Characterization of an indirect bldA target from Streptomyces coelicolor

Streptomycetes undergo both biochemical (antibiotic production) and morphological differentiation as part of their complex life cycle. These organisms are of great interest due to their ability to produce an extensive array of chemically diverse, commercially important secondary metabolites (which includes most of the antibiotics currently used in human and veterinary medicine). The bld (for bald) mutants of Streptomyces coelicolor are unable to complete the first stage of sporulation which involves the transition from a vegetative, substrate mycelium, non-antibiotic-producing mode of growth to an aerial mycelium, antibiotic-producing state.; The best characterized bld gene is bldA which encodes a leucyl tRNA that recognizes the rare UUA codon in the high G+C (70--74%) Streptomyces mRNA. This TTA codon for leucine is confined to a small number of Streptomyces genes that are predominantly involved in the regulation of morphogenesis and secondary metabolism.; Manipulation of antibiotic production relies on understanding the nature of the regulatory components involved in the induction of genes responsible for antibiotic biosynthesis. In an endeavor to advance current knowledge of the regulatory role that bldA plays in the process of differentiation, attempts were made to identify targets of bldA in Streptomyces coelicolor. bldA targets are TTA-containing genes that require the bldA-encoded tRNA for translation of their mRNAs. Since bldA acts at the level of translation, protein profiles from bldA+ and bldA- strains were compared. To narrow the search for the small number of expected bldA targets, in vitro protein phosphorylation was used. As a result of this analysis, a 32 kDa protein that exhibited a higher level of phosphorylation in surface culture cell-free extracts from a bldA- strain compared to a bldA + strain, was identified. Although the DNA sequence of the gene encoding this protein product was devoid of TTA codons, the apparent bldA-dependent phosphorylation status of this protein suggests it is an indirect bldA-target. Indirect bldA targets can be generally defined as proteins that do not possess TTA codon(s) but are regulated by a TTA-containing regulator. The use of a reverse genetics approach to clone the gene for this target identified a sequence that would encode the alpha (alpha) subunit of the succinyl-CoA synthetase (SCS) of the tricarboxylic acid (TCA) cycle. Western analysis with anti-phosphotyrosine antibodies indicated that the 32 kDa protein possessed phosphotyrosine. Furthermore, phosphoamino acid analysis of this protein confirmed the presence of phosphotyrosine and also demonstrated the presence of phosphoserine and phosphothreonine. Since attempts to construct a null mutation of the gene failed, the role played by this TCA cycle enzyme in the regulation of antibiotic production and morphological differentiation is not yet clear.