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

摘要

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 σ~(F)is replaced by the late-acting sigma factor σ~(G). The gene encoding σ~(G)( sigG ) is transcribed by σ~(F)and by σ~(G)itself in an autoregulatory loop; yet σ~(G)activity is not detected until σ~(F)-dependent gene expression is complete. This separation in σ~(F)and σ~(G)activities has been suggested to be due at least in part to a poorly understood intercellular checkpoint pathway that delays sigG expression by σ~(F). 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 σ~(F)and instead support a model in which sigG is transcribed by σ~(F)with 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 σ~(G)activity in the forespore during sporulation, as well as inappropriate σ~(G)activity during vegetative growth. Altogether, these data indicate that transcription and translation of the sigG gene is tuned to prevent vegetative expression of σ~(G)and to ensure the precise timing of the switch from σ~(F)to σ~(G)in the developing spore. Author summary Global changes in gene expression occur during normal cellular growth and development, as well as during cancer cell transformation and bacterial pathogenesis. In this study we have investigated the molecular mechanisms that drive the switch from early to late developmental gene expression during spore formation by the model bacterium Bacillus subtilis . At early times, gene expression in the developing spore is directed by the transcription factor σ~(F); at later times σ~(F)is replaced by σ~(G). An important, yet poorly understood aspect of this σ~(F)-to-σ~(G)transition is how σ~(G)activation is delayed until the early, σ~(F)-directed phase of gene expression is complete. Here we have carefully examined expression of the gene encoding σ~(G), sigG , and found that its transcription and translation are ordinarily dampened by several features of its regulatory and coding sequences. Moreover, we have found that this “tuning” of sigG expression is required for proper timing of the switch to σ~(G). These results reframe our understanding of how sigG is regulated during B . subtilis sporulation and, more broadly, advance our understanding of how global changes in gene expression can be precisely executed at the molecular/genetic level.