Cloning and functional studies of the chromosomal replication origin of Actinomadura yumaensis

Abstract:

The chromosomal replication origins (oriC) have been investigated in Gram-positive eubacteria actinomycetes，including Streptomyces，Mycobacterium and Amycolatopsis，and reveal various DnaA-boxes and AT-rich sequences between the conserved dnaA and dnaN genes. Actinomadura yumaensis NRRL12515 is a producer of anthelmintic polyether maduramicin. In this paper，cloning，sequencing and functional studies of its oriC have been carried out. A pair of oligo-nucleotide primers，based on the conserved sequences of dnaA and dnaN，was used to PCR amplification. A～1.3kb DNA band was detected on agarose gel. Subsequently cloning in an E. coli plasmid pBluescriptⅡ SK(+) and sequencing showed 1265bp，which contained 919bp between dnaA and dnaN genes. 14 DnaA-boxes with conserved 9bp sequence (T/C)(T/C)GTCC(A/C)CA and two 13bp AT-rich regions (GAAAAATCCCAAG，AAGAAAAAACTCA)，were found on the sequence，indicating the oriC of A. yumaensis NRRL12515. Phylogenetic trees based on the sequences of oriC and of 16S rRNA genes of the four actinomycetes species show a similar pattern，suggesting that oriC sequences also reflected well the relationship between actinomycetes species. An E. coli plasmid pOR1，containing the oriC，actinomycetes selection markers tsr and melC，was introduced into Streptomyces coelicolor M145 by conjugal transfer. Transformants were obtained，and plasmids DNA were isolated and detected as low copy number，suggesting a functional mini-chromosome in Streptomyces.