Overproduction of a P450 that metabolizes diazinon is linked to a loss-of-function in the chromosome 2 ali-esterase (Md alpha E7) gene in resistant houseflies

Up-regulation of detoxifying enzymes in insecticide-resistant strains of the house fly is a common mechanism for metabolic resistance. However, the molecular basis of this increased insecticide metabolism is not well understood. In the multiresistant Rutgers strain, several cytochromes P450 and glutathione S-transferases are constitutively overexpressed at the transcriptional level. Overexpression is the result of transregulation, and a regulatory gene has been located on chromosome 2. A Gly137 to Asp point mutation in alpha E7 esterase gene, leading to the loss of carboxylesterase activity, has been associated with organophosphate resistance in the house fly and the sheep blowfly. We show here that purified recombinant CYP6A1 is able to detoxify diazinon with a high efficiency. We also show that either the Gly137 to Asp point mutation In alpha E7 esterase gene or a deletion at this locus confer resistance and overproduction of the CYP6A1 protein. Based on these findings, we propose it is the absence of the wild-type Gly137 allele of the alpha E7 gene that releases the transcriptional repression of genes coding for detoxification enzymes such as CYP6A1, thereby leading to metabolic resistance to diazinon

Effects of Caste on the Expression of Genes Associated with Septic Injury and Xenobiotic Exposure in the Formosan Subterranean Termite

As social insects, termites live in densely populated colonies with specialized castes under conditions conducive to microbial growth and transmission. Furthermore, termites are exposed to xenobiotics in soil and their lignocellulose diet. Therefore, termites are valuable models for studying gene expression involved in response to septic injury, immunity and detoxification in relation to caste membership. In this study, workers and soldiers of the Formosan subterranean termite, Coptotermes formosanus, were challenged by bacterial injection or by no-choice feeding with a sublethal concentration (0.5%) of phenobarbital. Constitutive and induced expression of six putative immune response genes (two encoding for lectin-like proteins, one for a ficolin-precursor, one for the Down syndrome cell adhesion molecule, one for a chitin binding protein, and one for the gram-negative binding protein 2) and four putative detoxification genes (two encoding for cytochrome P450s, one for glutathione S-transferase, and one for the multi antimicrobial extrusion protein), were measured via quantitative real time polymerase chain reaction and compared within and among 1) colonies, 2) treatment types and 3) castes via ANOVA. Eight genes were inducible by septic injury, feeding with phenobarbital or both. Colony origin had no effect on inducibility or differential gene expression. However, treatment type showed significant effects on the expression of the eight inducible genes. Caste effects on expression levels were significant in five of the eight inducible genes with constitutive and induced expression of most target genes being higher in workers than in soldiers

Microbial Cytochromes P450

The microbial P450s perform an array of oxidative and other chemical reactions that are both crucial for the viability of the bacterial, archaeal, and fungal hosts, and which have numerous important applications. The soluble nature of the bacterial and archaeal P450s has facilitated their expression and purification in high yields, and has enabled the determination of the crystal structures of several important members of the P450 superfamily. Many of the major breakthroughs in our knowledge of the catalytic mechanisms of the P450s have been made through spectroscopic and transient kinetic studies on the microbial P450s, including recent research that has definitively identified the highly reactive P450iron–oxo species compound I and has demonstrated its catalytic potency. This chapter describes our current knowledge on the structural and functional properties of the microbial P450s, including their involvement in pathways for production of industrially important molecules such as antibiotics. Aspects such as engineering of these P450s for novel reaction chemistry are also detailed, along with emerging data for novel P450 enzymes fused to both redox and nonredox partner proteins, and novel systems that bypass the requirement for redox partner proteins altogether. The microbial P450s provide a rich source of catalysts that continue to provide new information on the versatility of the P450s as well as novel activitieswith applications in the biomedical, biofuels, and bioremediation fields. This chapter highlights recent breakthroughs and developments that will ensure that microbial P450s remain center stage for biotechnology applications in the coming years