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.

Engineering a recombinant Deinococcus radiodurans for organopollutant degradation in radioactive mixed waste environments.

Thousands of waste sites around the world contain mixtures of toxic chlorinated solvents, hydrocarbon solvents, and radionuclides. Because of the inherent danger and expense of cleaning up such wastes by physicochemical methods, other methods are being pursued for cleanup of those sites. One alternative is to engineer radiation-resistant microbes that degrade or transform such wastes to less hazardous mixtures. We describe the construction and characterization of recombinant Deinococcus radiodurans, the most radiation-resistant organism known, expressing toluene dioxygenase (TDO). Cloning of the tod genes (which encode the multicomponent TDO) into the chromosome of this bacterium imparted to the strain the ability to oxidize toluene, chlorobenzene, 3,4-dichloro-1-butene, and indole. The recombinant strain was capable of growth and functional synthesis of TDO in the highly irradiating environment (60 Gy/h) of a 137Cs irradiator, where 5x10(8)cells/ml degraded 125 nmol/ml of chlorobenzene in 150 min. D. radiodurans strains were also tolerant to the solvent effects of toluene and trichloroethylene at levels exceeding those of many radioactive waste sites. These data support the prospective use of engineered D. radiodurans for bioremediation of mixed wastes containing both radionuclides and organic solvents.

Engineering Deinococcus radiodurans for metal remediation in radioactive mixed waste environments.

We have developed a radiation resistant bacterium for the treatment of mixed radioactive wastes containing ionic mercury. The high cost of remediating radioactive waste sites from nuclear weapons production has stimulated the development of bioremediation strategies using Deinococcus radiodurans, the most radiation resistant organism known. As a frequent constituent of these sites is the highly toxic ionic mercury (Hg) (II), we have generated several D. radiodurans strains expressing the cloned Hg (II) resistance gene (merA) from Escherichia coli strain BL308. We designed four different expression vectors for this purpose, and compared the relative advantages of each. The strains were shown to grow in the presence of both radiation and ionic mercury at concentrations well above those found in radioactive waste sites, and to effectively reduce Hg (II) to the less toxic volatile elemental mercury. We also demonstrated that different gene clusters could be used to engineer D. radiodurans for treatment of mixed radioactive wastes by developing a strain to detoxify both mercury and toluene. These expression systems could provide models to guide future D. radiodurans engineering efforts aimed at integrating several remediation functions into a single host.

The University of Minnesota Biocatalysis/Biodegradation Database: emphasizing enzymes.

The University of Minnesota Biocatalysis/Biodegradation Database (UM-BBD, http://umbbd.ahc.umn.edu/) provides curated information on microbial catabolic enzymes and their organization into metabolic pathways. Currently, it contains information on over 400 enzymes. In the last year the enzyme page was enhanced to contain more internal and external links; it also displays the different metabolic pathways in which each enzyme participates. In collaboration with the Nomenclature Commission of the International Union of Biochemistry and Molecular Biology, 35 UM-BBD enzymes were assigned complete EC codes during 2000. Bacterial oxygenases are heavily represented in the UM-BBD; they are known to have broad substrate specificity. A compilation of known reactions of naphthalene and toluene dioxygenases were recently added to the UM-BBD; 73 and 108 were listed respectively. In 2000 the UM-BBD is mirrored by two prestigious groups: the European Bioinformatics Institute and KEGG (the Kyoto Encyclopedia of Genes and Genomes). Collaborations with other groups are being developed. The increased emphasis on UM-BBD enzymes is important for predicting novel metabolic pathways that might exist in nature or could be engineered. It also is important for current efforts in microbial genome annotation.

Biodegradation of low-molecular-weight halogenated hydrocarbons by methanotrophic bacteria.

Low-molecular-weight halogenated hydrocarbons are susceptible to degradation by anaerobic and aerobic bacteria. The methanotrophic bacterium Methylosinus trichosporium 0B3b degrades trichloroethylene more rapidly than other bacteria examined to date. Expression of soluble methane monooxygenase (MMO) is correlated with high rates of biodegradation. An analysis of 16 S rRNA sequences of 11 ribosomal RNAs from type I, type II and type X methanotrophs and methanol-utilizing bacteria have revealed four clusters of phylogenetically related methylotrophs. This information may be useful for the identification and enumeration of methylotrophs in bioreactors and other environments during remediation of contaminated waters.

Novel enzyme activities and functional plasticity revealed by recombining highly homologous enzymes.

BACKGROUND: Directed evolution by DNA shuffling has been used to modify physical and catalytic properties of biological systems. We have shuffled two highly homologous triazine hydrolases and conducted an exploration of the substrate specificities of the resulting enzymes to acquire a better understanding of the possible distributions of novel functions in sequence space. RESULTS: Both parental enzymes and a library of 1600 variant triazine hydrolases were screened against a synthetic library of 15 triazines. The shuffled library contained enzymes with up to 150-fold greater transformation rates than either parent. It also contained enzymes that hydrolyzed five of eight triazines that were not substrates for either starting enzyme. CONCLUSIONS: Permutation of nine amino acid differences resulted in a set of enzymes with surprisingly diverse patterns of reactions catalyzed. The functional richness of this small area of sequence space may aid our understanding of both natural and artificial evolution.

Co-metabolism: is the emperor wearing any clothes?

Co-metabolism is a term used for biochemically undefined observations in catabolic enzyme substrate specificity, the interplay between enzyme specificity and metabolic regulation, the metabolic interdependence of microorganisms, and co-substrate requirements in the catabolism of xenobiotic compounds. Recent findings in these four areas of microbial biochemistry necessitate a re-evaluation of the widespread use of the term.

Dehalogenation in environmental biotechnology.

During the past year, the field of dehalogenation has seen rapid progress in the identification of novel organisms, the sequencing of new genes, and the delineation of mechanisms for important enzymes. Newly identified anaerobic organisms are beginning to offer insights into a previously obscure, but important, group of bacteria involved in environmental dehalogenation. An important series of X-ray structure determinations have provided key knowledge for understanding and, ultimately, engineering biodehalogenation catalysis.

Environmental biotechnology.

There is an increasing interest in environmental biotechnology owing to a worldwide need to feed the world's growing population and to maintain clean soil, air and water. The major technological developments are in plant and microbial biology. Plants can be more readily engineered for resistances that enhance yield or produce new products whereas microorganisms are exploited for their catalytic diversity and ease of genetic engineering.

MIF protein are theta-class glutathione S-transferase homologs.

MIF proteins are mammalian polypeptides of approximately 13,000 molecular weight. This class includes human macrophage migration inhibitory factor (MIF), a rat liver protein that has glutathione S-transferase (GST) activity (TRANSMIF), and the mouse delayed early response gene 6 (DER6) protein. MIF proteins were previously linked to GSTs by demonstrating transferase activity and observing N-terminal sequence homology with a mu-class GST (Blocki, F.A., Schlievert, P.M., & Wackett, L.P., 1992, Nature 360, 269-270). In this study, MIF proteins are shown to be structurally related to the theta class of GSTs. This is established in three ways. First, unique primary sequence patterns are developed for each of the GST gene classes. The patterns identify the three MIF proteins as theta-like transferase homologs. Second, pattern analysis indicates that GST members of the theta class contain a serine residue in place of the N-terminal tyrosine that is implicated in glutathione deprotonation and activation in GSTs of known structure (Liu, S., et al., 1992, J. Biol. Chem. 267, 4296-4299). The MIF proteins contain a threonine at this position. Third, polyclonal antibodies raised against recombinant human MIF cross-react on Western blots with rat theta GST but not with alpha and mu GSTs. That MIF proteins have glutathione-binding ability may provide a common structural key toward understanding the varied functions of this widely distributed emerging gene family. Because theta is thought to be the most ancient evolutionary GST class, MIF proteins may have diverged early in evolution but retained a glutathione-binding domain.

Recruitment of co-metabolic enzymes for environmental detoxification of organohalides.

Polyhalogenated compounds are often environmentally persistent and toxic to mammals. Microorganisms that metabolize these compounds can detoxify contaminated environments. Different biochemical mechanisms are used to metabolize polyhalogenated compounds, but few naturally occurring bacteria have this capability. A recombinant bacterium was constructed to metabolize polyhalogenated compounds to nonhalogenated end products. Seven genes were expressed in Pseudomonas putida G786 to biosynthesize cytochrome P450CAM and toluene dioxygenase. Cytochrome P450CAM catalyzed reductive dechlorinated reactions and toluene dioxygenase catalyzed oxidative dechlorination. With pentachloroethane, reductive dechlorination yielded trichloroethylene, which was further oxidized to formate and glyoxylate. The sequential action of cytochrome P450CAM and toluene dioxygenase with polyhalogenated compounds constitutes a novel engineered metabolic pathway.

Directed evolution of new enzymes and pathways for environmental biocatalysis.

Biocatalysis is important in both natural and engineered environments. The major global reactions in the biospheric cycling of carbon, nitrogen, and other elements are catalyzed by microorganisms. The global carbon cycle includes millions of organic compounds that are made by plants, microorganisms, and organic chemists. Most of those compounds are transformed by microbial enzymes. Degradative metabolism is known as catabolism and yields principally carbon dioxide, methane, or biomass. Microbial catabolic enzymes are a great resource for biotechnology. They are the building blocks for engineering novel metabolic pathways and evolving improved enzymes in the laboratory. Two multicomponent bacterial oxygeneases, cytochrome P450cam and toluene dioxygenase, catalyze the dechlorination of polyhalogenated C2 compounds. Seven genes encoding those functional enzyme complexes were coexpressed in a Pseudomonas and shown to metabolize pentachloreothane to nonhalogenated organic acids that were metabolized further to carbon dioxide. In another example, the enzyme catalyzing the dechlorination of the herbicide atrazine was subjected to iterative DNA shuffling to produce mutations. By using a plate screening assay, mutated atrazine chlorohydrolase that catalyzed a more rapid dechlorination of atrazine was obtained. The mutant genes were sequences and found to encode up to 11 amino acid changes. Atrazine chlorohydrolase is currently being used in a model municipal water treatment system to test the feasibility of using enzymes for atrazine decontamination. These data suggest that the natural diversity of bacterial catabolic enzymes provides the starting point for improved biocatalytic systems that meet the needs of commercial applications.

[Site-directed mutagenesis of the dichloromethane dehalogenase gene from Methylophilus sp. strain DM11].

In order to investigate the role of different residues of Methylophilus sp. strain DM11 dichloromethane dehalogenase for substrate binding, glutathione affinity, and catalytic activity, site-directed mutagenesis studies of the gene encoding the enzyme were carried out. The conserved tryptophane residue at 103 region was respectively substituted by phenylalanine, valine or asparagine. The conserved arginine residue at 109 region was substituted by leucine. The conserved tryptophane residue at 117 region was respectively substituted by tyrosine or phenylalanine. Six mutant enzymes were produced. Among them three possess lower activities, other three do not possess activity. The properties of the mutant enzyme W117Y are very different from wild-type enzyme.

Biocatalysis, biodegradation and bioinformatics.

Biocatalysis, biodegradation and bioinformatics are prominent scientific fields in industrial microbiology and biotechnology. This paper describes developments in these fields with a focus on the role of David T Gibson as a researcher and mentor. He has pioneered studies on the mechanisms by which aerobic microorganisms transform aromatic hydrocarbons. In addition, his research has served as a model for further investigations into bacterial atrazine and dichloromethane catabolism described here. Microbial catabolism research requires information on organic chemistry, microorganisms, metabolic pathways, catabolic genes, and enzymes. These information needs are now being met more comprehensively by development of the University of Minnesota Biocatalysis/Biodegradation Database.

Degradation of trichloroethylene by toluene dioxygenase in whole-cell studies with Pseudomonas putida F1.

Toluene-induced cells of Pseudomonas putida F1 removed trichloroethylene from growth media at a significantly greater initial rate than the methanotroph Methylosinus trichosporium OB3b. With toluene-induced P. putida F1, the initial degradation rate varied linearly with trichloroethylene concentration over the range of 8 to 80 microM (1.05 to 10.5 ppm). At 80 microM (10.5 ppm) trichloroethylene and 30 degrees C, the initial rate was 1.8 nmol/min per mg of total cell protein, but the rate decreased rapidly with time. A series of mutant strains derived from P. putida F1 that are defective in the todC gene, which encodes the oxygenase component of toluene dioxygenase, failed to degrade trichloroethylene and to oxidize indole to indigo. A spontaneous revertant selected from a todC culture regained simultaneously the abilities to oxidize toluene, to form indigo, and to degrade trichloroethylene. The three isomeric dichloroethylenes were degraded by P. putida F1, but tetrachloroethylene, vinyl chloride, and ethylene were not removed from incubation mixtures.