Brian A. M. Rudd

Genetics of actinorhodin biosynthesis by Streptomyces coelicolor A3(2).

A series of 76 mutants of Streptomyces coelicolor A3(2) specifically blocked in the synthesis of the binaphthoquinone antibiotic actinorhodin were classified into seven phenotypic classes on the basis of antibiotic activity, accumulation of pigmented precursors or shunt products of actinorhodin biosynthesis, and cosynthesis of actinorhodin in pairwise combinations of mutants. The polarity of cosynthetic reactions, and other phenotypic properties, allowed six of the mutant classes to be arranged in the most probable linear sequence of biosynthetic blocks. One member of each mutant class was mapped unambigiguously to the chromosomal linkage map in the short segment between the hisD and guaA loci, suggesting that structural genes for actinorhodin biosynthesis may form an uninterrupted cluster of chromosomal genes.

A Pigmented Mycelial Antibiotic in Streptomyces coelicolor : Control by a Chromosomal Gene Cluster

Streptomyces coelicolor was found to produce a third secondary metabolite, in addition to the antibiotics methylenomycin A and actinorhodin previously described. This is a red pigmented, highly non-polar compound with antibiotic activity against certain Gram-positive bacteria. Mutants lacking the red compound fell into five cosynthetic classes. Representatives of each of the five classes were mapped to the chromosome of the producing organism, in a closely linked cluster. Genetic studies provided evidence that this new metabolite is distinct from actinorhodin and indicated that the two pigments do not share parts of the same biosynthetic pathway.

Cloning and expression in Escherichia coli of a Streptomyces coelicolor A3(2) argCJB gene cluster.

From a partial Sau3AI library of Streptomyces coelicolor A3(2) DNA in pIJ916, two hybrid plasmids pGX1 and pGX2 were isolated that complemented S. coelicolor A3(2) or S. lividans arginine auxotrophs. Subcloning DNA from pGX1 in the Escherichia coli expression vector pRK9 containing the Serratia marcescens trp promoter gave rise to one plasmid, pZC2, that complemented E. coli argB, C, E and H auxotrophs, and another, pZC1, that complemented only the first three. The plasmids were markedly unstable in the various complemented hosts, to varying extents; pZC1 was characterized further as providing the stablest host/plasmid combinations. In vitro deletion of part of the vectors trp promoter did not affect complementation of the argB and C auxotrophs, implying that the S. coelicolor A3(2) arg genes may be expressed from their own promoter. The trp promoter-less plasmids included isolates, such as pZC177, that had suffered extensive further deletion without loss of complementing ability. Extracts of an E. coli argE auxotroph carrying pZC177 showed ornithine acetyltransferase activity, indicating that the complementing gene is of the argJ type. The complementation properties of in vitro deletion derivatives of pZC177 indicated the gene order argC-J-B. Part of argC and the upstream region were sequenced; an ORF was identified whose predicted product showed appreciable homology with the E. coli and Bacillus subtilis ArgC polypeptide. Upstream of this ORF a consensus-type promoter and ribosome binding site could be discerned; overlapping its promoter was a sequence with homology to arginine operators in these two other organisms.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasmid effects on secondary metabolite production by a streptomycete synthesizing an anthelmintic macrolide

Transformation of the thermotolerant streptomycete, soil isolate S541, with plasmid cloning vectors of varying size, copy number, and parent replicon (derived from pIJ101, SCP2* and SLP1.2) depressed the biosynthesis of nemadectins (polyketide-derived secondary metabolites possessing anthelmintic activity). However, production of the chemically distinct 21-hydroxyl-oligomycin A, also produced by S541, was either unaffected or increased in plasmid-containing strains. A causal relationship between plasmid carriage and the changes in secondary metabolite yield was confirmed since cured strains were restored to normal production levels and their subsequent retransformation by plasmid DNA was followed by the same effects on nemadectin and oligomycin biosynthesis as before. All the plasmids tested were highly unstable in S541 and it was generally necessary to include an appropriate selective antibiotic (usually thiostrepton) in the growth medium. Thiostrepton was not responsible for the depressive effect, since this was also observed in plasmid-containing strains (i) when grown in antibiotic-free media and (ii) when alternative selective antibiotics such as neomycin were used. In addition, the plasmid-free strain produced both nemadectins and 21-hydroxyl-oligomycin A in the presence of sub-inhibitory levels of thiostrepton. The thiostrepton resistance gene, which was present on many of the plasmids tested, did not mediate the effect since plasmids carrying other selectable markers (pIJ58, neomycin, and pIJ355, viomycin) also depressed nemadectin but not 21-hydroxyl-oligomycin A production. No obvious recombination or integration events between S541 chromosomal DNA and any of the plasmids tested were revealed by DNA-DNA Southern hybridization.

The biosynthesis of nikkomycin X from histidine in Streptomyces tendae

Abstract L-Histidine (4) is the source in Streptomyces tendae for the imidazolone moiety (1) of nikkomycin X (2). It is deduced to be incorporated with stereospecific retention of the β - pro - S proton. Histamine (5), 4-formylimidazole (7) and 4-hydroxymethylimidazole (8) are not significant precursors. Urocanic acid (6) is a less efficient precursor than (4). Aldolase activity is identified in S.tendae which catalyses cleavage of threo -(4-hydroxyphenyl)serine (9) but not significantly of threo - or erythro - β -hydroxyhistidine.