Thermo-labile stability of sigma(H) (Spo0H) in temperature-sensitive spo0Hmutants of Bacillus subtilis can be suppressed by mutations in RNA polymerase beta subunit

We isolated novel temperature-sensitive mutants of spo0H, spo0H1 and spo0H5 , having E61K and G30E amino-acid substitutions within the sigma(H) protein , respectively, and located in the highly conserved region, "2", among prok aryotic sigma factors that participates in binding to core enzyme of RNA po lymerase. These mutants showed a sporulation-deficient phenotype at 43 degr ees C. Moreover, we successfully isolated suppressor mutants that were spon taneously generated from the spo0H mutants. Our genetic analysis of these s uppressor mutations revealed that the suppressor mutations are within the r poB gene coding for the beta subunit of RNA polymerase. The mutations cause d single amino-acid substitutions, E857A and P1055S, in rpoB18 and rpoB532 mutants that were generated from spo0H1 and spo0H5, respectively. Whereas t he sigma(H)-dependent expression of a spo0A-bgaB fusion was greatly reduced in both spo0H mutants, their expression was partially restored in the supp ressor mutants at 43 degrees C. Western blot analysis showed that the level of sigma(H) protein in the wild type increased between T-0 and T-2 and dec reased after T-3, while the level of sigma(H) protein in spo0H mutants was greatly reduced throughout growth, indicating that the mutant sigma(H) prot eins were rapidly degraded by some unknown proteolytic enzyme(s). The analy sis of the half-life of sigma(H) protein showed that the short life of sigm a(H) in spo0H mutants is prolonged in the suppressor mutants. These finding s suggest that, at least to some extent, the process of E-sigma(H) formatio n may be involved in stabilization of sigma(H) at the onset of sporulation. (C) 1999 Elsevier Science B.V. All rights reserved.