Pseudomonas Putida G7 Research Articles

Taxis-enhanced mineralization and co-metabolism of PAHs by bacteria in micrometer-scale environments

Polycyclic aromatic hydrocarbons (PAHs) are associated with micropores in sediments and soils. This limits the bioaccessibility of these compounds via existing bioremediation technologies, as biodegradation is strongly influenced by the ability of bacteria to access different sizes of pores. In this work, we employed naphthalene and pyrene as model contaminants to evaluate the transformation capacity of the soil bacterium Pseudomonas putida G7 (2 × 1 μm) via mineralization and co-metabolic activity, respectively. Under non-growing conditions and in the absence of hydraulic flow, we examined how the tactic behavior of this motile bacterium influenced biodegradation of these two PAHs when passing through membranes with micrometer-sized pores (3 and 5 μm). The bacteria were spontaneously retained by the membranes, which blocked the contaminants away from a passive dosing source. However, the cells were mobilized through 5 μm pores after the application plant root exudate components (γ-aminobutyric acid, citrate and fructose) as strong chemoeffectors, which enhanced the mineralization of naphthalene and co-metabolism of pyrene. The tactic-mediated biodegradation enhancement did not occur through 3 μm pores, possibly due a physical constrain to the gradient sensing mechanism. Our results suggest that bacterial transport by chemotaxis may enhance the biotransformation of poorly bioaccessible contaminants present in micro-meter scale environments.

Taxis-mediated bacterial transport and its implication for the cometabolism of pyrene in a model aquifer

One of the main problems in contaminated soils is that many toxic substances, such as PAHs, which are found in areas close to aquifers and groundwater, are difficult to access and degrade via traditional methods of remediation. The use of controlled bacterial mobility through chemotaxis has been shown to be efficient in increasing the dispersion of pollutant-degrading organisms, increasing the biodegradation rates of pollutants. In this study, using percolation columns as model aquifers, the mobilization of the Pseudomonas putida G7 strain to a distant pyrene source was demonstrated using γ-aminobutyric acid and artificial root exudates as strong chemoeffectors. An increase in the biodegradation rates of the pollutant was observed relative to columns in which the tactic effector was not added. The presence of different metabolites was detected via a fraction collector associated with an HPLC system, providing evidence for the cometabolic capacity of strain G7. The use of chemotactic organisms can be an effective approach for the remediation of polluted sediments associated with aquifers and groundwaters, offering new possibilities for the treatment of contaminated aqueous areas.

Risk reductions during pyrene biotransformation and mobilization in a model plant-bacteria-biochar system

The productive application of motile microorganisms for degrading hydrophobic contaminants in soil is one of the most promising processes in modern remediation due to its sustainability and low cost. However, the incomplete biodegradation of the contaminants and the formation of the intermediary metabolites in the process may increase the toxicity in soil during bioremediation, and motile inoculants may mobilize the pollutants through biosorption. Therefore, controlling these factors should be a fundamental part of soil remediation approaches. The aim of this study was to evaluate the sources of risk associated with the cometabolism-based transformation of 14C-labeled pyrene by inoculated Pseudomonas putida G7 and identify ways to minimize risk. Our model scenario examined the increase in bioaccessibility to a distant source of contamination facilitated by sunflower (Helianthus annuus L.) roots. A biochar trap for mobilized pollutant metabolites and bacteria has also been employed. The experimental design consisted of pots filled with a layer of sand with 14C-labeled pyrene (88 mg kg−1) as a contamination focus located several centimeters from the inoculation point. Half of the pots included a biochar layer at the bottom. The pots were incubated in a greenhouse with sunflower plants and P. putida G7 bacteria. Pots with sunflower plants showed a higher biodegradation of pyrene, its mobilization as metabolites through the percolate and the roots, and bacterial mobilization toward the source of contamination, also resulting in increased pyrene transformation. In addition, the biochar layer efficiently reduced the concentrations of pyrene metabolites collected in the leachates. Therefore, the combination of plants, motile bacteria and biochar safely reduced the risk caused by the biological transformation of pyrene.

Complete Genome Sequence of NAH7-Harboring Pseudomonas putida Strain G7.

ABSTRACTPseudomonas putida G7 (111) is an aerobic bacterial species discovered in soil known to harbor the naphthalene-degrading NAH7 plasmid. Here, we report the genome sequence of G7. The genome was assembled by Nanopore sequencing and consisted of a chromosome of 6.4 Mbp with a G+C content of 62.13%.

Chemotactic Bacteria Facilitate the Dispersion of Nonmotile Bacteria through Micrometer-Sized Pores in Engineered Porous Media.

Recent research has demonstrated that chemotactic bacteria can disperse inside microsized pores while traveling toward favorable conditions. Microbe–microbe cotransport might enable nonmotile bacteria to be carried with motile partners to enhance their dispersion and reduce their deposition in porous systems. The aim of this study was to demonstrate the enhancement in the dispersion of nonmotile bacteria (Mycobacterium gilvum VM552, a polycyclic aromatic hydrocarbon-degrader, and Sphingobium sp. D4, a hexachlorocyclohexane-degrader, through micrometer-sized pores near the exclusion-cell-size limit, in the presence of motile Pseudomonas putida G7 cells. For this purpose, we used bioreactors equipped with two chambers that were separated with membrane filters with 3, 5, and 12 μm pore sizes and capillary polydimethylsiloxane (PDMS) microarrays (20 μm × 35 μm × 2.2 mm). The cotransport of nonmotile bacteria occurred exclusively in the presence of a chemoattractant concentration gradient, and therefore, a directed flow of motile cells. This cotransport was more intense in the presence of larger pores (12 μm) and strong chemoeffectors (γ-aminobutyric acid). The mechanism that governed cotransport at the cell scale involved mechanical pushing and hydrodynamic interactions. Chemotaxis-mediated cotransport of bacterial degraders and its implications in pore accessibility opens new avenues for the enhancement of bacterial dispersion in porous media and the biodegradation of heterogeneously contaminated scenarios.

Role of tactic response on the mobilization of motile bacteria through micrometer-sized pores

A major cause of high bioremediation endpoints is the limited bioaccessibility to residual contaminants resting in soil pores with diameters close to the size exclusion limit of bacterial cells. Under nongrowing conditions and in the absence of hydraulic flow, we examined how the tactic behavior of motile, contaminant-degrading Pseudomonas putida G7 cells (2 × 1 μm) influenced passage through membranes with pores ranging in size from 1 μm to 12 μm. The bacteria were spontaneously retained by the membranes - even those with the largest pore size. However, the cells were mobilized through 5 μm and 12 μm pores after the application of an attractant (salicylate). Mobilization also occurred by attraction to the common root exudate constituents γ-aminobutyric acid and citrate and repellence (or negative taxis) to zero-valent iron nanoparticles. The observed pore size threshold for tactic mobilization (5 μm) and unaltered cell fluxes and effective cell diffusion against different chemoeffector strengths and concentrations suggest that there is a physical constraint on the gradient sensing mechanism at the pores that drives the tactic response. Our results indicate that chemically mediated, small-scale tactic reactions of motile bacteria may become relevant to enhance the bioaccessibility of the residual contaminants present in micrometer-sized soil pores.

Root-mediated bacterial accessibility and cometabolism of pyrene in soil

Partial transformation of pollutants and mobilization of the produced metabolites may contribute significantly to the risks resulting from biological treatment of soils polluted by hydrophobic chemicals such as polycyclic aromatic hydrocarbons (PAHs). Pyrene, a four-ringed PAH, was selected here as a model pollutant to study the effects of sunflower plants on the bacterial accessibility and cometabolism of this pollutant when located at a spatially distant source within soil. We compared the transformation of passively dosed 14C-labeled pyrene in soil slurries and planted pots that were inoculated with the bacterium Pseudomonas putida G7. This bacterium combines flagellar cell motility with the ability to cometabolically transform pyrene. Cometabolism of this PAH occurred immediately in the inoculated and shaken soil slurries, where the bacteria had full access to the passive dosing devices (silicone O-rings). Root exudates did not enhance the survival of P. putida G7 cells in soil slurries, but doubled their transport in column tests. In greenhouse-incubated soil pots with the same pyrene sources instead located centimeters from the soil surface, the inoculated bacteria transformed 14C-labeled pyrene only when the pots were planted with sunflowers. Bacterial inoculation caused mobilization of 14C-labeled pyrene metabolites into the leachates of the planted pots at concentrations of approximately 1 mg L−1, ten times greater than the water solubility of the parent compound. This mobilization resulted in a doubled specific root uptake rate of 14C-labeled pyrene equivalents and a significantly decreased root-to-fruit transfer rate. Our results show that the plants facilitated bacterial access to the distant pollutant source, possibly by increasing bacterial dispersal in the soil; this increased bacterial access was associated with cometabolism, which contributed to the risks of biodegradation.

Impact of bacterial motility on biosorption and cometabolism of pyrene in a porous medium

The risks of pollution by polycyclic aromatic hydrocarbons (PAHs) may increase in bioremediated soils as a result of the formation of toxic byproducts and the mobilization of pollutants associated to suspended colloids. In this study, we used the motile and chemotactic bacterium Pseudomonas putida G7 as an experimental model for examining the potential role of bacterial motility in the cometabolism and biosorption of pyrene in a porous medium. For this purpose, we conducted batch and column transport experiments with 14C-labelled pyrene loaded on silicone O-rings, which acted as a passive dosing system. In the batch experiments, we observed concentrations of the 14C-pyrene equivalents well above the equilibrium concentration observed in abiotic controls. This mobilization was attributed to biosorption and cometabolism processes occurring in parallel. HPLC quantification revealed pyrene concentrations well below the 14C-based quantifications by liquid scintillation, indicating pyrene transformation into water-soluble polar metabolites. The results from transport experiments in sand columns revealed that cometabolic-active, motile cells were capable of accessing a distant source of sorbed pyrene. Using the same experimental system, we also determined that salicylate-mobilized cells, inhibited for pyrene cometabolism, but mobilized due to their tactic behavior, were able to sorb the compound and mobilize it by biosorption. Our results indicate that motile bacteria active in bioremediation may contribute, through cometabolism and biosorption, to the risk associated to pollutant mobilization in soils. This research could be the starting point for the development of more efficient, low-risk bioremediation strategies of poorly bioavailable contaminants in soils.

Catalytic mechanism for the conversion of salicylate into catechol by the flavin-dependent monooxygenase salicylate hydroxylase.

Salicylate hydroxylase (NahG) is a flavin-dependent monooxygenase that catalyzes the decarboxylative hydroxylation of salicylate into catechol in the naphthalene degradation pathway in Pseudomonas putida G7. We explored the mechanism of action of this enzyme in detail using a combination of structural and biophysical methods. NahG shares many structural and mechanistic features with other versatile flavin-dependent monooxygenases, with potential biocatalytic applications. The crystal structure at 2.0 Å resolution for the apo form of NahG adds a new snapshot preceding the FAD binding in flavin-dependent monooxygenases. The kcat/Km for the salicylate reaction catalyzed by the holo form is >105 M-1 s-1 at pH 8.5 and 25 °C. Hammett plots for Km and kcat using substituted salicylates indicate change in rate-limiting step. Electron-donating groups favor the hydroxylation of salicylate by a peroxyflavin to yield a Wheland-like intermediate, whereas the decarboxylation of this intermediate is faster for electron-withdrawing groups. The mechanism is supported by structural data and kinetic studies at different pHs. The salicylate carboxyl group lies near a hydrophobic region that aids decarboxylation. A conserved histidine residue is proposed to assist the reaction by general base/general acid catalysis.

Soil Particles and Phenanthrene Interact in Defining the Metabolic Profile of Pseudomonas putida G7: A Vibrational Spectroscopy Approach.

In soil, organic matter and mineral particles (soil particles; SPs) strongly influence the bio-available fraction of organic pollutants, such as polycyclic aromatic hydrocarbons (PAHs), and the metabolic activity of bacteria. However, the effect of SPs as well as comparative approaches to discriminate the metabolic responses to PAHs from those to simple carbon sources are seldom considered in mineralization experiments, limiting our knowledge concerning the dynamics of contaminants in soil. In this study, the metabolic profile of a model PAH-degrading bacterium, Pseudomonas putida G7, grown in the absence and presence of different SPs (i.e., sand, clays and humic acids), using either phenanthrene or glucose as the sole carbon and energy source, was characterized using vibrational spectroscopy (i.e., FT-Raman and FT-IR spectroscopy) and multivariate classification analysis (i.e., PLS-DA). The different type of SPs specifically altered the metabolic profile of P. putida, especially in combination with phenanthrene. In comparison to the cells grown in the absence of SPs, sand induced no remarkable change in the metabolic profile of the cells, whereas clays and humic acids affected it the most, as revealed by the higher discriminative accuracy (R2, RMSEP and sensitivity) of the PLS-DA for those conditions. With respect to the carbon-source (phenanthrene vs. glucose), no effect on the metabolic profile was evident in the absence of SPs or in the presence of sand. On the other hand, with clays and humic acids, more pronounced spectral clusters between cells grown on glucose or on phenanthrene were evident, suggesting that these SPs modify the way cells access and metabolize PAHs. The macromolecular changes regarded mainly protein secondary structures (a shift from α-helices to β-sheets), amino acid levels, nucleic acid conformation and cell wall carbohydrates. Our results provide new interesting evidences that SPs specifically interact with PAHs in defining bacteria metabolic profiles and further emphasize the importance of studying the interaction of bacteria with their surrounding matrix to deeply understand PAHs degradation in soils.

Impact of Chemoeffectors on Bacterial Motility, Transport, and Contaminant Degradation in Sand-Filled Percolation Columns.

Chemoeffector-mediated bacterial motility and tactic swimming are major drivers for contaminant accessibility and biodegradation at submillimeter scales. In sand-filled percolated columns we tested how and to what degree chemoeffectors influenced bacterial transport and thereby promoted accessibility and degradation of distantly located 14C-naphthalene (NAH) at the centimeter scale. Sunflower root exudates and silver nanoparticles (AgNPs) were used as chemoeffectors to stimulate opposing effects of motility and tactic swimming of NAH-degrading Pseudomonas putida G7. Sunflower exudates prompted smooth bacterial movement and positive taxis, while AgNPs induced tortuous movement and repellent responses. Compared to chemoeffector-free controls exudates reduced deposition and stimulated bacterial transport during percolation experiments. AgNPs, however, provoked bacterial deposition and concomitant saturation of the collector surfaces (filter blocking) that led to progressively increased percolation of cells. Despite mechanistic differences, both motility patterns supported bacterial transport and promoted mineralization rates of NAH desorbing from a source placed at the column outlet. Observed mineralization rates in the presence of the chemoeffectors were 5-fold higher than those in their absence and similar to NAH-mineralization in well-stirred batch assays. Our results indicate that chemically mediated, small-scale bacterial motility patterns may become relevant for long-distance bacterial transport and the biodegradation of patchy contaminants at higher scales, respectively.

Chemotaxis Increases the Retention of Bacteria in Porous Media with Residual NAPL Entrapment.

Chemotaxis has the potential to decrease the persistence of nonaqueous phase liquid (NAPL) contaminants in aquifers by allowing pollutant-degrading bacteria to move toward sources of contamination and thus influence dissolution. This experimental study investigated the migratory response of chemotactic bacteria to a distribution of residual NAPL ganglia entrapped within a laboratory-scale sand column under continuous-flow at a superficial velocity of 0.05 cm/min. Naphthalene dissolved in a model NAPL 2,2,4,4,6,8,8-heptamethylnonane partitioned into the aqueous phase to create localized chemoattractant gradients throughout the column. A pulse mixture of equal concentrations of Pseudomonas putida G7, a strain chemotactic to naphthalene, and Pseudomonas putida G7 Y1, a nonchemotactic mutant, was introduced to the column and effluent bacterial concentrations were measured with time. Breakthrough curves (BTCs) for the two strains were noticeably different upon visual inspection. Differences in BTCs (compared to nonchemotactic controls) were quantified in terms of percent recovery and were statistically significant ( p < 0.01). Chemotaxis reduced percent recovery in the effluent by 45% thereby increasing the population of bacteria that were retained within the column in the vicinity of residual NAPL contaminants. An increase in flow rate to a superficial velocity of 0.25 cm/min did not diminish cell retention associated with the chemotactic effect.

Salicylate degradation by a cold-adapted Pseudomonas sp.

Pseudomonas sp. strain MC1 was characterized as a cold-adapted, naphthalene-degrading bacterium that is able to grow in a broad temperature range of 5–30°C. MC1 harbors a catabolic plasmid, designated pYIC1, which is almost identical to the archetypal NAH7 plasmid from the mesophilic bacterium Pseudomonas putida G7. On pYIC1, the catabolic genes for naphthalene degradation are clustered in two operons: nahAa-Ab-Ac-Ad-B-F-C-Q-E-D encoding the conversion of naphthalene to salicylate, and nahG-T-H-I-N-L-O-M-K-J encoding the conversion of salicylate through meta-cleavage pathway to pyruvate and acetyl CoA. NahH, the bona fide extradiol dioxygenase in MC1 salicylate metabolism, is thermolabile and is a cold-adapted enzyme. The thermal profiles of the NahH enzyme activities expressed in different hosts indicate the presence of a factor(s) or mechanism(s) to protect the thermolabile NahH enzyme (100% aa identity with MC1 counterpart) in G7. Overall, the results reported in the present work suggest that the thermolabile NahH might be a product of the cold-adaptation process of MC1 and thus contribute to the survival and growth ability of MC1 on salicylate and naphthalene in cold environments.

A combined approach for enhancing the stability of recombinant cis-dihydrodiol naphthalene dehydrogenase from Pseudomonas putida G7 allowed for the structural and kinetic characterization of the enzyme

The second enzyme of the naphthalene degradation pathway in Pseudomonas putida G7 is NahB, a dehydrogenase that converts cis-1,2-dihydroxy-1,2-dihydronaphthalene to 1,2-dihydroxynaphthalene. We report the cloning, optimization of expression, purification, kinetic studies and preliminary structural characterization of the recombinant NahB. The nahB gene was cloned into a T7 expression vector and the enzyme was overexpressed in Escherichia coli Rosetta (DE3) as an N-terminal hexa-histidine-tagged protein (6xHis-NahB). Using methods of enhancing protein stability in solution, we tested different expression, cell lysis, and purification protocols with and without ligand supplementation. The protein stability was evaluated by dynamic light scattering and circular dichroism spectroscopy assays. Best-derived protocols (expression at 18 °C, cell lysis with homogenizer, and three purification steps) were used to produce 20 mg of homogeneous 6xHis-NahB per liter of culture. The secondary and quaternary structures of 6xHis-NahB were assessed by circular dichroism and size-exclusion chromatography experiments, respectively. The enzyme was NAD+-dependent and active at pH 7.0 and 9.4 for the oxidation of the substrate. The Michaelis-Menten parameters determined at pH 7.0 and 25 °C for the substrate and cofactor, presented respective Km values of 6 and 350 μM, and a kcat value of 8.3 s−1. Furthermore, we identified conditions for the crystallization of 6xHis-NahB. X-ray diffraction data were collected from a single 6xHis-NahB crystal which diffracted to 2.21 Å. The crystal belongs to space group I222, with unit-cell parameters a = 63.62, b = 69.50, and c = 117.47 Å. The tertiary structure of 6xHis-NahB was determined using the molecular replacement method. Further structural refinement is currently underway.

Structural and Kinetic Properties of the Aldehyde Dehydrogenase NahF, a Broad Substrate Specificity Enzyme for Aldehyde Oxidation.

The salicylaldehyde dehydrogenase (NahF) catalyzes the oxidation of salicylaldehyde to salicylate using NAD(+) as a cofactor, the last reaction of the upper degradation pathway of naphthalene in Pseudomonas putida G7. The naphthalene is an abundant and toxic compound in oil and has been used as a model for bioremediation studies. The steady-state kinetic parameters for oxidation of aliphatic or aromatic aldehydes catalyzed by 6xHis-NahF are presented. The 6xHis-NahF catalyzes the oxidation of aromatic aldehydes with large kcat/Km values close to 10(6) M(-1) s(-1). The active site of NahF is highly hydrophobic, and the enzyme shows higher specificity for less polar substrates than for polar substrates, e.g., acetaldehyde. The enzyme shows α/β folding with three well-defined domains: the oligomerization domain, which is responsible for the interlacement between the two monomers; the Rossmann-like fold domain, essential for nucleotide binding; and the catalytic domain. A salicylaldehyde molecule was observed in a deep pocket in the crystal structure of NahF where the catalytic C284 and E250 are present. Moreover, the residues G150, R157, W96, F99, F274, F279, and Y446 were thought to be important for catalysis and specificity for aromatic aldehydes. Understanding the molecular features responsible for NahF activity allows for comparisons with other aldehyde dehydrogenases and, together with structural information, provides the information needed for future mutational studies aimed to enhance its stability and specificity and further its use in biotechnological processes.

Mobilization of Pollutant-Degrading Bacteria by Eukaryotic Zoospores.

The controlled mobilization of pollutant-degrading bacteria has been identified as a promising strategy for improving bioremediation performance. We tested the hypothesis whether the mobilization of bacterial degraders may be achieved by the action of eukaryotic zoospores. We evaluated zoospores that are produced by the soil oomycete Pythium aphanidermatum as a biological vector, and, respectively, the polycyclic aromatic hydrocarbon (PAH)-degrading bacteria Mycobacterium gilvum VM552 and Pseudomonas putida G7, acting as representative nonflagellated and flagellated species. The mobilization assay was performed with a chemical-in-capillary method, in which zoospores mobilized bacterial cells only when they were exposed to a zoospore homing inducer (5% (v/v) ethanol), which caused the tactic response and settlement of zoospores. The mobilization was strongly linked to a lack of bacterial motility, because the nonflagellated cells from strain M. gilvum VM552 and slightly motile, stationary-phase cells from P. putida G7 were mobilized effectively, but the actively motile, exponentially grown cells of P. putida G7 were not mobilized. The computer-assisted analysis of cell motility in mixed suspensions showed that the swimming rate was enhanced by zoospores in stationary, but not in exponentially grown, cells of P. putida G7. It is hypothesized that the directional swimming of zoospores caused bacterial mobilization through the thrust force of their flagellar propulsion. Our results suggest that, by mobilizing pollutant-degrading bacteria, zoospores can act as ecological amplifiers for fungal and oomycete mycelial networks in soils, extending their potential in bioremediation scenarios.

Chemotaxis Increases the Residence Time of Bacteria in Granular Media Containing Distributed Contaminant Sources.

The use of chemotactic bacteria in bioremediation has the potential to increase access to, and the biotransformation of, contaminant mass within the subsurface. This laboratory-scale study aimed to understand and quantify the influence of chemotaxis on the residence times of pollutant-degrading bacteria within homogeneous treatment zones. Focus was placed on a continuous-flow sand-packed column in which a uniform distribution of naphthalene crystals created distributed sources of dissolved-phase contaminant. A 10 mL pulse of Pseudomonas putida G7, which is chemotactic to naphthalene, and Pseudomonas putida G7 Y1, a nonchemotactic mutant strain, were simultaneously introduced into the sand-packed column at equal concentrations. Breakthrough curves obtained from experiments conducted with and without naphthalene were used to quantify the effect of chemotaxis on transport parameters. In the presence of the chemoattractant, longitudinal dispersion of PpG7 increased by a factor of 3, and percent recovery decreased by 43%. In contrast, PpG7 Y1 transport was not influenced by the presence of naphthalene. The results imply that pore-scale chemotaxis responses are evident at an interstitial velocity of 1.8 m/day, which is within the range of typical groundwater flow. Within the context of bioremediation, chemotaxis may work to enhance bacterial residence times in zones of contamination, thereby improving treatment.

Structural and kinetic characterization of recombinant 2-hydroxymuconate semialdehyde dehydrogenase from Pseudomonas putida G7

The first enzyme in the oxalocrotonate branch of the naphthalene-degradation lower pathway in Pseudomonas putida G7 is NahI, a 2-hydroxymuconate semialdehyde dehydrogenase which converts 2-hydroxymuconate semialdehyde to 2-hydroxymuconate in the presence of NAD+. NahI is in family 8 (ALDH8) of the NAD(P)+-dependent aldehyde dehydrogenase superfamily. In this work, we report the cloning, expression, purification and preliminary structural and kinetic characterization of the recombinant NahI. The nahI gene was subcloned into a T7 expression vector and the enzyme was overexpressed in Escherichia coli ArcticExpress as a hexa-histidine-tagged fusion protein. After purification by affinity and size-exclusion chromatography, dynamic light scattering and small-angle X-ray scattering experiments were conducted to analyze the oligomeric state and the overall shape of the enzyme in solution. The protein is a tetramer in solution and has nearly perfect 222 point group symmetry. Protein stability and secondary structure content were evaluated by a circular dichroism spectroscopy assay under different thermal conditions. Furthermore, kinetic assays were conducted and, for the first time, KM (1.3±0.3μM) and kcat (0.9s−1) values were determined at presumed NAD+ saturation. NahI is highly specific for its biological substrate and has no activity with salicylaldehyde, another intermediate in the naphthalene-degradation pathway.

Impact of dissolved organic matter on bacterial tactic motility, attachment, and transport.

Bacterial dispersal is a key driver of the ecology of microbial contaminant degradation in soils. This work investigated the role of dissolved organic matter (DOM) in the motility, attachment, and transport of the soil bacterium Pseudomonas putida G7 in saturated porous media. The study is based on the hypothesis that DOM quality is critical to triggering tactic motility and, consequently, affects bacterial transport and dispersal. Sunflower root exudates, humic acids (HA), and the synthetic oleophilic fertilizer S-200 were used as representatives of fresh, weathered, and artificially processed DOM with high nitrogen and phosphorus contents, respectively. We studied DOM levels of 16-130 mg L(-1), which are representative of DOM concentrations typically found in agricultural soil pore water. In contrast to its responses to HA and S-200, strain G7 exhibited a tactic behavior toward root exudates, as quantified by chemotaxis assays and single-cell motility observations. All DOM types promoted bacterial transport through sand at high concentrations (∼ 130 mg L(-1)). At low DOM concentrations (∼ 16 mg L(-1)), the enhancement occurred only in the presence of sunflower root exudates, and this enhancement did not occur with G7 bacteria devoid of flagella. Our results suggest that tactic DOM effectors strongly influence bacterial transport and the interception probability of motile bacteria by collector surfaces.

Development of eukaryotic zoospores within polycyclic aromatic hydrocarbon (PAH)-polluted environments: A set of behaviors that are relevant for bioremediation

In this study, we assessed the development (formation, taxis and settlement) of eukaryotic zoospores under different regimes of exposure to polycyclic aromatic hydrocarbons (PAHs), which imitated environmental scenarios of pollution and bioremediation. With this aim, we used an oomycete, Pythium aphanidermatum, as a source of zoospores and two PAH-degrading bacteria (Mycobacterium gilvum VM552 and Pseudomonas putida G7). The oomycete and both bacteria were not antagonistic, and zoospore formation was diminished only in the presence of the highest bacterial cell density (108–1010colony-formingunitsmL−1). A negative influence of PAHs on zoospore formation and taxis was observed when PAHs were exposed in combination with organic solutions and polar solvents. Co-exposure of PAHs with non-polar solvents [hexadecane (HD) and 2,2,4,4,6,8,8-heptamethylnonane (HMN)] did not affect zoospore settlement at the interfaces of the organic solvents and water. However, zoospores settled and created mycelial networks only at HD–water interfaces. Both bacteria diminished the toxic influence of PAHs on zoospore formation and taxis, and they did not interrupt zoospore settlement. The results suggest that zoospore development could be applicable for toxicity assessment of PAHs and enhancement of their bioavailability. Microbial interactions during both swimming modes and community formation at pollutant interfaces were revealed as major factors that have potential relevance to bioremediation.