Transcripts of the genes sacB, amyE, sacC and csn expressed in Bacillus subtilis under the control of the 5′ untranslated sacR region display different stabilities that can be modulated

When Bacillus subtilis levanase (SacC), alpha-amylase (AmyE) and chitosanase (Csn) structural genes were expressed under the regulated control of sacR, the inducible levansucrase (SacB) leader region in a degU32(Hy) mutant, it was observed that the production yields of the various extracellular proteins were quite different. This is mainly due to differences in the stabilities of their corresponding mRNAs which lead to discrepancies between the steady-state level of mRNA of sacB and csn on the one hand and amyE and sacC on the other. In contrast to levansucrase mRNA, the decay curves of alpha-amylase and levanase mRNAs obtained by Northern blotting analysis did not match the decay curves of their functional mRNA. This suggested that only a part of the population of the amyE and sacC transcripts was fully translated, while the others were possibly poorly bound to ribosomes and thus were only partially translated or not at all and consequently submitted to rapid endonuclease degradation. This hypothesis was substantiated by the finding that the introduction of a Shine-Dalgarno sequence upstream from the ribosome-binding site in the sacC transcript resulted in a fourfold increase in both the half-life of this transcript and the production of levanase. An additional cause of low-level levanase production is the premature release of mRNA by the polymerase. It was attempted to correlate this event with internal secondary structures of sacC mRNA.