Oxygenases and dehalogenases: molecular approaches to efficient degradation of chlorinated environmental pollutants

Microbial oxygenases and dehalogenases are key enzymes in the degradation of highly chlorinated compounds, which often become significant environmental pollutants. Oxygenases engineered by the methods of directed evolution exhibit enhanced degradation of PCBs and other chlorinated solvents such as trichloroethene and pentachloroethane. Dehalorespiration is an efficient dechlorination mechanism that is coupled with energy-yielding phosphorylation. Recently, a variety of chloroethene-dehalorespiring anaerobes have been isolated, and their reductive dehalogenases have been characterized in biochemical and genetic bases. This review describes our recent studies on dioxygenases and reductive dehalogenases.     

Engineering a catabolic pathway in plants for the degradation of 1,2-dichloroethane

Plants are increasingly being employed to clean up environmental pollutants such as heavy metals; however, a major limitation of phytoremediation is the inability of plants to mineralize most organic pollutants. A key component of organic pollutants is halogenated aliphatic compounds that include 1,2-dichloroethane (1,2-DCA). Although plants lack the enzymatic activity required to metabolize this compound, two bacterial enzymes, haloalkane dehalogenase (DhlA) and haloacid dehalogenase (DhlB) from the bacterium Xanthobacter autotrophicus GJ10, have the ability to dehalogenate a range of halogenated aliphatics, including 1,2-DCA. We have engineered the dhlA and dhlB genes into tobacco (Nicotiana tabacum 'Xanthi') plants and used 1,2-DCA as a model substrate to demonstrate the ability of the transgenic tobacco to remediate a range of halogenated, aliphatic hydrocarbons. DhlA converts 1,2-DCA to 2-chloroethanol, which is then metabolized to the phytotoxic 2-chloroacetaldehyde, then chloroacetic acid, by endogenous plant alcohol dehydrogenase and aldehyde dehydrogenase activities, respectively. Chloroacetic acid is dehalogenated by DhlB to produce the glyoxylate cycle intermediate glycolate. Plants expressing only DhlA produced phytotoxic levels of chlorinated intermediates and died, while plants expressing DhlA together with DhlB thrived at levels of 1,2-DCA that were toxic to DhlA-expressing plants. This represents a significant advance in the development of a low-cost phytoremediation approach toward the clean-up of halogenated organic pollutants from contaminated soil and groundwater.     

Molecular basis of biodegradation of chloroaromatic compounds

Chlorinated aromatic hydrocarbons are widely used in industry and agriculture, and comprise the bulk of environmental pollutants. Although simple aromatic compounds are biodegradable by a variety of degradative pathways, their halogenated counterparts are more resistant to bacterial attack and often necessitate evolution of novel pathways. An understanding of such evolutionary processes is essential for developing genetically improved strains capable of mineralizing highly chlorinated compounds. This article provides an overview of the genetic aspects of dissimilation of chloroaromatic compounds and discusses the potential of gene manipulation to promote enhanced evolution of the degradative pathways.     

Secretion and folding of human growth hormone in Escherichia coli

Abstract

Phytoremediation is the use of plants for the treatment of environmental pollution, including chlorinated organics. although conceptually very attractive, removal and biodegradation of chlorinated pollutants by plants is a rather slow and inefficient process resulting in incomplete treatment and potential release of toxic metabolites into the environment. In order to overcome inherent limitations of plant metabolic capabilities, plants have been genetically modified, following a strategy similar to the development of transgenic crops: genes from bacteria, fungi, and mammals involved in the metabolism of organic contaminants, such as cytochrome p-450 and glutathione substrate catabolic genes, natural or engineered, for the simultaneous remediation of a range of pollutants, such as usually found in contaminated sites, e.g., chlorinated solvent, metals, and nitroaromatics. In addition, biodegradation of many xenobiotics are catalyzed by similar, broad-substrate enzymes, such as cytochrome P-450 monoxygenases, glutathione S-transferases, and fungal peroxidases, that can potentially be used for the treatment of multiple pollutants. Moreover, the introduction of multiple transgenes involved in different phases of the metabolism of xenobiotics in plants, i.e., uptake by roots and the different phases of the green liver model, would allow enhancing both the removal and metabolism of several toxic compounds and could therefore help overcome a major limitation inherent to phytoremediation, i.e., the threat that accumulated toxic compounds would volatilize or otherwise contaminate the food chain. An important barrier to the application of transgenic plants for bioremediation in the field is associated with the true or perceived risk of horizontal gene transfer to related wild or cultivated plants. Therefore, it is likely that the next generation of transgenic plants will involve systems preventing such a transfer, for instance by the introduction of transgenes into chloroplastic DNA or the use of conditional lethality genes (Davison, 2005). Since bacteria naturally exchange plasmids via conjugation, endophytes that gain genes involved in pollutant degradation might not be considered ‘genetically modified’ and may be subject to fewer restrictions in usage.     

Phase I xenobiotic metabolic systems in plants

Phytoremediation uses living higher plants for the removal and biochemical decomposition of environmental pollutants. In this paper Phase I metabolic pathways in the biotransformation reactions of organic pollutants in plants are reviewed. These reactions result in the introduction of functional groups in the xenobiotic molecule or the exposure of preexisting functional groups and lead to the formation of more polar, more water-soluble, chemically more reactive and sometimes biologically more active derivatives. Phase I type reactions are most important in the phytoremediation of hydrophobic, chemically stable organic pollutants, such as polycyclic aromatic hydrocarbons and (poly)chlorinated aliphatic and aromatic hydrocarbons. Although Phase I reactions involve a wide range of chemical transformations from hydrolysis to reduction, oxidative processes catalyzed by cytochrome P450 containing monooxygenases are the most important. Transgenic plants with tailored Phase I enzymatic activities may play major roles in the removal of environmentally stable organic pollutants from contaminated fields.     

Biodegradation of halogenated organic compounds.

In this review we discuss the degradation of chlorinated hydrocarbons by microorganisms, emphasizing the physiological, biochemical, and genetic basis of the biodegradation of aliphatic, aromatic, and polycyclic compounds. Many environmentally important xenobiotics are halogenated, especially chlorinated. These compounds are manufactured and used as pesticides, plasticizers, paint and printing-ink components, adhesives, flame retardants, hydraulic and heat transfer fluids, refrigerants, solvents, additives for cutting oils, and textile auxiliaries. The hazardous chemicals enter the environment through production, commercial application, and waste. As a result of bioaccumulation in the food chain and groundwater contamination, they pose public health problems because many of them are toxic, mutagenic, or carcinogenic. Although synthetic chemicals are usually recalcitrant to biodegradation, microorganisms have evolved an extensive range of enzymes, pathways, and control mechanisms that are responsible for catabolism of a wide variety of such compounds. Thus, such biological degradation can be exploited to alleviate environmental pollution problems. The pathways by which a given compound is degraded are determined by the physical, chemical, and microbiological aspects of a particular environment. By understanding the genetic basis of catabolism of xenobiotics, it is possible to improve the efficacy of naturally occurring microorganisms or construct new microorganisms capable of degrading pollutants in soil and aquatic environments more efficiently. Recently a number of genes whose enzyme products have a broader substrate specificity for the degradation of aromatic compounds have been cloned and attempts have been made to construct gene cassettes or synthetic operons comprising these degradative genes. Such gene cassettes or operons can be transferred into suitable microbial hosts for extending and custom designing the pathways for rapid degradation of recalcitrant compounds. Recent developments in designing recombinant microorganisms and hybrid metabolic pathways are discussed.     

Biodegradation of phenol and its derivatives by engineered bacteria: current knowledge and perspectives

Biodegradation of phenolic compounds is a promising alternative to physical and chemical methods used to remove these toxic pollutants from the environment. The ability of various microorganisms to metabolize phenol and its derivatives (alkylphenols, nitrophenols and halogenated derivatives) has therefore been intensively studied. Knowledge of the enzymes catalyzing the individual reactions, the genes encoding these enzymes and the regulatory mechanisms involved in the expression of the respective genes in bacteria serves as a basis for the development of more efficient degraders of phenols via genetic engineering methods. Engineered bacteria which efficiently degrade phenolic compounds were constructed in laboratories using various approaches such as cloning the catabolic genes in multicopy plasmids, the introduction of heterologous genes or broadening the substrate range of key enzymes by mutagenesis. Efforts to apply the engineered strains in in situ bioremediation are problematic, since engineered strains often do not compete successfully with indigenous microorganisms. New efficient degraders of phenolic compounds may be obtained by complex approaches at the organism level, such as genome shuffling or adaptive evolution. The application of these engineered bacteria for bioremediation will require even more complex analysis of both the biological characteristics of the degraders and the physico-chemical conditions at the polluted sites.     

The little bacteria that can – diversity,genomics and ecophysiology of ‘Dehalococcoides’ spp. in contaminated environments

The fate and persistence of chlorinated organics in the environment have been a concern for the past 50 years. Industrialization and extensive agricultural activities have led to the accumulation of these pollutants in the environment, while their adverse impact on various ecosystems and human health also became evident. This review provides an update on the current knowledge of specialized anaerobic bacteria, namely ‘Dehalococcoides’ spp., which are dedicated to the transformation of various chlorinated organic compounds via reductive dechlorination. Advances in microbiology and molecular techniques shed light into the diversity and functioning of Dehalococcoides spp. in several different locations. Recent genome sequencing projects revealed a large number of genes that are potentially involved in reductive dechlorination. Molecular approaches towards analysis of diversity and expression especially of reductive dehalogenase-encoding genes are providing a growing body of knowledge on biodegradative pathways active in defined pure and mixed cultures as well as directly in the environment. Moreover, several successful field cases of bioremediation strengthen the notion of dedicated degraders such as Dehalococcoides spp. as key players in the restoration of contaminated environments.     

Bacterial degradation of aromatic pollutants: a paradigm of metabolic versatility.

Although most organisms have detoxification abilities (i.e mineralization, transformation and/or immobilization of pollutants), microorganisms, particularly bacteria, play a crucial role in biogeochemical cycles and in sustainable development of the biosphere. Next to glucosyl residues, the benzene ring is the most widely distributed unit of chemical structure in nature, and many of the aromatic compounds are major environmental pollutants. Bacteria have developed strategies for obtaining energy from virtually every compound under oxic or anoxic conditions (using alternative final electron acceptors such as nitrate, sulfate, and ferric ions). Clusters of genes coding for the catabolism of aromatic compounds are usually found in mobile genetic elements, such as transposons and plasmids, which facilitate their horizontal gene transfer and, therefore, the rapid adaptation of microorganisms to new pollutants. A successful strategy for in situ bioremediation has been the combination, in a single bacterial strain or in a syntrophic bacterial consortium, of different degrading abilities with genetic traits that provide selective advantages in a given environment. The advent of high-throughput methods for DNA sequencing and analysis of gene expression (genomics) and function (proteomics), as well as advances in modelling microbial metabolism in silico, provide a global, rational approach to unravel the largely unexplored potentials of microorganisms in biotechnological processes thereby facilitating sustainable development.     

Bacterial metabolism of polychlorinated biphenyls

Microbial metabolism is responsible for the removal of persistent organic pollutants including PCBs from the environment. Anaerobic dehalogenation of highly chlorinated congeners in aquatic sediments is an important process, and recent evidence has indicated that Dehalococcoides and related organisms are predominantly responsible for this process. Such anaerobic dehalogenation generates lower chlorinated congeners which are easily degraded aerobically by enzymes of the biphenyl upper pathway (bph). Initial biphenyl 2,3-dioxygenases are generally considered the key enzymes of this pathway which determine substrate range and extent of PCB degradation. These enzymes have been subject to different protein evolution strategies, and subsequent enzymes have been considered as crucial for metabolism. Significant advances have been made regarding the mechanistic understanding of these enzymes, which has also included elucidation of the function of BphK glutathione transferase. So far, the genomes of two important PCB-metabolizing organisms, namely Burkholderia xenovorans strain LB400 and Rhodococcus sp. strain RHA1, have been sequenced, with the rational to better understand their overall physiology and evolution. Genomic and proteomic analysis also allowed a better evaluation of PCB toxicity. Like all bph gene clusters which have been characterized in detail, particularly in strains LB400 and RHA1, these genes were localized on mobile genetic elements endowing single strains and microbial communities with a high flexibility and adaptability. However, studies show that our knowledge on enzymes and genes involved in PCB metabolism is still rather fragmentary and that the diversity of bacterial strategies is highly underestimated. Overall, metabolism of biphenyl and PCBs should not be regarded as a simple linear pathway, but as a complex interplay between different catabolic gene modules.     

Horizontal gene transfer and microbial adaptation to xenobiotics: new types of mobile genetic elements and lessons from ecological studies

The characterization of bacteria that degrade organic xenobiotics has revealed that they can adapt to these compounds by expressing 'novel' catabolic pathways. At least some of them appear to have evolved by patchwork assembly of horizontally transmitted genes and subsequent mutations and gene rearrangements. Recent studies have revealed the existence of new types of xenobiotic catabolic mobile genetic elements, such as catabolic genomic islands, which integrate into the chromosome after transfer. The significance of horizontal gene transfer and patchwork assembly for bacterial adaptation to pollutants under real environmental conditions remains uncertain, but recent publications suggest that these processes do occur in a polluted environment.     

Alkane hydroxylases involved in microbial alkane degradation

This review focuses on the role and distribution in the environment of alkane hydroxylases and their (potential) applications in bioremediation and biocatalysis. Alkane hydroxylases play an important role in the microbial degradation of oil, chlorinated hydrocarbons, fuel additives, and many other compounds. Environmental studies demonstrate the abundance of alkane degraders and have lead to the identification of many new species, including some that are (near)-obligate alkanotrophs. The availability of a growing collection of alkane hydroxylase gene sequences now allows estimations of the relative abundance of the different enzyme systems and the distribution of the host organisms.     

'Super bugs' for bioremediation

Chlorinated organic compounds are among the most significant pollutants in the world. Sequential use of anaerobic halorespiring bacteria, which are the key players in biological dehalogenation processes, and aerobic bacteria whose oxygenases are modified by directed evolution could lead to efficient and total degradation of highly chlorinated organic pollutants. Recently three interesting papers on halorespiration and polychlorinated biphenyl biodegradation were published.     

Mining <Emphasis Type="Italic">Xanthomonas</Emphasis> and <Emphasis Type="Italic">Streptomyces</Emphasis> genomes for new pectinase-encoding sequences and their heterologous expression in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Microbial genome sequencing has left a legacy of annotated yet uncharacterized genes or open reading frames, activities that may have useful applications in health and/or the environment. We are interested in the discovery and characterization of potentially new pectinolytic activities for the enzymatic retting of natural bast fibers such as hemp and flax. A highlight in this study is the discovery of a cold-active pectate lyase among five pectate-lyase-encoding sequences and two polygalacturonase-encoding sequences that we have cloned from the genomes of Xanthomonas campestris pv. campestris and Streptomyces coelicolor A3(2). Heterologous expression of these sequences as active pectate lyases and polygalacturonases required their subcloning in Escherichia coli Rosetta™ cells. The most active recombinant pectate lyase (XcPL NP_638163), a cold-active pectate lyase (XcPL NP_636037), and a polygalacturonase (XcPG NP_638805) were purified to near homogeneity and their kinetic parameters were determined. A significant amount of pectin degradation products was shown to be released by the two pectate lyases but not the polygalacturonase when hemp fiber pectin was used as substrate. Results of this study showed that genome data mining, besides an economical approach to new gene acquisition, may uncover new findings such as the discovery of a cold-active pectate-lyase-encoding sequence from X. campestris, a mesophilic microorganism.     

Bacterial catabolic transposons

The introduction of foreign organic hydrocarbons into the environment in recent years, as in the widespread use of antibiotics, has resulted in the evolution of novel adaptive mechanisms by bacteria for the biodegradation of the organic pollutants. Plasmids have been implicated in the catabolism of many of these complex xenobiotics. The catabolic genes are prone to undergo genetic rearrangement and this is due to their presence on transposons or their association with transposable elements. Most of the catabolic transposons have structural features of the class I (composite) elements. These include transposons for chlorobenzoate (Tn5271), chlorobenzene (Tn5280), the newly discovered benzene catabolic transposon (Tn5542), and transposons encoding halogenated alkanoates and nylon-oligomer-degradative genes. Transposons for the catabolism of toluene (Tn4651, Tn4653, Tn4656) and naphthalene (Tn4655) belong to class II (Tn3 family) elements. Many catabolic genes have been associated with insertion sequences, which suggests that these gene clusters could be rapidly disseminated among the bacterial populations. This greatly expands the substrate range of the microorganisms in the environment and aids the evolution of new and novel degradative pathways. This enhanced metabolic versatility can be exploited for and is believed to play a major part in the bioremediation of polluted environments. Received: 13 July 1998 / Received revision: 22 September 1998 / Accepted: 26 September 1998     

A family of human microRNA genes from miniature inverted-repeat transposable elements

While hundreds of novel microRNA (miRNA) genes have been discovered in the last few years alone, the origin and evolution of these non-coding regulatory sequences remain largely obscure. In this report, we demonstrate that members of a recently discovered family of human miRNA genes, hsa-mir-548, are derived from Made1 transposable elements. Made1 elements are short miniature inverted-repeat transposable elements (MITEs), which consist of two 37 base pair (bp) terminal inverted repeats that flank 6 bp of internal sequence. Thus, Made1 elements are nearly perfect palindromes, and when expressed as RNA they form highly stable hairpin loops. Apparently, these Made1-related structures are recognized by the RNA interference enzymatic machinery and processed to form 22 bp mature miRNA sequences. Consistent with their origin from MITEs, hsa-mir-548 genes are primate-specific and have many potential paralogs in the human genome. There are more than 3,500 putative hsa-mir-548 target genes; analysis of their expression profiles and functional affinities suggests cancer-related regulatory roles for hsa-mir-548. Taken together, the characteristics of Made1 elements, and MITEs in general, point to a specific mechanism for the generation of numerous small regulatory RNAs and target sites throughout the genome. The evolutionary lineage-specific nature of MITEs could also provide for the generation of novel regulatory phenotypes related to species diversification. Finally, we propose that MITEs may represent an evolutionary link between siRNAs and miRNAs.     

Versatility of soil column experiments to study biodegradation of halogenated compounds under environmental conditions

Soil column experiments were performed to obtain insight in the different biological and physico-chemical processes affecting biodegradation of halogenated compounds under natural conditions in a water infiltration site. Lower chlorinated aromatic compounds could be degraded under aerobic conditions, whereas highly chlorinated compounds and chlorinated aliphatic compounds were mainly transformed under anaerobic conditions. Microorganisms which derive energy from reductive dechlorination were enriched and characterized. It was found that microbes could adapt to using chlorinated benzenes by evolution of new enzyme specificities and by exchange of genetic material. For halogenated pollutants, which are generally hydrophobic, sorption processes control the concentration available for biodegradation. The effects of very low concentrations of halogenated compounds on their biodegradability are described. The use of isolated bacterial strains to enhance biodegradation was evaluated with respect to their temperature-related activity and to their adhesion properties.Abbreviations 3-CB 3-chlorobenzoate - DCB dichlorobenzene - HCH hexachlorocyclohexane - IS insertion sequence - PER tetrachloroethylene - S_min minimal substrate concentration for growth - TCB trichlorobenzene - TRI trichloroethylene - filtration coefficient     

Genome fragmentation is not confined to the peridinin plastid in dinoflagellates

When plastids are transferred between eukaryote lineages through series of endosymbiosis, their environment changes dramatically. Comparison of dinoflagellate plastids that originated from different algal groups has revealed convergent evolution, suggesting that the host environment mainly influences the evolution of the newly acquired organelle. Recently the genome from the anomalously pigmented dinoflagellate Karlodinium veneficum plastid was uncovered as a conventional chromosome. To determine if this haptophyte-derived plastid contains additional chromosomal fragments that resemble the mini-circles of the peridin-containing plastids, we have investigated its genome by in-depth sequencing using 454 pyrosequencing technology, PCR and clone library analysis. Sequence analyses show several genes with significantly higher copy numbers than present in the chromosome. These genes are most likely extrachromosomal fragments, and the ones with highest copy numbers include genes encoding the chaperone DnaK(Hsp70), the rubisco large subunit (rbcL), and two tRNAs (trnE and trnM). In addition, some photosystem genes such as psaB, psaA, psbB and psbD are overrepresented. Most of the dnaK and rbcL sequences are found as shortened or fragmented gene sequences, typically missing the 3'-terminal portion. Both dnaK and rbcL are associated with a common sequence element consisting of about 120 bp of highly conserved AT-rich sequence followed by a trnE gene, possibly serving as a control region. Decatenation assays and Southern blot analysis indicate that the extrachromosomal plastid sequences do not have the same organization or lengths as the minicircles of the peridinin dinoflagellates. The fragmentation of the haptophyte-derived plastid genome K. veneficum suggests that it is likely a sign of a host-driven process shaping the plastid genomes of dinoflagellates.     

Recent Findings on the Phytoremediation of Soils Contaminated with Environmentally Toxic Heavy Metals and Metalloids Such as Zinc,Cadmium, Lead,and Arsenic

Due to their immutable nature, metals are a group of pollutants of much concern. As a result of human activities such as mining and smelting of metalliferous ores, electroplating, gas exhaust, energy and fuel production, fertilizer and pesticide application, etc., metal pollution has become one of the most serious environmental problems today. Phytoremediation, an emerging cost-effective, non-intrusive, and aesthetically pleasing technology, that uses the remarkable ability of plants to concentrate elements and compounds from the environment and to metabolize various molecules in their tissues, appears very promising for the removal of pollutants from the environment. Within this field of phytoremediation, the utilization of plants to transport and concentrate metals from the soil into the harvestable parts of roots and above-ground shoots, i.e., phytoextraction, may be, at present, approaching commercialization. Improvement of the capacity of plants to tolerate and accumulate metals by genetic engineering should open up new possibilities for phytoremediation. The lack of understanding pertaining to metal uptake and translocation mechanisms, enhancement amendments, and external effects of phytoremediation is hindering its full scale application. Due to its great potential as a viable alternative to traditional contaminated land remediation methods, phytoremediation is currently an exciting area of active research.