Genetic characteristics and genome structure of Streptomyces coelicolor actinophages]

Actinophage phi C31 of Streptomyces coelicolor A3 (2) and two novel temperate actinophages phi C43 and phi C62 isolated from strains of blue actinomycetes group are homoimmune, serologically and functionally related. DNA molecules of phages phi C31, phi C43 and phi C62 have cohesive ends; sizes of DNAs of these phages and some mutants have been determined. The extent of homology between the DNAs of three phages is 93-96% as shown by heteroduplex analysis. The regions of non-homology are of a deletion-insertion type and of approximately 1500 base pairs in the length. Location of deletions in DNAs of mutant phages phi C31 vd and phi C31 c5 has been shown. Structural modifications in phage dnas have been found only to occur in the right part of molecules. Heteroduplex maps have been constructed for all phages studied.     

Genetic Analysis and Genome Structure in Streptomyces coelicolor

[This corrects the article on p. 388 in vol. 31.].     

Medium-dependent phenotypes of Streptomyces coelicolor with mutations in ftsI or ftsW

Streptomyces coelicolor A3(2) ftsI- and ftsW-null mutants produced aerial hyphae with no evidence of septation when grown on a traditional osmotically enhanced medium. This phenotype was partially suppressed when cultures were grown on media prepared without sucrose. We infer that functional FtsZ rings can form in ftsI- and ftsW-null mutants under certain growth conditions.     

Identification and characterization of CdgB, a diguanylate cyclase involved in developmental processes in Streptomyces coelicolor

We describe the identification and functional characterization of cdgB (SCO4281), a recently discovered target of BldD, a key regulator of morphological differentiation and antibiotic production in the mycelial bacteria of the genus Streptomyces. cdgB (cyclic dimeric GMP [c-di-GMP] B) encodes a GGDEF-containing protein that has diguanylate cyclase activity in vitro. Consistent with this enzymatic activity, heterologous expression of cdgB in Escherichia coli resulted in increased production of extracellular matrix in colonies and enhanced surface attachment of cells in standing liquid cultures. In Streptomyces coelicolor, both overexpression and deletion of cdgB inhibited aerial-mycelium formation, and overexpression also inhibited production of the antibiotic actinorhodin, implicating c-di-GMP in the regulation of developmental processes in Streptomyces.     

Role of phosphopantetheinyl transferase genes in antibiotic production by Streptomyces coelicolor

The phosphopantetheinyl transferase genes SCO5883 (redU) and SCO6673 were disrupted in Streptomyces coelicolor. The redU mutants did not synthesize undecylprodigiosin, while SCO6673 mutants failed to produce calcium-dependent antibiotic. Neither gene was essential for actinorhodin production or morphological development in S. coelicolor, although their mutation could influence these processes.     

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.     

Identification of Streptomyces coelicolor genes involved in regulation of antibiotic synthesis.

To define genetic elements that regulate antibiotic synthesis, we screened for mutations that visibly blocked synthesis of Streptomyces coelicolor's two pigmented antibiotics and found mutant strains in which all four antibiotics were blocked. The responsible mutations defined two loci, absA and absB. Two additional approaches to defining genes have been taken: isolation of cloned genes with a dominant negative effect on antibiotic synthesis and isolation of genes which, in multicopy, can compensate for specific mutational blocks. These genes apparently function in a global regulatory pathway (or network) for control of antibiotic synthesis.     

Sugar uptake and utilisation in Streptomyces coelicolor: a PTS view to the genome

Our research group is studying the phosphotransferase system (PTS) of Streptomyces coelicolor, which, in other bacteria, is centrally involved in carbon source uptake and regulation. We have surveyed the public available S. coelicolor genome sequence produced by the ongoing genome sequencing project for pts gene homologues (http://www.sanger.ac.uk/Projects/S_coelicolor/). Three genes encoding homologues of the general PTS components enzyme I (ptsI), HPr (ptsH), and enzyme IIA^Crr (crr; IIA^Glc-homologue) and six genes encoding homologues of sugar-specific PTS components were identified. The deduced primary sequences of the sugar-specific components shared significant similarities to PTS permeases of the mannitol/fructose family and of the glucose/sucrose family. A model is presented, in which possible functions of the novel described PTS homologues are discussed.     

High-multiplicity of chitinase genes in Streptomyces coelicolor A3(2).

Six different genes for chitinase from ordered cosmids of the chromosome of Streptomyces coelicolor A3(2) were identified by hybridization, using the chitinase genes from other Streptomyces spp. as probes, and cloned. The genes were sequenced and analyzed. The genes, together with an additional chitinase gene obtained from the data bank, can be classified into either family 18 or family 19 of the glycosyl hydrolase classification. The five chitinases that fall into family 18 show diversity in their multiple domain structures as well as in the amino acid sequences of their catalytic domains. The remaining two chitinases are members of family 19 chitinases, since their C-terminus shares more than 70% identity with the catalytic domain of ChiC of Streptomyces griseus, the sole gene for family 19 chitinase so far found in an organism other than higher plants.     

Genetic mapping, cloning and physiological aspects of the glucose kinase gene of Streptomyces coelicolor

Summary Glucose kinase in Streptomyces coelicolor has a molecular weight of about 110,000. In crude extracts, the enzyme exhibited apparent K_m values of 0.20 mM for ATP, 0.27 mM for glucose, and 2.2 mM for the glucose analogue 2-deoxyglucose. Mutations (glk) to 2-deoxyglucose-resistance, which greatly reduce glucose kinase activity and result in relief of glucose repression of utilisation of various carbon sources, were mapped between proA and hisA in the S. coelicolor linkage map. Glucose kinase activity, 2-deoxyglucose-sensitivity, glucose utilisation and glucose repression, were all restored to glk mutants by a 3.5 kb DNA fragment cloned from S. coelicolor into a phage vector (C31 KC515), and by larger (10–30 kb) fragments cloned into a low copy number plasmid vector (pIJ916). The glk gene was further localised to a 2.9 kb BclI fragment of the cloned DNA by sub-cloning. Part or all of this fragment was present in each of five primary plasmid clones tested.     

Genetic analysis of absB, a Streptomyces coelicolor locus involved in global antibiotic regulation.

The filamentous soil bacterium Streptomyces coelicolor is known to produce four antibiotics which are genetically and structurally distinct. An extensive search for antibiotic regulatory mutants led to the discovery of absB mutants, which are antibiotic deficient but sporulation proficient. Genetic analysis of the absB mutants has resulted in definition of the absB locus at 5 o'clock on the genetic map. Multiple cloned copies of the actII-ORF4 gene, an activator of synthesis of the antibiotic actinorhodin, restore actinorhodin biosynthetic capability to the absB mutants. These results are interpreted to mean that the failure of absB mutants to produce antibiotics results from decreased expression of the antibiotic genes. The absB gene is proposed to be involved in global regulation of antibiotic synthesis.     

Whole genome analysis of non-optimal codon usage in secretory signal sequences of Streptomyces coelicolor

Non-optimal (rare) codons have been suggested to reduce translation rate and facilitate secretion in Escherichia coli. In this study, the complete genome analysis of non-optimal codon usage in secretory signal sequences and non-secretory sequences of Streptomyces coelicolor was performed. The result showed that there was a higher proportion of non-optimal codons in secretory signal sequences than in non-secretory sequences. The increased tendency was more obvious when tested with the experimental data of secretory proteins from proteomics analysis. Some non-optimal codons for Arg (AGA, CGU and CGA), Ile (AUA) and Lys (AAA) were significantly over presented in the secretary signal sequences. It may reveal that a balanced non-optimal codon usage was necessary for protein secretion and expression in Streptomyces.     

Overexpression of the diguanylate cyclase CdgD blocks developmental transitions and antibiotic biosynthesis in Streptomyces coelicolor

Cyclic dimeric GMP(c-di-GMP) has emerged as the nucleotide second messenger regulating both development and antibiotic production in high-GC, Gram-positive streptomycetes. Here, a diguanylate cyclase(DGC), CdgD, encoded by SCO5345 from the model strain Streptomyces coelicolor, was functionally identified and characterized to be involved in c-di-GMP synthesis through genetic and biochemical analysis. cdgD overexpression resulted in significantly reduced production of actinorhodin and undecylprodigiosin, as well as completely blocked sporulation or aerial mycelium formation on two different solid media. In the cdgD-overexpression strain, intracellular c-di-GMP levels were 13-27-fold higher than those in the wild-type strain. In vitro enzymatic assay demonstrated that CdgD acts as a DGC, which could efficiently catalyze the synthesis of c-di-GMP from two GTP molecules. Heterologous overproduction of cdgD in two industrial Streptomyces strains could similarly impair developmental transitions as well as antibiotic biosynthesis. Collectively, our results combined with previously reported data clearly demonstrated that c-di-GMP-mediated signalling pathway plays a central and universal role in the life cycle as well as secondary metabolism in streptomycetes.