Files
Abstract
Salmonella enterica serovar Typhimurium is a gastrointestinal pathogen that must survive a variety of stress conditions throughout its infection cycle. One mechanism it utilizes to rapidly alter gene expression is the modification of RNA polymerase promoter recognition and activity via the alternative sigma factor, σ54. Transcription from σ54-dependent promoters cannot occur spontaneously; it requires interaction with a bacterial enhancer-binding protein (bEBP), which hydrolyzes ATP to fulfill the energetic requirements of transcription initiation. The bEBP becomes activated upon sensing a specific molecular signal; in this manner, genes of the σ54 regulon are tightly controlled.
The rsr-yrlBA-rtcBA operon was identified as part of the S. Typhimurium σ54 regulon; it is activated by the bEBP RtcR. This operon is called the RNA repair operon due to its purported function in RNA end repair and ligation, based on the homology of its components to genes found in metazoans and archaea and in vitro confirmation of RtcA and RtcB activities. Prior to this work, the conditions under which this operon is activated in any bacteria and the RtcR signaling pathway were totally unknown.
In this work, we characterize the regulation of the RNA repair operon in S. Typhimurium. Using qRT-PCR and promoter reporter fusion assays, we found that the operon is activated under conditions that cause nucleic acid damage. Using directed mutational analysis, we determined that RecA drives expression of the RNA repair operon via its activity as co-protease of the LexA repressor, but SOS response genes are largely uninvolved. Rather, strains that are cured of the Gifsy-1 prophage, whose induction is dependent on LexA cleavage, were deficient in RtcR-mediated activation of the operon promoter (rsrp). In a genome-wide mutagenesis screen, we found that the factor for inversion stimulation (Fis), which stimulates lambdoid prophage excision, is also essential for rsrp activation. Finally, we found that S. Typhimurium expressing CNPase is unable to activate rsrp under inducing conditions, providing support that the RtcR signaling ligand is an RNA cleavage product with a 2’,3’-cyclic phosphate terminus. These findings give significant insight into the regulation of this enigmatic operon and may offer clues for its physiological functions.
The rsr-yrlBA-rtcBA operon was identified as part of the S. Typhimurium σ54 regulon; it is activated by the bEBP RtcR. This operon is called the RNA repair operon due to its purported function in RNA end repair and ligation, based on the homology of its components to genes found in metazoans and archaea and in vitro confirmation of RtcA and RtcB activities. Prior to this work, the conditions under which this operon is activated in any bacteria and the RtcR signaling pathway were totally unknown.
In this work, we characterize the regulation of the RNA repair operon in S. Typhimurium. Using qRT-PCR and promoter reporter fusion assays, we found that the operon is activated under conditions that cause nucleic acid damage. Using directed mutational analysis, we determined that RecA drives expression of the RNA repair operon via its activity as co-protease of the LexA repressor, but SOS response genes are largely uninvolved. Rather, strains that are cured of the Gifsy-1 prophage, whose induction is dependent on LexA cleavage, were deficient in RtcR-mediated activation of the operon promoter (rsrp). In a genome-wide mutagenesis screen, we found that the factor for inversion stimulation (Fis), which stimulates lambdoid prophage excision, is also essential for rsrp activation. Finally, we found that S. Typhimurium expressing CNPase is unable to activate rsrp under inducing conditions, providing support that the RtcR signaling ligand is an RNA cleavage product with a 2’,3’-cyclic phosphate terminus. These findings give significant insight into the regulation of this enigmatic operon and may offer clues for its physiological functions.