Decontamination of vegetables sprayed with organophosphate pesticides by organophosphorus hydrolase and carboxylesterase (B1).

A genetically engineered Escherichia coli cell expressing both organophosphorus hydrolase (OPH) and carboxyl esterase (CaE) B1 intracellularly was constructed and cultivated. The harvested wet cells were vacuum dried, and the storage stability of the dried cell powder was determined in terms of OPH activity. Over a period of 5 mo, the dried cells showed no significant decrease in the activities of the detoxifying enzymes. The crude enzymes in 50 mM citrate-phosphate buffer (pH 8.0) were able to degrade approx 97% of the organophosphate pesticides sprayed on cabbage. The detoxification efficiency was superior to that of the treatments of water, detergent, and a commercially available enzyme product. Additionally, the products of pesticide hydrolysis generated by treatment with the enzyme extract were determined to be virtually nontoxic.

Genetic differentiation of Culex pipiens (Diptera: Culicidae) in China.

The population genetic structures of Culex pipiens Linnaeus were evaluated in China over a 2000 km transect that encompasses the two subspecies, C. p. pallens and C. p. quinquefasciatus. Four polymorphic allozyme loci were investigated in 1376 mosquitoes sampled from 20 populations across four provinces. These loci were not statistically dependent with no apparent heterozygote deficit or excess. On a regional scale (intra-province), a low (Fst=0.007-0.016) and significant genetic differentiation was found, with no clear geographical pattern. On a wider scale (inter-province), the genetic differentiation was higher (Fst=0.059), and an isolation by distance emerged. The results are compared with previous population genetic surveys of this mosquito species in different geographic areas over the world. The overall pattern suggests that Culex pipiens requires considerable distance (500-1000 km) to show isolation by distance, irrespective of the subspecies (C. p. pipiens, C. p. quinquefasciatus and C. p. pallens) or the geographic location.

[Cloning and fusion expression of detoxifying gene in Escherichia coli].

In insects, esterases play an important role in the degradation of organophosphate (OP) and Carbamate (CB) insecticides. The ability of esterase B1 to degrading OP and CB insecticides opens broad prospect of using it in programs to the insecticide pollution. OPs can be detoxified by hydrolysis of their phosphoester bonds, pyrethroids and some OPs like malathion, by hydrolysis of their carboxylester bonds, and diverse carbamate insecticides, herbicides and fungicides by hydrolysis of amide or other similar bonds. Most of the candidate bioremediation enzymes identified to date have been hydrolases. In recent years, we have focused our work on the transfer of a detoxifying esterase B1 gene from mosquito into E. coli and explored the possibility of using the detoxified enzymes in environmental protection. 5' initial of esterase B1 cDNA was cloned by RT-PCR and sequenced consequently. After the combination of 5' initial and 3' terminal fragment of esterase B1 cDNA, the recombinant vector pET-ESTB1 was constructed and transformed into E. coli BL21. A 60 kD protein was induced by IPTG and its expression was temperature-dependent. After 12 h induction, the target protein occupied 27% of the total protein. A pure recombinant protein was obtained by purification, and was detected with 10% SDS-PAGE. The results showed that 22.1% of malathion was degraded by crude detoxification enzyme in 15 mins, demonstrated a high degradation property. This research provides a novel approach, which takes the advantages of eucaryotes for bioremediation of pesticide pollutions.

[Cloning and high level expression of mosquito detoxifying gene].

The full-length cDNA of mosquito esterase B1 had been isolated, and subcloned into pBV220. The recombinant vector pBV220B1 was constructed and transformed into E. coli DH5 alpha. A 60 kD protein was induced by 42 degrees C and its expression was temperature-dependent. After 6 h induction, the target protein occupied 50% of the total protein. The expressed product existed in both inclusion body and soluble proteins in the cells. The amount of the soluble detoxifying enzyme increased along with the induction time. The data of detoxifying experiments indicated that the detoxifying enzyme in expression strain of E. coli can detoxified toxicity of organophosphate insecticides, it showed a clear detoxifying affect on hens poisoned by organophosphate insecticides.

Identification and characterization of novel organophosphate detoxifying esterase alleles in the Guangzhou area of China.

In the mosquito Culex pipiens, various alleles at the Ester locus provide insecticide resistance. These resistance alleles display a heterogeneous geographical distribution, particularly in China, where they are highly diverse. A new resistance allele, Ester9, coding for the overproduced esterases A9 and B9, is characterized and compared to the known resistant allele Ester8 isolated from the same southern China sample (from Guangzhou). Both alleles provide low but significant resistance to chlorpyrifos (relative synergism ratio [RSR] > 3) and temephos (RSR = 1.4), which is consistent with the low level of gene amplification they display (15 copies for Ester9 and 4 copies for Ester8). The full genomic sequence of the allele coding A8 and A9 is presented, which allowed us to set up a polymerase chain reaction assay to specifically identify these alleles. The peculiar situation in southern China, where numerous resistance alleles coexist, is discussed in comparison with the Mediterranean situation, the only one with a similar diversity of overproduced esterases.

Amplified esterases B1 and A2-B2 in field populations of Culex pipiens from China.

The main organophosphate (OP) resistance mechanism in the Culex pipiens complex is increased activity of esterases A and B. Fourth-stage larvae from 2 field populations of C. pipiens from Gaomi and Kunming, China, were compared for tolerance to parathion, dichlorovos (OP), and bassa (carbamate) insecticides. Both populations were resistant to OPs but not to bassa. Starch gel electrophoresis indicated that elevated esterase activity was correlated with OP resistance. High frequencies of amplified esterase genes B1 and A2-B2 (0.85 and 0.50) were discovered in Gaomi and Kunming, respectively. However, only low levels of gene amplification were detected.

[Cloning and expression of the detoxifying gene in cyanobacteria].

Plasmid pRL-B1 was constructed from detoxifying gene(called B1) of pesticide resistant Culex and from plasmid pRL-439 containing the strong promoter PpsbA. E. coli-cyanobacteria shuttle expression plasmid pDC-B1 was constructed from shuttle vector pDC-8 and from recombinant plasmid pRL-B1, then it was transferred into Synechococcus sp. PCC7942 by triparental conjugative transfer. The existence of B1 was detected by Southern analysis, and the expression of B1 was confirmed by enzyme activity analysis of detoxification of transgenic cyanobacteria. Experimental results indicated that the transgenic cyanobacteria could degrade beta-naphthyl acetate(beta-NA), a specific substrate of esterase. The enzyme activity of transgenic strain was higher than that of the wild type. It may be the first report on transformation of detoxify gene of pesticide resistant culex into Synechococcus strain.

New esterase enzymes involved in organophosphate resistance in Culex pipiens (Diptera: Culicidae) from Guang Zhou, China.

Organophosphate (OP) insecticides have been used widely to control Culex pipiens L. populations and this has led to the emergence of OP-resistance. Predominantly, resistance in Cx. pipiens is caused by over-production of nonspecific esterases, such as Est beta 1(1) and Est alpha 2(1)/beta 2(1). These esterases confer multiple resistance to organophosphorus and carbamate insecticides. To define the esterases in Chinese Cx. pipiens, restriction fragment-length polymorphism analysis was performed at the esterase beta locus. A new esterase haplotype (Est beta 8) was found. Starch gel electrophoresis indicated that Est beta 8 was coelevated with a novel Est alpha 8. This article reports Est alpha 8/beta 8 esterase-mediated resistance in Cx. pipiens complex.

A new esterase gene amplification involved in OP resistance in Culex pipiens mosquitoes from China.

Two overproduced esterases (A8 and B8) not previously described were found in southern China. They provide a low resistance level to organophosphate (OP) insecticides, and correspond to a coamplification of both esterase loci (Est-2 and Est-3) classically involved in OP resistance for this mosquito species. This coamplification is distinct from all other similar events thus far reported. The peculiar situation in southern China, where numerous OP resistance alleles at these two loci were found, is discussed in comparison with the Mediterranean situation, the only one with a similar diversity of overproduced esterases.

Esterase polymorphism in insecticide susceptible populations of the mosquito Culex pipiens.

Gene amplification involving a particular haplotype has been found at the esterase B locus of mosquitoes from various countries. This similarity has been explained by a unique amplification event followed by migration and selection by organophosphate (OP) insecticides. This assumes that the polymorphism of non-amplified esterase haplotypes is so large that the chance of independent amplification in two distinct populations is negligible. In order to test this assumption, three susceptible populations from northern Europe were sampled and analysed for esterase and haplotype polymorphism. At the protein level, 18 and 16 alleles were found for esterase A and B respectively in one French population (n = 74), and 16 and 14 in an English one (n = 50). At the DNA level, 24 alleles at the esterase B locus were detected in a sample of 72 mosquitoes from one population, with the use of only one restriction enzyme (EcoR V). Restriction maps of two non-amplified haplotypes randomly sampled from a single breeding site in Belgium were built with six restriction enzymes. 60% of all restriction sites were different among the two maps. The huge polymorphism found in northern Europe requires specific explanations for its stability, but it considerably strengthens the hypothesis of migration of amplified haplotypes.

The same esterase B1 haplotype is amplified in insecticide-resistant mosquitoes of the Culex pipiens complex from the Americas and China.

In Culex pipiens, overproduction of nonspecific esterases is a common mechanism of resistance to organophosphate insecticides. The esterases are attributed to closely linked loci named A and B, and overproduction of all esterases B is due to gene amplification. In order to determine if the esterase B1 identified by electrophoretic studies in Culex pipiens mosquitoes from different countries is overproduced due to the amplification of the same DNA haplotype, the amplified region encompassing the structural esterase B1 gene was characterized by restriction mapping and RFLP. The same amplified haplotype was found in mosquitoes with an esterase B1 protein, independently of their geographical origin: French Guiana, Venezuela, Puerto Rico, California and China. Large variations in amplification levels were observed. It is concluded that B1 amplification has a unique origin, either in America or in Asia, and has subsequently spread by migration. This migration is more limited than that of A2-B2 esterases, since B1 is confined to the Americas, the Caribbean and part of China, whereas the A2-B2 distribution now includes the Americas, the Caribbean, Asia, Africa, the Pacific Islands and Europe.

Molecular and functional properties of EF-Tu from Calderobacterium hydrogenophilum.

Protein synthesis elongation factor Tu (EF-Tu) was purified from an extreme thermophilic hydrogen-oxidizing bacterium Calderobacterium hydrogenophilum. The relative molecular mass of EF-Tu. GDP was 51,000. The factor was heat stable and lost only 50% of its activity after heating at 80 degrees C for 5 min. Native and reduced EF-Tu or EF-Tu. GDP contained one SH-reactive group. The elongation factors from C. hydrogenophilum and E. coli were shown to be immunologically identical. From the Southern hybridization analysis seems to suggest that chromosome DNA of C. hydrogenophilum has two tuf genes.

Elongation factor Tu of the extreme thermophilic hydrogen oxidizing bacterium Calderobacterium hydrogenophilum.

Protein synthesis elongation factor Tu has been purified from an extreme thermophilic hydrogen oxidizing bacterium Calderobacterium hydrogenophilum. The molecular mass of EF-Tu. GDP is 51,000. The factor is heat stable and loses only 50% of its activity after heating for 5 min at 80 degrees C. Under mild conditions trypsin cleaved EF-Tu. GDP to four main fragments. Only one fragment of Mr = 20,000 had a mobility similar to the trypsin fragment "B" of Escherichia coli EF-Tu. Other peptide fragments of E. coli and C. hydrogenophilum EF-Tu differed in size, but native preparations of both factors are immunologically similar.