Robyn J. Russell

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.

Biochemical Genetics and Genomics of Insect Esterases

Abstract Esterases are a major part of insect biochemical research and an important component of insects’ xenobiotic defense systems. This article reviews the biochemistry and genetics of insect esterases, focusing mainly on developments. It is written from a genomics perspective and the bulk of it is organized around the major clades of insect esterases as revealed by the comparative genomic analysis. Some of the historical functional definitions and classifications applied to insect esterases are briefly discussed.

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.

Evolution in agriculture: the application of evolutionary approaches to the management of biotic interactions in agro-ecosystems

Anthropogenic impacts increasingly drive ecological and evolutionary processes at many spatio‐temporal scales, demanding greater capacity to predict and manage their consequences. This is particularly true for agro‐ecosystems, which not only comprise a significant proportion of land use, but which also involve conflicting imperatives to expand or intensify production while simultaneously reducing environmental impacts. These imperatives reinforce the likelihood of further major changes in agriculture over the next 30–40 years. Key transformations include genetic technologies as well as changes in land use. The use of evolutionary principles is not new in agriculture (e.g. crop breeding, domestication of animals, management of selection for pest resistance), but given land‐use trends and other transformative processes in production landscapes, ecological and evolutionary research in agro‐ecosystems must consider such issues in a broader systems context. Here, we focus on biotic interactions involving pests and pathogens as exemplars of situations where integration of agronomic, ecological and evolutionary perspectives has practical value. Although their presence in agro‐ecosystems may be new, many traits involved in these associations evolved in natural settings. We advocate the use of predictive frameworks based on evolutionary models as pre‐emptive management tools and identify some specific research opportunities to facilitate this. We conclude with a brief discussion of multidisciplinary approaches in applied evolutionary problems.

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.

Carboxyl/cholinesterases: a case study of the evolution of a successful multigene family

The evolution of organismal diversity among the Metazoa is dependent on the proliferation of genes and diversification of functions in multigene families. Here we analyse these processes for one highly successful family, the carboxyl/cholinesterases. One key to the expansion of the functional niche of this group of enzymes is associated with versatile substrate binding and catalytic machinery. Qualitatively new functions can be obtained by substitution of one or a very few amino acids. This crudely adapted new functionality is then refined rapidly by a pulse of change elsewhere in the molecule; in one case about 13% amino acid divergence occurred in 5–10 million years. Furthermore, we postulate that the versatility of the substrate binding motifs underpins the recruitment of several family members to additional noncatalytic signal transduction functions. BioEssays 21:1031–1042, 1999.

Bridging the Synaptic Gap: Neuroligins and Neurexin I in Apis mellifera

Vertebrate studies show neuroligins and neurexins are binding partners in a trans-synaptic cell adhesion complex, implicated in human autism and mental retardation disorders. Here we report a genetic analysis of homologous proteins in the honey bee. As in humans, the honeybee has five large (31–246 kb, up to 12 exons each) neuroligin genes, three of which are tightly clustered. RNA analysis of the neuroligin-3 gene reveals five alternatively spliced transcripts, generated through alternative use of exons encoding the cholinesterase-like domain. Whereas vertebrates have three neurexins the bee has just one gene named neurexin I (400 kb, 28 exons). However alternative isoforms of bee neurexin I are generated by differential use of 12 splice sites, mostly located in regions encoding LNS subdomains. Some of the splice variants of bee neurexin I resemble the vertebrate α- and β-neurexins, albeit in vertebrates these forms are generated by alternative promoters. Novel splicing variations in the 3′ region generate transcripts encoding alternative trans-membrane and PDZ domains. Another 3′ splicing variation predicts soluble neurexin I isoforms. Neurexin I and neuroligin expression was found in brain tissue, with expression present throughout development, and in most cases significantly up-regulated in adults. Transcripts of neurexin I and one neuroligin tested were abundant in mushroom bodies, a higher order processing centre in the bee brain. We show neuroligins and neurexins comprise a highly conserved molecular system with likely similar functional roles in insects as vertebrates, and with scope in the honeybee to generate substantial functional diversity through alternative splicing. Our study provides important prerequisite data for using the bee as a model for vertebrate synaptic development.

The enzymatic basis for pesticide bioremediation.

Enzymes are central to the biology of many pesticides, influencing their modes of action, environmental fates and mechanisms of target species resistance. Since the introduction of synthetic xenobiotic pesticides, enzymes responsible for pesticide turnover have evolved rapidly, in both the target organisms and incidentally exposed biota. Such enzymes are a source of significant biotechnological potential and form the basis of several bioremediation strategies intended to reduce the environmental impacts of pesticide residues. This review describes examples of enzymes possessing the major activities employed in the bioremediation of pesticide residues, and some of the strategies by which they are employed. In addition, several examples of specific achievements in enzyme engineering are considered, highlighting the growing trend in tailoring enzymatic activity to a specific biotechnologically relevant function.

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.

cDNA cloning, baculovirus-expression and kinetic properties of the esterase, E3, involved in organophosphorus resistance in Lucilia cuprina

Resistance to organophosphorus insecticides (OPs) in the sheep blowfly, Lucilia cuprina, is associated with a non-staining phenotype of the carboxylesterase isozyme, E3 (E.C. 3.1.1.1). Here, we show that a member of alpha-esterase multigene family, Lc alpha E7, encodes E3. An Lc alpha E7 cDNA has been isolated from an OP-susceptible strain and expressed in a baculovirus. The expressed product is the same as E3 in its electrophoretic mobility and preference for alpha-over beta-naphthyl acetate as substrate. Its preference (kcat/K(m)) for a range of carboxylester substrates is alpha-naphthyl butyrate > alpha-naphthyl propionate > alpha-naphthyl acetate > methylthiobutyrate > p-nitrophenyl acetate. The enzyme is potently inhibited by OPs (ki [paraoxon] = 6.3 +/- 1.4 x 10(7)/M/min, ki [chlorfenvinphos] = 5.9 +/- 0.6 x 10(7)/M/min) and exhibits a high turnover of methylthiobutyrate (1009/s), consistent with its proposed homology to the ali-esterase that is thought to mutate to confer OP resistance in Musca domestica. E3 shares 64% amino acid identity with its Drosophila melanogaster homologue, Dm alpha E7, and is also closely related to other esterases involved in OP resistance such as the B1 esterase of Culex pipiens (38%) and E4 of Myzus persicae (30%).