Enhanced degradation of phenoxyacetic acid in soil by horizontal transfer of the tfdA gene encoding a 2,4-dichlorophenoxyacetic acid dioxygenase

Few studies have investigated the possible impact of in situ gene transfer on the degradation of xenobiotic compounds in natural environments. In this work we showed that horizontal transfer of the tfdA gene, carried on plasmid pRO103, to phenol degrading recipient strains significantly increased the degradation rate of phenoxyacetic acid in sterile and non-sterile soil microcosms. The tfdA gene encodes a 2,4-dichlorophenoxyacetic acid/2-oxoglutarate dioxygenase and by complementation with the phenol degradation pathway an expanded catabolic substrate range, now including phenoxyacetic acid, is evolved. Presence of selective pressure had a positive effect on the emergence of transconjugants. However, even in the absence of phenoxyacetic acid transconjugant populations were detected and were kept at a constant level throughout the experimental period. The residuesphere (interface between decaying plant material and soil matrix) of dry leaves of barley was shown to be a hot-spot for gene transfer and presence of barley straw increased the conjugation frequencies in soil microcosms to the same extent as presence of organic nutrients. The results of this study indicate that dissemination of catabolic plasmids is a possible mechanism of genetic adaptation to degradation of xenobiotic compounds in natural environments, and that complementation of catabolic pathways possibly plays an important role in the evolution of new degradative capabilities. The application of horizontal gene transfer as a possible tool in bioremediation of contaminated sites is discussed.     

Molecular mechanisms of genetic adaptation to xenobiotic compounds.

Microorganisms in the environment can often adapt to use xenobiotic chemicals as novel growth and energy substrates. Specialized enzyme systems and metabolic pathways for the degradation of man-made compounds such as chlorobiphenyls and chlorobenzenes have been found in microorganisms isolated from geographically separated areas of the world. The genetic characterization of an increasing number of aerobic pathways for degradation of (substituted) aromatic compounds in different bacteria has made it possible to compare the similarities in genetic organization and in sequence which exist between genes and proteins of these specialized catabolic routes and more common pathways. These data suggest that discrete modules containing clusters of genes have been combined in different ways in the various catabolic pathways. Sequence information further suggests divergence of catabolic genes coding for specialized enzymes in the degradation of xenobiotic chemicals. An important question will be to find whether these specialized enzymes evolved from more common isozymes only after the introduction of xenobiotic chemicals into the environment. Evidence is presented that a range of genetic mechanisms, such as gene transfer, mutational drift, and genetic recombination and transposition, can accelerate the evolution of catabolic pathways in bacteria. However, there is virtually no information concerning the rates at which these mechanisms are operating in bacteria living in nature and the response of such rates to the presence of potential (xenobiotic) substrates. Quantitative data on the genetic processes in the natural environment and on the effect of environmental parameters on the rate of evolution are needed.     

Evolution of a metabolic pathway for degradation of a toxic xenobiotic: the patchwork approach

The pathway for degradation of the xenobiotic pesticide pentachlorophenol in Sphingomonas chlorophenolica probably evolved in the past few decades by the recruitment of enzymes from two other catabolic pathways. The first and third enzymes in the pathway, pentachlorophenol hydroxylase and 2,6-dichlorohydroquinone dioxygenase, may have originated from enzymes in a pathway for degradation of a naturally occurring chlorinated phenol. The second enzyme, a reductive dehalogenase, may have evolved from a maleylacetoacetate isomerase normally involved in degradation of tyrosine. This apparently recently assembled pathway does not function very well: pentachlorophenol hydroxylase is quite slow, and tetrachlorohydroquinone dehalogenase is subject to severe substrate inhibition.     

Importance of unattached bacteria and bacteria attached to sediment in determining potentials for degradation of xenobiotic organic contaminants in an aerobic aquifer.

The bacterial abundance, distribution, and degradation potential (in terms of degradation versus lack of degradation) for four xenobiotic compounds in an aerobic aquifer sediment have been examined in laboratory and field experiments. The xenobiotic compounds studied were benzene, toluene, o-xylene, and naphthalene (all at concentrations of approximately 120 micrograms/liter). The aerobic degradation experiments ran for approximately 90 days at 10 degrees C, which corresponded to the groundwater temperature. At the end of the experiment, the major part of the microbial biomass, quantified as acridine orange direct counts, was attached to the groundwater sediment (18 x 10(6) to 25 x 10(6) cells per g [dry weight], and only a minor part was unattached in the groundwater (0.6 x 10(6) to 5.5 x 10(6) cells per ml). Experiments involving aquifer sediment suspensions showed identical degradation potentials in the laboratory and in the field. However, laboratory experiments involving only groundwater (excluding aquifer sediment) showed less degradation potential than in situ experiments involving only groundwater, indicating that the manipulation or approach of the laboratory experiments could affect the determination of the degradation potentials. No differences were observed between the groundwater-only and the sediment compartments in the in situ experiments in the ability to degrade the compounds, but the maximum degradation rates were substantially lower in the groundwater-only compartment. Preparations used in laboratory experiments for studying the degradation potential for xenobiotic organic contaminants should contain sediment to obtain the highest numbers of bacteria as well as the broadest and most stable degradation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Importance of unattached bacteria and bacteria attached to sediment in determining potentials for degradation of xenobiotic organic contaminants in an aerobic aquifer.

The bacterial abundance, distribution, and degradation potential (in terms of degradation versus lack of degradation) for four xenobiotic compounds in an aerobic aquifer sediment have been examined in laboratory and field experiments. The xenobiotic compounds studied were benzene, toluene, o-xylene, and naphthalene (all at concentrations of approximately 120 micrograms/liter). The aerobic degradation experiments ran for approximately 90 days at 10 degrees C, which corresponded to the groundwater temperature. At the end of the experiment, the major part of the microbial biomass, quantified as acridine orange direct counts, was attached to the groundwater sediment (18 x 10(6) to 25 x 10(6) cells per g [dry weight], and only a minor part was unattached in the groundwater (0.6 x 10(6) to 5.5 x 10(6) cells per ml). Experiments involving aquifer sediment suspensions showed identical degradation potentials in the laboratory and in the field. However, laboratory experiments involving only groundwater (excluding aquifer sediment) showed less degradation potential than in situ experiments involving only groundwater, indicating that the manipulation or approach of the laboratory experiments could affect the determination of the degradation potentials. No differences were observed between the groundwater-only and the sediment compartments in the in situ experiments in the ability to degrade the compounds, but the maximum degradation rates were substantially lower in the groundwater-only compartment. Preparations used in laboratory experiments for studying the degradation potential for xenobiotic organic contaminants should contain sediment to obtain the highest numbers of bacteria as well as the broadest and most stable degradation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Microbial dynamics in a continuous stirred tank bioreactor exposed to an alternating sequence of organic compounds

Microbial dynamics during aerobic biodegradation of an alternating mixture of organic compounds was investigated experimentally in a continuous stirred tank bioreactor (CSTB). A mathematical model describing this system was developed and tested using the experimental results. A model microbial culture consisting of Pseudomonas sp. JS150, a monochlorobenzene (MCB) degrader, and Xanthobacter autotrophicus GJ10, a 1,2-dichloroethane (DCE) degrader, each with exclusive degradation capabilities, was used. The CSTB was inoculated with both microbial strains and exposed to an alternating sequence of the two compounds at noninhibitory concentrations. Concentrations of each microbial strain, of each organic compound, and of degradation product evolved, as well as specific microbial activities via oxygen uptake tests, were monitored. Reduction of the residual DCE discharged from the bioreactor after an MCB to DCE transition was successfully achieved by continuously feeding a low flow of a concentrated solution of both compounds.     

Advances in development of transgenic plants for remediation of xenobiotic pollutants

Phytoremediation-the use of plants for cleaning up of xenobiotic compounds-has received much attention in the last few years and development of transgenic plants tailored for remediation will further enhance their potential. Although plants have the inherent ability to detoxify some xenobiotic pollutants, they generally lack the catabolic pathway for complete degradation/mineralization of these compounds compared to microorganisms. Hence, transfer of genes involved in xenobiotic degradation from microbes/other eukaryotes to plants will further enhance their potential for remediation of these dangerous groups of compounds. Transgenic plants with enhanced potential for detoxification of xenobiotics such as trichloro ethylene, pentachlorophenol, trinitro toluene, glycerol trinitrate, atrazine, ethylene dibromide, metolachlor and hexahydro-1,3,5-trinitro-1,3,5-triazine are a few successful examples of utilization of transgenic technology. As more genes involved in xenobiotic metabolism in microorganisms/eukaryotes are discovered, it will lead to development of novel transgenic plants with improved potential for degradation of recalcitrant contaminants. Selection of suitable candidate plants, field testing and risk assessment are important considerations to be taken into account while developing transgenic plants for phytoremediation of this group of pollutants. Taking advantage of the advances in biotechnology and 'omic' technologies, development of novel transgenic plants for efficient phytoremediation of xenobiotic pollutants, field testing and commercialization will soon become a reality.     

Sphingomonads involved in the biodegradation of xenobiotic polymers

Sphingomonads involved in the microbial degradation of xenobiotic polymers are introduced. The metabolism of polyethylene glycol was the primary focus of the study. Several others, including polyvinyl alcohol, polyethylene and polyaspartate were also studied. It is suggested that these xenobiotic polymers are metabolized by intracellular enzymes located in the periplasmic space or bound to membranes, indicating that transport of these polymers through outer membranes is requisite for their metabolism. Involvement of specific membrane structures of sphingomonads such as unusual sphingolipids is suggested for membrane transport of xenobiotic compounds, especially hydrophobic materials. Received 01 May 1999/ Accepted in revised form 17 July 1999     

Bacterial degradation of pyridine, indole, quinoline, and their derivatives under different redox conditions

Bacteria have evolved a diverse potential to transform and even mineralize numerous organic compounds of both natural and xenobiotic origin. This article describes the occurrence of N-heteroaromatic compounds and presents a review of the bacterial degradation of pyridine and its derivatives, indole, isoquinoline, and quinoline and its derivatives. The bacterial metabolism of these compounds under different redox conditions – by aerobic, nitrate-reducing, sulfate-reducing and methanogenic bacteria – is discussed. However, in natural habitats, various environmental factors, such as sorption phenomena, also influence bacterial conversion processes. Thus, both laboratory and field studies are necessary to aid our understanding of biodegradation in natural ecosystems and assist the development of strategies for bioremediation of polluted sites. Occurring predominantly near (former) wood-treatment facilities, creosote is a frequent contaminant of soil, subsoil, groundwater, and aquifer sediments. In situ as well as withdrawal-and-treatment techniques have been designed to remediate such sites, which are polluted with complex mixtures of aromatic and heterocyclic compounds. Received: 26 September 1997 / Received revision: 23 December 1997 / Accepted: 27 December 1997     

Transgenic rice plants expressing human p450 genes involved in xenobiotic metabolism for phytoremediation

Phytoremediation is the use of plants to remove xenobiotic compounds from the environment. Plants have the inherent ability to detoxify xenobiotic pollutants, but they are generally poor at degrading them. The introduction of genes involved in xenobiotic degradation is aimed at enhancing plants' potential further. Rice (Oryza sativa) is a good candidate for this purpose and has been transformed with genes encoding cytochrome P450 monooxygenases CYP1A1, CYP2B6, and CYP2C19. The transgenic plants were more tolerant to various herbicides than nontransgenic Nipponbare rice plants, owing to enhanced metabolism by the introduced P450 enzymes. Transgenic plants were able to remove atrazine and metolachlor from soil. Field testing and risk assessment are very important for developing transgenic plants for phytoremediation. Transgenic rice plants should become useful as herbicide-tolerant crops and for phytoremediation of xenobiotic pollutants in future.     

Genome and Phenotype Microarray Analyses of Rhodococcus sp. BCP1 and Rhodococcus opacus R7: Genetic Determinants and Metabolic Abilities with Environmental Relevance

In this paper comparative genome and phenotype microarray analyses of Rhodococcus sp. BCP1 and Rhodococcus opacus R7 were performed. Rhodococcus sp. BCP1 was selected for its ability to grow on short-chain n-alkanes and R. opacus R7 was isolated for its ability to grow on naphthalene and on o-xylene. Results of genome comparison, including BCP1, R7, along with other Rhodococcus reference strains, showed that at least 30% of the genome of each strain presented unique sequences and only 50% of the predicted proteome was shared. To associate genomic features with metabolic capabilities of BCP1 and R7 strains, hundreds of different growth conditions were tested through Phenotype Microarray, by using Biolog plates and plates manually prepared with additional xenobiotic compounds. Around one-third of the surveyed carbon sources was utilized by both strains although R7 generally showed higher metabolic activity values compared to BCP1. Moreover, R7 showed broader range of nitrogen and sulphur sources. Phenotype Microarray data were combined with genomic analysis to genetically support the metabolic features of the two strains. The genome analysis allowed to identify some gene clusters involved in the metabolism of the main tested xenobiotic compounds. Results show that R7 contains multiple genes for the degradation of a large set of aromatic and PAHs compounds, while a lower variability in terms of genes predicted to be involved in aromatic degradation was found in BCP1. This genetic feature can be related to the strong genetic pressure exerted by the two different environment from which the two strains were isolated. According to this, in the BCP1 genome the smo gene cluster involved in the short-chain n-alkanes degradation, is included in one of the unique regions and it is not conserved in the Rhodococcus strains compared in this work. Data obtained underline the great potential of these two Rhodococcus spp. strains for biodegradation and environmental decontamination processes.     

Laccase-mediator systems and their applications: a review

The mechanism of operation of laccase-mediator systems (LMSs) in xenobiotic degradation mediated by "true" redox mediators and laccase enhancing agents is considered. Structural formulae of most common laccase mediators and compounds that can be used as agents enhancing the enzyme operation are presented. Examples of LMS application in biotechnology are described.     

Biochemical and genetic bases of microbial degradation of polychlorinated biphenyls (PCBs)

The microbial degradation of polychlorinated biphenyls (PCBs) has been extensively conducted by many workers, and the following general results have been obtained. (1) PCBs are degraded oxidatively by aerobic bacteria and other microorganisms such as white rot fungi. PCBs are also reductively dehalogenated by anaerobic microbial consortia. (2) The biodegradability of PCBs is highly dependent on chlorine substitution, i.e., number and position of chlorine. The degradation and dehalogenation capabilities are also highly strain dependent. (3) Biphenyl-utilizing bacteria can cometabolize many PCB congeners to chlorobenzoates by biphenl-catabolic enzymes. (4) Enzymes involved in the PCB degradation were purified and characterized. Biphenyl dioxygenase, ring-cleavage dioxygenase, and hydrolase are crystallized, and two ring-cleavage dioxygenases are being solved by x-ray crystallography. (5) The bph gene clusters responsible for PCB degradation are cloned from a variety of bacterial strains. The structure and function are analyzed with respect to the evolutionary relationship. (6) The molecular engineering of biphenyl dioxygenases is successfully performed by DNA shuffling, domain exchange, and subunit exchange. The evolved enzymes exhibit wide and enhanced degradation capacities for PCBs and other aromatic compounds.     

Laccase-mediator systems and their applications: A review

The mechanism of operation of laccase-mediator systems (LMSs) in xenobiotic degradation mediated by “true” redox mediators and laccase enhancing agents is considered. Structural formulae of most common laccase mediators and compounds that can be used as agents enhancing the enzyme operation are presented. Examples of LMS application in biotechnology are described.     

High efficiency of a coupled aerobic-anaerobic recycling biofilm reactor system in the degradation of recalcitrant chloroaromatic xenobiotic compounds

Chloroaromatic compounds are xenobiotics that cause great concern. The degradation of a model molecule, 3,4-dichlorobenzoate (3,4-DCB), was studied using three aerobic (AE)-anaerobic (AN) biofilm reactor systems: a coupled aerobic-anaerobic recycle biofilm reactor (CAR) system, an in-series anaerobic-aerobic biofilm reactor (SAR) system; and an independent aerobic and anaerobic biofilm reactor (IAR) system. In all three systems the inlet substrate concentration was 2.0 g/l and the dilution rates ranged from 0.045 to 0.142 per hour. The results show that the degradation efficiency of the CAR system (expressed as dechlorination and xenobiotic disappearance efficiencies, and biomass yield), was higher at all dilution rates tested than in both SAR and IAR systems. Moreover, dechlorination and xenobiotic disappearance efficiencies for resting suspended aerobic and anaerobic cells or mixed aerobic-anaerobic growing cells under anaerobic conditions were higher than under aerobic conditions. These results suggest that a “cooperative metabolism” between aerobic and anaerobic bacteria (caused by an exchange of cells and metabolites between AE and AN reactors) in the CAR system overcame the metabolic and kinetic limitations of aerobic and anaerobic bacteria in the AE and AN reactors of IAR and SAR systems. Therefore, the degradation efficiency of persistent and recalcitrant chloroaromatic xenobiotic compounds could be enhanced by using a CAR system. Received: 1 March 1999 / Received revision: 11 May 1999 / Accepted: 16 May 1999     

Selection of Xenobiotic-Degrading Microorganisms in a Biphasic Aqueous-Organic System

Microbial selection on mixtures of chlorinated and nonchlorinated compounds that are poorly soluble in water and/or toxic to growing microbial cells was examined in both biphasic aqueous-organic and monophasic aqueous systems. A biphasic system in which silicone oil was used as the organic phase permitted the acceleration of acclimation, leading to rapid selection and to an increase in xenobiotic compound degradation. In contrast, acclimation, selection, and degradation were very slow in the monophasic aqueous system. The variation in microbial growth rate with the degree of dispersion (i.e., dispersion at different silicone oil concentrations and agitation rates), and cell adhesion to the silicone oil indicate that the performance of the biphasic aqueous-organic system is dependent on the interfacial area between the two phases and that microbial activity is important at this interface. Therefore, the biphasic water-silicone oil system could be used for microbial selection in the presence of xenobiotic compounds that are toxic and have low water solubility.     

The liver pharmacological and xenobiotic gene response repertoire

We have used a supervised classification approach to systematically mine a large microarray database derived from livers of compound‐treated rats. Thirty‐four distinct signatures (classifiers) for pharmacological and toxicological end points can be identified. Just 200 genes are sufficient to classify these end points. Signatures were enriched in xenobiotic and immune response genes and contain un‐annotated genes, indicating that not all key genes in the liver xenobiotic responses have been characterized. Many signatures with equal classification capabilities but with no gene in common can be derived for the same phenotypic end point. The analysis of the union of all genes present in these signatures can reveal the underlying biology of that end point as illustrated here using liver fibrosis signatures. Our approach using the whole genome and a diverse set of compounds allows a comprehensive view of most pharmacological and toxicological questions and is applicable to other situations such as disease and development.     

Second-Order Model to Predict Microbial Degradation of Organic Compounds in Natural Waters

The reliability of second-order rate constants for assessing microbial degradation kinetics in natural waters was examined by using three compounds that undergo hydrolytic degradation. The butoxyethyl ester of 2,4-dichlorophenoxyacetic acid was studied in water samples from 31 sites, malathion was examined in water from 14 sites, and chlorpropham was studied in samples from 11 sites. The coefficient of variation for rate constants for each compound was less than 65% over all sites. Additional studies indicated that the rate conformed to second-order kinetics; that is, the rate was proportional to both bacterial and xenobiotic concentrations.