Different alleles of the response regulator gene bldM arrest Streptomyces coelicolor development at distinct stages

whiK was one of five new whi loci identified in a recent screen of NTG-induced whi mutants and was defined by three mutants, R273, R318 and R655. R273 and R318 produce long, tightly coiled aerial hyphae with frequent septation. In contrast, R655 shows a more severe phenotype; it produces straight, undifferentiated aerial hyphae with very rare short chains of spores. Subcloning and sequencing showed that whiK encodes a member of the FixJ subfamily of response regulators, with a C-terminal helix-turn-helix DNA-binding domain and an apparently typical N-terminal phosphorylation pocket. Unexpectedly, a constructed whiK null mutant failed to form aerial mycelium, showing that different alleles of this locus can arrest Streptomyces coelicolor development at very distinct stages. As a consequence of the null mutant phenotype, whiK was renamed bldM. The bldM null mutant fits into the extracellular signalling cascade proposed for S. coelicolor and is a member of the bldD extracellular complementation group. The three original NTG-induced mutations that defined the whiK/bldM locus each affected the putative phosphorylation pocket. The mutations in R273 and in R318 were the same, replacing a highly conserved glycine (G-62) with aspartate. The more severe mutant, R655, carried a C-7Y substitution adjacent to the highly conserved DD motif at positions 8-9. However, although BldM has all the highly conserved residues associated with the phosphorylation pocket of conventional response regulators, aspartate-54, the putative site of phosphorylation, is not required for BldM function. Constructed mutant alleles carrying either D-54N or D-54A substitutions complemented the bldM null mutant in single copy in trans, and strains carrying the D-54N or the D-54A substitution at the native chromosomal bldM locus sporulated normally. BldM was not phosphorylated in vitro with either of the small-molecule phosphodonors acetyl phosphate or carbamoyl phosphate under conditions in which a control response regulator protein, NtrC, was labelled efficiently.