Toluene Elicits a Carbon Starvation Response in Pseudomonas putida mt-2 Containing the TOL Plasmid pWW0

Pseudomonas putida mt-2(pWWO) exhibited a carbon starvation response in the presence of toluene, a utilizable carbon source. When growth-supporting (4-mg/liter), inhibitory (130-mg/liter), and lethal (267-mg/ liter) levels of toluene were provided as the sole carbon source, P. putida responded by rapidly inhibiting protein synthesis and by producing 26 new proteins, 22 of which overlapped with those induced by carbon starvation. P. putida produced the same proteins when cultures were starved by depleting their carbon source or were downshifted into a carbon-free medium. Carbon supplementation of toluene-exposed cells suppressed the production of the toluene-induced proteins. The level of toluene provided as the sole carbon source influenced the length of time that this response was observed. Following 1.5 to 3 h in a basal salts medium with 4 mg of toluene per liter, protein synthesis increased, the production of the majority of the toluene-induced proteins ceased, and the cells began to grow. In cells provided with 130 mg of toluene per liter, protein synthesis remained inhibited over a 6.5-h experimental period. At this concentration, the production of 15 toluene-induced proteins was prolonged, with nine still detectable in the profiles at 6.5 h. In cells provided with 267 mg of toluene per liter, there was a rapid loss of viability and the toluene-induced proteins were detected prior to death. In cells provided with 4 mg of toluene per liter, the carbon starvation response is transient and likely reflects a period of induction and/or adaptation prior to growth on toluene. At the toluene concentrations which inhibit growth, P. putida exhibits a prolonged starvation response despite the presence of an excess of a utilizable carbon source.     

Oxidation of dimethyl sulfide byPseudomonas acidovorans DMR-11 isolated from peat biofilter

Summary Pseudomonas acidovorans DMR-11, capable of oxidizing dimethyl sulfide (DMS), was isolated from peat biofilter. DMS as a sole carbon or energy source was not degraded, but it was co-degraded in the medium containing organic carbon sources. The removal rate of DMS in heat-treated glucose medium was 1.12×10^–17 mole/h cell at 30 °C. Dimethyl sulfoxide (DMSO) was the only product of DMS oxidation and was formed stoichiometrically. DMS was reversibly evolved in excess of DMSO. The cell free extract of strain DMR-11 oxidized DMS in presence of NADPH.     

Plasmid instability inLactobacillus plantarum strain caTC2

The stability of plasmids inLactobacillus plantarum was investigated by extended incubation of bacterial cells in the presence of different carbohydrates. Strain caTC2, carrying a plasmid-encoded chloramphenicol-resistant (Cm^r) phenotype, was grown overnight (16–18 h) in MRS, MRS-L, and MRS-M broths containing 2% glucose, lactose, and maltose respectively at 30°C. The cultures were subsequently held at 30°C and room temperature (21±1°C) for an extended period (7 days). The total viable cell counts were assayed on MRS agar plates and tested for sensitivity to 30 g chloramphenicol/ml by replica plating. The plasmid profiles of the chloramphenicol-sensitive strains showed that there was a loss of the 8.5-kb plasmid, but not the 10.6 or 6.5 kb plasmids. Concomitant loss of the chloramphenicol resistance phenotype and plasmid at high frequency, particularly by using MRS-L at 21°C method, suggests that this would be a simple and efficient method for curing selected plasmids in lactobacilli.Contribution No. 2039 from the Centre for Food and Animal Research.     

Effect of nystatin,amphotericin B and amphotericin B methyl ester on Saccharomyces cerevisiae with different lipid composition

Saccharomyces cerevisiae was cultured under anaerobiosis in semi-complete medium to which either palmitoleic or oleic acid was added. Cells were grown at 20 °C or 30 °C. The levels of total lipids, total sterols, and phospholipids were higher in cells grown at 20 °C than at 30 °C. The effects of nystatin (NYS), amphotericin B (AMB), and amphotericin B methyl ester (AME) were evaluated by determining cell viability and liberation of intracellular compounds. The loss of cell viability is higher in the first 30 minutes of incubation with the drugs and is the same regardless of the type of cells obtained. Low molecular weight compounds and ions such as K⁺ are liberated a few minutes after incubation with the drugs whereas proteins and substances absorbing at 260 nm are liberated later. Phosphate liberation comes after K⁺ and before compounds of higher molecular weights.     

Effect of temperature on bacterial gellan production

The effect of temperature on the production of the polysaccharide gellan by the bacterium Sphingomonas paucimobilis ATCC 31461 was studied in relation to carbon source. When glucose served as the carbon source, gellan formation by the strain was highest after 72 h of growth at an incubation temperature of 30–31 °C. Polysaccharide production by the sphingomonad cells grown on corn syrup for 72 h was maximal at an incubation temperature of 31 °C. The highest cellular productivity in elaborating gellan was observed at 31 °C after 72 h of growth independent of the carbon source utilized.     

Temperature effects on solute accumulation by Candida utilis

Summary A study was made of the effect of temperature on accumulation of glucosamine and 2-aminoisobutyrate by Candida utilis NCYC 321 grown at 30° C or 10° C. Exponential-phase cells contained greater proportions of C_16:1 and C_18:3 acids, and smaller proportions of C_13:1 and C_18:2 acids, when grown in a defined medium at 10° C compared with 30° C. Cells grown at 30° C or 10° C were able to accumulate extracellular (10 mM) glucosamine and 2-aminoisobutyrate against concentration gradients. 2-Aminoisobutyrate was not metabolised by the cells; glucosamine was accumulated probably as a mixture of glucosamine 1- and 6-phosphates. Rates of accumulation of glucosamine and 2-aminoisobutyrate by cells grown at 30° C or 10° C decreased markedly when the test temperature was decreased from 30° C to 15° C. The rate of accumulation of glucosamine by cells grown at 10° C was considerably lower at each of the test temperatures compared with the corresponding rates for cells grown at 30° C; the rate of accumulation of 2-aminoisobutyrate was much less affected by the temperature at which the cells were grown and then only when measured at temperatures below about 20° C. Apparent K _m values for accumulation of glucosamine by cells grown at 30° C or 10° C decreased considerably when the test temperature was lowered from 20° C to 15° C. The extent of the decrease in K _m value was approximately the same for cells grown at 30° C or 10° C. Apparent K _m values for accumulation of 2-aminoisobutyrate were hardly affected by test temperature. Apparent V _max values for accumulation of glucosamine or 2-aminoisobutyrate were much lower when measured at 15° C than at 30° C. When measured at 30° C, apparent V _max values for accumulation of either solute were slightly lower with cells grown at 10° C compared with cells grown at 30° C; when measured at 15° C, the values were slightly greater with cells grown at 10° C. Net accumulation of glucosamine, at 30° C or 20° C, by cells grown at 30° C or 10° C ceased after 4–6 h. Cells grown at either temperature continued to accumulate 2-aminoisobutyrate at 30° C or 20° C for at least 12 h. The rate of efflux of glucosamine by cells grown at 30° C was slower when measured at 20° C compared with 30° C. With cells grown at 10° C, the rate of efflux at 30° C was slower than with cells grown at 30° C; when measured at 20° C, the rates were about equal. The temperature at which the cells were grown did not affect the ability of d-glucose, d-mannose or d-ribose to compete with d-glucosamine, or with the ability of l-alanine to compete with 2-aminoisobutyrate, when tested at 30° C or 20° C. Cells grown 30° C or 10° C had very similar ATP contents. The results are discussed in relation to the effect of temperature on the rate of solute accumulation by micro-organisms.Abbreviation AIB 2-Aminoisobutyrate     

Degradation of pyrene byRhodococcus sp. UW1

Summary A Rhodococcus species, designated strain UW1, was isolated from contaminated soil using conventional enrichment and isolation techniques. The isolate was able to use pyrene as sole source of carbon and energy; it mineralized 72% of the pyrene within 2 weeks. During growth a metabolite was detected in the culture fluid and further characterized by UV- and mass spectrometry. There is evidence that this metabolite resulted from a recyclization of the direct meta-ring-fission product of pyrene after dihydroxylation in either the 1,2- or 4,5-position. At pH 7.0 and 30°C Rhodococcus sp. UW1 showed a maximum degradation rate of 0.08 mg pyrene/ml per day, while growing with a doubling time of 30 h. The activity of the initial dioxygenase system was characterized by measuring the oxygen-consumption rates of pyrene-induced resting cells, the maxima of which occurred at pH 7.2 and 45°C. Rhodococcus sp. UW1 could also use phenathrene, anthracene, fluoranthene and chrysene as sole sources of carbon and energy, whereas naphthalene, dibenzofuran, fluorene and dibenzothiophene were only co-metabolized. Offprint requests to: U. Walter     

Characterization of Gluconobacter oxydans immobilized in calcium alginate

Summary Gluconobacter oxydans cells were immobilized in calcium alginate and the preparation was used for the oxidation of glycerol to dihydroxyacetone. The characterization was done according to the guidelines given by the Working Party on Immobilized Biocatalysts of the European Federation of Biotechnology. The pH optimum of the preparation was found to be 5.0 and the temperature optimum was 40°C. However, the operational stability was better at 30°C. The glycerol concentration required to obtain half the maximal reaction rate was about 5 mM for both immobilized and free cells. At low concentrations of glycerol and high concentrations of dihydroxyacetone a slight inhibition was noted. No loss of activity of the immobilized preparation was observed after storage for 68 days at +4°C. Investigation of the operational stability revealed a half-life of 5 days. Studies of the influence of particle size and cell densities as well as that of oxygen concentration revealed that the oxygen supply was the rate limiting step.     

Biodegradation of toluene by the new fungal isolates Paecilomyces variotii and Exophiala oligosperma

Two new fungal strains, namely Paecilomyces variotii and Exophiala oligosperma, were isolated on toluene as the sole carbon and energy source, mineralizing the substrate into carbon dioxide. Fungal strains isolated so far on such a pollutant and completely degrading it are very scarce. Both fungi degraded the pollutant over the pH range 3.9–6.9 and temperature range 23–40°C, but E. oligosperma was barely active at the highest temperature of 40°C. Fungal growth on alkylbenzenes at 40°C has not been reported before. Since the activity of the strains gradually decreased at pH values below 4.0, the use of nitrate instead of ammonium was tested. In the presence of toluene, nitrate was a suitable nitrogen source for the Exophiala strain, but not for the Paecilomyces strain. Nitrate rather than ammonium allowed the maintenance of a more constant pH.     

Response and tolerance of toxigenic Vibro cholerae O1 to cold temperatures

Survival and tolerance at cold temperatures, the differentially expressed cellular proteins, and cholera toxin (CTX) production were evaluated in Vibrio cholerae O1. Rapid loss of culturability and change to distinct coccoid morphology occurred when cultures of V. cholerae O1 were exposed to 5°C directly from 35°C. Also, cultures of V. cholerae first exposed to 15°C for 2 h and then maintained at 5°C failed to exhibit an adaptive response, instead a rapid loss of viable plate count was noticed. Results from Western blot experiments revealed the absence of a major cold shock protein, CS7.4. Also, a decreased level of CTX was noticed in V. cholerae O1 cultures exposed to 5 or 15°C after first being exposed to 15°C for 2 h, followed by transfer to 5°C. Reduced expression of CTX at cold temperatures, compared to the cultures maintained at 35°C, may be a result of decreased cellular metabolic activity. When V. cholerae O1 cultures were exposed to 15°C for 2 h, elevated expressions of 8, 26 and 194 kDa, and decreased expression of 28 and 183 kDa proteins occurred. It is suggested that these differentially expressed cold-responsive proteins are involved in regulating culturability and conversion to a coccoid cell morphology in V. cholerae O1.     

Production and stabilization of amylase preparations from rice bran solid medium

A low-cost amylase preparation of dried fermented bran was developed from rice bran solid cultures of Aspergillus oryzae supplemented with soya bean flour (SBF) and cassava starch (3:1) and dried at 50 °C for 4 h. Storage stability of preparations at 4 °C or 30 °C was significantly enhanced (P 0.05) by adding SBF or partially hydrolyzed starch (PHS). While amylase preparations without stabilizer retained 59 and 48% of their activity after 12 weeks storage at 4 and 30 °C respectively, the same preparations fortified with SBF (5% w/v) retained 95 and 94% stability respectively, during the same period. PHS at 5% (w/v) also gave a maximum stability of 94 and 91.8% at 4 and 30 °C, respectively. The unstabilized preparation retained only 42% of its activity compared to the stabilized forms, which retained 82–90% activity after 15 min incubation at 100 °C.     

Fructose production by immobilizedArthrobacter cells

Summary ImmobilizedArthrobacter cells (NRRL-B-3728) were used for continuous isomerization of glucose to fructose in a bioreactor system. The system utilized stationary phase (55h) cells (2.2×10⁹ CFU/ml saline) immobilized onto K-carrageenan (3% w/v) beads [cells were heated at 65°C for 10 min to inactivate endogenous proteolytic enzymes]. Immobilized-cell preparations were hardened using three different glutaraldehyde systems. Glutaraldehyde (0.2 M) treated-immobilized cells (pH 7.0, 5°C for 30 min) exhibited good gel strength and high glucose isomerase activities. Maximal bioreactor isomerization of 44% was achieved when a buffered feedstock containing 40% glucose was fed into the column (60°C) at a flow rate of 0.2 ml/min. The biological half-life of glucose isomerase activities in this system was 400 h. Scanning electron microscopy revealed large numbers of cells distributed within the beads. A thin layer surrounding the beads following glutaraldehyde treatment was mainly due to cross-linking reactions between cell proteins and glutaraldehyde. This layer prevented leaking of cells during continuous isomerization reaction.     

Characterization of the heat shock response inEnterococcus faecalis

We have characterized the general properties of the heat shock response of the Gram-positive hardy bacteriumEnterococcus faecalis. The heat resistance (60°C or 62.5°C, 30 min) of log phase cells ofE. faecalis grown at 37°C was enhanced by exposing cells to a prior heat shock at 45°C or 50°C for 30 min. These conditioning temperatures also induced ethanol (22%, v/v) tolerance. The onset of thermotolerance was accompanied by the synthesis of a number of heat shock proteins. The most prominent bands had molecular weights in the range of 48 to 94kDa. By Western blot analysis two of them were found to be immunologically related to the well known DnaK (72 kDa) and GroEL (63 kDa) heat shock proteins ofEscherichia coli. Four other proteins showing little or no variations after exposure to heat are related to DnaJ, GrpE and Lon (La)E. coli proteins and to theBacillus subtilis ⁴³ factor. Ethanol (2% or 4%, v/v) treatments elicited a similar response although there was a weaker induction of heat shock proteins than with heat shock.     

Covalent immobilization of laccase on activated carbon for phenolic effluent treatment

Summary Laccase was covalently immobilised to activated carbon using four derivatisation methods. The highest bound activity was obtained using diimide coupling of laccase to carboxyl groups on the carbon. The maximum bound activity was reached at 11.5 mg laccase/g carbon. The carbon-immobilised laccase (CIL) was stable at pH values from 4.0 to 9.0. CIL stored at 4°C lost 38± 5% activity in the first 4 days, then a further 22±5% in 126 days. CIL showed increased stability to low pH although the pH optimum was unchanged. The activation energy of CIL was lower than soluble laccase. Oxidation of 2,6-dimethoxyphenol (DMP) by CIL in a packed-bed system was only 30±10% of that in a fluidised bed system. Of the initial activity 10–30% was retained after oxidation of seven batches of DMP. CIL removed colour from two industrial effluents. Colour was removed from pulp mill bleach plant effluent at 115 colour units per enzyme unit per hour and the removal rate increased with increasing effluent concentration. Correspondence to: R. G. Burns     

Temperature dependence of horse heart cytochromec oxidation by membrane preparations ofAnacystis nidulans: Characterization of two distinct arrhenius discontinuities

The oxidation of reduced horse heart cytochromec by membranes isolated from the cyanobacteriumAnacystis nidulans after growth at different temperatures was studied between 4°C and 41°C in the light and the dark using both spectrophotometric and polarographic techniques. Arrhenius plots of the temperature dependence of cytochromec photooxidation showed a single discontinuity at 25°C, 15°C, and 12°C in membranes derived from cells grown at 40°C, 30°C, and 25°C, respectively. By contrast. Arrhenius plots of the temperature dependence of dark respiratory cytochromec oxidation always displayed two distinct breaks at 25 and 18°C, 15 and 8.5°C, and 12 and 5.5°C in membranes isolated from cells grown at 40°C, 30°C, and 25°C, respectively. The results are discussed in terms of the thermotropic lipid-phase transitions known to take place in the membranes ofA. nidulans. Special reference will be made to possibly distinct localizations of the membrane-bound cytochromec oxidase complexes in respiration and photosynthesis.     

Heat and cold shock responses in different strains of Thiobacillus ferrooxidans

Different strains of Thiobacillus ferrooxidans were examined for their ability to produce a heat shock and a cold shock response. Strain A1, heat shocked from 20° to 35°C, acquired thermotolerance, as it showed a 1000-fold reduction in cell mortality when exposed to the supermaximum temperature of 42°C, as compared to a non-heat-shocked control. A heat shock from 25° to 35°C yielded similar results, although a higher degree of thermotolerance was achieved for the shorter exposure times. Cultures heat shocked for 5 h showed a five-log reduction in viable counts after 41 h at 42°C, whereas non-heat-shocked cultures showed a similar reduction in viability in 28 h. Conferred thermotolerance was immediate and sustained for the duration of the exposure to 42°C. Heat-shocked cultures were not significantly protected against loss of viability due to freezing (-15°C for 24 h). Strain S2, cold shocked from 25° to 10°C, and strain D6, cold shocked from 25° to 5°C, were not protected against freezing at-15°C. An analysis of proteins extracted from heat-shocked cells of strain A1 showed the presence of at least one newly induced protein and eight hyper-induced proteins. The molecular weights of the heat shock proteins were in the range of 15–80.3 kDa.     

Regulation of alcohol-oxidizing capacity in chemostat cultures of Acetobacter pasteurianus

Acetobacter pasteurianus LMG 1635 was studied for its potential application in the enantioselective oxidation of alcohols. Batch cultivation led to accumulation of acetic acid and loss of viability. These problems did not occur in carbon-limited chemostat cultures (dilution rate = 0.05 h^–1) grown on mineral medium supplemented with ethanol, L-lactate or acetate. Nevertheless, biomass yields were extremely low in comparison to values reported for other bacteria. Cells exhibited high oxidation rates with ethanol and racemic glycidol (2,3-epoxy-1-propanol). Ethanol- and glycidol-dependent oxygen-uptake capacities of ethanol-limited cultures were higher than those of cultures grown on lactate or acetate. On all three carbon sources, A. pasteurianus expressed NAD-dependent and dye-linked ethanol dehydrogenase activity. Glycidol oxidation was strictly dye-linked. In contrast to the NAD-dependent ethanol dehydrogenase, the activity of dye-linked alcohol dehydrogenase depended on the carbon source and was highest in ethanol-grown cells. Cell suspensions from chemostat cultures could be stored at 4°C for over 30 days without significant loss of ethanol- and glycidol-oxidizing activity. It is concluded that ethanol-limited cultivation provides an attractive system for production of A. pasteurianus biomass with a high and stable alcohol-oxidizing activity.     

Genome-wide expression analysis of yeast response during exposure to 4°C

Adaptation to temperature fluctuation is essential for the survival of all living organisms. Although extensive research has been done on heat and cold shock responses, there have been no reports on global responses to cold shock below 10°C or near-freezing. We examined the genome-wide expression in Saccharomyces cerevisiae, following exposure to 4°C. Hierarchical cluster analysis showed that the gene expression profile following 4°C exposure from 6 to 48 h was different from that at continuous 4°C culture. Under 4°C exposure, the genes involved in trehalose and glycogen synthesis were induced, suggesting that biosynthesis and accumulation of those reserve carbohydrates might be necessary for cold tolerance and energy preservation. The observed increased expression of phospholipids, mannoproteins, and cold shock proteins (e.g., TIP1) is consistent with membrane maintenance and increased permeability of the cell wall at 4°C. The induction of heat shock proteins and glutathione at 4°C may be required for revitalization of enzyme activity, and for detoxification of active oxygen species, respectively. The genes with these functions may provide the ability of cold tolerance and adaptation to yeast cells.     

Maintenance of biodegradation capacities of aerobic bacteria during long-term preservation

Six strains of aerobic Gram negative bacteria degrading toluene, 2,4-dichlorophenoxyacetate, 2,2-dichloropropionate or 3-chlorobenzoate were freeze-dried and liquid-dried in the presence or absence of a protective agent. Survival and maintenance of the biodegradation capability was checked before and after drying, and after storage of the ampoules for one year at 4° or 25°C. In many cases, stability of the degradation potential was low although viability was high. Survival and stability of all strains was always highest after preservation by liquid drying in the presence of myo-inositol and activated charcoal as protective agents. Losses of biodegradation abilities were highest after freeze-drying using no protective agents. Cells grown on complex medium were less sensitive to drying than cells grown under selective pressure (on mineral medium with a special compound as the sole carbon source). A choice of the most appropriate preservation method and the use of an effective protectant is recommended to avoid genetic alterations, and to maintain biodegradation capacities during long-term preservation.