Secondary substrate utilization of methylene chloride by an isolated strain of Pseudomonas sp

Secondary substrate utilization of methylene chloride was analyzed by using Pseudomonas sp. strain LP. Both batch and continuously fed reactors demonstrated that this strain was capable of simultaneously consuming two substrates at different concentrations: the primary substrate at the higher concentration (milligrams per liter) and the secondary substrate at the lower concentration (micrograms per liter). The rate of methylene chloride utilization at trace concentrations was greater in the presence of the primary substrate, acetate, than without it. However, when the substrate roles were changed, the acetate secondary substrate utilization rate was less when methylene chloride was present. Thus, substrate interactions are important in the kinetics of secondary substrate utilization. Pseudomonas sp. strain LP showed a preference toward degrading methylene chloride over acetate, whether it was the primary or secondary substrate, providing it was below an inhibitory concentration of ca. 10 mg/liter.     

Secondary substrate utilization of methylene chloride by an isolated strain of Pseudomonas sp.

Secondary substrate utilization of methylene chloride was analyzed by using Pseudomonas sp. strain LP. Both batch and continuously fed reactors demonstrated that this strain was capable of simultaneously consuming two substrates at different concentrations: the primary substrate at the higher concentration (milligrams per liter) and the secondary substrate at the lower concentration (micrograms per liter). The rate of methylene chloride utilization at trace concentrations was greater in the presence of the primary substrate, acetate, than without it. However, when the substrate roles were changed, the acetate secondary substrate utilization rate was less when methylene chloride was present. Thus, substrate interactions are important in the kinetics of secondary substrate utilization. Pseudomonas sp. strain LP showed a preference toward degrading methylene chloride over acetate, whether it was the primary or secondary substrate, providing it was below an inhibitory concentration of ca. 10 mg/liter.     

Isolation and characterization of a Pseudomonas sp. strain PH1 utilizing meta-aminophenol

Pseudomonas sp. strain PH1 was isolated from soil contaminated with pharmaceutical and dye industry waste. The isolate PH1 could use m-aminophenol as a sole source of carbon, nitrogen, and energy to support the growth. PH1 could degrade up to 0.32 mM m-aminophenol in 120 h, when provided as nitrogen source at 0.4 mM concentration with citrate (0.5 mM) as a carbon source in the growth medium. The presence of ammonium chloride as an additional nitrogen source repressed the degradation of m-aminophenol by PH1. To identify strain PH1, the 16S rDNA sequence was amplified by PCR using conserved eubacterial primers. The FASTA program was used to analyze the 16S rDNA sequence and the resulting homology patterns suggested that PH1 is a Pseudomonas.     

Organophosphonate utilization by the wild-type strain of Pseudomonas fluorescens.

The wild-type strain of Pseudomonas fluorescens was found to utilize a range of structurally diverse organophosphonates as its sole carbon or nitrogen sources. Representative compounds included aminoalkylphosphonates, hydroxyalkylphosphonates, oxoalkylphosphonates, and phosphono dipeptides. Among them, amino(phenyl)methylphosphonate,2-aminoethylphosphonate, aminomethylphosphonate, diisopropyl 9-aminofluoren-9-ylphosphonate, and 2-oxoalkylphosphonates were used by P. fluorescens as its sole sources of phosphorus. Only slight growth was observed on the herbicide glyphosate (N-phosphonomethylglycine), which was metabolized to aminomethylphosphonate. Neither phosphinothricin nor its dialanyl tripeptide, bialaphos, supported growth of P. fluorescens. The possible mechanisms of organophosphonate degradation by this strain are discussed.     

Organophosphonate utilization by the wild-type strain of Pseudomonas fluorescens.

The wild-type strain of Pseudomonas fluorescens was found to utilize a range of structurally diverse organophosphonates as its sole carbon or nitrogen sources. Representative compounds included aminoalkylphosphonates, hydroxyalkylphosphonates, oxoalkylphosphonates, and phosphono dipeptides. Among them, amino(phenyl)methylphosphonate,2-aminoethylphosphonate, aminomethylphosphonate, diisopropyl 9-aminofluoren-9-ylphosphonate, and 2-oxoalkylphosphonates were used by P. fluorescens as its sole sources of phosphorus. Only slight growth was observed on the herbicide glyphosate (N-phosphonomethylglycine), which was metabolized to aminomethylphosphonate. Neither phosphinothricin nor its dialanyl tripeptide, bialaphos, supported growth of P. fluorescens. The possible mechanisms of organophosphonate degradation by this strain are discussed.     

Investigation on the regulation of aniline utilization in Pseudomonas multivorans strain An 1

Summary Pseudomonas multivorans strain An 1 was isolated from forest soil after enrichment in a medium containing 0.1% aniline as the sole source of carbon and energy. Increasing aniline concentrations increasingly inhibited bacterial growth. At pH 7, aniline concentrations greater than 16mM were toxic enough to completely arrest growth. The optimal pH for growth on aniline was 6.5.On binary mixtures of aniline and additional carbon sources, diauxic growth was observed. The addition carbon sources caused various degrees of repression of the aniline catabolizing enzyme system. The fastest induction of this system occurred at pH 4, suggesting that protonization of the aniline molecule is crucial.     

Impaired substrate utilization in mitochondria from strain 129 dystrophic mice

Mitochondria from skeletal muscle, heart and liver of strain 129/ReJ-dy dystrophic mice and their littermate controls were characterized with respect to their respiratory and phosphorylating activities. Skeletal muscle mitochondria from dystrophic mice showed significantly lower state 3 respiratory rates than controls with both pyruvate + malate and succinate as substrates (P < 0.01). ADP/O and Ca²⁺/O ratios were found to be normal. A decreased rate of NADH oxidation (0.01 <P < 0.05) by sonicated mitochondrial suspensions from dystrophic mice was also seen. High respiratory rates with ascorbate + phenazine methosulfate as substrates indicated that cytochrome oxidase was not rate limiting in the oxidation of either pyruvate + malate or succinate. Skeletal muscle mitochondria from dystrophic mice showed no deficiency in any of the cytochromes or coenzyme Q. Mg²⁺-stimulated ATPase activity was higher in dystrophic muscle mitochondria than in controls, but basal and oligomycin-insensitive activities were virtually identical to those of controls. A significant reduction in the intramitochondrial NAD⁺ content (0.01 <P < 0.02) was seen in dystrophic skeletal muscle as compared to controls. Heart mitochondria from dystrophic mice showed similar, though less extensive abnormalities while liver mitochondria were essentially normal. We concluded from these results that skeletal muscle mitochondria from strain 129 dystrophic mice possess impairments in substrate utilization which may result from (1) an abnormality in the transfer of electrons on the substrate side of coenzyme Q in the case of succinate oxidation; (2) a defect on the path of electron flow from NADH to cytochrome c, and (3) a deficiency of NAD⁺ in the case of NAD⁺-linked substrates.     

Numerical taxonomy of Pseudomonas based on published records of substrate utilization

Data published by R. Y. Stanier, N. J. Palleroni, M. Doudoroff and their colleagues on Pseudomonas have been analysed by numerical taxonomy. Records on 401 strains were used, representing 155 characters, mostly utilization of substrates as carbon-energy sources. Twenty-nine phenons were recognized, which included 394 strains: the remaining 7 remained unclustered. The results were in very good accord with the conclusions of these authors. Almost all phenons were well separated with very little overlap. Many of them corresponded to distinct species, and others corresponded to recognized biotypes. Some small groups may represent unnamed new species.Analyses by Gower's Coefficient showed five major groupings: A) the fluorescent pseudomonads; B) biochemically active species (Pseudomonas cepacia, P. pseudomallei and allies); D) P. solanacearum and allies; and E) P. mallei. P. diminuta does not appear to be clearly distinct from P. vesicularis, nor does P. alcaligenes appear clearly distinct from P. pseudoalcaligenes. There may, however, be some difference between P. multivorans and P. cepacia.Analyses using the Pattern Coefficient differed mainly in the relationships shown by a few of the metabolically active species. Of the two coefficients, the Pattern Coefficient gave results that were in somewhat better agreement with evidence from nucleic acids, but it showed an unexpectedly close relationship between P. solanacearum and P. cepacia.     

L-arginine utilization by Pseudomonas species

The utilization of arginine was studied in several different Pseudomonas species. The arginine decarboxylase and agmatine deiminase pathways were found to be characteristic of Pseudomonas species of group I as defined by Palleroni et al. (1974). Pseudomonas putida strains had three distinct arginine catabolic pathways initiated by arginine decarboxylase, arginine deiminase and arginine oxidase, respectively. The two former routes were also present in P. fluorescens and P. mendocina and in P. aeruginosa which also used arginine by a further unknown pathway. None of these pathways occurred in P. cepacia strains; agmatine catabolism seemed to follow an unusual route involving guanidinobutyrate as intermediate.     

Growth and phenol activity of Pseudomonas aeruginosa strain 101/1 in batch cultures

Strain 101/1, isolated from petroleum wastewater sediment was classified as Pseudomonas aeruginosa. In wild type condition the strain tolerated phenol in concentration 1,000 mg/L under aerobic conditions and 800 mg/L under denitrifying conditions. As a result of adaptation to phenol the resistance of the strain to the compound increased to 1,600 and 1,400 mg/L, respectively. Maximum phenol activity under aerobic and denitrifying conditions was 350 and 65 mg/L x day-1, respectively. Under denitrifying conditions a reduction in incubation temperature from 30 degrees C to 20 degrees C resulted in two-fold drop in phenol activity of the adapted strain and reduction in tolerance to phenol by 400 mg/L.     

Nitrogen control of atrazine utilization in Pseudomonas sp. strain ADP

Pseudomonas sp. strain ADP uses the herbicide atrazine as the sole nitrogen source. We have devised a simple atrazine degradation assay to determine the effect of other nitrogen sources on the atrazine degradation pathway. The atrazine degradation rate was greatly decreased in cells grown on nitrogen sources that support rapid growth of Pseudomonas sp. strain ADP compared to cells cultivated on growth-limiting nitrogen sources. The presence of atrazine in addition to the nitrogen sources did not stimulate degradation. High degradation rates obtained in the presence of ammonium plus the glutamine synthetase inhibitor MSX and also with an Nas(-) mutant derivative grown on nitrate suggest that nitrogen regulation operates by sensing intracellular levels of some key nitrogen-containing metabolite. Nitrate amendment in soil microcosms resulted in decreased atrazine mineralization by the wild-type strain but not by the Nas(-) mutant. This suggests that, although nitrogen repression of the atrazine catabolic pathway may have a strong impact on atrazine biodegradation in nitrogen-fertilized soils, the use of selected mutant variants may contribute to overcoming this limitation.     

Peptide utilization by Pseudomonas putida and Pseudomonas maltophilia.

Pseudomonas putida assimilates peptides and hydrolyses them with intracellular peptidases. Amino acid auxotrophs (his, trp, thr or met) grew on a variety of di- and tripeptides up to twice as slowly as with free amino acids. Pseudomonas putida has separate uptake systems for both dipeptides and oligopeptides (three or more residues). Although the dipeptide system transported a variety of structurally diverse dipeptides it did not transport peptides having either unprotonatable N-terminal amino groups, blocked C-terminal carboxyl groups, D-residues, three or more residues, N-methylated peptide bonds, or beta-amino acids. Oligopeptide uptake lacked amino acid side-chain specificity, required a free N-terminal L-residue and had an upper size limit. Glycylglycyl-D,L-p-fluorophenylalanine inhibited growth of P. putida. Uptake of glycylglycyl[I-14C]alanine was rapid and inhibited by 2,4-dinitrophenol. Both dipeptide and oligopeptide uptake were constitutive. Dipeptides competed with oligopeptides for oligopeptide uptake, but oligopeptides did not compete in the dipeptide system. Final bacterial yields were 5 to 10 times greater when P. putida his was grown on histidyl di- or tripeptides rather than on free histidine because the histidyl residue was protected from catabolism by L-histidine ammonia-lyase. Methionine peptides could satisfy the methionine requirements of P. maltophilia. Generation times on glycylmethionine and glycylmethionylglycine were equal to those obtained with free methionine. Methionylglycylmethionylmethionine gave a generation time twice that of free methionine. Growth of P. maltophilia was inhibited by glycylglycyl-D,L-p-fluorophenylalanine.     

Propachlor removal by Pseudomonas strain GCH1 in an immobilized-cell system

A bacterial strain capable of growing on propachlor (2-chloro-N-isopropylacetanilide) was isolated from soil by using enrichment and isolation techniques. The strain isolated, designated GCH1, was classified as a member of the genus Pseudomonas. Washed-cell suspensions of strain GCH1 accumulated N-isopropylacetanilide, acetanilide, acetamide, and catechol. Pseudomonas strain GCH1 grew on propachlor with a generation time of 4.2 h and a rate of substrate utilization of 1.75 +/- 0.15 micromol h(-1). Gene expression did not require induction but was subject to catabolite expression. Acetanilide was a growth substrate with a yield of 0.56 +/- 0.02 mg of protein micromol(-1). GCH1 strain cells were immobilized by adsorption onto a ceramic support and were used as biocatalysts in an immobilized cell system. Propachlor elimination reached 98%, with a retention time of 3 h and an initial organic load of 0.5 mM propachlor. The viability of immobilized cells increased 34-fold after 120 days of bioreactor operation.     

Growth Phase Dependent Substrate Utilization by PseudomonasStrain PH

Pseudomonas strain PH1 can utilize nitro-, chloro-, and aminophenols and was used in this study. The enzymes of two pathways, utilizing phenol and meta-aminophenol (MAP), were analyzed under different growth conditions. The enzymes responsible for phenol to catechol conversion followed by the ring cleavage enzyme for catechol, and also the enzymes responsible for MAP oxidation and hydroxylation of resorcinol, were studied. Enzyme and respirometric assays were carried out with cells harvested from log phase and stationary phase from medium with different carbon sources and nitrogen levels. It was observed that the first step for utilization of both the substrates requires the same physiological state of the cells, whereas, the subsequent step require different physiological states.     

The production and utilization of nitric oxide by a new,denitrifying strain of Pseudomonas aeruginosa

When a new strain of Pseudomonas aeruginosa was grown aerobically and then transferred to anaerobic conditions, cells reduced NO ₃ ^– quantitatively to NO ₂ ^– in NO ₃ ^– -respiration. In the absence of nitrate, NO ₂ ^– was immediately reduced to NO or N₂O but not to N₂ indicating that NO ₂ ^– -reductase but not N₂O-reductase was active. The formation of the products NO or N₂O depended on the pH in the medium and the concentration of NO ₂ ^– present. When P. aeruginosa was grown anaerobically for at least three davs N₂O-reductase was also active. Such cells reduced NO to N₂ via N₂O. The new strain generated a H⁺-gradient and grew by reducing N₂O to N₂ but not by converting NO to N₂O. For comparison, Azospirillum brasilense Sp7 showed the same pattern of NO-reduction. In contrast, Paracoccus denitrificans formed 3.5 H⁺/NO during the reduction of NO to N₂O in oxidant pulse experiments but could not grow in the presence of NO. Thus the NO-reduction pattern in P. denitrificans on one side and P. aeruginosa and A. brasilense on the other was very different. The mechanistic implications of such differences are discussed.     

Comparison of substrate utilization and growth kinetics between immobilized and suspended Pseudomonas cells

A methodology is described for measurement if immobilized and suspended cell growth and substrate utilization kinetics parameters. Substrate utilization and growth kinetics were compared between immobilized and suspended cells for toluene degrading Pseudomonas strains K3-2 and 2,4-dichlorophenoxyacetic acid (2,4-D) degrading strain DBO131(pR0101), respectively. Kinetic parameters were estimated using nonlinear parameter estimation methods and compared between the immobilized and suspended Pseudomonas cells to determine the effect of immobilization on cellular growth and substrate utilization. Factors influencing the experimental design included calculated oxygen flux rates, primary carbon substrate flux rates, and shear stresses on the immobilize cell. Statistical interpretation of the cellular reaction rate parameters indicates that only the growth kinetics of the toluene system were significantly altered upon immobilization. Substrate utilization kinetics remained unchanged upon immobilization. The substrate growth associated half-saturation constant (K(g)) for the toluene system increased by 30-fold and the maximum specific growth rate (mu(max)) decreased by 2-fold upon immobilization. Implication of these results for experimental determination of cellular kinetic parameters and for immobilization cell bioreactors design are discussed. (c) 1993 John Wiley & Sons, Inc.     

Growth substrate dependent localization of tetrachloroethene reductive dehalogenase in Sulfurospirillum multivorans

Sulfurospirillum multivorans is a dehalorespiring organism, which is able to utilize tetrachloroethene as terminal electron acceptor in an anaerobic respiratory chain. The localization of the tetrachloroethene reductive dehalogenase in dependence on different growth substrates was studied using the freeze-fracture replica immunogold labeling technique. When the cells were grown with pyruvate plus fumarate, a major part of the enzyme was either localized in the cytoplasm or membrane associated facing the cytoplasm. In cells grown on pyruvate or formate as electron donors and tetrachloroethene as electron acceptor, most of the enzyme was detected at the periplasmic side of the cytoplasmic membrane. These results were confirmed by immunoblots of the enzyme with and without the twin arginine leader peptide. Trichloroethene exhibited the same effect on the enzyme localization as tetrachloroethene. The data indicated that the localization of the enzyme was dependent on the electron acceptor utilized.