STRUCTURAL ALTERATIONS IN THE BACILLUS-SUBTILIS SPOOA REGULATORY PROTEIN WHICH SUPPRESS MUTATIONS AT SEVERAL SPOO LOCI

Secondary site mutations that restore sporulation to sporulation-defective spo0F or spo0B deletion mutants were found to reside in the spo0A gene. Sequence analysis of 23 such sof mutants showed that the sof mutations fell into six classes of missense codon changes, primarily in the conserved amino-terminal domain of the response regulator Spo0A protein. Changes were observed in codons 12, 14, 60, 92, and 121. The residues affected were predominantly located in the potential turn regions at one end of the amino-terminal conserved domain on the topological face as the active site aspartate residues. The ability of sof mutations to suppress deficiencies in the transmitter kinases, KinA and KinB, of two-component regulatory systems was tested. All of the sof mutations suppressed the sporulation deficiency of kinA mutants but only two classes among five tested suppressed kinB mutations. sof mutants segregated Spo- colonies at high frequency. Five of these Spo- mutants were found to result from mutations in the spo0A locus that reversed the effect of the sof mutation. One of these was sequenced and found to have the original sof mutation and a new mutation, sos, at codon 105. The accumulation of sos mutations in sof strains suggested that the sof mutations have a subtle, yet deleterious, effect on the growth of the cell. The results suggested that the sof mutations increase the avidity for or reactivity with transmitter kinases in an allele-specific manner, although in some cases it is possible that the sof mutations obviate the need for phosphorylation to activate the Spo0A protein. An alternative hypothesis is presented in which the sof mutations play the role of bypass mutations for kinases.