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.     

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.     

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.     

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.     

Misaminoacylation and tRNA-dependent transamidation in Streptomyces coelicolor A3(2)

Abstract Streptomyces coelicolor was found to be devoid of glutaminyl-tRNA synthetase. In this bacterium, tRNA^Gln is aminoacylated by glutamyl-tRNA synthetase to yield glutamyl-tRNA^Gln, followed by correction to glutaminyl-tRNA^Gln by a tRNA-dependent amidotransferase.     

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.     

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.     

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.     

Production of actinorhodin by Streptomyces coelicolor A3(2) grown in chemostat culture

Streptomyces coelicolor was grown in variously limited chemostat cultures and the specific rate of extracellular actinorhodin production (q(actinorhodin)) was measured. The highest q(actinorhodin) values were observed in glucose- or ammonia-limited cultures, whereas almost no actinorhodin was produced in sulfate-, phosphate-, potassium-, or magnesium-limited cultures. The effect of the dilution rate on actinorhodin production was studied in glucose-limited cultures. It was found that q(actinorhodin) was highest at D = 0.06h(-1), which was well below the maximal D value tested (0.14 h(-1)). This explains why, in batch cultures, actinorhodin production starts at the onset of the stationary phase. It was also found that the use of nitrilotriacetate instead of citrate as a chelating agent had a negative effect on actinorhodin production. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 577-582, 1997.     

SCP1-dependent Weigle activation of the VP5 phage in Streptomyces coelicolor A3(2)

The kinetics of induction of the UV-irradiated bacteriophage VP5 (Weigle reactivation) in Streptomyces coelicolor A3(2) strains with and without plasmid was investigated. Chloramphenicol (CAF) inhibits Weigle reactivation (WR) in UF strains (SCP1 absent) but not in SCP1+ strains of IF fertility (free plasmid). CAF, moreover, inhibits protein synthesis in non-irradiated UF and IF strains. In UV-irradiated IF strains, on the other hand, protein synthesis takes place irrespective of CAF. Weigle reactivation appears to require protein synthesis: the SCP1 plasmid, by protecting protein synthesis from CAF inhibition in UV-irradiated strains, allows WR. The proteins synthesized after UV induction during the pre-incubation period were investigated and the results suggest that a new UV-induced protein, coded by a gene localized on the plasmid, interacts with the cellular SOS system.     

Organisation of the ribosomal RNA genes in Streptomyces coelicolor A3(2)

Summary Using Southern hybridisation of radiolabelled purified ribosomal RNAs to genomic DNA the ribosomal RNA genes of Streptomyces coelicolor A3(2) were shown to be present in six gene sets. Each gene set contains at least one copy of the 5 S, 16 S and 23 S sequences and in at least two cases these are arranged in the order 16 S-23S-5S. Three gene sets, rrnB, rrnD and rrnF, were isolated by screening a library of S. coelicolor A3(2) DNA. The restriction map of one of these, rrnD, was determined and the nucleotide sequences corresponding to the three rRNAs were localised by Southern hybridisation. The gene order in rrnD is 16S-23S-5S.