Effects of glucanase treatment on the structure and mechanical properties of cell walls in the giant‐celled xanthophycean alga Vaucheria frigida

The cell wall of the tip‐growing cells of the giant‐cellular xanthophycean alga Vaucheria frigida is mainly composed of cellulose microfibrils (CMFs) arranged in random directions and the major matrix component into which the CMFs are embedded throughout the cell. The mechanical properties of a cell‐wall fragment isolated from the tip‐growing region, which was inflated by artificially applied pressure, were measured after enzymatic removal of the matrix component by using a protease; the results showed that the matrix component is involved in the maintenance of cell wall strength. Since glucose and uronic acid are present in the matrix component of Vaucheria cell walls, we measured the mechanical properties of the cell wall after treatment with endo‐1,3‐ß‐glucanase and observed the fine structures of its surfaces by atomic force microscopy. The major matrix component was partially removed from the cell wall by glucanase, and the enzyme treatment significantly weakened the cell wall strength without affecting the pH dependence of cell wall extensibility. The enzymatic removal of the major matrix component by using a protease released polysaccharide containing glucose and glucuronic acid. This suggests that the major matrix component of the algal cell walls contains both proteins (or polypeptides) and polysaccharides consisting of glucose and glucuronic acid as the main constituents.     

A cluster of five cell wall-associated receptor kinase genes,Wak1–5, are expressed in specific organs of Arabidopsis

WAK1 (wall-associated kinase 1) is a cytoplasmic serine/threonine kinase that spans the plasma membrane and extends into the extracellular region to bind tightly to the cell wall. The Wak1 gene was mapped and found to lie in a tight cluster of five highly similar genes (Wak1–5) within a 30 kb region. All of the Wak genes encode a cytoplasmic serine/threonine protein kinase, a transmembrane domain, and an extracytoplasmic region with several epidermal growth factor (EGF) repeats. The extracellular regions also contain limited amino acid identities to the tenascin superfamily, collagen, or the neurexins. RNA blot analysis with gene-specific probes revealed that Wak1, Wak3 and Wak5 are expressed primarily in leaves and stems of Arabidopsis. Wak4 mRNA is only detected in siliques, while Wak2 mRNA is found in high levels in leaves and stems, and in lower levels in flowers and siliques. A trace amount of Wak2 can also be detected in roots. Wak1 is induced by pathogen infection and salicylic acid or its analogue INA and is involved in the plant's response, and Wak2, Wak3 and Wak5 also can be greatly induced by salicylic acid or INA. The WAK proteins have the potential to serve as both linkers of the cell wall to the plasma membrane and as signaling molecules, and since Wak expression is organ-specific and the isoforms vary significantly in the cell wall associated domain this family of proteins may be involved in cell wall-plasma membrane interactions that direct fundamental processes in angiosperms.     

The effect of sorbic acid and esters of p-hydroxybenzoic acid on the protonmotive force in Escherichia coli membrane vesicles

The effect of three food preservatives, sorbic acid and methyl and butyl esters of p-hydroxybenzoic acid, on the protonmotive force in Escherichia coli membrane vesicles was investigated. Radioactive chemical probes were used to determine the two components of the protonmotive force: delta pH (pH difference) and delta psi (membrane potential). Both types of compound selectively eliminated delta pH across the membrane, while leaving delta psi much less disturbed indicating that transport inhibition by neutralization of the protonmotive force cannot be the only mechanism of action for the food preservatives tested.     

Fine structure of sporulation in Bacillus cereus grown in a chemically defined medium

Ellar, D. J. (Syracuse University, Syracuse, N.Y.), and D. G. Lundgren. Fine structure of sporulation in Bacillus cereus grown in a chemically defined medium. J. Bacteriol. 92:1748-1764. 1966.-A study was made of the fine structure of sporulating cells of Bacillus cereus grown in a chemically defined medium. The developmental stages of sporulation occurred in a fairly synchronous manner and were complete by 14 hr. This time period was shortened when spore wall peptide components were added to the medium, but the addition had no effect upon fine structure except to thicken the cell wall. Sporulation could be separated into six morphological stages which generally agreed with those published for other sporulating bacteria. The initiation of the spore (forespore) septum takes the form of an inward folding of the cytoplasmic membrane toward the pole of the cell. The inward folding forms a characteristic Y-shaped membrane structure enclosing an area within which vesicles are found. These vesicles comprise the perisporal mesosome of the cell. The membranes on opposite sides of the cell progress toward the cell center where they fuse to form the double unit membrane of the spore septum. As the proliferation of the spore septum continues, the vesicular areas move towards the pole. The end result is a double forespore membrane which completely encloses a part of the vegetative cell's chromatin. Sporal mesosomes, as well as membrane vesicles, are involved in the proliferation of the forespore. Vesicles are generally bounded by a single unit membrane, whereas in the sporal mesosomes several unit membranes are arranged concentrically. The latter become associated with the segregation of a portion of the nuclear material into the forespore region of the cell.     

The protonmotive force in bovine heart submitochondrial particles. Magnitude, sites of generation and comparison with the phosphorylation potential.

1. The magnitude of the protonmotive force in respiring bovine heart submitochondrial particles was estimated. The membrane-potential component was determined from the uptake of S14CN-ions, and the pH-gradient component from the uptake of [14C]methylamine. In each case a flow-dialysis technique was used to monitor uptake. 2. With NADH as substrate the membrane potential was approx. 145mV and the pH gradient was between 0 and 0.5 unit when the particles were suspended in a Pi/Tris reaction medium. The addition of the permeant NO3-ion decreased the membrane potential with a corresponding increase in the pH gradient. In a medium containing 200mM-sucrose, 50mM-KCl and Hepes as buffer, the total protonmotive force was 185mV, comprising a membrane potential of 90mV and a pH gradient of 1.6 units. Thus the protonmotive force was slightly larger in the high-osmolarity medium. 3. The phosphorylation potential (= deltaG0' + RT ln[ATP]/[ADP][Pi]) was approx. 43.1 kJ/mol (10.3kcal/mol) in all the reaction media tested. Comparison of this value with the protonmotive force indicates that more than 2 and up to 3 protons must be moved across the membrane for each molecule of ATP synthesized by a chemiosmotic mechanism. 4. Succinate generated both a protonmotive force and a phosphorylation potential that were of similar magnitude to those observed with NADH as substrate. 5. Although oxidation of NADH supports a rate of ATP synthesis that is approximately twice that observed with succinate, respiration with either of these substrates generated a very similar protonmotive force. Thus there seemed to be no strict relation between the size of the protonmotive force and the phosphorylation rate. 6. In the presence of antimycin and/or 2-n-heptyl-4-hydroxyquinoline N-oxide, ascorbate oxidation with either NNN'N'-tetramethyl-p-phenylenediamine or 2,3,5,6-tetramethyl-p-phenylenediamine as electron mediator generated a membrane potential of approx. 90mV, but no pH gradient was detected, even in the presence of NO3-. These data are discussed with reference to the proposal that cytochrome oxidase contains a proton pump.     

Cytoplasmic steps of peptidoglycan biosynthesis

The biosynthesis of bacterial cell wall peptidoglycan is a complex process that involves enzyme reactions that take place in the cytoplasm (synthesis of the nucleotide precursors) and on the inner side (synthesis of lipid-linked intermediates) and outer side (polymerization reactions) of the cytoplasmic membrane. This review deals with the cytoplasmic steps of peptidoglycan biosynthesis, which can be divided into four sets of reactions that lead to the syntheses of (1) UDP-N-acetylglucosamine from fructose 6-phosphate, (2) UDP-N-acetylmuramic acid from UDP-N-acetylglucosamine, (3) UDP-N-acetylmuramyl-pentapeptide from UDP-N-acetylmuramic acid and (4) D-glutamic acid and dipeptide D-alanyl-D-alanine. Recent data concerning the different enzymes involved are presented. Moreover, special attention is given to (1) the chemical and enzymatic synthesis of the nucleotide precursor substrates that are not commercially available and (2) the search for specific inhibitors that could act as antibacterial compounds.     

In vitro translocation of secretory proteins possessing no charges at the mature domain takes place efficiently in a protonmotive force-dependent manner.

The effect of charges existing on the mature domain of secretory proteins on the efficiency and protonmotive force dependence of translocation into everted membrane vesicles of Escherichia coli was studied. Model secretory proteins devoid of charges on the mature domain were constructed at the DNA level using proOmpF-Lpp as the starting protein. The chargeless presecretory proteins thus constructed were translocated and processed for the signal peptide much faster than proOmpF-Lpp and the rate of translocation was appreciably enhanced by imposition of the protonmotive force. Not only the membrane potential but also delta pH were effective in stimulating the rate of translocation of the chargeless proteins. The results indicate that the mature domain does not have to be charged for the secretory translocation and that the major requirement of the protonmotive force for the secretory translocation is not for the movement, including an electrophoretic one, of charged regions of the mature domain. All of the proOmpF-Lpp derivatives thus constructed were translocated efficiently into everted membrane vesicles in a SecA-dependent manner, irrespective of their size. The mature domain of the smallest one was 45 amino acid residues in length. Contrary to the views previously presented by other workers, these results suggest that there is no sharp boundary at the reported regions for the translocation of presecretory proteins across the cytoplasmic membrane or for the requirement of SecA.     

Early development of the kelp fly,Coelopa frigida (Diptera). II. Morphology of cleavage and blastoderm formation

Cleavage and blastoderm formation in Coelopa frigida are extremely rapid developmental processes. In short (6–7 minutes) successive cell cycles, nuclei multiply and spread out through the egg. The movement seems to be aided by endoplasmic vesicles and cisternae which are in direct contact with the nuclear membrane. The first cells to separate from the egg plasmodium in early superficial cleavage stages are the pole cells. Precursor material from multivesicular bodies forms the pole cell membranes. The primary nuclei from the posterior pole region are removed from the blastoderm by the pole cell segregation. Blastoderm nuclei from the regions adjacent to the posterior pole migrate into the residual periplasm after pole cell segregation has been completed and constitute the blastoderm nuclei in that region of the egg. Nucleoli are not revealed during internal cleavage. They appear in pole cells shortly after their segregation. The generation time of the blastoderm nuclei increases after the twelfth cleavage. Concurrently, nucleoli form in the blastoderm nuclei and permanent cell membranes separate individual blastoderm cells. After blastoderm cells have been separated from each other, they remain in contact with the interior yolk sac by means of cytoplasmic canals. This contact is maintained at least during the early phases of blastokinesis. Observations on nuclear migration and rapid membrane formation are discussed as examples of protein assembly from subunits as an alternative to de novo protein synthesis in early stages of development.     

MITOSIS,CYTOKINESIS, AND CELL ELONGATION IN THE DESMID,CLOSTERIUM LITTORALE1

Some details of interphase cell structure are given. At prophase the nuclear envelope breaks down and the nucleolus disperses; very small doubled chromosomes generally form a precisely aligned, metaphase plate with normal spindle microtubules present; 2 plates of chromatids separate during anaphase, the spindle becoming invaded, by (mucilage) vesicles. Telophase nuclei arc initially very hard to discern, until they increase in volume. Microtubules collect at each pole, becoming increasingly focused on one small region containing fine granular malarial, the microtubule center (MC). The septum, an annular ingrowth, begins forming at prophase and partitions the cell by telophase. At no stage were microtubules involved in this initial cross-wall formation. At telophase the spindle collapses and as the nuclei move back to the septum, increasing numbers of microtubules appear near this cross wall, all transversely aligned. An annular split deepens down the middle of the wall material in the septum, and the daughter cells begin to expand, stretching the new wall; the microtubules appearing near the septum now are transformed steadily into typical hooplike wall, microtubules, but strictly confined to the expanding wall (there are none near interphase cell walls). Meanwhile, the MC, has moved, to the side of the cell and begins migrating along one of the grooves in the chloroplast; a large number of parallel microtubules extends back to the nucleus, which becomes increasingly deformed as it begins to extend a long thin protrusion along these, microtubules. The MC keeps moving along the cell until it lodges in the cleavage developing in the chloroplast. Some microtubules extend still further up the cell, others appear in the chloroplast cleavage, but most en-sheathe the nucleus which by now is moving along the cell as a cylindrical structure tightly fitting in the chloroplast groove. The nuclear membrane is then drawn up into the deepening chloroplast constriction, and when the chloroplast is finally cut in 2, the nucleus lakes up its interphase position between the 2 halves. While all this is occurring, the whole cytoplasm is expanding into the new semicell being created by growth of the wall originally derived from the septum. Thus the interphase cell symmetry is reestablished after mitosis. These results are discussed in terms of more general phenomena of cell division and morphogenesis.     

The alpha-glucanase Agn1p is required for cell separation in Schizosaccharomyces pombe

BACKGROUND INFORMATION: In animal cells, cytokinesis occurs by constriction of an actomyosin ring. In fission yeast, ring constriction is followed by deposition of a multilayered division septum that must be cleaved to release the two daughter cells. Although many studies have focused on the actomyosin ring and septum assembly, little is known about the later steps involving the cleavage of the cell wall. RESULTS: We identified a novel gene in Schizosaccharomyces pombe, namely the agn1(+) gene that has homology to fungal 1,3-alpha-glucanases (mutanases). Disruption of the agn1(+) gene is not lethal to the cells, but does interfere with their separation, whereas overexpression of Agn1p is toxic and causes cell lysis. Agn1p levels reach a peak during septation and the protein localizes to the septum region before cell separation. Moreover, agn1(+) is responsible for the 1,3-alpha-glucanase activity, which shows a maximum at the end of septation. CONCLUSIONS: Our results clearly suggest the existence of a relationship between agn1(+), 1,3-alpha-glucanase activity and the completion of septation in S. pombe. Agn1p could be involved in the cleavage of the cylinder of the old wall that surrounds the primary septum, a region rich in alpha-glucans.     

Mechanical Response of Single Plant Cells to Cell Poking： A Numerical Simulation Model

Cell poking is an experimental technique that is widely used to study the mechanical properties of plant cells. A full understanding of the mechanical responses of plant cells to poking force Is helpful for experimental work. The aim of this study was to numerically investigate the stress distribution of the cell wall, cell turgor, and deformation of plant cells in response to applied poking force. Furthermore, the locations damaged during poking were analyzed. The model simulates cell poking, with the cell treated as a spherical, homogeneous, isotropic elastic membrane, filled with incompressible, highly viscous liquid. Equilibrium equations for the contact region and the non-contact regions were determined by using membrane theory. The boundary conditions and continuity conditions for the solution of the problem were found. The forcedeformation curve, turgor pressure and tension of the cell wall under cell poking conditions were obtained. The tension of the cell wall circumference was larger than that of the meridian. In general, maximal stress occurred at the equator around. When cell deformation increased to a certain level, the tension at the poker tip exceeded that of the equator. Breakage of the cell wall may start from the equator or the poker tip, depending on the deformation. A nonlinear model is suitable for estimating turgor, stress, and stiffness, and numerical simulation is a powerful method for determining plant cell mechanical properties.     

Differences in the formation of poles of Enterococcus and Bacillus.

The pole of Enterococcus hirae (Streptococcus faecium) is more pointed than that of Bacillus subtilis; i.e. the pole of the former is prolate and the latter is oblate. Both species form their poles by constructing annular additions on the inside surface. In both cases, the thick septum starts to split from the outside before the septum is complete. Physiochemical considerations dictate that the peptidoglycan must be unstretched as laid down. However, it later becomes stressed and may stretch to increase its surface area or to change its shape. Our earlier analysis for B. subtilis demonstrated that, without the addition of new peptidoglycan, the nascent wall is stretched after it is externalized to 1.51 times the original area. The wall of partially formed poles that is already exteriorized continues to deform with further development. For E. hirae, Higgins & Shockman's measurements showed that the completed pole has a surface area 2.18 times larger than a completed septal disk and the wall changes shape very little after exteriorization. A model is presented here for the streptococcus in which the septal wall does not increase its surface area on exteriorization either by expansion or by murein insertion. Instead, the septal wall as it is split and exteriorized twists to become oblique, increasing the inner radius of the incomplete septum. In consequence of this rotation, extra layers of peptidoglycan are added to the inside face of the developing septum. This additional murein forms the more pointed pole shape for E. hirae. This "split-and-splay" model thus refines and extends the surface stress theory of E. hirae developed a decade ago by proposing a source of the extra wall needed for the formation of its prolate, more pointed, pole.     

Immunoelectron microscopic localization of the F1F0 ATPase (ATP synthase) on the cytoplasmic membrane of alkalophilic Bacillus firmus RAB

Evidence that the F1F0 ATPase (ATP synthase) of alkalophilic Bacillus firmus RAB is localized exclusively on the cytoplasmic membrane was obtained by immunogold electron microscopy using a highly specific polyclonal antibody against the beta subunit of Escherichia coli F1F0 ATPase. The energetic problem faced by cells of B. firmus RAB growing oxidatively at pH 10.5 despite a low protonmotive force across the cytoplasmic membrane cannot, therefore, be circumvented by localization of energy transducing functions on hypothetical internal membranes.     

The residual protonmotive force in mitochondria after an oxygen pulse

Both from irreversible thermodynamics and from mass-action kinetics it can be derived that upon anaerobiosis in an oxygen-pulse experiment the protonmotive force across a mitochondrial membrane undergoes a sudden drop. Under representative conditions the protonmotive force after the drop (the residual protonmotive force) is shown to be less than 3 kJ . mol-1 as opposed to steady-state values for the protonmotive force of 19 kJ . mol-1. It is concluded that correction for proton leakage in pulse experiments by back extrapolation underestimate proton leakage. Consequently the observed H+/O stoichiometries must underestimate the true H+/O ratios.     

鱼精蛋白抗菌机制的研究

鱼精蛋白是一种存在于各类动物精巢组织中的多聚阳离子肽,其抗菌性很早就被所知,然而它的抗菌机理却一直未能得到很清楚的了解。现存在的机理有2种:一种认为鱼精蛋白与细菌细胞壁结合,通过破坏细胞壁的形成来达到抑菌效果;另一种认为鱼精蛋白破坏了细胞能量的转换、营养物质的吸收功能,细胞质膜是鱼精蛋白攻击的对象。事实上,作者认为,鱼精蛋白的抗菌效果可能是通过以上2种方式共同作用的结果,因而它的抗菌机理也可能是这两种机理的叠加,这还需进一步的研究证明。     

Touch and go: tying TonB to transport

The TonB system of Gram-negative bacteria appears to exist for the purpose of transducing the protonmotive force energy from the cytoplasmic membrane, where it is generated, to the outer membrane, where it is needed for active transport of iron siderophores, vitamin B12 and, in pathogens, iron from host-binding proteins. In this review, we bring the reader up to date on the developments in the field since the authors each wrote reviews in this journal in 1990.     

The protonmotive force in phosphorylating membrane vesicles from Paracoccus denitrificans. Magnitude, sites of generation and comparison with the phosphorylation potential

1. The magnitude of the protonmotive force in phosphorylating membrane vesicles from Paracoccus denitrificans was estimated. The membrane potential component was determined from the uptake of S(14)CN(-), and the transmembrane pH gradient component from the uptake of [(14)C]methylamine. In each case a flow-dialysis technique was used to monitor uptake. 2. With NADH as substrate, the membrane potential was about 145mV and the pH gradient was below 0.5 pH unit. The membrane potential was decreased by approx. 15mV during ATP synthesis, and was abolished on addition of carbonyl cyanide p-trifluoromethoxyphenylhydrazone. In the presence of KCl plus valinomycin the membrane potential was replaced by a pH gradient of 1.5 units. 3. Succinate oxidation generated a membrane potential of approx. 125mV and the pH gradient was below 0.5 pH unit. Oxidation of ascorbate (in the presence of antimycin) with either 2,3,5,6-tetramethyl-p-phenylenediamine or NNN'N'-tetramethyl-p-phenylenediamine as electron mediator usually generated a membrane potential of approx. 90mV. On occasion, ascorbate oxidation did not generate a membrane potential, suggesting that the presence of a third energy-coupling site in P. denitrificans vesicles is variable. 4. With NADH or succinate as substrate, the phosphorylation potential (DeltaG(p)=DeltaG(0)'+RTln[ATP]/ [ADP][P(i)]) was approx. 53.6kJ/mol (12.8kcal/mol). Comparison of this value with the protonmotive force indicates that more than 3 protons need to be translocated via the adenosine triphosphatase of P. denitrificans for each molecule of ATP synthesized by a chemiosmotic mechanism. In the presence of 10mm-KNO(3) the protonmotive force was not detectable (<60mV) but DeltaG(p) was not altered. This result may indicate either that there is no relationship between the protonmotive force and DeltaG(p), or that for an unidentified reason the equilibration of SCN(-) or methylamine with the membrane potential and the pH gradient is prevented by NO(3) (-) in this system.     

Variable proton conductance of submitochondrial particles.

The relationship between the rate of substrate oxidation and the protonmotive force (electrochemical proton gradient) generated by bovine heart submitochondrial particles has been examined. Unexpectedly, oxidation of succinate generated a higher protonmotive force than the oxidation of NADH, although the rate of proton translocation across the membrane was inferred to be considerably lower with succinate as substrate. The data suggest that the flow of electrons through site 1 of the respiratory chain may increase the conductance of the mitochondrial membrane for protons. Upon reduction of the rate of succinate oxidation by titration with malonate, the protonmotive force remained essentially constant until the extent of inhibition was greater than 75%. The general conclusion from this work is that a constant passive membrane conductance for protons cannot be assumed.     

Regulation of mitochondrial resting state respiration: slip, leak, heterogeneity?

The contribution of molecular slippage of proton pumps, of proton leak and of coupling heterogeneity of mitochondrial population to the well-known non-linear interrelationship between resting state respiration and the protonmotive force is discussed in view of the following experimental findings. (1) After blocking mitochondrial respiration with cyanide, the rate of dissipation of the membrane potential is non-linearly dependent on the actual membrane potential, similarly to the resting state respiration in mitochondria titrated with small amounts of an inhibitor. In contrast, delta pH dissipates proportionally to its actual value. (2) The rate of electron flow from succinate to ferricyanide depends upon the protonmotive force, similarly to the flow from succinate to oxygen. This strongly suggests that the H+/e- stoichiometry in complexes III and IV of the respiratory chain is constant. (3) Mitochondria 'in situ', in permeabilized Ehrlich ascites cells, exhibit the same non-linear flux/force relationship as isolated mitochondria. These results strongly suggest that the non-ohmic characteristics of the inner mitochondrial membrane, with respect to protons driven by the membrane potential but not by the concentration gradient, is the main factor responsible for the nonlinear flux/force relationship in resting state mitochondria.     

Isotope and thermal effects in chemiosmotic coupling to the membrane ATPase of Streptococcus

We measured rates of ATP synthesis by the proton-translocating ATPase of the motile Streptococcus strain V4051. Starved cells were energized artificially by exposing their membranes to a variable electrical potential difference (internal medium negative) and a fixed pH difference (internal medium alkaline). The initial rates of ATP synthesis increased exponentially with protonmotive force. The results were the same in D2O and H2O; there was no solvent isotope effect. At a fixed protonmotive force, the rates were strongly dependent on temperature, as expected for a reaction with a large enthalpy of activation. At a different protonmotive force, the rates varied with temperature in an identical fashion; there was no change in the enthalpy of activation. We conclude that protonation-deprotonation steps are not rate limiting and that the protons that cross the membrane drive ATP synthesis by mass action. The transmembrane electric field acts by changing the concentrations of the reactants, not by changing the configuration of the enzyme-substrate complex.