Ectopic integration of chromosomal genes in Streptococcus pneumoniae

When a DNA fragment containing a marker gene was ligated to random chromosomal fragments of Streptococcus pneumoniae and used to transform a recipient strain lacking that gene, the gene was integrated at various locations in the chromosome. Such ectopic integration was demonstrated for the malM gene, and its molecular basis was analyzed with defined donor molecules consisting of ligated fragments containing the malM and sul genes of S. pneumoniae. In a recipient strain deleted in the mal region of its chromosome, these constructs gave Mal+ transformants in which the malM and sul genes were now linked, with malM located between duplicate sul segments. Ectopic integration was unstable under nonselective conditions; mal(sul) ectopic insertions were lost at a rate of 0.05% per generation. Several possible mechanisms of ectopic integration were examined. The donor molecule is most likely to be a circular form of ligated homologous and nonhomologous fragments that, after entry into the cell, undergoes circular synapsis with the recipient chromosome at the site of homology, followed by repair and additive integration.