Evaluating the biodegradation of aromatic hydrocarbons by monitoring of several functional genes

Various microbial activities determine the effectiveness of bioremediation processes. In this work, we evaluated the feasibility of gene array hybridization for monitoring the efficiency of biodegradation processes. Biodegradation of ¹⁴C-labelled naphthalene and toluene by the aromatic hydrocarbon-degrading Pseudomonas putida F1, P. putida mt-2 and P. putida G7 was followed in mixed liquid culture microcosm by a preliminary, nylon membrane-based gene array. In the beginning of the study, toluene was degraded rapidly and increased amount of toluene degradation genes was detected by the preliminary gene array developed for the study. After toluene was degraded, naphthalene mineralization started and the amount of naphthalene degradation genes increased as biodegradation proceeded. The amount of toluene degradation genes decreased towards the end of the study. The hybridization signal intensities determined by preliminary gene array were in good agreement with mineralization of naphthalene and toluene and with the amount of naphthalene dioxygenase and toluene dioxygenase genes quantified by dot blot hybridization. The clear correlation between the results obtained by the preliminary array and the biodegradation process suggests that gene array methods can be considered as a promising tool for monitoring the efficiency of biodegradation processes.     

Catabolism of flavonol glucosides in plant cell suspension cultures

Catabolism of flavonol glucosides was investigated in plant cell suspension cultures using kaempferol 3-O-β-d-glucoside and kaempferol 7-O-β-d-glucoside labelled with ¹⁴C either in the glucose or in the flavonol moiety. Catabolic rates of glucosides were compared with those of free glucose and kaempferol. All substrates were degraded efficiently by cell cultures of mungbean, soybean, garbanzo bean and parsley. Based on ¹⁴CO₂-formation, glucose from position 3 of kaempferol is 3–5 times more rapidly metabolized than that from position 7. The flavonol nucleus from both isomers is, however, oxidized to the same extent with a considerable portion of the flavonol being incorporated into insoluble polymeric cell material.     

Molecular cloning and characterization of ARO7-OSM2, a single yeast gene necessary for chorismate mutase activity and growth in hypertonic medium

Summary The chorismate mutase structural gene, ARO7, which is necessary for both phenylalanine and tyrosine biosynthesis was cloned by complementation in yeast. Genetic analysis showed that ARO7 was identical to a gene necessary for growth in hypertonic medium, OSM2, which mapped nearby. After restriction mapping and subcloning of the plasmid, the cloned gene was used to detect mRNA levels in several growth conditions. Enzyme activities were measured in various genotypes. At our level of detection ARO7-OSM2 is a low level constitutively expressed gene.     

Catabolisme des steroides chez les Nocardia

Nocardia restrictus and N. corallina oxidize the A ring of 4-hydroxy-4-cholesten-3-one and a 3,5-seco-4-nor-3-keto-5-oic acid is formed. The enzymes necessary to this reaction are induced and their biosynthesis is suppressed by chloramphenicol. The catabolism of the aliphatic side chain at C-17 involves a cleavage between C-24 and C-25 and the liberation of propionic acid.     

A thermostable shikimate 5-dehydrogenase from the archaeon Archaeoglobus fulgidus

Shikimate 5-dehydrogenase (SKDH; EC 1.1.1.25) catalyzes the reversible reduction of 3-dehydroshikimate to shikimate and is a key enzyme in the aromatic amino acid biosynthesis pathway. The shikimate 5-dehydrogenase gene, aroE, from Archaeoglobus fulgidus was cloned and overexpressed in Escherichia coli. The recombinant enzyme purified as a homodimer and yielded a maximum specific activity of 732 U/mg at 87 degrees C (with NADP+ as coenzyme). Apparent Km values for shikimate, NADP+, and NAD+ were estimated at 0.17+/-0.03 mM, 0.19+/-0.01 mM, and 11.4+/-0.4 mM, respectively. The half-life of the A. fulgidus SKDH is 2 h at the assay temperature (87 degrees C) and 17 days at 60 degrees C. Addition of 1 M NaCl or KCl stabilized the enzyme's half-life to approximately 70 h at 87 degrees C and approximately 50 days at 60 degrees C. This work presents the first kinetic analysis of an archaeal SKDH.     

Desiccation resistance and water balance in southern African keratin beetles (Coleoptera, Trogidae): the influence of body size and habitat

Desiccation resistance and water balance were examined in the adults of seven trogid species, which differed both in body size and in the habitats from which they were collected. Body water contents (51–58% fresh mass) and desiccation rates at 27 °C (0.00026–0.00093 g h⁻¹) in these species were very similar to those of unrelated, similar-sized beetles from arid habitats. The keratin beetles differed markedly from many other adult Coleoptera by virtue of their very high haemolymph osmolality and inability to regulate haemolymph osmolality, and to catabolise lipids for water production, during desiccation. Like most other insects, the xeric trogid species had lower rates of water loss and longer survival times than trogids from mesic areas. This was due both to lower rates of water loss and to the larger body size of species from the more arid areas. Because absolute body water content was higher in large beetles than in small ones, larger body size conferred higher desiccation resistance on the very large Kalahari desert species. This suggests that there may be strong selection for large body size in such insects from arid areas. Most ecological and ecophysiological investigations of geographical variation in body size, and the species-body size distribution, have focused on temperature and metabolic rate as explanatory variables. This study suggests that attention should also be given to desiccation resistance. Accepted: 29 September 1997     

Enzymic arrangement and allosteric regulation of the aromatic amino acid pathway in Neisseria gonorrhoeae

The pathway construction and allosteric regulation of phenylalanine and tyrosine biosynthesis was examined in Neisseria gonorrhoeae. A single 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase enzyme sensitive to feedback inhibition by l-phenylalanine was found. Chorismate mutase and prephenate dehydratase appear to co-exist as catalytic components of a bifunctional enzyme, known to be present in related genera. The latter enzyme activities were both feedback inhibited by l-phenylalanine. Prephenate dehydratase was strongly activated by l-tyrosine. NAD⁺-linked prephenate dehydrogenase and arogenate dehydrogenase activities coeluted following ion-exchange chromatography, suggesting their identity as catalytic properties of a single broad-specificity cyclohexadienyl dehydrogenase. Each dehydrogenase activity was inhibited by 4-hydroxyphenylpyruvate, but not by l-tyrosine. Two aromatic aminotransferases were resolved, one preferring the l-phenylalanine:2-ketoglutarate substrate combination and the other preferring the l-tyrosine: 2-ketoglutarate substrate combination. Each aminotransferase was also able to transaminate prephenate. The overall picture of regulation is one in which l-tyrosine modulates l-phenylalanine synthesis via activation of prephenate dehydratase. l-Phenylalanine in turn regulates early-pathway flow through inhibition of DAHP synthase. The recent phylogenetic positioning of N. gonorrhoeae makes it a key reference organism for emerging interpretations about aromatic-pathway evolution.     

Specific rates of substrate oxidation and product formation in autotrophically growingChromatium vinosum cultures

Eighty-six species of fungi belonging to sixty-four genera were examined for their ability to metabolize naphthalene. Analysis by thin-layer and high pressure liquid chromatography revealed that naphthalene metabolism occurred in forty-seven species belonging to thirty-four genera from the major fungal taxa. All organisms tested from the order Mucorales oxidized naphthalene with species of Cunninghamella, Syncephalastrum and Mucor showing the greatest activity. Significant metabolism was also observed with Neurospora crassa, Claviceps paspali and four species of Psilocybe. The predominant metabolite formed by most organisms was 1-naphthol. Other products identified were, 4-hydroxy-1-tetralone, trans-1,2-dihydroxy-1,2-dihydronaphthalene, 2-naphthol, 1,2-and 1,4-naphthoquinone.