78f54b4108320c1bb6025f7b572c9cfc Perez-Varela_et_al._2026.pdf ed990ba75c2163e3ddeef6cb137ef18cf2d49d91 Perez-Varela_et_al._2026.pdf a0b1be3e8fdd888353737f836e99bf1afbe1ab084ddc1a90aefb597c8e9f2e75 Perez-Varela_et_al._2026.pdf Title: A phage repressor involved in the DNA-damage response of Francisella Subject: DOI: 10.1093/pnasnexus/pgag114; PNAS Nexus, 5, 5, 2026-04-17.; Abstract: Members of the S24 family of serine peptidases play central roles in bacterial and phage regulatory networks that respond to DNA damage. In most bacteria, the S24-family peptidase LexA functions as the master repressor of the SOS response, regulating the activity of genes involved in DNA repair and mutagenesis. LexA is known to be absent in some bacterial lineages, including genera of intracellular bacteria with substantial genomic reduction, but it has been shown that other S24-family peptidases can uptake its regulatory functions. In this work, we combine experimental and computational approaches to define the response to DNA damage in Francisella, a genus of facultative intracellular bacteria with moderate genome reduction that encodes a highly diverged LexA family protein. We use RNA sequencing to investigate the transcriptional response of Francisella hispaniensis to the DNA-damaging agent ciprofloxacin and reveal that canonical SOS genes are not induced upon DNA damage. Among the differentially expressed genes, we identify a gene encoding an S24-family peptidase that we term FddR (Francisella DNA-damage regulator). Electrophoretic mobility shift assays demonstrate that the product of this gene specifically binds to the palindromic sequence GTG-N11-CAC present in its promoter region, resulting in autoregulation. In silico analyses reveal that this S24-family peptidase is most likely a co-opted phage repressor that implements a DNA-damage response across the Francisella genus, regulating itself and divergently transcribed genes. Our results provide insights into the SOS repair system of the Francisella genus and its evolution, putting forward a mechanism for the gradual reconstruction of an SOS response network from self-contained divergently transcribed units. Keywords: Author: María Pérez-Varela; Miquel Sánchez-Osuna; Susana Campoy; Jesús Aranda; Jordi Barbé; Ivan Erill Creator: Servigistics Arbortext Advanced Print Publisher 11.1.4546/W-x64 Producer: PDFlib+PDI 9.0.7p3 (C++/Win64) CreationDate: Tue May 5 16:20:49 2026 CEST ModDate: Tue May 5 19:38:43 2026 CEST Custom Metadata: yes Metadata Stream: yes Tagged: yes UserProperties: no Suspects: no Form: AcroForm JavaScript: no Pages: 12 Encrypted: no Page size: 595.276 x 782.362 pts Page rot: 0 File size: 1375173 bytes Optimized: no PDF version: 1.6 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- TRQXXL+SourceSansPro-It Type 1C Custom yes yes yes 626 0 QXOWUS+SourceSansPro-Regular Type 1C Custom yes yes yes 623 0 JYMLFI+SourceSansPro-Bold Type 1C Custom yes yes yes 620 0 OGKNVK+SourceSansPro-Semibold Type 1C Custom yes yes yes 684 0 VRFITZ+SourceSansPro-BoldIt Type 1C Custom yes yes yes 681 0 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Title: A phage repressor involved in the DNA-damage response of Francisella Destination: head1 Children: Item: Title: Introduction Destination: secpgag114-s1 Item: Title: Results Destination: secpgag114-s2 Children: Item: Title: Transcriptomic response to DNA damage in Francisella species Destination: secpgag114-s2.1 Item: Title: Phylogeny of Francisella S24-family peptidase regulators Destination: secpgag114-s2.2 Item: Title: FddR regulates its own transcription by binding to its upstream promoter region Destination: secpgag114-s2.3 Item: Title: FddR targets a palindromic motif Destination: secpgag114-s2.4 Item: Title: FddR homologs define an SOS-like regulatory network in the Francisella genus Destination: secpgag114-s2.5 Item: Title: Discussion Destination: secpgag114-s3 Item: Title: Materials and methods Destination: secpgag114-s4 Children: Item: Title: Strains and growth conditions Destination: secpgag114-s4.1 Item: Title: Total RNA extraction and RT-qPCR Destination: secpgag114-s4.2 Item: Title: RNA-seq and analysis Destination: secpgag114-s4.3 Item: Title: FddR protein purification Destination: secpgag114-s4.4 Item: Title: Electrophoretic mobility shift assays Destination: secpgag114-s4.5 Item: Title: Motif discovery and regulon analysis Destination: secpgag114-s4.6 Item: Title: Phylogenetic inference Destination: secpgag114-s4.7 Item: Title: Acknowledgments Destination: acknow Item: Title: Supplementary Material Destination: secpgag114-s5 Item: Title: Competing Interest Destination: secpgag114-s6 Item: Title: Funding Destination: secpgag114-s7 Item: Title: Author Contributions Destination: secpgag114-s8 Item: Title: Data Availability Destination: secpgag114-s9 Item: Title: References Destination: reflist Filters: FilterPipeline: FlateDecode FilterPipeline: DCTDecode Images: Image: NisoImageMetadata: FormatName: image/jpg CompressionScheme: JPEG ImageWidth: 933 ImageHeight: 630 ColorSpace: RGB BitsPerSample: 8 BitsPerSampleUnit: integer Image: 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Differences: true ToUnicode: true Font: BaseFont: NPTYZV+SourceSansPro-Light FontSubset: true FirstChar: 1 LastChar: 75 FontDescriptor: FontName: NPTYZV+SourceSansPro-Light Flags: Nonsymbolic FontBBox: -169, -287, 1122, 939 FontFile3: true EncodingDictionary: BaseEncoding: WinAnsiEncoding Differences: true ToUnicode: true Font: BaseFont: QXOWUS+SourceSansPro-Regular FontSubset: true FirstChar: 1 LastChar: 95 FontDescriptor: FontName: QXOWUS+SourceSansPro-Regular Flags: Nonsymbolic FontBBox: -192, -323, 1160, 952 FontFile3: true EncodingDictionary: BaseEncoding: WinAnsiEncoding Differences: true ToUnicode: true CIDFontType2: Font: BaseFont: APKFVW+GentiumPlus CIDSystemInfo: Registry: Adobe Registry: Identity Supplement: 0 FontDescriptor: FontName: APKFVW+GentiumPlus Flags: Nonsymbolic FontBBox: -677, -464, 2149, 1270 FontFile2: true XMP: application/pdf 10.1093/pnasnexus/pgag114 en Oxford University Press A phage repressor involved in the DNA-damage response of Francisella María Pérez-Varela; Miquel Sánchez-Osuna; Susana Campoy; Jesús Aranda; Jordi Barbé; Ivan Erill Miquel Sánchez-Osuna Susana Campoy Jesús Aranda Jordi Barbé Ivan Erill DOI: 10.1093/pnasnexus/pgag114; PNAS Nexus, 5, 5, 2026-04-17.; Abstract: Members of the S24 family of serine peptidases play central roles in bacterial and phage regulatory networks that respond to DNA damage. In most bacteria, the S24-family peptidase LexA functions as the master repressor of the SOS response, regulating the activity of genes involved in DNA repair and mutagenesis. LexA is known to be absent in some bacterial lineages, including genera of intracellular bacteria with substantial genomic reduction, but it has been shown that other S24-family peptidases can uptake its regulatory functions. In this work, we combine experimental and computational approaches to define the response to DNA damage in Francisella, a genus of facultative intracellular bacteria with moderate genome reduction that encodes a highly diverged LexA family protein. We use RNA sequencing to investigate the transcriptional response of Francisella hispaniensis to the DNA-damaging agent ciprofloxacin and reveal that canonical SOS genes are not induced upon DNA damage. Among the differentially expressed genes, we identify a gene encoding an S24-family peptidase that we term FddR (Francisella DNA-damage regulator). Electrophoretic mobility shift assays demonstrate that the product of this gene specifically binds to the palindromic sequence GTG-N11-CAC present in its promoter region, resulting in autoregulation. In silico analyses reveal that this S24-family peptidase is most likely a co-opted phage repressor that implements a DNA-damage response across the Francisella genus, regulating itself and divergently transcribed genes. Our results provide insights into the SOS repair system of the Francisella genus and its evolution, putting forward a mechanism for the gradual reconstruction of an SOS response network from self-contained divergently transcribed units. © The Author(s) 2026. Published by Oxford University Press on behalf of National Academy of Sciences. PDFlib+PDI 9.0.7p3 (C++/Win64) 1 PStill version 1.84.42 Journal © The Author(s) 2026. 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