Transcription and translation of the sigG gene is tuned for proper execution of the switch from early to late gene expression in the developing Bacillus subtilis spore

Abstract

A cascade of alternative sigma factors directs developmental gene expression during spore formation by the bacterium Bacillus subtilis. As the spore develops, a tightly regulated switch occurs in which the early-acting sigma factor σFis replaced by the late-acting sigma factor σG. The gene encoding σG(sigG) is transcribed by σFand by σGitself in an autoregulatory loop; yet σGactivity is not detected until σF-dependent gene expression is complete. This separation in σFand σGactivities has been suggested to be due at least in part to a poorly understood intercellular checkpoint pathway that delays sigG expression by σF. Here we report the results of a careful examination of sigG expression during sporulation. Unexpectedly, our findings argue against the existence of a regulatory mechanism to delay sigG transcription by σFand instead support a model in which sigG is transcribed by σFwith normal timing, but at levels that are very low. This low-level expression of sigG is the consequence of several intrinsic features of the sigG regulatory and coding sequence—promoter spacing, secondary structure potential of the mRNA, and start codon identity—that dampen its transcription and translation. Especially notable is the presence of a conserved hairpin in the 5’ leader sequence of the sigG mRNA that occludes the ribosome-binding site, reducing translation by up to 4-fold. Finally, we demonstrate that misexpression of sigG from regulatory and coding sequences lacking these features triggers premature σGactivity in the forespore during sporulation, as well as inappropriate σGactivity during vegetative growth. Altogether, these data indicate that transcription and translation of the sigG gene is tuned to prevent vegetative expression of σGand to ensure the precise timing of the switch from σFto σGin the developing spore.