Protoplasts from pectinolytic fungi: isolation, regeneration and pectinolytic enzyme production

S. Solís, M.E. FLORES AND C. HUITRON. 1996. Protoplast release in pectinolytic strain mutants of Aspergillus sp. CH-Y-1043 (A13) and Aspergillus flavipes ATCC-16795 (F7) is described. Optimum yield of protoplasts A13 was obtained in a lapse of 1 h when commercially lytic enzymes of Trichoderma harzanium (2 mg ml⁻¹) were added in 0.05 mol 1⁻¹ citrate-phosphate buffer pH 5.0 containing 0.7 mol 1⁻¹ KCl and 10 mg ml⁻¹ BSA. Best results in F7 were obtained when the protoplasting system of A13 was supplemented with 10 mg ml⁻¹ Aureobasidium sp. lytic enzymes. Isolated protoplasts in A13 and F7 were capable of a high regeneration frequency of 87% and 53% when 0.7 mol 1⁻¹ KCl and sorbitol were used as osmotic stabilizers. Endo-P, Exo-P and pectin lyase production were not modified during the process of regeneration.     

CONTENT OF ATP IN CULTIVATED NEURONS AND ASTROCYTES EXPOSED TO BALANCED AND POTASSIUM-RICH MEDIA

Abstract— The content of ATP was measured in neurons from dorsal root ganglia and in cortical and spinal astrocytes with the luciferase method. No ATP could be demonstrated in the cortical cells but in the dorsal root neurons and spinal astrocytes an average amount of 7·9 and 11·9 × 10⁻¹⁵ mol per cell respectively was found. An increase in the external potassium concentration had no effect on the content of ATP in neurons (7·1 × 10⁻¹⁵ mol/cell) but led to a significant ( P < 0·005) decline to 6·4 × 10⁻¹⁵ mol per cell in the astrocytes.     

Electrofusion of protoplasts from Aspergillus nidulans

Abstract Electrical parameters were determined and quantified for the stimulation of the optimum alignment and fusion of Aspergillus nidulans protoplasts. In a non-homogeneous alternating electrical field A. nidulans protoplasts aligned to form pearl chains associated with the electrodes of the fusion chamber. Most protoplasts were in pearl chains in an alignment field frequency of 3.0 MHz but maximum pair formation occurred at 1.0 MHz. At a field strength between 100 and 1000 V · cm⁻¹ pearl chain formation occurred with minimal protoplast rotation or lysis. The application of DC pulses resulted in protoplast fusion. Most fusion events were observed after two 500 V · cm⁻¹ DC pulses with a 0.5 s interpulse period. Using 1 × 10³ protoplasts · cm⁻³ in a 7 μm fusion chamber a maximum of 17.2 ± 2.0% fusion events were achieved.     

Resistance to cadmium by pretreated rainbow trout alevins

A toxicity test with cadmium concentrations ranging from 0·1 to 100·0 mg Cd 1⁻¹ was used to assess the effect of cadmium pretreatment on rainbow trout ( Salmo gairdneri Richardson) alevins. The median period of survival for fish pretreated at 0·01 mg Cd 1⁻¹ was found to be increased at test concentrations up to 10mg Cd 1⁻¹ compared with alevins pretreated with dilution water. However, at concentrations above 10mg Cd 1⁻¹ pretreatment at 0·01 mg Cd 1⁻¹ reduced the median period of survival.     

Generation of a reproducible nutrient-depleted biofilm of Escherichia coli and Burkholderia cepacia

An in vitro method of growing bacteria as a defined nutrient-depleted biofilm is proposed. The medium was defined nutritionally in terms of the quantitative composition and by the total amount of nutrient required to achieve a defined population size. Escherichia coli and Burkholderia cepacia were incubated on a filter support placed on a defined volume of solid medium. The change of biomass of the biofilm population was compared with the change in a planktonic culture. The size of the population in stationary phase was proportional to the concentration of limiting substrate up to 40 μmol cm⁻² glucose for E. coli and up to 2·7 × 10⁻⁹ mol cm⁻² iron for B. cepacia . Escherichia coli growing exponentially had a growth rate of μ = 0·30 h⁻¹ in a biofilm and μ = 0·96 h⁻¹ in planktonic culture. The growth rate, μ, for exponentially growing B. cepacia in a biofilm was 1·12 h⁻¹ and in planktonic culture 0·78 h⁻¹. This method allows the limitation of the size of a biofilm population to a chosen value.     

Sucrose phosphorylase of the rumen bacterium Pseudobutyrivibrio ruminis strain A

Aims:  To verify the taxonomic affiliation of bacterium Butyrivibrio fibrisolvens strain A from our collection and to characterize its enzyme(s) responsible for digestion of sucrose.
Methods and Results:  Comparison of the 16S rRNA gene of the bacterium with GenBank showed over 99% sequence identity to the species Pseudobutyrivibrio ruminis . Molecular filtration, native electrophoresis on polyacrylamide gel, zymography and thin layer chromatography were used to identify and characterize the relevant enzyme. An intracellular sucrose phosphorylase with an approximate molecular mass of 52 kDa exhibiting maximum activity at pH 6·0 and temperature 45°C was identified. The enzyme was of inducible character and catalysed the reversible conversion of sucrose to fructose and glucose-1-P. The reaction required inorganic phosphate. The K _m for glucose-1-P formation and fructose release were 3·88 × 10⁻³ and 5·56 × 10⁻³ mol l⁻¹ sucrose, respectively – while the V _max of the reactions were −0·579 and 0·9  μ mol mg protein⁻¹ min⁻¹. The enzyme also released free glucose from glucose phosphate.
Conclusion:  Pseudobutyrivibrio ruminis strain A utilized sucrose by phosphorolytic cleavage.
Significance and Impact of the Study:  Bacterium P. ruminis strain A probably participates in the transfer of energy from dietetary sucrose to the host animal.     

Sample sizes for length and density estimation of 0+ fish when using point sampling by electrofishing

Standard lengths of cyprinids at several sites in the River Great Ouse, England, were compared between catches taken using a conventional 15 × 3 m micromesh seine (pore diameter 2 mm) and using point sampling by electrofishing (PSE). No statistically significant differences in sites were found in six out of 10 trials. In nine out of 10 trials, the difference between the mean lengths differed by <1 mm. No serial bias was found between PSE and seine netting for cyprinid fishes between 14 and 100 mm SL, although variation between size classes was high, owing to small sample sizes. The coefficient of variation in fish length with size tended to increase with the age of 0+ Rutilus rutilus . The relationship between sample size (n) and variance ( s² ) was explained by s ²=13·9 n ^−1·24. Sampling more than 30 fish resulted in little increase in precision. The relationship between the mean catch per site (̄) and the variance of the mean estimate was log s ²=1·6039 log ̄ +0·973. The number of samples required to estimate density within a given variance range was: n =9·33 ̄ ^1·6–2 CV_̄ ⁻². Given the high variance-mean ratio, great care should be taken when interpreting density data collected using PSE, and 50 point samples is the minimum required for density estimation.     

An efficient transformation system for Bifidobacterium spp.

This study describes a broad host transformation protocol that enables the uptake of plasmid DNA into 10 different species of Bifidobacterium , some of which have never been transformed before. The vector pNC7 (4·9 kb) was used to optimize the electroporation protocol. Transformation efficiencies ranged from 3·6×10⁻¹ to 1·2×10⁵ transformations per μg DNA. The impact of growth medium composition and electric field strength on transformation efficiency were independently optimized. Electrocompetent cells were grown in Iwata medium broth enriched with Actilight^RP 16%, harvested during the early exponential growth phase, and pulsed at 12·5 kV cm⁻¹, 100 Ω and 25 μF.     

Emission rate of amino acids and ammonia and their role in olfactory preference behaviour of juvenile Arctic charr, Salvelinus alpinus (L.)

The behaviour of juvenile Arctic charr, Salvelinus alpinus (L.) , to an abrupt concentration step of L-amino acids, L-alanine and ammonium chloride was studied by fluviarium technique. The emission rates of these substances were studied. Juvenile Arctic charr emit 8.0 × 10⁻⁴ mol total ammonia-N kg⁻¹ h⁻¹ and 3.3 × I0⁻⁵ mol amino acids kg⁻¹ h⁻¹. In behaviour tests the charr avoided 5.6x 10⁻⁶and 5.6 × 10⁻⁷ M ammonium chloride. The 17 L-amino acid mixture, ranked as observed in the analysis of emission, was avoided at 4.6 × 10⁻⁷ M, while 100 times dilution of this value gave neither avoidance nor attraction. The charr avoided L-alanine tested alone at the concentration of 4.6 × 10 ⁻⁷ M. Anosmic charr showed neither avoidance nor attraction to the mixture of 17 amino acids tested at 4.6 × 10⁻⁷ M. The results indicate that ammonia as well as emitted amino acids are not responsible for the olfactory mediated attraction to conspecific odour shown earlier in Arctic charr. On the contrary, these substances may have a negative effect by reducing the strength of attraction.     

Production of amylase by the intestinal microflora in cultured freshwater fish

The amylase-producing ability of the intestinal microflora in cultured specimens of ayu, carp, channel catfish, Japanese eel and tilapia was determined. Mean viable counts of aerobes and anaerobes ranged from 1·1×10⁶ to 3·7×10⁸ cfu g⁻¹ and from 1·3×10³ to 1·6×10⁸ cfu g⁻¹, respectively. Aeromonas spp. and Bacteroidaceae were predominant in four to five fish species. Of 206 strains examined, 65 (31·6%) produced ≥0·01 U amylase ml⁻¹. The percentage of producers differed among families and genera of bacteria and fish species. While 56% of the anaerobes produced amylase, only 20% of the aerobes did. More than 50% of Aeromonas , Bacteroidaceae and Clostridium strains produced amylase efficiently while Acinetobacter , coryneforms, Enterobacteriaceae, Moraxella , Plesiomonas and Streptococcus strains did not. High amylase production (≥0·05 U ml⁻¹) was found in 12 strains, 11 from Aeromonas and one Pseudomonas . The percentage of high amylase producers in Japanese eel was lower than the other four fish (2–30%). These results strongly suggest that the amylase produced by the intestinal microflora play an important role in the digestion of starch in freshwater fish to some extent.     

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.     

Conjugal plasmid transfer between lactococci on solid surface matings and during cheese making

Abstract The concept of deliberate use of genetically enginereed microorganisms in dairy products requires a clear understanding of their behaviour and of the dissemination of introduced DNA in these strains. Thus, transfer of a self-transmissible plasmid and a non-self-transmissible but mobilizable plasmid from an engineered strain of Lactococcus lactis subsp. lactis IL 1403 to wild-type strains of L. lactis subsp. lactis and subsp. cremoris of technological interest was studied on standard solid surface matings and in cheese during manufacture. On solid surface matings, transfer of the conjugative plasmid occurred at frequencies ranging from < 2.3 × 10⁻⁹ to 2.8 × 10⁻⁴. Mobilization of the non-conjugative plasmid was observed at a lower frequency (ca. 10⁻⁵) in only one recipient which was then selected along with another recipient strain (presenting intermediate transfer frequencies) for making Camembert cheese. During cheese making, only the transfer of the self-transmissible plasmid was observed. It occurred in the early stages of manufacturing. The transfer frequencies were 7.0 × 10⁻⁸ or 7.6 × 10⁻¹ 1, depending upon the recipient strain. These were about 3 to 4 orders of magnitude lower than on solid surface matings. Mobilization of the non-conjugative plasmid was never detected in cheese.     

Acute effects of the sodium salt of 2,4-D on the early developmental stages of bleak, Alburnm alburnus

The acute effects of a herbicide based on 2,4-D on embryos and larvae of bleak, Alburnus alburnus L. exposed to concentrations of 25–3200 mg 1⁻¹ for 12–48 hours were investigated. The 2,4-D delayed or stopped the development of embryos in their early stages, caused behavioural changes and morphological alterations. The L.C.₅₀ values for embryos varied between 12–9 and 15·9–4mg 1⁻¹ and for larvae from 51·6 to 111·2mg 1⁻¹ according to the time of exposure. The level of 2,4-D in Lake Balaton, or similar shallow lakes, without substantial injury of the littoral fish fauna should not surpass 0·5–1·0 mg 1⁻¹.     

Effects of river red gum, Eucalyptus camaldulensis, litter on golden perch, Macquaria ambigua

Aquaria with added river red gum, Eucalyptus camaldulensis , litter became hypoxic, with decreased pH and contained up to 30 mg 1⁻¹ tannin and lignin. Survival of golden perch, Macquaria ambigua , larvae in aquaria treated with a simulated annual litter density of 450 g m⁻² for 72 h was 14·9% for 15-day-old larvae and 0% for 8-day-old larvae. A litter density of 1223 g m⁻² resulted in total mortality for both age groups of larvae. Aeration increased survival of larvae to a minimum of 68·8% in 1223 g m⁻² litter treatments compared to 89·8% in aerated controls and 86·8% in non-aerated controls. A kinetic behavioural assay was used to detect alarm responses in golden perch larvae and juveniles exposed to leachates from river red gum bark, leaves and wood. Eight-day-old larvae exposed to bark and wood leachates (0·001–10 g 1⁻¹) exhibited an initial period of hyperactivity, followed by a concentration-dependent decrease in spontaneous activity. Larvae exposed to leaf leachates displayed only a decrease in spontaneous activity. Four-month-old juveniles exposed to wood leachates were also initially hyperactive, then progressively developed mild hypoactivity at increasing leachate concentrations. Juveniles exposed to wood leachates at 20g 1⁻¹ for 30min suffered 97·5% mortality in 96 h. Wood leachates induced dose-dependent lamellar fusion, epithelial dissociation and necrosis in the gills. The presence of toxic leachates and low oxygen availability in flooded river red gum forests may make these habitats unsuitable as nursery areas for native fish.     

Permeability of the Chara cell membrane for ethylene glycol, glycerol, meso-erythritol, xylitol and mannitol

The permeability of internodal cells of Chara australis R. Brown for polyol molecules was determined by using a turgor balance to measure the increase in the osmotic pressure of an internodal cell incubated in artificial pond water containing one of the polyol compounds tested. The permeabilities for ethylene glycol, glycerol, meso -erythritol, xylitol and mannitol were (4. 39 ± 0. 52) × 10⁻⁹, (1. 49 ± 0. 40) × 10¹⁰, (4. 92 ± 0. 27) × 10⁻¹⁰ (9. 9 ± 3. 4) × 10¹¹ and (7. 6 ± 4. 8) × 10⁻¹² m s⁻³, respectively. The permeability for glycerol was slightly smaller than that for meso -erythritol, whose molecular weight is larger than that of glycerol in this homologous series: but the reason for this is not clear.     

A Chromatographic Method for Analysis of the Gaseous Phase in Shake Flask Cultures and its Application to the Study of Methane Utilization

A simple gas chromatograph with a katharometer detector is described to determine O₂, N₂, methane and CO₂ in gas samples of 0·01–2·0 ml. The apparatus is inexpensive, and can be modified to determine other gases. The sensitivity to oxygen is 3 × 10⁻⁶ g. The use of the instrument is illustrated by a study of the growth kinetics of Methylococcus capsulatus grown on methane in shake flask experiments. The ratio of O₂ uptake to methane uptake is much lower in the stationary phase than in the growth phase of the culture.     

Retrospective approach to investigating the early life history of a diadromous fish: allis shad Alosa alosa (L.) in the Gironde–Garonne–Dordogne watershed

In this study, the identification of major ontogenetic events and the duration of the fresh water and estuarine phases before the seaward exit of young allis shad Alosa alosa were investigated retrospectively, using the otoliths of spawners. Daily growth increment periodicity in the otoliths was validated with an accuracy of 4 days (9·4%) for fish >30 days. The specific limits of the Sr:Ca ratios in the otoliths, indicating fish movements between salinity 'compartments', were up to 1·1 ×10⁻³ for the freshwater phase, 1·1 to 1·9 × 10⁻³ for the estuarine phase and 1·9 × 10⁻³ for the marine phase. In early life history, a major event occured at 22 days, which was marked on the otoliths by a change in the direction of the otolith growth axis. This event was related to a change in larval swimming behaviour. Juvenile freshwater residence time was between 58 and 123 days, with a median duration of 88 days. Contrary to what has been estimated in previous studies, the present study showed that the estuarine phase lasted from 4 to 36 days, with a median value of 11 days. Seaward exit occurred in fish aged from 63 to 150 days, with a median age of 99 days. These results have important implications for the sustainable management of this species.     

THE ASSESSMENT OF RELATIVE BACTERICIDAL ACTIVITY AMONG CERTAIN PHENOLS BY A PHYSICAL METHOD

SUMMARY: The addition of phenol, various alkyl phenols, or CTAB to a suspension of E. coli in distilled water caused the optical density to increase, the change being more marked as the concentration of a given compound was raised. Families of curves of similar shape were obtained with all the compounds when optical density was plotted against time for different concentrations, and figures expressing the activity relative to that of 1·2% phenol were obtained by dividing the concentration which produced the same curve as that concentration of phenol, into 1·2. These figures were very similar to those obtained for the bactericidal effects of the compounds relative to 1·2% phenol, using an 'end-point' method.
The presence of the chlorides of mono- and divalent metals at 10⁻⁴ M, or of the sulphates of trivalent metals at 10⁻⁵ M, did not affect the turbidity reaction, but higher concentrations of monovalent metal salts, e.g. 2 × 10⁻² M phosphate buffer (potassium) at pH 6·7, markedly retarded the change due to phenol. That concentration of the buffer also greatly reduced the bactericidal activity of 1·2% phenol. Much of the turbidity increase, however, occurred after all the cells of the suspensions had been killed, with phenol concentrations over 1·0%.     

Distribution of Synechococcus spp. determined by immunofluorescent assay

By using two polyclonal antisera against WH 7803 strain (Synechococcus sp.) and WH 5701 strain (Synechococcus bacillaris) it is possible to detect and to enumerate cells of the two cyanobacterial serogroups. The immunofluorescence technique was used to study the distribution of the two serogroups in the estuarine, coastal and upwelling waters of the Mediterranean Sea surrounding Messina. In the estuarine waters of the Alcantara River (Ionian Sea), the WH 7803 serogroup was present at a concentration in the order of 10² cells ml⁻¹ and the WH 5701 serogroup at a concentration of 5·5 × 10² cellsml⁻¹. In the coastal waters of Messina, where urban and industrial wastes are usuallydumped, the concentration of total phycoerythrin- Synechococcus ranged from 1·3 × 10² to 4·1 × 10³ cells ml⁻¹; the WH 7803 serogroup accounted for 50–94% of the totalpopulation in Ionian stations, whereas the WH 5701 serogroup ranged from1·4 × 10¹ to6·7 × 102cells ml⁻¹. In the upwelling area (Straits of Messina) bothserogroups were found. Vertical distribution of two Synechococcus strains had anopposite trend and their concentrations were of the order of 10¹–10²cells ml⁻¹. Theuse of the Scan laser system allows both autofluorescent and labelled organismsto be distinguished in a preparation for optical microscopy. It also allows false-positivecells to be distinguished.     

The relative abundance of brown trout in acidic softwater lakes in relation to water quality in tributary streams

The influence of the water quality of tributary streams on the relative abundance in benthic gillnet catches (catch per unit effort, cpue) of allopatric brown Salmo trutta was assessed in associated acidic, softwater lakes. The study was carried out over 6 years (1989–1994) in 15 lakes located at altitudes between 230–715 m a.s.l. in two Norwegian catchments. The water quality of the main inlets and outlets varied little, as indicated by their of pH range (4·93–5·51) and calcium concentrations (0·19–0·44 mg 1⁻¹), but varied more with respect to concentrations of inorganic, monomeric aluminium (7·0–41·0 μg l⁻¹). Most of the lakes were also fed by secondary streams with better water quality: a maximum pH of 6·56, calcium levels of up to 0·74mg 1⁻¹, and inorganic aluminium levels as low as l·0 μg 1⁻¹. The cpue was inversely correlated with lake altitude ( r ²=0·50), and thus was adjusted to a mean altitude. The calcium concentration in the richest secondary stream to each lake, its richness judged on the basis of its acid-neutralizing capacity, had the highest predictive power of the variability in cpue ( r ² = 0·49).The calcium content in the other secondary streams or in the main inlets and outlets did not correlate with cpue. Alkalinity in the main outlets correlated to some extent with cpue ( r ² = 0·27). It is suggested that secondary streams with good water quality provide important refuges for the recruitment of brown trout in acidic softwater lakes.