Metabolism of Halophenols by 2,4,5-trichlorophenoxyacetic acid-degrading Pseudomonas cepacia

Resting cells of 2,4,5-trichlorophenoxyacetic acid-grown Pseudomonas cepacia AC1100 were able to completely and rapidly dechlorinate several chlorine-substituted phenols, including 2,4,5-trichlorophenol, 2,3,4,6-tetrachlorophenol, and pentachlorophenol. Several other trichlorophenols were only partially dechlorinated. The evidence suggests that 2,4,5-trichlorophenol is an intermediate in the degradation of 2,4,5-trichlorophenoxyacetic acid by strain AC1100. Moreover, although strain AC1100 was isolated by selection for growth on a chlorinated aromatic compound, brominated and fluorinated analogs were efficiently dehalogenated by strain AC1100 resting cells, whereas an iodinated analog was poorly dehalogenated.     

Regulation of 2,4,5-trichlorophenoxyacetic acid and chlorophenol metabolism in Pseudomonas cepacia AC1100

The expression of the degradative genes encoding 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), 2,4,5-trichlorophenol (2,4,5-TCP), and pentachlorophenol (PCP) dechlorination in a 2,4,5-T-degrading strain of Pseudomonas cepacia was examined during growth on alternate carbon sources. The dechlorination mechanisms for all three compounds were expressed in 2,4,5-T- and 2,4,5-TCP-grown cells but were not expressed in cells grown on succinate, glucose, or lactate. The addition of 2,4,5-TCP or PCP to cells grown on succinate or lactate resulted in the expression of the 2,4,5-TCP dechlorination mechanism in resting cells after 1-h lag. This expression was prevented by the presence of chloramphenicol in the resting cell suspension. Succinate-plus-PCP-grown resting cells preincubated with 2,4,5-TCP fully induced the trichlorophenol dechlorination system and partially induced the PCP dechlorination system. Preincubation of succinate-plus-PCP-grown resting cells with PCP induced neither the 2,4,5-TCP nor the PCP dechlorinating system. Succinate-grown resting cells converted 2,4,5-T to 2,4,5-TCP even in the presence of chloramphenicol. Thus, the data indicate that the enzyme(s) which converts 2,4,5-T to 2,4,5-TCP is constitutively expressed, whereas those that convert 2,4,5-TCP to central intermediates are induced by 2,4,5-TCP but not by 2,4,5-T or PCP and are repressed in the presence of an alternate carbon source.     

Induction and Catabolite Repression of beta-Glucosidase Synthesis in Myceliophthora thermophila D-14 (= ATCC 48104)

beta-Glucosidase activity in Myceliophthora thermophila D-14 (= ATCC 48104) was inducible and was produced in culture filtrate during growth with various inducers, of which PNPG (p-nitrophenyl-beta-d-glucoside) was the most efficient. Induction of beta-glucosidase also occurred when the organism was grown in medium supplemented with different carbon sources. Carboxymethyl cellulose, cellobiose, and Solka-Floc were found effective for induction of enzyme biosynthesis. The addition of glucose to the culture medium severely repressed beta-glucosidase synthesis, which could not be reversed by exogenous cyclic AMP or dibutyryl cyclic AMP.     

Purification and characterization of guanosine diphospho-D-mannose dehydrogenase. A key enzyme in the biosynthesis of alginate by Pseudomonas aeruginosa

Alginate-producing Pseudomonas aeruginosa are usually associated with the cystic fibrosis lung environment and contribute to the high mortality rates observed among these patients. The present paper describes the purification and enzymatic properties of guanosine diphospho-D-mannose dehydrogenase (EC 1.1.1.132), a key enzyme in alginate biosynthesis by mucoid P. aeruginosa. The enzyme was overproduced using a plasmid vector containing algD (the gene encoding this enzyme) under control of the tac promoter. It was purified from cell-free lysates by lowering the pH to 5.0, heating the extract to 57.5 degrees C for 10 min, and discarding the protein pellet. The enzyme was selectively precipitated from the supernatant fraction with 45% acetone, resuspended in a 100 mM triethanolamine acetate buffer, pH 7.6, and ultimately purified by Bio-Sil TSK-400 gel filtration chromatography. The subunit molecular weight (Mr 48,000) as well as the N-terminal amino acid sequence corresponded to those predicted from the DNA sequence of algD. The native protein migrated as a hexamer of 290,000 molecular weight upon Bio-Gel A-1.5m gel filtration chromatography. Kinetic analysis demonstrated an apparent Km of 14.9 microM for the substrate GDP-D-mannose and 185 microM for the cofactor NAD+. GDP-D-mannuronic acid was identified as the enzyme reaction product. Several compounds (including GMP, ATP, GDP-D-glucose, and maltose) were found to inhibit enzymatic activity. GMP, the most potent of these inhibitors, exhibited competitive inhibition with an apparent Ki of 22.7 microM. Enzyme activity was also sensitive to the sulfhydryl group modifying agents iodoacetamide and p-hydroxymercuribenzoate. The addition of excess dithiothreitol restored enzyme activity, suggesting a possible involvement of cysteine residues in enzymatic activity.     

Law-medicine interfacing: patenting of human genes and mutations

Mutations, Single Nucleotide Polymorphisms (SNPs), deletions and genetic rearrangements in specific genes in the human genome account for not only our physical characteristics and behavior, but can lead to many in-born and acquired diseases. Such changes in the genome can also predispose people to cancers, as well as significantly affect the metabolism and efficacy of many drugs, resulting in some cases in acute toxicity to the drug. The testing of the presence of such genetic mutations and rearrangements is of great practical and commercial value, leading many of these genes and their mutations/deletions and genetic rearrangements to be patented. A recent decision by a judge in the Federal District Court in the Southern District of New York, has created major uncertainties, based on the revocation of BRCA1 and BRCA2 gene patents, in the eligibility of all human and presumably other gene patents. This article argues that while patents on BRCA1 and BRCA2 genes could be challenged based on a lack of utility, the patenting of the mutations and genetic rearrangements is of great importance to further development and commercialization of genetic tests that can save human lives and prevent suffering, and should be allowed.     

The dynamics of nutrient utilization and growth of apple root stock ‘M9 EMLA’ in temporary versus continuous immersion bioreactors

The present study investigated the dynamics of nutrient utilization and various growth and physiological parameters during in vitro proliferation of apple root stock ‘M9 EMLA’ in two different bioreactor systems, i.e. temporary and continuous immersions. Individual shoots obtained from temporary immersion system had higher dry mass and were of better quality than those obtained from continuous immersion. In continuous immersion bioreactor, apple shoots appeared to utilize more nutrients from liquid culture medium than that from temporary immersion. The shoot growth was limited by the availability of phosphate and nitrogen in continuous immersion system. The shoots produced in temporary immersion bioreactor showed higher photosynthetic rate, maximum quantum yield of photosystem-II and slow but steady rate of nutrient absorption, indicating the occurrence of higher photomixotrophic metabolism. The study also showed that high level of antioxidant scavenging enzymes in shoots grown in continuous immersion system induced physiological changes to foster adaptation to stresses.     

Nucleoside diphosphate kinase: role in bacterial growth, virulence, cell signalling and polysaccharide synthesis

Nucleoside diphosphate kinase (Ndk) is an important enzyme that generates nucleoside triphosphates (NTPs) or their deoxy derivatives by terminal phosphotransfer from an NTP such as ATP or GTP to any nucleoside diphosphate or its deoxy derivative. As NTPs, particularly GTP, are important for cellular macromolecular synthesis and signalling mechanisms, Ndk plays an important role in bacterial growth, signal transduction and pathogenicity. Specific examples of the role of Ndk in regulating growth, NTP formation and cell surface polysaccharide synthesis in two respiratory tract pathogens, Pseudomonas aeruginosa and Mycobacterium tuberculosis , are discussed.