ZouA, a putative relaxase, is essential for dna amplification in Streptomyces kanamyceticus.

Previously, we showed that a 145-kb DNA region, including the entire kanamycin biosynthetic gene cluster (with two kanamycin resistance genes), was tandemly amplified up to 36-fold in an industrial strain of Streptomyces kanamyceticus. Strain improvement had included the use of increased kanamycin resistance as an initial potential indicator of higher kanamycin productivity. We were able to recapitulate the DNA amplification by cultivating S. kanamyceticus under selection for kanamycin resistance. To identify the genes required for amplification, various chromosome deletions were constructed, and the DNA amplification was shown to depend on orf1082 (zouA), present in a putative mobile genetic element. ZouA consists of 1,481 amino acids and is homologous to the products of traA-like genes of some conjugative plasmids. These genes encode relaxases that initiate DNA transfer during conjugation by single-strand nicking at oriT. As in the original high-producing strain, DNA amplification occurred between 16-nucleotide (nt) sites (RsA and RsB) containing 14 identical nucleotides. Interestingly, RsA lies just 80 bp upstream of the initiation codon of zouA and is partially contained in an inverted repeat structure similar to those found in plasmid oriT sequences, suggesting that it might function in a manner similar to that of oriT. We therefore propose that DNA amplification in S. kanamyceticus is initiated by relaxase-mediated recombination between oriT-related sequences.