Streptomyces coelicolor A3(2) produces a new yellow pigment associated with the polyketide synthase Cpk

Streptomyces coelicolor A3(2) is an extensively studied model organism for the genetic studies of Streptomycetes - a genus known for the production of a vast number of bioactive compounds and complex regulatory networks controlling morphological differentiation and secondary metabolites production. We present the discovery of a presumptive product of the Cpk polyketide synthase. We have found that on the rich medium without glucose S. coelicolor A3(2) produces a yellow compound secreted into the medium. We have proved by complementation that production of the observed yellow pigment is dependent on cpk gene cluster previously described as cryptic type I polyketide synthase cluster. The pigment production depends on the medium composition, does not occur in the presence of glucose, and requires high density of spore suspension used for inoculation.     

A cryptic type I polyketide synthase (cpk) gene cluster in Streptomyces coelicolor A3(2)

The chromosome of Streptomyces coelicolor A3(2), a model organism for the genus Streptomyces, contains a cryptic type I polyketide synthase (PKS) gene cluster which was revealed when the genome was sequenced. The ca. 54-kb cluster contains three large genes, cpkA, cpkB and cpkC, encoding the PKS subunits. In silico analysis showed that the synthase consists of a loading module, five extension modules and a unique reductase as a terminal domain instead of a typical thioesterase. All acyltransferase domains are specific for a malonyl extender, and have a B-type ketoreductase. Tailoring and regulatory genes were also identified within the gene cluster. Surprisingly, some genes show high similarity to primary metabolite genes not commonly identified in any antibiotic biosynthesis cluster. Using western blot analysis with a PKS subunit (CpkC) antibody, CpkC was shown to be expressed in S. coelicolor at transition phase. Disruption of cpkC gave no obvious phenotype.     

Expression of a functional fungal polyketide synthase in the bacterium Streptomyces coelicolor A3(2).

The multifunctional 6-methylsalicylic acid synthase gene from Penicillium patulum was engineered for regulated expression in Streptomyces coelicolor. Production of significant amounts of 6-methylsalicylic acid by the recombinant strain was proven by nuclear magnetic resonance spectroscopy. These results suggest that it is possible to harness the molecular diversity of eukaryotic polyketide pathways by heterologous expression of biosynthetic genes in an easily manipulated model bacterial host in which prokaryotic aromatic and modular polyketide synthase genes are already expressed and recombined.     

Genetic analysis of A-factor synthesis in Streptomyces coelicolor A3(2) and Streptomyces griseus

A-factor is a potent pleiotropic effector produced by Streptomyces griseus and is essential for streptomycin production and spore formation in this organism. Its production is widely distributed among various actinomycetes including Streptomyces coelicolor A3(2). Genetic analysis of A-factor production was carried out with S. coelicolor A3(2), and two closely linked loci for A-factor mutations (afsA and B) were identified between cysD and leuB on the chromosomal linkage map. In contrast, genetic crosses of A-factor-negative mutants of S. griseus, using a protoplast fusion technique, failed to give a fixed locus for A-factor gene(s) and suggested involvement of an extrachromosomal or transposable genetic element in A-factor synthesis in this organism.     

New Sporulation Loci in Streptomyces coelicolor A3(2)

Sporulation mutants of Streptomyces coelicolor appear white because they are defective in the synthesis of the grey polyketide spore pigment, and such white (whi) mutants had been used to define eight sporulation loci, whiA, whiB, whiD, whiE, whiG, whiH, whiI, and whiJ (K. F. Chater, J. Gen. Microbiol. 72:9-28, 1972; N. J. Ryding, Ph.D. thesis, University of East Anglia, 1995). In an attempt to identify new whi loci, we mutagenized S. coelicolor M145 spores with nitrosoguanidine and identified 770 mutants with colonies ranging from white to medium grey. After excluding unstable strains, we examined the isolates by phase-contrast microscopy and chose 115 whi mutants with clear morphological phenotypes for further study. To exclude mutants representing cloned whi genes, self-transmissible SCP2*-derived plasmids carrying whiA, whiB, whiG, whiH, or whiJ (but not whiD, whiE, or whiI) were introduced into each mutant by conjugation, and strains in which the wild-type phenotype was restored either partially or completely by any of these plasmids were excluded from further analysis. In an attempt to complement some of the remaining 31 whi mutants, an SCP2* library of wild-type S. coelicolor chromosomal DNA was introduced into 19 of the mutants by conjugation. Clones restoring the wild-type phenotype to 12 of the 19 strains were isolated and found to represent five distinct loci, designated whiK, whiL, whiM, whiN, and whiO. Each of the five loci was located on the ordered cosmid library: whiL, whiM, whiN, and whiO occupied positions distinct from previously cloned whi genes; whiK was located on the same cosmid overlap as whiD, but the two loci were shown by complementation to be distinct. The phenotypes resulting from mutations at each of these new loci are described.     

Amplifying sequences in the Streptomyces coelicolor A3 (2) gene

The unstable feature of ristomycin resistance in S. coelicolor A3 (2) was studied. It was shown that the frequency of ristomycin-resistant derivatives was high in both chloramphenicol sensitive mutants and their resistant revertants. The 15- and 20-kb DNA sequences capable of amplifying were detected in the chloramphenicol resistant revertants. In the genomes of the studied strains they were represented by 50 and 40 copies, respectively.     

Functional replacement of genes for individual polyketide synthase components in Streptomyces coelicolor A3(2) by heterologous genes from a different polyketide pathway.

Streptomyces coelicolor A3(2) and Streptomyces violaceoruber Tü22 produce the antibiotics actinorhodin and granaticin, respectively. Both the aglycone of granaticin and the half-molecule of actinorhodin are derived from one acetyl coenzyme A starter unit and seven malonyl coenzyme A extender units via the polyketide pathway to produce benzoisochromane quinone moieties with identical structures (except for the stereochemistry at two chiral centers). In S. coelicolor and S. violaceoruber, the type II polyketide synthase (PKS) is encoded by clusters of five and six genes, respectively. We complemented a series of S. coelicolor mutants (act) defective in different components of the PKS (actI for carbon chain assembly, actIII for ketoreduction, and actVII for cyclization-dehydration) by the corresponding genes (gra) from S. violaceoruber introduced in trans on low-copy-number plasmids. This procedure showed that four of the act PKS components could be replaced by a heterologous gra protein to give a functional PKS. The analysis also served to identify which of three candidate open reading frames (ORFs) in the actI region had been altered in each of a set of 13 actI mutants. It also proved that actI-ORF2 (whose putative protein product shows overall similarity to the beta-ketoacyl synthase encoded by actI-ORF1 but whose function is unclear) is essential for PKS function. Mutations in each of the four complemented act genes (actI-ORF1, actI-ORF2, actIII, and actVII) were cloned and sequenced, revealing a nonsense or frameshift mutation in each mutant.     

Determination of Streptomyces coelicolor A3(2) resistance to erythromycin]

Resistance to erythromycin is genetically unstable in strains of Streptomyces coelicolor A3(2). The frequent loss of resistance as well as reversion of sensitive variants to the original unstable resistance phenotype excluded the possibility that plasmid elimination is involved. The spontaneous frequency of occurrence of sensitive clones was 0.14 to 1.5%, the rate of reversion ranging from 1.10(-6) to 1.10(-8). Resistance to erythromycin has been mapped on the chromosomes of two S. coelicolor A3(2) derivatives in different sites: between markers adeC (v 10) and ArgA1 in the strain A617, between pheA1 and SCP1 in the strain S18. It is suggested that genetic instability of erythromycin resistance determinants having chromosomal location is due to transposition of genetic material.