Factors Influencing In Vitro Skin Permeability Factor Production by Vibrio cholerae

The development of a new semisynthetic medium which stimulates in vitro production of the skin permeability factor (PF) by Vibrio cholerae is described. The effects of pH, aeration, temperature, and length of incubation on PF formation or release in strain 569B and several other strains, or both, are compared. Data are presented which show that maximal PF accumulation occurs during a transitional period of growth joining the exponential and stationary phases of the growth cycle. PF elaboration is completed well ahead of any visible signs of lysis in the culture and the PF activity appears to be proportional to the length of the linear growth phase. Possible mechanisms of toxigenicity and the nature of PF are discussed.     

Increase in intradermal vascular permeability caused by pertussis toxin from Bordetella pertussis

Rabbits that were injected intradermally with pertussis toxin (PT), produced from Bordetella pertussis, showed slight edema and erythema at the injection sites, but not hemorrhage nor necrosis. The edema lesions were stained blue by the intravenous injection of Pontamine Sky Blue 6B dye, suggesting that PT caused increased vascular permeability, similarly to the permeability factor (PF) of cholera toxin. The reaction of the PF of PT could be determined by measuring the diameter of the blue area. The diameter of the blue area bore a good linear relationship to the logarithm of the dose of PT. The activity of the PF was neutralized by anti-PT rabbit serum. Detoxification of PT with formalin did not increase the vascular permeability, but reverted pertussis toxoid showed a PF reaction in proportion to the reverted leukocytosis-promoting and histamine-sensitizing activities of PT. The supernate of a Bordetella pertussis culture also induced a PF reaction and the reaction could be made clear by heating the supernate at 56 C for 30 min, but the supernate of Bordetella bronchiseptica did not induce the reaction at all.     

Physiology of Batrachochytrium dendrobatidis, a chytrid pathogen of amphibians

Batrachochytrium dendrobatidis is a pathogen of amphibians that has been implicated in severe population declines on several continents. We investigated the zoospore activity, physiology and protease production of B. dendrobatidis to help understand the epidemiology of this pathogen. More than 95% of zoospores stopped moving within 24 h and swam less than 2 cm before encysting. Isolates of B. dendrobatidis grew and reproduced at temperatures of 4-25 C and at pH 4-8. Growth was maximal at 17-25 C and at pH 6-7. Exposure of cultures to 30 C for 8 d killed 50% of the replicates. B. dendrobatidis cultures grew on autoclaved snakeskin and 1% keratin agar, but they grew best in tryptone or peptonized milk and did not require additional sugars when grown in tryptone. B. dendrobatidis produced extracellular proteases that degraded casein and gelatin but had no measurable activity against keratin azure. The proteases were active against azocasein at temperatures of 6-37 C and in a pH range of 6-8, with the highest activity at temperatures of 23-30 C and at pH 8. The implications of these observations on disease transmission and development are discussed.     

Effect of nitrogen source on the levels of nitrate reductase in the yeast Hansenula anomala.

Levels of nitrate reductase (NR) protein in Hansenula anomala and Hansenula wingei were determined using specific antiserum raised against the enzyme from H. anomala. Extracts from nitrate-grown cells contained NR protein, while in those from cells grown on ammonium, glutamine or peptone, no cross-reacting material could be observed. Enzyme activity correlated with the levels of cross-reacting material. When nitrate was used as nitrogen source, NR was always present, even in cultures with ammonium, glutamine or peptone, although in these cases both the levels of activity and protein were lower. NR activity was consistently two to four times higher in cells grown in glucose than in cells grown in ethanol. Nitrate was required for NR induction, and deprivation of nitrate from nitrate-grown cells resulted in a rapid loss of NR activity.     

A nonhemolytic phospholipase C produced byPseudomonas cepacia

Pseudomonas cepacia Pc224c, a nonhemolytic strain originally isolated from the sputum of a cystic fibrosis patient, produced an extracellular, heat-labile phospholipase C activity, which was measured quantitatively on the synthetic substratep-nitrophenylphophorylcholine. Cell-free supernatants from cultures grown to late log phase in MOPS-minimal salts-Tryptose medium contained specific activity at least 38 times greater than that from cultures grown in Tryptose minimal medium, Tryptic Soy broth, or peptone medium. Production was inhibited by the presence of inorganic phosphate in the medium and enhanced by aeration of the culture. The PLC ofP. cepacia is nonhemolytic and does not exhibit lecithinase activity on egg-yolk agar.     

Phenomenological Background and Some Preliminary Trials of Automated Substrate Supply in pH-Stat Modal Fed-Batch Culture Using a Setpoint of High Limit

First, by considering all possible combination of methanol (as a carbon-energy source), peptone (as an organic carbon-nitrogen source), and ammonium sulfate (as an inorganic nitrogen source), five batch cultures of a methanol-assimilating bacterium, Protomonas extorquens, were done to elucidate the cause(s) of pH variations during the microbial cultivations. The batch cultures have been classified into five types in terms of stoichiometric equations of cell growth which involve the elements, C, H, O, and N. The equations explained the pH variation (drop and rise) of the batch cultures on the basis of the consumption and liberation of ammonium ion. Then, six fed-batch cultures using a setpoint of high limit were done by feeding either methanol only or methanol plus peptone. Growth rates could be controlled by the amount of substrate(s) fed per pulse. Supplying peptone in addition to methanol enhanced cell growth. Characteristic differences between pH-stat modal fed-batch cultures using a low limit and those using a high limit, and advantages of the pH-stat modal fed-batch culture using a setpoint of high limit are discussed.     

Factors affecting ureolytic activities ofPenicillium waksmanii isolated from sewage sludge

Twenty one fungal isolates belonging to 7 genera were screened for ureolytic activity. APenicillium waksmanii isolate was found to be the most potent and was selected for further study. No ammonia-nitrogen was detected inP. waksmanii cultures either urea-free or containing up to 1 g urea per L. The maximum extracellular urease production was recorded at a urea concentration of 15 g/L. It peaked after 6 d of incubation at 25°C when the initial pH of the glucose—peptone broth was adjusted to 6. On the other hand, the highest fungus biomass was detected at a concentration of 2 g urea per L after 4 d of incubation at 35°C when the pH of the medium was 8. The intracellular urease activity (measured in cell-free extract) was the highest at 12 mg urea per mL after 75-min incubation at 25°C at pH 8. Incubation temperature of 25°C favored both urease production and activity.     

The Effect of organic nitrogen sources on recombinant glucoamylase production by Aspergillus niger in chemostat culture

Aspergillus niger B1, a recombinant strain carrying 20 extra copies of the native glucoamylase gene, was grown in glucose-limited chemostat cultures supplemented with various organic nitrogen sources (dilution rate 0.12 +/- 0.01 h(-1), pH 5.4). In cultures supplemented with l-alanine, l-methionine, casamino acids, or peptone, specific glucoamylase (GAM) production rapidly decreased to less than 20% of the initial level. Reducing the pH of the culture to 4.0 resulted in stable GAM production for up to 400 h. Morphological mutants (a light brown and a dark brown mutant) appeared in each fermentation and generally displaced B1. Light brown mutants had higher selection coefficients relative to B1 than dark brown mutants and became the dominant strain in all fermentations except those maintained at pH 4.0. Several mutants isolated from these cultures had reduced ability to produce GAM in batch culture, although few had lost copies of the glaA gene. Some mutants had methylated DNA.     

Susceptibility of<Emphasis Type="Italic">Escherichia coli</Emphasis> to C<Subscript>2</Subscript>-C<Subscript>18</Subscript> fatty acids

The antimicrobial activity of C2-C18 fatty acids was determined in vitro in cultures of two strains of Escherichia coli grown on glucose. Antimicrobial activity was expressed as IC50 (a concentration at which only 50% of the initial glucose in the cultures was utilized). Utilization of glucose was inhibited by caprylic acid (IC50 0.30-0.85 g/L) and capric acid (IC50 1.25-2.03 g/L). Neither short-chain fatty acids (C2-C6) nor fatty acids with longer chain (C12-C18) influenced substrate utilization. Caproic acid, however, decreased cell yield in cultures of E. coli in a dose-dependent manner. No inhibition of glucose utilization was produced with unsaturated fatty acids, oleic and linoleic. Calcium ions added in excess reversed the antimicrobial effect of capric acid, but not that of caprylic acid. Antimicrobial activity of caprylic and capric acid decreased when the bacteria were grown in the presence of straw particles, or repeatedly subcultured in a medium containing these compounds at low concentrations. Counts of viable bacteria determined by plating decreased after incubation with caprylic and capric acid (30 min; 1 g/L) at pH 5.2 from > 10(9) to approximately 10(2)/mL. A reduction of a mere 0.94-1.96 log10 CFU was observed at pH 6.5-6.6. It can be concluded that caprylic acid, and to a lesser extent also capric acid, has a significant antimicrobial activity toward E. coli. Effects of other fatty acids were not significant or absent.     

Stopped-flow ESR study on the reactivity of vitamin E, vitamin C and its lipophilic derivatives towards Fremy's salt in micellar systems

The reaction between Fremy's salt and alpha-tocopherol (VE), ascorbic acid (VC) and its lipophilic derivatives ascorbyl-6-caprylate (VC-8), 6-laurate (VC-12) and 6-palmitate (VC-16) were studied by stopped-flow ESR spectroscopy in cetyl trimethylammonium bromide (CTAB) and sodium dodecyl sulfate (SDS) micelles, as a model reaction of these antioxidants with alkyl peroxy radicals in biological systems. The second order rate constants for the reaction of Fremy's salt with VE in CTAB and SDS micelles were found to be 7.9 x 10(3) and 2.2 M-1 s-1, respectively, with as high as a 3600-fold variation. Rate constants for VC, VC-8, VC-12 and VC-16 are 4.3, 35, 53 and 56 x 10(3) M-1 s-1 and 3.3, 2.7, 1.2 and 0.86 x 10(3) M-1 s-1 in CTAB and SDS micelles, respectively. The results demonstrate remarkable effects of the charge type of the micelles and the side-chain of the antioxidants on the antioxidation reactivity in the micelles. It reveals that the inter-micellar diffusion may be the rate-limiting step for antioxidation carried out in micelles.     

Characterization of an Enterotoxin Produced by Vibrio cholerae O139

A cholera-like enterotoxin was purified from Vibrio cholerae O139 strain AI-1841 isolated from a diarrheal patient in Bangladesh. Its characteristics were compared with that of cholera toxins (CTs) of classical strain 569B and El Tor strain KT25. Al-1841 produced as much toxin as O1 strains. The toxins were indistinguishable in terms of their migration profiles in conventional polyacrylamide gel disc electrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectrofocusing as well as their affinity for hydroxyapatite. The skin permeability factor activity and the fluid accumulation induced in rabbit ileal loops of the toxin of AI-1841 were identical to those of the CTs. Three toxins equally reacted against anti-569B CT antiserum in Western blotting, and their B subunits formed a precipitin line against any anti-B subunit antiserum by double gel immunodiffusion. Anti-569B CTB antibody neutralized the three toxins in their PF activities and enterotoxicities. The amino acid sequence of 1841 toxin B subunit was identical with that of KT25 CTB, corresponding to the DNA sequence of ctxB from El Tor strains of the seventh pandemic. We concluded 1841 toxin was identical to CT of the seventh pandemic El Tor vibrios.     

Mechanism of action of choleragen. Evidence for ADP-ribosyltransferase activity with arginine as an acceptor.

Choleragen catalyzed the hydrolysis of NAD to ADP-ribose and nicotinamide; nicotinamide production was dramatically increased by L-arginine methyl ester and to a lesser extent by D- or L-arginine, but not by other basic amino acids. Guanidine was also effective. Nicotinamide formation in the presence of L-arginine methyl ester was greatest under conditions previously shown to accelerate the hydrolysis of NAD by choleragen (Moss, J., Manganiello, V. C., and Vaughan, M. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 4424-4427). After incubation of [adenine-U14C]NAD and L[3H]arginine with coleragen, a product was isolated by thin layer chromatography that contained adenine and arginine in a 1:1 ratio and has been tentatively identified as ADP-ribose-L-arginine. Parallel experiments with [carbonyl-14C]NAD have demonstrated that formation of the ADP-ribosyl-L-arginine derivative was associated with the production of [carbonyl-14C]nicotinamide. As guanidine itself was active and D- and L-arginine was equally effective in promoting nicotinamide production, whereas citrulline, which possesses a ureido rather than a guanidino function, was inactive, it seems probable that the guanidino group rather than the alpha-amino moiety participated in the linkage to ADP-ribose. Based on the assumption that the ADP-ribosylation of L-arginine by choleragen is a model for the NAD-dependent activation of adenylate cyclase by choleragen, it is proposed that the active A protomer of choleragen catalyzes the ADP-ribosylation of an arginine, or related amino acid residue in a protein, which is the cyclase itself or is critical to its activation by choleragen.     

Optimization of culture conditions for the production of rifamycin oxidase by Curvularia lunata

Maximum activity (8.9 IU/ml) of rifamycin oxidase in Curvularia lunata, grown in shake-flask culture at 28°C and pH 6.5, was after 96 h. Nearly all the glucose was used in 72 h. An initial culture pH of 6.5 and 28°C were optimum for the growth and enzyme production. Among various carbon and organic nitrogen sources, carboxymethylcellulose and peptone were the most effective for enzyme yield. The rate of enzyme production was enhanced when yeast extract was also added to the medium. The optimum medium for the production of rifamycin oxidase contained 10 g each of yeast extract, peptone and carboxymethylcellulose/l and 0.04% (NH₄)₂SO₄.The author is with the Biochemical Engineering Research and Process Development Centre, Institute of Microbial Technology, Post Box 1304, Sector 39-A, Chandigarh 160 014, India     

3alpha-, 7alpha- and 12alpha-hydroxysteroid dehydrogenase activities from Clostridium perfringens.

25 strains of Clostridium perfringens were screened for hydroxysteroid dehydrogenase activity; 19 contained NADP-dependent 3alpha-hydroxysteroid dehydrogenase and eight contained NAD-dependent 12alpha-hydroxysteroid dehydrogenase active against conjugated and unconjugated bile salts. All strains containing 12alpha-hydroxysteroid dehydrogenase also contained 3alpha-hydroxysteroid dehydrogenase although 12alpha-hydroxysteroid dehydrogenase was invariably in lesser quantity than the 3alpha-hydroxysteroid dehydrogenase. In addition, 7alpha-hydroxysteroid dehydrogenase activity was evident only when 3alpha, 7alpha, 12alpha-trihydroxy-5beta-cholanoate was substrate but notably absent when 3alpha, 7alpha-dihydroxy-5beta-cholanoate was substrate. The oxidation product 12alpha-hydroxy-3, 7-diketo-5beta-cholanoate is rapidly further degraded to an unknown compound devoid of either 3alpha- or 7alpha-OH groups. Group specificity of these enzymes was confirmed by thin-layer chromatography studies of the oxidation products. These enzyme systems appear to be constitutive rather than inducible. In contrast to C. perfringens. Clostridium paraputrificum (five strains tested) contained no measurable hydroxysteroid dehydrogenase activity. pH studies of the C. perfringens enzymes revealed a sharp pH optimum at pH 11.3 and 10.5 for the 3alpha-OH- and 12alpha-OH-oriented activities, respectively. Kinetic studies gave Km estimates of approx. 5 X 10(-5) and 8 X 10(-4) M with 3alpha, 7a-dihydroxy-5beta-cholanoate and 3alpha, 12alpha-dihydroxy-5beta-cholanoate as substrates for two respective enzymes. 3alpha-hydroxysteroid dehydrogenase was active against 3alpha-OH-containing steroids such as androsterone regardless of the sterochemistry of the 5H (Both A/B cis and A/B trans steroides were substrates). There was no activity against 3beta-OH-containing steroids. The 3alpha- and 12alpha-hydroxysteroid dehydrogenase activities, although differing in cofactor requirements cannot be distinguished by their appearance in the growth curve, their mobility on disc gel electrophoresis, elution volume on passage through Sephadex G-200 or heat inactivation studies.     

Activities and partial purification of extracellular proteases of Bacteroides nodosus from virulent and benign footrot

In an attempt to differentiate virulent and benign strains of B. nodosus, the extracellular proteolytic activity of these cultures was assayed with elastin, casein and hide powder azure, and the stability to heating at 55 degrees C was determined. Broth cultures of both strains hydrolysed 125I-labelled elastin, indicating that this activity is not a unique marker of virulence. When cultures were grown in Trypticase-arginine-serine broth medium modified by omitting Na2CO3 and thioglycollic acid, the total proteolytic activity and its stability at 55 degrees C could be used to differentiate isolates causing virulent or benign footrot lesions. However, when other broth cultures were used, these parameters could no longer be used to make such a distinction. The proteases of a virulent and benign strain of B. nodosus were partially purified and characterized. Four to five closely related proteases were detected by polyacrylamide gel electrophoresis at pH 8.8 in both types of isolates. The proteases are serine-type enzymes requiring a divalent metal ion such as calcium for activity. The proteases of the benign strain were somewhat less stable to heat than the enzymes of the virulent strain. Differences in the relative mobilities of the proteases of virulent and benign strains of B. nodosus, on electrophoresis at pH 8.8, suggest that this property may be used to distinguish virulent and benign strains.     

Growth of bacteria on chitin, fungal cell walls and fungal biomass, and the effect of extracellular enzymes produced by these cultures on the antifungal activity of amphotericin B

Vibrio alginolyticus, Streptomyces griseus, Arthrobacter G12, Bacillus sp. and Cytophaga sp. were grown on solid and liquid media containing soluble and insoluble carbon sources. Arthrobacter G12, Bacillus sp. and Cytophaga sp. grew well on media which contained fungal cell walls or fungal biomass as the main carbon source. All bacteria produced extracellular proteases and all bacteria except Arthrobacter G12 produced extracellular chitinases. Growth of Cytophaga sp. on colloidal chitin was paralleled by the accumulated chitinase activity in the culture filtrate, and growth of Cytophaga sp. and Arthrobacter G12 on cell walls of Geotrichum candidum and cell walls of Candida pseudotropicalis was paralleled by the accumulation of laminarinase activity in the culture filtrate, but little or no extracellular chitinase activity was observed in these cultures. Mycolases purified from the culture filtrates of Cytophaga sp. grown on colloidal chitin on cell walls of C. pseudotropicalis potentiated the antifungal activity of amphotericin B.     

Lincomycin increases synthetic rate and periplasmic pool size for cholera toxin.

Increased enterotoxigenicity of Vibrio cholerae 569B grown with low concentrations of lincomycin, previously described in terms of increased extracellular biological activity (capillary permeability factor and fluid accumulation in ligated rabbit ileal loops), was further characterized. Polyacrylamide gel electrophoresis and single radial immunodiffusion showed that lincomycin-stimulated cells produced increased molar quantities of cholera toxin (CT) both extra- and intracellularly. The intracellular CT was released in comparable amounts by sonication, deoxycholate extraction, and polymyxin B treatment. Polymyxin B release of CT was nearly complete under conditions wherein only 6% of total cellular beta-galactosidase was released, implying a periplasmic pool of CT in stimulated cells. No intracellular choleragenoid (CT subunit B) was found in stimulated cells by polymyxin B release. No proteolysis of 14C-labeled CT was detected after prolonged incubation with sonicated nonstimulated cultures or sonicated concentrated cells. These data support the conclusion that the stimulatory effect of lincomycin involves an increase in the rate of synthesis of the CT molecule, and argue against alternative models involving inhibition of putative normal degradation of CT, increased release of otherwise cell-bound CT, or activation of inactive, or less active, forms of CT.     

Methanogens in the human intestinal tract and oral cavity

Curvularia lunata var.aeria was grown in YPD (yeast extract, peptone, and dextrose) medium (pH 6.5) at 28°C with varying concentrations (10–40 g/L) of glucose for the production of rifamycin oxidase. Enzyme activity and glucose concentration were found to be indirectly related to the production of black intracellular pigment by the organism. Depletion of glucose level and rise of culture pH initiate the synthesis of pigment. Carboxymethylcellulose (CMC) was used as a carbon source to improve the enzyme yield, but utilization of the substrate in the reactor was much less. Compared with 10 g/L of CMC in the medium, low or high concentrations of CMC did not yield any better result. Addition of glucose in YPC (yeast extract, peptone, and CMC) medium did not increase the enzyme activity, and glucose was rapidly utilized byC. lunata, forming pellets rather than mycelia.     

High-affinity maltose/trehalose transport system in the hyperthermophilic archaeon Thermococcus litoralis.

The hyperthermophilic marine archaeon Thermococcus litoralis exhibits high-affinity transport activity for maltose and trehalose at 85 degrees C. The K(m) for maltose transport was 22 nM, and that for trehalose was 17 nM. In cells that had been grown on peptone plus yeast extract, the Vmax for maltose uptake ranged from 3.2 to 7.5 nmol/min/mg of protein in different cell cultures. Cells grown in peptone without yeast extract did not show significant maltose or trehalose uptake. We found that the compound in yeast extract responsible for the induction of the maltose and trehalose transport system was trehalose. [14C]maltose uptake at 100 nM was not significantly inhibited by glucose, sucrose, or maltotriose at a 100 microM concentration but was completely inhibited by trehalose and maltose. The inhibitor constant, Ki, of trehalose for inhibiting maltose uptake was 21 nM. In contrast, the ability of maltose to inhibit the uptake of trehalose was not equally strong. With 20 nM [14C]trehalose as the substrate, a 10-fold excess of maltose was necessary to inhibit uptake to 50%. However, full inhibition was observed at 2 microM maltose. The detergent-solubilized membranes of trehalose-induced cells contained a high-affinity binding protein for maltose and trehalose, with an M(r) of 48,000, that exhibited the same substrate specificity as the transport system found in whole cells. We conclude that maltose and trehalose are transported by the same high-affinity membrane-associated system. This represents the first report on sugar transport in any hyperthermophilic archaeon.