New loci required for Streptomyces coelicolor morphological and physiological differentiation.

Streptomyces coelicolor colonies differentiate both morphologically, producing aerial spore chains, and physiologically, producing antibiotics as secondary metabolites. Single mutations, which block both aspects of differentiation, define bld (bald colony) genes. To identify new bld genes, mutagenized colonies were screened for blocks in the earliest stage of sporulation, the formation of aerial mycelia, and blocks in antibiotic synthesis. The mutations in 12 mutants were mapped; in each strain, the pleiotropic phenotype was due to a single mutation. Seven of the strains contained mutations in known bld loci, bldA and bldB. Three strains contained mutations in a new locus, bldG, and two contained mutations in another new locus, bldH. Like the previously defined bldA mutants, the bldG and bldH mutants were developmentally blocked on glucose. On a variety of carbon sources whose utilization was subject to glucose repression, the developmental blocks were partially relieved for bldG (and bldA) mutants and fully relieved for bldH mutants. These results are compatible with an hypothesis which suggests that there are two alternative controls on S. coelicolor differentiation, one of which is glucose repressible.     

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.     

The Streptomyces coelicolor A3(2) bldB region contains at least two genes involved in morphological development

Streptomyces coelicolor A3(2) bldB mutants are blocked in the formation of aerial hyphae. A phage library of wild-type S. coelicolor DNA was used to isolate recombinant phages which restore wild-type morphological development to several bldB mutants. Of several mutations, one, bld-28, previously mapped at bldB was not complemented by the cloned region, indicating that the bldB locus is composed of at least two distinct genes. Partial localization of bldB-complementing activity showed that a 1.5 kb fragment is sufficient for complementation of the bld-15 mutation whereas bld-17 requires the same region as well as additional sequences. Under stringent conditions, genomic DNA hybridizing to the cloned sequences was absent from other Streptomyces species, including the closely related Streptomyces lividans 66. DNA sequences causing marked plasmid structural instability in S. coelicolor, but not in S. lividans, are also located in this region.     

Changes in patterns of ADP-ribosylated proteins during differentiation of Streptomyces coelicolor A3(2) and its development mutants.

Mutants resistant to 3-aminobenzamide, a known inhibitor of ADP-ribosyltransferase, were obtained from Streptomyces coelicolor A3(2). One (strain 27) was analyzed in detail. Mutant 27 had a reduced ADP-ribosyl-transferase activity, exhibited substantial changes from the wild type in ADP-ribosylated protein profile during cell aging, and was defective in producing aerial mycelium and antibiotics. A 92-kDa ADP-ribosylated protein disappeared at the onset of differentiation in the parent strain but was present in mutant 27. Four ADP-ribosylated proteins (39, 41, 43, and 46 kDa) appeared at the onset of differentiation in the parent strain but were missing in mutant 27. Failure to ADP-ribosylate these four proteins was detected when the parent strain was grown in the presence of subinhibitory amounts of 3-aminobenzamide. Genetic analysis showed that the mutation, named brgA, conferring resistance to 3-aminobenzamide, cosegregated with the altered phenotypes (i.e., defects in ADP-ribosylation and aerial mycelium formation) and was mapped to a new locus near uraA. The brgA mutants were nonconditionally deficient in producing aerial mycelium and antibiotics, as determined by using various media, and had a morphological and physiological phenotype quite different from that of a bldG mutant carrying a mutation which was previously mapped near uraA. Among the known bld mutants, bldA, bldD, and bldG mutants exhibited a ADP-ribosylated protein profile similar to that of the wild type, while like mutant 27, bldB, bldC, and bldH mutants failed to ADP-ribosylate certain proteins.     

A morphological and genetic mapping study of bald colony mutants of Streptomyces coelicolor.

Twelve bld mutations of Streptomyces coelicolor resulting in a lack of visible aerial mycelium were mapped genetically. The mutants were classified into three groups on the basis of colony morphology, production of antibiotics and morphology on different carbon sources. Four map locations were found for the bld genes and three of these were very near the loci of whi genes, which are also involved in differentiation. Closely linked bld mutations had similar phenotypes.     

Critical residues and novel effects of overexpression of the Streptomyces coelicolor developmental protein BldB: evidence for a critical interacting partner

The bldB gene of Streptomyces coelicolor encodes the best-characterized member of a family of small proteins that have low isoelectric points but that lack any previously characterized sequence motifs. BldB is dimeric and is required for the efficient production of antibiotics and spore-forming cells, called aerial hyphae, by growing colonies. The mechanism of action of BldB and its relatives is unknown. Here, we have explored amino acids in BldB that either are highly conserved or have been implicated in function genetically. We show that five amino acids are important for its function at physiological expression levels. Mutations in three of these amino acids gave rise to proteins that were either monomeric or unstable in vivo, while two others are not. We find that overexpression of bldB in S. coelicolor blocks sporulation prior to sporulation-specific septation but permits the formation of aerial hyphae. Vegetative septation was apparently normal in both the bldB null mutant and the bldB overexpression strain. To our surprise, overexpression of the dimerization-competent but functionally defective alleles caused a dramatic acceleration of sporulation. Our results suggest that BldB makes at least one important contact with another subcellular constituent and that a loss or alteration of this interaction impairs the phenotypic properties of the organism.     

Accumulation of S-adenosylmethionine induced oligopeptide transporters including BldK to regulate differentiation events in Streptomyces coelicolor M145

S-Adenosylmethionine (SAM), the major methyl donor in diverse biological processes, was recently found to be involved in the regulation of differentiation in streptomycetes. Exogenous SAM, in a quantity as low as 2muM, enhanced antibiotic production and inhibited morphological development of Streptomyces coelicolor M145. Total protein profiling of S. coelicolor M145 revealed that SAM enhanced the expression of oligopeptide-binding components related to ABC transporters that included BldK, a well-known regulatory factor in S. coelicolor differentiation. A radiolabeled SAM feeding experiment verified that exogenous SAM can be imported into the cell, which is under the control of the bld cascade. This study substantiated that BldK serves as a transducer of the SAM signal and uncovered the possible role of oligopeptide import in the regulation of Streptomyces differentiation, particularly in relation to SAM.     

Differential stringent responses of Streptomyces coelicolor M600 to starvation of specific nutrients

This study focused on the involvement of the unusual nucleotide (p)ppGpp, a stringent factor, during the morphological and physiological differentiation of Streptomyces coelicolor. Two genes, relA and rshA, were disrupted to demonstrate the roles of the stringent factor in the differentiation. The intracellular concentration of (p)ppGpp in the wild-type (M600) and disrupted mutants was measured in relation to the intentional starvation of a specific nutrient such as carbon, nitrogen, and phosphate or the in situ depletion of nutrients in a batch culture. As a result, it was found that the morphological characteristic of the deltarelA mutant was a bld phenotype forming condensed mycelia, whereas the deltarshA mutant grew fast-forming spores and straightforward mycelia. In both mutants, the production of actinorhodin (Act) was completely abolished, yet the undecylprodigiosin (Red) production was increased. Intracellular (p)ppGpp was detected in the deltarelA mutant in the case of limited phosphate, yet not with limited carbon or nitrogen sources. In contrast, (p)ppGpp was produced in the deltarshA mutant under limited carbon and nitrogen conditions. Therefore, (p)ppGpp in S. coelicolor was found to be selectively regulated by either the RelA or RshA protein, which was differentially expressed in response to the specific nutrient limitation. These results were also supported by the in situ ppGpp production during a batch culture. Furthermore, it is suggested that RelA and RshA are bifunctional proteins that possess the ability to both synthesize and hydrolyze (p)ppGpp.     

Tn4563 transposition in Streptomyces coelicolor and its application to isolation of new morphological mutants.

The Tn3-like transposon Tn4556 (and its derivatives Tn4560 and Tn4563) has been used for insertion mapping of genetic loci cloned on plasmids, but it has been difficult to obtain chromosomal insertions, largely because of the lack of a strong selection against transposon donor molecules. In this communication, we report two efficient selection techniques for transposition and their use in the isolation of chromosomal insertion mutations. A number of independent Streptomyces coelicolor morphological mutants (bld and whi) were obtained. Two of the bld mutations were mapped to locations on the chromosome by SCP1-mediated conjugation; at least one mutation, bld-5m1, appears to define a novel locus involved in control of S. coelicolor morphogenesis and antibiotic production.     

A Streptomyces coelicolor Antibiotic Regulatory Gene, absB, Encodes an RNase III Homolog

Streptomyces coelicolor produces four genetically and structurally distinct antibiotics in a growth-phase-dependent manner. S. coelicolor mutants globally deficient in antibiotic production (Abs(-) phenotype) have previously been isolated, and some of these were found to define the absB locus. In this study, we isolated absB-complementing DNA and show that it encodes the S. coelicolor homolog of RNase III (rnc). Several lines of evidence indicate that the absB mutant global defect in antibiotic synthesis is due to a deficiency in RNase III. In marker exchange experiments, the S. coelicolor rnc gene rescued absB mutants, restoring antibiotic production. Sequencing the DNA of absB mutants confirmed that the absB mutations lay in the rnc open reading frame. Constructed disruptions of rnc in both S. coelicolor 1501 and Streptomyces lividans 1326 caused an Abs(-) phenotype. An absB mutation caused accumulation of 30S rRNA precursors, as had previously been reported for E. coli rnc mutants. The absB gene is widely conserved in streptomycetes. We speculate on why an RNase III deficiency could globally affect the synthesis of antibiotics.     

Structural and genetic analysis of the BldB protein of Streptomyces coelicolor

We have demonstrated that the bldB gene of Streptomyces coelicolor is required for the formation of aerial hyphae and the synthesis of antibiotics. We also found that BldB forms a higher-order complex (most likely a dimer) and that amino acid residues 20 to 78 are important for this interaction. This region is conserved in the BldB family, suggesting that dimer formation may be a common feature of these proteins.     

Identification and cloning of a umu locus in Streptomyces coelicolor A3(2)

The umuDC operon of Escherichia coli is required for efficient mutagenesis by UV and many other DNA-damaging agents. E. coli umu mutants are defective in mutagenesis and slightly more sensitive to DNA-damaging agents. The existence of a umuDC analogue in Streptomyces coelicolor was suggested by data of our previous works. We cloned from Streptomyces coelicolor a fragment of DNA homologous to the E. coli umuDC region that is able to complement the E coli umuC122::Tn5 mutation. Therefore our data suggest that S. coelicolor contains a functional umu-like operon.