Effect of ethylenediaminetetraacetic acid-Tris(hydroxymethyl)aminomethane on release of the acid-soluble nucleotide pool and on breakdown of ribosomal ribonucleic acid in Escherichia coli

Abstract

Brief treatment of Escherichia coli with 2 x 10(-4)m ethylenediaminetetraacetic acid (EDTA)-0.12 m tris(hydroxymethyl)aminomethane (Tris), pH 8.0, or 0.12 m Tris alone resulted in the release of the acid-soluble nucleotide pool at 3 or 23 C. Exposure to EDTA-Tris for up to 90 min at 3 C did not result in the release of increasing amounts of 260-mmu-absorbing material. At 23 and 37 C, EDTA-Tris resulted in a steady increase in acid-soluble 260-mmu-absorbing material. Previous growth environment did not alter the release. There appeared to be degradation of 23S ribonucleic acid (RNA) after 10 min of exposure at 23 C. In addition, there was degradation of nucleotides to nucleosides and bases. This occured either within the cells with altered permeability or in the periplasmic space. This occurred in the presence of EDTA and Tris but was not seen with EDTA-phosphate. The mechanism of this degradation is unclear, since it occurs in ribonuclease I-deficient strains. Exposure to Tris buffer for long periods of time at 23 C resulted in release of the nucleotide pool and in degradation of RNA and nucleotides. These studies point out that the EDTA-Tris effect on E. coli must be divided into two parts, an early (4 to 5 min) change in permeability and a later phase of actual RNA breakdown and nucleotide degradation. Studies utilizing EDTA and Tris as agents altering permeability must thus be viewed with caution. Although the cells are viable, they have lost their acid-soluble nucleotide pool and have undergone degradation of some ribosomal RNA.