Physiological characteristics of the strains of Cryptococcus deffluens--producers of penicillin acylase]

A fermentation medium balanced by the main components was developed for Cryptococcus diffluens strains producing penicillin-V-acylases (PA). It was shown that the culture needed for production of the enzyme was inductor, which was phenoxyacetic acid (POAA). Additional introduction of ethanol to the medium provided an increase in production of PA by 36 per cent and the culture growth by 25 per cent. Introduction of one of the following substances to the medium with POAA and ethanol i.e. (CN3COO)2Ca, FeSO4, proline or asparagine provided an additional increase in the production level of PA by 24 to 94 per cent. The use of the medium varieties will permit one to isolate highly productive cells of the culture.     

Alkaline phosphatase activity of rumen bacteria

Of the 54 strains of rumen bacteria examined for alkaline phosphatase (APase) production, 9 of 33 gram-negative strains and none of 21 gram-positive strains produced the enzyme. The APase of the cells of the three strains of Bacteroides ruminicola that produced significant amounts of the enzyme was located in the periplasmic area of the cell envelope, whereas the enzyme was located in the strains of Selenomonas ruminantium and Succinivibrio dextrinosolvens was associated with the outer membrane. The localization of APase production in the cells of natural populations of rumen bacteria from hay-fed sheep was accomplished by reaction product deposition, and both the proportion of APase-producing bacteria and the location of the enzyme in the cell envelope of the producing cells could be determined. We suggest that this procedure is useful in detecting shifts in the bacterial population and the release of cell-bound APase that accompany feedlot bloat and other sequelae of dietary manipulation in ruminants.     

Alkaline phosphatase activity of rumen bacteria.

Of the 54 strains of rumen bacteria examined for alkaline phosphatase (APase) production, 9 of 33 gram-negative strains and none of 21 gram-positive strains produced the enzyme. The APase of the cells of the three strains of Bacteroides ruminicola that produced significant amounts of the enzyme was located in the periplasmic area of the cell envelope, whereas the enzyme was located in the strains of Selenomonas ruminantium and Succinivibrio dextrinosolvens was associated with the outer membrane. The localization of APase production in the cells of natural populations of rumen bacteria from hay-fed sheep was accomplished by reaction product deposition, and both the proportion of APase-producing bacteria and the location of the enzyme in the cell envelope of the producing cells could be determined. We suggest that this procedure is useful in detecting shifts in the bacterial population and the release of cell-bound APase that accompany feedlot bloat and other sequelae of dietary manipulation in ruminants.     

Selection of phage-resistent clones of streptococcus group A and their properties]

In studying the effects induced by virulent phage CAI in the sensitive cultures of streptococcus, group A, a possibility was shown of selection of phage-resistant clones with the altered enzymatic activity. These clones lost their capacity to produce proteinase and retained residual lipoproteinase activity. This evidence correlates with literature data indicating that phage-resistant streptococci served as good producers of M-protein--the main virulence factor. Infection of the culture producing streptokinase with phage CAI with a definite infection multiplicity led to an increase of the enzyme activity in the culture fluid. This process was accompanied by selection of the resistant strains characterized by greater streptokinase production and greater enzyme stability. As suggested, the latter could result from the absence of proteolytic activity in the phage-resistant clone.     

Urokinase-type and tissue-type plasminogen activators are essential for in vitro invasion of human melanoma cells

This study evaluates the contribution of two types of plasminogen activators (PAs; tissue-type PA (tPA) versus urokinase-type PA (uPA) toward the invasiveness of human melanoma cells in a novel in vitro assay. We identified two human melanoma cell lines, MelJuso and MeWo, expressing uPA or tPA as shown at mRNA, protein, and enzyme activity level. MelJuso cells produced uPA as well as plasminogen activator inhibitor-1 (PAI-1). The latter was, however, not sufficient to neutralize the cell-associated or secreted uPA activity. MeWo cells secreted tPA, but the enzyme was not found to be cell-associated. PAI-1 production by these cells was not detectable. Plasminogen activation and fibrinolytic capacity of both cell lines were reduced by anticatalytic monoclonal antibodies specific for the respective type of PA or by aprotinin. In a novel in vitro invasion assay, antibodies to PA as well as aprotinin decreased the invasiveness of both cell lines into a fibrin gel, Matrigel, or intact extracellular matrix. Our results confirm the importance of uPA-catalyzed plasminogen activation in tumor cell invasiveness. Furthermore, we provide evidence that tPA, beyond its key role in thrombolysis, can also be involved in in vitro invasion of human melanoma cells.     

Application of a Genetically Encoded Biosensor for Live Cell Imaging of L-Valine Production in Pyruvate Dehydrogenase Complex-Deficient Corynebacterium glutamicum Strains

The majority of biotechnologically relevant metabolites do not impart a conspicuous phenotype to the producing cell. Consequently, the analysis of microbial metabolite production is still dominated by bulk techniques, which may obscure significant variation at the single-cell level. In this study, we have applied the recently developed Lrp-biosensor for monitoring of amino acid production in single cells of gradually engineered L-valine producing Corynebacterium glutamicum strains based on the pyruvate dehydrogenase complex-deficient (PDHC) strain C. glutamicum ΔaceE. Online monitoring of the sensor output (eYFP fluorescence) during batch cultivation proved the sensor''s suitability for visualizing different production levels. In the following, we conducted live cell imaging studies on C. glutamicum sensor strains using microfluidic chip devices. As expected, the sensor output was higher in microcolonies of high-yield producers in comparison to the basic strain C. glutamicum ΔaceE. Microfluidic cultivation in minimal medium revealed a typical Gaussian distribution of single cell fluorescence during the production phase. Remarkably, low amounts of complex nutrients completely changed the observed phenotypic pattern of all strains, resulting in a phenotypic split of the population. Whereas some cells stopped growing and initiated L-valine production, others continued to grow or showed a delayed transition to production. Depending on the cultivation conditions, a considerable fraction of non-fluorescent cells was observed, suggesting a loss of metabolic activity. These studies demonstrate that genetically encoded biosensors are a valuable tool for monitoring single cell productivity and to study the phenotypic pattern of microbial production strains.     

Patterns of plasminogen activator production in cultured normal embryonic cells

Cultured normal low-passage embryo fibroblasts, from a number of species, and two untransformed clones of a Balb/3T3 line elaborate increasing amounts of plasminogen activator (PA) as they approach confluence; the low-passage cells then lose this PA activity after reaching confluence, while the 3T3 cells retain it indefinitely. Even at their peaks, however, the PA activities of the low-passage cells remain well below those of the corresponding virally or spontaneously transformed cells. The PA increases in normal cells are probably a result of PA production rather than of adsorption of secreted PA to the cell surface, or of changes in cell-associated protease inhibitors. The elaboration of PA by normal cells is dependent upon their metabolic activity, such that the level of serum supplementation and the growth phase of the culture directly influence the level of cell-associated PA observed. In addition, there may be a component of serum which exerts a negative control on PA production and which is not an acid-labile protease inhibitor.     

Relative Rates of Lysis of Staphylococcal Cell Walls by Lytic Enzymes from Various Bacteriophage Types

Lytic enzymes were isolated from 14 strains of phage-infected Staphylococcus aureus. Cell walls were prepared from the same uninfected strains of bacteria. Comparison of the lytic rates was made for each enzyme, with each of the cell walls as substrate. Differences in the rate of substrate utilization of the various cell wall types exceeded 10-fold. Cell walls from strains 42E, 29, and 77 were the best substrates, whereas cell walls from strains 3C, 80, and 187 were the poorest substrates. The cell wall amino acid composition is discussed as related to lytic enzyme specificity. A possible explanation of phage typing of staphylococcal cells, based on enzyme activity and cell wall composition, is presented.     

Tumorigenicity of herpesvirus-transformed cells correlates with production of plasminogen activator.

Our studies first demonstrated that established hamster cell lines transformed in vitro by herpesviruses activate plasminogen more effectively than normal hamster fibroblasts. This ability is probably due to increased levels of the enzyme plasminogen activator (PA). In the studies described here, the 333-8-9 cell line, originally transformed by herpes simplex virus type 2 strain 333, was used to derive subclonal lines that maintained stable PA phenotypes over the course of long in vitro passage. We were interested in correlating tumor formation by the subclones with their fibrinolytic capacity. Cells were, therefore, single-cell subcloned twice, and resulting cultures were tested for ability to activate plasminogen in vitro. PA activity was then quantitated by [125I]fibrin lysis assay, and high- and low-activity subclones were isolated; these retained high- or low-activity phenotypes. Syngeneic newborn hamsters were inoculated with these subclones and observed for the appearance of palpable tumors. A strong correlation between enzyme activity and tumor formation was observed in four separate trials; animals receiving high-PA subclones developed tumors more rapidly than those inoculated with the parental cell line. Tumors were also excised from test animals, and the cell lines established from the tumors were tested in vitro at different passages for their ability to activate plasminogen. These tumor cells were then reinoculated into syngeneic animals to confirm the tumorigenicity of cell lines with high fibrinolytic activity. In these experiments, the positive correlation between PA production and tumorigenicity was confirmed.     

Effect of dimethyl sulfoxide on human carcinoma cells, inhibition of plasminogen activator synthesis, change in cell morphology, and alteration of response to cholera toxin.

Human carcinoma HEp-3 lost its tumorigenic and metastatic potential upon prolonged culture in vitro. This change was accompanied by a reduced production of plasminogen activator (PA) of the urokinase type (uPA), which is secreted by HEp-3 cells, a change in response to effectors that modulate uPA production, and an alteration of cell morphology. Similar but more rapid changes occurred when malignant HEp-3 cells were exposed to dimethyl sulfoxide (DMSO). uPA activity in the culture medium dropped below 50% of the control level within 6 h after the addition of DMSO and became undetectable after 24 h of treatment. This drop in uPA activity was not caused by an increased production of PA inhibitors. The cell-associated uPA decreased to 25 to 30% of the control level within 6 h of DMSO treatment and remained at this level for at least 96 h; the reduced uPA production was partially accounted for by a rapid decrease in the functional and chemical concentration of uPA mRNA. In contrast, the concentrations of most of the abundant mRNA species did not appear to be significantly affected, and cell growth was only slightly inhibited in the presence of DMSO. Malignant HEp-3 cells treated with DMSO responded to cholera toxin with an enhanced production of uPA, and their morphology became indistinguishable from that of nonmalignant HEp-3 cells grown in vitro for prolonged periods of time. All of the above changes were fully and rapidly reversible. The inhibitory effect of DMSO on PA production appears to be specific for uPA of human cell lines.     

爱德华氏菌产乳酸氧化酶的条件研究

在以前工作的基础上,对已获得的产乳酸氧化酶的5株菌进行复筛,对产酶量大的一株菌进行了分类鉴定,确定该菌株属迟钝爱德华氏菌生物群I(Edwardsiella tarda Biogroup I)。这与曾报道的产乳酸氧化酶分枝杆菌(Mycobacterium)和片球菌(Pediococcus)是不同的菌。分别研究了培养基中的培养初始pH、核黄素、乳酸钠以及硫酸铵对发酵产乳酸氧化酶的影响。这一酶源在酶法生产丙酮酸及医疗诊断和酶电极应用上有意义。     

Enzyme encapsulation in permeabilized Saccharomyces cerevisiae cells

The Saccharomyces cerevisiae cell wall provides a semipermeable barrier that can retain intracellular proteins but still permits small molecules to pass through. When S. cerevisiae cells expressing E. coli lacZ are treated with detergent to extract the cell membrane, beta-galactosidase activity in the permeabilized cells is approximately 40% of the activity of the protein in cell extract. However, the permeabilized cells can easily be collected and reused over 15 times without appreciable loss in activity. Cell wall composition and thickness can be modified using different cell strains for enzyme expression or by mutating genes involved in cell wall biosynthesis or degradation. The Sigma1278b strain cell wall is less permeable than the walls of BY4742 and W303 cells, and deleting EXG1, which encodes a 1,3-beta-glucanase, can further reduce permeability. A short Zymolyase treatment can increase cell wall permeability without rupturing the cells. Encapsulating multiple enzymes in permeabilized cells can offer kinetic advantages over the same enzymes in solution. Regeneration of ATP from AMP by adenylate kinase and pyruvate kinase encapsulated in the same cell proceeded more rapidly than regeneration using a cell extract. Combining permeabilized cells containing adenylate kinase with permeabilized cells containing pyruvate kinase can also regenerate ATP from AMP, but the kinetics of this reaction are slower than regeneration using cell extract or permeabilized cells expressing both enzymes.     

Production and excretion of cloacin DF13 by Escherichia coli harboring plasmid CloDF13.

The production and the mechanism of excretion of cloacin DF13 were investigated in noninduced and mitomycin C-induced cell cultures. A mitomycin C concentration was selected which did not cause lysis of cloacinogenic cells, but at the same time induced a maximal production of cloacin DF13. Native cloacin DF13, possessing killing activity, was first released into the cytoplasm. Shortly thereafter, the bacteriocin was transported through the cytoplasmic membrane and accumulated in the periplasm. Finally, cloacin DF13 was excreted into the culture medium. A small amount of cloacin DF13 remained associated with the cell surface. Producing cells did not become permeable for the cytoplasmic enzyme beta-galactosidase. Apparently the cloacin DF13 leaves the producing cells by an excretion process which is not similar to the mechanism proposed for bacterial secretory proteins. The processes of excretion by producing cells and of uptake by susceptible cells were also not identical because mutant cloacin DF13, which was not transported through the outer membrane into susceptible cells, was excreted like the wild-type cloacin DF13. The composition of the culture medium greatly affected production of cloacin DF13. The presence of sugars known to cause catabolite repression not only inhibited the production but also strongly reduced the excretion of cloacin DF13 into the culture medium.     

Two different types of fosfomycin resistance in clinical isolates of Klebsiella pneumoniae

Abstract The fosfomycin susceptibility of 100 clinical isolates of Klebsiella pneumoniae and the resistance mechanisms utilized by resistant strains were examined. Washed cells prepared from the strains demonstrating MICs of more than 8 μg ml⁻¹ of fosfomycin inactivated the drug. A crude extract from strain Tf129B, highly resistant to fosfomycin, was used to study the enzymatic properties of the drug-inactivating enzyme. The optimum pH for inactivation was 7.8 and the optimum temperature of the reaction was 37°C. Glutathione was shown to be effective as a cofactor in the inactivation. It was suggested that the inactivating enzyme of Klebsiella pneumoniae was fosfomycin: glutathione-S-transferase, a constitutive enzyme located in the periplasmic space. A good correlation was found between the specific activities of this enzyme and the MIC levels; however, certain strains showed a low level of fosfomycin: glutathione-S-transferase activity which could not account for the increased MIC. Strains Tf129B and Tf408E, both demonstrating MICs of more than 1024 μg ml⁻¹ of fosfomycin carried a transferable resistance plasmid. In strain Tf129B, the mechanism of fosfomycin resistance was due to a high level of enzymic activity. In strain Tf408E, it was determined to be mainly due to the reduced permeability of the cell membrane.     

Differences between fruit-bearing and non-bearing apple spurs in activity of an enzyme system decomposing phloridzin

The activity of an enzyme system decomposing phloridzin was investigated in fruitbearing and nonbearing spurs of apple trees, Landsberger Reinette cv., throughout vegetation. Acetone powder obtained from xylem sap of apple spurs was incubated with phloridzinsubstratum in citric buffer at pH 5.5 for 12, 18 and 24 h at 30 °C. A paper and thin-layer chromatography as well aa a spectrophotometric assay were employed for tentative identification of enzymic degradation products. Phloretic acid (PA), co-factor of IAA-oxidase, as well as phloretin (Pin), and phloroglucinol (PI) were found after the digestion of phloridzin. The chromatographed enzyme reaction products were measured densitometrically. The activity of the enzyme system was estimated by its efficiency in PA production and phloridzin disappearance. Obtained values, expressed in percentages, showed that the enzyme activity in fruitbearing spurs was much higher than in nonbearing ones; 30 and 10% of released PA in July, respectively. Because fruitbearing spurs of the apple tree are possibly additionally supplied with auxin translocated from developing seeds, an adaptive character of the enzyme system producing PA, a known auxin repressor, is suggested.     

Comparative characteristics of the metabolism of E. coli cells with various activity of penicillin acylase

A correlation between the synthesis and secretion of penicillin acylase (PA; EC 3.5.1.11) and the membrane phospholipid composition was observed in three E. coli strains. In cells with overproduction of PA, the phospholipid/protein ratio decreases, while the cardiolipin/phosphatidylglycerol ratio increases. The differences in the functioning of the electron transport system were revealed in cells with different levels of PA synthesis and secretion. The O2 consumption rate was 3 times lower in the cells with overproduction of PA than in those of less productive strains. On the contrary, membrane particles isolated from the cells of PA producers had no significant differences in the O2-reduction rate. The sensitivity of the strains to the inhibitor of terminal oxidases, sodium cyanide, and to the uncoupler of redox phosphorylation, chlorocarbonyl-phenylhydrazone, was different. Thus the E. coli cells with PA overproduction are characterized by significant changes in energetics and constructive metabolism. The interrelations between PA overproduction, phospholipid metabolism and the respiratory chain activity are discussed.     

Saccharomyces cerevisiae mutants selected for increased production of Trichoderma reesei cellulases

 Trichoderma reesei endoglucanase I (EGI) was used as a reporter enzyme for screening mutagenized yeast strains for increased ability to produce protein. Sixteen haploid Saccharomyces cerevisiae strains, transformed with a yeast multicopy vector pALK222, containing the EGI cDNA under the ADH1 promoter, produced EGI activity of 10^-5–10^-4 g/l. On the average 93% of the total activity was secreted into the culture medium. Two strains with opposite mating types were mutagenized, and several mutants were isolated possessing up to 45-fold higher EGI activity. The best mutants were remutagenized and a second-generation mutant, strain 2804, with an additional twofold increase in EGI activity was selected. The mutant strain 2804 grew more slowly and reached a lower final cell density than the parental strain. In the selective minimal medium, the 2804 strain produced 40 mg/l immunoreactive EGI protein, but only 2% was active enzyme. In the rich medium the secreted EGI enzyme stayed active, but without selection pressure the EGI production ceased after 2 days of cultivation, when the strain 2804 had produced 10 mg/l of EGI. A sevenfold difference was found between the parental and the 2804 strain in their total EGI production relative to cell density. The difference in favour of the mutant strain was also detected on the mRNA level. The 2804 mutant was found to be more active than the parental strain also in the production of T. reesei cellulases, cellobiohydrolase I, and cellobiohydrolase II. Received: 22 December 1995/Received revision: 26 February 1996/Accepted: 17 March 1996     

产碱性蛋白酶嗜碱芽孢杆菌的筛选及其研究

利用造纸黑液对土样进行富集,筛选出3株碱性蛋白酶酶活力较高的嗜碱芽孢杆菌X1、X2、X3。对它们的生长曲线,产酶曲线,在不同C、N源、pH值、盐浓度下的产酶活力进行的研究表明:3株嗜碱芽孢杆菌(Bacillussp.JBX1、X2、X5)酶活力较高(达到100U/mL),X2最高酶活可达140U/mL。最适pH值为9.5,碳源中的蔗糖,氮源中的酵母浸提物和硝酸钠均利于产酶。X1、X5两株嗜碱芽孢杆菌均表现出较强的耐盐耐高渗透压的能力。X1在11%的NaCl浓度下生长良好,酶活仍然达到80U/mL以上。而X2和X5对温度的耐受性比较强,在经70℃处理15min后依然保持了80%以上的酶活力,所产蛋白酶为高温碱性蛋白酶,从而为进一步的应用和研究奠定了基础。     

Gentle lysis of mucous producing cold-adapted bacteria by surfactant treatment combined with mechanical disruption

A procedure for the enhanced lysis of mucous producing psychrotrophic gram positive bacteria for subsequent enzyme studies is described. An initial washing of bacterial cells with Tween 80 was found to improve the degree of cell disruption in subsequent sonication or grinding with glass beads, resulting in about 20-200% increase in total soluble protein content. However, in terms of lactate dehydrogenase (LDH) activity present in the lysate, pretreatment with Tween 80 was more effective in combination with grinding, especially in the highly mucous producing strain GY11. The type of surfactant used in the pretreatment procedure before grinding strongly influenced the percentage lysis of tested strains, both in terms of released soluble protein and enzyme activity. Zymograms of LDH and glutamate dehydrogenase (GDH) activity present in the lysates also very well supported the results obtained by total protein and enzyme activity measurements.