SUPPRESSORS OF A SPO0A MISSENSE MUTATION AND THEIR EFFECTS ON SPORULATION IN BACILLUS-SUBTILIS

The spo0A gene product of Bacillus subtilis is a transcriptional regulator that is required for the initiation of sporulation. It has not been possible to isolate mutations that suppress the sporulation defect caused by spo0A null mutations. We describe the isolation and characterization of mutations that suppress the severe sporulation defect caused by a spo0A missense mutation (spo0A9V). Two suppressor mutations, spa2 and spa4, have been characterized in combination with, and separated from, the spo0A9V mutation. Both were located in the carboxyl half of Spo0A, in the putative DNA binding, transcriptional activation region. spa2 was in codon 174, causing a leucine to arginine change (spo0A174LR), and spa4 was in codon 162 (of 267), causing a histidine to arginine change (spo0A162HR). spa2 and spa4 significantly restored sporulation to the spo0A9V mutant, however, the appearance of heat resistant spores was delayed relative to wild-type. When separated from spo0A9V, that is, as single mutations in spo0A, spa4 caused a delay in sporulation, while spa2 allowed apparently normal sporulation. The spa mutations caused interesting phenotypes when combined with other early sporulation mutations. spa2 suppressed the sporulation defect caused by spo0E11. This was most easily seen in spo0E11 abrB double mutants, which had a much more severe sporulation defect than the spo0E11 single mutant. That is, spo0E11 and abrB mutations caused a synthetic (synergistic) sporulation phenotype. Both the spa2 spo0A9V and the spa4 spo0A9V alleles greatly enhanced the sporulation defect caused by mutations in spoIIJ, spo0J and spo0K. The significance of these synthetic sporulation defects is discussed.