Heat shock-mediated transformation in several Gram-negative bacteria such as Ralstonia pseudosolanacearum, Pseudomonas aeruginosa, Pseudomonas putida, and Enterobacter roggenkampii

Abstract

Transformation of foreign DNA, subjecting Escherichia coli to CaCl2 treatment followed by heat shock exposure, is a regular approach in recombinant DNA technology. However, in spite of its large popularity in E. coli, heat shock transformation is rarely reported in other Gram-negative bacteria. Instead, techniques such as natural transformation, conjugation, or electroporation are used in those bacteria. In this study, we have successfully implemented the heat shock transformation in four different Gram-negative bacteria, such as Ralstonia pseudosolanacearum, Pseudomonas aeruginosa, Pseudomonas putida, and Enterobacter roggenkampii. The standard heat shock transformation procedure used for E. coli DH5 has been modified. The pDSK-GFPuv plasmid bearing the green fluorescence gene was directly transferred to Ralstonia pseudosolanacearum by heat shock at 50 {o}C for 60 seconds. For Pseudomonas aeruginosa, Pseudomonas putida, and Enterobacter roggenkampii, the cells were made competent using CaCl2, followed by performing transformation by heat shock at 50 {o}C for 180 seconds. The transformants were resistant to kanamycin as well as exhibited fluorescence. These transformants were used to study the colonization pattern of tomato seedlings. Our study suggested that the heat shock transformation method can also be performed to introduce genes in other Gram-negative bacteria, other than E. coli.