Pseudomonas Putida G7 Research Articles

Effects of LB broth, naphthalene concentration, and acetone on the naphthalene degradation activities by Pseudomonas putida G7.

Luria-Bertani broth and acetone were usually used in naphthalene degradation experiments as nutrient and solvent. However, their effect on the degradation was seldom mentioned. In this work, we investigated the effect of LB, naphthalene concentration, and acetone on the degradation of naphthalene by Pseudomonas putida G7, which is useful for the degradation of naphthalene on future field remediation. By adding LB, the naphthalene degradation efficiencies and naphthalene dioxygenase were both decreased by 98%, while the catechol dioxygenase was decreased by 90%. Degradation of naphthalene was also inhibited when naphthalene concentration was 56 ppm and higher, which was accompanied with the accumulation of orange-colored metabolism products. However, acetone can stimulate the degradation of naphthalene, and the stimulation was more obvious when naphthalene concentration was lower than 2000 ppm. By assaying the enzyme activities of naphthalene dioxygenase and catechol dioxygenase, it was thought that the degradation efficiency was depending on the more sensitive enzymes on the complicated conditions.

Improving Rice Ability to Degrade Different Polycyclic Aromatic Hydrocarbons Through Multigene Transformation of a Hybrid Dioxygenase System

The widespread presence and potential harm conferred by polycyclic aromatic hydrocarbons (PAHs) to various organisms have raised increased research interest on these pollutants. Plants lack of efficient PAH degradation genes so that they had to suffer from PAH stress. To improve the ability of plants to tolerate various PAHs, four genes, namely nidA and nidB (encoding the large and small subunits of naphthalene dioxygenase of Mycobacterium vanbaalenii PYR-1) as well as nahAa and nahAb (encoding flavoprotein reductase and ferredoxin of the electron-transport chain of the Pseudomonas putida G7 naphthalene dioxygenase system), were ectopically expressed in rice. The hybrid multicomponent naphthalene dioxygenase can assemble correctly and break down PAHs powerfully. Transgenic rice plants exhibited enhanced tolerance toward two- to four-ring PAHs. PAH tolerance in transgenic plants was associated with high chlorophyll levels and photosynthesis efficiency, normal development, and minimal ion leakage under PAH stress. The naphthalene metabolic pathway in transgenic plants mainly involves the dioxygenase pathway. Our results indicate that grafting the naphthalene dioxygenase system into plants is useful to degrade PAHs efficiently in vivo and improve the plant tolerance to the pollutants. This study had introduced a suitable strategy to accelerate the bioremediation process of PAH-contaminated soil under waterlogged conditions and improve the food safety in contaminated area.

Metabolic engineering of Arabidopsis for remediation of different polycyclic aromatic hydrocarbons using a hybrid bacterial dioxygenase complex

The widespread presence of polycyclic aromatic hydrocarbons (PAHs) and their potential harm to various organisms has generated interest in efficiently eliminating these compounds from the environment. Phytoremediation is an efficient technology for cleaning up pollutants. However, unlike microorganisms, plants lack the catabolic pathway for complete degradation of these dangerous groups of compounds. One way to enhance the potential of plants for remediation of these compounds is by transferring genes involved in xenobiotic degradation from microbes to plants. In this paper, four genes, namely nidA and nidB (encoding the large and small subunits of naphthalene dioxygenase of Mycobacterium vanbaalenii PYR-1) as well as NahAa and NahAb (encoding flavoprotein reductase and ferredoxin of the electron-transport chain of the Pseudomonas putida G7 naphthalene dioxygenase system), were transferred and ectopically expressed in Arabidopsis thaliana. Transgenic Arabidopsis plants overexpressing the heterozygous naphthalene dioxygenase system exhibited enhanced tolerance toward 2–4 rings PAHs. Transgenic plants assimilated PAHs from the culture media faster and accumulated less in vivo than wild-type plants. Furthermore, examination of metabolic intermediates by gas chromatography–mass spectrometry revealed that the naphthalene metabolic pathway in transgenic plants mainly involves the dioxygenase pathway. Taken together, our findings suggest that grafting the naphthalene dioxygenase complex into plants is a possible strategy to breed PAH-tolerant plants to efficiently degrade PAHs in the environment.

Bioavailability of slow-desorbable naphthalene in a biological air sparging system

The bioavailability of sorbed organic contaminants is one of the most important factors used to determine their fate in the environment. This study was conducted to evaluate the bioavailability of slow-desorbable naphthalene in soils. An air sparging system was utilized to remove dissolved (or desorbed) naphthalene continuously and to limit the bacterial utilization of dissolved naphthalene. A biological air sparging system (air sparging system with bacteria) was developed to evaluate the bioavailability of the slow-desorption fraction in soils. Three different strains (Pseudomonas putida G7, Pseudomonas sp. CZ6 and Burkholderia sp. KM1) and two soils were used. Slow-desorbable naphthalene continuously decreased under air sparging; however, a greater decrease was observed in response to the biological air sparging system. Enhanced bioavailability was not observed in the Jangseong soil. Overall, the results of this study suggests that the removal rate of slow-desorbable contaminants may be enhanced by inoculation of degrading bacteria into an air sparging system during the remediation of contaminated soils. However, the enhanced bioavailability was found to depend more on the soil properties than the bacterial characteristics.

Chemical Effectors Cause Different Motile Behavior and Deposition of Bacteria in Porous Media

We tested the hypothesis whether chemically induced motility patterns of bacteria may affect their transport in porous media. Naphthalene-degrading Pseudomonas putida G7 cells were exposed to glucose, salicylate, and silver nanoparticles (AgNPs) and their motility was assessed by computer-assisted, quantitative swimming and capillary-based taxis determinations. Exposure to salicylate induced smooth movement with few acceleration events and positive taxis, whereas cells exposed to AgNPs exhibited tortuous movement and a repellent response. Although metabolized by strain G7, glucose did not cause attraction and induced a hyper-motile mode of swimming, characterized by a high frequency of acceleration events, high swimming speed (>60 μm s(-1)), and a high tortuosity in the trajectories. Chemically induced motility behavior correlated with distinct modes of attachment to sand in batch assays and breakthrough curves in percolation column experiments. Salicylate significantly reduced deposition of G7 cells in column experiments whereas glucose and AgNPs enhanced attachment and caused filter blocking that resulted in a progressive decrease in deposition. These findings are relevant for bioremediation scenarios that require an optimized outreach of introduced inoculants and in other environmental technologies, such as water disinfection and microbially enhanced oil recovery.

Expression, purification and preliminary crystallographic studies of NahF, a salicylaldehyde dehydrogenase from Pseudomonas putida G7 involved in naphthalene degradation.

Pseudomonas putida G7 is one of the most studied naphthalene-degrading species. The nah operon in P. putida, which is present on the 83 kb metabolic plasmid NAH7, codes for enzymes involved in the conversion of naphthalene to salicylate. The enzyme NahF (salicylaldehyde dehydrogenase) catalyzes the last reaction in this pathway. The nahF gene was subcloned into the pET28a(TEV) vector and the recombinant protein was overexpressed in Escherichia coli Arctic Express at 285 K. The soluble protein was purified by affinity chromatography followed by gel filtration. Crystals of recombinant NahF (6×His-NahF) were obtained at 291 K and diffracted to 2.42 Å resolution. They belonged to the hexagonal space group P6(4)22, with unit-cell parameters a = b = 169.47, c = 157.94 Å. The asymmetric unit contained a monomer and a crystallographic twofold axis generated the dimeric biological unit.

Glucose and plant exudate enhanced enumeration of bacteria capable of degrading polycyclic aromatic hydrocarbons

Enumerating environmental microbial isolates capable of polycyclic aromatic hydrocarbon (PAH) degradation can provide insight into the microbe-plant interactions that facilitate PAH removal. We examined a known PAH degrader ( Pseudomonas putida G7), a nondegrader ( Agrobacterium tumefaciens LBA4404), and several microorganisms isolated from the environment by using a PAH cocktail in an enumeration mediumwith or without 0.025% (m/v) glucose and (or) root exudates. Compared with the standard most probable number (MPN), the addition of glucose and root exudates in a modified MPN method resulted in a 3- to 11-fold enhancement of PAH degraders being enumerated among microorganisms found in PAH-contaminated soils. High-performance liquid chromatography analysis verified that PAH levels were reduced using this modified enumeration method. Low levels of glucose, perhaps in concert with other materials in exudates, may promote microbial metabolism, thereby enhancing PAH degradation.

An amperometric biosensor developed for detection of limonin levels in kinnow mandarin juices

The bitterness in kinnow mandarins (also in certain other citrus juices) has been ascribed to limonin that is gradually formed during processing of the juice, which lowers the consumer’s acceptability. Thus, detection of limonin at various stages of processing is crucial for the employment of suitable debittering interventions. Owing to the lack of rapid, reliable and economical method for determining limonin levels during the juice production process, an attempt has been made to develop an amperometric microbial biosensor using a mutant (lim+) of a strain Pseudomonas putida G7, that could selectively utilize limonin as a carbon source in the presence of other sugars in kinnow mandarin juice. Analytical determination was based on the respiratory activity of this stable, lim+ auxotrophic mutant in the presence of the analyte, limonin. The temperature of 30°C, pH 6.5 and the mid-log phase cells at concentrations of 106 CFU/ml were the most optimal operating conditions. A consistent correlation between limonin concentrations of 20, 25, 30 ppm (i.e. an optimal substrate–to-cell mass concentration) and the dissolved oxygen (DO) was established. Response times of approximately 20 min for the steady-state method and 12 min for the initial slope method were recorded. The calibration curve for limonin was linear in the 15–50 ppm range of limonin. The performance of the biosensor was reproducible and remained unaffected following intermittent storage and reuse for at least 1 month. Overall, this study suggests the possible application of the developed biosensor for monitoring the limonin content in citrus juices during processing.

Walking the tightrope of bioavailability: growth dynamics of PAH degraders on vapour‐phase PAH

SummaryMicrobial contaminant degradation may either result in the utilization of the compound for growth or act as a protective mechanism against its toxicity. Bioavailability of contaminants for nutrition and toxicity has opposite consequences which may have resulted in quite different bacterial adaptation mechanisms; these may particularly interfere when a growth substrate causes toxicity at high bioavailability. Recently, it has been demonstrated that a high bioavailability of vapour‐phase naphthalene (NAPH) leads to chemotactic movement of NAPH‐degrading Pseudomonas putida (NAH7) G7 away from the NAPH source. To investigate the balance of toxic defence and substrate utilization, we tested the influence of the cell density on surface‐associated growth of strain PpG7 at different positions in vapour‐phase NAPH gradients. Controlled microcosm experiments revealed that high cell densities increased growth rates close (< 2 cm) to the NAPH source, whereas competition for NAPH decreased the growth rates at larger distances despite the high gas phase diffusivity of NAPH. At larger distance, less microbial biomass was likewise sustained by the vapour‐phase NAPH. Such varying growth kinetics is explained by a combination of bioavailability restrictions and NAPH‐based inhibition. To account for this balance, a novel, integrated ‘Best Equation’ describing microbial growth influenced by substrate availability and inhibition is presented.

Enhancement of bioconversion efficiency of limonin by Pseudmonas putida G7

The biocatalytic activity of periplasmic dehydrogenase of Pseudomonas putida G7 strain to catalyse limonin was enhanced when whole cells were permeabilized with EDTA (1 μM) lysozyme (100 μg/ml). The treated cells were entrapped in dialysis membranes to increase the stability. Permeabilized cells (1 g dry weight) entrapped in dialysis membrane could biotransform 73.67% of limonin in unpasteurized mandarin juices in a single-batch cycle of 3 h. Furthermore, permeabilized cells stored for 45 days in phosphate buffered saline (at 4 or 30°C) retained enzyme activity and were reusable for up to eight batch cycles of limonin reduction. The results of this study suggest a potential application of permeabilized P. putida G7 cells for reducing limonin levels in mandarin juices.

Assessing biodegradation benefits from dispersal networks

The performance of biodegradation of organic pollutants in soil often depends on abiotic conditions and the bioavailability of these pollutants to degrading bacteria. In this context, bacterial dispersal is an essential aspect. Recent studies on the potential promotion of bacterial dispersal by fungal hyphae raised the idea of specifically applying fungal networks to accelerate bacterial degradation processes in situ. Our objective is to investigate these processes and their performance via simulation modelling and address the following questions: (1) Under what abiotic conditions can dispersal networks significantly improve bacterial degradation? and (2) To what extent does the spatial configuration of the networks influence the degradation performance? To answer these questions, we developed a spatially explicit bacterial colony model, which is applied to controlled laboratory experiments with Pseudomonas putida G7 organisms as a case study. Using this model, we analyzed degradation performance in response to different environmental scenarios and showed that conditions of limited bacterial dispersal also limit degradation performance. Under such conditions, dispersal networks have the highest potential for improving the bioavailability of pollutants to bacteria. We also found that degradation performance significantly varies with the spatial configuration of the dispersal networks applied and the time horizon over which performance is assessed. Regarding future practical applications, our results suggest that (1) fungal networks may dramatically improve initially adverse conditions for biodegradation of pollutants in soil, and (2) the network's spatial structure and accessibility are decisive for the success of such tasks.

Isothermal titration calorimetry — A new method for the quantification of microbial degradation of trace pollutants

The environmental fate and, in particular, biodegradation rates of hydrophobic organic compounds (HOC) are of high interest due to the ubiquity, persistence, and potential health effects of these compounds. HOC tend to interact with bioreactor materials and sampling devices and are frequently volatile, so that conventionally derived degradation parameters are often biased. We report on the development and validation of a novel calorimetric approach that serves to gain real time information on the kinetics and the physiology of HOC bioconversion in aqueous systems while overcoming weaknesses of conventional biodegradation experiments. Soil bacteria Mycobacterium frederiksbergense LB501T, Rhodococcus erythropolis K2 -3 and Pseudomonas putida G7 were exposed to pulsed titrations of dissolved anthracene, 4 -(2 ,4 -dichlorophenoxy)butyric acid or naphthalene, respectively, and the thermal responses were monitored. The combinations of strains and pollutants were selected as examples for complete and partial biodegradation and complete degradation with storage product formation, respectively. Heat production signals were interpreted thermodynamically and in terms of Michaelis–Menten kinetics. The half-saturation constant k D and the degradation rate r D Max were derived. Comparison with conventional methods shows the suitability to extract kinetic degradation parameters of organic trace pollutants from simple ITC experiments, while thermodynamic interpretation provided further information about the metabolic fate of HOC compounds.

Chemoeffectors Decrease the Deposition of Chemotactic Bacteria during Transport in Porous Media

Bacterial chemotaxis enables motile cells to move along chemical gradients and to swim toward optimal places for biodegradation. However, its potentially positive effects on subsurface remediation rely on the efficiency of bacterial movement in porous media, which is often restricted by high deposition rates and adhesion to soil surfaces. In well-controlled column systems, we assessed the influence of the chemo-effectors naphthalene, salicylate, fumarate, and acetate on deposition of chemotactic, naphthalene-degrading Pseudomonas putida G7 in selected porous environments (sand, forest soil, and clay aggregates). Our data showed that the presence of naphthalene in the pore water decreased deposition of strain 67 (but not of a derivative strain, P. putida 67.C1 (pHG100), nonchemotactic to naphthalene) by 50% in sand-filled columns, as calculated by the relative adhesion efficiency (at). Similar effects were observed with P. putida G7 strain for the other chemoeffectors. Deposition, however, depended on the chemoeffector's chemical structure, its interaction with the column packing material, and concomitantly its pore-water concentration. As the presence of the chemoeffectors had no influence on the physicochemical surface properties of the bacteria, we suggest that chemotactic sensing, combined with changed swimming modes, is likely to influence the deposition of bacteria in the subsurface, provided that the chemoeffector is dissolved at sufficient concentration in the pore water.

The effect of oxygen on chemotaxis to naphthalene by Pseudomonas putida G7

Chemotactic bacteria can be attracted to electron donors they consume. In systems where donor is heterogeneously distributed, chemotaxis can lead to enhanced removal of donor relative to that achieved in the absence of chemotaxis. However, simultaneous consumption of an electron acceptor may result in the formation of an acceptor gradient to which the bacteria also respond, thus diminishing the positive effect of chemotaxis. Depletion of an electron acceptor can also reduce the rate of electron donor consumption in addition to its effect on chemotaxis. In this study, we examined the effect of oxygen on chemotaxis to naphthalene and on naphthalene consumption by Pseudomonas putida G7. The organism was able to move up an oxygen gradient when there was a naphthalene gradient in the opposite direction. In the absence of an oxygen gradient, low levels of oxygen attenuated chemotaxis to naphthalene but did not affect random motility. The rate of naphthalene consumption decreased at dissolved oxygen concentrations similar to those at which chemotaxis was attenuated. These results suggest that low dissolved oxygen concentrations can reduce naphthalene removal by P. putida G7 in systems where naphthalene is heterogeneously distributed by simultaneously attenuating chemotactic motion toward naphthalene and decreasing the rate of naphthalene degradation.

A Quantitative Assay for Linking Microbial Community Function and Structure of a Naphthalene-Degrading Microbial Consortium

A comprehensive culture-independent assay, called Q-FAST, was developed for concurrent identification and quantification of active microorganisms involved a specific function in a given microbial community. The development of Q-FAST was achieved by integrating the concept of stable isotope probing technique into a new quantitative fingerprinting assay called real-time-t-RFLP for microbial community structure analysis. The Q-FAST was successfully validated by using a three-member artificial microbial community containing a known naphthalene-utilizing bacterium (Pseudomonas putida G7) and two nonnaphthalene-degrading bacteria (Escherichia coli and Bacillus thuringiensis). The application of Q-FAST to identify and quantify a guild of naphthalene-utilizing microorganisms in soils revealed the involvement of eight members, with six members relating to several phylogenetic groups of eubacteria (three in beta-proteobacteria, two in gamma-proteobacteria, and one in genera Intrasporangium of Gram-positive bacteria) and two members showing no close phylogenetic affiliation to any known bacterial sequences deposited in GenBank. The quantity of three members belonging to beta-proteobacteria accounted for 34% of total 16S rDNA copies measured from the "heavier" fraction of DNA that was contributed from the DNA of microorganisms capable of incorporating 13C-labeled naphthalene into their genetic biomarkers. The other five members composed 66% of total 16S rDNA copies of active naphthalene-utilizing populations measured. Offering a powerful tool for studying microbial ecology, Q-FAST thus opens a new avenue for deeper exploration of microbial-mediated processes, mainly the quantitative relationship between microbial diversity and microbial activity in a given environment.

Monitoring of accelerated naphthalene-biodegradation in a bioaugmented soil slurry

The effect of microbial inoculation on the mineralization of naphthalene in a bioslurry treatment was evaluated in soil slurry microcosms. Inoculation by Pseudomonas putida G7 carrying the naphthalene dioxygenase (nahA) gene resulted in rapid mineralization of naphthalene, whereas indigenous microorganisms in the PAH-contaminated soil required a 28 h adaptation period before significant mineralization occurred. The number of nahA-like gene copies increased in both the inoculated and non-inoculated soil as mineralization proceeded, indicating selection towards naphthalene dioxygenase producing bacteria in the microbial community. In addition, 16S rRNA analysis by denaturing gradient gel electrophoresis (DGGE) analysis showed that significant selection occurred in the microbial community as a result of biodegradation. However, the indigenous soil bacteria were not able to compete with the P. putida G7 inoculum adapted to naphthalene biodegradation, even though the soil microbial community slightly suppressed naphthalene mineralization by P. putida G7.

Efficiency of naphthalene biodegradation by Pseudomonas putida G7 in soil

AbstractThe efficiency of naphthalene degradation by Pseudomonas putida G7 in soil was assessed using a mathematical model. The number of microorganisms and the concentration of naphthalene in soil samples were monitored. The feasibility of a spectrofluorometric method for naphthalene assay in soil samples was compared with high pressure liquid chromatography. A proposed mathematical model described the growth of the naphthalene‐degrading strains and the consumption of substrates (naphthalene, naphthalene degradation intermediates and soil organic substances) in soil. To describe the growth kinetics of microorganisms having high affinity to substrates with low solubility, two differential equations with substrate exponent 2/3 were proposed. These equations were used to describe utilization of soil organic matter. The model parameters characterize the growth rates for different substrates and respective yield coefficients, specific bacterial death and adaptation rates, and also the rates of PAHs degradation and evaporation. These characteristics can be used in choosing the bacterial strains for biopreparations and efficient clean‐up biotechnology of polluted soils. Copyright © 2004 Society of Chemical Industry

Catechol 2,3-dioxygenase from Pseudomonas sp. strain ND6: gene sequence and enzyme characterization.

The catechol 2,3-dioxygenase (C23O) gene in naphthalene catabolic plasmid pND6-1 of Pseudomonas sp. ND6 was cloned and sequenced. The C23O gene was consisted of 924 nucleotides and encoded a polypeptide of molecular weight 36 kDa containing 307 amino acid residues. The C23O of Pseudomonas sp. ND6 exhibited 93% and 89% identities in amino acid sequence with C23Os encoded by naphthalene catabolic plasmid NAH7 from Pseudomonas putida G7 and the chromosome of Pseudomonas stutzeri AN10 respectively. The Pseudomonas sp. ND6 C23O gene was overexpressed in Escherichia coli DH 5alpha using the lac promoter of pUC18, and its gene product was purified by DEAE-Sephacel and Phenyl-Sepharose CL-4B chromatography. The enzymology experiments indicated that the specific activity and thermostability of C23O from Pseudomonas sp. ND6 were better than those of C23O from Pseudomonas putida G7.

Effect of additional carbon source on naphthalene biodegradation by Pseudomonas putida G7

Addition of a carbon source as a nutrient into soil is believed to enhance in situ bioremediation by stimulating the growth of microorganisms that are indigenous to the subsurface and are capable of degrading contaminants. However, it may inhibit the biodegradation of organic contaminants and result in diauxic growth. The objective of this work is to study the effect of pyruvate as another carbon source on the biodegradation of polynuclear aromatic hydrocarbons (PAHs). In this study, naphthalene was used as a model PAH, ammonium sulfate as a nitrogen source, and oxygen as an electron acceptor. Pseudomonas putida G7 was used as a model naphthalene-degrading microorganism. From a chemostat culture, the growth kinetics of P. putida G7 on pyruvate was determined. At concentrations of naphthalene and pyruvate giving similar growth rates of P. putida G7, diauxic growth of P. putida G7 was not observed. It is suggested that pyruvate does not inhibit naphthalene biodegradation and can be used as an additional carbon source to stimulate the growth of P. putida G7 that can degrade polynuclear aromatic hydrocarbons.

Expression of the nitroarene dioxygenase genes in Comamonas sp. strain JS765 and Acidovorax sp. strain JS42 is induced by multiple aromatic compounds.

This work reports a genetic analysis of the expression of nitrobenzene dioxygenase (NBDO) in Comamonas sp. strain JS765 and 2-nitrotoluene dioxygenase (2NTDO) in Acidovorax sp. strain JS42. Strains JS765 and JS42 possess identical LysR-type regulatory proteins, NbzR and NtdR, respectively. NbzR/NtdR is homologous to NahR, the positive salicylate-responsive transcriptional activator of the naphthalene degradation genes in Pseudomonas putida G7. The genes encoding NBDO and 2NTDO in each strain are cotranscribed, and transcription starts at the same site within identical promoter regions for each operon. Results from a lacZ reporter gene fusion demonstrated that expression of NBDO and 2NTDO is induced by multiple aromatic compounds, including an array of nitroaromatic compounds (nitrobenzene, 2-, 3-, and 4-nitrotoluene, 2,4- and 2,6-dinitrotoluene, and aminodinitrotoluenes), as well as salicylate and anthranilate. The nitroaromatic compounds appear to be the actual effector molecules. Analysis of beta-galactosidase and 2NTDO activities with strain JS42 demonstrated that NtdR was required for induction by all of the inducing compounds, high basal-level expression of 2NTDO, and complementation of a JS42 ntdR null mutant. Complementation with the closely related regulators NagR (from Ralstonia sp. strain U2) and NahR restored only induction by the archetype inducers, salicylate or salicylate and anthranilate, respectively, and did not restore the high basal level of expression of 2NTDO. The mechanism of 2NTDO gene regulation in JS42, and presumably that of NBDO gene regulation in JS765, appear similar to that of NahR-regulated genes in Pseudomonas putida G7. However, NbzR and NtdR appear to have evolved a broader specificity in JS42 and JS765, allowing for recognition of nitroaromatic compounds while retaining the ability to respond to salicylate and anthranilate. NtdR is also the first example of a nitroarene-responsive LysR-type transcriptional activator.