Biotransformations Of Fluoroaromatic Compounds: Accumulation Of Hydroxylated Products From 3-Fluorophthalic Acid Using Mutant Strains Of Pseudomonas Testosteroni

Biotransformations of 3-fluorophthalic acid have been investigated using blocked mutants of Pseudomonas testosteroni that are defective in the metabolism of phthalic acid (benzene-1,2-dicar-boxyfic acid). Mutant strains were grown with L-glutamic acid in the presence of 3-fluorophthalic acid as inducer of phthalic acid catabolic enzymes. Products that accumulated in the medium were isolated, purified and identified as the fluoroanalogues of those produced from phthalic acid by the same strains. The previously undescribed fluorochemicals cis-3-fluoro-4,5-dihydro-4,5-dihydroxyphthalic acid (VI) and 3-fluoro-4,5-dihydroxyphthalic acid (VII) have been obtained by biotransformation of 3-fluorophthalic acid, and 3-fluoro-5-hydroxyphthalic acid (X) from (VI) by freeze drying. In addition, samples of 2-fluoro-3,4-dihydroxybenzoic acid (2-fluoroprotocatechuic acid, VIII) and 3-fluoro-4,Sdi-hydroxybenzoic acid (5-fluoroprotocatechuic acid, IX) were obtained with a mutant deficient in the ring-fission enzyme, showing that the fluorine substituent in their precursor substrate (VII) is not recognized by the decarboxylase of the pathway, which shows no preference for which carboxyl group is removed. These studies of 3-fluorophthalic acid catabolism demonstrate the opportunities available for the production of novel fluorochemicals in reasonable yields by microbial transformations.     

Specificity of monohydric phenol oxidations by meta cleavage pathways in Pseudomonas aeruginosa T1

Summary The oxidation of several mono-hydric phenols by wild type and mutant strains of Pseudomonas aeruginosa T1 has been studied. The data suggest, that a non-specific enzyme sequence of the meta cleavage pathway is induced by all of these phenols, which can catalyze the oxidation of phenol and its analogues to pyruvate, a fatty acid and a carbonyl compound, according to the general scheme of Dagley et al. (1964). Mutants unable to grow on phenol (hydroxylase-negative), have been isolated, and they are also unable to grown on or oxidize the cresols and the xylenols. Revertants of these mutants regain the capacity to grow on all these phenols and are indistinguishable from the wild type. Induced-substrate relationships for the earlier enzymes of the pathway have been determined, e.g., phenol in addition to catechol and the methylcatechols is an inducer for catechol 2,3-oxygenase. Analysis of the enzymic content of cells grown in a variety of steadystate conditions shows (a) that the ratio of the specific activities of the phenol hydroxylase and catechol 2,3-oxygenase is constant for each of their analogous substrates; and (b) that induction and catabolite repression of catechol 2,3-oxygenase and the muconic semialdehyde hydrolyase are coordinate, but that control of the phenol hydroxylase is independent.Howard Hughes Medical Institute Investigator.     

Sucrose utilization by Zymomonas mobilis: formation of a levan

1. Molar growth-yield coefficients of Zymomonas mobilis for glucose, fructose, glucose plus fructose, and sucrose are reported. Yield coefficients for sucrose are appreciably lower than those for the equivalent concentrations of glucose plus fructose. 2. Only 2.6% of [U-(14)C]glucose supplied in the growth medium is incorporated into cell substance by Z. mobilis utilizing glucose as the energy source. 3. During growth on sucrose a levan is formed. It has been characterized and shown to resemble other bacterial levans. 4. Levan formation from sucrose could be demonstrated with both washed cell suspensions and cell extracts of Z. mobilis. 5. Sucrose phosphorylase could not be demonstrated in extracts of the organism.     

2-Naphthoate catabolic pathway in Burkholderia strain JT 1500.

Burkholderia strain (JT 1500), able to use 2-naphthoate as the sole source of carbon, was isolated from soil. On the basis of growth characteristics, oxygen uptake experiments, enzyme assays, and detection of intermediates, a degradation pathway of 2-naphthoate is proposed. The features of this pathway are convergent with those for phenanthrene. We propose a pathway for the conversion of 2-naphthoate to 1 mol (each) of pyruvate, succinate, and acetyl coenzyme A and 2 mol of CO2. During growth in the presence of 2-naphthoate, six metabolites were detected by thin-layer chromatography, high-performance liquid chromatography, and spectroscopy. 1-Hydroxy-2-naphthoate accumulated in the culture broth during growth on 2-naphthoate. Also, the formation of 2'-carboxybenzalpyruvate, phthalaldehydate, phthalate, protocatechuate, and beta-carboxy-cis,cis-muconic acid was demonstrated. (1R,2S)-cis-1,2-Dihydro-1,2-dihydroxy-2-naphthoate was thus considered an intermediate between 2-naphthoate and 1-hydroxy-2-naphthoate, but it was not transformed by whole cells or their extracts. We conclude that this diol is not responsible for the formation of 1-hydroxy-2-naphthoate from 2-naphthoate but that one of the other three diastereomers is not eliminated as a potential intermediate for a dehydration reaction.     

Stereochemical course of two arene-cis-diol dehydrogenases specifically induced in Pseudomonas putida.

Catabolism of nonphenolic arenes is frequently initiated by dioxygenases, yielding single isomer products with two adjacent hydroxylated asymmetric centers. The next enzymic reaction dehydrogenates these cyclic cis-diols, with aromatization yielding catechols for ring cleavage. There are two stereochemical questions to answer. (i) To which face of NAD is hydride transferred giving NADH? (ii) Which hydrogen of the arene-cis-diols is donated to NAD? We report the results of 1H nuclear magnetic resonance [1H NMR] experiments for two diol dehydrogenases induced during growth of Pseudomonas putida PaW1(TOL) and JT105 with p-xylene and p-toluate, respectively. per-[2H5]benzoate-1,2-dihydrodiol and per-[2H7]- and specifically [2H]p-toluate-2,3-dihydrodiols were the substrates used to examine this by 1H NMR, as the two protons of the prochiral center (C-4 of the nicotinamide ring) are easily distinguished in the region of 2.6 to 2.7 ppm. We found that with the partially purified dehydrogenases (i) 2H from the (2R) center of per-(1S,2R)-benzoate-1,2-dihydrodiol was donated to the Si-face of NAD to give (4S)-NAD2H; (ii) p-toluate-2,3-diol dehydrogenase also provided exclusively (4S)-NAD2H, but the 2H was transferred from both the 2- and 3-C atoms of (2S,3R)-p-toluate-2,3-dihydrodiol with specifically deuterated species in approximately equal amounts; and (iii) the unexpected lack of stereo- and regioselectivity of p-toluate-2,3-diol dehydrogenase was supported by kinetic isotope effect studies.     

The pericentriolar material in Chinese hamster ovary cells nucleates microtubule formation

The structure and function of the centrosomes from Chinese hamster ovary (CHO) cells were investigated by electron microscopy of negatively stained wholemount preparations of cell lysates. Cells were trypsinized from culture dishes, lysed with Triton X-100, sedimented onto ionized, carbon-coated grids, and negatively stained with phosphotungstate. The centrosomes from both interphase and dividing cells consisted of pairs of centrioles, a fibrous pericentriolar material, and a group of virus-like particles which were characteristic of the CHO cells and which served as markers for the pericentriolar material. Interphase centrosomes anchored up to two dozen microtubules when cells were lysed under conditions which preserved native microtubules. When Colcemid-blocked mitotic cells, initially devoid of microtubules, were allowed to recover for 10 min, microtubules formed at the pericentriolar material, but not at the centrioles. When lysates of Colcemid-blocked cells were incubated in vitro with micotubule protein purified from porcine brain tissue, up to 250 microtubules assembled at the centrosomes, similar to the number of microtubules that would normally form at the centrosome during cell division. A few microtubules could also be assembled in vitro onto the ends of isolated centrioles from which the pericentriolar material had been removed, forming characteristic axoneme- like bundles. In addition, microtubules; were assembled onto fragments of densely staining, fibrous material which was tentatively identified as periocentriolar material by its association of CHO can initiate and anchor microtubules both in vivo and in vitro.     

Committee for the study of the Scottish Flora

Background: Damage to alpine plant communities is likely to occur when hikers and pack animals trample vegetation. Currently, there is limited research that quantifies and compares impacts from these activities.

Aims: A manipulative experimental protocol was used to assess damage to alpine meadows by pack animals and hikers in the Aconcagua Provincial Park, Andes, Argentina.

Methods: Vegetation height, overall cover, cover of dominant species and species richness were measured immediately after, and 2 weeks after different numbers of passes (0, 25, 100 and 300) by hikers or pack animals in an experiment, using a randomised block design.

Results: Pack animals had two to three times the impact of hiking on the meadows, with greater reductions in plant height, the cover of one of the dominant sedges and declines in overall vegetation cover after 300 passes. Impacts of pack animals were also apparent at lower levels of use than for hikers. These differences occurred despite the meadow community having relatively high resistance to trampling due to the traits of one of the dominant sedges (Carex gayana).

Conclusions: Pack animals caused more damage than hikers to the alpine meadow, but the scale of the difference in short-term impacts depends on the characteristics of the plant community, the amount of use and the vegetation parameters measured. Use of the meadows by hikers and pack animals should be minimised as these meadows are scarce, and have high conservation values.     

A novel beta-diketone-cleaving enzyme from Acinetobacter johnsonii: acetylacetone 2,3-oxygenase

A novel Fe+Zn containing oxygenase from Acinetobacter johnsonii catalyses 2,3-cleavage of acetylacetone to acetate and methylglyoxal has been purified. The stoichiometry of reactants and products conforms to a classical dioxygenase. The pure protein is a homotetramer of 64kD with variable amounts of Fe(2+) and Zn(2+). Activity of the enzyme is more closely related to the Fe(2+) content than to the amount of protein. A purification of acetylacetone 2,3-oxygenase, some of its physical properties, and the preference for some analogous substrates are described.