would precipitate with con A at neutral pH in dilute buffer. The formation of a precipitate was inhibited by sugars which bind to the active site of con A. This observation suggested that it should be possible to purify the teichoic acid by affinity chromatography using insoluble con A as the affinity probe. Lloyd⁴ and Donnelly and Goldstein⁵ have successfully employed insoluble con A to purify polysaccharides and glycoproteins. In this communication, we describe conditions for the rapid purification of the alpha-glucosylated teichoic acid of B. subtilis 168. The teichoic acid prepared by this procedure appears to be less degraded than teichoic acids obtained by conventional methods.     

The role of teichoic acids in the regulation of biochemical processes in microorganisms]

The role of cell wall teichoic and lipoteichoic acids in the regulation of activity of some autolytic enzymes and in ion exchange, the role of lipoteichoic acids in biosynthesis of cell wall teichoic acids and some other functional features of these compounds in the bacterial cell are discussed.     

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 Bacillus subtilis extracytoplasmic-function sigmaX factor regulates modification of the cell envelope and resistance to cationic antimicrobial peptides

Bacillus subtilis contains seven extracytoplasmic-function sigma factors that activate partially overlapping regulons. We here identify four additional members of the sigma(X) regulon, pbpX (penicillin-binding protein), ywnJ, the dlt operon (D-alanylation of teichoic acids), and the pss ybfM psd operon (phosphatidylethanolamine biosynthesis). Modification of teichoic acids by esterification with D-alanine and incorporation of phosphatidylethanolamine into the cell membrane have a common consequence: in both cases positively charged amino groups are introduced into the cell envelope. The resulting reduction in the net negative charge of the cell envelope has been previously implicated as a resistance mechanism specific for cationic antimicrobial peptides. Consistent with this notion, we find that both sigX and dltA mutants are more sensitive to nisin than wild-type cells. We conclude that activation of the sigma(X) regulon serves to alter cell surface properties to provide protection against antimicrobial peptides.     

Bacteriophage SP50 as a marker for cell wall growth in Bacillus subtilis.

When grown under conditions of phosphate limitation, Bacillus subtilis W23 lacked wall teichoic acid and did not adsorb phage SP50. During transition from growth under conditions of phosphate limitation to those of potassium limitation, the bacteria developed an ability to adsorb phage which increased exponentially in relation to their content of wall teichoic acid. During transition in the reverse direction, the bacteria retained near-maximum phage-binding properties until their content of wall teichoic acid had fallen to a fairly low level. These observations suggest that newly incorporated wall material does not immediately appear at the cell surface in a structure to which phage can adsorb. Examination of the location of adsorbed phage particles showed that recently incorporated receptor material appeared at the cell surface first along the length of the cylindrical portion of the cell. The results are consistent with models of wall assembly in which newly synthesized wall material is intercalated at a large number of sites that are distributed along the length of the cell. This newly incorporated material may be located initially at a level underlying the surface of the cell and may become exposed at the surface only during subsequent growth. Incorporation of new material may also proceed rapidly into the developing septa, but new wall material is incorporated into existing polar caps more slowly, or perhaps not at all.     

Structure of the Staphylococcus aureus cell wall determined by the freeze-substitution method.

The fine structure of the Staphylococcus aureus cell wall was determined by electron microscopy with the new technique of rapid freezing and substitution fixation. The surface of the cell wall was covered with a fuzzy coat which consisted of fine fibers or an electron-dense mass. Morphological examination of the cell wall, which was treated sequentially with sodium dodecyl sulfate, trypsin, and trichloroacetic acid, revealed that this coat was partially removed by trypsin digestion and was completely removed by trichloroacetic acid extraction but was not affected by sodium dodecyl sulfate treatment, suggesting that the fuzzy coat consists mostly of a complex of teichoic acids and proteins. This was confirmed by the application of the concanavalin A-ferritin technique for teichoic acid and antiferritin immunoglobulin G technique for protein A.     

The occurrence of teichoic acids in streptomycetes

The presence of teichoic acids in a number of streptomycetes led to the conclusion that these biopolymers were widely spread in genus Streptomyces. The nature of the teichoic acid present in the mycelium was determined by extracting it with 10% trichloroacetic acid, precipitating it with ethanol and identifying the precipitated polymer by partial acid and alkali hydrolysis to alditol, alditol phosphates and glycosylalditol phosphates. Most strains examined in this survey contained glycerol or ribitol teichoic acids; in some cases neither type was detected.Structurally teichoic acids closely resemble those of other genera of gram-positive bacteria and in many cases represent poly(glycerol phosphate) and poly(ribitol phosphate) chains. The proportion of alditol residues bearing sugar substituents varied widely.Three species of genus Streptoverticillium contained glycerol teichoic acids. It is belived that some of the data presented in this paper might be used with some success in taxonomic studies of streptomycetes.     

A common linkage saccharide unit between teichoic acids and peptidoglycan in cell walls of Bacillus coagulans

Teichoic acid-glycopeptide complexes were isolated from lysozyme digests of the cell walls of Bacillus coagulans AHU 1631, AHU 1634, and AHU 1638, and the structure of the teichoic acid moieties and their linkage regions was studied. On treatment with hydrogen fluoride, each of the complexes gave a hexosamine-containing disaccharide, which was identified to be glucosyl(beta 1----4)N-acetylglucosamine, in addition to dephosphorylated repeating units of the teichoic acids, namely, galactosyl(alpha 1----2)glycerol and either galactosyl(alpha 1----2)[glucosyl(alpha 1----1/3)]glycerol (AHU 1638) or galactosyl(alpha 1----2)[glucosyl(beta 1----1/3)]glycerol (AHU 1631 and AHU 1634). From the results of Smith degradation, methylation analysis, and partial acid hydrolysis, the teichoic acids from these strains seem to have the same backbone chains composed of galactosyl(alpha 1----2)glycerol phosphate units joined by phosphodiester bonds at C-6 of the galactose residues. The presence of the disaccharide, glucosyl(beta 1----4)N-acetylglucosamine, in the linkage regions between teichoic acids and peptidoglycan was confirmed by the isolation of a disaccharide-linked glycopeptide fragment from each complex after treatment with mild alkali and of a teichoic acid-linked saccharide from each cell wall preparation after treatment with mild acid. Thus, it is concluded that despite structural differences in the glycosidic branches, the teichoic acids in the cell walls of the three strains are linked to peptidoglycan through a common linkage saccharide, glucosyl (beta 1----4) N-acetylglucosamine.     

Teichoic acids in the cell walls of Microbispora mesophila Ac-1953t and Thermobifida fusca Ac-1952t

The cell walls of Microbispora mesophila strain Ac-1953T (the family Streptosporangiaceae) and Thermobifida fusca Ac-1952T (the family Nocardiopsiceae) were found to contain teichoic acids of a poly(glycerol phosphate) nature. The teichoic acid of M. mesophila (formerly Thermomonospora mesophila) represents a poly(glycerol phosphate) containing 5% of substituent 2-acetamido-2-deoxy-alpha-galactosaminyl residues. The teichoic acid of such kind was found in actinomycetes for the first time. The cell wall of T. fusca (formerly Thermonospora fusca) contains two teichoic acids, namely, unsubstituted 1,3-poly(glycerol phosphate) and beta-glucosylated 1,3-poly(glycerol phosphate).