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Volume 144,
Issue 6,
1998
Volume 144, Issue 6, 1998
- Physiology And Growth
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Biodegradation kinetics of toluene, m-xylene, p-xylene and their intermediates through the upper TOL pathway in Pseudomonas putida (pWWO)
Pseudomonas putida mt-2, harbouring TOL plasmid pWWO, is capable of degrading toluene and a range of di- and tri-alkylbenzenes. In this study, chemostat-grown cells (D = 0.05 h-1, toluene or m-xylene limitation) of this strain were used to assess the kinetics of the degradation of toluene, m-xylene, p-xylene, and a number of their pathway intermediates. The conversion kinetics for the three hydrocarbons showed significant differences: the maximal conversion rates were rather similar [11-14 mmol h-1 (g dry wt)-1] but the specific affinity (the slope of the v vs s curve near the origin) of the cells for toluene [1300 I (g dry wt)-1 h-1] was only 5% and 14% of those found for m-xylene and p-xylene, respectively. Consumption kinetics of mixtures of the hydrocarbons confirmed that xylenes are strongly preferred over toluene at low substrate concentrations. The maximum flux rates of pathway intermediates through the various steps of the TOL pathway as far as ring cleavage were also determined. Supply of 0-5 mM 3-methylbenzyl alcohol or 3-methylbenzaidehyde to fully induced cells led to the transient accumulation of 3-methylbenzoate. Accumulation of the corresponding carboxylic acid (benzoate) was also observed after pulses of benzyl alcohol and benzaldehyde, which are intermediates in toluene catabolism. Analysis of consumption and accumulation rates for the various intermediates showed that the maximal rates at which the initial monooxygenation step and the conversion of the carboxylic acids by toluate 1,2-dioxygenase may occur are two- to threefold lower than those measured for the two intermediate dehydrogenation steps.
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Cyanide hydrolysis in a cyanide-degrading bacterium, Pseudomonas stutzeri AK61, by cyanidase
More LessThe cyanide-degrading bacterial strain AK61 was isolated from waste water at a metal-plating plant. The isolated strain was characterized by Gram-staining, quinone analysis, fatty acid profile and the API 20NE identification system, and identified as Pseudomonas stutzeri. Whole cells were able to degrade cyanide rapidly in a 1 mM solution containing no organic substances, and produced ammonia as a product. The induction of the cyanide-degrading activity of P. stutzeri AK61 did not depend on the presence of cyanide in the culture medium during growth. The cyanide-degrading enzyme was purified approximately 49-fold from a cell extract of P. stutzeri AK61. The enzyme had a Km of 1.7 mM for cyanide and a specific activity of 54.6 μmol ammonia produced min-1. The activity of the enzyme was optimal at 30 °C and pH 7.5. The results of SDS-PAGE, gel-filtration chromatography and NH2-terminal amino acid sequence analysis of the enzyme indicated that the functional enzyme was an aggregated protein consisting of a 38 kDa polypeptide. Like cyanidase (cyanide dihydratase), it was shown that the enzyme catalysed the hydrolysis of cyanide to ammonia and formate.
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Distribution of 14C-labelleed carbon from glucose and glutamate during anaerobic growth of Saccharomyces cerevisiae
More LessThe distribution of carbon from glucose and glutamate was studied using anaerobically grown Saccharomyces cerevisiae. The yeast was grown on glucose (20 g I-1) as the carbon/energy source and glutamic acid (3.5 g I1) as additional carbon and sole nitrogen source. The products formed were identified using labelled [U-14C]glucose or [U-14C]glutamic acid. A seldom-reported metabolite in S. cerevisiae, 2–hydroxyglutarate, was found in significant amounts. It is suggested that 2-hydroxyglutarate is formed from the reduction of 2-oxoglutarate in a reaction catalysed by a dehydrogenase. Succinate, 2-oxoglutarate and 2-hydroxyglutarate were found to be derived exclusively from glutamate. Based on radioactivity measurements, 55%, 17% and 14% of the labelled glutamate was converted to 2-oxoglutarate, succinate and 2-hydroxyglutarate, respectively, and 55%, 9% and 3% of the labelled glucose was converted to ethanol, glycerol and pyruvate, respectively. No labelled glucose was converted to 2-oxoglutarate, succinate or 2-hydroxyglutarate. Furthermore, very little of the evolved CO2 was derived from glutamate. Separation of the amino acids from biomass by paper chromatography revealed that the glutamate family of amino acids (glutamic acid, glutamine, proline, arginine and lysine) originated almost exclusively from the carbon skeleton of glutamic acid. It can be concluded that the carbon flow follows two separate paths, and that the only major reactions utilized in the tricarboxylic acid (TCA) cycle are those reactions involved in the conversion of 2-oxoglutarate to succinate.
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A novel quantitative mating assay for the fungal pathogen Cryptococcus neoformans provides insight into signalling pathways responding to nutrients and temperature
More LessCryptococcus neoformans is a fungal pathogen that causes a lethal meningitis in immunocompromised individuals. Several factors are associated with virulence of this fungus, including its mating type; however, the mechanism by which mating type affects virulence is unknown. C. neoformans is a basidiomycete that exists in two mating types called a and a α that can fuse to form an a/α dikaryon. A mating assay was developed that allowed a quantitative analysis of cryptococcal mating physiology. Interestingly, the efficiency of mating appeared to be dependent on temperature, being highest at 30 °C and almost completely absent at 37 °C. Thus, while mating type itself may be associated with virulence (which must occur at 37 °C), the ability to mate is probably not a virulence factor. Mating efficiency was increased by altering the carbon or nitrogen sources to give so-called starvation media. The addition of various drugs also seemed to alter the frequency of mating, depending on the composition of mating medium. The data suggested that cAMP, 8-bromo-cAMP and caffeine increased mating on starvation medium but only cAMP and 8-bromo-cAMP stimulated mating on rich medium; caffeine was unable to stimulate mating on rich medium. Aluminium fluoride, an activator of heterotrimeric GTP-binding proteins (G-proteins), was also found to stimulate mating, suggesting the involvement of a G-protein that may regulate the level of cAMP.
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- Systematics
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Psychroflexus torquis gen. nov., sp. nov. a psychrophilic species from Antarctic sea ice, and reclassification of Flavobacterium gondwanense (Dobson et al. 1993) as Psychroflexus gondwanense gen. nov., comb. nov.
A group of sea-ice-derived psychrophilic bacterial strains possessing the unusual ability to synthesize the polyunsaturated fatty acids eicosapentaenoic acid (20:5.3) and arachidonic acid (20:4.6) belong to the Family Flavobacteriaceae (Flexibacter-Bacteroides-Flavobacterium phylum), according to 16S rRNA sequence analysis. Surprisingly, the isolates were also found to cluster closely to the moderately halophilic and psychrotrophic species [Flavobacterium] gondwanense (sequence similarity 97-8-98-1 %). The whole-cell fatty acid profiles of this group and [Flavobacterium] gondwanense were very similar and distinct from other related flavobacteria. The sea ice strains and [Flavobacterium] gondwanense differed substantially in terms of ecophysiology, possibly representing divergent adaptations to sympagic and planktonic marine habitats, respectively. Evidence based on phylogeny and fatty acid profiles supports the conclusion that the taxa are close relatives distinct from other bacterial groups. It is thus proposed that the sea ice strains represent a novel taxon designated Psychroflexus torquis gen. nov., sp., nov. (type strain ACAM 623T) while [Flavobacterium] gondwanense becomes Psychroflexus gondwanense gen. nov., comb. nov.
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- Genome Analysis
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The yvsA-yvqA (293°-289°) region of the Bacillus subtilis chromosome containing genes involved in metal ion uptake and a putative sigma factor
The region between yvsA (293°) and yvqA (289°) of the Bacillus subtilis chromosome has been sequenced within the framework of the B. subtilis 168 international sequencing programme. A primary analysis of the 42 ORFs identified in this 43 kb region is presented. The region included a high proportion of genes that did not show homology with genes in other bacteria. The identified ORFs showed homology to proteins involved in the transport of metal ions, two-component signal transducers, ATP-binding-cassette-type transporters and a sigma factor.
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