Chemical basis for selectivity of metal ions by the Bacillus subtilis cell wall.

The use of equilibrium dialysis techniques established that isolated cell walls of Bacillus subtilis possess selective affinities for several cations. The binding of these cations to the cell wall was influenced by the presence of various functional groups in the peptidoglycan matrix. Selective chemical modification of the free carboxyl and amino groups showed that when amino groups were replaced by neutral, bulky, or negatively charged groups, the sites available for cation complexing generally increased. Introduction of positive charges into the wall resulted in a marked decrease in the numbers of metal binding sites and usually a decrease in the apparent association constants. Both teichoic acid and peptidoglycan contribute to the sites available for interaction with metals. Hill plots of equilibrium dialysis data suggest that metal binding to cell walls involves negative cooperativity. Competition between various metals for binding sites suggested that the cations complex with identical sites on the cell walls. When the hydrogen ion concentration was increased, the affinity of the walls for metals decreased, but the numbers of metal binding sites remained constant, suggesting that cations and protons also compete for the same sites.     

Ion-exchange properties of cell walls in chickpea cultivars with different sensitivities to salinity

Ion-exchange properties of polymeric matrices were compared for cell wall preparations isolated from roots and shoots of two cultivars of Cicer arietinum L. (cvs. Bivanij and ILC 482) with different sensitivities to salinity. Irrespective of growth conditions, the cell walls contained four types of ionogenic groups: amino groups, carboxyl groups of uronic and hydroxycinnamic acids, and phenolic hydroxyl groups. Regardless of the salt concentration in the medium, the cells walls of different chickpea cultivars and from different organs of the same plant were similar in qualitative composition of ionogenic groups, although quantities of ionogenic groups per unit dry wt of cell walls varied depending on external and internal factors. Irrespective of the external medium salinity, the cation-exchange capacity of cell walls, expressed per unit dry wt, decreased in a sequence: stem > root ∼ bottom leaves > upper leaves. The volume of chickpea cell walls was found to vary depending on ionic composition and pH of the incubation medium. The results were analyzed in the context of cell wall involvement in responses of C. arietinum to elevated salinity.     

Nonaqueous titration of amino groups in polymeric matrix of plant cell walls

Nonaqueous titration was used for detection of free amino groups in the polymeric matrix of plant cell walls. The content of amino groups varied in the range 0.54–0.91 and total nitrogen in the range 1.0–4.2 mmol per gram dry mass of cell walls depending on the plant species. However, these data on the high content of free amino groups do not correlate with the present day concept that the nitrogen fraction in charged amino groups in plant cell wall proteins, which are assumed to be mainly amino groups of lysine and arginine residues, is about 10%. It is supposed that most detected free amino groups belong to the hydroxy-amino acids hydroxyproline and tyrosine that can be bound at the hydroxyl group with the carbohydrate part of glycoprotein or another structural cell wall polymer.     

N-Glycyl-glucosamine: A novel constituent in the cell wall of Halococcus morrhuae

The dinitrophenyl-derivative of N-glycylglucosamine was isolated from partially hydrolyzed dinitrophenylated cell walls of Halococcus morrhuae CCM 859. To increase the yield of amino-terminal glycine residues, halococcal cell walls were treated with alkali or acid prior to dinitrophenylation. Authentic N-glycyl-glucosamine was used as a reference substance. A substitution of the amino group of glucosamine by an amino acid has so far not been found in any other wall of a pro- or eucaryotic cell. Since only 5% of the glycine residues reveal an unsubstituted carboxyl group within intact cell walls, glycine may play a role in connecting glycan strands through peptidic linkages between the amino group of glucosamine and the carboxyl group of an uronic acid or gulosaminuronic acid.Abbreviations DNP dinitrophenyl     

Relevance of charged groups for the integrity of the S-layer from Bacillus coagulans E38-66 and for molecular interactions.

In this paper, the importance of charged amino and carboxyl groups for the integrity of the cell surface layer (S-layer) lattice from Bacillus coagulans E38-66 and for the self-assembly of the isolated subunits was investigated. Amidination of the free amino groups which preserved their positive net charge had no influence on both. On the other hand, acetylation and succinylation, which converted the amino groups into either neutral or negatively charged groups, and amidation of carboxyl groups were accompanied by the disintegration or at least by the loss of the regular structure of the S-layer lattice. Treatment of S-layer monolayers with the zero-length cross-linker carbodiimide led to the introduction of peptide bonds between activated carboxyl groups and amino groups from adjacent subunits. This clearly indicated that in the native S-layer lattice the charged groups are located closely enough for direct electrostatic interactions. Under disrupting conditions in which the S-layer polypeptide chains were unfolded, 58% of the Asx and Glx residues could be amidated, indicating that they occur in the free carboxylic acid form. As derived from chemical modification of monolayer self-assembly products, about 90% of the lysine and 70% of the aspartic and glutamic acid residues are aligned on the surface of the S-layer protein domains. This corresponded to 45 amino groups and to 63 carboxyl groups per S-layer subunit. Labelling experiments with macromolecules with different sizes and charges and adsorption studies with ion-exchange particles revealed a surplus of free carboxyl groups on the inner and on the outer faces of the S-layer lattice. Since the carboxyl groups on the outer S-layer face were accessible only for protein molecules significantly smaller then the S-layer protomers or for positively charged, thin polymer chains extending from the surface of ion-exchange beads, the negatively charged sites must be located within indentations of the corrugated S-layer protein network. This was in contrast to the carboxyl groups on the inner S-layer face, which were found to be exposed on elevations of the S-layer protein domains (D. Pum, M. Sára, and U.B. Sleytr, J. Bacteriol. 171:5296-5303, 1989).     

Surface properties from the S-layer of Clostridium thermosaccharolyticum D120-70 and Clostridium thermohydrosulfuricum L111-69

Labelling experiments using a positively charged topographical marker for electron microscopy, polycationized ferritin, showed that the S-layers of two closely related clostridia Clostridium thermohydrosulfuricum L111-69 and C. thermosaccharolyticum D120-70 do not exhibit a net negative charge, as usually observed for bacterial cell surfaces. Chemical modification of reactive sites confirmed that amino and carboxyl groups are exposed on the S-layer surface of both strains. Amino-specific, bifunctional agents crosslinked both S-layer lattices. Studies with carbodiimides revealed that only the S-layer surface of C. thermohydrosulfuricum L111-69 had amino and carboxyl groups closely enough aligned to permit electrostatic interactions between the constituent protomers. The regular structure of this S-layer lattice was lost upon converting the carboxyl groups into neutral groups by amidation. Disintegration of both S-layer lattices occurred upon N-acetylation or N-succinylation of the free amino groups. Adhesion experiments showed that in neutral and weakly alkaline environment whole cells of C. thermosaccharolyticum D120-70 exhibited a stronger tendency to bind to charged surfaces than whole cells of C. thermohydrosulfuricum L111-69, but showed a lower tendency to bind to hydrophobic materials.     

Ion-exchange properties of cell walls of red seaweed Phyllophora crispa

Research into ion-exchange properties of cell walls isolated from thallus of red seaweed Phyllophora crispa was carried out. Ion-exchange capacity and the swelling coefficient of the red alga cell walls were estimated at various pH values (from 2 to 12) and at constant ionic strength of a solution (10 mM). It was established that behavior of cell walls as ion-exchangers is caused by the presence in their matrix of two types of cation-exchange groups and amino groups. The amount of the functional group of each type was estimated, and the corresponding values of pK(a) were calculated. It can be assumed that ionogenic groups with pK(a) -5 are carboxyl groups of uronic acids, and ionogenic groups with pK(a) -7.5 are carboxyl groups of the proteins. Intervals of pH in which cation-exchange groups are ionized and can take part in exchange reactions with cations in the environment are defined. It was found that protein was a major component of cell wall polymeric matrix because its content was 36%.     

Charge distribution on the S layer of Bacillus stearothermophilus NRS 1536/3c and importance of charged groups for morphogenesis and function.

The distribution and functional significance of charged groups on the outer and inner faces of the S layer from Bacillus stearothermophilus NRS 1536/3c was investigated. Chemical modification of the exposed amino or carboxyl groups was performed on whole cells, isolated S layers self-assembled in vitro, and cell wall fragments (S layer attached to the peptidoglycan-containing sacculus). Without chemical modification, S layer self-assembly products could be labeled with polycationic ferritin, while S layers on whole cells could not. Following treatment with glutaraldehyde, whole cells were uniformly labeled with polycationic ferritin. Whole cells treated with glutaraldehyde and glycine methyl ester in the presence of carbodiimide did not bind polycationic ferritin significantly above background. Treatment of cell wall fragments with amino-specific, homobifunctional cross-linkers or with carbodiimide alone rendered the S layer protein nonextractable with sodium dodecyl sulfate. After amidation of the accessible carboxyl groups, the modified, guanidine hydrochloride-extractable S layer protomers did not self-assemble into regularly structured lattices. N-Amidination with ethylacetimidate did not interfere with the self-assembly of the isolated protomers. N-Acetylation resulted in a considerable destabilization of the S layer lattice, as seen by the release of a large amount of modified protomers during the reaction. N-Succinylation led to a complete disintegration of the protein lattice. These results indicated that only the inner face of the S layer carried a net negative charge. On both faces, free amino and carboxyl groups of adjacent protomers were arranged in proximity so as to contribute by electrostatic interactions to the cohesion of the protomers in the two-dimensional array. The native charge of the protomers was required for both the in vitro self-assembly of the isolated subunits and the maintenance of the structural integrity of the S layer lattice. Among other functions, the biological significance of the S layers may be in masking the electronegative charge of the cell wall proper.     

Role of charged groups in factor XI/XIa activity

To elucidate the role of charged groups in expression of factor XI coagulant activity, the charged groups of purified human blood coagulation factor XI/XIa containing 125I-XI/XIa were derivatized: free amino groups by succinylation, guanido groups of arginine by reaction with phenylglyoxal hydrate, and free carboxyl groups by reaction with ethylenediamine. The modified proteins were tested for: 1) ability to adsorb to glass, 2) ability to be cleaved by trypsin or factor XII-high molecular weight kininogen, 3) coagulant activity. The amino group-modified factor XI had a significantly decreased ability to bind to glass; modification of arginine or carboxyl groups did not affect adsorption. Trypsin cleaved factor XI with modified free amino, guanido, or carboxyl groups. Factor XII-high molecular weight kininogen could cleave only the arginine-modified factor XI. Amino group-modified factor XI and carboxyl group-modified factor XI lost all their factor XI assay activity, whereas arginine-modified factor XI retained 50% of the original activity. Amino group-modified factor XI could not be activated by trypsin, but arginine-modified and carboxyl group-modified factor XI could be activated by trypsin to 50% of the original activity. Succinylation of the amino groups of factor XIa destroyed all its factor XIa activity. Arginine-modified and carboxyl group-modified factor XIa retained 50% of their factor XIa activity. We conclude that epsilon-amino groups are essential for adsorption; activation by factor XII-high molecular weight kininogen requires free amino and carboxyl but not guanido groups; free amino, carboxyl, and guanido groups in factor XIa all appear to be critical for interaction of factor XIa with factor IX.     

Reassembly of a regularly arranged protein in the cell wall of Lactobacillus buchneri and its reattachment to cell walls: chemical modification studies

Regularly arranged protein (RA protein) isolated from the cell wall of Lactobacillus buchneri was chemically modified by amidination, acetylation, succinylation, and amidation. The modified RA proteins were examined for their ability to reassemble into a regular array and to reattach to the cell walls from which the regular array had been detached. Only amidinated RA protein could be either reassembled into a regular array or reattached to the cell walls; RA proteins modified by the other methods lost the ability for both reassembly and reattachment. The unmodified RA protein could be reattached to periodate-oxidized cell walls, but not to methylated ones. These results suggest that the positive charge of the amino group as well as the negative charge of the carboxyl group of RA protein plays an important role(s) in morphogenesis of the hexagonal array and in its attachment to the underlying cell wall layer. The periodate-insensitive lone hydroxyl groups of the neutral polysaccharide molecule in the cell wall seem to be the receptor sites for RA protein in the attachment to the cell wall.     

Characterization of various functional groups present in the capsule of Microcystis and study of their role in biosorption of Fe, Ni and Cr

This study demonstrates highest biosorption of Fe followed by Ni and Cr by Microcystis in single, bi and trimetallic combination. Fe was not only preferentially adsorbed from the metal mixtures but Ni and Cr failed to decrease its biosorption. The agreement of the data of Fe biosorption with the Langmuir model suggested monolayer sorption and existence of constant sorption energy during the experimental conditions. In contrast to Fe biosorption, Ni and Cr sorption followed the Freundlich isotherm; this demonstrates a multilayer biosorption of the two metals. IR analysis of Microcystis cells confirmed the presence of a large number of -COO(-) and some amino groups in the Microcystis cell wall. The oxygen and nitrogen donor atoms from carboxyl and amino groups were found to play a vital role in metal biosorption by Microcystis cell walls, and ion exchange mechanisms were involved in the biosorption of test metals. Extra peaks present in Ni and Cr treated cells implied that amino groups are more responsible for Ni and Cr biosorption.     

Ion-exchange properties of the cell wall of reindeer lichen <Emphasis Type="Italic">Cladonia rangiferina</Emphasis>

Ion-exchange properties of cell walls were investigated in reindeer lichen Cladonia rangiferina (L.) F. H. Wigg. In order to isolate cell walls, we used living parts of podetia as well as young parts (four upper internodes of podetia) and old parts (from the 4th to the 8th internode). We studied functional dependences of cell wall ion-exchange capacity on pH in the range from 2 to 12 and constant ionic strength of solution equal to 10 mM. It was found that three-dimensional structure of C. rangiferina cell walls comprised three types of ionogenic groups, which determine ion-exchange properties of the cell walls. They are amino groups with pK_a of about 3, carboxyl groups with pK_a of about 7, and phenolic OH-groups with pK_a of about 10. The content of groups of each type and their ionization constants were determined, and it was shown that, in the cell walls of young parts, the content of amino groups and carboxylic groups was greater than in old parts of podetia (by 1.5 and 2.0 times, respectively). It was found that with age the content of nitrogen and the proportion of deacetylated amino groups in the cell walls changed from 34% (young parts of podetia) to 40% (old parts of podetia). It was shown that in C. rangiferina N-acetyl glucosamine and glucosamine are not the main monomers of cell wall polymers because both in thalli and in the cell walls isolated therefrom the content of total nitrogen was less than 1%.     

Features of ionogenic group composition in polymeric matrix of lily pollen wall

The composition of ionogenic groups and ion-exchange capacity were studied in the polymeric matrix of cell walls isolated from the pollen grain and tissues of vegetative organs (leaves and stems) of Lilium longiflorum Thunb. The ion-exchange capacity was evaluated at different pH values and ionic strength of 100 mM. In the two-layered pollen wall and the somatic cell walls four types of ionogenic groups were found: amino groups, two carboxyl groups (represented by residues of uronic and hydroxycinnamic acids), and phenolic OH-groups. The groups of all four types are present in the intine, whereas the exine contains one type of anion-exchange and two types of cation-exchange groups. The contents of each type group and their ionization constants were determined. The qualitative and quantitative compositions of structural polymers of the pollen intine and somatic cell walls are significantly different. It is suggested that hydroxycinnamic acids should be involved in cross-linking of polysaccharide chains in both the intine and somatic cell primary walls, and such cross-links play a crucial role in the structural organization and integrity of the pollen grain wall.     

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.     

Positive Charges on Lysine Residues of the Extrinsic 18 kDa Protein Are Important to Its Electrostatic Interaction with Spinach Photosystem Ⅱ Membranes

To determine the contribution of charged amino acids to binding with the photosystem II complex （PSII）, the amino or carboxyl groups of the extrinsic 18 kDa protein were modified with N- succinimidyl propionate （NSP） or glycine methyl ester （GME） in the presence of a water-soluble carbodiimide, respectively. Based on isoelectric point shift, 4-10 and 10-14 amino groups were modified in the presence of 2 and 4 mM NSP, respectively. Similarly, 3-4 carboxyl groups were modified by reaction with 100 mM GME. Neutralization of negatively charged carboxyl groups with GME did not alter the binding activity of the extrinsic 18 kDa protein. However, the NSP-modified 18 kDa protein, in which the positively charged amino groups had been modified to uncharged methyl esters, failed to bind with the PSII membrane in the presence of the extrinsic 23 kDa protein. This defect can not be attributed to structural or conformational alterations imposed by chemical modification, as the fluorescence and circular dichroism spectra among native, GME- and NSP-modified extrinsic 18 kDa proteins were similar. Thus, we have concluded that the positive charges of lysyl residues in the extrinsic 18 kDa protein are important for its interaction with PSII membranes in the presence of the extrinsic 23 kDa protein. Furthermore, it was found that the negative charges of carboxyl groups of this protein did not participate in binding with the extrinsic 23 kDa protein associated with PSII membranes.     

Effect of nitrogen deficiency on the ion-exchange properties of cell wall polymers from wheat roots

The ion-exchange properties of cell wall polymers isolated from the roots of wheat (Triticum aestivum L.) plants grown on either nitrate-free (N-deficient) or nitrate-containing (+N) hydroponic nutrient medium have been investigated. Irrespective of the nitrogen nutrition regimen, the studied cell walls contained four types of ion-exchange groups: primary amino groups of structural proteins (pK_a < 3), carboxyl groups of polygalacturonic acid in pectin (pK _a ~4.7), carboxyl groups of hydroxycinnamic acids (pK _a ~7.3), and phenolic OH-groups of lignin (pK_a ~10.2). The quantitative ratio between these types of ion-exchange groups, the mass fraction of cell walls in the dry weight of roots, and the swelling coefficient of cell walls depended on the nitrate presence in the growing medium. Compared to the +N variant, the N-deficient variant was characterized by a 2.4 times higher content of phenolic OH-groups in cell walls and 1.24 times higher mass fraction of cell walls; at the same time, the swelling coefficient for this variant was lower by 10%. The obtained data indicate that nitrogen deficiency results in a formation of thicker root cell walls with a higher degree of polymer cross-linking that may be caused by the increased lignin content.     

Extracellular Lytic Enzymes of Myxococcus virescens

An easy and rapid method for the purification of a bacteriolytic endopeptidase produced by Myxococcus virescens is described. The bacteria were grown in casitone media and the cells were sedimented by centrifugation. About 1.2 g of montmorillonite were added per liter of cell-free culture solution. The clay was sedimented by centrifugation and the enzyme was then eluted by 0.05 M Na-phosphate buffer pH 6.0, containing 0.4 M NaCl. The enzyme was diluted with water and chromatographed on carboxymethyl-cellulose columns. The purified enzyme liberated free amino groups but no reducing sugars or N-acetylhexosamines when acting on purified N-acetylated cell walls of Micrococcus lysodeikticus. Analysis of N- and C-terminal amino acids in the digestion products showed that the enzyme had liberated about 110 nmoles of lysine ε-amino groups and 60 nmoles of alanine carboxyl groups per mg of cell wall. When it acted on a bisdisaccharide pentapeptide dimer isolated from M. lysodeikticus cell walls, it cleaved about 30% of the alanyl-lysine linkages. Consequently the enzyme was an alanyl-lysine endopeptidase. It had no muramyl-alanine amidase activity.     

Use of regularly structured bacterial cell envelope layers as matrix for the immobilization of macromolecules

Summary Carboxyl groups present on the outer face of the hexagonally ordered S-layer lattices from Bacillus stearothermophilus PV72 and Clostridium thermohydrosulfuricum L111-69 were activated with carbodiimide. The reaction of the activated carboxyl groups with free amino groups of low molecular weight nucleophiles was controlled by labelling with polycationized ferritin, a net positively charged topographical marker for electron microscopy, which densely binds to S-layers possessing free carboxyl groups. Carbodiimide-activated carboxyl groups were also allowed to react with amino groups of ferritin (MW 440 000) and invertase (MW 270 000). Covalent attachment of ferritin was examined by electron microscopy. Using invertase, approximately 1 mg enzyme was bound per mg S-layer protein indicating a high packing density of invertase molecules on the outer face of the S-layer lattice. The immobilized invertase retained 70% of its original activity.     

Ion-exchange properties of cell walls of Spinacia oleracea L. roots under different environmental salt conditions

Ion-exchange properties of the polymeric matrix of cell walls isolated from roots of 55-day-old Spinacia oleracea L. (Matador cv.) plants grown in nutrient solution in the presence of 0.5, 150, and 250 mM NaCl and from roots of Suaeda altissima L. Pall plants of the same age grown in the presence of 0.5 and 250 mM NaCl were studied. The ion-exchange capacity of the spinach cell walls was determined at pH values from 2 to 12 and different ionic strength of the solution (10 and 250 mM NaCl). In the structure of the root cell walls, four types of ionogenic groups were found: amine, two types of carboxyl (the first being galacturonic acid residue), and phenolic groups. The content of each type of group and their ionization constants were evaluated. The ion-exchange properties of spinach and the halophyte Suaeda altissima L. Pall were compared, and the qualitative composition of the ion-exchange groups in the cell walls of roots of these plants appeared to be the same and not depend on conditions of the root nutrition. The content of carboxyl groups of polygalacturonic acid changed in the cell walls of the glycophyte and halophyte depending on the salt concentration in the medium. These changes in the composition of functional groups of the cell wall polymers seemed to be a response of these plants to salt and were more pronounced in the halophyte. A sharp increase in the NaCl concentration in the medium caused a decrease in pH in the extracellular water space as a result of exchange reactions between sodium ions entering from the external solution and protons of carboxyl groups of the cell walls. The findings are discussed from the standpoint of involvement of root cell walls of different plant species in response to salinity.     

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.