The EmhABC efflux pump in Pseudomonas fluorescens LP6a is involved in naphthalene tolerance but not efflux

The EmhABC efflux pump in Pseudomonas fluorescens LP6a effluxes polycyclic aromatic hydrocarbons (PAHs) such as phenanthrene and anthracene but not naphthalene. We previously showed that the presence of EmhABC decreased the efficiency of phenanthrene biodegradation. In this study, we determined whether P. fluorescens LP6a tolerance to naphthalene is a function of the EmhABC efflux pump and how its presence affects the efficiency of naphthalene biodegradation. Growth, membrane fatty acid (FA) composition, and cell morphology showed that 5-mmol?L^?1 naphthalene is inhibitory to P. fluorescens LP6a strains. The deleterious effect of naphthalene is suppressed in the presence of EmhABC, which suggests that, although naphthalene is not effluxed by EmhABC, this efflux pump is involved in tolerance of naphthalene toxicity. LP6a mutants lacking the EmhB efflux pump were unable to convert cis-unsaturated FAs to cyclopropane FAs, indicating that naphthalene interferes with the formation of cyclopropane FAs and supporting the proposal that EmhABC is involved in FA turnover in P. fluorescens LP6a strains. The EmhABC efflux pump increases the efficiency of naphthalene metabolism in strain LP6a, which may make naphthalene efflux unnecessary. Thus, the activity of hydrocarbon efflux pumps may be an important factor to consider when selecting bacterial strains for bioremediation or biocatalysis of PAHs.     

Uptake and Active Efflux of Polycyclic Aromatic Hydrocarbons by Pseudomonas fluorescens LP6a

The mechanism of transport of polycyclic aromatic hydrocarbons (PAHs) by Pseudomonas fluorescens LP6a, a PAH-degrading bacterium, was studied by inhibiting membrane transport and measuring the resulting change in cellular uptake. Three cultures were used: wild-type LP6a which carried a plasmid for PAH degradation, a transposon mutant lacking the first enzyme in the pathway for PAH degradation, and a cured strain without the plasmid. Washed cells were mixed with aqueous solutions of radiolabelled PAH; then the cells were removed by centrifugation, and the concentrations of PAH in the supernatant and the cell pellet were measured. The change in the pellet and supernatant concentrations after inhibitors of membrane transport (azide, cyanide, or carbonyl cyanide m-chlorophenyl hydrazone) were added indicated the role of active transport. The data were consistent with the presence of two conflicting transport mechanisms: uptake by passive diffusion and an energy-driven efflux system to transport PAHs out of the cell. The efflux mechanism was chromosomally encoded. Under the test conditions used, neither uptake nor efflux of phenanthrene by P. fluorescens LP6a was saturated. The efflux mechanism showed selectivity since phenanthrene, anthracene, and fluoranthene were transported out of the cell but naphthalene was not.     

Saturable, Energy-Dependent Uptake of Phenanthrene in Aqueous Phase by Mycobacterium sp. Strain RJGII-135

The mechanism of uptake of phenanthrene by Mycobacterium sp. strain RJGII-135, a polycyclic hydrocarbon-degrading bacterium, was examined with cultures grown on phenanthrene (induced for phenanthrene metabolism) and acetate (uninduced). Washed cells were suspended in aqueous solutions of [9-¹⁴C]phenanthrene, and then the cells were collected by filtration. Low-level steady-state ¹⁴C concentrations in uninduced cells were achieved within the first 15 s of incubation. This immediate uptake did not show saturation kinetics and was not susceptible to inhibitors of active transport, cyanide and carbonyl cyanide m-chlorophenylhydrazone. These results indicated that phenanthrene enters rapidly into the cells by passive diffusion. However, induced cells showed cumulative uptake over several minutes. The initial uptake rates followed saturation kinetics, with an apparent affinity constant (K_t) of 26 ± 3 nM (mean ± standard deviation). Uptake of phenanthrene by induced cells was strongly inhibited by the inhibitors. Analysis of cell-associated ¹⁴C-labeled compounds revealed that the concurrent metabolism during uptake was rapid and was not saturated at the substrate concentrations tested, suggesting that the saturable uptake observed reflects membrane transport rather than intracellular metabolism. These results were consistent with the presence of a saturable, energy-dependent mechanism for transport of phenanthrene in induced cells. Moreover, the kinetic data for the cumulative uptake suggested that phenanthrene is specifically bound by induced cells, based on its saturation with an apparent dissociation constant (K_d) of 41 ± 21 nM (mean ± standard deviation). Given the low values of K_t and K_d, Mycobacterium sp. strain RJGII-135 may use a high-affinity transport system(s) to take up phenanthrene from the aqueous phase.     

PAH Degradation Capacity of Soil Microbial Communities—Does It Depend on PAH Exposure?

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous pollutants of the environment. But is their microbial degradation equally wide in distribution? We estimated the PAH degradation capacity of 13 soils ranging from pristine locations (total PAHs ≈ 0.1 mg kg^?1) to heavily polluted industrial sites (total PAHs ≈ 400 mg kg^?1). The size of the pyrene- and phenanthrene-degrading bacterial populations was determined by most probable number (MPN) enumeration. Densities of phenanthrene degraders reflected previous PAH exposure, whereas pyrene degraders were detected only in the most polluted soils. The potentials for phenanthrene and pyrene degradation were measured as the mineralization of ¹⁴C-labeled spikes. The time to 10% mineralization of added ¹⁴C phenanthrene and ¹⁴C pyrene was inversely correlated with the PAH content of the soils. Substantial ¹⁴C phenanthrene mineralization in all soils tested, including seven unpolluted soils, demonstrated that phenanthrene is not a suitable model compound for predicting PAH degradation in soils. ¹⁴C pyrene was mineralized by all Danish soil samples tested, regardless of whether they were from contaminated sites or not, suggesting that in industrialized areas the background level of pyrene is sufficient to maintain pyrene degradation traits in the gene pool of soil microorganisms. In contrast, two pristine forest soils from northern Norway and Ghana mineralized little ¹⁴C pyrene within the 140-day test period. Mineralization of phenanthrene and pyrene by all Danish soils suggests that soil microbial communities of inhabited areas possess a sufficiently high PAH degradation capacity to question the value of bioaugmentation with specific PAH degraders for bioremediation.     

Identification of a Novel Metabolite in the Degradation of Pyrene by Mycobacterium sp. Strain AP1: Actions of the Isolate on Two- and Three-Ring Polycyclic Aromatic Hydrocarbons

Mycobacterium sp. strain AP1 grew with pyrene as a sole source of carbon and energy. The identification of metabolites accumulating during growth suggests that this strain initiates its attack on pyrene by either monooxygenation or dioxygenation at its C-4, C-5 positions to give trans- or cis-4,5-dihydroxy-4,5-dihydropyrene, respectively. Dehydrogenation of the latter, ortho cleavage of the resulting diol to form phenanthrene 4,5-dicarboxylic acid, and subsequent decarboxylation to phenanthrene 4-carboxylic acid lead to degradation of the phenanthrene 4-carboxylic acid via phthalate. A novel metabolite identified as 6,6′-dihydroxy-2,2′-biphenyl dicarboxylic acid demonstrates a new branch in the pathway that involves the cleavage of both central rings of pyrene. In addition to pyrene, strain AP1 utilized hexadecane, phenanthrene, and fluoranthene for growth. Pyrene-grown cells oxidized the methylenic groups of fluorene and acenaphthene and catalyzed the dihydroxylation and ortho cleavage of one of the rings of naphthalene and phenanthrene to give 2-carboxycinnamic and diphenic acids, respectively. The catabolic versatility of strain AP1 and its use of ortho cleavage mechanisms during the degradation of polycyclic aromatic hydrocarbons (PAHs) give new insight into the role that pyrene-degrading bacterial strains may play in the environmental fate of PAH mixtures.     

Identification and characterization of the emhABC efflux system for polycyclic aromatic hydrocarbons in Pseudomonas fluorescens cLP6a

The hydrocarbon-degrading environmental isolate Pseudomonas fluorescens LP6a possesses an active efflux mechanism for the polycyclic aromatic hydrocarbons phenanthrene, anthracene, and fluoranthene but not for naphthalene or toluene. PCR was used to detect efflux pump genes belonging to the resistance-nodulation-cell division (RND) superfamily in a plasmid-cured derivative, P. fluorescens cLP6a, which is unable to metabolize hydrocarbons. One RND pump, whose gene was identified in P. fluorescens cLP6a and was designated emhB, showed homology to the multidrug and solvent efflux pumps in Pseudomonas aeruginosa and Pseudomonas putida. The emhB gene is located in a gene cluster with the emhA and emhC genes, which encode the membrane fusion protein and outer membrane protein components of the efflux system, respectively. Disruption of emhB by insertion of an antibiotic resistance cassette demonstrated that the corresponding gene product was responsible for the efflux of polycyclic aromatic hydrocarbons. The emhB gene disruption did not affect the resistance of P. fluorescens cLP6a to tetracycline, erythromycin, trimethoprim, or streptomycin, but it did decrease resistance to chloramphenicol and nalidixic acid, indicating that the EmhABC system also functions in the efflux of these compounds and has an unusual selectivity. Phenanthrene efflux was observed in P. aeruginosa, P. putida, and Burkholderia cepacia but not in Azotobacter vinelandii. Polycyclic aromatic hydrocarbons represent a new class of nontoxic, highly hydrophobic compounds that are substrates of RND efflux systems, and the EmhABC system in P. fluorescens cLP6a has a narrow substrate range for these hydrocarbons and certain antibiotics.     

Polyphasic characterization of four soil-derived phenanthrene-degrading Acidovorax strains and proposal of Acidovorax carolinensis sp. nov.

Four bacterial strains identified as members of the Acidovorax genus were isolated from two geographically distinct but similarly contaminated soils in North Carolina, USA, characterized, and their genomes sequenced. Their 16S rRNA genes were highly similar to those previously recovered during stable-isotope probing (SIP) of one of the soils with the polycyclic aromatic hydrocarbon (PAH) phenanthrene. Heterotrophic growth of all strains occurred with a number of organic acids, as well as phenanthrene, but no other tested PAHs. Optimal growth occurred aerobically under mesophilic temperature, neutral pH, and low salinity conditions. Predominant fatty acids were C_16:1ω7c/C_16:1ω6c, C_16:0, and C_18:1ω7c, and were consistent with the genus. Genomic G + C contents ranged from 63.6 to 64.2%. A combination of whole genome comparisons and physiological analyses indicated that these four strains likely represent a single species within the Acidovorax genus. Chromosomal genes for phenanthrene degradation to phthalate were nearly identical to highly conserved regions in phenanthrene-degrading Delftia, Burkholderia, Alcaligenes, and Massilia species in regions flanked by transposable or extrachromosomal elements. The lower degradation pathway for phenanthrene metabolism was inferred by comparisons to described genes and proteins. The novel species Acidovorax carolinensis sp. nov. is proposed, comprising the four strains described in this study with strain NA3^T as the type strain (=LMG 30136, =DSM 105008).     

Effects of Soil Water Content on Biodegradation of Phenanthrene in a Mixture of Organic Contaminants

Phenanthrene biodegradation was investigated at different soil water contents [0.11, 0.22, 0.33, 0.44 g H₂O (g soil)^?1] to determine the effects of water availability on biodegradation rate. A subsurface horizon of Kennebec silty loam soil was used in this study. [9-¹⁴C] phenanthrene was dissolved in a mixture of organic contaminants that consisted of 76% decane, 6% ρ-xylene, 6% phenanthrene, 6% pristane, and 6% naphthalene, and then added to the soil. The highest rate of mineralization, in which 0.23% of the [9-¹⁴C] phenanthrene degraded to ¹⁴CO₂ after 66 days of incubation, was observed at the soil water content of 0.44 g H₂O/g dry soil. Most of the ¹⁴C remained in the soil as the parent compound or as nonextractable compounds by acetonitrile at the highest water content. Concentrations of nonextractable compounds increased with water content, but residual extractable phenanthrene decreased significantly with increasing water content, which presumably indicates that bio-transformation occurred. The mineralization analysis of radiolabeled 9th carbon in phenanthrene underestimated phenanthrene biodegradation. The strong adsorption and low solubility of phenanthrene contributed to the low mineralization of phenanthrene 9th carbon. The other components were subject to higher biological and abiotic dissipation processes with increasing soil water content.     

Insights into the genome and proteome of <Emphasis Type="Italic">Sphingomonas paucimobilis</Emphasis> strain 20006FA involved in the regulation of polycyclic aromatic hydrocarbon degradation

In order to study the mechanisms regulating the phenanthrene degradation pathway and the intermediate-metabolite accumulation in strain S. paucimobilis 20006FA, we sequenced the genome and compared the genome-based predictions to experimental proteomic analyses. Physiological studies indicated that the degradation involved the salicylate and protocatechuate pathways, reaching 56.3% after 15 days. Furthermore, the strain degraded other polycyclic aromatic hydrocarbons (PAH) such as anthracene (13.1%), dibenzothiophene (76.3%), and fluoranthene. The intermediate metabolite 1-hydroxy-2-naphthoic acid (HNA) accumulated during phenanthrene catabolism and inhibited both bacterial growth and phenanthrene degradation, but exogenous-HNA addition did not affect further degradation. Genomic analysis predicted 126 putative genes encoding enzymes for all the steps of phenanthrene degradation, which loci could also participate in the metabolism of other PAH. Proteomic analysis identified enzymes involved in 19 of the 23 steps needed for the transformation of phenanthrene to trichloroacetic-acid intermediates that were upregulated in phenanthrene cultures relative to the levels in glucose cultures. Moreover, the protein-induction pattern was temporal, varying between 24 and 96 h during phenanthrene degradation, with most catabolic proteins being overexpressed at 96 h—e. g., the biphenyl dioxygenase and a multispecies (2Fe–2S)-binding protein. These results provided the first clues about regulation of expression of phenanthrene degradative enzymes in strain 20006FA and enabled an elucidation of the metabolic pathway utilized by the bacterium. To our knowledge the present work represents the first investigation of genomic, proteomic, and physiological studies of a PAH-degrading Sphingomonas strain.     

Degradation of phenanthrene by the endophytic fungus <Emphasis Type="Italic">Ceratobasidum stevensii</Emphasis> found in <Emphasis Type="Italic">Bischofia polycarpa</Emphasis>

Some strains of white rot fungi, non-lignolytic fungi and litter-decomposing basidiomycetes have been recognized as PAH degraders. The purpose of our research was to enlarge the scope of PAH-degrading fungi and explore the huge endophytic microorganism resource for bioremediation of PAHs. In this study, phenanthrene was used as a model PAHs compound. Nine strains of endophytic fungi isolated from four kinds of plant from Eupharbiaceae were screened for degradation of phenanthrene. The endophytic fungus Ceratobasidum stevensii (strain B6) isolated from Bischofia polycarpam showed high degradation efficiency and was selected for further studies. Into the fungal culture, 100 mg l⁻¹ phenanthrene was added, and after 10 days of incubation, about 89.51% of the phenanthrene was removed by strain B6. Extracellular ligninolytic enzyme activities of strain B6 were tested. The results showed that manganese peroxidase [MnP] was the predominant ligninolytic enzyme and that its production was greatly induced by the presence of phenanthrene. To confirm the involvement of MnP in phenanthrene degradation, promotion and inhibition studies on MnP in different concentration level of Mn²⁺ and NaN₃ were performed. Additionally, fungal mycelium-free and resuspended experiments were carried out. The results showed no apparent correlation between MnP activity and phenanthrene degradation. The mycelium and fresh medium were the crucial factors affecting the degradation of phenanthrene. To date, this is the first report on PAH degradation by Ceratobasidum stevensii. This study suggests that endophytic fungi might be a novel and important resource for microorganisms that have PAH-degrading capabilities.     

Isolation of Soil Bacteria Adapted To Degrade Humic Acid-Sorbed Phenanthrene

The goal of these studies was to determine how sorption by humic acids affected the bioavailability of polynuclear aromatic hydrocarbons (PAHs) to PAH-degrading microbes. Micellar solutions of humic acid were used as sorbents, and phenanthrene was used as a model PAH. Enrichments from PAH-contaminated soils established with nonsorbed phenanthrene yielded a total of 25 different isolates representing a diversity of bacterial phylotypes. In contrast, only three strains of Burkholderia spp. and one strain each of Delftia sp. and Sphingomonas sp. were isolated from enrichments with humic acid-sorbed phenanthrene (HASP). Using [¹⁴C]phenanthrene as a radiotracer, we verified that only HASP isolates were capable of mineralizing HASP, a phenotype hence termed “competence.” Competence was an all-or-nothing phenotype: noncompetent strains showed no detectable phenanthrene mineralization in HASP cultures, but levels of phenanthrene mineralization effected by competent strains in HASP and NSP cultures were not significantly different. Levels and rates of phenanthrene mineralization exceeded those predicted to be supported solely by the metabolism of phenanthrene in the aqueous phase of HASP cultures. Thus, competent strains were able to directly access phenanthrene sorbed by the humic acids and did not rely on desorption for substrate uptake. To the best of our knowledge, this is the first report of (i) a selective interaction between aerobic bacteria and humic acid molecules and (ii) differential bioavailability to bacteria of PAHs sorbed to a natural biogeopolymer.     

Impact of Irradiation and Polycyclic Aromatic Hydrocarbon Spiking on Microbial Populations in Marine Sediment for Future Aging and Biodegradability Studies

Experiments were carried out to develop methods to generate well-characterized, polycyclic aromatic hydrocarbon (PAH)-spiked, aged but minimally altered sediments for fate, biodegradation, and bioavailability experiments. Changes in indigenous bacterial populations were monitored in mesocosms constructed of relatively clean San Diego Bay sediments, with and without exposure to gamma radiation, and then spiked with five different PAHs and hexadecane. While phenanthrene and chrysene degraders were present in the unspiked sediments and increased during handling, PAH spiking of nonirradiated sediments led to dramatic increases in their numbers. Phenotypic characterization of isolates able to grow on phenanthrene or chrysene placed them in several genera of marine bacteria: Vibrio, Marinobacter or Cycloclasticus, Pseudoalteromonas, Marinomonas, and Halomonas. This is the first time that marine PAH degraders have been identified as the latter two genera, expanding the diversity of marine bacteria with this ability. Even at the highest irradiation dose (10 megarads), heterotrophs and endospore formers reappeared within weeks. However, while bacteria from the unirradiated sediments had the capacity to both grow on and mineralize ¹⁴C-labeled phenanthrene and chrysene, irradiation prevented the reappearance of PAH degraders for up to 4 months, allowing spikes to age onto the sediments, which can be used to model biodegradation in marine sediments.     

Effect of rapeseed oil on the degradation of polycyclic aromatic hydrocarbons in soil by Rhodococcus wratislaviensis

The effect of rapeseed oil (0, 0.1 and 1% w/w) on the degradation of polycyclic aromatic hydrocarbons (PAH) by Rhodococcus wratislaviensis was studied in soils artificially contaminated with phenanthrene, anthracene, pyrene and benzo(a)pyrene (50 mg kg⁻¹ each), during 49 days at 30 °C. Without or with 0.1% of rapeseed oil, R. wratislaviensis degraded >90% of phenanthrene and anthracene in 14 days and mineralised approx. 23% of ¹⁴C-phenanthrene. The native microflora degraded pyrene (90% degradation; 75% mineralisation) and benzo(a)pyrene (30% degradation, no mineralisation). With 1% rapeseed oil, R. wratislaviensis degraded only 66% of the phenanthrene and mineralised 12.4%, and had no effect on other PAH, while degradation by the native microflora was inhibited. On the other hand, the addition of 1% oil promoted degradation of benzo(a)pyrene (75%) and anthracene (90%) and anthraquinone was produced at high concentrations and accumulated. Two distinct processes gave degradation of PAH, one biological and one abiotic. Biological processes mainly degraded phenanthrene and pyrene, either by R. wratislaviensis or by the indigenous microflora. Benzo(a)pyrene was degraded mainly by an abiotic process in the presence of 1% rapeseed oil. Anthracene was degraded by a combination of both processes.PAH are often found in contaminated soils and there is the need of developing techniques that can be applied in the remediation of these sites, where PAH, specially those with high molecular weight, pose health and environmental risks. There is a continuous search for efficient microorganisms able to degrade these pollutants and for methods to enhance their degradation and bioavailability, e.g. by the use of vegetable oils. This paper presents a novel process for the degradation of PAH by a combined biological/abiotic system.     

Two-Stage Mineralization of Phenanthrene by Estuarine Enrichment Cultures

The polycyclic aromatic hydrocarbon phenanthrene was mineralized in two stages by soil, estuarine water, and sediment microbial populations. At high concentrations, phenanthrene was degraded, with the concomitant production of biomass and accumulation of Folin-Ciocalteau-reactive aromatic intermediates. Subsequent consumption of these intermediates resulted in a secondary increase in biomass. Analysis of intermediates by high-performance liquid chromatography, thin-layer chromatography, and UV absorption spectrometry showed 1-hydroxy-2-naphthoic acid (1H2NA) to be the predominant product. A less pronounced two-stage mineralization pattern was also observed by monitoring ¹⁴CO₂ production from low concentrations (0.5 mg liter⁻¹) of radiolabeled phenanthrene. Here, mineralization of ¹⁴C-labeled 1H2NA could explain the incremental ¹⁴CO₂ produced during the later part of the incubations. Accumulation of 1H2NA by isolates obtained from enrichments was dependent on the initial phenanthrene concentration. The production of metabolites during polycyclic aromatic hydrocarbon biodegradation is discussed with regard to its possible adaptive significance and its methodological implications.     

Study on the biochemical basis of mefloquine resistant Plasmodium falciparum

Increase in drug detoxification and alteration of drug uptake and efflux of Plasmodium falciparum were investigated for their possible association with mefloquine (MQ) resistance in five different clones of P. falciparum from Thailand (T994b₃, K1CB₂, PR70CB₁, PR71CB₂ and TM₄CB8-2.2.3). Fifty percent inhibitory concentration (IC₅₀) values from these five clones varied between 30- and 50-fold. Regarding the detoxification mechanism, the ability of P. falciparum clones to biotransform MQ was shown in vitro by parasite microsomal protein prepared from parasite infected red blood cells protein (30 μg), NADPH (1 nM) and phosphate buffer pH 7.4, carried out at 37 °C with agitation. Radiolabelled unmetabolized MQ and possible metabolite(s) generated from the reaction was extracted into ethylacetate and separated by radiometric-HPLC after 1 h. All clones were capable of converting MQ into carboxymefloquine (CMQ), which is the main metabolite in human plasma. In addition, another unidentified metabolite eluted at 4.2 min on the chromatograph could be detected from the incubation reaction. This metabolite has never been detected in human liver microsomes before. There was no significant difference in the percentages of CMQ formed in the resistant (T994_b3, PR₇₀CB₁, PR₇₁CB₂) and sensitive (TM₄CB8-2.2.3, K1CB₂) clones. Another possible mechanism, i.e., alteration in the accumulation of MQ in the parasites was investigated in vitro using [¹⁴C]MQ as a tracer. The time courses of [¹⁴C]MQ uptake and efflux were generally characterized by two phases. A trend of increased efflux of [¹⁴C]MQ was observed in the resistant compared with sensitive clones.     

Rhizoremediation of Trichloroethylene by a Recombinant, Root-Colonizing Pseudomonas fluorescens Strain Expressing Toluene ortho-Monooxygenase Constitutively

Trichloroethylene (TCE) was removed from soils by using a wheat rhizosphere established by coating seeds with a recombinant, TCE-degrading Pseudomonas fluorescens strain that expresses the tomA⁺ (toluene o-monooxygenase) genes from Burkholderia cepacia PR1₂₃(TOM_23C). A transposon integration vector was used to insert tomA⁺ into the chromosome of P. fluorescens 2-79, producing a stable strain that expressed constitutively the monooxygenase at a level of 1.1 nmol/min · mg of protein (initial TCE concentration, 10 μM, assuming that all of the TCE was in the liquid) for more than 280 cell generations (36 days). We also constructed a salicylate-inducible P. fluorescens strain that degraded TCE at an initial rate of 2.6 nmol/min · mg of protein in the presence of 10 μM TCE [cf. B. cepacia G4 PR1₂₃(TOM_23C), which degraded TCE at an initial rate of 2.5 nmol/min · mg of protein]. A constitutive strain, P. fluorescens 2-79TOM, grew (maximum specific growth rate, 0.78 h⁻¹) and colonized wheat (3 × 10⁶ CFU/cm of root) as well as wild-type P. fluorescens 2-79 (maximum specific growth rate, 0.77 h⁻¹; level of colonization, 4 × 10⁶ CFU/cm of root). Rhizoremediation of TCE was demonstrated by using microcosms containing the constitutive monooxygenase-expressing microorganism, soil, and wheat. These closed microcosms degraded an average of 63% of the initial TCE in 4 days (20.6 nmol of TCE/day · plant), compared to the 9% of the initial TCE removed by negative controls consisting of microcosms containing wild-type P. fluorescens 2-79-inoculated wheat, uninoculated wheat, or sterile soil.     

Plasmalemma Redox Chain and H Extrusion: II. Respiratory and Metabolic Changes Associated with Fusicoccin-Induced and with Ferricyanide-Induced H Extrusion

Ferricyanide reduction by Elodea densa leaves is associated with a release of protons in the cytoplasm, a fraction of the increase in protons being then extruded by the ATP-driven proton pump (20). The data presented here show that ferricyanide induces a marked increase in O₂ uptake, additive to that induced by fusicoccin plus K⁺, and here interpreted as depending on the utilization of ATP by the H⁺ pump. Glucose 6-phosphate and malate levels are markedly increased by fusicoccin plus K⁺. The simultaneous presence of ferricyanide reduces by about 50% the increase of malate, while it completely suppresses that of glucose 6-phosphate. The ferricyanide-induced decrease of malate is interpreted as due to the acidification of the cytosol associated with ferricyanide reduction, while the more marked decrease of glucose 6-phosphate might depend in part on the pH change and in part on a faster oxidation of this substrate. In fact, ferricyanide reduction is accompanied by a marked decrease of the incorporation into RNA ribose of C-1 as compared with C-2 of [¹⁴C]glucose. This suggests a stimulation of the release of C-1 as CO₂ at the level of the glucose 6-phosphate oxidation pathway, as expected if NADPH was the electron donor for ferricyanide reduction. These results are interpreted as confirming that the H⁺ efflux associated with ferricyanide reduction depends on the activation of the ATP-driven plasmalemma H⁺ pump. They also suggest that NADPH is used as an electron donor to some initial component of the plasmalemma redox system.     

Analysis of Indole-3-Acetic Acid Metabolites from Dalbergia dolichopetala by High Performance Liquid Chromatography-Mass Spectrometry

A mixture of [2-¹⁴C₁] and [¹³C₆]indole-3-acetic acid was applied to the cotyledons of 6-day-germinated seeds of “jacarandá do cerrado” (Dalbergia dolichopetala) and after 8 hours the seeds were extracted. Analysis of the fractionated extract by reversed-phase high performance liquid chromatography-radiocounting revealed the presence of five radiolabeled metabolite peaks (I-V). After further purification, the individual peaks of radioactivity were analyzed by combined high performance liquid chromatography-steel filter-fast atom bombardment-mass spectrometry. The metabolite fraction V was found to contain [¹⁴C₁, ¹³C₆]indole-3-acetylas-partic acid and unlabeled indole-3-acetylglutamic acid. Analysis of the metabolite fraction II revealed the presence of dioxindole-3-acetylaspartic acid and putative dioxindole-3-acetylglutamic acid as well as putative benzene ring-hydroxylated derivatives of oxindole-3-acetylaspartic acid and oxindole-3-acetylglutamic acid. There was no evidence of significant incorporation of label from [2′-¹⁴C₁] or [¹³C₆]indole-3-acetic acid into any of these conjugated indoles.     

Degradation of phenanthrene by different bacteria: evidence for novel transformation sequences involving the formation of 1-naphthol

Four polycyclic aromatic hydrocarbon (PAH)- degrading bacteria, namely Arthrobacter sulphureus RKJ4, Acidovorax delafieldii P4-1, Brevibacterium sp. HL4 and Pseudomonas sp. DLC-P11, capable of utilizing phenanthrene as the sole source of carbon and energy, were tested for its degradation using radiolabelled phenanthrene. [9-¹⁴C]Phenanthrene was incubated with microorganisms containing 100 mg/l unlabelled phenanthrene and the evolution of ¹⁴CO₂ was monitored: within 18 h of incubation, 30.1, 35.6, 26.5 and 2.1% of the recovered radiolabelled carbon was degraded to ¹⁴CO₂ by RKJ4, P4-1, HL4 and DLC-P11, respectively. When mixtures of other PAHs such as fluorene, fluoranthene and pyrene, in addition to phenanthrene, were added as additional carbon sources, there was a 36.1 and 20.6% increase in ¹⁴CO₂ production from [9-¹⁴C]phenanthrene in the cases of RKJ4 and HL4, respectively, whereas P4-1 and DLC-P11 did not show any enhancement in ¹⁴CO₂ production. Although, a combination of many bacteria enhances the degradation of organic compounds, no enhancement in the degradation of [9-¹⁴C]phenanthrene was observed in mixed culture involving all four microorganisms together. However, when different PAHs, as indicated above, were used in mixed culture, there was a 68.2% increase in ¹⁴CO₂ production. In another experiment, the overall growth rate of P4-1 on phenanthrene could be enhanced by adding the non-ionic surfactant Triton X-100, whereas RKJ4, HL4 and DLC-P11 did not show any enhancement in growth. Pathways for phenanthrene degradation were also analysed by thin-layer chromatography, gas chromatography and gas chromatography-mass spectrometry. Common intermediates such as o-phthalic acid and protocatechuic acid were detected in the case of RKJ4 and o-phthalic acid was detected in the case of P4-1. A new intermediate, 1-naphthol, was detected in the cases of HL4 and DLC-P11. HL4 degrades phenanthrene via 1-hydroxy-2-naphthoic acid, 1-naphthol and salicylic acid, whereas DLC-P11 degrades phenanthrene via the formation of 1-hydroxy-2-naphthoic acid, 1-naphthol and o-phthalic acid. Both transformation sequences are novel and have not been previously reported in the literature. Mega plasmids were found to be present in RKJ4, HL4 and DLC-P11, but their involvement in phenanthrene degradation could not be established. Received: 25 May 1999 / Received revision: 16 July 1999 / Accepted: 1 August 1999