The molecular biology of nonactin biosynthesis and resistance in Streptomyces griseus ETH A7796

Plater, Richard William (1991) The molecular biology of nonactin biosynthesis and resistance in Streptomyces griseus ETH A7796. University of Southampton, Doctoral Thesis.

Abstract

The Gl clone, which had been isolated from a lambda library of S.griseus ETH A7796 genomic DNA by its ability to hybridise to the octl probe, was shown to contain adjacent regions of actl and acflll homology; actl and actlll are sequences known to encode enzymes involved in the early steps of actinorhodin biosynthesis by S.coelicolor A(3)2. The Gl clone, therefore, possibly encodes the polyicetide synthase (PKS) for nonactin, a polyketide metabolite of S.griseus. Hybridisation analysis showed that Gl contains only a portion of the unique actl homologous fragment identified within the S.griseus genome at high stringency. The remainder of this actl homologous fragment was contained in four new clones (G2 to G5) isolated in a chromosome walk from the act homologous end of Gl. The clones Gl to G5 represent 31.8kb from the S.griseus genome within which the actl and actlll homologous sequences occupy a central 6.7kb region.

A more detailed analysis used as Southern probes actl, actlll and sequences believed to encode the two subunits of a heterodimeric /?-ketoacyl synthase and an acyl carrier protein (ACP) from the granaticin PKS of S.violaceoruber Tu22. The arrangement of homologies found was strongly suggestive of a PKS including ketoacyl reductase, ACP and /8-ketoacyl synthase components. The presence of the ketoacyl reductase component was confirmed by the complementation of the acllll mutant strain, S.coelicolor TK18. DNA sequencing confirmed the presence of the ACP component and suggested that the cloned PKS has a type II organisation.

A second approach was taken aimed at the cloning of nonactin biosyn-thetic genes. This used the often observed linkage between antibiotic resistance and biosynthetic genes. Tests established that S.lividans is sensitive to 10^g/ml tetranactin, a macrotetrolide related to nonactin and also produced by S.griseus, on media containing 200mM KC1. Consistent with this sensitivity being due to the ionophore action of tetranactin, no inhibition was observed when KC1 was omitted from the medium. A S.griseus genomic library was constructed in the plasmid vector pIJ699 and screened using this assay. Seventeen positive clones were isolated. Restriction analysis and an imperfect resistant phenotype conferred by two of the clones on re-transformation of S.lividans showed these clones to be structurally unstable. The inserts from six of the clones were rescued by subcloning into pBR329. Restriction mapping showed these inserts to represent 9kb of the S.griseus genome with a central 3.4kb common to all six. Southern hybridisation experiments established that all of the sixteen unstable pIJ699 clones contained inserts overlapping this 3.4kb region. Subcloning of the 3.4kb region into the low copy number plasmid vector pIJ943 did not result in stable constructs.

The nucleotide sequence of the 3.4kb region revealed three complete open reading frames (ORFs) and a fourth only partially represented. Sub-cloning allowed the assignment of one of the complete ORFs as that which encodes the resistance determinant. Comparisons with protein sequence databases suggested that the product of this macrotetrolide resistance gene possibly acts by cleaving the ester linkages within nonactin and the other macrotetrolides. The partially sequenced ORF may encode a protein involved in macrotetrolide biosynthesis.

Hybridisation between the entire 9kb represented by the mapped resistance

clone and the lambda Gl to G5 showed that there is no overlap between

these two loci.

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