Effect of physiological conditions on the autolysis ofStaphylococcus aureus strains

The effect of physiological conditions on autolysis and autolytic activity in various strains ofStaphylococcus aureus was determined. The rate of whole cell autolysis ofS. aureus was growth phase dependent and a maximum rate was observed in early stationary phase cultures. However, the autolysins extracted by the freeze-thaw method (cell-wall bound autolytic activity) did not show any significant increase in activity. The addition of NaCl to the growth medium enhanced the rate of autolysis with the highest rate being displayed by cultures grown in 1.5 M NaCl. However, lower autolytic activity was found in the freeze-thaw extracts of cultures grown at higher concentrations of NaCl. The rate of autolysis of cultures grown at 30°C was higher than cultures grown at 37 or 43°C. Thus, the rate of autolysis seems to be independent of the bacterial growth rate. Cultures grown in slightly acidic conditions showed a faster rate of autolysis compared to cultures grown under alkaline conditions. SDS-polyacrylamide gel containing 0.2% crude cell-wall ofS. aureus did not show any obvious correlation with the appearance of any particular lytic band in the zymogram to autolytic activity or rate of autolysis of cultures grown under various environmental conditions. A nonhemolytic phenotype, mutations in the accessory gene regulator, and lysogeny (phages ø11, ø12, ø13) had no obvious effect either on the rate of autolysis or on the pattern of lytic bands in the zymograms.     

Phenotypic changes in the chemistry of Aspergillus nidulans: Influence of culture conditions on mycelial composition

A quantitative study was made of macromolecular (nucleic acids, protein), carbohydrate and mineral (magnesium, potassium and phosphorus) components of Aspergillus nidulans in glucose limited chemostat cultures, under varying conditions of dilution rate, temperature, pH and NaCl concentration.The overall mineral content showed greatest variation in response to changes in culture salinity, which also affected the mycelial carbohydrate content. Concomitant and opposite changes in the conent of cations and carbohydrates under conditions of increasing salinity may be interpreted in terms of mycelial osmoregulation. Slight variations in DNA content but gross fluctuations in the level of RNA were noted under the different cultural conditions examined. Co-ordinate changes in RNA and Mg²⁺ contents were evident only under certain conditions: dilution rate from 0.05–0.07 h^-1 or temperature from 22–30° C. The constant molar stoichiometry between RNA and Mg²⁺ characteristic of unicellular microorganisms was not a feature of fungal growth. The protein content was most affected by shifts of temperature and reached minimal values at 25 and 50° C.The growth environment had a marked influence on the protein synthesising activity of RNA, which increased eightfold as the dilution rate was increased from 0.02–0.175 h^-1, doubled within the temperature range 20–30° C and fell by 50% between 40 and 50° C. These observations are discussed in the context of the constant ribosomal efficiency in protein synthesis hypothesis.     

Life history and growth in culture of the endemic New Zealand kelp <Emphasis Type="Italic">Lessonia variegata</Emphasis> J. Agardh in response to differing regimes of temperature,photoperiod and light

Lessonia variegata J. Agardh (Laminariales, Phaeophyta) is endemic to New Zealand, where it occurs in subtidal kelp forests on wave exposed coasts in the North, South and Stewart Islands. This is the first account of the growth in culture and life history of L. variegata. Microscopic gametophytes alternate with macroscopic sporophytes, characteristic of members of the order Laminariales. The life history was completed in culture within 14 days under growth conditions of 12 °C, 12:12 (L:D) and 15 °C, 15:9 (L:D). Maximum growth of sporophytes occurred at 15 °C, 15:9, and slowest growth at 10 °C, 9:15. Under low light conditions (8–9 μmol photon m⁻² s⁻¹) filamentous growth of gametophytes predominated, and both the proportion of gametogenesis and the growth of sporophytes after 30 days was much reduced from equivalent cultures grown under conditions of higher light (16–17.5 μmol photon m⁻² s⁻¹). Interest in this species relates both to its potential for commercial utilisation as well as in the role it plays in coastal rocky reef ecosystems.     

Enhanced production and characterization of a highly thermostable alkaline protease from Bacillus sp. P-2

An obligatory alkalophilic Bacillus sp. P-2, which produced a thermostable alkaline protease was isolated by selective screening from water samples. Protease production at 30 °C in static conditions was highest (66 U/ml) when glucose (1% w/v) was used with combination of yeast extract and peptone (0.25% w/v, each), in the basal medium. Protease production by Bacillus sp. P-2 was suppressed up to 90% when inorganic nitrogen sources were supplemented in the production medium. Among the various agro-byproducts used in different growth systems (solid state, submerged fermentation and biphasic system), wheat bran was found to be the best in terms of maximum enhancement of protease yield as compared to rice bran and sunflower seed cake. The protease was optimally active at pH 9.6, retaining more than 80% of its activity in the pH range of 7–10. The optimum temperature for maximum protease activity was 90 °C. The enzyme was stable at 90 °C for more than 1h and retained 95 and 37% of its activity at 99 °C and 121 °C, respectively, after 1 h. The half-life of protease at 121 °C was 47 min.     

The effect of temperature on growth and cellulase (β-1,4-endoglucanase) production in the compost fungusChaetomium thermophile var.dissitum

Chaetomium thermophile var.dissitum, isolated from an experimental urban refuse compost, had the following growth characteristics: Minimum temperature, 27±1°C; optimum, 45–50°C; maximum, 57±1°C; pH optimum 5.5–6.0.A number of carbohydrates could be used for growth, but cellulase formation measured with carboxymethylcellulose as substrate was initiated only on cellulose or xylan. With cellulose as the carbon source, cellulase accumulation in the culture filtrate followed closely that of growth, when the temperature was varied. pH optimum for the cellulase system was 5.0.The optimum temperature for cellulase activity with carboxymethylcellulose as substrate varied between 77°C with 1/2 h incubation time and 58°C with 10 h incubation time.With cotton as substrate, the optimum temperature was 58°C regardless of incubation time. Carboxymethylcellulose had a higher stabilizing effect on the enzyme than cotton. The temperature stability of the cellulase was highest at pH 6.0.     

A thermostable lipase produced by a newly isolated <Emphasis Type="Italic">Geotrichum</Emphasis>-like strain,R59

Growth and production of lipase by a new Geotrichum-like strain, R59, were studied. Production of extracellular lipase was substantially enhanced when the initial pH of the culture medium, types of carbon and nitrogen sources, substances probably stimulating the lipase biosynthesis, the temperature, and time of growth were optimized. Sucrose and triolein were the most effective carbon sources for lipase production. Maximum lipase activity (146 U/ml^–1) was obtained with urea as the nitrogen source. Growth at 30°C, an initial pH of 6.0 and incubation time of 48 h were found as optimum conditions for cell growth and production of lipase by Geotrichum-like strain R59. The enzyme was thermostable and exhibited very high activity after 1 h incubation at 60°C.     

Upper temperature limits for growth and diazotrophy in the thermophilic cyanobacterium HTF Chlorogloeopsis

The effect of temperature and oxygen on diazotrophic growth of the thermophilic cyanobacterium HTF (High Temperature Form) Chlorogloeopsis was investigated using cells grown in light-limited continuous culture at a dilution rate of 0.02 h^-1. Diazotrophy was more sensitive to elevated temperatures than growth with combined nitrogen. The maximum temperature for growth of cultures gassed with CO₂-enriched air was more than 55 °C but less than 60 °C with N₂ as the sole nitrogen source, but between 60°C and 65°C when nitrate was present in the medium. The effect of temperature on nitrogenase activity, photosynthesis and respiration in the dark was determined using cells grown at 55°C. Maximal rates of all three processes were observed at 55°C and rates at 60°C during shortterm incubations were not less than 75% of the maximum. However, nitrogenase activity at 60°C was unstable and decayed at a rate of 2.2 h^-1 under air and at 0.3 h^-1 under argon. Photosynthesis and respiration were more stable at 60°C than anoxic nitrogen fixation. The upper temperature limits for diazotrophic growth thus seem to be set by the stability of nitrogenase.Abbreviations chl chlorophyll a - DCMU N-(3,4-dichlorophenyl) N,N-dimethylurea - Taps N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid     

Cylindrospermopsin production and release by the potentially invasive cyanobacterium Aphanizomenon ovalisporum under temperature and light gradients

The growth rates, production and release of the potent cytotoxin cylindrospermopsin (CYN) were studied in batch and semi-continuous cultures of Aphanizomenon ovalisporum (Cyanobacteria; Nostocaceae) strains UAM 289 and UAM 290 from Spain, over a gradient of temperatures (10–40 °C) and irradiances (15–340 μE m⁻² s⁻¹). This species grew in temperatures ranging from 15 °C to 35 °C as well as under all irradiances assayed. The growth rates ranged from 0.08 d⁻¹ to 0.35 d⁻¹, and the maximum growth was recorded above 30 °C and at 60 μE m⁻² s⁻¹. CYN was produced under all conditions where net growth occurred. Total CYN reached up to 6.4 μg mg⁻¹ dry weight, 2.4 μg mm⁻³ biovolume, 190.6 fg cell⁻¹ and 0.5 μg μg⁻¹ chlorophyll a. Although CYN concentrations varied only 1.9-fold within the 15–30 °C range, a drastic 25-fold decrease was observed at 35 °C. The irradiance induced up to 4-fold variations, with maximum total CYN measured at 60 μE m⁻² s⁻¹. An elevated extracellular CYN share ranging from 20% to 35% was observed during the exponential growth phase in most experiments, with extreme temperatures (15 and 35 °C) being related to the highest release (63% and 58%, respectively) and without remarkable influence of irradiance. Growth did not have a direct influence on either CYN production or release throughout the entire range of experimental conditions. Our study demonstrates a strong and stable production and release of CYN by A. ovalisporum along field-realistic gradients of temperature and light, thus becoming a predictive tool useful for the management of water bodies potentially affected by this ecologically plastic cyanobacterium.     

Extracellular lipolytic activity in Phoma glomerata

Several properties of the lipolytic activity exhibited by the conidial fungus Phoma glomerata were studied. Lipolytic activity in an aqueous buffer medium was measured on triacylglycerol, phosphoglyceride and cholesterol ester under different experimental conditions. The effect of storage temperature on the stability of the hydrolytic activity, and optimal conditions of temperature and time of maximal activity were determined. The optimal conditions for maximal lipolytic activity were found to be 40–50 °C and 1 h. The activity released to the medium by 1 mg cells for 1 h at 40 °C was stated as the enzyme released unit (ERU). The protein fraction of MW > 50 kDa obtained by ultrafiltration of the medium, was active on the three substrates assayed, and it showed a non-specific hydrolytic activity on both the 1- and 2-acyl esters either in the neutral glyceride or in the phosphoglyceride. A protein of M _r approx. 75 kDa was the only one that showed esterase activity. The crude medium, stored at –15 °C, maintained its initial hydrolytic activity on triacylglycerol for at least 42 days, though when it was kept for 10 days at 4 °C, the activity fell to 50%. Kinetic parameters using substrates such as triolein (TO), dipalmitoyl phosphatidylcholine (DPPC) and cholesteryl oleate (ChoO), were comparatively evaluated. The activity of the enzyme in the hydrolysis of TO showed the highest values, whereas the maximal specific activities were less when the enzyme was assayed against DPPC and ChoO.     

Influence of root temperature on growth of Pinus sylvestris,Fagus sylvatica,Tilia cordata and Quercus robur

One-year-old tree seedlings were incubated in a greenhouse from April to July, under natural daylight conditions, with their root systems at constant temperatures of 5, 10, 15, 20, 25, 30 and 35 °C and with the above ground parts kept at a constant air temperature of 18–20 °C. The course of height growth, total mass increment, root, shoot and leaf weight as well as leaf areas were measured. The results indicate that clear differences exist in the optimal root zone temperatures for various growth parameters in different tree species. Pinus sylvestris had a maximal height increment at about 5–10 °C and maximal total mass increment at 15 °C root temperature. In contrast, the optimum for Quercus robur was at 25 °C. Tilia cordata and Fagus sylvatica had their optima for most growth parameters at 20 °C. The root temperature apparently indirectly influenced photosynthesis (dry weight accumulation) and respiration loss. From the observed symptoms and indications in the literature it seems probable that a change in hormone levels is involved as the main factor in the described effects. Variation of root temperature had only an insignificant effect on bud burst and the time at which the shoots sprouted. Apparently species of northern origin seem to have lower root temperature optima than those of more southern origin. This is to be verified by investigation of other tree species.     

Fruit-body production of an ectomycorrhizal fungus in genus <Emphasis Type="Italic">Boletus</Emphasis> in pure culture

Mycelial growth and fruit-body production of an ectomycorrhizal Boletus sp. were examined in pure culture. Mycelia of the strain Bo1 grew well on a medium consisting of sawdust and barley grains. Mature fruit bodies bearing basidiospores were produced after incubation at 22°C for 90 days in the dark, followed by incubation at 26°C for 30–46 days under conditions of high humidity and illumination. The addition of porous stone as a casing on the medium increased fruit-body yield. Deposited spores germinated well on an agar medium and formed mycelial colonies, thus completing the life cycle of Bo1 without a host plant and under axenic conditions. The ability of Bo1 to form ectomycorrhizas was confirmed by axenic resynthesis of mycorrhizas on Quercus serrata. Cultured fruit bodies of Bo1 resembled Gyroporus castaneus and Boletus subcinnamomeus, but its taxonomic position was not elucidated at the species level.     

Temperature control of nitrogen fixation in Klebsiella pneumoniae

At growth temperatures above 37°C, Klebsiella pneumoniae does not grow in a medium containing N₂ or NO ₃ ^- as nitrogen sources. However, both the growth in the presence of other nitrogen sources as well as the in vitro nitrogenase activity are not affected at this temperature. The inability to fix N₂ at high temperature is due to the failure of the cells to synthesize nitrogenase and other nitrogen fixation (nif) gene encoded proteins. When cells grown under nitrogen fixing conditions at 30°C were shifted to 39°C, there was a rapid decrease of the rate of de novo biosynthesis of nitrogenase (component 1), nitrogenase reductase (component 2), and the nifJ gene product. There was no degradation of nitrogenase at the elevated temperature since preformed enzyme remained stable over a period of at least 3 h at 39°C. Thus, temperature seems to represent a third control system, besides NH ₄ ⁺ and O₂, governing the expression of nif genes of K. pneumoniae.     

Control of early seedling growth in varietal lines of hexaploid wheat (Triticum aestivum), durum wheat (Triticum durum), and barley (Hordeum vulgare) in response to the phthalimide growth regulant,AC 94,377

AC 94,377 caused elongation of seedlings of Triticum aestivum, Triticum durum, and Hordeum vulgare when applied to the soil, or the soil plus seed at planting. Affected were the leaf sheathes and the coleoptiles, and at high compound rates there was premature elongation of the stem internodes. As exemplified by the response of T. aestivum var. Fidel, the influence on coleoptile elongation was greatest under conditions whereby the coleoptile was naturally stimulated to elongate, i.e., when growth was in the dark and temperatures were cool (15°C). All of the stem internodes were capable of elongation except the one below the coleoptile node. The effect on leaf sheath elongation was prolonged when compared to activity of gibberellic acid.Several varieties of the three cereal species were examined in the greenhouse for sensitivity to AC 94,377 in order to evaluate the extent of the response. All of the barley varieties examined were sensitive to AC 94,377, elongating regardless of the planting conditions. Two such conditions were established, including incubation under warm (28/20°C) conditions following planting the grains 1 cm deep, and incubation under cool (22/16°C) conditions following planting the grains 6 cm deep. Wheat varieties distributed into two general categories, those which were sensitive and those which were not. The insensitivity correlated well to the presence of the reduced height (Rht) and GA-insensitive (Gai) genes in Triticum aestivum and Triticum durum, respectively. Thus, AC 94,377 can be used conveniently to evaluate varietal lines for the presence of this phenotype. This correlation also lends support to the notion that the Rht/Gai mutations in wheat are either at the level of a gibberellin receptor or at a step in the signal transduction pathway.     

Effect of temperature on the growth rate of Griffithsia tenuis C. Agardh (rhodophyta: ceramiales)

Clonal cultures of Griffithsia tenuis were grown for 18 days (Erdschreiber solution, LD 12 : 12, 2200 lux) at 13, 18, 22 and 25°C. The optimum temperature for growth (increase in number of cells) under these conditions was 22°C.

     

Production of a cellulase-free xylanase from agricultural waste materials by a thermotolerant Streptomyces sp.

1444 microorganisms were isolated from soil samples from the northern Thai and screened at 55 °C by using basal medium supplemented with 1% carboxymethyl cellulose as a sole carbon source. One isolate, Streptomyces Ab106, had a high activity of a cellulase-free xylanase also without mannanase activity. The maximum cellulase-free xylanase activities of 3.5, 3.3, 3.1 and 2.7 IU were after growth of the organism with 1% (w/v) corn hull, corncob, bagasse and oat spelt xylan, respectively, at 55 °C for 6 days, respectively. The activity was more than 5 times higher than that at 35 °C.     

The respiratory chain of the facultative thermophile,Bacillus coagulans

Two oxidases were found to be present in membranes from the facultative thermophile Bacillus coagulans grown at 55°C, compared to one in cells grown at 37°C. Cytochrome spectra and inhibitors of the respiratory chain identified them as cytochrome oxidases aa ₃ and d. Both were present in membranes from 55°C grown cells, but only cytochrome oxidase aa ₃ was found in membranes from 37°C grown cells. The presence of cytochrome d in 55°C grown cultures was found to be due to decreased oxygen tension and not to the high growth temperature. This was confirmed by (a) induction of cytochrome d at 37°C under conditions of oxygen limitation and (b) its repression at 55°C under conditions of high aeration and its subsequent induction on lowering the dissolved oxygen concentration in chemostat cultures. Two cytochromes b (_max 558 and _max 562) were present in both 37°C and 55°C grown cells. Results from the inhibition of substrate oxidation by membranes suggested different pathways of electron transport by the respiratory chain.     

Production of a raw starch saccharifying amylase byBacillus alvei grown on different agricultural substrates

Maximum activity of the amylase ofBacillus alvei was attained after growth of the organism on sorghum starch. Rice, corn, yam, cassava and potato starch gave high enzyme activities as did soluble starch. Glucose, maltose and glycerol were less effective. Optimum conditions for both growth and enzyme production were pH 6.8 at 40°C.     

Reduction of the nodulation barrier inMedicago laciniata by alteration of the root temperature

Medicago laciniata, an annual leguminous plant of Saharo-Sindian origin, is particularly refractory to root nodulation by most strains ofRhizobium meliloti. Using a series of such bacterial strains belonging to the 8 groups of Brockwell and Hely, and a variety of environmental conditions, it was noted that several normally non-nodulating strains (at 20°C) produced ineffective nodules at root temperatures of 24°C to 28°C. Nodulation at 20°C failed to occur in the presence of a wide variety of test compounds and physical conditions. No phytoalexins or anti-Rhizobium growth inhibitors were isolated from inoculated root tissue at any temperature. Temperature shift experiments indicated no infection of the root hairs at 20°C, and infection threads produced at the permissive root temperature failed to elongate after transfer to 20°C. However, if meristematic activity had been initiated in the inner root-cortical cells as a result of infection thread penetration at 28°C, no blockage of nodule maturation occurred upon subsequent transfer to 20°C root temperature. Nodules produced at 28°C were completely devoid of nitrogenase activity, although the apical (but not the distal) regions contained normal-appearing bacteriods, surrounded by enclosing membranes, and possessed a fully functional leghaemoglobin. A shortage of metabolic energy did not appear to be involved in the ineffective response. A hypothesis to explain the nodulation phenomenon observed was based on the observation in the roots of 2 factors present at 20°C but not at 28°C.     

Development Models for the Filth Fly ParasitoidsSpalangia gemina, S. cameroni,andMuscidifurax raptor(Hymenoptera: Pteromalidae) under Constant and Variable Temperatures

Development rates were determined for three pteromalid parasitoids of houseflies under constant and varying temperatures from 15 to 35°C.Muscidifurax raptorGirault and Sanders was the fastest developing species, with females completing development in 13.8 days at 32.5°C and 66.5 days at 15°C.Spalangia geminaBoucek females completed development in 20.8 days at 30.0°C and 161 days at 15.0°C, whereasS. cameroniPerkins females completed development in 20.6 days at 30.0°C and 155.5 days at 15.0°C. Male development times were 90.3% of those for femaleS. geminaand 92.7 and 88.6% of those for femaleS. cameroniandM. raptor,respectively. Parasitoid survival was very low at 35°C for all species and noSpalangiasurvived constant exposure to 15.0°C. Exposure to these lethal temperatures for shorter periods indicated that the parasitoids can tolerate them well under conditions more typical of the field. Development rates were modeled using biophysical and degree-day models and the models were tested for their ability to predict development under fluctuating conditions (24–36°C). Neither model was superior for all three species because of interspecific differences in the parasitoids' responses to high temperatures. Agreement between predicted and observed development times for all three species was achieved by small empirical adjustments of a key parameter in the biophysical model.