Fractional protein makeup of Candida utilis yeast protoplasts in the process of their growth and development

Candida utilis IBFMY-405 was grown in a synthetic medium with glucose. Cells taken at the logarithmic phase of growth were studied. The cells were treated with the enzyme from Helix pomatia to prepare protoplasts which were separated by differential centrifugation into groups according to their size. Three protein fractions were isolated from each group and the amino acid composition of the proteins was determined. Proteins of the first fraction (cytoplasmic) prevailed in all of the protoplast groups while the content of proteins of the second fraction (intermediate or myosin-like) was the lowest. As the size of protoplasts increased, difference in the quantitative content of proteins from the first and second fractions became less pronounced. The content of proteins of the third fraction was 3.6 and 2.4 times higher in the protoplasts of the medium size than in the largest protoplasts. The amino acid composition of each protein fraction differed quantitatively and qualitatively in all of the protoplast groups.     

Purification of a GSH-affinity binding protein from Bacteroides fragilis devoid of glutathione transferase activity

For the location of the aminoglycoside-(3)-N-acetyltransferase isoenzyme II (AAC(3)-II) in the bacterial cell, two strains were studied: Escherichia coli HB101(pJV03), producing the 31-kDa AAC (3)-II enzyme, and E. coli HB101, which served as a control. From each strain five protein fractions were prepared: culture supernatant, and proteins occurring in the periplasm, cytoplasm, inner membrane and outer membrane. All fractions were tested for enzymatic activity of AAC(3)-II. Most of the acetylating activity was found in the cytoplasmic fraction. The distribution of marker enzymes showed a good separation between the periplasmic and the cytoplasmic fraction.     

甘蔗和烟草幼叶原生质体的核酸含量与核酸酶活性及其影响因素

与幼叶组织相比,酶法新鲜分离的甘蔗和烟草幼叶原生质体内的ＲＮＡ、ＤＮＡ及总核酸含量均降低。其原因可能是刚游离的原生质体内酸性和碱性ＲＮＡ酶与ＤＮＡ酶等活性提高所致。甘蔗叶原生质体内的核酸降低量和ＲＮＡ酶与ＤＮＡ酶活性的增加程度均高于烟草。随用作渗透压稳定剂的甘露醇浓度增加,甘蔗和烟草叶原生质体的ＲＮＡ酶和ＤＮＡ酶活性均相应提高。其中以甘蔗叶原生质体的核酸酶活性增加水平较明显。在细胞壁降解产物的作用下,除了甘蔗原生质体内的ＲＮＡ酶活性略被促进外,其ＤＮＡ酶和烟草叶原生质体内的核酸酶均不受影响     

Subcellular localization of spermidine synthase in the protoplasts of chinese cabbage leaves

Previous studies on the presence of spermidine synthase (EC 2.5.1.16) in the protoplasts of Chinese cabbage (Brassica pekinensis var Pak Choy) leaves had detected a small but significant fraction of the enzyme in a crude chloroplast fraction (Cohen, Balint, Sindhu 1981 Plant Physiol 68: 1150-1155). To establish whether this enzyme is truly a chloroplast component, we have isolated purified intact chloroplasts from protoplasts by density gradient centrifugation in silica sols (Ludox AM). Such chloroplasts contained all of the diaminopimelate decarboxylase (EC 4.1.1.20) of the protoplasts, but were essentially devoid of spermidine synthase. Control experiments showed that the latter had not been inactivated under conditions of isolation, purification, and assay of the intact chloroplasts. Isolation and assay of protoplast vacuoles in a further examination of the supernatant fluid containing the enzyme revealed a significant fraction of the enzyme in the vacuole fraction. However this fraction was found to contain similar proportions of a soluble enzyme, glucose 6-phosphate dehydrogenase. It has been concluded that vacuolar fractions are difficultly separable from soluble cytoplasmic material, which is probably the only compartment containing spermidine synthase.     

Granular layer in the periplasmic space of gram-positive bacteria and fine structures of Enterococcus gallinarum and Streptococcus gordonii septa revealed by cryo-electron microscopy of vitreous sections

High-resolution structural information on optimally preserved bacterial cells can be obtained with cryo-electron microscopy of vitreous sections. With the help of this technique, the existence of a periplasmic space between the plasma membrane and the thick peptidoglycan layer of the gram-positive bacteria Bacillus subtilis and Staphylococcus aureus was recently shown. This raises questions about the mode of polymerization of peptidoglycan. In the present study, we report the structure of the cell envelope of three gram-positive bacteria (B. subtilis, Streptococcus gordonii, and Enterococcus gallinarum). In the three cases, a previously undescribed granular layer adjacent to the plasma membrane is found in the periplasmic space. In order to better understand how nascent peptidoglycan is incorporated into the mature peptidoglycan, we investigated cellular regions known to represent the sites of cell wall production. Each of these sites possesses a specific structure. We propose a hypothetic model of peptidoglycan polymerization that accommodates these differences: peptidoglycan precursors could be exported from the cytoplasm to the periplasmic space, where they could diffuse until they would interact with the interface between the granular layer and the thick peptidoglycan layer. They could then polymerize with mature peptidoglycan. We report cytoplasmic structures at the E. gallinarum septum that could be interpreted as cytoskeletal elements driving cell division (FtsZ ring). Although immunoelectron microscopy and fluorescence microscopy studies have demonstrated the septal and cytoplasmic localization of FtsZ, direct visualization of in situ FtsZ filaments has not been obtained in any electron microscopy study of fixed and dehydrated bacteria.     

Electron microscopic studies on protoplast release from I.C.A.-1.65 line (I.F.C.-1.65 line) of Bacillus subtilis cells.

Under lysozyme action a minicell-forming line (I.C.A.-1.65) of B. subtilis releases protoplasts. The main cytologic events which proceed protoplast releasing are described. Different areas of the cell wall prove a remarkable difference in their sensitivity to enzymatic lysozyme action. Central areas of the cell wall are most sensitive and the polar areas are most resistant. Mesosomal vesicles and tubules are extruded and released together with other cytoplasmic extrusion during the protoplasting process. The cell wall of minicells does not prove resistance particularities to lysozyme action. The minicells release protoplasts.     

Ultrastructural and biochemical aspects of cell wall reconstitution in recalcitrant (grapevine) and regenerating (tobacco) leaf protoplasts

Summary To identify possible reasons that may contribute to recalcitrance in plant protoplasts, the time course of new cell wall deposition was studied by scanning electron microscopy in protoplasts of a recalcitrant species, the grapevine. Results showed that microfibrils were developed after 2 days of culture, that complete cell wall formation occurred on Day 6 to 7 of protoplast culture, and its ultrastructural appearance was identical to that of grapevine leaf-derived callus cells. In addition, a comparative study was undertaken on [U-¹⁴C]glucose uptake and incorporation in ethanol-soluble, cellulosic, and noncellulosic polysaccharide fractions in protoplasts of grapevine and of a readily regenerating species, tobacco, during culture. There was a significantly higher [U-¹⁴C]glucose uptake by tobacco than by grapevine protoplasts. The label distribution in the ethanol-soluble, cellulosic, and noncellulosic fractions of newly synthesized cell walls differed quantitatively between the two species. In particular, the labeled glucose incorporated in the noncellulosic cell wall fraction was threefold greater in tobacco than in grapevine protoplasts. Differences were also revealed in the monosaccharide composition of this fraction between the two species. Addition of dimethyl sulfoxide to the culture medium resulted in a dramatic increase in [U-¹⁴C]glucose uptake by grapevine protoplasts, whereas it exhibited a limited effect in tobacco protoplasts. It showed no effect on the ultrastructural characteristics of new cell wall nor on the incorporation rate of labeled glucose in the cellulosic and noncellulosic cell wall fractions.     

Development and characterization of membrane surface display system using molecular chaperon, prsA, of Bacillus subtilis

We report a new membrane surface display system based on molecular chaperon, prsA, of Bacillus subtilis. Clostridium thermocellum cellulase, celA, was fused to C-terminal end of PrsA. Cellulase activity of B. subtilis protoplast, which expressed PrsA-CelA was 15 times higher compared to control strain. More than 85% of total cellulase activity was observed in surface displayed format and less than 15% of total cellulase activity was found in supernatant. Flow cytometric analysis of protoplast of PrsA-CelA fusion expressing bacteria provided another proof of uniform expression of fusion protein onto cytoplasmic membrane of B. subtilis. Without lysozyme treatment, only part of cellulase activity (10%) was observed in whole cell fraction.     

The transport of group 2 capsular polysaccharides across the periplasmic space in Escherichia coli. Roles for the KpsE and KpsD proteins

The cell surface expression of group 2 capsular polysaccharides involves the translocation of the polysaccharide from its site of synthesis on the inner face of the cytoplasmic membrane onto the cell surface. The transport process is independent of the repeat structure of the polysaccharide, and translocation across the periplasm requires the cytoplasmic membrane-anchored protein KpsE and the periplasmic protein KpsD. In this paper we establish the topology of the KpsE protein and demonstrate that the C terminus interacts with the periplasmic face of the cytoplasmic membrane. By chemical cross-linking we show that KpsE is likely to exist as a dimer and that dimerization is independent of the other Kps proteins or the synthesis of capsular polysaccharide. No interaction between KpsD and KpsE could be demonstrated by chemical cross-linking, although in the presence of both KpsE and Lpp, KpsD could be cross-linked to a 7-kDa protein of unknown identity. In addition, we demonstrate that KpsD is present not only within the periplasm but is also in both the cytoplasmic and outer membrane fractions and that the correct membrane association of KpsD was dependent on KpsE, Lpp, and the secreted polysaccharide molecule. Both KpsD and KpsE showed increased proteinase K sensitivity in the different mutant backgrounds, reflecting conformational changes in the KpsD and KpsE proteins as a result of the disruption of the transport process. Collectively the data suggest that the trans-periplasmic export involves KpsD acting as the link between the cytoplasmic membrane transporter and the outer membrane with KpsE acting to facilitate this transport process.     

Structure-functional analysis of the Dictyoglomus cell envelope

Several closely related strains of the thermophilic bacterium Dictyoglomus have been isolated from various hot springs on the Philippine archipelago. These strains as well as Dictyoglomus thermophilum H-6-12 were analyzed in view of the structure-functional relationships of the cell envelopes. All envelopes of Dictyoglomus strains show several peculiar features that are apparently either unique for the genus or common for other phylogenetically related Thermotogales. The filamentous cells develop pili at the cell poles, guided by large columnar protein assemblies that traverse the periplasm. Filamentous protein complexes span the periplasmic space at the longitudinal sides of the cell. By the end of the exponential growth phase, Dictyoglomus strains form multicellular aggregates ("rotund bodies") inside a compartment surrounded by a single, continuous outer envelope. The formation of these rotund bodies which are also found in some other deeply branching thermophilic phyla (Thermotoga, Thermus) was studied in detail. The transition between unicellular and multicellular lifestyle can be explained by the partial detachment of a protoplast from the outer envelope during cell division. When the outer envelope is partially detached from the protoplast, mechanical forces generated by protoplast elongation may drive cell rearrangement of daughter cells inside the compartment. During the following rounds of cell division, the overall shape of the compartment changes from spindle-like to globular geometry. Analysis of subcellular fractions of Dictyoglomus cells shows that glucan hydrolases are associated with the compartment. This feature is discussed in view of the multicellular life style of Dictyoglomus.     

Release from protoplasts of the Bacillus subtilis protease inhibitor

Abstract Conversion of Bacillus subtilis to protoplasts resulted in the release of 70–80% of the total protease inhibitor activity. Inhibitor fractions contained a polypeptide of approx. 15 kDa which reacted with inhibitor antibody. There was no release of protease inhibitor into the medium by sporulating cells, by osmotic shock of cells nor by washing with high concentrations of salt. The release of inhibitor activity was selective in that only 10–20% of the total protein, and < 10% of the glutamine synthetase activity was found in the protoplast supernatant. The inhibitor could be localized near the cell surface and function in cell protection.     

A comparative study of submicroscopic structures of protoplasts of various yeast species

A submicroscopic structure was studied of protoplasts of five different yeast species multiplying by budding, formation of cross septum and by a division typical for apiculate yeasts. The protoplasts retain their species specificity. Most considerable changes typical for the conversion of a cell to protoplast are found in membrane cell systems. The reduction of membranes of the endoplasmic reticulum is particularly striking. Both membrane units are frequently separated from each other by lenticular pseudovacuoles. Mitochondria in protoplasts are swollen and their number is reduced approximately two-fold. Defects are often observed in a nuclear membrane. The perinuclear space is usually extended by lenticular pseudovacuoles. A large number of vacuoles is observed in the basic protoplast cytoplasm. The surface of the protoplasts of all species studied is formed only by a cytoplasmic membrane. A partially digested original cell wall often adheres to protoplasts ofSchizosaccharomyces pombe.     

Localization of competence-induced proteins in Streptococcus pneumoniae.

Intracellular locations of 11 proteins associated with the development of competence in Streptococcus pneumoniae were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of subcellular fractions prepared from protoplasts. Controls showed that the competence-induced proteins were stable during the formation of protoplasts at 25 degrees C even though some had a half-life of only 8 min at 37 degrees C. Five competence-induced proteins p38, p27, p19.5, p16, and p14.5, were found in the cytoplasm. Two, p52 and p41, were associated with the membrane, and one, p10, was extracellular. Three others, p50, p36, and p29, were recovered in both cytoplasmic and membrane fractions. No competence-induced protein was detected in the periplasmic fraction except under conditions where leakage of all components was occurring, a phenomenon that was seen in many preparations. Similar fractionation of competent cells soon after uptake of [3H]DNA showed the "eclipse complex" of single-stranded DNA and p19.5 was associated approximately one-third with membranes and two-thirds with cytoplasmic fractions, with almost none in the periplasm. This result suggests strongly that at the time the donor DNA entered the cytosol it was in single-stranded form and it had not yet paired with the recipient DNA.     

Localization of ribonucleases in the mycelium of the fungus Aspergillus clavatus

The localization of ribonucleases in Aspergillus clavatus mycelium was studied using differential centrifugation of mycelial homogenates, preparation of protoplasts, by cytochemical and immunochemical studies. The RNase activity was represented mainly by guanylic and nonspecific RNases and was not associated with the intracellular structures. The cytochemical and immunochemical observations revealed the presence of a nonspecific RNase activity in the periplasm, mitochondria, perinuclear space and vesicular structures. The presence of this RNase in the periplasmic space and vesicular structures of A. clavatus suggests that it may be secreted through a fusion of the vesicular membranes with the cytoplasmic membrane, and then from the periplasm through the cell wall into the surrounding medium.     

Molecular forms of AlpA and AlpB lytic endopeptidases from <Emphasis Type="Italic">Lysobacter</Emphasis> sp. Xl1: immunochemical determination of their intra- and extracellular localization

Homologous endopeptidases AlpA and AlpB are components of the secreted complex of lytic enzymes of the Gram-negative bacterium Lysobacter sp. ХL1. These enzymes are synthesized as precursors that consist of a signal peptide, propeptide, and proteolytically active mature part. To understand the topogenetic features of these proteins, bacterial cell fractions were investigated by a sensitive sandwich enzymelinked immunosorbent assay and immunoblot analysis with the use of monoclonal antibodies recognizing unique epitopes of proteins’ mature forms and their propeptides. Only mature forms of the enzymes, without propeptides, were shown to be released outside the cell into the environment. AlpA significantly exceeds AlpB in the production level at the early stationary growth stage. The AlpB precursor was revealed in the cytoplasmic and periplasmic fractions, and the AlpA precursor was found only in the cytoplasmic fraction. The periplasmic fraction was also found to contain the mature forms of both enzymes and their propeptides. These results indicate that AlpA and AlpB are released into the environment through different mechanisms. AlpA is translocated across the cell envelope without being interrupted in the periplasm. The homologous AlpB enzyme, on the contrary, accumulates in the periplasmic space and is captured by outer membrane vesicles in the process of their formation.     

Cellular Location of Degradative Enzymes in Staphylococcus aureus

Staphylococus aureus, ATCC 6538P, was fractionated into protoplast membranes, mesosomal vesicles, periplasm, and cytoplasm. These fractions and the culture fluid were then assayed for various degradative enzyme activities. They were not restricted to a single fraction nor dispersed homogeneously, but were distributed predominantly (on the basis of specific activity) as follows: nuclease in the culture fluid; alkaline phosphatase, 5'-nucleotidase, and acid phosphatase in the periplasm; adenosine triphosphatase in the protoplast membrane; and protease (low levels) in mesosomal vesicles. No significant esterase nor cell wall hydrolytic activity was found in any fraction. S. aureus 80/81 was studied for penicillinase activity after induction with benzyl penicillin; this enzyme was localized in the mesosomal vesicles. Electron microscopy did not reveal any ultrastructural changes associated with secretion of the extracellular fraction. Overall, these studies demonstrate that degradative enzymes are located in several surface compartments and that, therefore, the mesosome does not function as a prototype lysosome in S. aureus.     

Age-Dependent Modifications and Further Localization of the CX 26-like Protein from Vicia faba L.

A strong age dependency together with alterations in the cellular distribution of CX 26 immunorelated protein(s) was found for differently developed leaves of Vicia faba L. With increasing age, an immunoreactive 40 kD band was observed in the soluble and microsomal fraction. In the cell wall protein preparation of young and fully differentiated leaves the 40 kD band was the minor constituent. A 33 kD polypeptide was dominantly localized in the microsomal fractions of all developmental stages and in SDS-extracts of total cell proteins of young leaves. A 21 kD protein together with a 16 kD polypeptide was associated with the cell wall fraction. The 21 kD protein, assumed to represent a plasmodesmatal constituent, was reduced with age. In SDS extracts, prepared from the different developmental stages of the leaves and of mesophyll protoplasts, the age-dependent appearance of the several immunostained bands was most obvious. A correlation of the 16, 33, and 40 kD bands to a turnover of the 21 kD protein is suggested. The reduced amount of the 21 kD protein with increasing age may be contemplated as an indication for a relative decrease of symplastic connections between cells of maturing leaves. This is in agreement with the results obtained by immunofluorescence studies using guard cell protoplasts. Here, observations pointed also to a reduction and final loss of CX 26-related protein at the protoplast surfaces.     

Isolation and Characterization of Protoplasts from Saccharomyces rouxii

Cells of the osmotolerant yeast Saccharomyces rouxii were transformed to protoplasts in good yield (85%) by digesting cell walls with snail-gut enzyme in the presence of 10 mM dithioerythritol, 0.1 M sodium phosphate buffer (pH 6.8), and 2.0 M KCl. The requirement for 2.0 M KCl compares with that for S. bisporus var. mellis (another osmotolerant species) and contrasts with the 0.3 to 0.8 M KCl concentrations used in the preparation of most yeast protoplasts. Short digestions (60 min or less) produced mostly spheroplasts; longer incubations (90 min or more) yielded mostly protoplasts as judged by electron micrographs. These protoplasts could be transferred to 1.0 M KCl or 2.0 M sorbitol without lysing, but lysis was pronounced in 0.5 M KCl or 1.0 M mannitol and complete in 0.02 M KCl. Protoplasts were separated from isolated cell wall remnants and debris by centrifugation on a linear gradient of Ficoll 400 (35 to 17.5%, wt/vol) containing 2.0 M KCl. Both crude and fractionated protoplast preparations contained vesicles which were identified with the periplasmic bodies of whole cells. Some of the periplasmic bodies were connected to protoplasts by fine pedicels; others appeared free. Independent degeneracy of periplasmic bodies was occasionally observed. beta-Fructofuranosidase (EC 3.2.1.26) activity is cryptic (physically) in cells of S. rouxii in contrast to the expressed enzyme (periplasmic space) of other Saccharomyces species. This enzyme remains cryptic in protoplast preparations of S. rouxii but is expressed upon lysis. The same specific activities were found per unit cell or protoplast. The possible association of the cryptic enzyme with periplasmic bodies is discussed.     

Immunofluorescence of the microtubular skeleton in growing and drug-treated yeast protoplasts

The microtubular system in growing protoplasts of Saccharomyces uvarum was visualized by immunofluorescence using the monoclonal antitubulin antibody TU 01. We confirmed the coexistence of regular spindle configuration and extensive cytoplasmic networks in growing protoplasts and also observed a distinct distortion of cytoplasmic microtubules in association with wall removal. After a short period for recovery of protoplasts in nutrient medium a restitution of cytoplasmic microtubules and their resumed contact with the protoplast surface was observed. Treatment of growing protoplasts with nocodazole resulted in the disappearance of spindle and cytoplasmic microtubules in the relevant fraction of the protoplast population. In carbendazime (MBC)-arrested protoplasts spindle microtubules were absent but cytoplasmic microtubules associated with spindle pole bodies were clearly visible. Microtubule reassembly on spindle pole bodies occurred within 30 min after washing out nocodazole as well as carbendazime. The approach using protoplasts suggests a simple way in which the differential effect of antimicrotubule agents can be experimentally tested and the microtubule organizing activity of yeast protoplasts visualized at the population level.