Comparative Physiology of Dimethyl Sulfide Production by Dimethylsulfoniopropionate Lyase in Pseudomonas doudoroffii and Alcaligenes sp. Strain M3A

Dimethylsulfoniopropionate (DMSP) lyase enzymatically cleaves DMSP, an algal metabolite, to produce acrylate, a proton, and dimethyl sulfide (DMS), the most abundant volatile sulfur compound emitted from oceans. The physiology of DMS production by DMSP lyase was studied in vivo in an Alcaligenes-like organism, strain M3A, a salt marsh bacterial isolate, and in a marine strain, Pseudomonas doudoroffii. Enzymes from both strains were induced at optimum rates by 1 mM DMSP and vigorous aeration. P. doudoroffii was very sensitive to continued aeration and lost activity rapidly; the enzyme was more stable when aeration ceased. In addition to DMSP, acrylate and several of its analogs acted as inducers of DMSP lyase in Alcaligenes sp. strain M3A but not in P. doudoroffii. Turnover of DMSP by P. doudoroffii was enhanced by 3.5% NaCl or seawater, whereas the Alcaligenes sp. strain M3A enzyme was not salt dependent and salt did not greatly affect its activity. The pH profile showed two peaks of DMSP lyase activity (6.5 and 8.8) for Alcaligenes sp. strain M3A and a single peak at pH 8 for P. doudoroffii. Enzyme activity in both organisms was inhibited by methyl-3-mercaptopropionate and homocysteine. Cyanide, azide and p-chloromercuribenzoate inhibited only the P. doudoroffii DMSP lyase. The apparent K(infm) values for DMSP for cell cultures of Alcaligenes sp. strain M3A and P. doudoroffii were ca. 2 mM and <20 (mu)M, respectively. The differences in the physiology of DMSP metabolism in these two bacterial isolates may enable them to exist in diverse ecological niches.     

Isolation, screening and characterization of thermophilic Bacillus species isolated from dairy products

Proteolytic thermophilic bacterial cultures (171 strains) were isolated from different milk and milk products. After screening these isolates for protease production in a liquid medium, fifty that exhibited enzyme activity in excess of 100 units/ml were selected and identified. Twenty-nine were Bacillus stearothermophilus (constituting 58% of the total), twelve were B. coagulans , five were B. circulans and four were B. licheniformis . Skim milk powder contributed the maximum number of B. stearothermophilus (64.7%) followed by raw milk (63.2%) and pasteurized milk (44.4%). When the culture supernatant liquids from the selected isolates were given heat treatment, five cultures retained 100% protease activity at 65°C for 30 min. Protease of B. stearothermophilus RM-67 had the maximum heat resistance because it retained 87.5% of its activity at 70°C for 30 min.     

Effect of Temperature on the Formation of Microsclerotia of Verticillium dahliae

Microsclerotium formation by six isolates of Verticillium dahliae was studied at different temperatures both in vitro and in Arabidopsis thaliana . In vitro mycelial growth was optimal at 25°C, but microsclerotium formation was greatest at 20°C (two isolates) or 15–20°C (one isolate). Seedlings of A. thaliana were root-dipped in a conidial suspension, planted, and either placed at 5, 10, 15, or 25°C, or left at 20°C until the onset of senescence, after which some of the plants were placed at 5, 10, 15, or 25°C. The amount of microsclerotia per unit of shoot weight was assessed in relation to isolate and temperature. The optimal temperature for production of microsclerotia was 15–25°C. Two isolates each produced about 10 times more microsclerotia than each of the other four isolates. For these isolates, high R ²_adj.-values of 0.77 and 0.66 were obtained, with temperature and its square as highly significant (P   < 0.001) independent variables. R ²_adj.-values for the other isolates varied between 0.28 and 0.39. Moving plants to different temperatures at the onset of senescence led to microsclerotial densities that were intermediate between densities on plants that had grown at constantly 20°C and plants grown at other temperatures. This suggests that vascular colonization rate and rate of microsclerotium formation are similarly affected by temperature. The senescence rate of plants appeared unimportant except for plants grown at 25°C, which showed the highest amounts of microsclerotia per unit of plant weight in the most rapidly senescing plants.     

Production and characterization of thermostable cellulases from Streptomyces transformant T3-1

A total of 26 thermophilic isolates, selected from a compost of agricultural waste, which was mostly composed of vegetable, corncob and rice straw, were cultivated at 50 °C for further studies of thermostable cellulase production. The thermostable cellulase gene from the chromosomal DNA of actinomycetes isolate no. 10 was shotgun-cloned and transformed into Streptomyces sp. IAF 10-164. A transformant, T3-1, was found to be a good strain for the production of thermostable cellulases. Cultivation of T3-1 in modified Mandels–Reese broth containing 1% carboxymethylcellulose (CMC)-sodium salt and the optimal condition for microbial growth were studied. Batch cultivation in a flask revealed that CMCase and Avicelase production reached the maximum between the third to fifth day, whereas maximum -glucosidase production occurred on the ninth day. Microbial biomass increased from the first day to the fifth day and then decreased. The crude enzyme had the highest activity at 50 °C and at pH 6.5. The enzyme was shown to be a thermostable cellulase whose activities were stable at 50 °C for more than 7 days.     

Nisin induces changes in membrane fatty acid composition of Listeria monocytogenes nisin-resistant strains at 10°C and 30°C

Listeria monocytogenes isolates resistant to 10⁵ IU ml^-1 nisin were obtained at 30°C (NR30) and at 10°C (NR10). Nisin prolonged the lag phase of isolate NR30 at 10°C. Isolates NR30 and NR10 did not produce a nisinase. Protoplasts of isolate NR30 were unaffected by exposure to nisin. The fatty acid composition from the wild-type strain and NR isolates was determined. As expected, temperature-induced differences in the C15/C17 fatty acid ratios were found. Growth of the NR strains in the presence of nisin resulted in significantly different C15/C17 ratios and a significant increase in the percentage of C16:0, C16: 1, C18:0 and C18: 1 fatty acids at 10°C and 30°C. Both the NR10 and NR30 isolates had similar growth rates at low temperatures, but these were slower than the wild-type strain. These results indicate that 'nisin resistance'is an environmentally defined phenotype and that nisin induces changes in the fatty acid composition of the membrane in L. monocytogenes nisin-resistant isolates regardless of the growth temperature.     

Isolation and characterization of bacteria from soil contaminated with diesel oil and the possible use of these in autochthonous bioaugmentation

Two bacterial species (isolates N and O) were isolated from a paddy soil microcosm that had been artificially contaminated with diesel oil to which extrinsic Pseudomonas aeruginosa strain WatG, had been added exogenously. One bacterial species (isolate J) was isolated from a similar soil microcosm that had been biostimulated with Luria–Bertani (LB) medium. Isolates N and O, which were tentatively identified as Stenotrophomonas sp. and Ochromonas sp., respectively, by sequencing of their 16 S rRNA genes had no ability to degrade diesel oil on their own in any liquid medium. When each strain was cocultivated with P. aeruginosa strain WatG in liquid mineral salts medium (MSM) containing 1% diesel oil, isolate N enhanced the degradation of diesel oil by P. aeruginosa strain WatG, but isolate O inhibited it. In contrast, isolate J, which was tentatively identified as a Rhodococcus sp., degraded diesel oil contained not only in liquid LB and MSM, but also in paddy soil microcosms supplemented with LB medium. The bioaugmentation capacity of isolate J in soil microcosms contaminated with diesel oil was much higher than that of P. aeruginosa strain WatG. The possibility of using isolate J for autochthonous bioaugmentation is discussed.     

Effects of temperature and food quality on life-history parameters in Parameletus chelifer and P. minor (Ephemeroptera): a laboratory study

SUMMARY. 1. Growth rate of Parameletus minor was greatest between 10.8 and 19.8°C, survival rate peaked at 5.9°C, developmental time was shortest at 14.6°C, and adult size and fecundity reached maximum values between 5.9 and 10.8°C. Growth rate of P. chelifer was greatest between 14.6 and 19.8°C, survival rate peaked at 5.9°C, and developmental time was shortest at 14.6°C. A large adult size was found at 10.8°C, and highest fecundity between 10.8 and 14.6°C.
2. Food quality significantly affected growth rate, developmental time, adult size and fecundity of both species. Both P. chelifer and P minor attained highest growth rate, largest adult size and highest fecundity when the C/N ratio of food was 5.95. Developmental time was shortest at a C/N ratio between 5.95 and 12.8.
3. Nymphs of P. chelifer had a higher temperature 'optimum' for growth than nymphs of P. minor . Growth rate of nymphs of P. chelifer reared on detritus from a seasonal stream (C/N ratio 12.8) was about 3 times that of nymphs reared on detritus from a river margin (C/N ratio 20.9). The corresponding growth rate difference for nymphs of P. minor was only about 1.5.
4. When all life-history parameters are taken into consideration, P. chelifer had a higher temperature 'optimum' than P. minor .     

Thermolabile ribonucleases from antarctic psychrotrophic bacteria: detection of the enzyme in various bacteria and purification from Pseudomonas fluorescens

Abstract Thirteen terrestrial psychrotrophic bacteria from Antarctica were screened for the presence of a thermolabile ribonuclease (RNAase-HL). The enzyme was detected in three isolates of Pseudomonas fluorescens and one isolate of Pseudomonas syringae . It was purified from one P. fluorescens isolate and the molecular mass of the enzyme as determined by SDS-PAGE was 16 kDa. RNAase-HL exhibited optimum activity around 40°C at pH 7.4. It could hydrolyse Escherichia coli RNA and the synthetic substrates poly(A), poly(C), poly(U) and poly(A-U). Unlike the crude RNAase from mesophilic P. fluorescens and pure bovine pancreatic RNAase A which were active even at 65°C, RNAase-HL was totally and irreversibly inactivated at 65°C.     

Characterization of a novel thermophilic, cellulose-degrading bacterium Paenibacillus sp. strain B39

Aims:  The aims of this study were to identify and characterize the novel thermophilic, cellulose-degrading bacterium Paenibacillus sp. strain B39.
Methods and Results:  Strain B39 was closely related to Paenibacillus cookii in 16S rRNA gene sequence. Nonetheless, this isolate can be identified as a novel Paenibacillus sp. with respect to its physiological characteristics, biochemical reactions, and profiles of fatty acid compositions. A cellulase with both CMCase and avicelase activities was secreted from strain B39 and purified by ion-exchange chromatography. By sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis, the molecular weight of B39 cellulase was determined as 148 kDa, which was much higher than other cellulases currently reported from Paenibacillus species. The enzyme showed a maximum CMCase activity at 60°C and pH 6·5. Addition of 1 mmol l⁻¹ of Ca²⁺ markedly enhanced both CMCase and avicelase activities of the enzyme.
Conclusions:  We have identified and characterized a novel thermophilic Paenibacillus sp. strain B39 which produced a high-molecular weight cellulase with both CMCase and avicelase activities.
Significance and Impact of the Study:  Based on the ability to hydrolyse CMC and avicel, the cellulase produced by Paenibacillus sp. strain B39 would have potential applications in cellulose biodegradation.     

Isolation and characterization of plant growth-promoting strain Pantoea NII-186. From Western Ghat Forest soil, India

Aims:  To isolate plant growth-promoting bacterium from Western Ghat forests in India.
Methods and Results:  A Gram-negative, rod shaped, cream white coloured strain Pantoea NII-186 isolated from Western Ghat soil sample. The taxonomic position of the bacterium was confirmed by sequencing of 16S rRNA and phylogenetic analysis. A strain grew at a wide range of temperature ranging from 5–40°C, but optimum growth was observed at 28–30°C. It showed multiple plant growth-promoting attributes such as phosphate solubilization activity, indole acetic acid (IAA) production, siderophore production and HCN production. It was able to solubilize (28 μg of Ca₃PO₄ ml⁻¹ day⁻¹), and produce IAA (59 μg) at 28°C. The solubilization of insoluble phosphate was associates with a drop in the pH of the culture medium. Pantoea sp. NII-186 tolerate to different environmental stresses like 5–40°C, 0–7% salt concentration and 4–12 pH range.
Conclusions:  The 16S rRNA gene sequence confirmed that the isolate NII-186 was belongs to Pantoea genus and showed considerable differences in physiological properties with previously reported species of this genus. Isolate NII-186 possessed multiple attributes of plant growth-promoting activity.
Significance and Impact of the Study:  Hence in the context it is proposed that Pantoea sp. NII-186, could be deployed as an inoculant to attain the desired plant growth-promoting activity in agricultural environment.     

Hemicellulase activity of antarctic microfungi

The mannanase (endo-β-1,4-mannanase; E.C. 3.2.1.78) and xylanase (endo-β-1,4-xylanase; E.C. 3.2.1.8) activity of five microfungal isolates from Antarctica were characterized at different temperatures and pH. In general, the hemicellulase activity of the antarctic strains occurred at least 10 °C and as much as 30 °C lower than that of a mesophilic reference strain. At 0 °C, two strains, a Phoma and a Penicillium , produced in excess of 40% of their measured maximum activity of mannanase. All strains had maximum hemicellulase activity in the range pH 4–5, with Penicillium , Phoma and Alternaria strains exhibiting high (in excess of 80% of maximum) mannanase activity at pH 10. Three of the antarctic isolates exhibited high levels of xylanase activity over a pH range of 3–11.     

A thermotolerant and high acetic acid-producing bacterium Acetobacter sp. I14–2

A thermotolerant bacterium with high production of acetic acid was isolated from spoiled banana in Taiwan. The isolate, I14–2 ,was considered to be an Acetobacter sp. according to phenotypic and chemotaxonomic characteristics. Optimal cultural conditions for Acetobacter sp. I14–2 to produce acetic acid were studied under cultivation in a medium containing 2 mg l⁻¹ acetic acid and 5% ethanol at 30 °C. Acetic acid productivity by Acetobacter sp. I14–2 was almost two and three times the amount produced by Acet. aceti IFO3283 and Acetobacter sp. CCRC 12326, respectively. The isolate retained 22% residual acetic acid-producing activity after 3 d incubation in a medium containing 8% ethanol, and produced acetic acid in a medium containing 10 g l⁻¹ acetic acid. This bacterium is thermotolerant and retained 97% and 68% of acetic acid-producing activity after 3 d incubation at 35 °C and 37 °C, respectively, compared with that when incubated at 30 °C.     

Cold active β-galactosidase from Thalassospira sp. 3SC-21 to use in milk lactose hydrolysis: a novel source from deep waters of Bay-of-Bengal

The cold active β-galactosidase from psychrophilic bacteria accelerate the possibility of outperforming the current commercial β-galactosidase production from mesophilic sources. The present study is carried out to screen and isolate a cold active β-galactosidase producing bacterium from profound marine waters of Bay-of-Bengal and to optimize the factors for lactose hydrolysis in milk. Isolated bacterium 3SC-21 was characterized as marine psychrotolerant, halophile, gram negative, rod shaped strain producing an intracellular cold active β-galactosidase enzyme. Further, based upon the 16S rRNA gene sequence, bacterium 3SC-21 was identified as Thalassospira sp. The isolated strain Thalassospira sp. 3SC-21 had shown the enzyme activity between 4 and 20?°C at pH of 6.5 and the enzyme was completely inactivated at 45?°C. The statistical method, central composite rotatable design of response surface methodology was employed to optimize the hydrolysis of lactose and to reveal the interactions between various factors behind this hydrolysis. It was found that maximum of 80.18?% of lactose in 8?ml of raw milk was hydrolysed at pH of 6.5 at 20?°C in comparison to 40?% of lactose hydrolysis at 40?°C, suggesting that the cold active β-galactosidase from Thalassospira sp. 3SC-21would be best suited for manufacturing the lactose free dairy products at low temperature.     

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.     

Endo-(1 → 3)β-D-glucanase activity secreted by Bacillus sp. no. 215

The production of an extracellular endo-(1 → 3)-β-D-glucanase by Bacillus sp. no. 215 was induced during growth with (1 → 3)-β-D-glucan (curdlan) from Cellulomonas flavigena strain KU as carbon and energy source. Maximum levels of activity (0.26 U ml^-1 resp. 1.40 U mg^-1) were detected in cell-free culture supernatant fluid after 25 h of aerobic growth at 55°C. The cells secreted an endo-(1 → 3)-β-D-glucanase with low electrophoretic mobility that used curdlan from C. flavigena strain KU and from Agrobacterium sp. (formerly Alcaligenes faecalis var. myxogenes ) as substrates. The enzyme activity was highest at pH 7.0 and 55°C. It exhibited a remarkably low thermal stability with a half-life of 14 min at 55°C in the presence of substrate. Divalent metal cations were required for enzyme activity.     

Current Production by a Deep-Sea Strain <Emphasis Type="Italic">Shewanella</Emphasis> sp. DS1

Electricity-generating bacteria have attracted intensive studies in recent years as an important resource for energy generation. In this investigation, a bacterial isolate capable of producing electric current during fermentation processes was obtained from a deep-sea hydrothermal field in the Atlantic Ocean for the first time. The strain, assigned to Shewanella sp. DS1 based on its 16S-rRNA sequence analysis, grew at the optimum temperature of 30°C and optimum pH of 6.5. The results showed that the electric current generated by the strain increased during the first few hours and eventually reached the maximum (0.29 mA) at approximately 15 hours after inoculation. The electric current, however, decreased slowly as time increased. Thus, our study demonstrated that the deep sea promises to be a good reservoir for screening electricity-generating microbes.     

Remarkably low temperature optima for extracellular enzyme activity from Arctic bacteria and sea ice

Extracellular degradative enzymes released by psychrophilic marine bacteria (growing optimally at or below 15°C and maximally at 20°C) typically express activity optima at temperatures well above the upper growth limit of the producing strain. In the present study, we investigated whether or not near-zero Arctic environments contain extracellular enzymes with activity optimized to temperatures lower than previously reported. By applying fluorescently tagged substrate analogues to measure leucine-aminopeptidase and chitobiase activity, the occurrence of extracellular enzymatic activity (EEA) with remarkably low temperature optima (15°C) was documented in sea-ice samples. An extremely psychrophilic bacterial isolate, strain 34H, yielded an extract of cell-free protease with activity optimized at 20°C, the lowest optimum yet reported for cell-free EEA from a pure culture. The use of zymogram gels revealed the presence of three proteolytic bands (between 37 and 45 kDa) in the extract and the release of the greatest quantities of the proteases when the strain was grown at −1°C, suggesting a bacterial strategy for counteracting the effects of very cold temperatures on the catalytic efficiency of released enzymes. The detection of unusually cold-adapted EEA in environmental samples has ramifications not only to polar ecosystems and carbon cycling but also to protein evolution, biotechnology and bioremediation.     

Effect of water activity and temperature on growth and fumonisin B1 and B2 production by Fusarium proliferatum and F. moniliforme on maize grain

S. MARIN, V. SANCHIS, I. VINAS, R. CANELA AND N. MAGAN. 1995. The effect of different water activities ( a _w, 0.968, 0.956, 0.944, 0.925) and temperature (25°C and 30°C) on colonization and production of fumonisin B₁ (FB₁) and B₂ (FB₂) on sterile layers of maize by Fusarium proliferatum and F. moniliforme isolates was determined over periods of 6 weeks. Generally, both F. moniliforme and F. proliferatum grew faster with increasing a _w and best at 30°C. All three isolates produced more FB 1 than FB₂ regardless of a _w or temperature. Very little FB₁ and FB₂ were produced at 0.925 a _w, with maximum produced at 0.956 and 0.968 a _w at both temperatures tested. Most FB₁ and FB₂ were produced by F. moniliforme (25N), followed by F. proliferatum isolates (73N and 131N). At all a _w levels and both temperatures there was an increase in FB₁ and FB₂ concentration with time. Statistical analyses of a _w, temperature, time, two- and three-way interactions showed some significant differences between isolates and FB₁ and FB₂ production.     

Antimicrobial activity of food-related Penicillium sp. against pathogenic bacteria in laboratory media and a cheese model system

Food-related Penicillium species ( n ep fy1 = rs 34) and Geotrichum candidum ( n = 11) grown on Czapek Dox and brie agar were tested for their ability to suppress growth of pathogenic bacteria. Ten out of 13 P. camemberti showed antagonistic activity while the other species did not interact significantly with the bacterial growth. The order of inhibition was: Gram-negative bacteria and Bacillus cereus > Listeria monocytogenes , Lactococcus sp. > Micrococcus sp. whereas Lactobacillus sp., Staphylococcus aureus and some Micrococcus sp. were unaffected. When Salmonella typhimurium was inoculated together with P. camemberti P25 in brie agar, bacterial growth was inhibited during the first 6 d of incubation whereafter growth started. The inhibition of L. monocytogenes was similar but less pronounced. The antimicrobial activity produced by P. camemberti P25 and L84 was enhanced with increasing amount of sucrose in the medium. The activity was increased at low pH and destroyed at pH above 8. It was detectable at 15°C but not at 37°C indicating that volatile metabolites might be involved. No significant accumulation of organic acids and no secondary metabolites such as mycotoxins were detected. HSGC-MS analysis indicated that acetaldehyde, benzaldehyde, 3-methylbutanal and 1-octen-3-ol were produced by P. camemberti during the period when inhibitory activity was observed. Pure acetaldehyde and benzaldehyde were shown to be inhibitory to L. monocytogenes and Salm. typhimurium when grown at 15°C and pH 5·5 and 7·0.     

Isolation and characterization of nisin-producing Lactococcus lactis subsp. lactis from bean-sprouts

Bacterial isolates from bean-sprouts were screened for anti- Listeria monocytogenes bacteriocins using a well diffusion method. Thirty-four of 72 isolates inhibited the growth of L.monocytogenes Scott A. One, HPB 1688, which had the biggest inhibition zone against L.monocytogenes Scott A, was selected for subsequent analysis. Both ribotyping and DNAsequencing of 16S ribosomal RNA gene demonstrated that the isolate was Lactococcus lactis subsp. lactis . Polymerase chain reaction and nucleotide sequencing revealed that thegenomic DNA of the bean-sprout isolates contained a nisin Z structural gene. In MRS broth,bean-sprout isolate HPB 1688 survived at 3–4·5°C for at least 20 d, grew at 4°Cand produced anti-listerial compoundsat 5°C. When co-cultured with L. monocytogenes in MRS broth, the isolate inhibited thegrowth of L. monocytogenes at 4°C after 14d and at 10°C after 2 d. When co-inoculatedwith 10²cells g⁻¹ of L.monocytogenes on fresh-cut ready-to-eat Caesar salad, L. lactis subsp. lactis (10⁸cells g⁻¹) was able to reduce the number of L. monocytogenes by 1–1·4 logs after storage for 10 d at 7° and 10°C. A bacteriocin-producing Enterococcusfaecium was also able to reduce the numbers of L. monocytogenes onCaesar salad, butdid not act synergistically when co-inoculated with L. lactis subsp. lactis .