Recombinant protein excretion in Escherichia coli JM103[pUC8]: Effects of plasmid content, ethylenediaminetetraacetate, and phenethyl alcohol on cell membrane permeability

The presence of a high copy number plasmid (pUC8) was found to affect integrity of the cell envelope of Escherichia coli JM103, causing in turn significant release of the plasmid-encoded protein (beta-lactamase). The alterations in cell membrane permeability were evident from the increased susceptibility of recombinant cells to deoxycholic acid and methylene blue, which did not have appreciable effect on plasmid-free cells. The deteriorated cell membrane structure also resulted in a substantial reduction in specific growth rate and mass yield of plasmid-bearing cells. Further enhancement in beta-lactamase excretion was achieved by permeabilizing cell membrane with ethylenediaminetetraacetate (EDTA) and phenethyl alcohol (PEA). Unlike other commonly used physical and chemical methods for releasing the enzymes accumulated in the cells, application of EDTA and PEA at appropriate concentrations neither led to cell death nor interrupted synthesis of the plasmid-encoded protein. While in situ application of PEA was complicated due to interference with beta-lactamase activity, in situ application of EDTA was found to be an efficient way of releasing the recombinant protein without sacrificing its productivity. The experimental results demonstrate that the presence of EDTA and PEA can substantially reduce the growth rate differential between plasmid-free and plasmid-bearing cells, suggesting possible improvement of plasmid stability by application of these cell membrence-permeabilizing agents on a periodic basis.     

Mechanism of Action of Phenethyl Alcohol: Breakdown of the Cellular Permeability Barrier

Phenethyl alcohol (PEA) caused Escherichia coli to take up greatly increased amounts of acriflavine, a compound to which healthy growing cells are impermeable. PEA also caused an increased rate of efflux (leakage) of cellular potassium under conditions which do not greatly alter the influx of potassium via the energy-dependent potassium pump. We therefore propose that the primary effect of PEA is a limited breakdown of the cell membrane. The inhibition of deoxyribonucleic acid synthesis and other cellular functions would then be secondary consequences of the alteration in the membrane structure.     

Expression and transport of cellulase in avocado mesocarp during ripening

Summary Cellulase localization in the mesocarp of ripening avocado fruits (Persea americana Mill. cv. Hass) was studied by a variety of immunological methods. As localized by immunodetection on whole fruit tissue blots, cellulase first appeared near the stylar end of the fruit during the late portion of the rise in climacteric respiration. Cellulase appearance subsequently expanded outward and upward, reaching the peduncle end of the fruit by the day after the climacteric peak. Cellulase expression in cells surrounding vascular bundles was delayed relative to expression in the adjacent mesocarp. Immuno-labeled frozen sections, viewed with the light microscope, showed that cellulase appeared in both parenchyma and oil cells concom-mitantly with wall breakdown. Immunogold detection of cellulase by electron microscopy revealed labeling associated with the endoplasmic reticulum, plasmodesmata, and cell wall during the period between the late portion of the climacteric rise and the day after the climacteric peak. Cellulase appeared in the nucleus during all stages of ripening after the early portion of the climacteric rise. Immunoblot analysis of organelle fractions, isolated from avocado fruit at the climacteric peak of respiration, revealed three molecular weight forms of cellulase; a light and a heavy form found in endoplasmic reticulum-enriched fractions, and an intermediate form found in Golgi and plasma membrane-enriched fractions.Abbreviations Endo-H endoglyosidase H - Tris tris(hydroxymethyl)-aminomethane - MeOH methyl alcohol - EtOH ethyl alcohol - BSA bovine serum albumin - PM plasmamembrane - ATPase adenosine 5-triphosphatase - Pi inorganic phosphate - IDPase inosine 5-di-phosphatase - ER endoplasmic reticulum The work presented here has been submitted in partial fulfillment of the requirements for the Ph.D. degree.     

A new role for veratryl alcohol: regulation of synthesis of lignocellulose-degrading enzymes in the ligninolytic ascomyceteous fungus, Botryosphaeria sp.; influence of carbon source

A new physiological role for veratryl alcohol in fungi important in the biodegradation of the lignified plant cell wall is presented. Botryosphaeria sp., grown on starch, pectin, cellulose or xylan produced amylase, pectinase, cellulase, xylanase and laccase, whereas glucose and xylose repressed the synthesis of cellulase and xylanase, but not laccase. When cultured on each of these substrates in the presence of veratryl alcohol, laccase activity increased but the activities of amylase, pectinase, cellulase and xylanase significantly decreased. Basal medium containing softwood kraft lignin in the presence of veratryl alcohol induced laccases above constitutive levels. Ethyl alcohol also stimulated laccase production.     

Cytochemical localization of cellulase in glandular trichomes ofcannabis (cannabaceae)

Cellulase reaction product was localized cytochemically at the ultrastructural level in the cell wall of disc cells, the secretory cavity and in the subcuticular wall of glands inCannabis. Cellulase reaction product was evident in the less dense region of the disc cell wall prior to secretory cavity formation. Reactivity in this region was associated with separation of an outer zone, forming the subcuticular wall, from the inner wall zone adjacent to the plasma membrane of the disc cells. Reaction product was associated with the disc cell wall and fibrillar matrix extending from it into the secretory cavity. Reactivity remained evident over the subcuticular wall throughout enlargement of the secretory cavity. Reaction product also was present over fibrillar matrix in the secretory cavity associated with both the inner wall and the subcuticular wall. The distribution of cellulase reaction product supports an interpretation that cellulase is involved in formation of the secretory cavity and subsequent redistribution of wall products to form the subcuticular wall during development of the secretory cavity.     

The mode of action of cell wall-degrading enzymes and their interference with Nod factor signalling in <Emphasis Type="Italic">Medicago sativa</Emphasis> root hairs

Medicago sativa L. (alfalfa) root hairs respond to Nod factors [NodRm-IV(C16:2,S)] in a host-specific manner with depolarization and rapid ion fluxes. Protoplasts prepared from these cells using the cell wall-digesting enzymes pectolyase and cellulase do not, or to a rather small extent, respond to Nod factors. In an effort to understand this activity loss we analyzed the mode of action of both enzymes with respect to their effects on the root hairs as well as their interference with the Nod factor response. (i) In the presence of the enzymes, Nod factor at saturating concentrations neither depolarized the plasma membrane of root hairs nor caused ion fluxes. Even after removal of the enzymes, Nod factor responses were strongly refractory. (ii) After a lag-phase of 12-18 s, pectolyase depolarized the plasma membrane, alkalized the external space, acidified the cytosol and increased the cytosolic Ca(2+) activity. (iii) Cellulase, without a lag-phase, depolarized the plasma membrane, acidified the cytosol, but only marginally increased the cytosolic Ca(2+) activity. Unlike pectolyase, the cellulase response was only weakly refractory to a second addition. (iv) Neither enzyme increased the membrane conductance, but pectolyase inhibited the H(+)-pump. (v) Pectolyase shows all the signs of an elicitor, while cellulase yields a mixed response. (vi) Denatured enzymes yielded strong effects similar to those of untreated enzymes. We conclude that the effects shown do not originate from enzymatic activity, but from interactions of the proteins with cell wall or plasma membrane constituents. It is further concluded that these enzymes (pectolyase more so than cellulase) trigger defense-related signal pathways, which makes protoplasts prepared with such enzymes unsuitable for studies of symbiotic or defense-related signalling.     

CYTOCHEMICAL LOCALIZATION OF CELLULASE ACTIVITY IN ARTICULATED,ANASTOMOSING LATICIFERS OF PAPAVER SOMNIFERUM L. (PAPAVERACEAE)

Cellulase activity was localized at the ultrastructural level in the articulated, anastomosing laticifers of Papaver somniferum. Electron-dense crystalline deposits indicating the presence of cellulase activity were restricted to discrete patches along the laticifer wall in regions of recently formed perforations. This report presents the first direct evidence for the involvement of cellulases in the cell wall perforation process in articulated laticifers.     

Cellulase activity and localization during induced abscission of Coleus blumei

Treatment with dimethipin (2,3-dihydro-5,6-dimethyl-1,4-dithiin 1,1,4,4 tetroxide) inhibited the increase in cellulase activity and decrease in breakstrength associated with the normal course of abscission in Coleus. Application of the surfactant UBI-1126 (Emery OAL 20 in isopropyl alcohol) increased cellulase activity and accelerated the process of abscission in Coleus expiants within 24 h of application. Cellulase activity was localized histochemically at the electron microscopic level in surfactant-treated tissue. The enzyme activity was localized primarily in the cell wall, middle lamella, and paramural bodies of abscission zone cells.     

Multinucleation in the conidia of polyploids derived fromTrichoderma reesei QM 9414 by colchicine treatment

Conidia ofTrichoderma reesei QM 9414 were treated with colchicine in order to obtain polyploids (diploids; tetraploids). Cellulase production by diploids (mononucleate conidia) was almost twice as great as that of the original strain, but that of tetraploids (binucleate conidia) was not increased. When these latter conidia were re-treated with 2.0% (w/v) colchicine, multiple nuclei were produced in each conidium, and their diameter was almost the same as that of the original nucleus. Cellulase production of the diploid was almost the same in either mononucleate or multinucleate nature. However, cellulase production by the tetraploid which produced multinucleate conidia was greater than that of the binucleate tetraploid and that of the diploid. The multinucleation technique can contribute to enhancing cellulase production.     

The Activity of a Wall-Bound Cellulase Is Required for and Is Coupled to Cell Cycle Progression in the Dinoflagellate Crypthecodinium cohnii

Cellulose synthesis, but not its degradation, is generally thought to be required for plant cell growth. In this work, we cloned a dinoflagellate cellulase gene, dCel1, whose activities increased significantly in G₂/M phase, in agreement with the significant drop of cellulose content reported previously. Cellulase inhibitors not only caused a delay in cell cycle progression at both the G₁ and G₂/M phases in the dinoflagellate Crypthecodinium cohnii, but also induced a higher level of dCel1p expression. Immunostaining results revealed that dCel1p was mainly localized at the cell wall. Accordingly, the possible role of cellulase activity in cell cycle progression was tested by treating synchronized cells with exogenous dCelp and purified antibody, in experiments analogous to overexpression and knockdown analyses, respectively. Cell cycle advancement was observed in cells treated with exogenous dCel1p, whereas the addition of purified antibody resulted in a cell cycle delay. Furthermore, delaying the G₂/M phase independently with antimicrotubule inhibitors caused an abrupt and reversible drop in cellulase protein level. Our results provide a conceptual framework for the coordination of cell wall degradation and reconstruction with cell cycle progression in organisms with cell walls. Since cellulase activity has a direct bearing on the cell size, the coupling between cellulase expression and cell cycle progression can also be considered as a feedback mechanism that regulates cell size.     

Inhibition of Growth, Synthesis, and Permeability in Neurospora crassa by Phenethyl Alcohol.

Lester, Gabriel (Reed College, Portland, Ore.). Inhibition of growth, synthesis, and permeability in Neurospora crassa by phenethyl alcohol. J. Bacteriol. 90: 29-37. 1965.-Inhibition of the growth of Neurospora crassa in still culture was detected at 0.05% and was complete at a level of 0.2% phenethyl alcohol (PEA). Benzyl alcohol was less inhibitory, and 3-phenyl-1-propanol and phenol were more inhibitory, than PEA; benzylamine and phenethylamine were less inhibitory than the analogous hydroxylated compounds. Inhibition by PEA was not reversed by synthetic mixtures of purines and pyrimidines or vitamins, or by casein digests, yeast extract, or nutrient broth. The germination of conidia was inhibited by PEA, but after an exposure of 8.5 hr no loss of viability was observed. The addition of PEA to growing shake cultures caused a simultaneous inhibition of growth and of the syntheses of ribonucleic and deoxyribonucleic acids and protein; the relationships of these compounds to mycelial dry weight and to one another were constant in growing mycelia, and PEA did not significantly affect these relationships. PEA partially inhibited the uptake of glucose, but severely restricted the accumulation of l-leucine, l-tryptophan, or alpha-aminoisobutyric acid in germinated conidia. The efflux of alpha-aminoisobutyric acid from germinated conidia was somewhat enhanced by PEA, but this effect was not so pronounced as the (complete) inhibition of alpha-aminoisobutyric acid accumulation by PEA. It is suggested that PEA affects primarily the initial influx of alpha-aminoisobutyric acid rather than the subsequent retention of alpha-aminoisobutyric acid.     

Differential effects of procaine and phenethyl alcohol on excision repair of DNA in u.v.-irradiated Escherichia coli

Experiments were performed to investigate the involvement of the cell membrane in the excision DNA repair process in Escherichia coli. Two membrane-binding drugs, procaine and phenethyl alcohol (PEA), inhibited liquid-holding recovery (LHR) in u.v.-irradiated E. coli wild-type and recA strains. In uvrB and polA strains where, after u.v.-irradiation, LHR was absent the two drugs had no effect. Both drugs markedly reduced the removal of u.v.-induced thymine dimers in the DNA of wild-type cells (H/r30). Analysis by alkaline sucrose gradients revealed that PEA inhibited the incision step in excision repair. In contrast, procaine had no effect on incision but apparently inhibited the late steps in excision repair. PEA dissociated DNA from the cell membrane, whereas procaine did not. The results suggest that the two drugs PEA and procaine inhibit LHR and the excision repair process operating on u.v.-induced damage in E. coli by at least two different mechanisms each of which may involve the cell membrane.     

Maintenance energy demand affects biomass synthesis but not cellulase production by a mesophilicClostridium

Summary Secretion of cellulolytic activity by the mesophilClostridium strain C7 was studied while the bacterium underwent progressive carbon/energy starvation and the ensuing continuous decline in growth rate. In the slowest range of growth rates studied the organism was in full response to the global regulation imposed by guanosine 5, 3-bispyrophosphate (ppGpp). The exoenzymes of the cellulase complex were produced at the same volumetric rate whether or not the response was active. However, the volumetric rate of biomass synthesis was reduced 45% or more by the response. Energy necessary to maintain the ppGpp-regulated state (i.e., maintenance energy) was, therefore, diverted from energy going to synthesis of biomass but not from that going to exoenzyme synthesis, making the yield of cellulase activity per mole of carbon-energy substrate independent of growth rate and the exoenzyme complex produced from the substrate with equal efficiency at all growth rates. The primary consideration in improving exoenzyme productivity by bacteria with this type of energy distribution between secretion, growth, and maintenance is simply increasing yield per mole of carbon-energy substrate, with growth rate effects on yield a secondary and minimum concern.     

Postharvest Variation in Cellulase, Polygalacturonase, and Pectinmethylesterase in Avocado (Persea americana Mill, cv. Fuerte) Fruits in Relation to Respiration and Ethylene Production

Cellulase, polygalacturonase (PG), pectinmethylesterase (PME), respiration, and ethylene production were determined in single “Fuerte” avocado fruits from the day of harvest through the start of fruit breakdown. PME declined from its maximum value at the time of picking to a low level early in the climacteric. PG activity was not detectable in the preclimacteric stage, increased during the climacteric, and continued to increase during the postclimacteric phase to a level three times greater than when the fruit reached the edible soft stage. Cellulase activity was low in the preclimacteric fruit, started to increase just as respiration increased, and reached a level two times greater than at the edible soft stage. Cellulase activity started to increase 3 days before PG activity could be detected. Increased production of ethylene followed the increase in respiration and cellulase activity by about 1.5 days. These results indicate that a close relation exists between the rapid increase in the cell wall-depolymerizing enzymes and the rise in respiration and ethylene production and refocused attention on the role of the cell wall and the associated plasma membrane in the early events of fruit ripening.     

Cell elongation and metabolic turnover of the cell wall as affected by auxin and cell wall degrading enzymes

A cell wall fraction (pectic substances) of oat coleoptile segmentsfed with ¹⁴C-glucose contained more radioactivity under theeffect of auxin than did the control. When labeled segmentswere grown for 6 hr in auxin or glucanase solution the labelin the hemicellulose fraction decreased as growth increased.ß-1,3-Glucanase prepared from the culture of a fungus,Sclerotinia libertiana, induces elongation of segments of thepea stem and the oat coleoptile. Traces of cellulase and pectinmethylesterase contaminating the enzyme preparation are notresponsible for the stimulatory effect. Cellulase seemed tobe rather inhibitory and pectin methylesterase showed only aslight effect on coleoptile elongation. A possible relationshipbetween the metabolic turnover of hemicellulosic polysaccharideand cell wall extension is suggested. (Received February 5, 1968; )     

Effects of phenethyl alcohol on yeast cells

An investigation of the physiological effects of phenethyl alcohol (PEA) on exponentially growing yeast cells is reported. RNA, DNA, protein and aminoimidazole ribotide syntheses and glucose uptake and incorporation are inhibited by PEA at concentrations of 0.1% to 0.3%. Two classes of response curves are found and the sensitivities of processes in each class to PEA differ. Glucose incorporation and RNA synthesis are the most sensitive processes in their respective classes. The effects of PEA at 0.3% or less are largely or completely reversible. It is deduced that PEA inhibits intracellular processes as well as the cell membrane.     

Derepressed synthesis of cellulase by Cellulomonas.

A Cellulomonas sp. was isolated from the soil which hydrolyzed cellulose, as shown by clear-zone formation on cellulose agar medium. Catabolite repression of cellulase synthesis occurred when moderate levels of glucose were added to the medium. A stable mutant that no longer exhibits catabolite repression was produced through treatment of the wild-type organism with N-methyl-N'-nitro-N-nitrosoguanidine. Both enzyme concentration and specific activity, as determined by the rate of hydrolysis of carboxymethylcellulose, were greater with the mutant than with the wild-type organism under various test conditions. The wild type had no measurable cellulase activity when grown in the presence of either 1.0% glucose or cellobiose. Cellobiose, but not glucose, inhibited enzyme activity towards both cellulose and carboxymethylcellulose. Cellobiose, cellulose, and sophorose at low concentrations induced cellulase synthesis in both the wild-type and the mutant organism. Cellulase regulation appears to depend upon a complex relationship involving catabolite repression, inhibition, and induction.     

Cellulase production by a thermophilic clostridium species

Strain M7, a thermophilic, anaerobic, terminally sporing bacterium (0.6 by 4.0 μm) was isolated from manure. It degraded filter paper in 1 to 2 days at 60 C in a minimal cellulose medium but was stimulated by yeast extract. It fermented a wide variety of sugars but produced cellulase only in cellulose or carboxymethyl-cellulose media. Cellulase synthesis not only was probably repressed by 0.4% glucose and 0.3% cellobiose, but also cellulase activity appeared to be inhibited by these sugars at these concentrations. Both C₁ cellulase (degrades native cellulose) and C_x cellulase (β-1,4-glucanase) activities in strain M7 cultures were assayed by measuring the liberation of reducing sugars with dinitrosalicylic acid. Both activities had optima at pH 6.5 and 67 C. One milliliter of a 48-h culture of strain M7 hydrolyzed 0.044-meq of glucose per min from cotton fibers. The cellulase(s) from strain M7 was extracellular, produced during exponential growth, but was not free in the growth medium until approximately 30% of the cellulose was hydrolyzed. Glucose and cellobiose were the major soluble products liberated from cellulose by the cellulase. ZnCl₂ precipitation appeared initially to be a good method for the concentration of cellulase activity, but subsequent purification was not successful. Isoelectric focusing indicated the presence of four C_x cellulases (pI 4.5, 6.3, 6.8, and 8.7). The rapid production and high activity of cellulases from this organism strongly support the basic premise that increased hydrolysis of native cellulose is possible at elevated temperature.     

柿果实采后软化过程中细胞壁组分代谢和超微结构的变化

柿果实采后果胶酯酶活性迅速上升,其活性与果实硬度的下降呈明显的负相关。多聚半乳糖醛酸酶活性增加缓慢,但其活性与果实硬度的下降无明显相关性。β-半乳糖苷酶活性迅速增加,其活性与果实硬度的下降呈明显的负相关。纤维素酶活性呈逐渐上升趋势,与果实硬度的下降也呈明显的负相关。伴随着细胞壁水解酶活性的增加,果实原果胶和纤维素含量迅速下降,而水溶性果胶含量则迅速上升。柿果刚采收时细胞壁结构完整,3d后细胞壁中胶层基本被溶解,甚至初生壁也局部发生降解。