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The DNA damage checkpoint protein DisA and the branch migration translocase RecG are implicated in the preservation of genome integrity in reviving haploid Bacillus subtilis spores. DisA synthesizes the essential cyclic 3′, 5′-diadenosine monophosphate (c-di-AMP) second messenger and such synthesis is suppressed upon replication perturbation. In vitro, c-di-AMP synthesis is suppressed when DisA binds DNA structures that mimic stalled or reversed forks (gapped forks or Holliday junctions [HJ]). RecG, which does not form a stable complex with DisA, unwinds branched intermediates, and in the presence of a limiting ATP concentration and HJ DNA, it blocks DisA-mediated c-di-AMP synthesis. DisA pre-bound to a stalled or reversed fork limits RecG-mediated ATP hydrolysis and DNA unwinding, but not if RecG is pre-bound to stalled or reversed forks. We propose that RecG-mediated fork remodeling is a genuine in vivo activity, and that DisA, as a molecular switch, limits RecG-mediated fork reversal and fork restoration. DisA and RecG might provide more time to process perturbed forks, avoiding genome breakage.
Rubén Torres; Carolina Gándara; Begoña Carrasco; Ignacio Baquedano; Silvia Ayora; Juan Alonso. DisA Limits RecG Activities at Stalled or Reversed Replication Forks. Cells 2021, 10, 1357 .
AMA StyleRubén Torres, Carolina Gándara, Begoña Carrasco, Ignacio Baquedano, Silvia Ayora, Juan Alonso. DisA Limits RecG Activities at Stalled or Reversed Replication Forks. Cells. 2021; 10 (6):1357.
Chicago/Turabian StyleRubén Torres; Carolina Gándara; Begoña Carrasco; Ignacio Baquedano; Silvia Ayora; Juan Alonso. 2021. "DisA Limits RecG Activities at Stalled or Reversed Replication Forks." Cells 10, no. 6: 1357.
Rok, a Bacillus subtilis nucleoid‐associated protein (NAP), negatively regulates competence development and silences xenogeneic genes. We show that rok inactivation increases rpoB482 natural intraspecies chromosomal transformation (CT) and plasmid transformation to a different extent. In ΔaddAB, ΔrecO, recF15, ΔrecU, ΔruvAB or rec+ cells intraspecies CT significantly increases, but the ΔrecD2 mutation reduces, and the ΔrecX, ΔradA or ΔdprA mutation further decreases CT in the Δrok context when compared to rok+ cells. These observations support the idea that rok inactivation, by altering the topology of the recipient DNA, differentially affects the integration of homologous DNA in rec‐deficient strains, and in minor extent the competent subpopulation size. The impairment of other NAP (Hbsu or LrpC) also increased intra‐ and interspecies CT (nonself‐DNA, ~8% nucleotide sequence divergence) in rec+ cells, but differentially reduced both types of CTs in certain rec‐deficient strains. We describe that rok inactivation significantly stimulates intra and interspecies CT, but differentially reduces them in transformation‐deficient cells, perhaps by altering the nucleoid architecture. We extend the observation to other NAPs (Hbsu, LrpC).
Ester Serrano; Rubén Torres; Juan C. Alonso. Nucleoid‐associated Rok differentially affects chromosomal transformation on Bacillus subtilis recombination‐deficient cells. Environmental Microbiology 2021, 23, 3318 -3331.
AMA StyleEster Serrano, Rubén Torres, Juan C. Alonso. Nucleoid‐associated Rok differentially affects chromosomal transformation on Bacillus subtilis recombination‐deficient cells. Environmental Microbiology. 2021; 23 (6):3318-3331.
Chicago/Turabian StyleEster Serrano; Rubén Torres; Juan C. Alonso. 2021. "Nucleoid‐associated Rok differentially affects chromosomal transformation on Bacillus subtilis recombination‐deficient cells." Environmental Microbiology 23, no. 6: 3318-3331.
Bacillus subtilis PcrA interacts with the RNA polymerase and might contribute to mitigate replication–transcription conflicts (RTCs). We show that PcrA depletion lethality is partially suppressed by rnhB inactivation, but cell viability is significantly reduced by rnhC or dinG inactivation. Following PcrA depletion, cells lacking RnhC or DinG are extremely sensitive to DNA damage. Chromosome segregation is not further impaired by rnhB or dinG inactivation but is blocked by rnhC or recA inactivation upon PcrA depletion. Despite our efforts, we could not construct a ΔrnhC ΔrecA strain. These observations support the idea that PcrA dismantles RTCs. Purified PcrA, which binds single-stranded (ss) DNA over RNA, is a ssDNA-dependent ATPase and preferentially unwinds DNA in a 3′→5′direction. PcrA unwinds a 3′-tailed RNA of an RNA-DNA hybrid significantly faster than that of a DNA substrate. Our results suggest that a replicative stress, caused by mis-incorporated rNMPs, indirectly increases cell viability upon PcrA depletion. We propose that PcrA, in concert with RnhC or DinG, contributes to removing spontaneous or enzyme-driven R-loops, to counteract deleterious trafficking conflicts and preserve to genomic integrity.
María Moreno-Del Álamo; Begoña Carrasco; Rubén Torres; Juan Alonso. Bacillus subtilis PcrA Helicase Removes Trafficking Barriers. Cells 2021, 10, 935 .
AMA StyleMaría Moreno-Del Álamo, Begoña Carrasco, Rubén Torres, Juan Alonso. Bacillus subtilis PcrA Helicase Removes Trafficking Barriers. Cells. 2021; 10 (4):935.
Chicago/Turabian StyleMaría Moreno-Del Álamo; Begoña Carrasco; Rubén Torres; Juan Alonso. 2021. "Bacillus subtilis PcrA Helicase Removes Trafficking Barriers." Cells 10, no. 4: 935.
Toxin–antitoxin (TA) systems, which are ubiquitously present in plasmids, bacterial and archaeal genomes, are classified as types I to VI, according to the nature of the antitoxin and to the mode of toxin inhibition
Juan Alonso. Toxin–Antitoxin Systems in Pathogenic Bacteria. Toxins 2021, 13, 74 .
AMA StyleJuan Alonso. Toxin–Antitoxin Systems in Pathogenic Bacteria. Toxins. 2021; 13 (2):74.
Chicago/Turabian StyleJuan Alonso. 2021. "Toxin–Antitoxin Systems in Pathogenic Bacteria." Toxins 13, no. 2: 74.
Toxin-antitoxin (TA) modules are ubiquitous in bacteria, but their biological importance in stress adaptation remains a matter of debate. The inactive ζ-ε2-ζ TA complex is composed of one labile ε2 antitoxin dimer flanked by two stable ζ toxin monomers. Free toxin ζ reduces the ATP and GTP levels, increases the (p)ppGpp and c-di-AMP pool, inactivates a fraction of uridine diphosphate-N-acetylglucosamine, and induces reversible dormancy. A small subpopulation, however, survives toxin action. Here, employing a genetic orthogonal control of ζ and ε levels, the fate of bacteriophage SPP1 infection was analyzed. Toxin ζ induces an active slow-growth state that halts SPP1 amplification, but it re-starts after antitoxin expression rather than promoting abortive infection. Toxin ζ-induced and toxin-facilitated ampicillin (Amp) dormants have been revisited. Transient toxin ζ expression causes a metabolic heterogeneity that induces toxin and Amp dormancy over a long window of time rather than cell persistence. Antitoxin ε expression, by reversing ζ activities, facilitates the exit of Amp-induced dormancy both in rec+ and recA cells. Our findings argue that an unexploited target to fight against antibiotic persistence is to disrupt toxin-antitoxin interactions.
María Moreno-Del Álamo; Chiara Marchisone; Juan C. Alonso. Antitoxin ε Reverses Toxin ζ-Facilitated Ampicillin Dormants. Toxins 2020, 12, 801 .
AMA StyleMaría Moreno-Del Álamo, Chiara Marchisone, Juan C. Alonso. Antitoxin ε Reverses Toxin ζ-Facilitated Ampicillin Dormants. Toxins. 2020; 12 (12):801.
Chicago/Turabian StyleMaría Moreno-Del Álamo; Chiara Marchisone; Juan C. Alonso. 2020. "Antitoxin ε Reverses Toxin ζ-Facilitated Ampicillin Dormants." Toxins 12, no. 12: 801.
Natural chromosomal transformation (CT) plays a major role in prokaryote evolution; yet factors that govern the integration of DNA from related species remain poorly understood. We show that in naturally competent Bacillus subtilis cells the acquisition of homeologous sequences is governed by sequence divergence (SD). Integration initiates in a minimal efficient processing segment via homology‐directed CT, and its frequency decreases log‐linearly with increased sequence divergence up to 15%. Beyond this and up to 23% SD the interspecies boundaries prevail, the CT frequency marginally decreases, and short (<10‐nucleotides) segments are integrated via homology‐facilitated micro‐homologous integration. Both mechanisms are RecA dependent. We identify the other recombination proteins required for the acquisition of homeologous DNA. The absence of AddAB, RecF, RecO, RuvAB or RecU, crucial for repair‐by‐recombination, did not affect CT. However, dprA, radA, recJ, recX or recD2 inactivation strongly decreased intraspecies and interspecies CT. Interspecies CT was not detected beyond ~8% SD in ΔdprA, ~10% in ΔrecJ, ΔradA, ΔrecX and ~14% in ΔrecD2 cells. We propose that DprA, RecX, RadA/Sms, RecJ and RecD2 accessory proteins are important for the generation of genetic diversity. Together with RecA, they facilitate gene acquisition from bacteria of related species.
Ester Serrano; Cristina Ramos; Juan C. Alonso; Silvia Ayora. Recombination proteins differently control the acquisition of homeologousDNAduringBacillus subtilisnatural chromosomal transformation. Environmental Microbiology 2020, 23, 512 -524.
AMA StyleEster Serrano, Cristina Ramos, Juan C. Alonso, Silvia Ayora. Recombination proteins differently control the acquisition of homeologousDNAduringBacillus subtilisnatural chromosomal transformation. Environmental Microbiology. 2020; 23 (1):512-524.
Chicago/Turabian StyleEster Serrano; Cristina Ramos; Juan C. Alonso; Silvia Ayora. 2020. "Recombination proteins differently control the acquisition of homeologousDNAduringBacillus subtilisnatural chromosomal transformation." Environmental Microbiology 23, no. 1: 512-524.
The DisA diadenylate cyclase (DAC), the DNA helicase RadA/Sms and the RecA recombinase are required to prevent a DNA replication stress during the revival of haploidBacillus subtilisspores. Moreover,disA, radAandrecAare epistatic among them in response to DNA damage. We show that DisA inhibits the ATPase activity of RadA/Sms C13A by competing for single-stranded (ss) DNA. In addition, DisA inhibits the helicase activity of RadA/Sms. RecA filamented onto ssDNA interacts with and recruits DisA and RadA/Sms onto branched DNA intermediates. In fact, RecA binds a reversed fork and facilitates RadA/Sms-mediated unwinding to restore a 3′-fork intermediate, but DisA inhibits it. Finally, RadA/Sms inhibits DisA DAC activity, but RecA counters this negative effect. We propose that RecA, DisA and RadA/Sms interactions, which are mutually exclusive, limit remodelling of stalled replication forks. DisA, in concert with RecA and/or RadA/Sms, indirectly contributes to template switching or lesion bypass, prevents fork breakage and facilitates the recovery of c-di-AMP levels to re-initiate cell proliferation. Subject Categories Genomic stability & Dynamics
Rubén Torres; Juan C. Alonso. DisA limits RecA- and RadA/Sms-mediated replication fork remodelling to prevent genome instability. 2020, 1 .
AMA StyleRubén Torres, Juan C. Alonso. DisA limits RecA- and RadA/Sms-mediated replication fork remodelling to prevent genome instability. . 2020; ():1.
Chicago/Turabian StyleRubén Torres; Juan C. Alonso. 2020. "DisA limits RecA- and RadA/Sms-mediated replication fork remodelling to prevent genome instability." , no. : 1.
Bacillus subtilis PcrA abrogates replication-transcription conflicts in vivo and disrupts RecA nucleoprotein filaments in vitro. Inactivation of pcrA is lethal. We show that PcrA depletion lethality is suppressed by recJ (involved in end resection), recA (the recombinase), or mfd (transcription-coupled repair) inactivation, but not by inactivating end resection (addAB or recQ), positive and negative RecA modulators (rarA or recX and recU), or genes involved in the reactivation of a stalled RNA polymerase (recD2, helD, hepA, and ywqA). We also report that B. subtilis mutations previously designated as recL16 actually map to the recO locus, and confirm that PcrA depletion lethality is suppressed by recO inactivation. The pcrA gene is epistatic to recA or mfd, but it is not epistatic to addAB, recJ, recQ, recO16, rarA, recX, recU, recD2, helD, hepA, or ywqA in response to DNA damage. PcrA depletion led to the accumulation of unsegregated chromosomes, and this defect is increased by recQ, rarA, or recU inactivation. We propose that PcrA, which is crucial to maintain cell viability, is involved in different DNA transactions.
María Moreno-Del Alamo; Rubén Torres; Candela Manfredi; José A. Ruiz-Masó; Gloria Del Solar; Juan Carlos Alonso. Bacillus subtilis PcrA Couples DNA Replication, Transcription, Recombination and Segregation. Frontiers in Molecular Biosciences 2020, 7, 140 .
AMA StyleMaría Moreno-Del Alamo, Rubén Torres, Candela Manfredi, José A. Ruiz-Masó, Gloria Del Solar, Juan Carlos Alonso. Bacillus subtilis PcrA Couples DNA Replication, Transcription, Recombination and Segregation. Frontiers in Molecular Biosciences. 2020; 7 ():140.
Chicago/Turabian StyleMaría Moreno-Del Alamo; Rubén Torres; Candela Manfredi; José A. Ruiz-Masó; Gloria Del Solar; Juan Carlos Alonso. 2020. "Bacillus subtilis PcrA Couples DNA Replication, Transcription, Recombination and Segregation." Frontiers in Molecular Biosciences 7, no. : 140.
In naturally competentBacillus subtiliscells the acquisition of closely related genes occursviahomology-directed chromosomal transformation (CT), and its frequency decreases log-linearly with increased sequence divergence (SD) up to 15%. Beyond this and up to 23% SD the interspecies boundary prevails, the CT frequency marginally decreases, and short (viahomology-facilitated micro-homologous integration. Both poorly known CT mechanisms are RecA-dependent. Here we identify the recombination proteins required for the acquisition of interspecies DNA. The absence of AddAB, RecF, RecO, RuvAB or RecU, crucial for repair-by-recombination, does not affect CT. However, inactivation ofdprA, radA, recJ, recXorrecD2strongly interfered with CT. Interspecies CT was abolished beyond ~8% SD in ΔdprA, ~10% in ΔrecJ, ΔradA, ΔrecXand 14% in ΔrecD2cells. We propose that DprA, RecX, RadA/Sms, RecJ and RecD2 help RecA to unconstrain speciation and gene flow. These functions are ultimately responsible for generating genetic diversity and facilitate CT and gene acquisition from bacteria of the same genus.
Ester Serrano; Cristina Ramos; Juan Carlos Alonso; Silvia Ayora. Recombination proteins differently control the acquisition of homeologous DNA duringBacillus subtilisnatural chromosomal transformation. 2020, 1 .
AMA StyleEster Serrano, Cristina Ramos, Juan Carlos Alonso, Silvia Ayora. Recombination proteins differently control the acquisition of homeologous DNA duringBacillus subtilisnatural chromosomal transformation. . 2020; ():1.
Chicago/Turabian StyleEster Serrano; Cristina Ramos; Juan Carlos Alonso; Silvia Ayora. 2020. "Recombination proteins differently control the acquisition of homeologous DNA duringBacillus subtilisnatural chromosomal transformation." , no. : 1.
Ubiquitous RarA AAA+ ATPases play crucial roles in the cellular response to blocked replication forks in pro- and eukaryotes. Here, we provide evidence that absence of RarA reduced the viability of ΔrecA, ΔrecO, and recF15 cells during unperturbed growth. The rarA gene was epistatic to recO and recF genes in response to H2O2- or MMS-induced DNA damage. Conversely, the inactivation of rarA partially suppressed the HR defect of mutants lacking end-resection (ΔaddAB, ΔrecJ, ΔrecQ, ΔrecS) or branch migration (ΔruvAB, ΔrecG, ΔradA) activity. RarA contributes to RecA thread formation, that are thought to be the active forms of RecA during homology search. The absence of RarA reduced RecA accumulation, and the formation of visible RecA threads in vivo upon DNA damage. When ΔrarA was combined with mutations in genuine RecA accessory genes, RecA accumulation was further reduced in ΔrarA ΔrecU and ΔrarA ΔrecX double mutant cells, and was blocked in ΔrarA recF15 cells. These results suggest that RarA contributes to the assembly of RecA nucleoprotein filaments onto single-stranded DNA, and possibly antagonizes RecA filament disassembly.
Hector Romero; Ester Serrano; Rogelio Hernandez-Tamayo; Begoña Carrasco; Paula P. Cárdenas; Silvia Ayora; Peter L. Graumann; Juan C. Alonso. Bacillus subtilis RarA Acts as a Positive RecA Accessory Protein. Frontiers in Microbiology 2020, 11, 92 .
AMA StyleHector Romero, Ester Serrano, Rogelio Hernandez-Tamayo, Begoña Carrasco, Paula P. Cárdenas, Silvia Ayora, Peter L. Graumann, Juan C. Alonso. Bacillus subtilis RarA Acts as a Positive RecA Accessory Protein. Frontiers in Microbiology. 2020; 11 ():92.
Chicago/Turabian StyleHector Romero; Ester Serrano; Rogelio Hernandez-Tamayo; Begoña Carrasco; Paula P. Cárdenas; Silvia Ayora; Peter L. Graumann; Juan C. Alonso. 2020. "Bacillus subtilis RarA Acts as a Positive RecA Accessory Protein." Frontiers in Microbiology 11, no. : 92.
A proteolyzed bacteriophage (phage) might release its DNA into the environment. Here, we define the recombination functions required to resurrect an infective lytic phage from inactive environmental viral DNA in naturally competent Bacillus subtilis cells. Using phage SPP1 DNA, a model that accounts for the obtained data is proposed: i) the DNA uptake apparatus takes up environmental SPP1 DNA, fragments it, and incorporates into the cytosol different linear single‐stranded (ss) DNA molecules shorter than genome‐length; ii) the SsbA‐DprA mediator loads RecA onto any fragmented linear SPP1 ssDNA, but negative modulators (RecX and RecU) promote a net RecA disassembly from these ssDNAs not homologous to the host genome; iii) single strand annealing (SSA) proteins, DprA and RecO, anneal the SsbA‐ or SsbB‐coated complementary strands, yielding tailed SPP1 duplex intermediates; iv) RecA polymerized on these tailed intermediates invades a homologous region in another incomplete molecule, and in concert with RecD2 helicase, reconstitutes a complete linear phage genome with redundant regions at the ends of the molecule; and v) DprA, RecO or viral G35P SSA, may catalyze the annealing of these terminally redundant regions, alone or with the help of an exonuclease, to produce a circular unit‐length duplex viral genome ready to initiate replication. This article is protected by copyright. All rights reserved.
Ester Serrano; Cristina Ramos; Silvia Ayora; Juan C. Alonso. Viral SPP1 DNA is infectious in naturally competentBacillus subtiliscells: inter‐ and intramolecular recombination pathways. Environmental Microbiology 2019, 22, 714 -725.
AMA StyleEster Serrano, Cristina Ramos, Silvia Ayora, Juan C. Alonso. Viral SPP1 DNA is infectious in naturally competentBacillus subtiliscells: inter‐ and intramolecular recombination pathways. Environmental Microbiology. 2019; 22 (2):714-725.
Chicago/Turabian StyleEster Serrano; Cristina Ramos; Silvia Ayora; Juan C. Alonso. 2019. "Viral SPP1 DNA is infectious in naturally competentBacillus subtiliscells: inter‐ and intramolecular recombination pathways." Environmental Microbiology 22, no. 2: 714-725.
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.
Rubén Torres; Begoña Carrasco; Carolina Gándara; Amit K Baidya; Sigal Ben-Yehuda; Juan C Alonso. Bacillus subtilis DisA regulates RecA-mediated DNA strand exchange. Nucleic Acids Research 2019, 47, 5141 -5154.
AMA StyleRubén Torres, Begoña Carrasco, Carolina Gándara, Amit K Baidya, Sigal Ben-Yehuda, Juan C Alonso. Bacillus subtilis DisA regulates RecA-mediated DNA strand exchange. Nucleic Acids Research. 2019; 47 (10):5141-5154.
Chicago/Turabian StyleRubén Torres; Begoña Carrasco; Carolina Gándara; Amit K Baidya; Sigal Ben-Yehuda; Juan C Alonso. 2019. "Bacillus subtilis DisA regulates RecA-mediated DNA strand exchange." Nucleic Acids Research 47, no. 10: 5141-5154.
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.
Hector Romero; Rubén Torres; Rogelio Hernández-Tamayo; Begoña Carrasco; Silvia Ayora; Peter L. Graumann; Juan C. Alonso. Bacillus subtilis RarA acts at the interplay between replication and repair-by-recombination. DNA Repair 2019, 78, 27 -36.
AMA StyleHector Romero, Rubén Torres, Rogelio Hernández-Tamayo, Begoña Carrasco, Silvia Ayora, Peter L. Graumann, Juan C. Alonso. Bacillus subtilis RarA acts at the interplay between replication and repair-by-recombination. DNA Repair. 2019; 78 ():27-36.
Chicago/Turabian StyleHector Romero; Rubén Torres; Rogelio Hernández-Tamayo; Begoña Carrasco; Silvia Ayora; Peter L. Graumann; Juan C. Alonso. 2019. "Bacillus subtilis RarA acts at the interplay between replication and repair-by-recombination." DNA Repair 78, no. : 27-36.
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 (K104 A and K104R) or KNRFG (K255 A 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 K255 A, K255R and wild type RadA/Sms reduced the diadenylate cyclase activity of DisA, whereas RadA/Sms K104 A 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.
Rubén Torres; Ester Serrano; Kristina Tramm; Juan C. Alonso. Bacillus subtilis RadA/Sms contributes to chromosomal transformation and DNA repair in concert with RecA and circumvents replicative stress in concert with DisA. DNA Repair 2019, 77, 45 -57.
AMA StyleRubén Torres, Ester Serrano, Kristina Tramm, Juan C. Alonso. Bacillus subtilis RadA/Sms contributes to chromosomal transformation and DNA repair in concert with RecA and circumvents replicative stress in concert with DisA. DNA Repair. 2019; 77 ():45-57.
Chicago/Turabian StyleRubén Torres; Ester Serrano; Kristina Tramm; Juan C. Alonso. 2019. "Bacillus subtilis RadA/Sms contributes to chromosomal transformation and DNA repair in concert with RecA and circumvents replicative stress in concert with DisA." DNA Repair 77, no. : 45-57.
RarA is a widely conserved protein proposed to be involved in recombination-dependent replication. We present a cell biological approach to identify functional connections between RarA and other proteins using single molecule tracking. We found that 50% of RarA molecules were static, mostly close to replication forks and likely DNA-bound, while the remaining fraction was highly dynamic throughout the cells. RarA alternated between static and dynamic states. Exposure to H2O2 increased the fraction of dynamic molecules, but not treatment with mitomycin C or with methyl methanesulfonate, which was exacerbated by the absence of RecJ, RecD2, RecS and RecU proteins. The ratio between static and dynamic RarA also changed in replication temperature-sensitive mutants, but in opposite manners, dependent upon inhibition of DnaB or of DnaC (pre)primosomal proteins, revealing an intricate function related to DNA replication restart. RarA likely acts in the context of collapsed replication forks, as well as in conjunction with a network of proteins that affect the activity of the RecA recombinase. Our novel approach reveals intricate interactions of RarA, and is widely applicable for in vivo protein studies, to underpin genetic or biochemical connections, and is especially helpful for investigating proteins whose absence does not lead to any detectable phenotype.
Hector Romero; Thomas C. Rösch; Rogelio Hernandez-Tamayo; Daniella Lucena; Silvia Ayora; Juan C. Alonso; Peter L. Graumann. Single molecule tracking reveals functions for RarA at replication forks but also independently from replication during DNA repair in Bacillus subtilis. Scientific Reports 2019, 9, 1 -13.
AMA StyleHector Romero, Thomas C. Rösch, Rogelio Hernandez-Tamayo, Daniella Lucena, Silvia Ayora, Juan C. Alonso, Peter L. Graumann. Single molecule tracking reveals functions for RarA at replication forks but also independently from replication during DNA repair in Bacillus subtilis. Scientific Reports. 2019; 9 (1):1-13.
Chicago/Turabian StyleHector Romero; Thomas C. Rösch; Rogelio Hernandez-Tamayo; Daniella Lucena; Silvia Ayora; Juan C. Alonso; Peter L. Graumann. 2019. "Single molecule tracking reveals functions for RarA at replication forks but also independently from replication during DNA repair in Bacillus subtilis." Scientific Reports 9, no. 1: 1-13.
The efficiency of horizontal gene transfer, which contributes to acquisition and spread of antibiotic resistance and pathogenicity traits, depends on nucleotide sequence and different mismatch-repair (MMR) proteins participate in this process. To study how MutL and MutS MMR proteins regulate recombination across species boundaries, we have studied natural chromosomal transformation with DNA up to ∼23% sequence divergence. We show that Bacillus subtilis natural chromosomal transformation decreased logarithmically with increased sequence divergence up to 15% in wild type (wt) cells or in cells lacking MutS2 or mismatch repair proteins (MutL, MutS or both). Beyond 15% sequence divergence, the chromosomal transformation efficiency is ∼100-fold higher in ΔmutS and ΔmutSL than in ΔmutS2 or wt cells. In the first phase of the biphasic curve (up to 15% sequence divergence), RecA-catalyzed DNA strand exchange contributes to the delineation of species, and in the second phase, homology-facilitated illegitimate recombination might aid in the restoration of inactivated genes. To understand how MutS modulates the integration process, we monitored DNA strand exchange reactions using a circular single-stranded DNA and a linear double-stranded DNA substrate with an internal 77-bp region with ∼16% or ∼54% sequence divergence in an otherwise homologous substrate. The former substrate delayed, whereas the latter halted RecA-mediated strand exchange. Interestingly, MutS addition overcame the heterologous barrier. We propose that MutS assists DNA strand exchange by facilitating RecA disassembly, and indirectly re-engagement with the homologous 5′-end of the linear duplex. Our data supports the idea that MutS modulates bidirectional RecA-mediated integration of divergent sequences and this is important for speciation.
Begoña Carrasco; Ester Serrano; Alejandro Martín-González; Fernando Moreno-Herrero; Juan C. Alonso. Bacillus subtilis MutS Modulates RecA-Mediated DNA Strand Exchange Between Divergent DNA Sequences. Frontiers in Microbiology 2019, 10, 237 .
AMA StyleBegoña Carrasco, Ester Serrano, Alejandro Martín-González, Fernando Moreno-Herrero, Juan C. Alonso. Bacillus subtilis MutS Modulates RecA-Mediated DNA Strand Exchange Between Divergent DNA Sequences. Frontiers in Microbiology. 2019; 10 ():237.
Chicago/Turabian StyleBegoña Carrasco; Ester Serrano; Alejandro Martín-González; Fernando Moreno-Herrero; Juan C. Alonso. 2019. "Bacillus subtilis MutS Modulates RecA-Mediated DNA Strand Exchange Between Divergent DNA Sequences." Frontiers in Microbiology 10, no. : 237.
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, Pi, 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.
María Moreno-Del Álamo; Mariangela Tabone; Juan Muñoz-Martínez; José R. Valverde; Juan C. Alonso. Toxin ζ Reduces the ATP and Modulates the Uridine Diphosphate-N-acetylglucosamine Pool. Toxins 2019, 11, 29 .
AMA StyleMaría Moreno-Del Álamo, Mariangela Tabone, Juan Muñoz-Martínez, José R. Valverde, Juan C. Alonso. Toxin ζ Reduces the ATP and Modulates the Uridine Diphosphate-N-acetylglucosamine Pool. Toxins. 2019; 11 (1):29.
Chicago/Turabian StyleMaría Moreno-Del Álamo; Mariangela Tabone; Juan Muñoz-Martínez; José R. Valverde; Juan C. Alonso. 2019. "Toxin ζ Reduces the ATP and Modulates the Uridine Diphosphate-N-acetylglucosamine Pool." Toxins 11, no. 1: 29.
During natural transformation Bacillus subtilis RecA, polymerized onto the incoming single-stranded (ss) DNA, catalyses DNA strand invasion resulting in a displacement loop (D-loop) intermediate. A null radA mutation impairs chromosomal transformation, and RadA/Sms unwinds forked DNA in the 5'→3' direction. We show that in the absence of RadA/Sms competent cells require the RecG translocase for natural chromosomal transformation. RadA/Sms tetracysteine motif (C13A and C13R) variants, which fail to interact with RecA, are also deficient in plasmid transformation, but this defect is suppressed by inactivating recA. The RadA/Sms C13A and C13R variants bind ssDNA, and this interaction stimulates their ATPase activity. Wild-type (wt) RadA/Sms interacts with and inhibits the ATPase activity of RecA, but RadA/Sms C13A fails to do it. RadA/Sms and its variants, C13A and C13R, bound to the 5'-tail of a DNA substrate, unwind DNA in the 5'→3' direction. RecA interacts with and loads wt RadA/Sms to promote unwinding of a non-cognate 3'-tailed or 5'-fork DNA substrate, but RadA/Sms C13A or C13R fail to do it. We propose that wt RadA/Sms interaction with RecA is crucial to recruit the former onto D-loop DNA, and both proteins in concert catalyse D-loop extension to favour integration of ssDNA during chromosomal transformation.
Rubén Torres; Ester Serrano; Juan C Alonso. Bacillus subtilis RecA interacts with and loads RadA/Sms to unwind recombination intermediates during natural chromosomal transformation. Nucleic Acids Research 2019, 47, 9198 -9215.
AMA StyleRubén Torres, Ester Serrano, Juan C Alonso. Bacillus subtilis RecA interacts with and loads RadA/Sms to unwind recombination intermediates during natural chromosomal transformation. Nucleic Acids Research. 2019; 47 (17):9198-9215.
Chicago/Turabian StyleRubén Torres; Ester Serrano; Juan C Alonso. 2019. "Bacillus subtilis RecA interacts with and loads RadA/Sms to unwind recombination intermediates during natural chromosomal transformation." Nucleic Acids Research 47, no. 17: 9198-9215.
Natural plasmid transformation plays an important role in the dissemination of antibiotic resistance genes in bacteria. During this process, Bacillus subtilis RecA physically interacts with RecU, RecX, and DprA. These three proteins are required for plasmid transformation, but RecA is not. In vitro, DprA recruits RecA onto SsbA-coated single-stranded (ss) DNA, whereas RecX inhibits RecA filament formation, leading to net filament disassembly. We show that a null recA (ΔrecA) mutation suppresses the plasmid transformation defect of competent ΔrecU cells, and that RecU is essential for both chromosomal and plasmid transformation in the ΔrecX context. RecU inhibits RecA filament growth and facilitates RecA disassembly from preformed filaments. Increasing SsbA concentrations additively contributes to RecU-mediated inhibition of RecA filament extension. DprA is necessary and sufficient to counteract the negative effect of both RecU and SsbA on RecA filament growth onto ssDNA. DprA-SsbA activates RecA to catalyze DNA strand exchange in the presence of RecU, but this effect was not observed if RecU was added prior to RecA. We propose that DprA contributes to RecA filament growth onto any internalized SsbA-coated ssDNA. When the ssDNA is homologous to the recipient, DprA antagonizes the inhibitory effect of RecU on RecA filament growth and helps RecA to catalyze chromosomal transformation. On the contrary, RecU promotes RecA filament disassembly from a heterologous (plasmid) ssDNA, overcoming an unsuccessful homology search and favoring plasmid transformation. The DprA–DprA interaction may promote strand annealing upon binding to the complementary plasmid strands and facilitating thereby plasmid transformation rather than through a mediation of RecA filament growth.
Ester Serrano; Begoña Carrasco; Jamie L. Gilmore; Kunio Takeyasu; Juan C. Alonso. RecA Regulation by RecU and DprA During Bacillus subtilis Natural Plasmid Transformation. Frontiers in Microbiology 2018, 9, 1514 .
AMA StyleEster Serrano, Begoña Carrasco, Jamie L. Gilmore, Kunio Takeyasu, Juan C. Alonso. RecA Regulation by RecU and DprA During Bacillus subtilis Natural Plasmid Transformation. Frontiers in Microbiology. 2018; 9 ():1514.
Chicago/Turabian StyleEster Serrano; Begoña Carrasco; Jamie L. Gilmore; Kunio Takeyasu; Juan C. Alonso. 2018. "RecA Regulation by RecU and DprA During Bacillus subtilis Natural Plasmid Transformation." Frontiers in Microbiology 9, no. : 1514.
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 ssDNA 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 to maintain genome integrity. Through its interaction with SsbA and with a preprimosomal component, RarA might impede the assembly of the replicative helicase, to prevent that recombination intermediates contribute to pathological DNA replication restart.
Begoña Carrasco; Elena M Seco; María López-Sanz; Juan C Alonso; Silvia Ayora. Bacillus subtilis RarA modulates replication restart. Nucleic Acids Research 2018, 46, 7206 -7220.
AMA StyleBegoña Carrasco, Elena M Seco, María López-Sanz, Juan C Alonso, Silvia Ayora. Bacillus subtilis RarA modulates replication restart. Nucleic Acids Research. 2018; 46 (14):7206-7220.
Chicago/Turabian StyleBegoña Carrasco; Elena M Seco; María López-Sanz; Juan C Alonso; Silvia Ayora. 2018. "Bacillus subtilis RarA modulates replication restart." Nucleic Acids Research 46, no. 14: 7206-7220.