The use of a rare codon specifically during development?

A range of circumstantial evidence suggests that in Streptomyces spp., genes required for vegetative growth do not contain the leucine codon TTA. Instead, the codon seems to be confined to a few genes necessary during differentiation, when the colonies begin to produce aerial hyphae and antibiotics. Thus, mutations in bldA, the structural gene for tRNATTALeu, do not retard vegetative growth, but they prevent normal aerial mycelium and antibiotic production. Most of the known TTA-containing genes specify regulatory or resistance proteins associated with antibiotic-production clusters. Possibly the ability to translate the UUA codons in mRNA from such genes is confined to late stages of colony development. Factors that might have contributed to the evolution of this unusual situation are discussed.     

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.     

Changes in the extracellular proteome caused by the absence of the bldA gene product, a developmentally significant tRNA, reveal a new target for the pleiotropic regulator AdpA in Streptomyces coelicolor

The extracellular proteome of Streptomyces coelicolor grown in a liquid medium was analyzed by using two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization-time of flight peptide mass fingerprint analysis. Culture supernatants became protein rich only after rapid growth had been completed, supporting the idea that protein secretion is largely a stationary phase phenomenon. Out of about 600 protein spots observed, 72 were characterized. The products of 47 genes were identified, with only 11 examples predicted to be secreted proteins. Mutation in bldA, previously known to impair the stationary phase processes of antibiotic production and morphological differentiation, also induced changes in the extracellular proteome, revealing even greater pleiotropy in the bldA phenotype than previously known. Four proteins increased in abundance in the bldA mutant, while the products of 11 genes, including four secreted proteins, were severely down-regulated. Although bldA encodes the only tRNA capable of efficiently translating the rare UUA (leucine) codon, none of the latter group of genes contains an in-frame TTA. SCO0762, a serine-protease inhibitor belonging to the Streptomyces subtilisin inhibitor family implicated in differentiation in other streptomycetes, was completely absent from the bldA mutant. This dependence was shown to be mediated via the TTA-containing regulatory gene adpA, also known as bldH, a developmental gene that is responsible for the effects of bldA on differentiation. Mutation of the SCO0762 gene abolished detectable trypsin-protease inhibitory activity but did not result in any obvious morphological defects.     

Novel genes that influence development in Streptomyces coelicolor

Filamentous soil bacteria of the genus Streptomyces carry out complex developmental cycles that result in sporulation and production of numerous secondary metabolites with pharmaceutically important activities. To further characterize the molecular basis of these developmental events, we screened for mutants of Streptomyces coelicolor that exhibit aberrant morphological differentiation and/or secondary metabolite production. On the basis of this screening analysis and the subsequent complementation analysis of the mutants obtained we assigned developmental roles to a gene involved in methionine biosynthesis (metH) and two previously uncharacterized genes (SCO6938 and SCO2525) and we reidentified two previously described developmental genes (bldA and bldM). In contrast to most previously studied genes involved in development, the genes newly identified in the present study all appear to encode biosynthetic enzymes instead of regulatory proteins. The MetH methionine synthase appears to be required for conversion of aerial hyphae into chains of spores, SCO6938 is a probable acyl coenzyme A dehydrogenase that contributes to the proper timing of aerial mycelium formation and antibiotic production, and SCO2525 is a putative methyltransferase that influences various aspects of colony growth and development.     

Spatial and temporal regulation of protein expression by bldA within a Streptomyces lividans colony

The bldA gene encodes the only tRNA for the UUA codon that, although dispensable in genes important for primary vegetative growth of Streptomyces spp., is important in genes that serve a regulatory purpose in the differentiation. To investigate this role further, the spatial and temporal expression profiles of the bldA-regulated and unregulated genes within a Streptomyces colony were examined using modified genes for the green fluorescent protein (gfp) as an expression-tag. A comparative study, based on computer-assisted quantitative analysis of the GFP fluorescence, revealed that the presence of TTA codons in gfp results in a temporal delay of translation and, consequently, changed the spatial pattern of the GFP expression within a colony, especially during early differentiation. The delay of GFP expression was undetectable at 60 h post-inoculation. These results provide the first extensive evidence that the bldA does indeed play a significant regulatory role during colony differentiation.     

Complex extracellular interactions of proteases and a protease inhibitor influence multicellular development of Streptomyces coelicolor

Streptomyces coelicolor produces an extracellular protease inhibitor protein, STI (Streptomyces trypsin inhibitor). We show that post-growth elimination of STI is needed for colonies to develop aerial mycelium efficiently. Inactivation of STI, and thus the normal progression of colony development, at least partly involves an extracellular protease specified by gene SCO5913. Two-hybrid analysis identified two possible targets of STI inhibition (the products of SCO1355 and SCO5447), both extracellular proteases containing a domain homologous with the P-domain of eukaryotic convertases, proteases that mediate the processing of many precursors with important cellular or developmental roles. At least the SCO1355 protease is needed for the normal progression of development. Two components of the proposed cascade are dependent on the tRNA for the rare UUA (leucine) codon, which is specified by the developmental gene bldA. A model is presented that links intracellular regulatory events with an extracellular protease cascade to facilitate normal development.     

Multilevel regulation of Streptomyces differentiation

Streptomyces species are unusual among bacteria in their extensive colony differentiation, which involves the development of spore-bearing aerial hyphae on mycelial colonies. This process, which is usually accompanied by antibiotic production, is controlled by factors as diverse as pheromones, a transfer RNA and a sigma factor dedicated to differentiation, and perhaps by storage compounds and substances related to antibiotics.     

An oligopeptide permease responsible for the import of an extracellular signal governing aerial mycelium formation in Streptomyces coelicolor

Morphological differentiation in the filamentous bacterium Streptomyces coelicolor is believed to involve a mechanism of extracellular signalling that culminates with the formation of an aerial mycelium. We have identified a gene cluster designated bldK in which insertional and deletion mutations cause a block in aerial mycelium formation. Extracellular complementation experiments indicate that bldK defines a step in a cascade of extracellular signals; colonies of a bldK -mutant strain extracellularly complement bld261 -mutant colonies, and are themselves extracellularly complemented by bldA -and bldH -mutant colonies. The bldK locus, which is located at 5 o'clock on the genetic map and within Ase  I fragment 'N' on the physical map, consists of five adjacent open reading frames. These genes specify homologues of the subunits of the oligopeptide-permease family of ATP-binding cassette (ABC) membrane-spanning transporters. Because bldK mutations confer resistance to the toxic tripeptide bialaphos, it is inferred that BldK is an oligopeptide importer. We propose that the BldK transporter is responsible for the import of an extracellular signalling molecule produced under the control of the wild-type product of the bld261 gene. The BldK-imported signal, in turn, causes the production of a second extracellular signal molecule that depends on the products of bldA and bldH for its action.     

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.     

Evolutionary flux of potentially <Emphasis Type="Italic">bldA</Emphasis>-dependent <Emphasis Type="Italic">Streptomyces</Emphasis> genes containing the rare leucine codon TTA

Previous studies have shown that one of the six leucine codons, UUA, is rare in Streptomyces, and that, while the gene for the UUA-specific tRNA, bldA, can generally be inactivated in diverse streptomycetes without impairing vegetative growth, bldA mutants are typically defective in reproductive aerial growth and in antibiotic production. Here, four complete genome sequences and 143 gene clusters for antibiotic biosynthesis from diverse streptomycetes were analysed in order to evaluate the evolution and function of genes whose possession of TTA codons makes them dependent on bldA. It was deduced that the last common ancestor of the four sequenced genomes, possibly 220 million years ago, already possessed the bldA system, together with perhaps 200 TTA-containing target genes. Some 33 of these genes are retained by the modern descendants, though only three of them retain a TTA in all occurrences. Nearly all of these 33, as well as many of the TTA-containing genes with orthologues in two or three of the four genomes, have the same location on the chromosomes as in their common ancestor. However, the majority of TTA-containing genes (61% overall in the four genomes) are species-specific, and were probably acquired by comparatively recent horizontal gene transfer. Most of these genes are of unknown function, and it is likely that many of them confer specialised ecological benefits. On the other hand, one class of species-specific, functionally recognisable, horizontally acquired genes--the gene clusters for antibiotic production--very often contain TTA codons; and nearly half of them have TTA codons in their pathway-specific regulatory genes.     

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.     

Mutations in a new Streptomyces coelicolor locus which globally block antibiotic biosynthesis but not sporulation

Streptomyces coelicolor produces four known antibiotics. To define genetic elements that regulate antibiotic synthesis, we screened for mutations that visibly blocked synthesis of the two pigmented antibiotics and found that the mutant strains which we recovered were of two classes--double mutants and mutants in which all four antibiotics were blocked. The mutations in these multiply blocked strains define a new locus of S. coelicolor which we have named absA. The genetic location of absA, at 10 o'clock, is distinct from the locations of the antibiotic gene clusters and from other known mutations that affect antibiotic synthesis. The phenotype of the absA mutants suggests that all S. coelicolor antibiotic synthesis genes are subject to a common global regulation that is at least in part distinct from sporulation and that absA is a genetic component of the regulatory mechanism.