Role of nucleases in the isolation of plasmid deoxyribonucleic acid from Pseudomonas cepacia 4G9.

Brij 58-cleared lysates of Pseudomonas cepacia 4G9 contain both exonucleolytic and endonucleolytic activities. Endonuclease activity was unaffected by 125 mM ethylenediaminetetraacetic acid, whereas the exonuclease activity was inhibited. In contrast, Sarkosyl NL97 inhibited only the endonuclease. Sodium dodecyl sulfate inhibited all nuclease activity in in vitro assays, but plasmid deoxyribonucleic acid added to P. cepacia 4G9 spheroplasts during sodium dodecyl sulfate lysis was degraded. Irreproducible plasmid isolation from P. cepacia 4G9 may be due to this nucleolytic activity.     

Monitoring trichloroethylene mineralization by Pseudomonas cepacia G4 PR1

To analyze the extent of mineralization of trichloroethylene (TCE) without disturbing an actively growing biofilm, a minimal growth medium was formulated that reduces the concentration of chloride ions to the extent that the chloride ions generated from TCE mineralization may be detected with a chloride-ion-specific electrode. By substituting chloride salts with phosphates and nitrates, a chloride-free minimal medium was produced that yields a specific growth rate for Pseudomonas cepacia G4 PR1 which was 93% of that in chloride-ion-containing minimal medium. Furthermore, TCE degradation by resting cell suspensions was similar in both media (85% of 75 M TCE degraded in 6 h), and complete mineralization of TCE was slightly superior in the chloride-free minimal medium (77% compared to 60% of 75 M TCE mineralized in 6 h). In addition, indole-containing, minimal-medium agar plates were developed to indicate the presence of the TCE-degrading enzyme toluene ortho-monooxygenase (fire-engine-red colonies) as well as to distinguish this enzyme from other TCE-degrading enzymes (toluene dioxygenase and toluene para-monooxygenase).     

Degradation of trichloroethylene by Pseudomonas cepacia G4 and the constitutive mutant strain G4 5223 PR1 in aquifer microcosms.

Pseudomonas cepacia G4 degrades trichloroethylene (TCE) via a degradation pathway for aromatic compounds which is induced by substrates such as phenol and tryptophan. P. cepacia G4 5223 PR1 (PR1) is a Tn5 insertion mutant which constitutively expresses the toluene ortho-monooxygenase responsible for TCE degradation. In groundwater microcosms, phenol-induced strain G4 and noninduced strain PR1 degraded TCE (20 and 50 microM) to nondetectable levels (< 0.1 microM) within 24 h at densities of 10(8) cells per ml; at lower densities, degradation of TCE was not observed after 48 h. In aquifer sediment microcosms, TCE was reduced from 60 to < 0.1 microM within 24 h at 5 x 10(8) PR1 organisms per g (wet weight) of sediment and from 60 to 26 microM over a period of 10 weeks at 5 x 10(7) PR1 organisms per g. Viable G4 and PR1 cells decreased from approximately 10(7) to 10(4) per g over the 10-week period.     

Mutants of Pseudomonas cepacia G4 defective in catabolism of aromatic compounds and trichloroethylene

Pseudomonas cepacia G4 possesses a novel pathway of toluene catabolism that is shown to be responsible for the degradation of trichloroethylene (TCE). This pathway involves conversion of toluene via o-cresol to 3-methylcatechol. In order to determine the enzyme of toluene degradation that is responsible for TCE degradation, chemically induced mutants, blocked in the toluene ortho-monooxygenase (TOM) pathway of G4, were examined. Mutants of the phenotypic class designated TOM A- were all defective in their ability to oxidize toluene, o-cresol, m-cresol, and phenol, suggesting that a single enzyme is responsible for conversion of these compounds to their hydroxylated products (3-methylcatechol from toluene, o-cresol, and m-cresol and catechol from phenol) in the wild type. Mutants of this class did not degrade TCE. Two other mutant classes which were blocked in toluene catabolism, TOM B-, which lacked catechol-2,3-dioxygenase, and TOM C-, which lacked 2-hydroxy-6-oxoheptadienoic acid hydrolase activity, were fully capable of TCE degradation. Therefore, TCE degradation is directly associated with the monooxygenation capability responsible for toluene, cresol, and phenol hydroxylation.     

Phenol and trichloroethylene degradation by Pseudomonas cepacia G4: kinetics and interactions between substrates.

Intact cells of Pseudomonas cepacia G4 completely degraded trichloroethylene (TCE) following growth with phenol. Degradation kinetics were determined for both phenol, used to induce requisite enzymes, and TCE, the target substrate. Apparent Ks and Vmax values for degradation of phenol by cells were 8.5 microM and 466 nmol/min per mg of protein, respectively. At phenol concentrations greater than 50 microM, phenol degradation was inhibited, yielding an apparent second-order inhibitory value, KSI, of 0.45 mM as modeled by the Haldane expression. A partition coefficient for TCE was determined to be 0.40 +/- 0.02, [TCEair]/[TCEwater], consistent with Henry's law. To eliminate experimental problems associated with TCE volatility and partitioning, a no-headspace bottle assay was developed, allowing for direct and accurate determinations of aqueous TCE concentration. By this assay procedure, apparent Ks and Vmax values determined for TCE degradation by intact cells were 3 microM and 8 nmol/min per mg of protein, respectively. Following a transient lag period, P. cepacia G4 degraded TCE at concentrations of at least 300 microM with no apparent retardation in rate. Consistent with Ks values determined for degradation, TCE significantly inhibited phenol degradation.     

TOM, a new aromatic degradative plasmid from Burkholderia (Pseudomonas) cepacia G4.

Burkholderia (Pseudomonas) cepacia PR1(23) has been shown to constitutively express to toluene catabolic pathway distinguished by a unique toluene ortho-monooxygenase (Tom). This strain has also been shown to contain two extrachromosomal elements of < 70 and > 100 kb. A derivative strain cured of the largest plasmid, PR1(23) Cure, was unable to grow on phenol or toluene as the sole source of carbon and energy, which requires expression of the Tom pathway. Transfer of the larger plasmid from strain G4 (the parent strain inducible for Tom) enabled PR1(23) Cure to grow on toluene or phenol via inducible Tom pathway expression. Conjugal transfer of TOM23c from PR1(23) to an antibiotic-resistant derivative of PR1(23) Cure enabled the transconjugant to grow with either phenol or toluene as the sole source of carbon and energy through constitutive expression of the Tom pathway. A cloned 11.2-kb EcoRI restriction fragment of TOM23c resulted in the expression of both Tom and catechol 2,3-dioxygenase in Escherichia coli, as evidenced by its ability to oxidize trichloroethylene, toluene, m-cresol, o-cresol, phenol, and catechol. The largest resident plasmid of PR1 was identified as the source of these genes by DNA hybridization. These results indicate that the genes which encode Tom and catechol 2,3-dioxygenase are located on TOM, an approximately 108-kb degradative plasmid of B. cepacia G4.     

Chemotaxis of Pseudomonas stutzeri OX1 and Burkholderia cepacia G4 toward chlorinated ethenes

The chemotactic responses of Pseudomonas putida F1, Burkholderia cepacia G4, and Pseudomonas stutzeri OX1 were investigated toward toluene, trichloroethylene (TCE), tetrachloroethylene (PCE), cis-1,2-dichloroethylene (cis-DCE), trans-1,2-dichloroethylene (trans-DCE), 1,1-dichloroethylene (1,1-DCE), and vinyl chloride (VC). P. stutzeri OX1 and P. putida F1 were chemotactic toward toluene, PCE, TCE, all DCEs, and VC. B. cepacia G4 was chemotactic toward toluene, PCE, TCE, cis-DCE, 1,1-DCE, and VC. Chemotaxis of P. stutzeri OX1 grown on o-xylene vapors was much stronger than when grown on o-cresol vapors toward some chlorinated ethenes. Expression of toluene-o-xylene monooxygenase (ToMO) from touABCDEF appears to be required for positive chemotaxis attraction, and the attraction is stronger with the touR (ToMO regulatory) gene.     

Performance characterization of a model bioreactor for the biodegradation of trichloroethylene by Pseudomonas cepacia G4.

Pseudomonas cepacia G4 grown in chemostats with phenol demonstrated constant specific degradation rates for both phenol and trichloroethylene (TCE) over a range of dilution rates. Washout of cells from chemostats was evident at a dilution rate of 0.2 h-1 at 28 degrees C. Increased phenol concentrations in the nutrient feed led to increased biomass production with constant specific degradation rates for both phenol and TCE. The addition of lactate to the phenol feed led to increased biomass production but lowered specific phenol and TCE degradation rates. The maximum potential for TCE degradation was about 1.1 g per day per g of cell protein. Cell growth and degradation kinetic parameters were used in the design of a recirculating bioreactor for TCE degradation. In this reactor, the total amount of TCE degraded increased as either reaction time or biomass was increased. TCE degradation was observed up to 300 microM TCE with no significant decreases in rates. On the average, this reactor was able to degrade 0.7 g of TCE per day per g of cell protein. These results demonstrate the feasibility of TCE bioremediation through the use of bioreactors.     

Cometabolic degradation of trichloroethylene by Pseudomonas cepacia G4 in a chemostat with toluene as the primary substrate.

Pseudomonas cepacia G4 is capable of cometabolic degradation of trichloroethylene (TCE) if the organism is grown on certain aromatic compounds. To obtain more insight into the kinetics of TCE degradation and the effect of TCE transformation products, we have investigated the simultaneous conversion of toluene and TCE in steady-state continuous culture. The organism was grown in a chemostat with toluene as the carbon and energy source at a range of volumetric TCE loading rates, up to 330 mumol/liter/h. The specific TCE degradation activity of the cells and the volumetric activity increased, but the efficiency of TCE conversion dropped when the TCE loading was elevated from 7 to 330 mumol/liter/h. At TCE loading rates of up to 145 mumol/liter/h, the specific toluene conversion rate and the molar growth yield of the cells were not affected by the presence of TCE. The response of the system to varying TCE loading rates was accurately described by a mathematical model based on Michaelis-Menten kinetics and competitive inhibition. A high load of 3,400 mumol of TCE per liter per h for 12 h caused inhibition of toluene and TCE conversion, but reduction of the TCE load to the original nontoxic level resulted in complete recovery of the system within 2 days. These results show that P. cepacia can stably and continuously degrade toluene and TCE simultaneously in a single-reactor system without biomass retention and that the organism is more resistant to high concentrations and shock loadings of TCE than Methylosinus trichosporium OB3b.     

Phenol and trichloroethylene degradation by Pseudomonas cepacia G4: kinetics and interactions between substrates

Intact cells of Pseudomonas cepacia G4 completely degraded trichloroethylene (TCE) following growth with phenol. Degradation kinetics were determined for both phenol, used to induce requisite enzymes, and TCE, the target substrate. Apparent Ks and Vmax values for degradation of phenol by cells were 8.5 microM and 466 nmol/min per mg of protein, respectively. At phenol concentrations greater than 50 microM, phenol degradation was inhibited, yielding an apparent second-order inhibitory value, KSI, of 0.45 mM as modeled by the Haldane expression. A partition coefficient for TCE was determined to be 0.40 +/- 0.02, [TCEair]/[TCEwater], consistent with Henry's law. To eliminate experimental problems associated with TCE volatility and partitioning, a no-headspace bottle assay was developed, allowing for direct and accurate determinations of aqueous TCE concentration. By this assay procedure, apparent Ks and Vmax values determined for TCE degradation by intact cells were 3 microM and 8 nmol/min per mg of protein, respectively. Following a transient lag period, P. cepacia G4 degraded TCE at concentrations of at least 300 microM with no apparent retardation in rate. Consistent with Ks values determined for degradation, TCE significantly inhibited phenol degradation.     

Resistance of a strain of Pseudomonas cepacia to esters of p-hydroxybenzoic acid.

Cells of a strain of Pseudomonas cepacia were isolated from an oil-in-water emulsion containing methyl and propyl p-hydroxybenzoates (methylparaben and propylparaben) as preservative additives. This strain demonstrated the ability to destroy these additives, to utilize the propyl ester as sole carbon source, and to hydrolyze the methyl ester. When the isolate was grown on Eugon agar, exposure to the methyl ester killed 99.9% of the inoculum, but the surviving cells grew logarithmically. On the other hand, cells grown on media containing propylparaben were less susceptible when subsequently exposed to emulsions containing methylparaben. These observations demonstrate one mechanism by which microorganisms develop resistance to antimicrobial preservatives.     

Resistance of a strain of Pseudomonas cepacia to esters of p-hydroxybenzoic acid.

Cells of a strain of Pseudomonas cepacia were isolated from an oil-in-water emulsion containing methyl and propyl p-hydroxybenzoates (methylparaben and propylparaben) as preservative additives. This strain demonstrated the ability to destroy these additives, to utilize the propyl ester as sole carbon source, and to hydrolyze the methyl ester. When the isolate was grown on Eugon agar, exposure to the methyl ester killed 99.9% of the inoculum, but the surviving cells grew logarithmically. On the other hand, cells grown on media containing propylparaben were less susceptible when subsequently exposed to emulsions containing methylparaben. These observations demonstrate one mechanism by which microorganisms develop resistance to antimicrobial preservatives.     

Performance characterization of a model bioreactor for the biodegradation of trichloroethylene by Pseudomonas cepacia G4

Pseudomonas cepacia G4 grown in chemostats with phenol demonstrated constant specific degradation rates for both phenol and trichloroethylene (TCE) over a range of dilution rates. Washout of cells from chemostats was evident at a dilution rate of 0.2 h-1 at 28 degrees C. Increased phenol concentrations in the nutrient feed led to increased biomass production with constant specific degradation rates for both phenol and TCE. The addition of lactate to the phenol feed led to increased biomass production but lowered specific phenol and TCE degradation rates. The maximum potential for TCE degradation was about 1.1 g per day per g of cell protein. Cell growth and degradation kinetic parameters were used in the design of a recirculating bioreactor for TCE degradation. In this reactor, the total amount of TCE degraded increased as either reaction time or biomass was increased. TCE degradation was observed up to 300 microM TCE with no significant decreases in rates. On the average, this reactor was able to degrade 0.7 g of TCE per day per g of cell protein. These results demonstrate the feasibility of TCE bioremediation through the use of bioreactors.     

Selective medium for Pseudomonas cepacia containing 9-chloro-9-(4-diethylaminophenyl)-10-phenylacridan and polymyxin B sulfate.

Contamination of solutions and lotions with Pseudomonas cepacia is a growing concern among health professionals. The identification of P. cepacia usually requires a long series of biochemical tests. In an effort to develop a more direct method, we evaluated plate count agar containing 9-chloro-9-(4-diethylaminophenyl)-10-phenylacridan and polymyxin B sulfate at respective concentrations of 1 and 75 micrograms/ml as a medium for selectively isolating P. cepacia. The medium inhibited the growth of all gram-negative bacilli and gram-positive cocci tested except P. cepacia and Serratia marcescens. These two microorganisms could easily be differentiated by their colony morphology and their reactions in the oxidase test. When nonsterilized water samples were inoculated with P. cepacia and spread or streaked on the selective medium, all P. cepacia organisms were recovered. These results demonstrate the usefulness of 9-chloro-9-(4-diethylaminophenyl)-10-phenylacridan and polymyxin B sulfate in the detection of P. cepacia. We believe that this selective medium could be useful in isolating P. cepacia from mixed bacterial flora that might be present in environmental water and water-related samples, such as solutions and lotions.     

Nah plasmids of the IncP-9 group in natural Pseudomonas strains

Polymerase chain reaction studies showed that naphthalene-utilizing bacteria isolated from various localities of Belarus most often contained Nah plasmids of the P-9 incompatibility group and plasmids of indefinite systematics. The conventional incompatibility test and restriction enzyme analysis revealed three new IncP-9 subgroups: ζ, η, and IncP-9-like. In addition to the known nucleotide sequences of nahG and nahAc, two novel nahG variants were revealed by a restriction enzyme analysis of amplification products. An amplified rDNA restriction enzyme analysis (ARDRA) demonstrated that the native hosts of IncP-9 Nah plasmids were fluorescent bacteria of the genus Pseudomonas (P. fluorescens, P. putida, P. aeruginosa, and Pseudomonas sp.) and nonfluorescent bacteria of indefinite systematics.     

Cytotoxicity associated with trichloroethylene oxidation in Burkholderia cepacia G4

The effects of trichloroethylene (TCE) oxidation on toluene 2-monooxygenase activity, general respiratory activity, and cell culturability were examined in the toluene-oxidizing bacterium Burkholderia cepacia G4. Nonspecific damage outpaced inactivation of toluene 2-monooxygenase in B. cepacia G4 cells. Cells that had degraded approximately 0.5 micromol of TCE (mg of cells(-1)) lost 95% of their acetate-dependent O(2) uptake activity (a measure of general respiratory activity), yet toluene-dependent O(2) uptake activity decreased only 35%. Cell culturability also decreased upon TCE oxidation; however, the extent of loss varied greatly (up to 3 orders of magnitude) with the method of assessment. Addition of catalase or sodium pyruvate to the surfaces of agar plates increased enumeration of TCE-injured cells by as much as 100-fold, indicating that the TCE-injured cells were ultrasensitive to oxidative stress. Cell suspensions that had oxidized TCE recovered the ability to grow in liquid minimal medium containing lactate or phenol, but recovery was delayed substantially when TCE degradation approached 0.5 micromol (mg of cells(-1)) or 66% of the cells' transformation capacity for TCE at the cell density utilized. Furthermore, among B. cepacia G4 cells isolated on Luria-Bertani agar plates from cultures that had degraded approximately 0.5 micromol of TCE (mg of cells(-1)), up to 90% were Tol(-) variants, no longer capable of TCE degradation. These results indicate that a toxicity threshold for TCE oxidation exists in B. cepacia G4 and that once a cell suspension has exceeded this toxicity threshold, the likelihood of reestablishing an active, TCE-degrading biomass from the cells will decrease significantly.     

Cytotoxicity Associated with Trichloroethylene Oxidation in Burkholderia cepacia G4

The effects of trichloroethylene (TCE) oxidation on toluene 2-monooxygenase activity, general respiratory activity, and cell culturability were examined in the toluene-oxidizing bacterium Burkholderia cepacia G4. Nonspecific damage outpaced inactivation of toluene 2-monooxygenase in B. cepacia G4 cells. Cells that had degraded approximately 0.5 μmol of TCE (mg of cells⁻¹) lost 95% of their acetate-dependent O₂ uptake activity (a measure of general respiratory activity), yet toluene-dependent O₂ uptake activity decreased only 35%. Cell culturability also decreased upon TCE oxidation; however, the extent of loss varied greatly (up to 3 orders of magnitude) with the method of assessment. Addition of catalase or sodium pyruvate to the surfaces of agar plates increased enumeration of TCE-injured cells by as much as 100-fold, indicating that the TCE-injured cells were ultrasensitive to oxidative stress. Cell suspensions that had oxidized TCE recovered the ability to grow in liquid minimal medium containing lactate or phenol, but recovery was delayed substantially when TCE degradation approached 0.5 μmol (mg of cells⁻¹) or 66% of the cells' transformation capacity for TCE at the cell density utilized. Furthermore, among B. cepacia G4 cells isolated on Luria-Bertani agar plates from cultures that had degraded approximately 0.5 μmol of TCE (mg of cells⁻¹), up to 90% were Tol⁻ variants, no longer capable of TCE degradation. These results indicate that a toxicity threshold for TCE oxidation exists in B. cepacia G4 and that once a cell suspension has exceeded this toxicity threshold, the likelihood of reestablishing an active, TCE-degrading biomass from the cells will decrease significantly.     

Pseudomonas cepacia mutants blocked in the Entner-Doudoroff pathway.

Pseudomonas cepacia mutants deficient in either 6-phosphogluconate (6PGA) dehydratase (Edd-) or 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase (Eda-) failed to utilize glucose or gluconate despite the prominence of of 6-phosphogluconate dehydrogenase (6PGAD) ii this bacterium and the potential for utilizing the pentose shunt suggested by its growth on ribitol and xylose. The Eda- strains grew normally on glucuronic acid, indicating that in P. cepacia its degradation does not depend upon KDPG aldolase as it does in Escherichia coli. Both 6PGA dehydratase and KDPG aldolase were inducible enzymes, with 6PGA rather than gluconate the apparent inducer. Edd- as well as Eda- strains were sensitive to growth inhibition by glucose, gluconate, fructose, and related carbohydrates when these substrates were present in combination with alternate carbon sources such as citrate or phthalate, presumably as a consequence of accumulation and toxicity of 6PGA, KDPG, or both. Edd- mutants were somewhat less sensitive to such inhibition than were Eda- strains. Certain derivatives of the Edd- strains we examined were able to utilize gluconate despite their deficiency of 6PGA dehydratase. Such mutants formed higher levels of 6PGAD than did the wild type. It is likely that the elevated levels of 6PGAD in these strains prevents accumulation of toxic levels of 6PGA that would otherwise result from a block in he Entner-Doudoroff pathway. The results suggest that P. cepacia can mutate to grow slowly on gluconate utilizing only the pentose shunt.     

Structure of the O9 antigen from Burkholderia (Pseudomonas) cepacia

Abstract The O9 antigen of Burkholderia (Pseudomonas) cepacia has the following disaccharide repeating-unit: → 4)-α-D-Glc-p-(1 → 3)-α-L-Rha p-(1 → The same unit is present in the O antigens of Serratia marcescens strain S1254 and serogroup O4 (predominantly acetylated at O-2 of rhamnose in the latter case).     

Guanidine hydrochloride-induced denaturation of Pseudomonas cepacia lipase.

The guanidine hydrochloride-induced denaturation of Pseudomonas cepacia lipase (PCL) was studied at pH 7 by monitoring the changes in the fluorescence and circular dichroism of the enzyme. The denaturation was irreversible as a whole, and the addition of Ca2+ ions decreased the velocity of the denaturation. The denaturation process was well explained consistently by a two-step mechanism, as follows: [see equation in text] where N is the native state of PCL, D(I) an intermediate denatured-state which can be refolded into the native state, and D(F) the final denatured-state that can not be renatured. Ethanol (10%) increased the denaturation velocity by decreasing the refolding step, D(I) + Ca2+ --> N x Ca2+, which would be caused by the stabilization of D(I) by ethanol.