Actinorhodin production by Streptomyces coelicolor A3(2) in iron-restricted media

Production of the polyketide antibiotic actinorhodin by Streptomyces coelicolor A3(2) was investigated using a defined medium with or without iron supplementation. Iron limitation was found to enhance the intracellular production and export of the pigmented antibiotic. The effect of iron deficiency was particularly pronounced when the bacterium was grown with nitrate instead of ammonium. Analysis of the excreted pigment led to the identification of the lactone form of actinorhodin, gamma-actinorhodin.     

Synthetic RNA Silencing of Actinorhodin Biosynthesis in Streptomyces coelicolor A3(2)

We demonstrate the first application of synthetic RNA gene silencers in Streptomyces coelicolor A3(2). Peptide nucleic acid and expressed antisense RNA silencers successfully inhibited actinorhodin production. Synthetic RNA silencing was target-specific and is a new tool for gene regulation and metabolic engineering studies in Streptomyces.     

Effect of inoculum type on actinorhodin production by Streptomyces coelicolor A3(2)

Summary The production of actinorhodin by Streptomyces coelicolor in a defined medium was examined using spore and vegetative inocula. The spore inoculum yielded higher concentrations of biomass and actinorhodin as well as a higher maximum specific growth rate compared with the vegetative inoculum. Nevertheless, the productivity of the batch culture for actinorhodin formation with vegetative inoculum was higher than that with spore inoculum.     

Streptomyces coelicolor oxidase (SCO2837p): a new free radical metalloenzyme secreted by Streptomyces coelicolor A3(2)

The SCO2837 open-reading frame is located within the conserved central core region of the Streptomyces coelicolor A3(2) genome, which contains genes required for essential cellular functions. SCO2837 protein (SCO2837p) expressed by Pichia pastoris is a copper metalloenzyme, catalyzing the oxidation of simple alcohols to aldehydes and reduction of dioxygen to hydrogen peroxide. Distinct optical absorption spectra are observed for oxidized and one-electron reduced holoenzyme, and a free radical EPR signal is present in the oxidized apoprotein, characteristic of the Tyr-Cys redox cofactor previously reported for fungal secretory radical copper oxidases, galactose oxidase and glyoxal oxidase, with which it shares weak sequence similarity. SCO2837p was detected in the growth medium of both S. coelicolor and a recombinant expression host (Streptomyces lividans TK64) by Western blotting, with the expression level dependent on the nature of the carbon source. This represents the first characterized example of a prokaryotic radical copper oxidase.     

S-Adenosyl-L-methionine Activates Actinorhodin Biosynthesis by Increasing Autophosphorylation of the Ser/Thr Protein Kinase AfsK in Streptomyces coelicolor A3(2)

S-Adenosyl-L-methionine (SAM) is one of the major methyl donors in all living organisms. The exogenous treatment with SAM leads to increased actinorhodin production in Streptomyces coelicolor A3(2). In this study, mutants from different stages of the AfsK-AfsR signal transduction cascade were used to test the possible target of SAM. SAM had no significant effect on actinorhodin production in afsK, afsR, afsS, or actII-open reading frame 4 (ORF4) mutant. This confirms that afsK plays a critical role in delivering the signal generated by exogenous SAM. The afsK-pHJL-KN mutant did not respond to SAM, suggesting the involvement of the C-terminal of AfsK in binding with SAM. SAM increased the in vitro autophosphorylation of kinase AfsK in a dose-dependent manner, and also abolished the effect of decreased actinorhodin production by a Ser/Thr kinase inhibitor, K252a. In sum, our results suggest that SAM activates actinorhodin biosynthesis in S. coelicolor M130 by increasing the phosphorylation of protein kinase AfsK.     

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.     

Hopanoids are formed during transition from substrate to aerial hyphae in Streptomyces coelicolor A3(2)

Streptomyces coelicolor A3(2) contains a cluster of putative isoprenoid and hopanoid biosynthetic genes. The strain does not produce the pentacyclic hopanoids in liquid culture but produces them on solid medium when sporulating. Mutants defective in the formation of aerial mycelium and spores (bld), with the exception of bldB, do not synthesize hopanoids, whereas mutants, which form aerial mycelium but no spores (whi), do. The membrane condensing hopanoids possibly may alleviate stress in aerial mycelium by diminishing water permeability across the membrane.     

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.     

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.