Quantification of chemotaxis to naphthalene by Pseudomonas putida G7.

The capillary assay was used to quantify the chemotactic response of Pseudomonas putida G7 to naphthalene. Experiments were conducted in which the cell concentration in the assay chamber, the naphthalene concentration in the capillary, or the incubation time was varied. Data from these experiments were evaluated with a model that accounted for the effect of diffusion on the distribution of substrate and the transport of cells from the chamber through the capillary orifice. By fitting a numerical solution of this model to the data, it was possible to determine the chemotactic sensitivity coefficient, chi0. The mean of the best-fit values for chi0 from the three types of experiments was 7.2 x 10(-5) cm2/s. A less computationally intensive model based on earlier approaches that ignore cell transport in the chamber resulted in chi0 values that were approximately three times higher. The models evaluated in the present study could simulate the results of capillary assays only at low chamber cell concentrations, for which the effect of consumption on the distribution of substrate was negligible. Results from this work suggest that it is possible to use the capillary assay to quantify taxis towards environmentally relevant chemoeffectors that have low aqueous solubility.     

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.     

Stability of toluene oxidation by Pseudomonas putida under nutrient deprivation

Toluene-induced cells of Pseudomonas putida NCIMB 11767 lost their ability to oxidise toluene within 300 h under conditions of carbon/energy or nitrogen deprivation at 30°C, while incubation at 4°C improved the stability of this activity. Provision of inducing substrates (toluene or phenol) to nitrogen-deprived cells at 30°C also enhanced the stability of toluene oxidation, whereas provision of a non-inducing carbon/energy source (ethanol) led to a total loss of toluene oxidation within 160 h. Disappearance of toluene-induced proteins, at different rates accompanied the loss of toluene oxidation in carbon-deprived cells. The data suggest that degradation of one or more of the major proteins of toluene metabolism determines the stability of toluene oxidation in carbon-deprived cells. Around 40% of the whole-cell toluene oxidation rate was recoverable after cryopreservation (–20°C under glycerol) of toluene-induced cells but most of this recovered activity (86%) was associated with dead cells. These observations may have important implications for the application of these toluene-induced cells as in situ bioremediation catalysts.     

Growth and plasmid-encoded naphthalene catabolism of Pseudomonas putida in batch culture

Abstract The growth characteristics of Pseudomonas putida plasmid-harbouring strains which catabolize naphthalene via various pathways in batch culture with naphthalene as the sole source of carbon and energy have been investigated. The strains under study were constructed using the host strain P. putida BS394 which contained various naphthalene degradation plasmids. The highest specific growth rate was ensured by the plasmids that control naphthalene catabolism through the meta-pathway of catechol oxidation. The strains metabolizing catechol via the ortho -pathway grew at a lower rate. The lowest growth rate was observed with strain BS291 harbouring plasmid pBS4 which controls naphthalene catabolism via the gentisic acid pathway. Various pathways of naphthalene catabolism appear to allow these strains to grow at various rates which should be taken into account when constructing efficient degraders of polycyclic aromatic compounds.     

Changes in fatty acid composition in Pseudomonas putida and Pseudomonas stutzeri during naphthalene degradation

The effects of naphthalene on the whole cell-derived fatty acid composition of Pseudomonas putida and Pseudomonas stutzeri during naphthalene degradation were investigated. These strains differed in their abilities to degrade naphthalene and in 1,2-catechol dioxygenase activities. The cells of both strains reacted to the addition of naphthalene with an increase in the saturated/unsaturated ratio. The dynamic changes comprised also alterations in the percentage of hydroxy, cyclopropane and branched fatty acids. Upon the exposure of naphthalene, new fatty acids were detected.     

Quantification of Chemotaxis to Naphthalene by Pseudomonas putida G7

The capillary assay was used to quantify the chemotactic response of Pseudomonas putida G7 to naphthalene. Experiments were conducted in which the cell concentration in the assay chamber, the naphthalene concentration in the capillary, or the incubation time was varied. Data from these experiments were evaluated with a model that accounted for the effect of diffusion on the distribution of substrate and the transport of cells from the chamber through the capillary orifice. By fitting a numerical solution of this model to the data, it was possible to determine the chemotactic sensitivity coefficient, χ₀. The mean of the best-fit values for χ₀ from the three types of experiments was 7.2 × 10⁻⁵ cm²/s. A less computationally intensive model based on earlier approaches that ignore cell transport in the chamber resulted in χ₀ values that were approximately three times higher. The models evaluated in the present study could simulate the results of capillary assays only at low chamber cell concentrations, for which the effect of consumption on the distribution of substrate was negligible. Results from this work suggest that it is possible to use the capillary assay to quantify taxis towards environmentally relevant chemoeffectors that have low aqueous solubility.     

Toluene degradation kinetics for planktonic and biofilm-grown cells of Pseudomonas putida 54G

Toluene degradation kinetics by biofilm and planktonic cells of Pseudomonas putida 54G were compared in this study. Batch degradation of (14)C toluene was used to evaluate kinetic parameters for planktonic cells. The kinetic parameters determined for toluene degradation were: specific growth rate, mu(max) = 10.08 +/- 1.2/day; half-saturation constant, K(S) = 3.98 +/- 1.28 mg/L; substrate inhibition constant, K(I) = 42.78 +/- 3.87 mg/L. Biofilm cells, grown on ceramic rings in vapor phase bioreactors, were removed and suspended in batch cultures to calculate (14)C toluene degradation rates. Specific activities measured for planktonic and biofilm cells were similar based on toluene degrading cells and total biomass. Long-term toluene exposure reduced specific activities that were based on total biomass for both biofilm and planktonic cells. These results suggest that long-term toluene exposure caused a large portion of the biomass to become inactive, even though the biofilm was not substrate limited. Conversely, specific activities based on numbers of toluene-culturable cells were comparable for both biofilm and planktonically grown cultures. Planktonic cell kinetics are often used in bioreactor models to model substrate degradation and growth of bacteria in biofilms, a procedure we found to be appropriate for this organism. For superior bioreactor design, however, changes in cellular activity that occur during biofilm development should be investigated under conditions relevant to reactor operation before predictive models for bioreactor systems are developed. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 535-546, 1997.     

Plasmid characteristics of naphthalene and salicylate biodegradation in Pseudomonas putida

The object of this work was to study the physico-chemical and biological properties of DNAs of the biodegradation plasmids NAH and SAL. A comparative analysis of the physico-chemical parameters for these DNAs made it possible to detect a number of identical properties in them: the same sedimentation profile for covalently-closed circular DNA forms, 68--70 S; the molecular weight of ca. 50 MD; a roughly equal number of fragments (up to 23) was found when the DNAs of NAH and SAL were restricted by EcoRI endonuclease. The transformation of the plasmidless strain PpGI was done.     

Structure and carbohydrate analysis of the exopolysaccharide capsule of Pseudomonas putida G7

The aromatic hydrocarbon-degrading bacterium, Pseudomonas putida G7, produces exopolymers of potential interest in biotechnological applications. These exopolymers have been shown to have significant metal-binding ability . To initiate the study of the metal–polymer interactions, we explored the physical and chemical nature of the P. putida G7 exopolysaccharide, a major component of the exopolymer. A capsular structure was observed by light microscopy surrounding both planktonic and attached cells in biofilms after immunofluorescence staining with polyclonal antiserum raised against planktonic cells. Further work with planktonic cells showed that the immunostained capsule remained associated with young (log phase) cells, whereas older (stationary phase) cells lost their capsular material to the external milieu. Visualization of frozen, hydrated stationary phase cells by cryo-field emission scanning electron microscopy (cryoFESEM) revealed highly preserved extracellular material. In contrast, conventional scanning electron microscopy (SEM) of stationary phase cells showed rope-like material that most probably results from dehydrated and collapsed exopolymer. Both capsular and released exopolymers were separated from cells, and the released extracellular polysaccharide (EPS) was purified. Deoxycholate–polyacrylamide gel electrophoresis (PAGE) and silver/alcian blue staining of the partially purified material showed that it contained both EPS and lipopolysaccharide (LPS). Further purification of the EPS using a differential solubilization technique to remove LPS yielded highly purified EPS. Gas chromatography–mass spectrometry revealed that the purified EPS contained the monosaccharides, glucose, rhamnose, ribose, N-acetylgalactosamine and glucuronic acid. The structural and chemical properties of the P. putida EPS described here increase our understanding of the mechanisms of toxic metal binding by this well-known Proteobacterium.     

Evidence for plasmid-mediated chemotaxis of Pseudomonas putida towards naphthalene and salicylate

A naphthalene (Nap) and salicylate (Sal) degrading microorganism, Pseudomonas putida RKJ1, is chemotactic towards these compounds. This strain carries a 83 kb plasmid. A 25 kb EcoRI fragment of the plasmid contains the genes responsible for Nap degradation through Sal. RKJ5, the plasmid-cured derivative of RKJ1, is neither capable of degradation nor is chemotactic towards Nap or Sal. The recombinant plasmid pRKJ3, which contained a 25 kb EcoRI fragment, was transferred back into the plasmid-free wild-type strain RKJ5, and the transconjugant showed both degradation and chemotaxis. The recombinant plasmid pRKJ3 was also transferred into motile, plasmid-free P. putida KT2442. The resulting transconjugant (RKJ15) showed chemotaxis towards both Nap and Sal. Two mutant strains carrying deletions in pRKJ3 (in KT2442) with phenotypes Nap- Sal+ and Nap- Sal-, were also tested for chemotaxis. It was found that the Nap- Sal+ mutant strain showed chemotaxis towards Sal only, whereas the Nap- Sal- mutant strain is non-chemotactic towards both the compounds. These results suggest that the metabolism of Nap and Sal may be required for the chemotactic activity.     

Cloning of genes for naphthalene metabolism in Pseudomonas putida.

Plasmid pIG7 DNA cloned in Pseudomonas putida with the broad-host-range vectors pRK290 and pKT240 expresses the genes encoding nephthalene oxidation in the presence of the intermediate substrate, salicylate, or the gratuitous inducer, anthranilate. Two operons, nahAF and nahGK, cloned from the EcoRI fragment A (25 kilobases) are under wild-type regulation by the nahR locus. Deletion plasmids provide a restriction map of both operons. Double transformants containing structural and regulatory cistron nahR in trans are used to demonstrate positive control of expression.     

Growth and survival of Pseudomonas putida strains degrading naphthalene in soil model systems with different moisture levels

The effect of different moisture levels (from 20 to 70%) on the growth and survival of Pseudomonas putida strains G7 and BS3701 degrading naphthalene was studied in soil model systems. P. putida G7 contains plasmid NAH7 and P. putida BS3701 harbours plasmids pBS1141 and pBS1142. A mathematical model is proposed to describe the observed dynamics of the number of viable bacterial cells. Naphthalene and soil organic matter were considered as substrates available to bacteria. Data fitting allowed the estimation of model parameters characterizing microbial growth rate, utilization rate of substrates, specific maintenance rate and yield coefficient. Both the maximum bacterial concentration and the highest yield coefficient were observed at a soil moisture level of 40%. This optimal moisture level is close to but less than the water capacity (48%) of the soil used.     

Modeling of permeabilization process in Pseudomonas putida G7 for enhanced limonin bioconversion

A facile process of enhanced whole cell biotransformation to debitter the triterpenoid limonin in citrus juices was optimized in this work. To maximize bioconversion, permeabilization conditions were modeled using response surface methodology. A central composite rotatable design with four significant variables (concentration, temperature, pH, and treatment time) was employed. The second order polynomial equations with R2 values above 0.9 showed good correspondence between experimental and predicted values. The concentration, temperature, pH, and treatment time as well as their interactions had significant effects (p?相似文献   

Evidence of indigenous NAH plasmid of naphthalene degrading Pseudomonas putida PpG7 strain implicated in limonin degradation

A well characterized naphthalene-degrading strain, Pseudomonas putida PpG7 was observed to utilize limonin, a highly-oxygenated triterpenoid compound as a sole source of carbon and energy. Limonin concentrations evidenced a 64% reduction over 48 h of growth in batch cultures. Attempts were made to acquire a plasmid-less derivative via various methods (viz. Ethidium Bromide, SDS, elevated temperature & mitomycin C), among which the method involving mitomycin C (20 ug/ml) proved successful. Concomitant with the loss of plasmid in P. putida PpG7 strain, the cured derivative was identified as a lim- phenotype. The lim+ phenotype could be conjugally transferred to the cured derivative. Based on the results of curing with mitomycin C, conjugation studies and presence of ndo gene encoding naphthalene 1,2 dioxygenase, it was demonstrated that genes for the limonin utilization were encoded on an 83 kb indigenous transmissible Inc. P9 NAH plasmid in Pseudomonas putida PpG7 strain.     

Initial reactions in the oxidation of naphthalene by Pseudomonas putida.

A strain of Pseudomonas putida that can utilize naphthalene as its sole source of carbon and energy was isolated from soil. A mutant strain of this organism, P. putida 119, when grown on glucose in the presence of naphthalene, accumulates optically pure (+)-cis-1(R),2(S)-dihydroxy-1,2-dihydronaphthalene in the culture medium. The cis relative stereochemistry in this molecule was established by nuclear magnetic resonance spectrometry. Radiochemical trapping experiments established that this cis dihydrodiol is an intermediate in the metabolism of naphthalene by P. Fluorescens (formerly ATCC, 17483), P. putida (ATCC, 17484), and a Pseudomonas species (NCIB 9816), as well as the parent strain of P. putida described in this report. Formation of the cis dihydrodiol is catalyzed by a dioxygenase which requires either NADH or NADPH as an electron donor. A double label procedure is described for determining the origin of oxygen in the cis dihydrodiol under conditions where this metabolite would not normally accumulate. Several aromatic hydrocarbons are oxidized by cell extracts prepared from naphthalene-grown cells of P. putida. The cis dihydrodiol is converted to 1,2-dihydroxynaphthalene by an NAD+-dependent dehydrogenase. This enzyme is specific for the (+) isomer of the dihydrodiol and shows a primary isotope effect when the dihydrodiol is substituted at C-2 with deuterium.     

Physiological stress in batch cultures of Pseudomonas putida 54G during toluene degradation

Physiological stress associated with toluene exposure in batch cultures of Pseudomonas putida 54G was investigated. P. putida 54G cells were grown using a continuous vapor phase feed stream containing 150 ppmv or 750 ppmv toluene as the sole carbon and energy source. Cells were enumerated on non-selective (R2A agar plates) and a selective minimal medium incubated in the presence of vapor phase toluene (HCMM2). Differential recovery on the two media was used to evaluate bacterial stress, culturability and loss of toluene-degrading capability. A majority of the bacteria were reversibly stressed and could resume active colony formation on selective medium after passage on non-selective medium. A small fraction of the bacterial cells suffered an irreversible loss of toluene degradation capability and were designated as Tol⁻ variants. Numbers of stressed organisms increased with duration of toluene exposure and toluene concentration and coincided with accumulation of metabolic intermediates from incomplete toluene degradation. Respiring cell numbers in the batch cultures decreased as injury increased, indicating a possible relationship between respiring and injured cells. Rate expressions for injury, for formation of Tol⁻ variants and for growth of Tol⁻ variants were determined by calibrating a theoretical model to the results obtained. These rate expressions can be used to calibrate bioreactor models, and provide a basis for better design and control of bioremediation systems. Received 01 July 1996/ Accepted in revised form 25 March 1997     

Multi-substrate growth kinetics of Pseudomonas putida for phenol removal

The biodegradation of phenol by a pure culture of Pseudomonas putida was investigated in a continuously fed stirred-tank reactor, under aerobic conditions. The dilution rate was varied between 0.0174 h⁻¹ and 0.278 h⁻¹, covering a wide range of dissolved oxygen and the inhibition region of phenol. Through non-linear analysis of the data, a dual-substrate growth kinetics, Haldane kinetics for phenol and Monod kinetics for oxygen, was derived with high correlation coefficients. Respective biokinetic parameters were evaluated as μ_m = 0.569 h⁻¹, K _p = 18.539 mg/l, K _i = 99.374 mg/l, K _o = 0.048 mg/l, Y _x/p = 0.521 g microorganism/g phenol and Y _x/o = 0.338 g microorganism/g oxygen, being in good agreement with other studies in the literature. Maintenance factors for both phenol and oxygen were calculated for the first time for P. putida while the saturation coefficient for oxygen, K _o, was genuinely evaluated from the constructed model, not imported or adapted from other studies as reported in the literature. All pertinent biokinetic parameters for P. putida have been calculated from continuous system data, which are most appropriate for use in continuous bioprocess applications. Received: 29 July 1996 / Received revision: 18 November 1996 / Accepted: 23 November 1996     

Enhanced tolerance to naphthalene and enhanced rhizoremediation performance for Pseudomonas putida KT2440 via the NAH7 catabolic plasmid

In this work, we explore the potential use of the Pseudomonas putida KT2440 strain for bioremediation of naphthalene-polluted soils. Pseudomonas putida strain KT2440 thrives in naphthalene-saturated medium, establishing a complex response that activates genes coding for extrusion pumps and cellular damage repair enzymes, as well as genes involved in the oxidative stress response. The transfer of the NAH7 plasmid enables naphthalene degradation by P. putida KT2440 while alleviating the cellular stress brought about by this toxic compound, without affecting key functions necessary for survival and colonization of the rhizosphere. Pseudomonas putida KT2440(NAH7) efficiently expresses the Nah catabolic pathway in vitro and in situ, leading to the complete mineralization of [(14)C]naphthalene, measured as the evolution of (14)CO(2), while the rate of mineralization was at least 2-fold higher in the rhizosphere than in bulk soil.