Irene Horne

Identification of an opd (Organophosphate Degradation) Gene in an Agrobacterium Isolate

ABSTRACT We isolated a bacterial strain, Agrobacterium radiobacter P230, which can hydrolyze a wide range of organophosphate (OP) insecticides. A gene encoding a protein involved in OP hydrolysis was cloned from A. radiobacter P230 and sequenced. This gene (called opdA) had sequence similarity to opd, a gene previously shown to encode an OP-hydrolyzing enzyme in Flavobacterium sp. strain ATCC 27551 and Brevundimonas diminuta MG. Insertional mutation of the opdA gene produced a strain lacking the ability to hydrolyze OPs, suggesting that this is the only gene encoding an OP-hydrolyzing enzyme in A. radiobacter P230. The OPH and OpdA proteins, encoded by opd and opdA, respectively, were overexpressed and purified as maltose-binding proteins, and the maltose-binding protein moiety was cleaved and removed. Neither protein was able to hydrolyze the aliphatic OP malathion. The kinetics of the two proteins for diethyl OPs were comparable. For dimethyl OPs, OpdA had a higher kcat than OPH. It was also capable of hydrolyzing the dimethyl OPs phosmet and fenthion, which were not hydrolyzed at detectable levels by OPH.

Enrichment of an endosulfan-degrading mixed bacterial culture

ABSTRACT An endosulfan-degrading mixed bacterial culture was enriched from soil with a history of endosulfan exposure. Enrichment was obtained by using the insecticide as the sole source of sulfur. Chemical hydrolysis was minimized by using strongly buffered culture medium (pH 6.6), and the detergent Tween 80 was included to emulsify the insecticide, thereby increasing the amount of endosulfan in contact with the bacteria. No growth occurred in control cultures in the absence of endosulfan. Degradation of the insecticide occurred concomitant with bacterial growth. The compound was both oxidized and hydrolyzed. The oxidation reaction favored the alpha isomer and produced endosulfate, a terminal pathway product. Hydrolysis involved a novel intermediate, tentatively identified as endosulfan monoaldehyde on the basis of gas chromatography-mass spectrometry and chemical derivatization results. The accumulation and decline of metabolites suggest that the parent compound was hydrolyzed to the putative monoaldehyde, thereby releasing the sulfite moiety required for growth. The monoaldehyde was then oxidized to endosulfan hydroxyether and further metabolized to (a) polar product(s). The cytochrome P450 inhibitor, piperonyl butoxide, did not prevent endosulfan oxidation or the formation of other metabolites. These results suggest that this mixed culture is worth investigating as a source of endosulfan-hydrolyzing enzymes for use in enzymatic bioremediation of endosulfan residues.

ENZYMATIC BIOREMEDIATION: FROM ENZYME DISCOVERY TO APPLICATIONS

1. Enzymatic bioremediation is potentially a rapid method of removing environmental pesticide residues. Applications include the treatment of residues resulting from agricultural production and processing industries, such as the treatment of irrigation waters, surface‐contaminated fruit and vegetables and spent dip liquors.

A single monooxygenase, ese, is involved in the metabolism of the organochlorides endosulfan and endosulfate in an Arthrobacter sp.

ABSTRACT In this paper we describe isolation of a bacterium capable of degrading both isomers of the organochloride insecticide endosulfan and its toxic metabolite, endosulfate. The bacterium was isolated from a soil microbial population that was enriched with continuous pressure to use endosulfate as the sole source of sulfur. Analysis of the 16S rRNA sequence of the bacterium indicated that it was an Arthrobacter species. The organochloride-degrading activity was not observed in the presence of sodium sulfite as an alternative sulfur source, suggesting that the activity was part of the sulfur starvation response of the strain. A gene, ese, encoding an enzyme capable of degrading both isomers of endosulfan and endosulfate was isolated from this bacterium. The enzyme belongs to the two-component flavin-dependent monooxygenase family whose members require reduced flavin for activity. Nuclear magnetic resonance analyses identified the metabolite of endosulfan as endosulfan monoalcohol and the metabolite of endosulfate as endosulfan hemisulfate. The ese gene was located in a cluster of 10 open reading frames encoding proteins with low levels of sulfur-containing amino acids. These open reading frames were organized into two apparent divergently orientated operons and a gene encoding a putative LysR-type transcriptional regulator. The operon not containing ese did contain a homologue whose product exhibited 62% amino acid identity to the ese-encoded protein.

Comparative and functional genomics of lipases in holometabolous insects.

Lipases have key roles in insect lipid acquisition, storage and mobilisation and are also fundamental to many physiological processes underpinning insect reproduction, development, defence from pathogens and oxidative stress, and pheromone signalling. We have screened the recently sequenced genomes of five species from four orders of holometabolous insects, the dipterans Drosophila melanogaster and Anopheles gambiae, the hymenopteran Apis mellifera, the moth Bombyx mori and the beetle Tribolium castaneum, for the six major lipase families that are also found in other organisms. The two most numerous families in the insects, the neutral and acid lipases, are also the main families in mammals, albeit not in Caenorhabditis elegans, plants or microbes. Total numbers of the lipases vary two-fold across the five insect species, from numbers similar to those in mammals up to numbers comparable to those seen in C. elegans. Whilst there is a high degree of orthology with mammalian lipases in the other four families, the great majority of the insect neutral and acid lipases have arisen since the insect orders themselves diverged. Intriguingly, about 10% of the insect neutral and acid lipases have lost motifs critical for catalytic function. Examination of the length of lid and loop regions of the neutral lipase sequences suggest that most of the insect lipases lack triacylglycerol (TAG) hydrolysis activity, although the acid lipases all have intact cap domains required for TAG hydrolysis. We have also reviewed the sequence databases and scientific literature for insights into the expression profiles and functions of the insect neutral and acid lipases and the orthologues of the mammalian adipose triglyceride lipase which has a pivotal role in lipid mobilisation. These data suggest that some of the acid and neutral lipase diversity may be due to a requirement for rapid accumulation of dietary lipids. The different roles required of lipases at the four discrete life stages of holometabolous insects may also contribute to the diversity of lipases required by insects. In addition, insects use lipases to perform roles for which there are no correlates in mammals, including as yolk and male accessory gland proteins.

Enrichment of a microbial culture capable of degrading endosulphate, the toxic metabolite of endosulfan

Aims: The aim of this study was to isolate a source of enzymes capable of degrading endosulphate (endosulfan sulphate), the toxic metabolite of the pesticide endosulfan.

Isolation and characterization of a Mycobacterium strain that metabolizes the insecticide endosulfan

Aim: The aim of this study was to isolate and characterize a bacterium capable of metabolizing endosulfan. 
Methods and Results: A endosulfan‐degrading bacterium (strain ESD) was isolated from soil inoculum after repeated culture with the insecticide as the sole source of sulfur. Analysis of its 16S rRNA gene sequence, and morphological and physiological characteristics revealed it to be a new fast‐growing Mycobacterium, closely related to other Mycobacterium species with xenobiotic‐degrading capabilities. Degradation of endosulfan by strain ESD involved both oxidative and sulfur‐separation reactions. Strain ESD did not degrade endosulfan when sulfite, sulphate or methionine were present in the medium along with the insecticide. Partial degradation occurred when the culture was grown, with endosulfan, in the presence of MOPS (3‐(N‐morpholino)propane sulphonic acid), DMSO (dimethyl sulfoxide), cysteine or sulphonane and complete degradation occurred in the presence of gutathione. When both beta‐endosulfan and low levels of sulphate were provided as the only sources of sulfur, biphasic exponential growth was observed with endosulfan metabolism being restricted to the latter phase of exponential growth. 
Conclusions: This study isolated a Mycobacterium strain (strain ESD) capable of metabolizing endosulfan by both oxidative and sulfur‐separation reactions. The endosulfan‐degrading reactions are a result of the sulfur‐starvation response of this bacterium. 
Significance and Impact of the Study: This describes the isolation of a Mycobacterium strain capable of degrading the insecticide endosulfan. This bacterium is a valuable source of enzymes for use in enzymatic bioremediation of endosulfan residues.

Gene Cloning and Molecular Characterization of a Two-Enzyme System Catalyzing the Oxidative Detoxification of β-Endosulfan

ABSTRACT The gram-positive bacterium Mycobacterium sp. strain ESD is able to use the cyclodiene insecticide endosulfan as a source of sulfur for growth. This activity is dependent on the absence of sulfite or sulfate in the growth medium. A cosmid library of strain ESD DNA was constructed in a Mycobacterium-Escherichia coli shuttle vector and screened for endosulfan-degrading activity in Mycobacterium smegmatis, a species that does not degrade endosulfan. Using this method, we identified a single cosmid that conferred sulfur-dependent endosulfan-degrading activity on the host strain. An open reading frame (esd) was identified within this cosmid that, when expressed behind a constitutive promoter in a mycobacterial expression vector, conferred sulfite- and sulfate-independent β-endosulfan degradation activity on the recombinant strain. The translation product of this gene (Esd) had up to 50% sequence identity with an unusual family of monooxygenase enzymes that use reduced flavins, provided by a separate flavin reductase enzyme, as cosubstrates. An additional partial open reading frame was located upstream of the Esd gene that had sequence homology to the same monooxygenase family. A flavin reductase gene, identified in the M. smegmatis genome, was cloned, expressed, and used to provide reduced flavin mononucleotide for Esd in enzyme assays. Thin-layer chromatography and gas chromatography analyses of the enzyme assay mixtures revealed the disappearance of β-endosulfan and the appearance of the endosulfan metabolites, endosulfan monoaldehyde and endosulfan hydroxyether. This suggests that Esd catalyzes the oxygenation of β-endosulfan to endosulfan monoaldehyde and endosulfan hydroxyether. Esd did not degrade either α-endosulfan or the metabolite of endosulfan, endosulfan sulfate.

Isolation and functional characterization of two independently-evolved fatty acid Δ12-desaturase genes from insects

We report the first isolation and characterization of insect fatty acid Δ12‐desaturase genes, AdD12Des from house cricket (Acheta domesticus) and TcD12Des from the red flour beetle (Tribolium castaneum), responsible for the production of linoleic acid from oleic acid. Sequence analysis shows the cricket and flour beetle Δ12‐desaturase genes have evolved independently from all previously known Δ12‐desaturases and are much more closely related to the archetypal stearoyl‐Coenzyme A‐acting desaturase from rat than to the phospholipid‐acting Δ12‐desaturases widely reported in plants. Phylogenetic and functional analysis indicates the cricket AdD12Des gene may have evolved from an ancestral Δ9‐desaturase. By contrast, the beetle Δ12‐desaturase is distantly related to the cricket genes and beetle Δ9‐desaturases suggesting evolution by an independent route. Linoleic acid has key physiological roles in insects and this is the first report of genes capable of producing this essential fatty acid in higher animals.

Greatly enhanced detection of a volatile ligand at femtomolar levels using bioluminescence resonance energy transfer (BRET)

Our goal is to develop a general transduction system for G-protein coupled receptors (GPCRs). GPCRs are present in most eukaryote cells and transduce diverse extracellular signals. GPCRs comprise not only the largest class of integral membrane receptors but also the largest class of targets for therapeutic drugs. In all cases studied, binding of ligand to a GPCR leads to a sub-nanometer intramolecular rearrangement. Here, we report the creation of a novel chimaeric BRET-based biosensor by insertion of sequences encoding a bioluminescent donor and a fluorescent acceptor protein into the primary sequence of a GPCR. The BRET(2)-ODR-10 biosensor was expressed in membranes of Saccharomyces cerevisiae. Assays conducted on isolated membranes indicated an EC(50) in the femtomolar range for diacetyl. The response was ligand-specific and was abolished by a single point mutation in the receptor sequence. Novel BRET-GPCR biosensors of this type have potential application in many fields including explosive detection, quality control of food and beverage production, clinical diagnosis and drug discovery.