Cloning of Streptococcus pneumoniae DNA: Its use in pneumococcal transformation and in studies of mismatch repair

Abstract

EcoRI fragments of the amiA locus in Streptococcus pneumoniae were cloned either into a derivative of X or into pBR325 plasmid. Mutations in the amiA locus confer resistance to aminopterin. Pneumococcal DNA fractions were enriched for the desired EcoKI fragments by agarose gel electrophoresis. Recombinant clones were detected directly by transformation with DNA from X plaques or from single-colony lysates containing pBR325. The use of cloned DNA in pneumococcal transformation has revealed a number of features pertinent to transformation in general, and also to the mismatch repair process. High transformation levels can be achieved, from 40 to 80% of a competent culture. These high levels of transformation with cloned DNA made in a foreign host are taken to confirm the absence of restriction effects on transformation in S. pneumoniae. At saturation, similar transformation levels are obtained with hybrid phage or hybrid plasmid DNAs, but the DNA amount required is 20 to 25 times lower for hybrid plasmid than for hybrid phage, probably because plasmid DNA is 10 times shorter than phage DNA. There is no “end effect” with intact hybrid DNA i.e. similar transformation levels are achieved for markers whatever their map position on the cloned pneumococcal fragment. Cloned DNA has been used to study the action of the mismatch repair process ( hex system). The presence of two mismatches in the same cell is not enough to saturate the hex system, and is not enough to kill the colony-forming center (cfc).