The Mechanism of Action of the Extracellular Bacteriolytic Enzymes of Lysobacter sp. on Gram-Positive Bacteria: The Role of the Cell Wall Anionic Polymers of Target Bacteria

The study of the extracellular bacteriolytic enzymes of Lysobacter sp. showed that they can efficiently hydrolyze the peptidoglycan of gram-positive bacteria provided that there is an electrostatic interaction of these enzymes with the cell wall anionic polymers, teichoic and teichuronic acids in particular. The hydrolytic action of bacteriolytic enzymes on the cell wall largely depends on the negative charge of the teichoic and teichuronic acids rather than on their chemical composition.     

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.     

Electrical properties and interaction with silicon dioxide particles of Bacillus subtilis cells

It was shown that, depending on the quantity of phosphate in cultivation media, the surface layer of the Bacillus subtilis cell wall can mainly consist of either teichoic or teichuronic acids. It was found that the addition of silicon dioxide particles to the bacterial suspension induces an increase in the negative charge of the cell. This cell response to contact interaction with solid particles is observed in bacteria whose surface components are teichoic acids.     

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.     

A balancing act times two: sensing and regulating cell envelope homeostasis in Bacillus subtilis

Bacterial cell wall homeostasis is an intricately coordinated process that ensures that envelope integrity is maintained during cell growth and division, but can also adequately respond to growth‐limiting conditions such as phosphate starvation. In Bacillus subtilis, biosynthesis of the two major cell wall components, peptidoglycan and anionic polymers, is controlled by a pair of paralogous two‐component systems, WalRK and PhoPR respectively. Favorable growth conditions allow for a fast rate of cell wall biosynthesis (WalRK‐ON) and the incorporation of the phosphate‐containing anionic polymer teichoic acids (PhoPR‐OFF). In contrast, growth‐restricted cells under phosphate‐limiting conditions reduce the incorporation of peptidoglycan building blocks (WalRK‐OFF) and switch from the phosphate‐containing teichoic acids to the phosphate‐free anionic polymer teichuronic acid (PhoPR‐ON). Botella et al. (2014) deepen our knowledge on the PhoPR system by identifying one signal that is perceived by its histidine kinase PhoR. In fast‐growing cells, intracellular intermediates of teichoic acid biosynthesis are sensed by the cytoplasmic Per‐Arnt‐Sim domain as an indicator of favorable conditions, thereby inhibiting the autokinase activity of PhoR and keeping the system inactive. Depletion of teichoic acid building blocks under phosphate‐limiting conditions relieves this inhibition, activates PhoPR‐dependent signal transduction and hence the switch to teichuronic acid biosynthesis.     

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.     

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.     

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.     

Selective extraction of polymers from cell walls of Gram-positive bacteria (Short Communication)

Brief heating of Bacillus Licheniformis cell walls at 100 degrees C in aqueous buffers of pH3.0-4.0 removes some polymers but not others from the mucopeptides. For example, relatively undegraded teichuronic acid can be extracted at 100 degrees C in 20min at pH3.0 whereas the teichoic acids are not removed. Similar specificity can be shown with walls from three other species of micro-organism.     

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.     

Characterization of the Bacillus subtilis CwbA protein which stimulates cell wall lytic amidases

The Bacillus subtilis cell wall binding protein, CwbA, stimulated the cell wall lytic activities of the B. subtilis and B. licheniformis autolysins (CwlA and CwlM, respectively) in addition to that of the major B. subtilis autolysin (CwlB). Even though the substrate for the enzyme reaction was changed from B. subtilis cell wall containing a teichoic acid to Micrococcus luteus cell wall containing a teichuronic acid, the stimulatory effect of CwbA on CwlA activity was observed.     

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.     

Comparative in vivo nitrogen-15 nuclear magnetic resonance study of the cell wall components of five Gram-positive bacteria.

The proton-decoupled 9.12 MHz 15N NMR spectra of 15N-labeled Bacillus subtilis, Bacillus licheniformis, Staphylococcus auresu, Streptococcus faecalis, and Micrococcus lysodeikticus intact cells, isolated cells walls, and cell wall digests have been examined. The general characteristics of Gram-positive bacteria 15N NMR spectra and described and spectral assignments are provided, which allow in vivo 15N NMR to be applied to a wide range of problems in bacterial cell wall research. The qualitative similarity of the intact cell and cell wall spectra found in each bacteria allowed the 15 N resonances observed in the proton broad-band noise-decoupled 15N NMR spectra of intact cells to be assigned to cell wall components. Each of the five Gram-positive bacteria displayed a unique set of cell wall 15N resonances, which reflected variations in the primary structure of peptidoglycans and the amounts of teichoic acid and teichuronic acid in the cell wall, as well as the dynamic properties of the cell wall polymers. Spectral assignments of cell wall 15 N resonances assigned to teichoic D-Ala residues, teichuronic acid and acetamido groups, and peptidoglycan acetamido, amide, peptide, and free amino groups have been made on the basis of specific isotopic labeling and dilution experiments, comparison of chemical shifts to literature values, determination of pH titration shifts, cell wall fractionation experiments, and comparative analysis of the cell wall lysozyme digest spectra in terms of the known primary sequences of peptide chains. All the peptidoglycan 15N peptide resonances observed in the intact cells and isolated cell walls could be accounted for by residues in the bridge or crossbar regions of the peptide chains, which indicated that only the cross-linking groups had a high degree of motional freedom. Thermal- and pH-induced conformational changes around the cross-linking D-Ala residues were detected in the B. licheniformis cell wall lysozyme digest products. Comparison of the proton broad-band noise-decoupled and gated decoupled intact cell and cell wall 15N spectra indicated that broad-band proton decoupling resulted in nulling of cytoplasmic resonances and enhancement of the cell wall resonances by the 15N [1H5 nuclear Overhauser effect.     

The biosynthesis and functionality of the cell-wall of lactic acid bacteria

The cell wall of lactic acid bacteria has the typical Gram-positive structure made of a thick, multilayered peptidoglycan sacculus decorated with proteins, teichoic acids and polysaccharides, and surrounded in some species by an outer shell of proteins packed in a paracrystalline layer (S-layer). Specific biochemical or genetic data on the biosynthesis pathways of the cell wall constituents are scarce in lactic acid bacteria, but together with genomics information they indicate close similarities with those described in Escherichia coli and Bacillus subtilis, with one notable exception regarding the peptidoglycan precursor. In several species or strains of enterococci and lactobacilli, the terminal D-alanine residue of the muramyl pentapeptide is replaced by D-lactate or D-serine, which entails resistance to the glycopeptide antibiotic vancomycin. Diverse physiological functions may be assigned to the cell wall, which contribute to the technological and health-related attribut es of lactic acid bacteria. For instance, phage receptor activity relates to the presence of specific substituents on teichoic acids and polysaccharides; resistance to stress (UV radiation, acidic pH) depends on genes involved in peptidoglycan and teichoic acid biosynthesis; autolysis is controlled by the degree of esterification of teichoic acids with D-alanine; mucosal immunostimulation may result from interactions between epithelial cells and peptidoglycan or teichoic acids.     

The effect of the extracellular bacteriolytic enzymes of Lysobacter sp. on gram-negative bacteria

The effect of the extracellular bacteriolytic enzymes of Lysobacter sp. on gram-negative bacteria was studied. These enzymes were found to be able to hydrolyze the peptidoglycan that was isolated from the gram-negative bacteria, the hydrolysis being completely inhibited by the cell wall lipopolysaccharide of these bacteria. The native cells of the gram-negative bacteria became susceptible to the bacteriolytic enzymes after the permeability of the outer membrane of the cells had been altered by treating them with polymyxin B.     

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.     

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 effects of growth conditions on cell wall composition and cell morphology in a temperature-sensitive tag-B mutant of Bacillus subtilis

The relationship between wall anionic polymer synthesis and cell morphology has been studied in Bacillus subtilis 168 and its temperature-sensitive tagB mutant strain BR19-200B. The amount and type of anionic polymer synthesized varied under different growth conditions, as did the morphology of the bacteria. Anionic polymer synthesis was affected by the phosphate supply. It was also found that teichuronic acid synthesis was temperature-sensitive in wild-type bacteria. Teichuronic acid synthesis was affected by the tagB lesion, previously thought to affect only teichoic acid synthesis. A relationship was observed between synthesis of the alternative polymers, such that suppression of teichuronic acid synthesis is accompanied by an increase in the synthesis of teichoic acid. Variation in anionic polymer content was accompanied by variation in cell shape. Differences in shape were related to differences in total anionic polymer rather than to differences in individual polymer type.     

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.