2-D PAGE analysis of pesticide-induced stress proteins of E. coli

Abstract

Logarithmically growing batch cultures of Escherichia coli were exposed to sublethal concentrations of pyrethroid and carbamate pesticides of four different technical grades. This induced 17–20 stress proteins, as observed using two-dimensional polyacrylamide gel electrophoresis. An E. coli culture growing exponentially in Luria Bertani medium (cell density ~2.3×10⁹ cells/ml) was exposed to predetermined sublethal doses of individual pesticides. The cells were harvested after 30 minutes of induction and the stress response was developed in fresh LB medium for three hours under the same growth conditions. Cell pellets were obtained and stored in sonication buffer. Two-dimensional polyacrylamide gel electrophoresis was performed to resolve the proteins. Visualization of the protein spots by rapid silver staining showed 17–20 stress proteins which were absent in the standard protein profile of E. coli. On average 29% of these stress proteins were unique to the pollutant, while the remaining stress proteins overlapped with those of other pesticides. The iso-electric points (PIs) and molecular weights of the proteins were determined by comparing with protein markers with known PIs and molecular weights. Furthermore, upon comparing the pesticide-induced proteins within the same class and between the two different classes (pyrethroid and carbamate), it was apparent that the general nature of the stress remained the same throughout, which indirectly proved that the gene or set of genes responsible for stress expression are also the same, irrespective of the chemical nature of the substituents of the pesticides.