Abstract
Laboratory strains of Bacillus subtilis encode many alternative sigma factors, each dedicated to expressing a unique regulon such as those involved in stress resistance, sporulation, and motility. The ancestral strain of B. subtilis also encodes an additional sigma factor homolog, ZpdN, not found in lab strains due to being encoded on the large, low-copy-number plasmid pBS32, which was lost during domestication. DNA damage triggers pBS32 hyperreplication and cell death in a manner that depends on ZpdN, but how ZpdN mediates these effects is unknown. Here, we show that ZpdN is a bona fide sigma factor that can direct RNA polymerase to transcribe ZpdN-dependent genes, and we rename ZpdN SigN accordingly. Rend-seq (end-enriched transcriptome sequencing) analysis was used to determine the SigN regulon on pBS32, and the 5' ends of transcripts were used to predict the SigN consensus sequence. Finally, we characterize the regulation of SigN itself and show that it is transcribed by at least three promoters: PsigN1 , a strong SigA-dependent LexA-repressed promoter; PsigN2 , a weak SigA-dependent constitutive promoter; and PsigN3 , a SigN-dependent promoter. Thus, in response to DNA damage SigN is derepressed and then experiences positive feedback. How cells die in a pBS32-dependent manner remains unknown, but we predict that death is the product of expressing one or more genes in the SigN regulon.IMPORTANCE Sigma factors are utilized by bacteria to control and regulate gene expression. Some sigma factors are activated during times of stress to ensure the survival of the bacterium. Here, we report the presence of a sigma factor that is encoded on a plasmid that leads to cellular death after DNA damage.
Highlights
Laboratory strains of Bacillus subtilis encode many alternative sigma factors, each dedicated to expressing a unique regulon such as those involved in stress resistance, sporulation, and motility
SigN is a sigma factor homolog encoded on the plasmid pBS32 that is necessary and sufficient for pBS32-mediated cell death [17]
Consistent with previous results, treatment of cells deleted for the PBSX and SP prophages [14, 18,19,20,21] with the DNA-damaging agent mitomycin C (MMC) caused a 3-fold decrease in optical density (OD) from peak absorbance, and the decrease in OD was abolished in cells deleted for sigN [17] (Fig. 1A)
Summary
Laboratory strains of Bacillus subtilis encode many alternative sigma factors, each dedicated to expressing a unique regulon such as those involved in stress resistance, sporulation, and motility. The ancestral strain of B. subtilis encodes an additional sigma factor homolog, ZpdN, not found in lab strains due to being encoded on the large, low-copy-number plasmid pBS32, which was lost during domestication. DNA damage triggers pBS32 hyperreplication and cell death in a manner that depends on ZpdN, but how ZpdN mediates these effects is unknown. How cells die in a pBS32-dependent manner remains unknown, but we predict that death is the product of expressing one or more genes in the SigN regulon. Approximately one-third of the pBS32 sequence encodes a cryptic prophagelike element, and cell death is triggered in a pBS32-dependent manner following treatment with the DNA-damaging agent mitomycin C (MMC) [7, 14,15,16,17]. How ZpdN is activated by the presence of DNA damage and the mechanism by which ZpdN promotes cell death are unknown
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