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Alterations of the structure and activity of the enzyme acetylcholinesterase (AChE) leading to resistance to organophosphate insecticides have been examined in the oriental fruit fly, Bactrocera dorsalis (Hendel), an economic pest of great economic importance in the Asia-Pacific region. We used affinity chromatography to purify AChE isoenzymes from heads of insects from lines showing the phenotypes of resistance and sensitivity to insecticide treatments. The AChE enzyme from a strain selected for resistance to the insecticide fenitrothion shows substantially lower catalytic efficiency for various substrates and 124-, 373- and 5810-fold less sensitivity to inhibition by paraoxon, eserine and fenitroxon, respectively, compared to that of the fenitrothion susceptible line. Using peptide mass fingerprinting, we also show how specific changes in the structure of the AChE enzymes in these lines relate to the resistant and sensitive alleles of the AChE (ace) gene characterized previously in this species (described in Hsu, J.-C., Haymer, D.S., Wu, W.-J., Feng, H.-T., 2006. Mutations in the acetylcholinesterase gene of Bactrocera dorsalis associated with resistance to organophosphorus insecticides. Insect Biochem. Mol. Biol. 36, 396-402). Polyclonal antibodies specific to the purified isoenzymes and real-time PCR were also used to show that both the amount of the isoenzyme present and the expression levels of the ace genes were not significantly different between the R and S lines, indicating that quantitative changes in gene expression were not significantly contributing to the resistance phenotype. Overall, our results support a direct causal relationship between the mutations previously identified in the ace gene of this species and qualitative alterations of the structure and function of the AChE enzyme as the basis for the resistance phenotype. Our results also provide a basis for further comparisons of insecticide resistance phenomena seen in closely related species, such as Bactrocera oleae, as well as in a wide range of more distantly related insect species.

An acetylcholinesterase (AChE, EC 3.1.1.7) was purified from the head of the insecticide susceptible oriental fruit fly, Bactrocera dorsalis (Hendel), by affinity chromatography of Triton X-100 extract. The degree of purification was about 8183-fold with recoveries of 52%. The molecular mass of purified AChE was 116 kDa for its native protein (nonreduced form) and 61 kDa for its subunits (reduced form) as revealed on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), suggesting that the homodimer of AChE linked with disulfide bonds. Nondenaturing PAGE of the purified AChE revealed only one molecular form. The maximum velocities (V(max)) for hydrolyzing acetylthiocholine (ATC), propionylthiocholine, and S-butyrylthiocholine were 833.3, 222.2, and 57.5 micromol/min/mg, and the Michaelis constants (K(m)) were 87.9, 26.9, and 195.3 microM, respectively. More than 97% of AChE activity was inhibited by 10 microM eserine or BW284C51, but only 53% of the activity was inhibited by ethopropazine at the same concentration. On the basis of the substrate and inhibitor specificities, the purified enzyme appeared to be a true AChE. Nevertheless, the purified AChE exhibited some distinctive characteristics including (i) a lack of the substrate inhibition phenomenon when using ATC as the hydrolyzing substrate and (ii) a higher V(m) value for ATC than AChE from other insect species. These biochemical properties may show that AChE purified from the oriental fruit fly may have structural differences from those of other insect species.

In Xanthomonas campestris, the causative agent of black rot in crucifers, the endoglucanase level is greatly decreased in the mutant deficient in Clp, a homologue of cyclic AMP receptor protein (CRP). It is established that Clp has the same DNA binding specificity as CRP at positions 5, 6, and 7 (GTG motif) of the DNA half site. In this study, the engA transcription initiation site was determined by the 5′ RACE method, and two consensus Clp-binding sites, site I and site II centered at −69.5 and −42.5, respectively, were located. Transcriptional fusion assays indicated that Clp greatly activates engA transcription. Site-directed mutagenesis indicated that position 5 of GTG motif in site II is essential for both DNA-protein complex formation in electrophoretic mobility shift assays and engA transcription in vivo. In addition, mutation at position 5 of site I drastically reduces the promoter activity, indicating that binding of Clp to site I exerts a synergistic effect on the transcription activation by site II. engA appears to be the first X. campestris gene known to be activated by Clp via a direct binding to the promoter.