Transformation In Escherichia coli: Studies On The Role Of The Heat Shock In Induction Of Competence

Abstract

Escherichia coli can be rendered competent for DNA uptake by exposure to a heat shock in the presence of divalent cations. We have studied the influence of variations of the incubation temperature in the competence regimen on the efficiency of competence induction, i.e. the efficiency of uptake of DNA into a DNAase resistant form. For cells grown at 37 "C DNA uptake occurs (1) during a heat shock from 0 "C to temperatures between 15 "C and 42 "C (optimal, 30 "C) and (2) after a heat shock from 0 "C to temperatures between 20 "C and 42 "C (optimal, 32 "C) and a subsequent cold shock to 0 "C. Under the latter conditions DNA uptake occurs during incubation of the transformation mixture at 0 "C after the heat shock. In both cases the efficiency of DNA uptake increases as the incubation temperature during the heat shock increases from about 18 "C to about 32 "C. When recipient cells are grown at 22 "C instead of at 37 "C, the temperature range at which competence induction occurs is shifted by 5 "C to lower temperatures. These results indicate that phase transitions of membrane lipids may play a critical role in induction of competence. When recipient cells are shocked from high temperature to low temperature, leakage of the periplasmic P-lactamase occurs ; the degree of leakage and the efficiency of competence induction are affected similarly by the temperature range of the shock. This observation indicates that phase transition of membrane lipids causes damage to the outer membrane, and that this damage may be essential for induction of competence.

Natural DNA Uptake by Escherichia coli

“…When this DNA recombines and changes the cell's genotype, the cell is said to be transformed. Natural competence has never been directly demonstrated in Escherichia coli , and most transformation instead relies on artificial permeabilisation to bring DNA into cells [2], [3], [4], [5], [6], [7], [8], [9], [10], [11], [12]. Though this DNA can recombine with the host chromosome using the cell's recombination machinery if RecBCD is disabled, higher levels of recombination can be attained by controlled expression of phage recombination proteins (recombineering) [2], [6], [13], [14], [15].…”

Natural Genetic Transformation of Clinical Isolates of Escherichia coli in Urine and Water

“…Laboratory strains of Escherichia coli were shown to be transformable by a nonphysiological Ca 2ϩ concentration of 100 mM (9,22). Recombination-and DNase-deficient laboratory strains are generally used, with a temperature shift from 0 to 37°C after the addition of free DNA (9,22,27). Such conditions are never encountered in the original biotope of E. coli (i.e., the mammalian gastrointestinal and urogenital tracts).…”

A genetically engineered protein domain binding to bacterial murein, archaeal pseudomurein, and fungal chitin cell wall material

“…Primers LR and PF contain homologous nucleotide sequences to the PMB MTH719 domain and the LysM AcmA domain, respectively, to facilitate overlap PCR. Plasmids annealed with DNA fragments of individual constructs of LysM AcmA , PMB MTH719 domains, and the M–P fusion protein were used for transformation of Escherichia coli Rosetta gami 2 (Novagen, Darmstadt, Germany) using the heat shock method (van Die et al 1983). Transformed cells were plated on selective TY 1.5% ( w / v ) agar plates and transformants were checked by colony PCR and plasmid DNA sequencing (ServiceXS, Leiden, the Netherlands) for the correct sequence and right orientation of the desired gene products.…”