Surface of lactic acid bacteria: relationships between chemical composition and physicochemical properties

The surface chemical composition and physicochemical properties (hydrophobicity and zeta potential) of two lactic acid bacteria, Lactococcus lactis subsp. lactis bv. diacetilactis and Lactobacillus helveticus, have been investigated using cells harvested in exponential or stationary growth phase. The surface composition determined by X-ray photoelectron spectroscopy (XPS) was converted into a molecular composition in terms of proteins, polysaccharides, and hydrocarbonlike compounds. The concentration of the last was always below 15% (wt/wt), which is related to the hydrophilic character revealed by water contact angles of less than 30 degrees. The surfaces of L. lactis cells had a polysaccharide concentration about twice that of proteins. The S-layer of L. helveticus was either interrupted or crossed by polysaccharide-rich compounds; the concentration of the latter was higher in the stationary growth phase than in the exponential growth phase. Further progress was made in the interpretation of XPS data in terms of chemical functions by showing that the oxygen component at 531.2 eV contains a contribution of phosphate in addition to the main contribution of the peptide link. The isoelectric points were around 2 and 3, and the electrophoretic mobilities above pH 5 (ionic strength, 1 mM) were about -3.0 x 10(-8) and -0.6 x 10(-8) m(2) s(-1) V(-1) for L. lactis and L. helveticus, respectively. The electrokinetic properties of the latter reveal the influence of carboxyl groups, while the difference between the two strains is related to a difference between N/P surface concentration ratios, reflecting the relative exposure of proteins and phosphate groups at the surface.     

Surface of Lactic Acid Bacteria: Relationships between Chemical Composition and Physicochemical Properties

The surface chemical composition and physicochemical properties (hydrophobicity and zeta potential) of two lactic acid bacteria, Lactococcus lactis subsp. lactis bv. diacetilactis and Lactobacillus helveticus, have been investigated using cells harvested in exponential or stationary growth phase. The surface composition determined by X-ray photoelectron spectroscopy (XPS) was converted into a molecular composition in terms of proteins, polysaccharides, and hydrocarbonlike compounds. The concentration of the last was always below 15% (wt/wt), which is related to the hydrophilic character revealed by water contact angles of less than 30°. The surfaces of L. lactis cells had a polysaccharide concentration about twice that of proteins. The S-layer of L. helveticus was either interrupted or crossed by polysaccharide-rich compounds; the concentration of the latter was higher in the stationary growth phase than in the exponential growth phase. Further progress was made in the interpretation of XPS data in terms of chemical functions by showing that the oxygen component at 531.2 eV contains a contribution of phosphate in addition to the main contribution of the peptide link. The isoelectric points were around 2 and 3, and the electrophoretic mobilities above pH 5 (ionic strength, 1 mM) were about −3.0 × 10⁻⁸ and −0.6 × 10⁻⁸ m² s⁻¹ V⁻¹ for L. lactis and L. helveticus, respectively. The electrokinetic properties of the latter reveal the influence of carboxyl groups, while the difference between the two strains is related to a difference between N/P surface concentration ratios, reflecting the relative exposure of proteins and phosphate groups at the surface.     

Surface properties of the conidiospores of Phanerochaete chrysosporium and their relevance to pellet formation.

The conidiospores of the white rot basidiomycete Phanerochaete chrysosporium tend to aggregate during swelling and germination in agitated liquid medium; as time passes, the initial aggregates tend to associate together and to capture conidiospores that remain isolated. The surface chemical compositions of the conidiospores and of developed hyphae were analyzed by X-ray photoelectron spectroscopy. The data were interpreted by modelling the surface in terms of proteins, polysaccharides and hydrocarbonlike compounds. The surface molecular composition of the dormant conidiospores was estimated to be about 45% proteins, 20% carbohydrates, and 35% hydrocarbonlike compounds. There was an increase in the polysaccharide content during germination. Later, when the hyphae were developed, the polysaccharide content became still higher, and the protein content dropped. The initial step of aggregation is attributed to polysaccharide bridging; its occurrence cannot be explained by a change of the overall hydrophobicity or electrical properties of the conidiospores.     

Biochemical and immunochemical properties of the cell surface of Renibacterium salmoninarum.

The biochemical composition of the cell envelope of Renibacterium salmoninarum was investigated in a total of 13 strains isolated from different salmonid fish species at various geographical locations of the United States, Canada, and Europe. A marked similarity with the type strain R. salmoninarum ATCC 33209 was found both in the peptidoglycan and the cell wall polysaccharide. The primary structure of the peptidoglycan was found to be consistent with lysine in the third position of the peptide subunit, a glycyl-alanine interpeptide bridge between lysine and D-alanine of adjacent peptide subunits, and a D-alanine amide substituent at the alpha-carboxyl group of D-glutamic acid in position 2 of the peptide subunit. The cell wall polysaccharide contained galactose as the major sugar component which was accompanied by rhamnose, N-acetylglucosamine, and N-acetylfucosamine. The polysaccharide amounted to more than 60% of the dry weight of the cell walls. It was found to be covalently linked to the peptidoglycan and was released by hot formamide treatment. On gel filtration chromatography the extracted polysaccharide behaved like a homogeneous polymeric compound. The purified cell wall polysaccharide showed antigenic activity with antiserum obtained by immunization of rabbits with heat-inactivated trypsinized cells of R. salmoninarum. Immunoblotting experiments with nontrypsinized cell walls and antisera raised against R. salmoninarum cells revealed that antigenic proteins were attached to the cell walls.     

Comparative characterization of spirosomes isolated from Lactobacillus brevis, Lactobacillus fermentum, and Lactobacillus buchneri

Spirosomes, cytoplasmic fine spirals, were isolated and purified from Lactobacillus brevis ATCC 8287, L. fermentum F-1, and L. buchneri ATCC 4005, and their morphological, biochemical, and immunological properties were investigated. The spirosomes of these lactobacilli were morphologically indistinguishable from one another, and they had the same buoyant density of 1.320 g/cm3 in CsCl. All of the spirosomes were composed of a single protein, spirosin, with an apparent molecular weight of about 95,000 for L. brevis and L. fermentum and of about 96,000 for L. buchneri as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The spirosins from the three lactobacilli were compared by peptide mapping on SDS-PAGE after cleavage with N-chlorosuccinimide and limited proteolysis with Staphylococcus aureus V8 protease. The peptide map of the L. brevis spirosin was identical with that of the L. fermentum spirosin, whereas it was markedly different from the L. buchneri spirosin. The amino acid composition of the L. brevis spirosin was almost similar to that of the L. fermentum spirosin, while it differed appreciably from the L. buchneri spirosin. Using antiserum against the L. brevis spirosin, immunodiffusion test revealed that the antigenicity of the spirosomes from L. brevis was identical with that from L. fermentum, whereas it was partially different from that from L. buchneri.     

Composition and properties of the outer cell wall layer in Bacillus brevis--the producer of gramicidin S

Two membrane antigens were found by cross immunoelectrophoresis in the cell walls of Bacillus brevis var. G.-B., R form, which started to synthesize gramicidin S (20 mg per 1 ml of cultural broth). The cell wall contained no membrane components in cells at the beginning of the logarithmic growth phase. The protein with a molecular mass of 100 kDa is a component of the cell wall outer layer. The protein is not digested by trypsin or pronase when it comprises the cell walls of cells synthesizing gramicidin S. In the preparation of isolated cell walls, this protein becomes susceptible to the action of the above proteases only when the peptidoglycan layer is broken down by lysozyme. Electron microscopy of cells treated with proteases and shadowed with a metal revealed that many cells lacked the cytoplasm. Therefore, the outer layer of B. brevis R cell wall contains small regions susceptible to the action of protease along with regions composed of the 100 kDa protein and resistant to these enzymes. It is possible that the small regions contain membrane components.     

Cell wall and lipid composition of Isosphaera pallida, a budding eubacterium from hot springs.

Isosphaera pallida is an unusual gliding, budding eubacterium recently isolated from North American hot springs. Electron micrographs of ultrathin sections revealed a cell wall atypical of eubacteria: two electrondense layers separated by an electron-transparent layer, with no evident peptidoglycan layer. Growth was not inhibited by penicillin. Cell walls were isolated from sheared cells by velocity sedimentation. The rigid-layer fraction, prepared from cell walls by treatment with boiling 10% sodium dodecyl sulfate, was hydrolyzed and chemically analyzed for muramic acid. This essential component of peptidoglycan was absent. Amino acid analysis demonstrated a proteinaceous wall structure. Pitlike surface structures seen in negatively stained whole cells and thin sections were correlated with periodically spaced perforations of the rigid sacculus. An analysis of the lipid composition of I. pallida revealed typical ester-linked lipids with unbranched fatty acids, in contrast to the isoprenyl ether-linked lipids of archaebacteria, which also have proteinaceous cell walls. Capnoids, unusual sulfonolipids which are present in gliding bacteria of the Cytophaga-Flexibacter group, were absent.     

Immunochemical analysis of O-specific polysaccharides from the soil nitrogen-fixing bacteria Azospirillum brasilense

Immunochemical reactivity of O-specific polysaccharide and the monosaccharide composition of O-antigenic determinants of the lipopolysaccharide isolated from the type strain Sp7 of Azospirillum brasilense were studied. An original modification of the method of spectroturbidimetry for disperse biological systems and a nonstandard procedure for the preparation of monospecific antibodies against cell surface antigens were used. The polysaccharide fraction, which contained residues of galactose, rhamnose, and galacturonic acid, was able to bind about 50% of the antibodies raised against whole bacterial cells. Twelve immunodeterminant groups were shown to be present in its molecule. Galactose and, less effectively, rhamnose but not galacturonic acid inhibited the antigen-antibody reactions. It is concluded that the serotype of the strain studied is determined by galactose residues.     

Depth profiling of the elemental surface composition of the oral microorganism S. salivarius HB and fibrillar mutants by X-ray photoelectron spectroscopy.

X-ray photoelectron spectroscopy (XPS) on microbial cell surfaces requires freeze-drying of cells, and as a result, the cell surface appendages flatten out on the cell surface and form a collapsed fibrillar mass. At present, it is unclear how the density, length and composition of these fibrils influence the elemental surface composition as probed by XPS. The sampling depth of XPS can be varied by changing the electron take-off angle. In this article, we made a depth profiling of the collapsed fibrillar mass of Streptococcus salivarius HB and fibril-deficient mutants by angle-dependent XPS. Methylamine tungstate negative staining and ruthenium red staining followed by sectioning revealed distinct classes of fibrils with various lengths on each of the strains. Interpretation of the angle dependence of the oxygen/carbon (O/C) and phosphorus/carbon (P/C) surface concentration ratios of these strains was difficult. However, the angle dependence of the nitrogen/carbon (N/C) surface concentration ratio could be fully interpreted: N/C did not vary with sampling depth on a bald strain, S. salivarius HBC12 and on S. salivarius HB7, a strain with a dense array of fibrils of uniform length. N/C decreased with sampling depth in case of a sparsely fibrillated strain, S. salivarius HBV51 and eventually reached the value observed for the bald strain, HBC12. A high N/C at small sampling depth was observed for S. salivarius HB with protruding, protein rich fibrils. We conclude that elemental depth profiling of microbial cell surfaces by XPS can be interpreted to coincide with structural and biochemical information on the cell surface as obtained by electron microscopy and can therefore be considered as a useful technique to study structural features of cell surfaces in combination with electron microscopy.     

Electroosmosis in Membranes: Effects of Unstirred Layers and Transport Numbers: II. Experimental

In an earlier paper, it was shown that the differences in transport numbers in membranes and adjacent solutions will result in a depletion and enhancement of the local concentration profiles at the appropriate interfaces. These should, in general, cause both current-induced volume flows and transient changes in membrane potential difference (PD). The predicted concentration changes were measured near an isolated segment of plant cell wall just after a current pulse. The current-induced volume flows observed were separated into a “transport number component” and an instantaneous, electroosmotic one for both cell walls and whole cells. For walls, the electroosmotic component contributed about 53 moles · Faraday^-1 to a total coefficient of 112 moles · Faraday^-1. For whole cells, the average electroosmotic component (for both hyperpolarizing and depolarizing currents) contributed about 38 moles · Faraday^-1 to a total of about 100 moles · Faraday^-1. There was good agreement between the magnitudes and time courses of the flows and membrane PD's predicted from the theory in the previous paper, and those measured in both cell walls and whole cells.     

Passive Electrical Properties of Microorganisms: III. Conductivity of Isolated Bacterial Cell Walls

The dielectric properties of isolated Micrococcus lysodeikticus cell walls have been studied to establish more firmly the view that wall-associated ions play a major role in the conduction of low frequency electric current by intact bacterial cells. The conductivity of isolated walls was found to be about 0.40 mho/m. If counterions associated with fixed, ionized groups in the wall have average mobilities equal to that of sodium ions in free solution, the fixed charge concentration required to account for the measured conductivity is between 75 and 95 meq/liter of wet wall volume. Estimates of the numbers of titratable amino and carboxyl groups in wall polymers indicate that conductivity is more closely related to net wall charge than to total wall charge. The measured wall conductivity was used to predict a value of 0.15 ± 0.03 mho/m for whole cell conductivity. This prediction is close to the measured value of 0.25 ± 0.05 mho/m and it is thought that much of the disparity in values is related to changes in wall structure and composition during the isolation procedures.     

Depth profiling of the elemental surface composition of the oral microorganismS. salivarius HB and fibrillar mutants by X-ray photoelectron spectroscopy

X-ray photoelectron spectroscopy (XPS) on microbial cell surfaces requires freeze-drying of cells, and as a result, the cell surface appendages flatten out on the cell surface and form a collapsed fibrillar mass. At present, it is unclear how the density, length and composition of these fibrils influence the elemental surface composition as probed by XPS. The sampling depth of XPS can be varied by changing the electron take-off angle. In this article, we made a depth profiling of the collapsed fibrillar mass ofStreptococcus salivarius HB and fibril-deficient mutants by angle-dependent XPS. Methylamine tungstate negative staining and ruthenium red staining followed by sectioning revealed distinct classes of fibrils with various lengths on each of the strains. Interpretation of the angle dependence of the oxygen/carbon (O/C) and phosphorus/carbon (P/C) surface concentration ratios of these strains was difficult. However, the angle dependence of the nitrogen/carbon (N/C) surface concentration ratio could be fully interpreted: N/C did not vary with sampling depth on a bald strain,S. salivarius HBC12 and onS. salivarius HB7, a strain with a dense array of fibrils of uniform length. N/C decreased with sampling depth in case of a sparsely fibrillated strain,S. salivarius HBV51 and eventually reached the value observed for the bald strain, HBC12. A high N/C at small sampling depth was observed forS. salivarius HB with protruding, protein rich fibrils. We conclude that elemental depth profiling of microbial cell surfaces by XPS can be interpreted to coincide with structural and biochemical information on the cell surface as obtained by electron microscopy and can therefore be considered as a useful technique to study structural features of cell surfaces in combination with electron microscopy.     

Biosorption of cadmium,copper and lead by isolated mother cell walls and whole cells of Chlorella fusca

Using a new method for the isolation of released mother cell walls of Chlorella fusca, the biosorption of cadmium, copper and lead by purified cell wall isolates and whole cell suspensions was comparatively characterized. In all cases whole cells accumulated more metal ions than isolated cell walls. Both the Langmuir and Freundlich isotherm models were suitable for describing the short-term adsorption of cadmium, copper and lead by cell walls and the cadmium and copper adsorption by whole cells. However, neither model could sufficiently explain the lead accumulation by whole cells. The feasibility of a practical use of whole cells or isolated cell walls as biosorbents is discussed.     

Comparative study on the cell wall structure of protein-producing Bacillus brevis

Abstract Among eight strains of protein-producing Bacillus brevis , three morphological groups have been identified according to the structure of the cell walls.

• (I)

  Cell wall consisting of a peptidoglycanlayer
• (II)

  Two-layered cell wall consisting of a peptidoglycan-layer and an S-layer
• (III)

  Three-layered cell wall consisting of a peptidoglycan-layer and two S-layers

Group I and group II cell walls have not been described yet for protein-producing bacteria. The S-layers observed in this study all had hexagonal symmetry and lattice constants of approximately 18 nm. The immunological relation between the S-layer proteins of the newly isolated B. brevis strains and those of B. brevis 47 has been examined using antisera against both S-layer-proteins of B. brevis 47. S-layers from protein-producing B. brevis strains, which were adjacent to the peptidoglycan-layer, were similar to each other, whether they were the outermost cell wall layer (group II) or not (group III). However, no similarity was found between these layers and the outermost S-layer of B. brevis 47 (group III).     

The role of cell wall in plant embryogenesis

This review presents recent data about cell wall involvement in plant embryogenesis. During plant development, the cell wall is subjected to precise regulation. During this process a bidirectional information exchange between the cell wall and the protoplast is observed. The cell wall also mediates in the cell-cell (apoplastic) and cell to cell (symplastic) information flow. Especially some products derived from the hydrolysis of specific cell wall compounds can act as short distance signal transduction molecules during the development. Oligosaccharins are a group of such products. Their activity and sources focused the researchers' attention on the biochemical composition of the cell wall and the activity of some cell wall enzymes. The dramatic influence on the embryo body shape has also the cell wall synthesis machinery, including vesicular secretion pathways. Moreover, the interplay between the turgor pressure and counteracting cell walls and neighbouring cells (in higher organisms) creates the specific mechanical forces influencing the development of the whole plant. We conclude that discovering factors which can influence cell wall physiology and architecture is crucial for a better understanding of plant embryogenesis. In this review we summarize some recent experimental data reporting plant cell wall involvement in embryogenesis, putting special emphasis on somatic embryogenesis.     

Uptake of chromium(III) and chromium(VI) compounds in the yeast cell structure

The study presented in this article investigated the influence of different Cr(III) and Cr(VI) compounds in the cultivation medium on the uptake and localization of chromium in the cell structure of the yeast Candida intermedia. The morphology of the yeast cell surface was observed by the scanning electron microscopy. Results demonstrated that the growth inhibitory concentration of Cr(III) in the cultivation medium induced changes in the yeast cell shape and affected the budding pattern, while inhibitory concentration of Cr(VI) did not cause any visible effects on morphological properties of the yeast cells. The amount of total accumulated chromium in yeast cells and the distribution of chromium between the yeast cell walls and spheroplasts were determined by atomic absorption spectroscopy. No significant differences were found neither in total chromium accumulation nor in the distribution of chromium in yeast cell walls and spheroplasts between the two of Cr(VI) compounds. Conversely, substantial differences between Cr(III) compounds were demonstrated in the total uptake as well as the localization of chromium in yeast cells.     

Structuro-functional properties of the bacteria Bacillus brevis in relation to the accumulation of gramicidin S in cells

The culture of Bacillus brevis var. G-B R-form was grown in the presence of beta-phenyl-beta-alanine, the inhibitor of gramicidin S synthesis, is characterized by enhanced endogenous respiration and the DPI-reductase activity as compared to the culture synthezising antibiotic. The increased synthesis of the antibiotic in the region of the culture transition from the logarithmic growth phase to the linear one is associated with a decrease in the number of viable cells despite the fact that the culture on the whole does not die but continues to grow. The membranes prepared from young gramicidin S-free cells and from the cells enriched with the antibiotic possess identical electron micrograph images, IR spectra and protein sets as determined by polyacrylamide gel electrophoresis in a Na-DS system. However, in young cell membranes NADH and succinate dehydrogenase are insensitive to gramicidin S and only malate dehydrogenase is inhibited by this antibiotic. In aged cell membranes the activities of all mentioned dehydrogenases are suppressed. Malate dehydrogenase from young cells is weakly inhibited by thyrotrycin obtained from Bac. brevis ATCC 10068; succinate dehydrogenase is entirely insensitive to this antibiotic, while NADH-dehydrogenase is almost completely inhibited by it. The specificity of action on the respiratory chain of peptide antibiotics synthesized by the cells of one strain of Bac. brevis is suggestive of a possible regulatory role of these peptides in the metabolism of the producent. Hence the accumulation of gramicidin S which is adsorbed on the membrane and destroys the respiratory chain function to the cause of the low rate of oxygen uptake by the culture of Bac. brevis var. G-B R-form and of the low activities of DPI-reductases.     

Cell wall composition in the ascomycete sphaerostilbe repens at different developmental stages

Changes in the biochemical composition of isolated cell walls were analysed during the differentiation of coremia and rhizomorphs in Sphaerostilbe repens.Differentiation was accompanied by exclusively quantitative variations of the wall components: the content in carbohydrates, chitin and free amino sugars increased; on the contrary, amino acids, uronic acids, lipids and mineral substances decreased.Carbohydrates were composed of glucose, galactose and mannose; glucosamine was the main component of amino sugars. The predominant amino acid in the walls was cysteine the amount of which increased during hyphal aggregation, while quantities of the sixteen other determined amino acids decreased.Mineral matter was present in large quantities in the walls of the fungus, especially in vegetative mycelium. Iron, phosphorus and calcium were the most abundant elements.Possible relations between the variations in chemical composition of the wall and the capability of hyphae to aggregate are discussed.     

Relations between hydrophobicity tested by three methods and surface chemical composition of Escherichia coli

The cell surface hydrophobicity of three strains of Escherichia coli cultured in liquid medium and on solid medium was measured using various methods including adsorption to pxylene, partition of cells in a polyethylene glycol/dextran (PEG/DEX) two phase system and contact angle measurements. The percentage adsorbed to pxylene ranged from 1.6% to 67% and the percentage of cells in polyethylene glycol phase ranged from 19% to 64%. The contact angle data of less than 40 degrees C revealed a hydrophylic character of the E. coli strains studied here. No relations were found between paraxylene/water partitioning, PEG/DEX partioning and water contact angles. The linear correlation coefficients between the results of the three hydrophobicity assays and the elemental concentration ratios obtained by X-ray photoelectron spectroscopy (XPS) were calculated. A linear correlation was found between the contact angles and the O/C ratios (r=0.91) and the N/C ratios (0.67). The adsorption to pxylene correlates better with N/C ratios (0.88) but does not correlate with O/C ratios (0.46). However, this test correlates with N/P ratios (0.79). No relation was obtained between partition in PEG/DEX system and any elemental concentration ratios. The surface composition determined by XPS was converted into a molecular composition in terms of proteins, polysaccharides, and hydrocarbon-like compounds. The proteins/polysaccharides and the hydrocarbons/polysaccharides seems to determine the contact angle of E. coli but not the adsorption to paraxylene or partition in the PEG/DEX system.