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Journal of Bacteriology, December 2008, p. 7957-7965, Vol. 190, No. 24
0021-9193/08/$08.00+0     doi:10.1128/JB.00698-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Alanylclavam Biosynthetic Genes Are Clustered Together with One Group of Clavulanic Acid Biosynthetic Genes in Streptomyces clavuligerus{triangledown} ,§

Nathan J. Zelyas, Hui Cai, Thomas Kwong, and Susan E. Jensen*

Department of Biological Sciences, University of Alberta, Edmonton, Alberta T6G 2E9, Canada

Received 16 May 2008/ Accepted 7 October 2008

Streptomyces clavuligerus produces at least five different clavam metabolites, including clavulanic acid and the methionine antimetabolite, alanylclavam. In vitro transposon mutagenesis was used to analyze a 13-kb region upstream of the known paralogue gene cluster. The paralogue cluster includes one group of clavulanic acid biosynthetic genes in S. clavuligerus. Twelve open reading frames (ORFs) were found in this area, and mutants were generated in each using either in vitro transposon or PCR-targeted mutagenesis. Mutants with defects in any of the genes orfA, orfB, orfC, or orfD were unable to produce alanylclavam but could produce all of the other clavams, including clavulanic acid. orfA encodes a predicted hydroxymethyltransferase, orfB encodes a YjgF/YER057c/UK114-family regulatory protein, orfC encodes an aminotransferase, and orfD encodes a dehydratase. All of these types of proteins are normally involved in amino acid metabolism. Mutants in orfC or orfD also accumulated a novel clavam metabolite instead of alanylclavam, and a complemented orfC mutant was able to produce trace amounts of alanylclavam while still producing the novel clavam. Mass spectrometric analyses, together with consideration of the enzymes involved in its production, led to tentative identification of the novel clavam as 8-OH-alanylclavam, an intermediate in the proposed alanylclavam biosynthetic pathway.

* Corresponding author. Mailing address: Department of Biological Sciences, University of Alberta, Edmonton, AB T6G 2E9, Canada. Phone: (780) 492-0830. Fax: (780) 492-9234. E-mail: susan.jensen{at}ualberta.ca

{triangledown} Published ahead of print on 17 October 2008.

§ Supplemental material for this article may be found at http://jb.asm.org/.

Journal of Bacteriology, December 2008, p. 7957-7965, Vol. 190, No. 24
0021-9193/08/$08.00+0     doi:10.1128/JB.00698-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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