Improvements of polymerase chain reaction and capillary electrophoresis single-strand conformation polymorphism methods in microbial ecology: toward a high-throughput method for microbial diversity studies in soil.

The molecular signature of bacteria from soil ecosystems is an important tool for studying microbial ecology and biogeography. However, a high-throughput technology is needed for such studies. In this article, we tested the suitability of available methods ranging from soil DNA extraction to capillary electrophoresis single-strand conformation polymorphism (CE-SSCP) for high-throughput studies. Our results showed that the extraction method does not dramatically influence CE-SSCP profiles, and that DNA extraction of a 0.25 g soil sample is sufficient to observe overall bacterial diversity in soil matrices. The V3 region of the 16S rRNA gene was amplified by PCR, and the extension time was found to be critical. We have also found that proofreading DNA polymerases generate a better signal in CE-SSCP profiles. Experiments performed with different soil matrices revealed the repeatability, efficiency, and consistency of CE-SSCP. Studies on PCR and CE-SSCP using single-species genomic DNA as a matrix showed that several ribotypes may migrate at the same position, and also that single species can produce double peaks. Thus, the extrapolation between number of peaks and number of species remains difficult. Additionally, peak detection is limited by the analysis software. We conclude that the presented method, including CE-SSCP and the analyzing step, is a simple and effective technique to obtain the molecular signature of a given soil sample.

Stimulation of pyrene mineralization in freshwater sediments by bacterial and plant bioaugmentation.

As a means to study the fate of polycyclic aromatic hydrocarbons (PAHs) in freshwater sediments, pyrene mineralization was examined in microcosms spiked with [14C]pyrene. Some microcosms were planted with reeds (Phragmites australis) and/or inoculated with a pyrene-degrading strain, Mycobacterium sp. 6PY1. Mineralization rates recorded over a 61 d period showed that reeds promoted a significant enhancement of pyrene degradation, which possibly resulted from a root-mediated increase of oxygen diffusion into the sediment layer, as indicated by in situ redox measurements. In inoculated microcosms, mineralization reached a higher level in the absence (8.8%) than in the presence of plants (4.4%). Mineralization activity was accompanied by the release of water-soluble pyrene oxidation products, the most abundant of which was identified as 4,5-diphenanthroic acid. Pyrene was recovered from plant tissues, including stems and leaves, at concentrations ranging between 40 and 240 microg/g of dry mass. Plants also accumulated labeled oxidation products likely derived from microbial degradation. Pyrene-degrading strains were 35-70-fold more abundant in inoculated than in noninoculated microcosms. Most of the pyrene-degrading isolates selected from the indigenous microflora were identified as Mycobacterium austroafricanum strains. Taken together, the results of this study show that plants or PAH-degrading bacteria enhance pollutant removal, but their effects are not necessarily cumulative.

Identification of pyrene-induced proteins in Mycobacterium sp. strain 6PY1: evidence for two ring-hydroxylating dioxygenases.

In this study, the enzymes involved in polycyclic aromatic hydrocarbon (PAH) degradation were investigated in the pyrene-degrading Mycobacterium sp. strain 6PY1. [(14)C]pyrene mineralization experiments showed that bacteria grown with either pyrene or phenanthrene produced high levels of pyrene-catabolic activity but that acetate-grown cells had no activity. As a means of identifying specific catabolic enzymes, protein extracts from bacteria grown on pyrene or on other carbon sources were analyzed by two-dimensional gel electrophoresis. Pyrene-induced proteins were tentatively identified by peptide sequence analysis. Half of them resembled enzymes known to be involved in phenanthrene degradation, with closest similarity to the corresponding enzymes from Nocardioides sp. strain KP7. The genes encoding the terminal components of two distinct ring-hydroxylating dioxygenases were cloned. Sequence analysis revealed that the two enzymes, designated Pdo1 and Pdo2, belong to a subfamily of dioxygenases found exclusively in gram-positive bacteria. When overproduced in Escherichia coli, Pdo1 and Pdo2 showed distinctive selectivities towards PAH substrates, with the former enzyme catalyzing the dihydroxylation of both pyrene and phenanthrene and the latter preferentially oxidizing phenanthrene. The catalytic activity of the Pdo2 enzyme was dramatically enhanced when electron carrier proteins of the phenanthrene dioxygenase from strain KP7 were coexpressed in recombinant cells. The Pdo2 enzyme was purified as a brown protein consisting of two types of subunits with M(r)s of about 52,000 and 20,000. Immunoblot analysis of cell extracts from strain 6PY1 revealed that Pdo1 was present in cells grown on benzoate, phenanthrene, or pyrene and absent in acetate-grown cells. In contrast, Pdo2 could be detected only in PAH-grown cells. These results indicated that the two enzymes were differentially regulated depending on the carbon source used for growth.

Fungal flora and ochratoxin a production in grapes and musts from france.

Eleven samples of grapes and musts used in red table wines were investigated for the occurrence of potential ochratoxin A (OTA)-producing molds. From these samples, 59 filamentous fungi and 2 yeasts were isolated. Among the 30 genera isolated, Deuteromycetes were the most frequent (70%) followed by Ascomycetes (10%). Six of the eleven grapes samples were contaminated by potentially ochratoxinogenic strains (Penicillium chrysogenum and Aspergillus carbonarius). When cultivated in vitro on solid complex media, the 14 strains of A. carbonarius produced OTA. No other species produced OTA under the same conditions. Among must samples, eight of eleven were found to be contaminated by OTA (concentrations from <10 to 461 ng/L). There is a strong correlation between the presence of ochratoxin-producing strains on grapes and OTA in musts. These findings should be connected with the OTA contamination of human blood in these areas and in France.