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-indu ced whi mutants and was defined by three mutants, R273, R318 and R655. R273 and R318 produce long, tightly coiled aerial hyphae with frequent septatio n. In contrast, R655 shows a more severe phenotype; it produces straight, u ndifferentiated aerial hyphae with very rare short chains of spores. Subclo ning and sequencing showed that whiK encodes a member of the FixJ subfamily of response regulators, with a C-terminal helix-turn-helix DNA-binding dom ain and an apparently typical N-terminal phosphorylation pocket. Unexpected ly, a constructed whiK null mutant failed to form aerial mycelium, showing that different alleles of this locus can arrest Streptomyces coelicolor dev elopment at very distinct stages. As a consequence of the null mutant pheno type, whiK was renamed bldM. The bldM null mutant fits into the extracellul ar signalling cascade proposed for S. coelicolor and is a member of the bld D extracellular complementation group. The three original NTG-induced mutat ions that defined the whiK/bldM locus each affected the putative phosphoryl ation pocket. The mutations in R273 and in R318 were the same, replacing a highly conserved glycine (G-62) with aspartate. The more severe mutant, R65 5, carried a C-7Y substitution adjacent to the highly conserved DD motif at positions 8-9. However, although BldM has all the highly conserved residue s associated with the phosphorylation pocket of conventional response regul ators, aspartate-54, the putative site of phosphorylation, is not required for BldM function. Constructed mutant alleles carrying either D-54N or D-54 A 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 chro mosomal bldM locus sporulated normally. BldM was not phosphorylated in vitr o with either of the small-molecule phosphodonors acetyl phosphate or carba moyl phosphate under conditions in which a control response regulator prote in, NtrC, was labelled efficiently.