Demonstration of the physiological role of autolysis by a comparative study with a wild-type and its non-autolytic mutant of Micrococcus lysodeikticus (luteus) cultivated with externally added proteolytic enzymes

The log phase cells of autolytic Microccus lysodeikticus (luteus) IFO 3333 did not autolyze when grown in the presence of trypsin although the growth curve and morphology of the cells were not influenced. A non-autolytic mutant was obtained by subculture of the wild-type strain IFO 3333 on an agar slant containing 1% glucose. The mutant (strain MT) was wild-type IFO 3333 which occurred singly or in irregular masses. The mutant MT grown in a culture medium containing trypsin caused remarkable alteration in cell morphology: large cell packets consisting of a number of "unit tetrads" arranged regularly in three dimensions were formed by the addition of trypsin to the medium. The findings suggest that inhibition of the separation of divided cells is brought about by inactivation or suppression of a cell wall autolytic enzyme which plays an important role in the separation step and is accessible to externally added trypsin in the mutant cells but not in the wild-type cells. The possibility that there are two kinds or phases of autolytic enzymes "a physiological autolytic enzyme" and "a useless autolytic enzyme", is discussed.     

Cell wall-bridge maintaining three dimensional structure of cell packets formed by the localized suppression of cell separation of a Micrococcus lysodeikticus (luteus) mutant.

Cell packets of Micrococcus lysodeikticus (luteus) mutant strain MT grown in medium supplemented with trypsin consisted of a tetrad as the unit structure. An interstice was observed between the unit-tetrads, and a three dimensional structure of cell packets was maintained by the cell wall-bridge along the rim of the cell packets which linked each unit-tetrad. This unique structure of strain MT cell packets seemed to occur when the cell separation was suppressed locally, i.e., when the cross wall inside the initial site of cell separation was cut off, while the wall outside the initial site of separation was not cut off but remained as a joint of the daughter cells. The mechanism of cell wall-bridge formation is discussed in connection with cell separation.     

Process of Consecutive Cell Divisions and Separations in a Regular Tetrads-Forming Mutant of Micrococcus lysodeikticus (luteus)1

A mutant MT of Micrococcus lysodeikticus (luteus) IFO 3333, whose minimum growing unit is not a single cell, but a tetrad unlike the wild-type divides by binary fission of each monococcus, and then separates first into two daughter tetrads, second into four tetrads and third into eight tetrads. The three planes of either the cell division or the cell separation are equivalent to one another and oriented at right angles in three dimensions, respectively. The process of consecutive cell divisions and separations of the mutant tetrads was schematically illustrated.     

Cell surface of a tetrads-forming mutant of Micrococcus luteus: chemical treatment of the cells and teichuronic acids on the surface

Tetrads-forming mutant MT cells of Micrococcus luteus, both treated with chemical reagents and non-treated, were observed with a scanning electron microscope (SEM). The agglutinability of the cells with antiserum containing anti-teichuronic acid antibody was examined. The binding of protein A-gold particles to the cells, mediated with the antiserum, was also observed with SEM. A tetrad surface, not surface of each of four "unit monococci" constituting a tetrad, consisted of two or three smooth areas with borders. The difference in the surface features between M. luteus wild-type IFO 3333 (Monodane et al, Microbiol. Immunol. 33: 165-174, 1989) and the mutant MT cells is discussed.     

Cell surface of Micrococcus luteus: chemical treatment of the cells and teichuronic acids on the surface

Micrococcus luteus IFO 3333 cells, both treated with chemical reagents and non-treated, were observed with a scanning electron microscope (SEM). The agglutinability of the cells with antiserum containing anti-teichuronic acid antibody was examined. The binding of protein A-gold particles to the cells, mediated with the antiserum, was also observed with SEM. The surface of a M. luteus cell consisted of two or three areas with borders--the rough and the smooth areas, or the rough, the slightly rough, and the smooth areas; fluffy materials were clearly seen in the rough area. Gold particles were observed uniformly and densely on the whole cell surface. However, either mild acid treatment or mild Smith degradation of the cells altered the fluffy rough area to a rough one, and extremely decreased the agglutinability and the binding of protein A-gold particles. Teichuronic acids appeared to be distributed uniformly on the whole cell surface of M. luteus IFO 3333.     

Cell wall autolysis in log phase cells of Micrococcus lysodeikticus (luteus).

Log phase cells of Micrococcus lysodeikticus (luteus) IFO 3333 autolyzed when incubated at 37 C in 0.01 M sodium-phosphate buffer pH 7.5. The enzyme involved in the autolysis was recovered mainly in an aqueous phase from cytoplasmic membranes and cytoplasmic materials treated with n-butanol, and proved to be an N-acetylmuramyl-L-alanine amidase. The autolysis of log phase cells suspended in autolyzing buffer was depressed by the addition of trypsin to the buffer.     

Chemical treatments of the cell packets induced from a Micrococcus luteus mutant and teichuronic acids on the packet surface

Cell packets (MT packets) induced from a tetrads-forming mutant (strain MT) of Micrococcus luteus, both treated with chemical reagents and non-treated, were observed with a scanning electron microscope (SEM). The agglutinability of MT packets with antiserum containing anti-teichuronic acid antibody was examined. The binding of protein A-gold particles to the MT packets, mediated with the antiserum, was also observed with SEM. Gold particles were observed uniformly on the whole packet surface and also on the bridging structure formed by the outermost layer of the cell wall. Mild acid treatment, NaIO4-NaBH4 treatment and mild Smith degradation of the MT packets extremely decreased the agglutinability and binding of protein A-gold particles. The treatments gave a little influence on the surface feature and appreciably destroyed the regular packet structure. It was supposed that teichuronic acids distributed uniformly on the whole packet surface, naturally on the surface of the bridging structure too, and appreciably participated in the maintenance of the regular packet structure.     

Antigenicity of Candida tropicalis strain cells cultured at 27 and 37 degrees C

All cells of four Candida tropicalis strains IFO 0199 (Ct-0199), IFO 0587 (Ct-0587), IFO 1400 (Ct-1400), and IFO 1647 (Ct-1647), obtained by cultivation at 27 and 37 degrees C for 48 h in yeast extract-added Sabouraud liquid medium, showed the shapes of typical budding yeast and the same agglutination patterns against factor sera 1, 4, 5 and 6 in the commercially available kit 'Candida Check'. The cells of the C. tropicalis IFO 0589 strain display the same properties at 27 degrees C but formed hyphae at 37 degrees C. The cell wall mannan (Ct-0589-37-M) obtained from the strain cells cultured at 37 degrees C had lost most of its reactivity against factor sera 4, 5 and 6 in an enzyme-linked immunosorbent assay, in contrast to the mannan (Ct-0589-27-M) at 27 degrees C. The 1H-nuclear magnetic resonance patterns of the mannans obtained from the cells of the four C. tropicalis strains IFO 0199, IFO 0587, IFO 1400, and IFO 1647, obtained by cultivation at 37 degrees C, did not change compared to those at 27 degrees C. By contrast, the Ct-0589-37-M had significantly lost the beta-1,2-linked mannopyranose units, corresponding to the serum factors 5 and 6. These results show that the IFO 0589 strain is an unusual strain among the general C. tropicalis strains studied.     

Distribution of capsular materials on the cell wall surface of strain Smith diffuse of Staphylococcus aureus.

The fine structure of the capsule of Staphylococcus aureus Smith diffuse was examined by the technique of freeze-substitution and immunoelectron microscopy. The cell surface was covered with a thick layer consisting of fine fibrous structures which were absent from an unencapsulated strain, Smith compact. Anti-teichoic acid antibody did not react with this surface layer but reacted with the surface of strain Smith compact. Anti-capsular antibody, made from the serum of a rabbit immunized with strain Smith diffuse and specific absorption with unencapsulated strain Wood 46, reacted with the fibrous layer of the Smith diffuse strain. Since the anti-teichoic acid antibody did not react with the encapsulated strain Smith diffuse, the capsular layer acts as a barrier to penetration of the anti-teichoic acid antibody through the capsular layer. A portion of a few cell surfaces of the encapsulated strain remained accessible to the anti-teichoic acid antibody. The capsular layer in this portion of the cell surface was thin, and this surface seemed to be a new cell wall surface created by the cell separation.     

Opening angle and residual strain in a three-layered model of pig oesophagus

Studies of various biological tissues have shown that residual strains are important for tissue function. Since a force balance exists in whole wall thickness specimens cut radially, it is evident that layer separation is an important procedure in the understanding of the meaning of residual stresses and strains. The present study investigated the zero-stress state and residual strain distribution in a three-layer model of the pig oesophagus. The middle part of the oesophagus was obtained from six slaughterhouse pigs. Four 3-mm-wide rings were serially cut from each oesophagus. Two of them were used for separating the wall into mucosa-submucosa, inner and outer muscle layers. The remaining two rings were kept as intact rings. The inner and outer circumferences and wall thickness of different layers in intact and separated rings were measured from the digital images in the no-load state and zero-stress state. The opening angle was measured and the residual strain at the inner and outer surface of different layers and the intact wall were computed. Compared with intact sectors (62.8+/-9.8 degrees ), the opening angles were smaller in the inner muscle sectors (37.2+/-11.4 degrees , P<0.01), whereas the opening angles of mucosa-submucosa (63.9+/-6.8 degrees ) and outer muscle sectors (63.9+/-6.8 degrees ) did not differ (P>0.1). Referenced to the zero-stress state of the intact sectors, the inner and outer residual strains of the intact rings was -0.128+/-0.043 and outer residual strain was 0.308+/-0.032. Referenced to the "true" zero-stress state of separated three-layered sectors, the inner residual strain of intact rings were -0.223+/-0.021 (P<0.01) and 0.071+/-0.022 (P<0.01). Referenced to the "true" zero-stress state, the residual strain distribution of different layers in intact rings was shown that the inner surface residual strain was negative at mucosa-submucosa and inner muscle layers and was positive at outer muscle layer, whereas the outer surface residual strain was negative at the mucosa-submucosa layer and positive at the inner and outer muscle layers. For the separated different layered rings, the inner residual strain was negative and outer residual strain was positive; however, the absolute values did not differ (P>0.1). In conclusion, it is possible to microsurgically separate the oesophagus into three layers, i.e., mucosa-submucosa, inner muscle and outer muscle layers, the residual strain differ between the layers, and the residual strain distribution was more uniform after the layers were separated.     

Ultrastructure of the cell wall of a Synechocystis strain

The ultrastructure of the cell wall of a Synechocystis strain, isolated from the Gulf of Finland, was studied using several electron microscopic techniques. This cyanobacterium has numerous projections which were observed to penetrate the cell wall complex. An additional layer (AL) was associated with the outer membrane. An additional external wall layer (EL) was connected to the outer membrane complex by thin fibers as revealed by ruthenium red staining. A hexagonal arrangement of the subunits in the additional external wall layer with a lattice constant of 15.5 nm was found.     

Changes in fine morphological structures of Escherichia coli, Staphylococcus aureus and spores of Bacillus anthracis vaccine strain STI under the action of the disinfectant "Veltolen"

Changes in fine morphological structures of E. coli, S. aureus and spores of B. anthracis vaccine strain STI under the action of the disinfectant "Veltolen" manufactured by the Closed Joint-Stock Company "Velt", were evaluated. When used at concentrations of 0.0025-0.025%, the preparation induced the loosening of the cell wall in all microorganisms under study, the intensive formation of bubbles on the cell wall surface with their subsequent separation from the cell wall and the formation of "rosettes". In case of more prolonged exposure (up to 60 minutes) and higher concentrations of the preparation these phenomena became more intensive and finally led to the destruction of bacterial cells.     

Essential Roles of Cell Surface Protein and Carbohydrate Components in Flocculation of a Brewer’s Yeast

Co-flocculation between cells of beer yeast IFO 2018, a flocculent strain, and non-flocculent strains was investigated by means of a chemical modification method. Treatment with periodate deprived non-flocculent cells, but not flocculent cells, of the ability to co-flocculate. Treatment with mercaptoethanol or photo-irradiation in the presence of methylene blue deprived flocculent cells, but not non-flocculent cells, of the co-flocculating ability. Mercaptoethanol-treated or photoirradiated flocculent cells (beer yeast IFO 2018) co-flocculated with periodate-treated flocculent cells, but periodate-treated cells subsequently subjected to mercaptoethanol treatment or photoirradiation neither flocculated by themselves nor co-flocculated with other cells. Thus, it is likely that both protein and carbohydrate components of the yeast cell surface play important roles in the mutual recognition and intercellular interaction involved in flocculation. It is strongly suggested that the essential carbohydrate which is widely distributed among Saccharomyces species is the mannan fraction on the cell wall, and that a flocculent yeast strain produces surface protein component(s) which recognize and bind the mannan component of adjacent cells.     

Transfer of 'Thermoactinomyces glaucus' IFO 12530 and 'Thermoactinomyces monosporus' IFO 14050 to the genus Saccharomonospora as members of Saccharomonospora glauca

Two available strains of 'Thermoactinomyces glaucus' and 'Thermoactinomyces monosporus', 'T. glaucus' IFO 12530 and 'T. monosporus' IFO 14050, were considered not to be members of the genus Thermoactinomyces and that they belonged to the genus Saccharomonospora on the basis of the colors of colonies and 16S rDNA sequences. Some chemotaxonomic characteristics also showed that the two strains belong to the genus Saccharomonospora. The two strains contained meso-diaminopimelic acid, galactose, and arabinose in the cell wall and MK-9(H(4)) as the predominant menaquinone. The genomic DNAs of the two strains had a G+C content of 69 mol%. The 16S rDNAs of 'T. glaucus' IFO 12530 and 'T. monosporus' IFO 14050 showed only 1 and 2 bp sequence differences, respectively, from that of the type strain of Saccharomonospora glauca. Furthermore, the two strains of 'T. glaucus' and 'T. monosporus' and the type strain of S. glauca shared identical 16S-23S rDNA ITS sequences. The levels of DNA-DNA relatedness confirm that the two strains of 'T. glaucus' and 'T. monosporus' are members of Saccharomonospora glauca. Therefore it is proposed that 'T. glaucus' IFO 12530 and 'T. monosporus' IFO 14050 should be considered as strains belonging to Saccharomonospora glauca.     

Comparison of two kinds of cell packets of Micrococcus luteus by scanning electron microscopy: outermost layer maintaining the packet structure

Two kinds of cell packets of Micrococcus luteus, one having teichuronic acids (TUA) in the cell wall and the other lacking TUA, have been independently reported by two groups of workers. A comparison by scanning electron microscopy of these packets provided a possibly consistent interpretation for the seemingly conflicting opinions whether TUA were involved in packet induction. It was strongly suggested that the packets having TUA in the wall were rigidly maintained by a bridging structure of the outermost layer of the peripheral wall, while the packets lacking TUA showed low contribution of the outermost layer to the bridging structure probably due to the absence of TUA.     

Cell wall structure of secreted laccase-silenced strain in Lentinula edodes

Laccase1 (Lcc1) is abundantly secreted from vegetative mycelia into culture medium by Lentinula edodes. Down-regulation of lcc1 in L. edodes results in abnormal hyphal structure and thinner cell wall in mycelia. In this study, we observed the effects of Lcc1 on the hyphal morphology and cell wall structure of L. edodes. A thick cell wall and fibrous layer were clearly observed in the lcc1-silenced strain ivrL1#32, when purified Lcc1 (0.1 mU/mL) was added to the culture medium. The ratio of cell wall polysaccharide contents was compared between the ivrL1#32 strain and the wild-type (WT) strain SR-1, revealing that levels of the alkali soluble β-1,3-1,6-glucan were significantly lower in the lcc1-silenced strain than in the WT strain. Chronological analysis revealed that chitin content in the cell wall did not increase over time, but that the alkali soluble β-1,3-1,6-glucan content increased after Lcc1 secretion in the WT. Taken together, these data suggest that the increased level of β-1,3-1,6-glucan induced by Lcc1 in the mycelial cell wall contributes to increased cell wall thickness and strength.     

A planar microtubule-organizing zone in guard cells of Allium: experimental depolymerization and reassembly of microtubules

The generation of the unique radial array of microtubules (MTs) in stomatal guard cells raises questions about the location and activities of relevant MT-organizing centers. By using tubulin immunofluorescence microscopy, we studied the pattern of depolymerization and reassembly of MTs in guard cells of Allium cepa L. Chilling at 0°C reduces the MTs to small remnants that surround the nuclear surface of cells in the early postcytokinetic stage, or form a dense layer along the central portion of the ventral wall in older guard cells. A rapid reassembly on rewarming restores either MTs extending from the nuclear surface randomly throughout the cytoplasm in very young cells, or an array of MTs radiating from the dense layer at the ventral wall later in development. A similar pattern of depolymerization and reassembly is achieved by incubation with 100 M colchicine followed by a brief irradiation with ultraviolet (UV) light. Incubation with 200 M colchicine leads to a complete depolymerization that leaves only a uniform, diffuse cytoplasmic fluorescence. Nonetheless, UV irradiation of developing guard cells induces the regeneration of a dense layer of MTs at the ventral wall. The layer is again positioned centrally along the wall, even if the nucleus has been displaced by centrifugation in the presence of cytochalasin D. Neither the regenerated layer nor the perinuclear MTs seen earlier are related to the staining pattern of serum 5051, which reportedly binds to centrosomal material in animal and plant cells. The results support the view that, soon after cytokinesis, a planar MT-organizing zone is established in the cortex along the central portion of the ventral wall, which then generates the radial MT array.Abbreviations GC guard cell - MT microtubule - MTOC microtubule-organizing center - UV ultraviolet To whom correspondence should be addressed.     

Influence of muskmelon cell wall polysaccharides on roridin E production by a pathogenic strain of Myrothecium roridum

Addition of fruit cell wall extracts from two muskmelon cultivars into liquid media affected mycotoxin production by a strain of Myrothecium roridum pathogenic to muskmelon. Cell wall extracts from a susceptible cultivar (Iroquois) significantly increased toxin production while cell wall extracts from a resistant cultivar (Hales Best) significantly inhibited toxin production. Media containing 0.1 or 1.0 mg ml^–1 stimulated toxin production more than media containing 10 or 100 mg ml^–1 of cell wall extracts. Previous studies in our laboratory suggest that roridin E may be involved in virulence or pathogenicity of M. roridum; the present study indicates that cell wall polysaccharides as well as other materials present in cell wall preparations from susceptible host tissue provide a better substrate for toxin production than cell wall preparation from resistant host tissue.     

Control of microtubule assembly by extracellular matrix and externally applied strain

A number of studies have suggested that externally applied mechanical forces and alterations in the intrinsic cell-extracellular matrix (ECM) force balance equivalently induce changes in cell phenotype. However, this possibility has never been directly tested. To test this hypothesis, we directly investigated the response of the microtubule (MT) cytoskeleton in smooth muscle cells to both mechanical signals and alterations in the ECM. A tensile force that resulted in a positive 10% step change in substrate strain increased MT mass by 34 +/- 10% over static controls, independent of the cell adhesion ligand and tyrosine phosphorylation. Conversely, a compressive force that resulted in a negative 10% step change in substrate strain decreased MT mass by 40 +/- 6% over static controls. In parallel, increasing the density of the ECM ligand fibronectin from 50 to 1,000 ng/cm(2) in the absence of any applied force increased the amount of polymeric tubulin in the cell from 59 +/- 11% to 81 +/- 13% of the total cellular tubulin. These data are consistent with a model in which MT assembly is, in part, controlled by forces imposed on these structures, and they suggest a novel control point for MT assembly by altering the intrinsic cell-ECM force balance and applying external mechanical forces.     

Purification and characterization of an epsilon-poly-L-lysine-degrading enzyme from an epsilon-poly-L-lysine-producing strain of Streptomyces albulus

An epsilon-poly-L-lysine-degrading enzyme of an epsilon-poly-L-lysine-producing strain of Streptomyces albulus was purified and characterized. The enzyme was tightly bound to the cell membrane. After solubilization with NaSCN, the enzyme was purified to homogeneity by phenyl-Sepharose CL-4B column chromatography. The subunit molecular mass of the purified enzyme was 54 kDa. Enzyme activity was inhibited by o-phenanthroline, and could be restored in the presence of 1 mM Mg(2+), Ca(2+), Fe(3+) or Zn(2+). The mode of epsilon-poly-L-lysine degradation was of the exo-type, and the enzyme released N-terminal L-lysines one by one. The enzyme acted on various peptides possessing L-lysine residues at the N-terminus and was classified as an aminopeptidase. Epsilon-Poly-L-lysine-degrading activity was found in the membrane fraction of some other Streptomyces strains as well as that of Streptomyces albulus. Streptomyces virginiae IFO 12827 and Streptomyces norsei IFO 15452 exhibited high epsilon-poly-L-lysine-degrading activity, and both strains could produce epsilon-poly-L-lysine, indicating a correlation between the distribution of membrane-bound epsilon-poly-L-lysine-degrading enzyme and epsilon-poly-L-lysine-producing activity.