Autoradiographic method to screen for soil microorganisms which accumulate zinc.

An autoradiographic method was developed to screen for and isolate soil microorganisms which accumulate zinc (Zn). Diluted soil samples (Rubicon fine sand, Entic Haplorthods [pH 5.9]) were plated on soil extract-glucose agar containing radioactive 65Zn. After 7 days of incubation, individual colonies which accumulated sufficient 65Zn could be detected by autoradiography. These colonies were isolated and confirmed as Zn accumulators in pure culture by using the autoradiographic plate technique. Most Zn accumulators were filamentous fungi, identified as Penicillium janthinellum, Aspergillus fumigatus, and Paecilomyces sp. Isolates of Penicillium janthinellum were the most common Zn accumulators. The most abundant Zn-accumulating bacteria were Bacillus spp. The validity of the autoradiographic plate technique to differentiate soil microbes which accumulate Zn was examined independently by energy dispersive X-ray analysis in a scanning electron microscope. This method confirmed that fungal isolates which gave positive autoradiographic responses in the plate assay bioaccumulated more Zn in their biomass than fungal isolates from the same soil sample which gave negative autoradiographic responses. Thus, this technique can be applied to specifically screen for and isolate microbes from the environment which bioaccumulate Zn.     

A plate method to screen for microorganisms producing xylose isomerase

A plate method was developed to screen for xylose isomerase-producing microorganisms based on the use of 2,3,5-triphenyltetrazolium as an indicator of D-xylulose, the D-xylose isomerization product. The use of this method allows microorganisms to be differentiated by the character of the enzyme synthesis (inducible or constitutive).     

Conditions used with a continuous cultivation system to screen for d-hydantoinase-producing microorganisms

The continuous cultivation technique has been used to screen for microorganisms producing d-hydantoinase, a biocatalyst involved in the production of optically active amino acids. Pseudomonas putida strain DSM 84 was used as a model hydantoinase producer to establish selective culture conditions through the addition of various pyrimidines, dihydropyrimidines, hydantoins and 5-monosubstituted hydantoins. Thymine induced more activity than all cyclic amides tested. Addition of thymine as a non-metabolised inducer at a concentration of 0.05 g l^–1 in a continuous culture of P. putida stimulated hydantoinase production up to 80 times the basal level. Using continuous culture conditions established with the model strain, a different strain of P. putida having hydantoinase activity was isolated from commercial mixed cultures of microorganisms. DNA fingerprinting revealed that this new isolate was distinct from strain DSM 84. When used as a probe, the d-hydantoinase gene of strain DSM 84 hybridized with the DNA of the new P. putida isolate.     

Improved methodology for isolating soil microorganisms

Polyurethane foam cylinders and a replica-stamp technique were used for plating soil samples. This in conjunction with the addition of NaCl to the isolation medium substantially increased the variety of isolates recovered.     

A rapid method to screen fungi for resistance to toxic chemicals

Although many species of fungi are able to degrade highly toxic chemicals, only a few species have been evaluated for resistance to toxic effects of these chemicals. In this paper we demonstrate the successful application of a method to rapidly screen several species of fungi for toxicity to chemicals or mixtures of chemicals using pentachlorophenol (PCP) as a model toxic compound. Cellulose antibiotic assay disks were soaked in solutions containing different concentrations of PCP (5, 10, 25, 50, and 80 mg l^–1) and then placed in a triangular pattern outside the growing edge of the mycelia of eighteen species of white rot fungi. The plates were incubated and observed for development of inhibition zones (non-growth areas) around the disks. The short-term (24 h) growth of all eighteen species of fungi was inhibited by 5–10 mg-PCP l^–1, a range similar to that observed using previously reported techniques. Long-term growth studies using this screening method were not useful since PCP diffused from the disk into the agar, decreasing the applied dose.     

A method for the detection and analysis of growth patterns of microorganisms in soil.

A fluorescence-staining technique using the magnesium salt of 8-anilino-1-naphthalene sulfonic acid is described and used to follow the changes in the distribution patterns of microorganisms in soils. A statistical procedure was used to determine the occurrence of significant differences in clumping of bacteria (i.e., production of colonies) in different regions of artificial soil-aggregate systems treated with nutrient solutions and also with a herbicide, Linuron. The response of soil microorganisms to glucose amendment was most marked in the aerobic, outer zone of aggregates. Linuron inhibited colony formation in aggregates treated with the herbicide. The method allows continued observations to be made on the same soil sample at intervals during incubation and os can be used to determine growth rates, inhibitory effects of chemicals, distribution patterns in soils, effects of added nutrients, and other effects where growth in situ is important.     

Competition for nitrogen between plants and soil microorganisms

Experiments suggest that plants and soil microorganisms are both limited by inorganic nitrogen, even on relatively fertile sites. Consequently, plants and soil microorganisms may compete for nitrogen. While past research has focused on competition for inorganic nitrogen, recent studies have found that plants/mycorrhizae in a wide range of ecosystems can use organic nitrogen. A new view of competitive interactions between plants and soil microorganisms is necessary in ecosystem where plant uptake of organic nitrogen is observed.     

Insecticidal effects on soil microorganisms and their biochemical processes related to soil fertility

Among the four insecticides under study, hexachlorocyclohexane (BHC) followed by phorate significantly stimulated the populations of (total) bacteria, actinomycetes, fungi, aerobic non-symbiotic N2-fixing bacteria and P-solubilizing microorganisms in soil. Carbofuran significantly stimulated total as well as N2-fixing bacteria. Fenvalerate had no effect on P-solubilizers. All the insecticides stimulated the proportion of Penicillium in soil. Similarly, Pseudomonas with BHC, Sarcina with phorate, Corynebacterium, Azotobacter and Streptomyces with fenvalerate were also stimulated. On the other hand, Erysipelothrix with BHC, Staphylococcus with phorate, Staphylococcus, Nocardia and Fusarium with fenvalerate were inhibited. Almost all the insecticides reduced the proportions of Micrococcus and Rhizopus in soil. Insecticides also augmented the non-symbiotic N2-fixing and P-solubilizing capacities of the soil and the augmentation was more pronounced with BHC followed by phorate.     

A new method to screen polysaccharide cleavage enzymes

The activity of polysaccharide cleavage enzymes has usually been evaluated by qualitative plate screening methods and quantitative colorimetric or chromatographic assays. The recent development of protein engineering has shown the limits of these techniques when applied to high throughput screening. Here we propose a microplate method to measure the activity of polysaccharide cleavage enzymes through small variations in viscosity. Polysaccharide solutions are co-incubated with magnetic particles in enzyme buffers. The cleavage action of polymer-degrading enzymes increases the mobility of the particles in a magnetic field, even at low levels of enzyme activities. This reproducible, sensitive technique was used to evaluate enzymatic specificity towards substrates. BioFilm indices (BFI) determined by associated software were used to follow enzyme kinetics and measure the usual variables.     

A method for determining phospholipases in microorganisms

A method is suggested for the determination of bacteria phospholipases in dense nutrient medium. The medium contains the egg-yolk solution, buffer pH 9.2, meat extract, calcium chloride and toluidine blue. The enzyme activity is estimated by the diameter value of the medium clarification zone around the bacterial mass inoculation by injection into an agar plate. It is possible to study 6-8 strains on one Petri dish. This method is a simple one and thus it can be used in the bacteriological practice when determining phospholipases of bacteria, especially in strains of those species where this enzyme is a pathogenicity factor.     

Screening for novel lipolytic enzymes from uncultured soil microorganisms

The construction and screening of metagenomic libraries constitute a valuable resource for obtaining novel biocatalysts. In this work, we present the construction of a metagenomic library in Escherichia coli using fosmid and microbial DNA directly isolated from forest topsoil and screened for lipolytic enzymes. The library consisted of 33,700 clones with an average DNA insert size of 35 kb. Eight unique lipolytic active clones were obtained from the metagenomic library on the basis of tributyrin hydrolysis. Subsequently, secondary libraries in a high-copy-number plasmid were generated to select lipolytic subclones and to characterize the individual genes responsible for the lipolytic activity. DNA sequence analysis of six genes revealed that the enzymes encoded by the metagenomic genes for lipolytic activity were novel with 34–48% similarity to known enzymes. They had conserved sequences similar to those in the hormone-sensitive lipase family. Based on their deduced amino acid similarity, the six genes encoding lipolytic enzymes were further divided into three subgroups, the identities among which ranged from 33% to 45%. The six predicted gene products were successfully expressed in E. coli and secreted into the culture broth. Most of the secreted enzymes showed a catalytic activity for hydrolysis of p-nitrophenyl butyrate (C₄) but not p-nitrophenyl palmitate (C₁₆).     

Techniques for the observation and isolation of soil microorganisms

The Botanical Review -     

Models for predicting soil zinc availability for barley

Regression models are proposed for the prediction of soil zinc availability within different pH ranges. The relationship between zinc concentration in barley plants, zinc content in soils and soil pH was described using a two-dimensional diagram. Results show that the soil zinc availability is significantly affected by pH changes at pH above 6.5. Below this point, the pH effect becomes gradually less important. In the low pH range, the soil zinc availability is mainly dependent on soil zinc quantity.     

Novel screening method for extracellular phytase-producing microorganisms

A bioassay was developed to screen extracellular phytase-producing microorganisms. Washed cells of Corynebacterium glutamicum, which cannot use sodium phytate as source of phosphate, were mixed with phytate-minimal agar as indicator strain. By this method, we could easily obtain phytase-producing strains from soil samples and 71 % of the isolates had phytase activities above 0.01 U/ml when they were grown in modified phytase screening medium. © Rapid Science Ltd. 1998