Conditions Affecting Transformation of a Group H Streptococcus

Abstract

A defined transforming medium (DTM) containing buffer and 5 to 10,g per ml of deoxyribonucleic acid was developed to study the physical and chemical requirements for optimal transformation in streptococcal strain SBE. Optimal transformation in DTM occurred at pH 7.5 and 7.0 in 0.07 M sodium phosphate buffer and 0.05 M tris(hydroxymethyl)aminomethane buffer, respectively. In the presence of either a monovalent or a divalent cation, transformation was stimulated maximally by Mn+2 (108 M) and K+ (0.05 M). Other cations tested (Na+, Mg+2, Ca+2) were less stimulatory. A mixture of K+ and Mn+2 stimulated transformation to a level higher than either cation alone. Kinetic studies showed that the stimulating effect of cations was greatest during the early part of the transformation reaction and decreased with time. Transformation was inhibited by CU+2 (105 M) and Mn+2 (102 M). Ethylenediaminetetraacetic acid (EDTA) inhibited transformation at 10-M. The inhibition by EDTA could be overcome by Mn+2 during the early part of the transformation reaction. Genetic transformation of bacteria by deoxyribonucleic acid (DNA) is a multiphasic process involving the attachment of DNA to a microorganism and its subsequent transport into the cell where it is incorporated into the recipient cell genome. Although DNA attachment and uptake by competent cells has been studied extensively, many of these studies were done under poorly defined conditions. Studies in which Bacillus subtilis (16), Haemophilus influenzae (1), and B. licheniformis (8) were used in a defined environment to study DNA attachment and uptake demonstrated that there are very definite conditions necessary for this process to proceed rapidly. Both monovalent and divalent cations, as well as pH, temperature, and inhibitors, affect this phase of transformation. This investigation was conducted to determine the ability of various physical and chemical environments to enhance the transformation of streptococcal strain SBE, and to study the mechanisms involved. A preliminary report of these data was presented (Schlissel and Sword, Bacteriol. Proc., p. 36, 1966). MATERIALS AND METHODS Cultures. The transformable strain of group H streptococcus, SBE, used exclusively in this study was 1357