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.Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous contaminants in soils and sediments and are of environmental concern because of their mutagenic and/or carcinogenic effects. While low-molecular-weight PAHs (composed of two or three rings) are readily degraded by bacteria, PAHs consisting of four rings or more are recalcitrant to biodegradation and persist in the environment (6, 41). The biodegradation of lowmolecular-weight PAHs, especially naphthalene, has been extensively studied with pseudomonads, leading to a good understanding of the bacterial catabolic pathway (42). On the other hand, relatively little information is available on the metabolism of high-molecular-weight PAHs (20). A number of bacterial isolates capable of pyrene mineralization have been described. Most of them are actinomycetes and belong to the genus Mycobacterium (2, 7, 37), Rhodococcus (4, 40), or Gordonia (21). A few pyrene-degrading strains have been identified as gram-negative species, including Stenotrophomonas maltophilia, Pseudomonas fluorescens (3), Sphingomonas paucimobilis (22), and Burkholder...

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.