Bacillus subtilis bacteriophage SPP1 is a lytic siphovirus first described 50 years ago. Its complete DNA sequence was reported in 1997. Here we present an updated annotation of the 44,016 bp SPP1 genome and its correlation to different steps of the viral multiplication process. Five early polycistronic transcriptional units encode phage DNA replication proteins and lysis functions together with less characterized, mostly non-essential, functions. Late transcription drives synthesis of proteins necessary for SPP1 viral particles assembly and for cell lysis, together with a short set of proteins of unknown function. The extensive genetic, biochemical and structural biology studies on the molecular mechanisms of SPP1 DNA replication and phage particle assembly rendered it a model system for tailed phages research. We propose SPP1 as the reference species for a new SPP1-like viruses genus of the Siphoviridae family., Research in our teams was funded by the CNRS, INRA, MINECO grant BFU2015-67065-P (to S.A.), ANR grants 09-BLAN-0149-0 (to P.T.), ANR-12-Blanc-BSV3-0021 (to P.T. and R.C.L.), ANR-15-CE11-0 010-01 (to P.T. and R.C.L.), and an “Equipe de la Fondation Médicale (FRM)” grant (to P.T.)., Peer reviewed

Toxin ζ expression triggers a reversible state of dormancy, diminishes the pool of purine nucleotides, promotes (p)ppGpp synthesis, phosphorylates a fraction of the peptidoglycan precursor uridine diphosphate-N-acetylglucosamine (UNAG), leading to unreactive UNAG-P, induces persistence in a reduced subpopulation, and sensitizes cells to different antibiotics. Here, we combined computational analyses with biochemical experiments to examine the mechanism of toxin ζ action. Free ζ toxin showed low affinity for UNAG. Toxin ζ bound to UNAG hydrolyzed ATP·Mg²⁺, with the accumulation of ADP, P_i, and produced low levels of phosphorylated UNAG (UNAG-P). Toxin ζ, which has a large ATP binding pocket, may temporally favor ATP binding in a position that is distant from UNAG, hindering UNAG phosphorylation upon ATP hydrolysis. The residues D67, E116, R158 and R171, involved in the interaction with metal, ATP, and UNAG, were essential for the toxic and ATPase activities of toxin ζ; whereas the E100 and T128 residues were partially dispensable. The results indicate that ζ bound to UNAG reduces the ATP concentration, which indirectly induces a reversible dormant state, and modulates the pool of UNAG., This research was funded by the Spanish Ministerio de Economía y Competitividad grant BFU2015-67065-P to J.C.A. This research received no external funding., We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI), Peer reviewed

Bacillus subtilis diadenylate cyclase DisA converts two ATPs into c-di-AMP, but this activity is suppressed upon interaction with sites of DNA damage. DisA forms a rapid moving focus that pauses upon induction of DNA damage during spore development. We report that DisA pausing, however, was not observed in the absence of the RecO mediator or of the RecA recombinase, suggesting that DisA binds to recombination intermediates formed by RecA in concert with RecO. DisA, which physically interacts with RecA, was found to reduce its ATPase activity without competing for nucleotides or ssDNA. Furthermore, increasing DisA concentrations inhibit RecA-mediated DNA strand exchange, but this inhibition failed to occur when RecA was added prior to DisA, and was independent of RecA-mediated nucleotide hydrolysis or increasing concentrations of c-di-AMP. We propose that DisA may preserve genome integrity by downregulating RecA activities at several steps of the DNA damage tolerance pathway, allowing time for the repair machineries to restore genome stability. DisA might reduce RecA-mediated template switching by binding to a stalled or reversed fork., Ministerio de Economía, Industria y Competitividad (MINECO)/FEDER) [BFU2015-67065-P, PGC2018–097054-B-I00 to J.C.A.]; R.T. is a PhD fellow of La Caixa Foundation International Fellowship Program (La Caixa/CNB) and his stay in Jerusalem was supported by an EMBO Short-Term Fellowship [7425; S.B.Y. is supported by the European Research Council Advanced Grant [339984]. Funding for open access charge: MINECO)/FEDER [BFU2015-67065-P]., Peer reviewed

Bacillus subtilis radA is epistatic to disA and recA genes in response to methyl methane sulfonate- and 4-nitroquinoline-1-oxide-induced DNA damage. We show that ΔradA cells were sensitive to mitomycin C- and H2O2-induced damage and impaired in natural chromosomal transformation, whereas cells lacking DisA were not. RadA/Sms mutants in the conserved H1 (K104A and K104R) or KNRFG (K255A and K255R) motifs fail to rescue the sensitivity of ΔradA in response to the four different DNA damaging agents. A RadA/Sms H1 or KNRFG mutation impairs both chromosomal and plasmid transformation, but the latter defect was suppressed by inactivating RecA. RadA/Sms K255A, K255R and wild type RadA/Sms reduced the diadenylate cyclase activity of DisA, whereas RadA/Sms K104A and K104R blocked it. Single-stranded and Holliday junction DNA are preferentially bound over double-stranded DNA by RadA/Sms and its variants. Moreover, RadA/Sms ATPase activity was neither stimulated by a variety of DNA substrates nor by DisA. RadA/Sms possesses a 5´→3´ DNA helicase activity. The RadA/Sms mutants neither hydrolyze ATP nor unwind DNA. Thus, we propose that RadA/Sms has two activities: to modulate DisA and to promote RecA-mediated DNA strand exchange. Both activities are required to coordinate responses to replicative stress and genetic recombination., This work was partially supported by the Spanish Ministerio de Economía y Competitividad grant BFU2015-67065-P to J.C.A. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We thank S. Ayora for providing us with G40P protein, and for comments on the manuscript. We are grateful to C. Marchisone for technical assistance. RT is a PhD fellow of the International Fellowship Programme of La Caixa Foundation (La Caixa-CNB), and ES thanks MINECO (BES-2013-063433) for the fellowship.

Bacterial RarA is thought to play crucial roles in the cellular response to blocked replication forks. We show that lack of Bacillus subtilis RarA renders cells very sensitive to H2O2, but not to methyl methane sulfonate or 4-nitroquinoline-1-oxide. RarA is epistatic to RecA in response to DNA damage. Inactivation of rarA partially suppressed the DNA repair defect of mutants lacking translesion synthesis polymerases. RarA may contribute to error-prone DNA repair as judged by the reduced frequency of rifampicin-resistant mutants in ΔrarA and in ΔpolY1 ΔrarA cells. The absence of RarA strongly reduced the viability of dnaD23ts and dnaB37ts cells upon partial thermal inactivation, suggesting that ΔrarA cells are deficient in replication fork assembly. A ΔrarA mutation also partially reduced the viability of dnaC30ts and dnaX51ts cells and slightly improved the viability of dnaG40ts cells at semi-permissive temperature. These results suggest that RarA links re-initiation of DNA replication with repair-by-recombination by controlling the access of the replication machinery to a collapsed replication fork., This work was supported by the Ministerio de Economía, Industria y Competitividad (MINECO/FEDER) BFU2015-67065-P to J.C.A and S.A., and by LOEWE funding to SYNMIKRO (state of Hessen) and by the Philipps Universität Marburg to P.L.G.

The ubiquitous RarA/Mgs1/WRNIP protein plays a crucial, but poorly understood role in genome maintenance.
We show that Bacillus subtilis RarA, in the apo form, preferentially binds single-stranded (ss) over double-stranded (ds) DNA. SsbA bound to ss-DNA loads RarA, and for such recruitment the amphipathic C-terminal domain of SsbA is required. RarA is a DNA-dependent ATPase strongly stimulated by ssDNA–dsDNA junctions and SsbA, or by dsDNA ends. RarA, which may interact with PriA, does not stimulate PriA DNA unwinding. In a reconstituted PriA-dependent DNA replication system, RarA inhibited initiation, but not chain elongation.
The RarA effect was not observed in the absence of SsbA, or when the host-encoded preprimosome and the DNA helicase are replaced by proteins from the SPP1 phage with similar function. We propose that RarA assembles at blocked forks tomaintain genome integrity. Through its interaction with SsbA and with a preprimosomal component, RarAmight impede the assembly of the replicative helicase, to prevent that recombination intermediates contribute to pathological DNA replication restart., Ministerio de Economía, Industria y Competitividad
(MINECO/FEDER) (BFU2015-67065-P), Peer reviewed

Virus infection causes major rearrangements in the subcellular architecture of eukaryotes, but its impact in prokaryotic cells was much less characterized. Here, we show that infection of the bacterium Bacillus subtilis by bacteriophage SPP1 leads to a hijacking of host replication proteins to assemble hybrid viral–bacterial replisomes for SPP1 genome replication. Their biosynthetic activity doubles the cell total DNA content within 15 min. Replisomes operate at several independent locations within a single viral DNA focus positioned asymmetrically in the cell. This large nucleoprotein complex is a self-contained compartment whose boundaries are delimited neither by a membrane nor by a protein cage. Later during infection, SPP1 procapsids localize at the periphery of the viral DNA compartment for genome packaging. The resulting DNA-filled capsids do not remain associated to the DNA transactions compartment. They bind to phage tails to build infectious particles that are stored in warehouse compartments spatially independent from the viral DNA. Free SPP1 structural proteins are recruited to the dynamic phage-induced compartments following an order that recapitulates the viral particle assembly pathway. These findings show that bacteriophages restructure the crowded host cytoplasm to confine at different cellular locations the sequential processes that are essential for their multiplication., Peer reviewed