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.     

Effect of trichloroethylene (TCE) and toluene concentrations on TCE and toluene biodegradation and the population density of TCE and toluene degraders in soil.

Toluene is one of several cosubstrates able to support the cometabolism of trichloroethylene (TCE) by soil microbial communities. Indigenous microbial populations in soil degraded TCE in the presence, but not the absence, of toluene after a 60- to 80-h lag period. Initial populations of toluene and TCE degraders ranged from 0.2 x 10(3) to 4 x 10(3) cells per g of soil and increased by more than 4 orders of magnitude after the addition of 20 micrograms of toluene and 1 microgram of TCE per ml of soil solution. The numbers of TCE and toluene degraders and the percent removal of TCE increased with an increase in initial toluene concentration. As the initial TCE concentration was increased from 1 to 20 micrograms/ml, the numbers of toluene and TCE degraders and the rate of toluene degradation decreased, and no TCE degradation occurred. No toluene or TCE degradation occurred at a TCE concentration of 50 micrograms/ml.     

Microbial Succession during a Field Evaluation of Phenol and Toluene as the Primary Substrates for Trichloroethene Cometabolism

Microbial community composition and succession were studied in an aquifer that was amended with phenol, toluene, and chlorinated aliphatic hydrocarbons to evaluate the effectiveness of these aromatic substrates for stimulating trichloroethene (TCE) bioremediation. Samples were taken after the previous year's field studies, which used phenol as the primary substrate, and after three successive monthly treatments of phenol plus 1,1-dichloroethene (1,1-DCE) plus TCE, phenol plus TCE, and toluene plus TCE. Dominant eubacteria in the community were assessed after each of the four treatments by characterizing isolates from the most dilute most-probable-number tubes and by extracting DNA from aquifer samples. The succession of dominant phenol- and toluene-degrading strains was evaluated by genomic fingerprinting, cellular fatty acid methyl ester (FAME) analysis, and amplified ribosomal DNA restriction analysis (ARDRA). 1,1-DCE was found to drastically reduce microbial growth and species richness, which corresponded to the reduction in bioremediation effectiveness noted previously for this treatment (G. D. Hopkins and P. L. McCarty, Environ. Sci. Technol. 29:1628-1637, 1995). Only a few gram-positive isolates could be obtained after treatment with 1,1-DCE, and these were not seen after any other treatments. Microbial densities returned to their original levels following the subsequent phenol-TCE treatment, but the original species richness was not restored until after the subsequent toluene-TCE treatment. Genomic fingerprinting and FAME analysis indicated that six of the seven originally dominant microbial groups were still dominant after the last treatment, indicating that the community is quite resilient to toxic disturbance by 1,1-DCE. FAME analysis indicated that six microbial taxa were dominant: three members of the (beta) subclass of the class Proteobacteria (Comamonas-Variovorax, Azoarcus, and Burkholderia) and three gram-positive groups (Bacillus, Nocardia, and an unidentified group). ARDRA revealed that the dominant community members were stable during the three nontoxic treatments and that virtually all of the bands could be accounted for by isolates from five of the dominant taxa, indicating that the isolation protocol used likely recovered most of the dominant members of this community.     

Isolation, characterization, and distribution of denitrifying toluene degraders from a variety of habitats.

Enrichments capable of toluene degradation under O2-free denitrifying conditions were established with diverse inocula including agricultural soils, compost, aquifer material, and contaminated soil samples from different geographic regions of the world. Successful enrichment was strongly dependent on the initial use of relatively low toluene concentrations, typically 5 ppm. From the enrichments showing positive activity for toluene degradation, 10 bacterial isolates were obtained. Fingerprints generated by PCR-amplified DNA, with repetitive extragenic palindromic sequence primers, showed that eight of these isolates were different. Under aerobic conditions, all eight isolates degraded toluene, five degraded ethylbenzene, three consumed benzene, and one degraded chlorobenzene, meta-Xylene was the only other substrate used anaerobically and was used by only one isolate. All isolates were motile gram-negative rods, produced N2 from denitrification, and did not hydrolyze starch. All strains but one fixed nitrogen as judged by ethylene production from acetylene, but only four strains hybridized to the nifHDK genes. All strains appeared to have heme nitrite reductase since their DNA hybridized to the heme (nirS) but not to the Cu (nirU) genes. Five strains hybridized to a toluene ortho-hydroxylase catabolic probe, and two of those also hybridized to a toluene meta-hydroxylase probe. Partial sequences of the 16S rRNA genes of all isolates showed substantial similarity to 16S rRNA sequences of Azoarcus sp. Physiological, morphological, fatty acid, and 16S rRNA analyses indicated that these strains were closely related to each other and that they belong to the genus Azoarcus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Trichloroethylene mineralization in a fixed-film bioreactor using a pure culture expressing constitutively toluene ortho -monooxygenase

An aerobic, single-pass, fixed-film bioreactor was designed for the continuous degradation and mineralization of gas-phase trichloroethylene (TCE). A pure culture of Burkholderia cepacia PR1(23)(TOM(23C)), a Tn5transposon mutant of B. cepacia G4 that constitutively expresses the TCE-degrading enzyme, toluene ortho-monooxygenase (TOM), was immobilized on sintered glass (SIRANtrade mark carriers) and activated carbon. The inert open-pore structures of the sintered glass and the strongly, TCE-absorbing activated carbon provide a large surface area for biofilm development (2-8 mg total cellular protein/mL carrier with glucose minimal medium that lacks chloride ions). At gas-phase TCE concentrations ranging from 0.04 to 2.42 mg/L of air and 0.1 L/min of air flow, initial maximum TCE degradation rates of 0.007-0.715 nmol/(min mg protein) (equivalent to 8.6-392.3 mg TCE/L of reactor/day) were obtained. Using chloride ion generation as the indicator of TCE mineralization, the bioreactor with activated carbon mineralized an average of 6.9-10.3 mg TCE/L of reactor/day at 0.242 mg/L TCE concentration with 0.1 L/min of air flow for 38-40 days. Although these rates of TCE degradation and mineralization are two- to 200-fold higher than reported values, TOM was inactivated in the sintered-glass bioreactor at a rate that increased with increasing TCE concentration (e.g., in approximately 2 days at 0.242 mg/L and <1 day at 2.42 mg/L), although the biofilter could be operated for longer periods at lower TCE concentrations. Using an oxygen probe and phenol as the substrate, the activity of TOM in the effluent cells of the bioreactor was monitored; the loss of TOM activity of the effluent cells corroborated the decrease in the TCE degradation and mineralization rates in the bioreactor. Repeated starving of the cells was found to restore TOM activity in the bioreactor with activated carbon and extended TCE mineralization by approximately 34%. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 674-685, 1997.     

Microcosm and in situ field studies of enhanced biotransformation of trichloroethylene by phenol-utilizing microorganisms.

The ability of different aerobic groundwater microorganisms to cometabolically degrade trichloroethylene (TCE), 1,2-cis-dichloroethylene (c-DCE), and 1,2-trans-dichloroethylene (t-DCE) was evaluated both in groundwater-fed microcosms and in situ in a shallow aquifer. Microcosms amended with phenol or toulene were equally effective in removing c-DCE (> 90%) followed by TCE (60 to 70%), while the microcosm fed methane was most effective in removing t-DCE (> 90%). The microcosm fed ammonia was the least effective. None of the microcosms effectively degraded 1,1,1-trichloroethane. At the Moffett Field groundwater test site, in situ removal of c-DCE and TCE coincided with biostimulation through phenol and oxygen injection and utilization, with c-DCE removed more rapidly than TCE. Greater TCE and c-DCE removal was observed when the phenol concentration was increased. Over 90% removal of c-DCE and TCE was observed in the 2-m biostimulated zone. This compares with 40 to 50% removal of c-DCE and 15 to 25% removal of TCE achieved by methane-grown microorganisms previously evaluated in an adjacent in situ test zone. The in situ removal with phenol-grown microorganisms agrees qualitatively with the microcosm studies, with the rates and extents of removal ranked as follows: c-DCE > TCE > t-DCE. These studies demonstrate the potential for in situ TCE bioremediation using microorganisms grown on phenol.     

Trichloroethylene cometabolic degradation by Rhodococcus sp. L4 induced with plant essential oils

Cometabolic degradation of TCE by toluene-degrading bacteria has the potential for being a cost-effective bioremediation technology. However, the application of toluene may pose environmental problems. In this study, several plant essential oils and their components were examined as alternative inducer for TCE cometabolic degradation in a toluene-degrading bacterium, Rhodococcus sp. L4. Using the initial TCE concentration of 80 muM, lemon and lemongrass oil-grown cells were capable of 20 +/- 6% and 27 +/- 8% TCE degradation, which were lower than that of toluene-grown cells (57 +/- 5%). The ability of TCE degradation increased to 36 +/- 6% when the bacterium was induced with cumin oil. The induction of TCE-degrading enzymes was suggested to be due to the presence of citral, cumin aldehyde, cumene, and limonene in these essential oils. In particular, the efficiency of cumin aldehyde and cumene as inducers for TCE cometabolic degradation was similar to toluene. TCE transformation capacities (T (c)) for these induced cells were between 9.4 and 15.1 mug of TCE mg cells(-1), which were similar to the known toluene, phenol, propane or ammonia degraders. Since these plant essential oils are abundant and considered non-toxic to humans, they may be applied to stimulate TCE degradation in the environment.     

Enumeration of petroleum-degrading marine and estuarine microorganisms by the most probable number method.

Several media designed for use in a most probable number (MPN) determination of petroleum-degrading microorganisms were compared. The best results, i.e., largest numbers, were obtained using a buffered (32 mM PO4=) liquid medium containing 1% hydrocarbon substrate. Of 104 presumptive oil degraders tested, 20 grew on oil agar medium but did not utilize oil or a mixture of pure paraffinic hydrocarbons (C10 to C16 n-alkanes) in liquid (MPN) medium. Visible turbidity in the liquid medium was correlated with hydrocarbon utilization. Counts of petroleum degraders obtained using liquid medium (MPN) were in most cases higher than those obtained on an oil-amended silica gel medium. Both procedures yield an estimation of oil degraders, and the oil-amended agar permits growth of organisms which do not degrade crude oil. All strains of oil-degrading microorganisms examined in this study were lipolytic, but the converse was not always true.     

Trichloroethylene metabolism by microorganisms that degrade aromatic compounds.

Trichloroethylene (TCE) was metabolized by the natural microflora of three different environmental water samples when stimulated by the addition of either toluene or phenol. Two different strains of Pseudomonas putida that degrade toluene by a pathway containing a toluene dioxygenase also metabolized TCE. A mutant of one of these strains lacking an active toluene dioxygenase could not degrade TCE, but spontaneous revertants for toluene degradation also regained TCE-degradative ability. The results implicate toluene dioxygenase in TCE metabolism.     

Trichloroethylene metabolism by microorganisms that degrade aromatic compounds

Trichloroethylene (TCE) was metabolized by the natural microflora of three different environmental water samples when stimulated by the addition of either toluene or phenol. Two different strains of Pseudomonas putida that degrade toluene by a pathway containing a toluene dioxygenase also metabolized TCE. A mutant of one of these strains lacking an active toluene dioxygenase could not degrade TCE, but spontaneous revertants for toluene degradation also regained TCE-degradative ability. The results implicate toluene dioxygenase in TCE metabolism.     

Aquifer Protist Response and the Potential for TCE Bioremediation with Burkholderia cepacia G4 PR1

Abstract Bacterivorous protists have been recovered from pristine and contaminated aquifer environments, but the ecological role of these organisms in bioremediation strategies has not been well defined. Burkholderia cepacia G4 PR1 constitutively expresses a toluene ortho-monooxygenase (tom) due to a secondary transposition of a Tn5 transposable element in a trichloroethylene (TCE) degradative plasmid (TOM). Groundwater and sediment from a potential site for a TCE bioremediation field demonstration were used in laboratory microcosms to test the survival of this organism. In nonsterile aquifer sediment slurries, the bacterium was eliminated in a logrithmic decay concomitant with an increase in bacterivorous protists. A half-life for the organism calculated from extinction coefficients increased logarithmically with increasing inoculation density above 1 × 10⁶ PR1 ml⁻¹. For inoculation densities below this level, the half-life of PR1 increased exponentially with decreasing inoculation density. The lowest half-lives corresponded to densities of bacteria that stimulate response of bacterivores. In a column system designed to incorporate aquifer flow, repeated addition of PR1 resulted in a buildup of bacterivore populations and reduced half-life of the bacterium. Addition of TCE and growth substrate in the eluent resulted in prolonged survival of PR1 and apparent mineralization of TCE. The results indicate significant but predictable losses due to native bacterivores would occur within and beyond a treatment zone where PR1 would be added to the aquifer, and mineralization of TCE in contaminated groundwater might be possible with repeated inoculation and addition of nutrients. Received: November 1999; Accepted: February 2000; Online Publication: 28 August 2000     

Prevalence of monochloroacetate degrading genotypes among soil isolates of Pseudomonas

This study reports the isolation of Pseudomonas sp strains with monochloroacetate (MCA) degradation function, from uncontaminated soil, and the use of Southern blot hybridization technique to detect MCA degrading catabolic genes and their divergence. Based on their capacity to remove Cl^- from MCA in a minimal medium containing 185 ppm Cl^-, the strains were classified into three groups: poor degraders (Cl^- release between 0–15 ppm), medium degraders (Cl^- release between 16–30 ppm), and high degraders (Cl^- release between 31–45 ppm).We have applied a gene probe assay for determining the diversity of MCA degradative genotypes of 61 strains. Two different gene probes, dehCI and dehCII were used in Southern blot hybridization assays. Majority of the DNA samples that produced signals on the membrane blots (18 out of 24)hybridized with only dehCI DNA probe, while 6strains hybridized with only dehCII probe. On the other hand, 37 isolates did not hybridize to either of the gene probes used. The results indicated the high specificity of the DNA hybridization method and the divergence of metabolic functions and/or genotypes among the native MCA-degrading Pseudomonas sp. populations in the soil. This revised version was published online in August 2006 with corrections to the Cover Date.     

Group-specific monitoring of phenol hydroxylase genes for a functional assessment of phenol-stimulated trichloroethylene bioremediation

The sequences of the largest subunit of bacterial multicomponent phenol hydroxylases (LmPHs) were compared. It was found that LmPHs formed three phylogenetic groups, I, II, and III, corresponding to three previously reported kinetic groups, low-K(s) (the half-saturation constant in Haldane's equation for trichloroethylene [TCE]), moderate-K(s), and high-K(s) groups. Consensus sequences and specific amino acid residues for each group of LmPH were found, which facilitated the design of universal and group-specific PCR primers. PCR-mediated approaches using these primers were applied to analyze phenol/TCE-degrading populations in TCE-contaminated aquifer soil. It was found that the aquifer soil harbored diverse genotypes of LmPH, and the group-specific primers successfully amplified LmPH fragments affiliated with each of the three groups. Analyses of phenol-degrading bacteria isolated from the aquifer soil confirmed the correlation between genotype and phenotype. Competitive PCR assays were used to quantify LmPHs belonging to each group during the enrichment of phenol/TCE-degrading bacteria from the aquifer soil. We found that an enrichment culture established by batch phenol feeding expressed low TCE-degrading activity at a TCE concentration relevant to the contaminated aquifer (e.g., 0.5 mg liter(-1)); group II and III LmPHs were predominant in this batch enrichment. In contrast, group I LmPHs overgrew an enrichment culture when phenol was fed continuously. This enrichment expressed unexpectedly high TCE-degrading activity that was comparable to the activity expressed by a pure culture of Methylosinus trichosporium OB3b. These results demonstrate the utility of the group-specific monitoring of LmPH genes in phenol-stimulated TCE bioremediation. It is also suggested that phenol biostimulation could become a powerful TCE bioremediation strategy when bacteria possessing group I LmPHs are selectively stimulated.     

Biodegradation of trichloroethylene and toluene by indigenous microbial populations in soil.

The biodegradation of trichloroethylene (TCE) and toluene, incubated separately and in combination, by indigenous microbial populations was measured in three unsaturated soils incubated under aerobic conditions. Sorption and desorption of TCE (0.1 to 10 micrograms ml-1) and toluene (1.0 to 20 micrograms ml-1) were measured in two soils and followed a reversible linear isotherm. At a concentration of 1 micrograms ml-1, TCE was not degraded in the absence of toluene in any of the soils. In combination, both 1 microgram of TCE ml-1 and 20 micrograms of toluene ml-1 were degraded simultaneously after a lag period of approximately 60 to 80 h, and the period of degradation lasted from 70 to 90 h. Usually 60 to 75% of the initial 1 microgram of TCE ml-1 was degraded, whereas 100% of the toluene disappeared. A second addition of 20 micrograms of toluene ml-1 to a flask with residual TCE resulted in another 10 to 20% removal of the chemical. Initial rates of degradation of toluene and TCE were similar at 32, 25, and 18 degrees C; however, the lag period increased with decreasing temperature. There was little difference in degradation of toluene and TCE at soil moisture contents of 16, 25, and 30%, whereas there was no detectable degradation at 5 and 2.5% moisture. The addition of phenol, but not benzoate, stimulated the degradation of TCE in Rindge and Yolo silt loam soils, methanol and ethylene slightly stimulated TCE degradation in Rindge soil, glucose had no effect in either soil, and dissolved organic carbon extracted from soil strongly sorbed TCE but did not affect its rate of biodegradation.     

Diversity in kinetics of trichloroethylene-degrading activities exhibited by phenol-degrading bacteria

Whole-cell kinetics of phenol- and trichloroethylene (TCE)-degrading activities expressed by 13 phenol-degrading bacteria were analyzed. The Ks (apparent affinity constant in Haldane's equation) values for TCE were unexpectedly diverse, ranging from 11 microM to over 800 microM. The Vmax/Ks values for phenol were three orders of magnitude higher than the values for TCE in all bacteria analyzed, suggesting that these bacteria preferentially degrade phenol rather than TCE. A positive correlation between Ks for phenol and Ks for TCE was found, i.e., bacteria exhibiting high Ks values for phenol showed high Ks values for TCE, and vice versa. A comparison of the Ks values allowed grouping of these bacteria into three types, i.e., low-, moderate- and high-Ks types. Pseudo-first-order degradation-rate constants for TCE at 3.8 microM were found to be adequate to rapidly discriminate among the three types of bacteria. When bacteria were grown on phenol at the initial concentration of 2 mM, Comamonas testosteroni strain R5, a representative of low-Ks bacteria, completely degraded TCE at 3.8 microM, while strain P-8, a representative of high-Ks bacteria, did not. A mixed culture of these two bacteria poorly degraded TCE under the same conditions, where P-8 outgrew R5. These results suggest that low-Ks bacteria should be selectively grown for effective bioremediation of TCE-contaminated groundwater.     

Degradation of Toluene and Trichloroethylene by Burkholderia cepacia G4 in Growth-Limited Fed-Batch Culture

Burkholderia (Pseudomonas) cepacia G4 was cultivated in a fed-batch bioreactor on either toluene or toluene plus trichloroethylene (TCE). The culture was allowed to reach a constant cell density under conditions in which the amount of toluene supplied equals the maintenance energy demand of the culture. Compared with toluene only, the presence of TCE at a toluene/TCE ratio of 2.3 caused a fourfold increase in the specific maintenance requirement for toluene from 22 to 94 nmol mg of cells (dry weight)(sup-1) h(sup-1). During a period of 3 weeks, approximately 65% of the incoming TCE was stably converted to unidentified products from which all three chlorine atoms were liberated. When toluene was subsequently omitted from the culture feed while TCE addition continued, mutants which were no longer able to grow on toluene or to degrade TCE appeared. These mutants were also unable to grow on phenol or m- or o-cresol but were still able to grow on catechol and benzoate. Plasmid analysis showed that the mutants had lost the plasmid involved in toluene monooxygenase formation (pTOM). Thus, although strain G4 is much less sensitive to TCE toxicity than methanotrophs, deleterious effects may still occur, namely, an increased maintenance energy demand in the presence of toluene and plasmid loss when no toluene is added.     

Group-Specific Monitoring of Phenol Hydroxylase Genes for a Functional Assessment of Phenol-Stimulated Trichloroethylene Bioremediation

The sequences of the largest subunit of bacterial multicomponent phenol hydroxylases (LmPHs) were compared. It was found that LmPHs formed three phylogenetic groups, I, II, and III, corresponding to three previously reported kinetic groups, low-K_s (the half-saturation constant in Haldane's equation for trichloroethylene [TCE]), moderate-K_s, and high-K_s groups. Consensus sequences and specific amino acid residues for each group of LmPH were found, which facilitated the design of universal and group-specific PCR primers. PCR-mediated approaches using these primers were applied to analyze phenol/TCE-degrading populations in TCE-contaminated aquifer soil. It was found that the aquifer soil harbored diverse genotypes of LmPH, and the group-specific primers successfully amplified LmPH fragments affiliated with each of the three groups. Analyses of phenol-degrading bacteria isolated from the aquifer soil confirmed the correlation between genotype and phenotype. Competitive PCR assays were used to quantify LmPHs belonging to each group during the enrichment of phenol/TCE-degrading bacteria from the aquifer soil. We found that an enrichment culture established by batch phenol feeding expressed low TCE-degrading activity at a TCE concentration relevant to the contaminated aquifer (e.g., 0.5 mg liter⁻¹); group II and III LmPHs were predominant in this batch enrichment. In contrast, group I LmPHs overgrew an enrichment culture when phenol was fed continuously. This enrichment expressed unexpectedly high TCE-degrading activity that was comparable to the activity expressed by a pure culture of Methylosinus trichosporium OB3b. These results demonstrate the utility of the group-specific monitoring of LmPH genes in phenol-stimulated TCE bioremediation. It is also suggested that phenol biostimulation could become a powerful TCE bioremediation strategy when bacteria possessing group I LmPHs are selectively stimulated.     

Genetic exchange in soil between introduced chlorobenzoate degraders and indigenous biphenyl degraders.

Pseudomonas aeruginosa JB2, a chlorobenzoate degrader, was inoculated into soil having indigenous biphenyl degraders but no identifiable 2-chlorobenzoate (2CBa) or 2,5-dichlorobenzoate (2,5DCBa) degraders. The absence of any indigenous chlorobenzoate degraders was noted by the failure to obtain enrichment cultures with the addition of 2CBa, 3CBa, or 2,5DCBa and by the failure of soil DNA to hybridize to the tfdC gene, which encodes ortho fission of chlorocatechols. In contrast, DNA extracted from inoculated soils hybridized to this probe. Bacteria able to utilize both biphenyl and 2CBa as growth substrates were absent in uninoculated soil, but their presence increased with time in the inoculated soils. This increase was related kinetically to the growth of biphenyl degraders. Pseudomonas sp. strain AW, a dominant biphenyl degrader, was selected as a possible parental strain. Eight of nine recombinant strains, chosen at random, had high phenotypic similarity (90% or more) to the inoculant; the other, strain JB2-M, had 78% similarity. Two hybrid strains, P. aeruginosa JB2-3 and Pseudomonas sp. JB2-M, were the most effective of all strains, including strain AW, in metabolizing polychlorinated biphenyls (Aroclor 1242). Repetitive extragenic palindromic-PCR analysis of putative parental strains JB2 and AW and the two recombinant strains JB2-3 and JB2-M showed similar fragments among the recombinants and JB2 but not AW. These results indicate that the bph genes were transferred to the chlorobenzoate-degrading inoculant from indigenous biphenyl degraders.     

Activity and stability of a recombinant plasmid-borne TCE degradative pathway in suspended cultures

The retention and expression of the plasmid-borne, TCE degradative toluene-ortho-monooxygenase (TOM) pathway in suspended continuous cultures of transconjugant Burkholderia cepacia 17616 (TOM31c) were studied. Acetate growth and TCE degradation kinetics for the transconjugant host are described and utilized in a plasmid loss model. Plasmid maintenance did not have a significant effect on the growth rate of the transconjugant. Both plasmid-bearing and plasmid-free strains followed Andrews inhibition growth kinetics when grown on acetate and had maximum growth rates of 0.22 h-1. The transconjugant was capable of degrading TCE at a maximum rate of 9.7 nmol TCE/min. mg protein, which is comparable to the rates found for the original plasmid host, Burkholderia cepacia PR131 (TOM31c). The specific activity of the TOM pathway was found to be a linear function of growth rate. Plasmid maintenance was studied at three different growth rates: 0.17/h, 0.1/h, and 0.065/h. Plasmid maintenance was found to be a function of growth rate, with the probability of loss ranging from 0.027 at a growth rate of 0.065/h to 0.034 at a growth rate 0.17/h.     

Oxidation of benzene to phenol, catechol, and 1,2,3-trihydroxybenzene by toluene 4-monooxygenase of Pseudomonas mendocina KR1 and toluene 3-monooxygenase of Ralstonia pickettii PKO1

Aromatic hydroxylations are important bacterial metabolic processes but are difficult to perform using traditional chemical synthesis, so to use a biological catalyst to convert the priority pollutant benzene into industrially relevant intermediates, benzene oxidation was investigated. It was discovered that toluene 4-monooxygenase (T4MO) of Pseudomonas mendocina KR1, toluene 3-monooxygenase (T3MO) of Ralstonia pickettii PKO1, and toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4 convert benzene to phenol, catechol, and 1,2,3-trihydroxybenzene by successive hydroxylations. At a concentration of 165 microM and under the control of a constitutive lac promoter, Escherichia coli TG1/pBS(Kan)T4MO expressing T4MO formed phenol from benzene at 19 +/- 1.6 nmol/min/mg of protein, catechol from phenol at 13.6 +/- 0.3 nmol/min/mg of protein, and 1,2,3-trihydroxybenzene from catechol at 2.5 +/- 0.5nmol/min/mg of protein. The catechol and 1,2,3-trihydroxybenzene products were identified by both high-pressure liquid chromatography and mass spectrometry. When analogous plasmid constructs were used, E. coli TG1/pBS(Kan)T3MO expressing T3MO formed phenol, catechol, and 1,2,3-trihydroxybenzene at rates of 3 +/- 1, 3.1 +/- 0.3, and 0.26 +/- 0.09 nmol/min/mg of protein, respectively, and E. coli TG1/pBS(Kan)TOM expressing TOM formed 1,2,3-trihydroxybenzene at a rate of 1.7 +/- 0.3 nmol/min/mg of protein (phenol and catechol formation rates were 0.89 +/- 0.07 and 1.5 +/- 0.3 nmol/min/mg of protein, respectively). Hence, the rates of synthesis of catechol by both T3MO and T4MO and the 1,2,3-trihydroxybenzene formation rate by TOM were found to be comparable to the rates of oxidation of the natural substrate toluene for these enzymes (10.0 +/- 0.8, 4.0 +/- 0.6, and 2.4 +/- 0.3 nmol/min/mg of protein for T4MO, T3MO, and TOM, respectively, at a toluene concentration of 165 microM).