Isolation and nucleotide sequencing of an aminocyclitol acetyltransferase gene from Streptomyces rimosus forma paromomycinus.

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RESUMO

A gene (aacC7) encoding an aminocyclitol 3-N-acetyltransferase type VII [AAC(3)-VII] from Streptomyces rimosus forma paramomycinus NRRL 2455 was cloned in the Streptomyces plasmid pIJ702 and expressed in Streptomyces lividans 1326. Subcloning experiments located the aacC7 structural gene on a 1.05-kilobase DNA sequence. The direction of transcription of aacC7 was determined by using riboprobes synthesized in vitro from a DNA fragment internal to the gene. A DNA segment encoding the AAC(3)-VII activity and comprising 1,495 base pairs was sequenced. The aacC7 gene was located in an open reading frame of 864 base pairs that encoded a polypeptide of Mr 31,070, consistent with the Mr (32,000) of the AAC(3)-VII enzyme as determined by physicochemical methods. High-resolution S1 nuclease mapping suggested that transcription starts at or near the A residue of the ATG initiator codon. A DNA fragment from the 5' region of aacC7 had promoter activity in the promoter-probe plasmid pIJ486. The -10 and -35 regions of this fragment showed limited sequence resemblance to other Streptomyces promoters. The primary structure of the AAC(3)-VII enzyme showed strong homology with those of the AAC(3)-III and AAC(3)-IV enzymes encoded by plasmids in gram-negative bacterial genera. Upstream of the aacC7 gene was an open reading frame of 357 nucleotides which did not appear to be involved in controlling the expression of the aacC7 gene.

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