Biodegradation of polyfluorinated biphenyl in bacteria

Fluorinated aromatic compounds are significant environmental pollutants, and microorganisms play important roles in their biodegradation. The effect of fluorine substitution on the transformation of fluorobiphenyl in two bacteria was investigated. Pseudomonas pseudoalcaligenes KF707 and Burkholderia xenovorans LB400 used 2,3,4,5,6-pentafluorobiphenyl and 4,4??-difluorobiphenyl as sole sources of carbon and energy. The catabolism of the fluorinated compounds was examined by gas chromatography?Cmass spectrometry and fluorine-19 nuclear magnetic resonance spectroscopy (¹⁹F NMR), and revealed that the bacteria employed the upper pathway of biphenyl catabolism to degrade these xenobiotics. The novel fluorometabolites 3-pentafluorophenyl-cyclohexa-3,5-diene-1,2-diol and 3-pentafluorophenyl-benzene-1,2-diol were detected in the supernatants of biphenyl-grown resting cells incubated with 2,3,4,5,6-pentafluorobiphenyl, most likely as a consequence of the actions of BphA and BphB. 4-Fluorobenzoate was detected in cultures incubated with 4,4??-difluorobiphenyl and ¹⁹F NMR analysis of the supernatant from P. pseudoalcaligenes KF707 revealed the presence of additional water-soluble fluorometabolites.     

Abscisic Acid Translocation and Metabolism in Soybeans following Depodding and Petiole Girdling Treatments

It was found earlier that depodding and girdling treatments which obstruct translocation, result in increased leaf AbA levels and partial stomatal closure. In the present work (±) [2-¹⁴C]abscisic acid (AbA) was introduced into leaves and the mass, and radioactivity of AbA and AbA-metabolites were analyzed following translocation obstruction to determine whether the increased AbA was due to higher rates of synthesis, or lower rates of catabolism or export. The (±) [2-¹⁴C]AbA was introduced into soybean (Merr.) leaves by injection into the petiole region. AbA and AbA-metabolites (phaseic acid [PA], dihydrophaseic acid [DPA], AbA-conjugate, and an unknown metabolite) were separated with preparative high performance liquid chromatography. Methyl esters of AbA (free and that released after hydrolysis of AbA-conjugate), PA and DPA were determined with gas chromatography using electron capture detection.     

Abscisic Acid and photosynthesis in isolated leaf mesophyll cell

Abscisic acid (AbA) treatments of concentrations of up to 135 micromolar did not inhibit photosynthesis in enzymatically isolated leaf mesophyll cells of Phaseolus vulgaris, Nicotiana tabacum, and Lycopersicum esculentum over periods of up to 5 hours. Thin slices of leaves preincubated in hypertonic solutions identical to those used to isolate cells were shown to synthesize AbA rapidly, although accumulation of AbA in the cells was low due to extensive release of the newly synthesized AbA into the medium. The levels of endogenously made AbA in leaf cells of Phaseolus vulgaris rose from a low of 0.27 micromolar to a high of 6.74 micromolar during 2 hours preincubation. Exogenously applied AbA can be taken up by the cells as was demonstrated using ¹⁴[C]AbA. Thus, AbA applied at concentrations 19 times higher than endogenous levels does not change the rate of photosynthesis.     

The P450-4 Gene of Gibberella fujikuroi Encodes ent-Kaurene Oxidase in the Gibberellin Biosynthesis Pathway

At least five genes of the gibberellin (GA) biosynthesis pathway are clustered on chromosome 4 of Gibberella fujikuroi; these genes encode the bifunctional ent-copalyl diphosphate synthase/ent-kaurene synthase, a GA-specific geranylgeranyl diphosphate synthase, and three cytochrome P450 monooxygenases. We now describe a fourth cytochrome P450 monooxygenase gene (P450-4). Gas chromatography-mass spectrometry analysis of extracts of mycelia and culture fluid of a P450-4 knockout mutant identified ent-kaurene as the only intermediate of the GA pathway. Incubations with radiolabeled precursors showed that the metabolism of ent-kaurene, ent-kaurenol, and ent-kaurenal was blocked in the transformants, whereas ent-kaurenoic acid was metabolized efficiently to GA₄. The GA-deficient mutant strain SG139, which lacks the 30-kb GA biosynthesis gene cluster, converted ent-kaurene to ent-kaurenoic acid after transformation with P450-4. The B1-41a mutant, described as blocked between ent-kaurenal and ent-kaurenoic acid, was fully complemented by P450-4. There is a single nucleotide difference between the sequence of the B1-41a and wild-type P450-4 alleles at the 3′ consensus sequence of intron 2 in the mutant, resulting in reduced levels of active protein due to a splicing defect in the mutant. These data suggest that P450-4 encodes a multifunctional ent-kaurene oxidase catalyzing all three oxidation steps between ent-kaurene and ent-kaurenoic acid.     

A study on Candida biofilm growth characteristics and its susceptibility to aureobasidin A

Background

Biofilm is known to contribute to the antifungal resistance of Candida yeasts. Aureobasidin A (AbA), a cyclic depsipeptide targeting fungal sphingolipid biosynthesis, has been shown to be effective against several Candida species.

ABA 9'-hydroxylation is catalyzed by CYP707A in Arabidopsis

Abscisic acid (ABA) catabolism is important for regulating endogenous ABA levels. To date, most effort has focused on catabolism of ABA to phaseic acid (PA), which is generated spontaneously after 8′-hydroxylation of ABA by cytochrome P450s in the CYP707A subfamily. Neophaseic acid (neoPA) is another well-documented ABA catabolite that is produced via ABA 9′-hydroxylation, but the 9′-hydroxylase has not yet been defined. Here, we show that endogenous neoPA levels are reduced in loss-of-function mutants defective in CYP707A genes. In addition, in planta levels of both neoPA and PA are reduced after treatment of plants with uniconazole-P, a P450 inhibitor. These lines of evidence suggest that CYP707A genes also encode the 9′-hydroxylase required for neoPA synthesis. To test this, in vitro enzyme assays using microsomal fractions from CYP707A-expressing yeast strains were conducted and these showed that all four Arabidopsis CYP707As are 9′-hydroxylases, although this activity is minor. Collectively, our results demonstrate that ABA 9′-hydroxylation is catalyzed by CYP707As as a side reaction.     

Sphingolipid biosynthesis is required for polar growth in the dimorphic phytopathogen Ustilago maydis

Sphingolipids participate in different biological processes such as cell growth, differentiation, virulence, etc. In recent years these lipids are becoming prime targets for antifungal research. In the dimorphic phytopathogen Ustilago maydis inhibition of sphingolipid biosynthesis with the antifungal drug aureobasidin A (AbA) did not alter cell cycle but caused loss of cell polarity and growth inhibition. The addition of AbA had effects on the assembly of sterol-rich domains (SRDs), which concentrate at the sites of active growth in U. maydis, the actin cytoskeleton, FM4-64 trafficking, and the localization of the motor protein Myo5 and the septin Sep1. However, AbA did not affect the localization of the scaffold proteins Bem1 and Spa2. The septin Sep1 and SRDs showed interdependent partial colocalization suggesting that both act in conjunction to maintain the stability of the polar axis. Time course experiments suggest that AbA provokes cell depolarization in three steps: disassembly of SRDs from the tips, delocalization of some polarity proteins, and lastly appearance of medial division septa.     

Isolation and characterization of thermophilic bacteria capable of degrading dehydroabietic acid.

Using a semi-continuous enrichment method, we isolated two thermophilic bacterial strains, which could completely degrade abietane resin acids, including dehydroabietic acid (DhA). Strain DhA-73, isolated from a laboratory-scale bioreactor treating bleached kraft mill effluent at 55 degrees C, grew on DhA as sole carbon source; while DhA-71, isolated from municipal compost, required dilute tryptic soy broth for growth on DhA. DhA-71 grew on DhA from 30 degrees C to 60 degrees C with maximum growth at 50 degrees C; while, DhA-73 grew on DhA from 37 degrees C to 60 degrees C with maximum growth at 55 degrees C. At 55 degrees C, the doubling times for DhA-71 and DhA-73 were 3.3 and 3.7 h, respectively. DhA-71 and DhA-73 had growth yields of 0.26 and 0.19 g of protein per g of DhA, respectively. During growth on DhA, both strains converted DhA to CO2, biomass, and dissolved organic carbon. Analyses of the 16S-rDNA sequences of these two strains suggest that they belong to two new genera in the Rubrivivax subgroup of the beta subclass of the Proteobacteria. Strains DhA-71 and DhA-73 are the first two bacteria isolated and characterized that are capable of biodegradation of resin acids at high temperatures. This study provided direct evidence for biodegradation of resin acids and feasibility for biotreatment of pulp mill effluent at elevated temperatures.     

Effect of Abscisic Acid and Other Plant Hormones on Growth of Apical and Lateral Buds of Seedlings

The effect of abscisic acid (AbA) on the growth of lateral and apical buds was studied in seedlings of Pisum sativum and some other species. The hormone was applied in three different ways: 1) directly to the lateral bud on the second node of decapitated pea seedlings as 5 μI droplets in an ethanolic solution; 2) to the cut surface of decapitated seedlings: 3) to the apical bud of intact plants. AbA directly applied in amounts of 5 to 0.1 μg to the lateral bud of the second node of decapitated seedlings had a strong inhibitory effect on the bud. Application to the cut surface of seedlings decapitated about 5 mm above the second node resulted in slight inhibition of the lateral bud on the second node and in growth promotion of the bud on the first node. When AbA at 10 to 0.1 μg was applied to the apical bud of intact seedlings, the growth of this bud was inhibited but the lateral buds grew out. It is concluded that the release of the lateral buds from apícal dominance is the result of the inhibitory effect of AbA on growth of the apical bud and of low transport of AbA. This conclusion is supported by application of GA₃ and IAA, individually and each combined with AbA.     

AUR1, a novel gene conferring aureobasidin resistance onSaccharomyces cerevisiae: a study of defective morphologies in Aur1p-depleted cells

Aureobasidin A (AbA), a cyclic depsipeptide produced byAureobasidium pullulans R106, is highly toxic to fungi includingSaccharomyces cerevisiae. We isolated several dominant mutants ofS. cerevisiae which are resistant to more than 25 µg/ml of AbA. From a genomic library of one suchAUR1 mutant, theAUR1 ^R (foraureobasidinresistant) mutant gene was isolated as a gene that confers resistance to AbA on wild-type cells. Its nucleotide sequence showed that the predicted polypeptide is a hydrophobic protein composed of 401 amino acids, which contains several possible transmembrane domains and at least one predicted N-linked glycosylation site. Comparison of the mutant gene with the wild-typeaur1 ⁺ gene revealed that the substitution of Phe at position 158 by Tyr is responsible for acquisition of AbA resistance. We suggest that the gene product of the wild-typeaur1 ⁺ is a target for AbA on the basis of following results. Firstly, cells that overexpress the wild-typeaur1 ⁺ gene become resistant to AbA, just as cells with anAUR1 ^R mutation do. Secondly, disruption of theaur1 ⁺ gene demonstrated that it is essential for growth. Thirdly, in the cells with a disruptedaur1 locus, pleiotropic morphological changes including disappearance of microtubules, degradation of tubulin and abnormal deposition of chitin were observed. Some of these abnormalities are also observed when wild-type cells are treated with AbA. The abnormality in microtubules suggests that the Aur1 protein is involved in microtubule organization and stabilization.     

Bacteria obtained from a sequencing batch reactor that are capable of growth on dehydroabietic acid.

Eleven isolates capable of growth on the resin acid dehydroabietic acid (DhA) were obtained from a sequencing batch reactor designed to treat a high-strength process stream from a paper mill. The isolates belonged to two groups, represented by strains DhA-33 and DhA-35, which were characterized. In the bioreactor, bacteria like DhA-35 were more abundant than those like DhA-33. The population in the bioreactor of organisms capable of growth on DhA was estimated to be 1.1 x 10(6) propagules per ml, based on a most-probable-number determination. Analysis of small-subunit rRNA partial sequences indicated that DhA-33 was most closely related to Sphingomonas yanoikuyae (Sab = 0.875) and that DhA-35 was most closely related to Zoogloea ramigera (Sab = 0.849). Both isolates additionally grew on other abietanes, i.e., abietic and palustric acids, but not on the pimaranes, pimaric and isopimaric acids. For DhA-33 and DhA-35 with DhA as the sole organic substrate, doubling times were 2.7 and 2.2 h, respectively, and growth yields were 0.30 and 0.25 g of protein per g of DhA, respectively. Glucose as a cosubstrate stimulated growth of DhA-33 on DhA and stimulated DhA degradation by the culture. Pyruvate as a cosubstrate did not stimulate growth of DhA-35 on DhA and reduced the specific rate of DhA degradation of the culture. DhA induced DhA and abietic acid degradation activities in both strains, and these activities were heat labile. Cell suspensions of both strains consumed DhA at a rate of 6 mumol mg of protein-1 h-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Whole-cell hydroxylation of n-octane by Escherichia coli strains expressing the CYP153A6 operon

CYP153A6 is a well-studied terminal alkane hydroxylase which has previously been expressed in Pseudomonas putida and Escherichia coli by using the pCom8 plasmid. In this study, CYP153A6 was successfully expressed in E. coli BL21(DE3) by cloning the complete operon from Mycobacterium sp. HXN-1500, also encoding the ferredoxin reductase and ferredoxin, into pET28b(+). LB medium with IPTG as well as auto-induction medium was used to express the proteins under the T7 promoter. A maximum concentration of 1.85?μM of active CYP153A6 was obtained when using auto-induction medium, while with IPTG induction of LB cultures, the P450 concentration peaked at 0.6–0.8?μM. Since more biomass was produced in auto-induction medium, the specific P450 content was often almost the same, 0.5–1.0?μmol P450 g _DCW ^?1 , for both methods. Analytical scale whole-cell biotransformations of n-octane were conducted with resting cells, and it was found that high P450 content in biomass did not necessarily result in high octanol production. Whole cells from LB cultures induced with IPTG gave higher specific and volumetric octanol formation rates than biomass from auto-induction medium. A maximum of 8.7?g octanol L _BRM ^?1 was obtained within 24?h (0.34?g L _BRM ^?1 ?h^?1) with IPTG-induced cells containing only 0.20?μmol P450 g _DCW ^?1 , when glucose (22?g L _BRM ^?1 ) was added for cofactor regeneration.