Thermal properties and oxygenase activity of ribulose-1,5-bisphosphate carboxylase from the thermophilic purple bacterium, Chromatium tepidum

Abstract Ribulose-1,5-biphosphate carboxylase (RuBPCase) partially purified from the thermophilic purple bacterium Chromatium tepidum displayed maximum carboxylase activity at 50°C, while enzyme from a related mesophilic species, Chromatium vinosum , was completely inactive at 50°C. RuBPCase from C. tepidum showed ribulose-1,5- bisphosphate-dependent oxygenase activity, and, in addition, O₂ was found to partially destroy carboxylase activity. It is concluded that thermophilic purple bacteria produce heat-stable RuBPCase and that all RuBPCases, even those from an obligate anaerobe such as C. tepidum , have associated oxygenase activity.     

Isolation and partial characterization of bacteria in an anaerobic consortium that mineralizes 3-chlorobenzoic Acid

A methanogenic consortium able to use 3-chlorobenzoic acid as its sole energy and carbon source was enriched from anaerobic sewage sludge. Seven bacteria were isolated from the consortium in mono- or coculture. They included: one dechlorinating bacterium (strain DCB-1), one benzoate-oxidizing bacterium (strain BZ-2), two butyrate-oxidizing bacteria (strains SF-1 and NSF-2), two H(2)-consuming methanogens (Methanospirillum hungatei PM-1 and Methanobacterium sp. strain PM-2), and a sulfate-reducing bacterium (Desulfovibrio sp. strain PS-1). The dechlorinating bacterium (DCB-1) was a gram-negative, obligate anaerobe with a unique "collar" surrounding the cell. A medium containing rumen fluid supported minimal growth; pyruvate was the only substrate found to increase growth. The bacterium had a generation time of 4 to 5 days. 3-Chlorobenzoate was dechlorinated stoichiometrically to benzoate, which accumulated in the medium; the rate of dechlorination was ca. 0.1 pmol bacterium day. The benzoate-oxidizing bacterium (BZ-2) was a gram-negative, obligate anaerobe and could only be grown as a syntroph. Benzoate was the only substrate observed to support growth, and, when grown in coculture with M. hungatei, it was fermented to acetate and CH(4). One butyrate-oxidizing bacterium (NSF-2) was a gram-negative, non-sporeforming, obligate anaerobe; the other (SF-1) was a gram-positive, sporeforming, obligate anaerobe. Both could only be grown as syntrophs. The substrates observed to support growth of both bacteria were butyrate, 2-dl-methylbutyrate, valerate, and caproate; isobutyrate supported growth of only the sporeforming bacterium (SF-1). Fermentation products were acetate and CH(4) (from butyrate, isobutyrate, or caproate) or acetate, propionate, and CH(4) (from 2-dl-methylbutyrate or valerate) when grown in coculture with M. hungatei. A mutualism among at least the dechlorinating, benzoate-oxidizing, and methane-forming members was apparently required for utilization of the 3-chlorobenzoate substrate.     

Bacillus polymyxa stimulates increased Rhizobium etli populations and nodulation when co-resident in the rhizosphere of Phaseolus vulgaris

Abstract A Gram-negative sporulating thermophilic anaerobe, designated AB11Ad, was isolated from the heated waters of the Great Artesian Basin of Australia. It grew on a variety of carbohydrates including glucose, starch, and dextran and produced a thermostable and thermoactive extracellular endo-dextranase. The enzyme was produced more actively under pH controlled continuous culture conditions than under batch conditions. Ammonium sulfate precipitated crude dextranase exhibited a temperature optimum of 70 °C and a pH optimum between 5 and 6. The half life was ~ 6.5 h at 75 °C and 2 h at 80 °C at pH 5.0 and in the absence of added dextran. 16S rRNA sequence analysis indicated that isolate AB1 lAd was a member of the genus Thermoanaerobacter .     

The protective effect of some food ingredients on Staphylococcus aureus MF31

The upper limiting temperature of growth of Staphylococcus aureus MF31 in heart infusion broth (HI) was about 44°C but addition of monosodium glutamate (MSG) and soy sauce permitted the organism to grow above this temperature. This effect is similar to that of NaCl. Tomato ketchup, Worcestershire and HP sauces added to HI did not allow growth at the non-permissive temperature of 46°C but death was delayed. Staphylococcus aureus died in unsupplemented chicken meat slurry at 46°C but grew at 48°C in slurry supplemented with 5.8% NaCl and survived incubation for 18 h at 50°C in slurry supplemented with 5.8% NaCl and 5% MSG. Cultures grown at 37°C had a D ₆₀ value of 2 min in 50 mmol/l Tris (pH 7.2) buffer. Cultures grown at 46°C in HI containing 5.8% NaCl had a D ₆₀ value of 8 min in Tris buffer. Addition of 5.8% NaCl plus 5% MSG to the buffer increased the D ₆₀ by a factor of about 7 for both cultures. In storage experiments at room temperature, the culture grown at 37°C and at 46°C plus 5.8% NaCl died at about the same rate in salami. In milk powder, however, the count of 37°C culture decreased from 10⁹/g to 10⁶/g in 5 weeks while the count of 46°C culture remained unchanged. In cottage cheese, freeze-dried rice and macaroni, the 37°C cultures also died more rapidly. It is suggested that cultures grown at 46°C plus 5.8% NaCl may be suitable for experiments with artificially contaminated foods.     

Anaerobic bacteria that dechlorinate perchloroethene.

In this study, we identified specific cultures of anaerobic bacteria that dechlorinate perchlorethene (PCE). The bacteria that significantly dechlorinated PCE were strain DCB-1, an obligate anaerobe previously shown to dechlorinate chlorobenzoate, and two strains of Methanosarcina. The rate of PCE dechlorination by DCB-1 compared favorably with reported rates of trichloroethene bio-oxidation by methanotrophs. Even higher PCE dechlorination rates were achieved when DCB-1 was grown in a methanogenic consortium.     

Anaerobic Aryl Reductive Dehalogenation of Halobenzoates by Cell Extracts of "Desulfomonile tiedjei"

We studied the transformation of halogenated benzoates by cell extracts of a dehalogenating anaerobe, "Desulfomonile tiedjei." We found that cell extracts possessed aryl reductive dehalogenation activity. The activity was heat labile and dependent on the addition of reduced methyl viologen, but not on that of reduced NAD, NADP, flavin mononucleotide, flavin adenine dinucleotide, desulfoviridin, cytochrome c(3), or benzyl viologen. Dehalogenation activity in extracts was stimulated by formate, CO, or H(2), but not by pyruvate plus coenzyme A or by dithionite. The pH and temperature optima for aryl dehalogenation were 8.2 and 35 degrees C, respectively. The rate of dehalogenation was proportional to the amount of protein in the assay mixture. The substrate specificity of aryl dehalogenation activity for various aromatic compounds in "D. tiedjei" cell extracts was identical to that of whole cells, except differences were observed in the relative rates of halobenzoate transformation. Dehalogenation was 10-fold greater in "D. tiedjei" extracts prepared from cells cultured in the presence of 3-chlorobenzoate, suggesting that the activity was inducible. Aryl reductive dehalogenation in extracts was inhibited by sulfite, sulfide, and thiosulfate, but not sulfate. Experiments with combinations of substrates suggested that cell extracts dehalogenated 3-iodobenzoate more readily than either 3,5-dichlorobenzoate or 3-chlorobenzoate. Dehalogenation activity was found to be membrane associated. This is the first report characterizing aryl dehalogenation activity in cell extracts of an obligate anaerobe.     

Anaerobic bacteria that dechlorinate perchloroethene

In this study, we identified specific cultures of anaerobic bacteria that dechlorinate perchlorethene (PCE). The bacteria that significantly dechlorinated PCE were strain DCB-1, an obligate anaerobe previously shown to dechlorinate chlorobenzoate, and two strains of Methanosarcina. The rate of PCE dechlorination by DCB-1 compared favorably with reported rates of trichloroethene bio-oxidation by methanotrophs. Even higher PCE dechlorination rates were achieved when DCB-1 was grown in a methanogenic consortium.     

Strain DCB-1 conserves energy for growth from reductive dechlorination coupled to formate oxidation

Strain DCB-1 is a strict anaerobe capable of the reductive dechlorination of chlorobenzoates. The effect of dechlorination on the yield of pure cultures of DCB-1 was tested. Cultures were incubated with formate or H₂ as electron donors and CO₂ as a putative carbon source. Relative to control cultures with benzoate, cultures which dechlorinated 3-chlorobenzoate and 3,5-dichlorobenzoate had higher yields measured both as protein and cell density. On the media tested the apparent growth yield was 1.7 to 3.4 g cell protein per mole Cl^- removed. Dechlorination also stimulated formate oxidation by growing cultures. Resuspended cells required an electron donor for dechlorination activity, with either formate or elemental iron serving this function. Resuspended cells did not require an electron acceptor for formate consumption, but reductive dechlorination of 3CB to benzoate stoichiometrically stimulated oxidation of formate to CO₂. These results indicate that DCB-1 conserves energy for growth by coupling formate, and probably, H₂ oxidation to reductive dechlorination.Non-standard abbreviations 3CB 3-chlorobenzoate - 35DCB 3,5-dichlorobenzoate - PCF Propionibacterium sp. culture fluid     

Growth of Listeria monocytogenes at refrigeration temperatures

The growth of three strains of Listeria monocytogenes at refrigeration temperatures (-0.5 to 9.3°C) in chicken broth and/or UHT milk was determined using a rocking temperature gradient incubator. Minimum growth temperatures ranged from -0.1 to -0.4°C for the three strains. Lag times of 1–3 d and 3 to >34 d were observed with incubation at 5 and 0°C respectively. Corresponding generation times ranged from 13–24 h at 5°C and 62–131 h at 0°C. The type of culture medium had an influence on both the rate and extent of growth. Incubation of cultures at 4°C before inoculation caused a marked reduction in the lag time when compared with cultures which had been previously incubated at 30°C.     

Effect of culture age, pre-incubation at low temperature and pH on the thermal resistance of Aeromonas hydrophila

S. CONDON, M.L. GARCIA, A. OTERO AND F.J. SALA. 1992. The thermal resistance of Aeromonas hydrophila strain NCTC 8049 was determined within the range 48°-65°C with a thermoresistometer TR-SC and McIlvaine buffer. The effects of culture age, pre-incubation at 7°C and the pH of the heating menstruum were evaluated. The pattern of thermal death was dependent on culture age. Cells heated in the late logarithmic growth phase (15 h at 30°C) were twice as resistant as those in the early stage (5 h at 30°C), and the maximum D -value was obtained after 72 h incubation (5.5 total increase). The age of the cells did not affect z -values significantly. The heat resistance of cells incubated for 48 h at 30°C increased (twice) after holding at 7°C for 72 h. Pre-incubation at low temperature of older cultures (72 h, 30°C) did not influence their D -values. Maximum heat resistance was found at pH 6.0 and minimal at pH 4.0. Decreasing the pH from 6.0 4.0 reduced D -values by a factor of 5. Although the strain studied was heat-sensitive ( D _55°C= 0.17 min; z = 5.11°C), survivor curves of cultures older than 50 h showed a significant tailing. Organisms surviving in the tails were only slightly more resistant than were the original population.     

Resuscitation of Vibrio cholerae O1 strain TSI-4 from a viable but nonculturable state by heat shock

Abstract Vibrio cholerae strain TSI-4 was incubated in an M9 salt solution at 15 °C for more than 100 days. The plate counts showed no viable cells on day 30, but a broth culture from that day showed the growth of bacteria. However, after 35 days the bacteria entered the nonculturable state, based on the assessment of both the plate counts and broth culture. A portion of the culture was heated at 45 °C for 1 min in a water bath and subsequently plated onto a nutrient agar plate. More than 1000 colonies were recovered after this heat-shock treatment. The recovered cells showed the same chromosomal DNA pattern in the restriction map and the same outer membrane protein pattern in SDS-PAGE. Recovery of viable cells by heat-shock was achieved in cultures grown on M9 salt but not from cultures grown in phosphate-buffered saline. This suggests that the presence of NH₄Cl in the M9 salt solution may support the growth of the bacteria in a low nutrient medium, while also playing an important role in resuscitation.     

Tetrachloroethene and 3-chlorobenzoate dechlorination activities are co-induced inDesulfomonile tiedjei DCB-1

Desulfomonile tiedjei, a strict anaerobe capable of reductively dechlorinating 3-chlorobenzoate, also dechlorinates tetrachloroethene and trichloroethene. It is not known, however, if the aryl and aliphatic dechlorination activities are catalyzed by the same enzymatic system. Cultures induced for 3-chlorobenzoate activity dechlorinated tetrachloroethene and trichloroethene to lower chlorinated products while uninduced parallel cultures did not dechlorinate either substrate. The observed rate of PCE dechlorination in induced cultures was 22 µmol h^–1 g protein^–1, which is considerably faster than previous rates obtained with defined cultures of this organism. These results show that both dechlorination activities are co-induced and therefore, that the dechlorination mechanisms may share at least some components.Abbreviations PCE tetrachloroethene - TCE trichloroethene - cis-DCE cis-dichloroethene - trans-DCE trans-dichloroethene - 3FBz 3-fluorobenzoate - 3ClBz 3-chlorobenzoate     

Incidence of Enterotoxigenic Clostridium perfringens in Healthy Humans in relation to the Enhancement of Enterotoxin Production by Heat Treatment

Enterotoxin production was greatly enhanced in two of five food poisoning strains of Clostridium perfringens subjected to heat treatment prior to incubation in Duncan and Strong sporulation medium. Heating was carried out on three successive cultures of each strain, the optimum temperature for treatment being 85 °C for one strain and 95 °C for another: on each occasion cultures were heated for 20 min. The triple heat treatment procedure was used in testing strains of Cl. perfringens isolated from faeces of healthy human subjects for production of enterotoxin. Eleven of 35 (31%) individuals were found to be carriers of enterotoxigenic strains, the isolates producing more than 0·1 μ/ml of enterotoxin. Six of the 11 enterotoxigenic strains were killed by heating at 95 °C but one isolate produced more enterotoxin following treatment at this temperature than after heating at 75 °C.     

BINDING CHARACTERISTICS OF 125I-LABELLED TETANUS TOXIN TO PRIMARY TISSUE CULTURES FROM MOUSE EMBRYONIC CNS

Abstract— The interaction of ¹²⁵I-labelled tetanus toxin with cells in tissue cultures derived from embryonic CNS has been studied.
The optimum toxin binding occurs about 2–3 weeks after transfer of the cells to culture conditions. The amount of label bound per culture was doubled at this time in comparison to the fourth day nfter inoculation.
The amount of toxin bound depends on the concentration applied. It reaches its maximum 8 h after application then decreases slowly. Low amounts of radioactivity were still detectable 97 h after washing off the unbound toxin. Up to 80% of the label can be replaced by simultaneous application of'cold'toxin. Fixation of the toxin is higher at 4°C than at 37°C.
Preincubation of the cultures with neuraminidase prevents about 75% of the binding. The presence of cytochalasin B leads to a small but reproducible decrease of binding, whereas colchicine had no measurable effect.
The radioactive (¹²⁵1) material was identified by a double-isotope technique in disc gel electrophoresis before and after reductive cleavage of its disulphide bonds. In every test is was indistinguishable from ¹³¹I-labelled toxin added as standard.
Our results largely parallel those obtained with synaptosomes and other systems. They suggest that gangliosides might be the acceptor molecules, and that the culture system will be suitable for studying the actions of this toxin in vitro.     

A comparative study of the formation of extracellular proteins by Aeromonas salmonicida at two different temperatures

Aeromonas salmonicida was grown in a supplemented 3% (w/v) tryptone soya broth medium at 10°C, a temperature at the lower end of the range over which furunculosis has been observed to occur in the field, and 25°C, the optimum temperature for growth. Similar bacterial densities in the range 2.35 × 0.05 mg dry wt/ml were achieved in the two cultures at the beginning of the stationary phase of the growth cycle, after 125 h at 10°C and 18 h at 25°C. At this point, at the higher temperature 1.5 times more exoprotein was formed, 80 × 2.8 μg/ml compared with 54 × 1.7 μg/ml. Exoprotein contained the same proportion of haemolysin at both temperatures and twice as much protease at the higher temperature. The most marked difference was in an unidentified 100 kD protein which was formed in a 10-fold greater amount at 10°C.     

The effect of temperature, pH and medium in a surface adhesion immunofluorescent technique for detection of Listeria monocytogenes

A rapid surface adhesion-based immunofluorescence technique was used to detect Listeria monocytogenes from inoculated culture systems. The effect of culture type (pure, mixed and meat), pH (7·00, 6·40, 4·76 and 3·13), acids (citric and HCl) and temperature (25°, 30° and 37°C) on the adhesion of Listeria to the polycarbonate membrane used in this technique was determined. It was found that pH had a significant effect ( P < 0·05) with higher numbers of Listeria adhering at low pH values (4·76). Culture type was also important with significantly higher numbers of Listeria ( P < 0·05) adhering to membranes immersed in meat cultures than in pure or mixed cultures. This effect was seen at 30°C but not at 25° or 37°C. The total viable count (TVC) on the membrane was unaffected by pH but temperature had an influence with optimum adhesion occurring at 25°C. The reasons for observed differences and their implications for the surface adhesion immunofluorescent rapid method are discussed.     

The effect of temperature on the growth of strains of Kloeckera apiculata and Saccharomyces cerevisiae in apple juice fermentation

The influence of temperature (10°C and 25°C) on the survival and growth of Saccharomyces cerevisiae and Kloeckera apiculata was examined in mixed and pure cultures during fermentation in apple juice. The growth reached by S. cerevisiae did not seem to be affected by temperature and the presence of K. apiculata . However, the growth and survival of K. apiculata , both in single and mixed cultures, were substantially enhanced at 10°C. The highest amount of ethyl acetate was produced by K. apiculata in pure culture at 10°C. Nevertheless, this concentration was lowest when both yeasts were fermented together at 10°C and 25°C.     

Biodegradation of linear alkylbenzenesulphonates (LAS) by mixed methanotrophic-heterotrophic cultures

The biodegradation of undecylbenzenesulphonate (C₁₁LAS) was studied in shake flasks at 21°C using two mixed bacterial cultures. The first culture, MM1, contained a type II methanotroph and four heterotrophs, and was enriched from a groundwater aquifer. The second culture, MC, consisted of five heterotrophic strains, most of them belonging to the genus Pseudomonas , and was isolated from the wastewater of a detergent plant. Methane, carbon dioxide and oxygen concentrations were determined by gas chromatography. Concentrations of C₁₁LAS and the aromatic intermediates were determined by reversed-phase HPLC. In spite of faster transformation of the alkyl side-chain by the culture MC, the culture MM1 containing type II methanotroph was capable of further degradation of C₁₁LAS aromatic intermediates (sulphophenylalkanoates). The most probable mechanism for the degradation of the alkyl part of the C₁₁LAS molecule by both cultures was β-oxidation of the terminal methyl group followed by β-oxidation. Studies of methane utilization demonstrated an approximately three times higher second-order rate coefficient for methane consumption ( k _max/ K _s) in the absence of C₁₁LAS. This indicates a possible metabolic activity of methanotrophs in the transformation of the complex LAS molecule due to the methane monooxygenase enzyme system.     

Anaerobic Aryl Reductive Dehalogenation of Halobenzoates by Cell Extracts of “Desulfomonile tiedjei”

We studied the transformation of halogenated benzoates by cell extracts of a dehalogenating anaerobe, “Desulfomonile tiedjei.” We found that cell extracts possessed aryl reductive dehalogenation activity. The activity was heat labile and dependent on the addition of reduced methyl viologen, but not on that of reduced NAD, NADP, flavin mononucleotide, flavin adenine dinucleotide, desulfoviridin, cytochrome c₃, or benzyl viologen. Dehalogenation activity in extracts was stimulated by formate, CO, or H₂, but not by pyruvate plus coenzyme A or by dithionite. The pH and temperature optima for aryl dehalogenation were 8.2 and 35°C, respectively. The rate of dehalogenation was proportional to the amount of protein in the assay mixture. The substrate specificity of aryl dehalogenation activity for various aromatic compounds in “D. tiedjei” cell extracts was identical to that of whole cells, except differences were observed in the relative rates of halobenzoate transformation. Dehalogenation was 10-fold greater in “D. tiedjei” extracts prepared from cells cultured in the presence of 3-chlorobenzoate, suggesting that the activity was inducible. Aryl reductive dehalogenation in extracts was inhibited by sulfite, sulfide, and thiosulfate, but not sulfate. Experiments with combinations of substrates suggested that cell extracts dehalogenated 3-iodobenzoate more readily than either 3,5-dichlorobenzoate or 3-chlorobenzoate. Dehalogenation activity was found to be membrane associated. This is the first report characterizing aryl dehalogenation activity in cell extracts of an obligate anaerobe.     

Moist-heat disinfection of pathogenic Acanthamoeba cysts

The effect of moist-heat in the disinfection of pathogenic Acanthamoeba cysts was investigated. Temperatures of 56°C or 60°C were not effective in killing cysts of A. polyphaga even after a contact time of 60 min, A 4 log reduction in viability was achieved within 15 min at 65°C and 2 min at 70°C giving decimal reduction rates (D-values) of 3·75 min and 30 s respectively. The ability of a commercial moist-heat contact lens disinfection unit to kill 1 times 10⁵ cysts of Acanthamoeba isolated from contact lens storage cases was also shown. Holding temperatures inside the cases of 65°C for 5·5 min and 70°C (the maximum temperature obtained) for 1 min were recorded during the disinfection cycle.