A new approach to isolating siderophore-producing actinobacteria.

AIMS: This study was conducted to investigate the application of 2,2'-dipyridyl as a new approach to isolating siderophore-producing actinobacteria. METHODS AND RESULTS: Isolation of actinobacteria from soil was conducted by a soil dilution plate technique using starch-casein agar. Iron starvation was fostered by the incorporation of the iron chelator 2,2'-dipyridyl in the isolation medium. Pretreatment of the samples at an elevated temperature (40°C) ensured that the majority of nonsporulating bacteria were excluded. The survivors of this treatment were largely actinobacteria. Of the viable cultures grown in the presence of 2,2'-dipyridyl, more than 78-88% (average of three separate studies) were reported to produce siderophore-like compounds compared to 13-18% (average of three separate studies) when grown on the basic media in the absence of the chelating agent. The most prolific producers as assessed by the chrome azurol sulphate (CAS) assay were further characterized and found to belong to the genus Streptomyces. CONCLUSIONS: Selective pressure using 2,2'-dipyridyl as an iron-chelating agent in starch-casein media increased the isolation of siderophore-producing actinobacteria compared to the unamended medium. SIGNIFICANCE AND IMPACT OF THE STUDY: The study described represents a new approach to the isolation of siderophore-producing actinobacteria using a novel procedure that places a selection on cell population based upon the incorporation of a chelating agent in the medium.

An extracellular glucoamylase produced by endophytic fungus EF6.

A strain of endophytic fungus EF6 isolated from Thai medicinal plants was found to produce higher levels of extracellular glucoamylase. This strain produced glucoamylase of culture filtrate when grown on 1% soluble starch. The enzyme was purified and characterized. Purification steps involved (NH4)2SO4 precipitation, anion exchange, and gel filtration chromatography. Final purification fold was 14.49 and the yield obtained was 9.15%. The enzyme is monomeric with a molecular mass of 62.2 kDa as estimated by SDS-PAGE, and with a molecular mass of 62.031 kDa estimated by MALDI-TOF spectrometry. The temperature for maximum activity was 60 degrees C. After 30 min for incubation, glucoamylase was found to be stable lower than 50 degrees C. The activity decrease rapidly when residual activity was retained about 45% at 55 degrees C. The pH optimum of the enzyme activity was 6.0, and it was stable over a pH range of 4.0-7.0 at 50 degrees C. The activity of glucoamylase was stimulated by Ca2+, Co2+, Mg2+, Mn2+, glycerol, DMSO, DTT and EDTA, and strongly inhibited by Hg2+. Various types of starch were test, soluble starch proved to be the best substrate for digestion process. The enzyme catalyzes the hydrolysis of soluble starch and maltose as the substrate, the enzyme had Km values of 2.63, and 1.88 mg/ml and Vmax, values of 1.25, and 2.54 U/min/mg protein, and Vmax/Km values of 0.48 and 1.35, respectively. The internal amino acid sequences of endophytic fungus EF6 glucoamylase; RALAN HKQVV DSFRS have similarity to the sequence of the glucoamylase purified form Thermomyces lanuginosus. From all results indicated that this enzyme is a glucoamylase (1,4-alpha-D-glucan glucanohydrolase).

Elucidation of distribution patterns and possible infection routes of the neurotropic black yeast Exophiala dermatitidis using AFLP.

Distribution of populations of the opportunistic black yeast Exophiala dermatitidis was studied using AFLP. This fungus has been hypothesized to have a natural habitat in association with frugivorous birds and bats in the tropical rain forest, and to emerge in the human-dominated environment, where it occasionally causes human pulmonary or fatal disseminated and neurotropic disease. The hypothesis of its natural niche was investigated by comparing a set of 178 strains from natural and human-dominated environments in Thailand with a worldwide selection of 107 strains from the reference collection of the CBS Fungal Biodiversity Centre, comprising 75.7% clinical isolates. Many isolates had unique AFLP patterns and were too remote for confident comparison. Eight populations containing multiple isolates could be distinguished, enabling determination of geographic distributions of these populations. Some of the populations were confined to Thailand, while others occurred worldwide. The local populations from Thailand contained strains from natural and urban environments, suggesting an environmental jump of the fungus. Strains from human brain belonged to widely dispersed populations. In some cases cerebral isolates were identical to isolates from the human intestinal tract. The possibility of cerebral infection through intestinal translocation was thus not excluded.

Rapid screening for genotypes as possible markers of virulence in the neurotropic black yeast Exophiala dermatitidis using PCR-RFLP.

A simple method for fungal genotype screening was developed for the black yeast Exophiala dermatitidis based on RFLP of ribosomal ITS regions currently used as potential virulence markers. In a study set of 502 strains of the species, two main genotypes were recognized. Only 0.97% of lanes were difficult to interpret as they did not clearly present one of the expected genotypes. Twenty strains were deviating and proved to be E. spinifera after sequencing. Eight common, related species (based on SSU data) with clinical significance yielded different patterns with TaqI digestion, and thus the method is also usable for routine diagnostics.