Development of a two-part transcription probe to determine the completeness of temporal and spatial compartmentalization of gene expression during bacterial development

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The National Academy of Sciences

RESUMO

We have developed a two-part test, using the Bacillus subtilis sacB/SacY transcription antitermination system, to evaluate the completeness of temporal and spatial compartmentalization of gene expression during bacterial cell development. Transcription of sacY(1–55) (encoding a constitutively active form of the antiterminator, SacY) is directed by one promoter, whereas transcription of sacB′-′lacZ (the target of SacY action) is directed by the same or another promoter. To obtain β-galactosidase activity, SacY(1–55) needs to be present when sacB′-′lacZ is being transcribed. We tested the system by analyzing the spatial compartmentalization of the activities of RNA polymerase σ factors, which are tightly regulated during sporulation of B. subtilis: σF and then σG in the prespore, σE and then σK in the mother cell. We have confirmed that the activities of σF and σE are spatially compartmentalized. We have demonstrated that there is also sharp temporal compartmentalization, with little or no overlap in the activities of σF and σG or of σE and σK. In contrast, we found no compartmentalization of the activity of the main vegetative factor, σA, which continued to be active alongside all of the sporulation-specific σ factors. We also found no temporal compartmentalization of expression of loci that are activated during the development of competent cells of B. subtilis, a developmental program distinct from spore formation. A possible mechanism to explain the temporal compartmentalization of σF and σG activities is that the anti-sigma factor SpoIIAB transfers from σG to σF.

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