Persistence of detectable insecticidal proteins from Bacillus thuringiensis (Cry) and toxicity after adsorption on contrasting soils.

Insecticidal Cry, or Bt, proteins are produced by the soil-endemic bacterium, Bacillus thuringiensis and some genetically modified crops. Their environmental fate depends on interactions with soil. Little is known about the toxicity of adsorbed proteins and the change in toxicity over time. We incubated Cry1Ac and Cry2A in contrasting soils subjected to different treatments to inhibit microbial activity. The toxin was chemically extracted and immunoassayed. Manduca sexta was the target insect for biotests. Extractable toxin decreased during incubation for up to four weeks. Toxicity of Cry1Ac was maintained in the adsorbed state, but lost after 2 weeks incubation at 25 °C. The decline in extractable protein and toxicity were much slower at 4 °C with no significant effect of soil sterilization. The major driving force for decline may be time-dependent fixation of adsorbed protein, leading to a decrease in the extraction yield in vitro, paralleled by decreasing solubilisation in the larval gut.

Cloning of two pectate lyase genes from the marine Antarctic bacterium Pseudoalteromonas haloplanktis strain ANT/505 and characterization of the enzymes.

A marine Antarctic psychrotolerant bacterium (strain ANT/505), isolated from sea ice-covered surface water from the Southern Ocean, showed pectinolytic activity on citrus pectin agar. The sequencing of the 16S rRNA of isolate ANT/505 indicates a taxonomic affiliation to Pseudoalteromonas haloplanktis. The supernatant of this strain showed three different pectinolytic activities after growth on citrus pectin. By activity screening of a genomic DNA library of isolate ANT/505 in Escherichia coli, two different pectinolytic clones could be isolated. Subcloning and sequencing revealed two open reading frames (ORF) of 1,671 and 1,968 nt, corresponding to proteins of 68 and 75 kDa, respectively. The deduced amino acid sequence of the two ORFs showed homology to pectate lyases from Erwinia chrysanthemi and Aspergillus nidulans. The pectate lyases contain signal peptides of 17 and 26 amino acids that were correctly processed after overexpression in E. coli BL21. Both enzymes were purified by anionic exchange chromatography. Maximal enzymatic activities for both pectate lyases were observed at 30 degrees C and a pH range of 9 to 10. The Km values of both lyases for pectate and citrus pectin were 1 g l(-1) and 5 g l(-1), respectively. Calcium was required for activity on pectic substrates, whereas the addition of 1 mM ethylenediaminetetraacetic acid (EDTA) resulted in complete inhibition of the enzymes. These two enzymes represent the first pectate lyases isolated and characterized from a cold-adapted marine bacterium.