Genetic and transcriptional analysis of a regulatory region in streptococcal conjugative transposon Tn5252.

In an attempt to increase our understanding of the mechanisms of conjugal transposition among gram-positive bacteria, we analyzed the genetic and structural properties of a 1.2-kb DNA fragment at the left end of the streptococcal conjugative transposon Tn5252. The sequence data revealed four short open reading frames. Polypeptides likely to correspond to two of these genes were identified. Transcriptional start sites and the promoter sequences of three transfer-related genes in the left terminal region of the element were identified. The deduced amino acid sequence of one of these, ORF3, was found to be similar to that of several prokaryotic transcriptional regulator proteins. Insertion mutagenesis at this locus reduced the transfer of the element by three orders of magnitude. The presence of a multicopy plasmid carrying ORF3 in a donor cell carrying Tn5252 with a mutated copy of ORF3 or an unaltered element also reduced the transfer frequency of the element similarly. Gel mobility shift assays showed that the ORF3 protein was capable of binding to not only other discrete sites at the left end of the element but also its own promoter, suggesting autoregulation. These results indicate that the ORF3 protein is involved in the regulation of the conjugative transposition of the element. Copyright 2000 Academic Press.