Nucleotide sequence of the kanamycin resistance determinant of the pneumococcal transposon Tn1545: Evolutionary relationships and transcriptional analysis of aphA-3 genes

Summary

The nucleotide sequence of the kanamycin resistance determinant aphA-3 encoded by transposon Tn1545 from Streptococcus pneumoniae was determined and compared to those of plasmids pJH1 and pIP1433 from Streptococcus faecalis and Campylobacter coli, respectively. The three sequences were found to be identical and differed by two substitutions and the deletion of a codon from that of plasmid pSH2 from Staphylococcus aureus. Comparison of the 5′ noncoding sequences indicated that the regions containing the aphA-3 gene in pJH1 and in Tn1545 evolved independently by deletion from a sequence similar to that found in pIP1433. In the latter plasmid, aphA-3 is transcribed from a promoter, P1, which is flanked by two 12-base pair direct repeats. The rearrangement observed in pJH1 removed one of these recombinogenic sites and altered the-10 and 3′ flanking sequences of P1. The promoter thus generated, P1′, allows expression of similar level of kanamycin resistance as P1. However, fusion experiments carried out with a promotorless chloramphenicol acetyltransferase gene indicated that the canonical promoter P1 is significantly less efficient than P1′. From analysis of the thermodynamic properties of these promoters, we conclude that this difference in strength reflects the melting properties of the-10 sequences. The transition from pIP1433 to pJH1 may correspond to the progression of a molecule structurally unstable to a more stable one combined with the need to maintain an efficient promoter upstream of the aphA-3 gene. The deletion event in Tn1545, which occurred between the two 12-base pair directly repeated sequences, removed P1 in its entirety. Transcription of aphA-3 is then initiated from a downstream promoter P2. This rearrangement behaves as a down mutation since P2 is weak, compared to P1 and P1′. It is suggested that P2, which is also present in pIP1433 and in pJH1, is occluded by P1 and P1′, respectively.