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.     

Effect of Iysozyme concentration, heating at 90°C, and then incubation at chilled temperatures on growth from spores of non-proteolytic Clostridium botulinum

The heat treatment necessary to inactivate spores of non-proteolytic Clostridium botulinum in refrigerated, processed foods may be influenced by the occurrence of lysozyme in these foods. Spores of six strains of non-proteolytic Cl. botulinum were inoculated into tubes of an anaerobic meat medium, to give 10⁶ spores per tube. Hen egg white lysozyme (0–50 μg ml^-1) was added, and the tubes were given a heat treatment equivalent to 19·8 min at 90°C, cooled, and incubated at 8°, 12°, 16° and 25°C for up to 93 d. In the absence of added lysozyme, neither growth nor toxin formation were observed. A 6–D inactivation was therefore achieved. In tubes to which lysozyme (5–50 μg ml^-1) had been added prior to heating, growth and toxin formation were observed. With lysozyme added at 50 μg ml^-1, growth was first observed after 68 d at 8°C, 31 d at 12°C, 24 d at 16°C, and 9 d at 25°C. Thus, in these circumstances, a heat treatment equivalent to 19·8 min at 90°C was not sufficient, on its own, to give a 6–D inactivation. A combination of the heat treatment, maintenance at less than 12°C, and a shelf-life not more than 4 weeks reduced the risk of growth of non-proteolytic Cl. botulinum by a factor of 10⁶.     

Interaction between isolation source, cellular fatty acid composition and stress tolerance in Saccharomyces cerevisiae and its subspecies

The physiological properties and fatty acid content of 59 strains of Saccharomyces cerevisiae isolated from soft-drink factories, a fruit puree factory, a fuel-alcohol distillery and a winery were compared. Discriminant analysis of the results allocated the strains to four groups according to their source. Resistance to preservatives and temperature stress were correlated with differences in fatty acid composition. The fatty acid C18: 1Δ11, growth at pH 2 and in the presence of 200–600 mg 1^-1 benzoate or sorbate, and maximal growth rate at 42°C were characteristics associated with yeasts from particular environments. However, tolerance of thermal stress and content of the C18: 2 fatty acid were associated with subspecies: the former species S. capensis, S. chevalieri , etc. The relative content of C10 : 0, C12 : 0 and C18 : 0 acids varied according to both isolation source and subspecies.     

Thermoregulation and metabolism in the Cape golden mole (Insectivora: Chrysochloris asiatica)

The Cape golden mole, Chrysochloris asiatica is an insectivore which excavates superficial foraging burrows as it searches for its food. It has a mean (±S.D.) resting metabolic rate (RMR) when newly captured of 1–17±0.17 cm³ O₂g^-1 h^-1 ( n = 14), within the thermoneutral zone (TNZ) of 30–32°C.
The body temperature (Tb) of the mole in the TNZ is low 32.9 ± 0.36 ( n = 14) and remains stable at ambient temperatures (Tas) from 28–32°C. Above 32°C (range 34–37°C), Tb increases albeit slightly to 36 ± 1.75°C ( n = 14). The conductance is high 0.27 ± 006cm³ O₂g^-1 h^-l°C^-1 ( n = 46) at the lower limit of thermoneutrality. The mean RMR at 9°C (the lowest Ta tested) was 4.82±11 cm³ O₂g^-1h^-1, which is 4.1 times that of the RMR in the TNZ.
At an ambient temperature of 9°C, three of the golden moles entered a state of torpor where the RMR was reduced from 5.9±0.56 to 10 1.0 ± 0.69cm³O₂g^-1h^-1.     

Cellulolytic activity of Actinomycetes isolated from termites (Termitidae) gut

Abstract Cellulolytic actinomycetes were isolated from the hindgut of four different termites: Macrotermes, Armitermes, Odontotermes and Microcerotermes spp.
The isolated actinomycetes ( Streptomyces sp. and Micromonospora sp.) were grown on cellulosic substrates and their extracellular cellulase (C_l, C_x and cellobiase) activity evaluated; using filter paper as a substrate for C_l, carboxymethylcellulose (CMC) for C_x and d -cellobiose for cellobiase, all strains were shown to degrade soluble and insoluble cellulose; optimum pH for growth was 6.2–6.7 at 28°C; three strains could grow at 48°C on cellulosic substrates.
Some strains exhibited high cellulase activity, constant for 5–7 days, but inhibition by glucose was a common feature for almost all isolates.     

The Isolation and Some Characteristics of Photosynthetic Bacteria (Chromatiaceae and Chlorobiaceae) from Antarctic Marine Sediments

Sediment samples taken at depths from 5 to 50 m in Borge Bay, Signy Island, Antarctica yielded purple ( Chromatiaceae ) and green ( Chlorobiaceae ) phototrophic bacteria when inoculated into Winogradsky columns or directly into Pfennig's medium. Pure cultures of Thiocapsa roseopersicina (2 strains), Thiocapsa pfennigii, Chromatium gracile, Chromatium vinosum, Thiocystis violaceae, Chlorobium limicola, Chlorobium vibrioforme and Prostaecochloris aestuarii were subsequently isolated from these enrichments. Thiocapsa roseopersicina and Chromatium vinosum were the species isolated most commonly. None of the isolates showed evidence of temperature adaptation; optimum growth was achieved between 25–30°C but all grew readily at low white light levels (spectral irradiance 160 μW/cm²) with mean generation times ranging from 8·9 to 14·2 h at 23°C depending upon the isolate. The green phototrophs grew readily in blue light (440–510 nm) of low irradiance (126 μW/cm²) which penetrates to a depth of 40–50 m in coastal waters surrounding Signy Island. Under these conditions Chlorobium limicola and Chlorobium vibrioforme had mean generation times of 22 and 12·2 h respectively compared with 10·3 and 8·9 when grown in white light of similar total irradiance. None of the purple phototrophs grew under blue light but they grew under yellow light (peak transmission 590 nm, irradiance 137 μW/cm²). Chromatium gracile and Thiocapsa roseopersicina had mean generation times of 22·3 and 18·3 h respectively compared with 14·2 and 12·3 h in white light of similar total irradiance.     

Influence of water temperature and feeding regime on otolith growth in Anguilla japonica glass eels and elvers: does otolith growth cease at low temperatures?

The influences of water temperature and feeding regime on otolith growth in Anguilla japonica glass eels and elvers were investigated using individuals reared at 5, 10, 15, 20, 25 and 30° C and in fed or unfed conditions at salinity 32 after their otoliths were marked with alizarin complexone (ALC). To eliminate the difficulty of observing the edges of otoliths with optical (OM) or scanning electron (SEM) microscopes, three to 10 individuals were sampled from each tank at 10, 20 and 30 days during the experiment and reared for an additional 10 days at 25° C after their otoliths were marked a second time. Otolith growth and the number of increments were measured using both OM and SEM. Most A. japonica commenced feeding after 10 days at 20–30° C or after 20 days at 15° C, but no feeding occurred at 5 and 10° C. No otolith growth occurred at 5 and 10° C except in two individuals with minimal increment deposition at 10° C. Otolith growth was proportional to water temperature within 15–25° C and not different between 25 and 30° C. At 15, 25 and 30° C, the mean otolith growth rate in fed conditions was higher than in unfed conditions. The number of increments per day was significantly different among water temperatures (0·00–0·01 day⁻¹ at 5 and 10° C, 0·43–0·48 day⁻¹ at 15° C and 0·94–1·07 day⁻¹ at 20–30° C). These results indicated that otolith growth in A. japonica glass eels and elvers was affected by temperature and ceased at ≤10° C under experimental conditions. Hence, future studies analysing the otoliths of wild-caught A. japonica glass eels and elvers need to carefully consider the water temperatures potentially experienced by the juveniles in the wild.     

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.     

Reappraisal of the effect of temperature on the growth kinetics of Aeromonas salmonicida

The effect of temperature (1–34 °C ) on the maximum specific growth rate of Aeromonas salmonicida could not be described by the classical growth models ; for some strains, two optimal temperatures at 23 °C and 30 °C were observed, as well as an unexpected increase in the pseudolag time above 27 °C. This could be explained by the presence of two subsets, notably S-layer⁺ and S-layer⁻ sub-populations. The A⁻ cells had higher growth parameters (T_opt and μ_opt) than the A⁺ cells and were selected by subcultures above 30 °C. Yet the relative proportion of A⁺ cells did not explain all the variation of μ_max versus temperature, and the growth kinetics of an Aer. salmonicida isolate remained unpredictable.     

Antibacterial effect of protamine assayed by impedimetry

Impedimetric measurements were used to assay the antibacterial effect of protamine. A good linear correlation between the impedance detection time and the initial cell counts was obtained ( r = 0.99, n = 2). As basic peptides may cause clumping of cells, this correlation curve was used when estimating the cell number after protamine treatment, rather than colony counts.
Protamine from salmon killed growing Gram-positive bacteria and significantly inhibited growth of Gram-negative bacteria in Tryptone Soy Broth (TSB) at 25°C. In general Gram-positive bacteria were more sensitive to protamine than Gram-negative bacteria; the minimum inhibitory concentrations (MIC) determined for Gram-positive strains varied from 20 to 1000 μ ml^-1 and for Gram-negative strains from 500 μ ml^-1 to more than 4000 μ ml^-1.
The effect of protamine on non-growing Listeria monocytogenes Scott A suspended in buffer was not lethal as was the effect on growing cells; however, protamine (50–500 μg ml^-1) killed the Gram-negative fish spoilage bacteria Shewanella putrefaciens when the live cells were suspended in buffer.     

Recognition of pathogenic Yersinia enterocolitica by crystal violet binding and polymerase chain reaction

Cefsulodin-Irgasan-Novobiocin (CIN) agar is used for the selective isolation and enumeration of Yersinia enterocolitica from clinical specimens and food. The medium contains crystal violet and about 1 mmol l^-1 calcium and can be used for the phenotypic characterization of strains that carry a virulence plasmid. At 32°C, irrespective of pathogenicity, colonies are translucent with a pale pink centre surrounded by a transparent border ('bullseye'), while at 37°C pathogenic strains grow as calcium-dependent microcolonies which, because of crystal violet binding, are intensely coloured. These results were confirmed by the polymerase chain reaction with primers directed at the vir F gene, which is present only in pathogenic strains of Y. enterocolitica. Pathogenic strains of Y. enterocolitica can be recognized by growth at 37°C on Yersinia selective agar.     

Influence of growth temperature on the production of extracellular virulence factors and pathogenicity of environmental and human strains of Aeromonas hydrophila

The biochemical properties, virulence for mice and trout, and the extracellular virulence factors at 28° and 37°C of 11 environmental and nine human strains of Aeromonas hydrophila were compared. All the environmental isolates and four of the human group were virulent for trout at 3 x 10⁷ cfu, but only human strains were able to cause death or lesions in mice by the intramuscular route. Extracellular virulence factors such as haemolysins, cytotoxins and proteases were also investigated in supernatant fluids of cultures grown at 28°C and 37°C. The production of haemolysins, caseinases, elastases and growth yields of environmental strains decreased sharply during cultivation at 37°C but cytotoxins were produced to the same extent, or slightly less, than at 28°C. The human strains differed from the environmental strains in response to growth temperatures: protease activity decreased at 37°C, although growth yield was not affected, but more haemolysins and cytotoxins were produced by the virulent strains at this temperature than at 28°C. Sodium caseinate SDS-PAGE of culture supernatant fluids of selected human strains revealed that temperature selectively inhibited the production of certain proteases.     

The interaction of temperature and fish size on growth of juvenile halibut

Growth rate of individually tagged juvenile halibut was influenced significantly by the interaction of temperature and fish size. The results suggest an optimum temperature for growth of juvenile halibut in the size range 5–70 g between 12 and 15° C. Overall growth rate was highest at 13° C (1·62% day ⁻¹). At c. 5 g at the beginning of the experiment, fish at 16° C had the highest growth rate (3·2% day ⁻¹), but reduced this rate as they grew bigger. At 9 and 11°p C, growth rates were equal or only slightly lower during the later stages of the experiment, while the fish at 6° C showed significantly lower overall growth rate (0·87% day⁻¹). Optimal temperature for growth decreased rapidly with increasing size, indicating an ontogenetic reduction in optimum temperature for growth. Moreover, a more flattened parabolic regression curve between growth and temperature as size increased indicated reduced temperature dependence with size. Although individual growth rates varied significantly at all times within the experimental temperatures, significant size rank correlations were maintained during the experiment. This indicated an early establishment of a stable size hierarchy within the fish groups. Haematocrit was highest at the highest temperature while Na⁺/K⁺-ATPase activity was inversely related to temperature. There was no difference in plasma Na⁺, Cl⁻ and K⁺ concentrations among the temperature groups.     

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 .     

Major outer membrane proteins of Prochlorothrix hollandica (Prochloraceae)

Abstract Rhizobium sp. isolated from Lablab purpureus utilized catechol as sole carbon and energy source, a property which is plasmid encoded. The heat curable (39–41°C) plasmid, designated as pAMG1, was transferred to cat⁻ strains of Rhizobium sp. with a transfer frequency of 2.6 × 10⁻⁶ transconjugants/donor cell.     

Purification and characterization of the major β-1,4-endoglucanase from Thermomonospora curvata

The major β-1,4-endoglucanase (EG) of the thermophilic actinomycete, Thermomonospora curvata , contributed over 80% of the total EG activity recovered from cell-free culture fluid after growth on cellulose. The enzyme was purified to electrophoretic homogeneity by ammonium sulphate precipitation, ion-exchange chromatography and size exclusion HPLC. This monomeric enzyme had a specific activity of 750 IU mg⁻¹ when assayed with 2.5% (w/v) carboxymethyl cellulose (CMC) at 70°C, pH 6.0. Highest activity was observed on CMC with a degree of polymerization of 3200. The EG was stable for 48 h at 60°C, pH 6.0 and had a half-life of 30 min at 80°C; temperature and pH optima were 70–73°C and 6.0–6.5, respectively. The mol. wt was 100000 and the pI was 4.0. The K _m and V _max values were 7.33 mg ml⁻¹ and 833 μmol min⁻¹, respectively. EG activity was inhibited by Fe^{2 +}, Hg^{2 +}, Ag⁺ and Pb^{2 +}, and enhanced by dithiothreitol and Zn^{2 +}. The first 12 amino acid residues at the N -terminus were: Asp-Glu-Val-Asp-Glu-Ile-Arg-Asn-Gly-Asp-Phe-Ser. Glutamic and aspartic acid constituted 24% of the total amino acid composition; no amino sugar was found.     

Inhibition and inactivation of pathogenic serogroups of Yersinia enterocolitica by a bacteriocin produced by Yersinia kristensenii

Growth of Yersinia enterocolitica strains representing serogroups O: 3, O: 5, 27, O:6, 30, O:8, O:9 (human isolates) and O:6, 31 (food isolate) were inhibited in the presence of a bacteriocin produced by Yersinia kristensenii at high initial cell count of 10⁶ ml^-1. Complete (100%) inactivation of most Y. enterocolitica cells of different serotypes was observed within 24 h at low initial cell counts of 10⁴ ml^-1. Complete injury of the cells was observed within 4–8 h, with all the serotypes at 10°C and 28°C. The degree of susceptibility to the injury and the recovery of cells from the injury varied from serogroup to serogroup.     

Energy and ration requirements of juvenile Pacific halibut (Hippoglossus stenolepis) based on energy consumption and growth rates

Growth of captive juvenile Pacific halibut was linearly related to energy consumption (J g⁻¹ day⁻¹) at 4°C by the following equation: growth (% body weight (b.w.) day⁻¹)=0–007 (consumption J g⁻¹ day⁻¹)– 0.192; r² =0.81. Weight gain was independent of size for fish between 9 and 7000 g when growth was expressed as a function of consumption in J g⁻¹ day⁻¹. Maintenance ration determined in feeding–growth experiments averaged 27.4 J g⁻¹ day⁻¹ at 4–0°C. Small halibut ate significantly more food than large fish. Single meals following 2 day fasts averaged 4.1% b.w. for halibut under 100 g, 1.72% b.w. for 1.2 kg fish and 1.1% B.W. for 6.8 kg fish. Both large and small size categories of halibut tended to evacuate their meal in about 3 days even though small fish ate relatively larger meals. Minimum estimates for daily ration to achieve growth rates observed in the Gulf of Alaska were approximately 0.5 to 2.4% b.w. day⁻¹ depending on fish size and whether northern shrimp or yellowfin sole were their prey.     

Respiration and energetics of the bumblebee Bombus terrestris queen

Carbon dioxide production by the Bombus terrestris queen was measured at different temperatures (10–30°C) and during different activities of the bumblebee. During flight the CO₂ production averaged 50 ml g⁻¹ (fresh weight) h⁻¹ and was only slightly affected by temperature. During rest (with a readiness to fly) and incubation the respiration rate clearly increased with decreasing temperature (5–40 and 13–56 ml g⁻¹ h⁻¹, respectively), whilst during torpor it increased with temperature (0.1–1.7 ml g⁻¹ h⁻¹ at temperatures from 10 to 30°C).
The expenditure of energy as calculated from the continuous respiration measurements agreed well with the amount of energy obtained from food (discrepancy 6–19%). The energy budget of an incubating queen was correctly predicted using the measured respiratory functions, prevailing temperatures, and the behaviour of the queen. The number of flower visits needed to fulfil the daily energy requirements of an incubating queen is discussed.     

C4 photosynthesis in Cyperus longus L., a species occurring in temperate climates

Abstract. Cyperus longus L. , which has a widespread but disjunct distribution throughout Europe and extends northwards into Britain, was found to be a C₄ species based upon its Kranz leaf anatomy, low CO₂ compensation point and the labelling of malate as an early product of ¹⁴CO₂ fixation. The photosynthetic characteristics of C. longus are similar to many other C₄ species with a high maximum rate of photosynthesis (> 1.5 mg CO₂ m ⁻² s ⁻¹) and a relatively high temperature optimum (30–35°C), but unlike many C₄ species the rate of photosynthesis does not decline rapidly below the optimum temperature and a substantial rate (0.6 mgCO₂ m⁻²s⁻¹)occursat 15°C. Leaf extension is very slow at 15°C and shows a curvilinear response to temperatures between 15 and 25°C. Leaves extend at a rate of almost 4 cm d⁻¹ at 25°C.