Phytoremediation: a novel strategy for the removal of toxic metals from the environment using plants.

Toxic metal pollution of waters and soils is a major environmental problem, and most conventional remediation approaches do not provide acceptable solutions. The use of specially selected and engineered metal-accumulating plants for environmental clean-up is an emerging technology called phytoremediation. Three subsets of this technology are applicable to toxic metal remediation: (1) Phytoextraction--the use of metal-accumulating plants to remove toxic metals from soil; (2) Rhizofiltration--the use of plant roots to remove toxic metals from polluted waters; and (3) Phytostabilization--the use of plants to eliminate the bioavailability of toxic metals in soils. Biological mechanisms of toxic metal uptake, translocation and resistance as well as strategies for improving phytoremediation are also discussed.

Designing pathways for environmental purposes.

Genetic modifications intended to improve the properties of enzymes or entire metabolic pathways involved in hazardous waste treatment have resulted in the discovery of promising approaches for expanding the utility of biotreatment. Construction of hybrid metabolic pathways as well as hybrid enzymes, site-directed mutagenesis, and gene amplification have all facilitated the desired outcome of more complete and rapid contaminant removal and considerably broadened substrate specificity.

A gas lift bioreactor for removal of contaminants from the vapor phase.

The cometabolic degradation of trichloroethylene (TCE) as a vapor by two aromatic-metabolizing pseudomonads was evaluated in an airlift reactor. These microorganisms were able to degrade 90 to 95% of TCE in air at concentrations at the reactor inlet of 300 to 4,000 mug/liter. Although exposure of the cells to high inlet concentrations of TCE (4 mg/liter) caused a decline in enzyme-specific activity and TCE removal efficiency, this loss in activity could be prevented or delayed by increasing the rate of cosubstrate addition. Under the appropriate operating conditions, the microorganisms were able to degrade even high concentrations of TCE and activity of the cells in the reactor could be maintained for periods of at least 2 weeks.

Sequences of genes encoding naphthalene dioxygenase in Pseudomonas putida strains G7 and NCIB 9816-4.

The multicomponent enzyme, naphthalene dioxygenase, initiates the metabolism of naphthalene by Pseudomonas putida strains G7 (PpG7) and NCIB 9816-4 (Pp9816-4). The genes involved (nahAaAbAcAd) are encoded by the NAH7 and pDTG1 plasmids, respectively, and form part of the nah operon. The locations of the structural genes were determined on previously cloned fragments of DNA. The nucleotide (nt) sequences were determined for nahAaAb from Pp9816-4 and for nahAaAbAcAd from PpG7. The appropriate open reading frames were identified using N-terminal amino acid sequences determined from the purified proteins. The two nt sequences showed 93% homology, with the least homology seen upstream from the promoter region.

Construction of metabolic operons catalyzing the de novo biosynthesis of indigo in Escherichia coli.

The efficient production of the textile dye indigo by fermentation has been a goal since the early 1980's when the first bacterial strains capable of this synthesis were constructed. We report here the development of a recombinant microorganism that directly synthesizes indigo from glucose. This construction involved the cloning and genetic manipulation of at least 9 genes and modifications of the fermentation medium to help stabilize the biosynthetic activity. Directed genetic changes in two operons caused significant increases in reaction rates and in the stability of the catalytic enzymes. This example of whole cell catalysis by a recombinant Escherichia coli represents a novel and environmentally sound approach to the synthesis of a high value specialty chemical.

Expression of naphthalene oxidation genes in Escherichia coli results in the biosynthesis of indigo.

A fragment of plasmid NAH7 from Pseudomonas putida PpG7 has been cloned and expressed in Escherichia coli HB101. Growth of the recombinant Escherichia coli in nutrient medium results in the formation of indigo. The production of this dye is increased in the presence of tryptophan or indole. Several bacteria that oxidize aromatic hydrocarbons to cis-dihydrodiols also oxidize indole to indigo. The results suggest that indigo formation is due to the combined activities of tryptophanase and naphthalene dioxygenase.

Naphthalene dioxygenase: purification and properties of a terminal oxygenase component.

Naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816 is a multicomponent enzyme system that oxidized naphthalene to cis-(1R, 2S)-dihydroxy-1,2-dihydronaphthalene. The terminal oxygenase component B was purified to homogeneity by a three-step procedure that utilized ion-exchange and hydrophobic interaction chromatography. The purified enzyme oxidized naphthalene only in the presence of NADH, oxygen, and partially purified preparations of components A and C. An estimated Mr of 158,000 was obtained by gel filtration. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed the presence of two subunits with molecular weights of ca. 55,000 and 20,000, indicative of an alpha 2 beta 2 quaternary structure. Absorption spectra of the oxidized enzyme showed maxima at 566 (shoulder), 462, and 344 nm, which were replaced by absorption maxima at 520 and 380 nm when the enzyme was reduced anaerobically by stoichiometric quantities of NADH in the presence of the other two components of the naphthalene dioxygenase system. Component B bound naphthalene. Enzyme-bound naphthalene was oxidized to product upon the addition of components A and C, NADH, and O2. These results, together with the detection of the presence of 6.0 g-atoms of iron and 4.0 g-atoms of acid-labile sulfur per mol of the purified enzyme, suggest that component B of the naphthalene dioxygenase system is an iron-sulfur protein which functions in the terminal step of naphthalene oxidation.

Oxidation of naphthalene by a multicomponent enzyme system from Pseudomonas sp. strain NCIB 9816.

The initial reactions in the oxidation of naphthalene by Pseudomonas sp. strain NCIB 9816 involves the enzymatic incorporation of one molecule of oxygen into the aromatic nucleus to form (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene. The enzyme catalyzing this reaction, naphthalene dioxygenase, was resolved into three protein components, designated A, B, and C, by DEAE-cellulose chromatography. Incubation of naphthalene with components A, B, and C in the presence of NADH resulted in the formation of (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene. The ratio of oxygen and NADH utilization to product formation was 1:1:1. NADPH also served as an electron donor for naphthalene oxygenation. However, its activity was less than 50% of that observed with NADH. Component A showed NAD(P)H-cytochrome c reductase activity which was stimulated by the addition of flavin adenine dinucleotide and flavin mononucleotide. A similar stimulation was observed when these flavin nucleotides were added to the naphthalene dioxygenase assay system. These preliminary observations indicate that naphthalene dioxygenase has properties in common with both monooxygenase and dioxygenase multicomponent enzyme systems.

Effects of growth substrate and respiratory chain composition on bioenergetics in Acinetobacter sp. strain HO1-N.

The relationship between respiratory chain composition and efficiency of coupling phosphorylation to electron transport was examined in Acinetobacter sp. strain HO1-N. Cells containing only cytochrome o as a terminal oxidase displayed the same stoichiometries of adenosine 5'-triphosphate synthesis and proton extrusion as cells which contained both cytochromes o and d as terminal oxidases. In addition, CO inhibition and photo-relief of cytochromes o or d did not alter the efficiency of energy coupling. These findings indicate that adenosine 5'-triphosphate synthesis is coupled to electron transport through both cytochromes o and d in Acinetobacter.

Influences of growth substrates and oxygen on the electron transport system in Acinetobacter sp. HO1-N.

The electron transport system of Acinetobacter sp. HO1-N was studied to determine the specific cytochromes and to measure changes in the composition of the respiratory system due to growth in various concentrations of oxygen or types of growth substrates. Spectrophotometric analysis revealed that the quantity and types of cytochromes changed in response to growth under various concentrations of oxygen. Growth on alkane and nonalkane substrates resulted in only minor differences in cytochrome composition or oxidase activities. Membranes prepared from cells grown under oxygen-limiting conditions contained at least one b-type cytochrome, cytochrome o, cytochrome d, and slight traces of cytochrome a1, whereas membranes prepared from cells grown in the presence of high oxygen concentrations contained only low levels of cytochromes b and o. Polarographic measurements, electron transport inhibitor studies, and photoaction spectrum analyses indicated that cytochromes o, a1, and d were potentially capable of functioning as terminal oxidases in this organism. These experiments also revealed that all three cytochromes may be involved in the oxidation of reduced nicotinamide adenine dinucleotide, succinate, or N,N,N',N'-tetramethyl-p-phenylenediamine.