A missense mutation in ftsZ differentially affects vegetative and developmentally controlled cell division in Streptomyces coelicolor A3(2)

Streptomyces coelicolor A3(2) undergoes at least two kinds of cell division: vegetative septation leading to cross-walls in the substrate mycelium; and developmentally regulated sporulation septation in aerial hyphae. By isolation and characterization of a non-sporulating ftsZ mutant, we demonstrate a difference between the two types of septation. The ftsZ17(Spo) allele gave rise to a classical white phenotype. The mutant grew as well as the parent on plates, and formed apparently normal hyphal cross-walls, although with a small reduction in frequency. In contrast, sporulation septation was almost completely abolished, resulting in a phenotype reminiscent of whiH and ftsZdelta2p mutants. The ftsZ17(Spo) allele was partially dominant and had no detectable effect on the cellular FtsZ content. As judged from both immunofluorescence microscopy of FtsZ and translational fusion of ftsZ to egfp, the mutation prevented correct temporal and spatial assembly of Z rings in sporulating hyphae. Homology modelling of S. coelicolor FtsZ indicated that the mutation, an A249T change in the C-terminal domain, would be expected to alter the protein on the lateral face of FtsZ protofilaments. The results suggest that cytokinesis may be developmentally controlled at the level of Z-ring assembly during sporulation of S. coelicolor A3(2).     

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.     

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.     

Spore colour in Streptomyces coelicolor A3(2) involves the developmentally regulated synthesis of a compound biosynthetically related to polyketide antibiotics

Streptomyces coelicolor produces spores whose development of a grey colour requires the activity of the whiE locus. The cloned whiE locus was identified after mobilization into a whiE mutant of a library of S. coelicolor DNA inserted into a transmissible plasmid vector. The whiE region of the cloned DNA was localized both by subcloning and by mutagenesis of the cloned DNA with the Streptomyces transposon Tn4560. Nucleotide sequencing of this region revealed seven open reading frames, of which six show homology at the level of deduced gene products with genes involved in the synthesis of polyketide antibiotics. A previously described S. coelicolor DNA segment encoding biosynthesis of a brown pigment (Horinouchi and Beppu, 1985) corresponds to the cloned whiE DNA. It is proposed that whiE is normally expressed only in the aerial hyphae, where the biosynthetic product is responsible for spore colour.     

CDA is a new chromosomally-determined antibiotic from Streptomyces coelicolor A3(2)

Mutations (cda) leading to non-production of the new calcium-dependent antibiotic (CDA) of Streptomyces coelicolor A3(2) were closely linked on the chromosome. One representative mutation (cda-1) was mapped precisely between nicA and adeC. No cosynthesis of CDA was found in any pairwise combinations of 14 cda mutants. Mutations lacking aerial mycelium (bald mutations), mapping to the four previously described loci (bldA-D), were pleiotropically defective in production of CDA.     

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.