J. Alonso | CSIC (Consejo Superior de Investigaciones Científicas-Spanish National Research Council) (original) (raw)

Papers by J. Alonso

Research paper thumbnail of Toxin–Antitoxin Systems in Pathogenic Bacteria

Toxins

Toxin–antitoxin (TA) systems, which are ubiquitously present in plasmids, bacterial and archaeal ... more 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 [...]

Research paper thumbnail of Bacillus subtilis PcrA Helicase Removes Trafficking Barriers

Cells

Bacillus subtilis PcrA interacts with the RNA polymerase and might contribute to mitigate replica... more 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 increase...

Research paper thumbnail of DisA Limits RecG Activities at Stalled or Reversed Replication Forks

Cells

The DNA damage checkpoint protein DisA and the branch migration translocase RecG are implicated i... more 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 reve...

Research paper thumbnail of Antitoxin ε Reverses Toxin ζ-Facilitated Ampicillin Dormants

Toxins

Toxin-antitoxin (TA) modules are ubiquitous in bacteria, but their biological importance in stres... more 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...

Research paper thumbnail of Bacillus subtilis RarA Acts as a Positive RecA Accessory Protein

Frontiers in Microbiology

Ubiquitous RarA AAA + ATPases play crucial roles in the cellular response to blocked replication ... more 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 recA, recO, and recF15 cells during unperturbed growth. The rarA gene was epistatic to recO and recF genes in response to H 2 O 2-or MMS-induced DNA damage. Conversely, the inactivation of rarA partially suppressed the HR defect of mutants lacking end-resection (addAB, recJ, recQ, recS) or branch migration (ruvAB, recG, radA) 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 rarA was combined with mutations in genuine RecA accessory genes, RecA accumulation was further reduced in rarA recU and rarA recX double mutant cells, and was blocked in rarA 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.

Research paper thumbnail of Toxin ζ Reduces the ATP and Modulates the Uridine Diphosphate-N-acetylglucosamine Pool

Toxins

Toxin ζ expression triggers a reversible state of dormancy, diminishes the pool of purine nucleot... more 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 E1...

Research paper thumbnail of RecA Regulation by RecU and DprA During Bacillus subtilis Natural Plasmid Transformation

Frontiers in Microbiology

Natural plasmid transformation plays an important role in the dissemination of antibiotic resista... more 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 singlestranded (ss) DNA, whereas RecX inhibits RecA filament formation, leading to net filament disassembly. We show that a null recA (recA) mutation suppresses the plasmid transformation defect of competent recU cells, and that RecU is essential for both chromosomal and plasmid transformation in the recX 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.

Research paper thumbnail of Toxin ζ Triggers a Survival Response to Cope with Stress and Persistence

Frontiers in microbiology, 2017

Bacteria have evolved complex regulatory controls in response to various environmental stresses. ... more Bacteria have evolved complex regulatory controls in response to various environmental stresses. Protein toxins of the ζ superfamily, found in prominent human pathogens, are broadly distributed in nature. We show that ζ is a uridine diphosphate-N-acetylglucosamine (UNAG)-dependent ATPase whose activity is inhibited in vitro by stoichiometric concentrations of ε2 antitoxin. In vivo, transient ζ expression promotes a reversible multi-level response by altering the pool of signaling purine nucleotides, which leads to growth arrest (dormancy), although a small cell subpopulation persists rather than tolerating toxin action. High c-di-AMP levels (absence of phosphodiesterase GdpP) decrease, and low c-di-AMP levels (absence of diadenylate cyclase DisA) increase the rate of ζ persistence. The absence of CodY, a transition regulator from exponential to stationary phase, sensitizes cells to toxin action, and suppresses persisters formed in the ΔdisA context. These changes, which do not affec...

Research paper thumbnail of The N-Terminal Region of the RecU Holliday Junction Resolvase Is Essential for Homologous Recombination

Journal of Molecular Biology, Jun 1, 2009

The RecU Holliday junction (HJ)-resolving enzyme is highly conserved in the Firmicutes phylum of ... more The RecU Holliday junction (HJ)-resolving enzyme is highly conserved in the Firmicutes phylum of bacteria. In Bacillus subtilis, the recU gene has two putative initiation codons, at positions 1 and 33. In rec + cells, only the fulllength RecU polypeptide (206 residues, 23.9 kDa) was detected even after different stress treatments. To address the relevance of the flexible Nterminus, we constructed mutant variants. Experiments in vivo revealed that recUΔ1-32 (which initiates at Met33 and encodes RecUΔ1-32) and recU31 (the conserved Arg31 residue was substituted with alanine to give RecUR31A) are genuine RecU mutants, rendering cells impaired in DNA repair and chromosomal segregation. RecU has three activities: It (i) cleaves HJs, (ii) anneals complementary strands and (iii) modulates RecA activities. RecUR31A binds and cleaves HJ DNA in vitro as efficiently as wild-type RecU, but RuvB•ATPγS•Mg 2+ fails to stimulate the RecUR31A cleavage reaction. In contrast, RecUΔ1-32 forms unstable complexes with DNA and fails to cleave HJs. RecU and its variants are capable of promoting DNA strand annealing and exert a negative effect on deoxy-ATP-dependent RecAmediated DNA strand exchange. This study shows that the flexible Nterminus of RecU is essential for protein activity.

Research paper thumbnail of ParAB Partition Dynamics in Firmicutes: Nucleoid Bound ParA Captures and Tethers ParB-Plasmid Complexes

PLOS ONE, 2015

In Firmicutes, small homodimeric ParA-like (δ 2) and ParB-like (ω 2) proteins, in concert with ci... more In Firmicutes, small homodimeric ParA-like (δ 2) and ParB-like (ω 2) proteins, in concert with cis-acting plasmid-borne parS and the host chromosome, secure stable plasmid inheritance in a growing bacterial population. This study shows that (ω:YFP) 2 binding to parSfacilitates plasmid clustering in the cytosol. (δ:GFP) 2 requires ATP binding but not hydrolysis to localize onto the cell's nucleoid as a fluorescent cloud. The interaction of (δ:CFP) 2 or δ 2 bound to the nucleoid with (ω:YFP) 2 foci facilitates plasmid capture, from a very broad distribution, towards the nucleoid and plasmid pairing. parS-bound ω 2 promotes redistribution of (δ: GFP) 2 , leading to the dynamic release of (δ:GFP) 2 from the nucleoid, in a process favored by ATP hydrolysis and protein-protein interaction. (δD60A:GFP) 2 , which binds but cannot hydrolyze ATP, also forms unstable complexes on the nucleoid. In the presence of ω 2 , (δD60A:GFP) 2 accumulates foci or patched structures on the nucleoid. We propose that (δ: GFP) 2 binding to different nucleoid regions and to ω 2-parS might generate (δ:GFP) 2 gradients that could direct plasmid movement. The iterative pairing and unpairing cycles may tether plasmids equidistantly on the nucleoid to ensure faithful plasmid segregation by a mechanism compatible with the diffusion-ratchet mechanism as proposed from in vitro reconstituted systems.

Research paper thumbnail of Toxin ζ Reversible Induces Dormancy and Reduces the UDP-N-Acetylglucosamine Pool as One of the Protective Responses to Cope with Stress

Toxins, 2014

Toxins of the δ/PezT family, found in the genome of major human pathogens, phosphorylate the pept... more Toxins of the δ/PezT family, found in the genome of major human pathogens, phosphorylate the peptidoglycan precursor uridine diphosphate-N-acetylglucosamine (UNAG) leading to unreactive UNAG-3P. Transient over-expression of a PezT variant impairs cell wall biosynthesis and triggers autolysis in Escherichia coli. Conversely, physiological levels of δ reversibly induce dormancy produce a sub-fraction of membrane-compromised cells, and a minor subpopulation of Bacillus subtilis cells become tolerant of toxin action. We report here that purified δ is a strong UNAG-dependent ATPase, being GTP a lower competitor. In vitro, δ toxin phosphorylates a fraction of UNAG. In vivo, δ-mediated inactivation of UNAG by phosphorylation does not deplete the active UNAG pool, because expression of the toxin enhances the efficacy of genuine cell wall inhibitors (fosfomycin, vancomycin or ampicillin). Transient δ expression together with fosfomycin treatment halt cell proliferation, but ε 2 antitoxin expression facilitates the exit of δ-induced dormancy, suggesting that there is sufficient UNAG for growth. We propose that δ induces diverse cellular responses to cope with stress, being the reduction of the UNAG pool one among them. If the action of δ is not inhibited, e.g., by de novo ε 2 antitoxin synthesis, the toxin markedly enhances the efficacy of antimicrobial treatment without massive autolysis in Firmicutes.

Research paper thumbnail of The Interplay between Different Stability Systems Contributes to Faithful Segregation: Streptococcus pyogenes pSM19035 as a Model

Microbiology Spectrum, 2014

The Streptococcus pyogenes pSM19035 low-copynumber θ-replicating plasmid encodes five segregation... more The Streptococcus pyogenes pSM19035 low-copynumber θ-replicating plasmid encodes five segregation (seg) loci that contribute to plasmid maintenance. These loci map outside of the minimal replicon. The segA locus comprises β 2 recombinase and two six sites, and segC includes segA and also the γ topoisomerase and two ssiA sites. Recombinase β 2 plays a role both in maximizing random segregation by resolving plasmid dimers (segA) and in catalyzing inversion between two inversely oriented six sites. segA, in concert with segC, facilitates replication fork pausing at ssiA sites and overcomes the accumulation of "toxic" replication intermediates. The segB1 locus encodes ω, ε, and ζ genes. The short-lived ε 2 antitoxin and the long-lived ζ toxin form an inactive ζε 2 ζ complex. Free ζ toxin halts cell proliferation upon decay of the ε 2 antitoxin and enhances survival. If ε 2 expression is not recovered, by loss of the plasmid, the toxin raises lethality. The segB2 locus comprises δ and ω genes and six parS sites. Proteins δ 2 and ω 2 , by forming complexes with parS and chromosomal DNA, pair the plasmid copies at the nucleoid, leading to the formation of a dynamic δ 2 gradient that separates the plasmids to ensure roughly equal distribution to daughter cells at cell division. The segD locus, which comprises ω 2 (or ω 2 plus ω2 2) and parS sites, coordinates expression of genes that control copy number, better-than-random segregation, faithful partition, and antibiotic resistance. The interplay of the seg loci and with the rep locus facilitates almost absolute plasmid stability.

Research paper thumbnail of Role of Toxin ζ and Starvation Responses in the Sensitivity to Antimicrobials

PLoS ONE, 2014

A fraction of otherwise antimicrobial-sensitive Bacillus subtilis cells, called persisters, are p... more A fraction of otherwise antimicrobial-sensitive Bacillus subtilis cells, called persisters, are phenotypically tolerant of antimicrobial treatment. We report that, independently of B. subtilis' growth phase, transient f toxin expression induces a dormant state and alters cellular responses so that cells are more sensitive to antimicrobials with different modes of action. This outcome is modulated by fine tuning (p)ppGpp and GTP levels: i) in the presence of low ''dysregulated'' (p)ppGpp levels (as in relA 2 cells) hyper-tolerance to both toxin and antimicrobials was observed; ii) physiological or low (p)ppGpp levels (as in the wild-type, sasA 2 , sasB 2 and relA 2 sasA 2 context) show a normal toxin and antimicrobial tolerance; and iii) lower levels (in relA 2 sasB 2) or absence of (p)ppGpp (in the relA 2 sasA 2 sasB 2 context), in concert with elevated GTP levels, potentiate the efficacy of both toxin and antimicrobial action, rendering tolerance vulnerable to eradication.

Research paper thumbnail of The RecU Holliday junction resolvase acts at early stages of homologous recombination

Nucleic Acids Research, 2008

Homologous recombination is essential for DNA repair and generation of genetic diversity in all o... more Homologous recombination is essential for DNA repair and generation of genetic diversity in all organisms. It occurs through a series of presynaptic steps where the substrate is presented to the recombinase (RecA in bacteria). Then, the recombinase nucleoprotein filament mediates synapsis by first promoting the formation of a D-loop and later of a Holliday junction (HJ) that is subsequently cleaved by the HJ resolvase. The coordination of the synaptic step with the late resolution step is poorly understood. Bacillus subtilis RecU catalyzes resolution of HJs, and biochemical evidence suggests that it might modulate RecA. We report here the isolation and characterization of two mutants of RecU (recU56 and recU71), which promote resolution of HJs, but do not promote RecA modulation. In vitro, the RecU mutant proteins (RecUK56A or RecUR71A) bind and cleave HJs and interact with RuvB. RecU interacts with RecA and inhibits its single-stranded DNA-dependent dATP hydrolysis, but RecUK56A an...

Research paper thumbnail of Molecular anatomy of the Streptococcus pyogenes pSM19035 partition and segrosome complexes

Nucleic Acids Research, 2011

Vancomycin or erythromycin resistance and the stability determinants, du and uef, of Enterococci ... more Vancomycin or erythromycin resistance and the stability determinants, du and uef, of Enterococci and Streptococci plasmids are genetically linked. To unravel the mechanisms that promoted the stable persistence of resistance determinants, the early stages of Streptococcus pyogenes pSM19035 partitioning were biochemically dissected. First, the homodimeric centromere-binding protein, u 2 , bound parS DNA to form a short-lived partition complex 1 (PC1). The interaction of PC1 with homodimeric d [d 2 even in the apo form (Apo-d 2)], significantly stimulated the formation of a long-lived u 2 • parS complex (PC2) without spreading into neighbouring DNA sequences. In the ATP • Mg 2+ bound form, d 2 bound DNA, without sequence specificity, to form a transient dynamic complex (DC). Second, parS bound u 2 interacted with and promoted d 2 redistribution to co-localize with the PC2, leading to transient segrosome complex (SC, parS • u 2 • d 2) formation. Third, d 2 , in the SC, interacted with a second SC and promoted formation of a bridging complex (BC). Finally, increasing u 2 concentrations stimulated the ATPase activity of d 2 and the BC was disassembled. We propose that PC, DC, SC and BC formation were dynamic processes and that the molar u 2 :d 2 ratio and parS DNA control their temporal and spatial assembly during partition of pSM19035 before cell division.

Research paper thumbnail of Bacillus subtilis RecA and its accessory factors, RecF, RecO, RecR and RecX, are required for spore resistance to DNA double-strand break

Nucleic Acids Research, 2014

Bacillus subtilis RecA is important for spore resistance to DNA damage, even though spores contai... more Bacillus subtilis RecA is important for spore resistance to DNA damage, even though spores contain a single non-replicating genome. We report that inactivation of RecA or its accessory factors, RecF, RecO, RecR and RecX, drastically reduce survival of mature dormant spores to ultrahigh vacuum desiccation and ionizing radiation that induce single strand (ss) DNA nicks and double-strand breaks (DSBs). The presence of non-cleavable LexA renders spores less sensitive to DSBs, and spores impaired in DSB recognition or end-processing show sensitivities to X-rays similar to wild-type. In vitro RecA cannot compete with SsbA for nucleation onto ssDNA in the presence of ATP. RecO is sufficient, at least in vitro, to overcome SsbA inhibition and stimulate RecA polymerization on SsbA-coated ssDNA. In the presence of SsbA, RecA slightly affects DNA replication in vitro, but addition of RecO facilitates RecA-mediated inhibition of DNA synthesis. We propose that repairing of the DNA lesions genera...

Research paper thumbnail of The N-Terminal Region of the RecU Holliday Junction Resolvase Is Essential for Homologous Recombination

Journal of Molecular Biology, 2009

The RecU Holliday junction (HJ)-resolving enzyme is highly conserved in the Firmicutes phylum of ... more The RecU Holliday junction (HJ)-resolving enzyme is highly conserved in the Firmicutes phylum of bacteria. In Bacillus subtilis, the recU gene has two putative initiation codons, at positions 1 and 33. In rec + cells, only the fulllength RecU polypeptide (206 residues, 23.9 kDa) was detected even after different stress treatments. To address the relevance of the flexible Nterminus, we constructed mutant variants. Experiments in vivo revealed that recUΔ1-32 (which initiates at Met33 and encodes RecUΔ1-32) and recU31 (the conserved Arg31 residue was substituted with alanine to give RecUR31A) are genuine RecU mutants, rendering cells impaired in DNA repair and chromosomal segregation. RecU has three activities: It (i) cleaves HJs, (ii) anneals complementary strands and (iii) modulates RecA activities. RecUR31A binds and cleaves HJ DNA in vitro as efficiently as wild-type RecU, but RuvB•ATPγS•Mg 2+ fails to stimulate the RecUR31A cleavage reaction. In contrast, RecUΔ1-32 forms unstable complexes with DNA and fails to cleave HJs. RecU and its variants are capable of promoting DNA strand annealing and exert a negative effect on deoxy-ATP-dependent RecAmediated DNA strand exchange. This study shows that the flexible Nterminus of RecU is essential for protein activity.

Research paper thumbnail of Bacillus subtilis RecO Nucleates RecA onto SsbA-coated Single-stranded DNA

Journal of Biological Chemistry, 2008

Research paper thumbnail of Bacillus subtilis RecU protein cleaves Holliday junctions and anneals single-stranded DNA

Proceedings of the …, 2004

Research paper thumbnail of The cell pole: the site of cross talk between the DNA uptake and genetic recombination machinery

Critical Reviews in Biochemistry and Molecular Biology, 2012

Natural transformation is a programmed mechanism characterized by binding of free double-stranded... more Natural transformation is a programmed mechanism characterized by binding of free double-stranded (ds) DNA from the environment to the cell pole in rod-shaped bacteria. In Bacillus subtilis some competence proteins, which process the dsDNA and translocate single-stranded (ss) DNA into the cytosol, recruit a set of recombination proteins mainly to one of the cell poles. A subset of single-stranded binding proteins, working as "guardians", protects ssDNA from degradation and limit the RecA recombinase loading. Then, the "mediators" overcome the inhibitory role of guardians, and recruit RecA onto ssDNA. A RecA•ssDNA filament searches for homology on the chromosome and, in a process that is controlled by "modulators", catalyzes strand invasion with the generation of a displacement loop (D-loop). A D-loop resolvase or "resolver" cleaves this intermediate, limited DNA replication restores missing information and a DNA ligase seals the DNA ends. However, if any step fails, the "rescuers" will repair the broken end to rescue chromosomal transformation. If the ssDNA does not share homology with resident DNA, but it contains information for autonomous replication, guardian and mediator proteins catalyze plasmid establishment after inhibition of RecA. DNA replication and ligation reconstitute the molecule (plasmid transformation). In this review, the interacting network that leads to a cross talk between proteins of the uptake and genetic recombination machinery will be placed into prospective.

Research paper thumbnail of Toxin–Antitoxin Systems in Pathogenic Bacteria

Toxins

Toxin–antitoxin (TA) systems, which are ubiquitously present in plasmids, bacterial and archaeal ... more 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 [...]

Research paper thumbnail of Bacillus subtilis PcrA Helicase Removes Trafficking Barriers

Cells

Bacillus subtilis PcrA interacts with the RNA polymerase and might contribute to mitigate replica... more 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 increase...

Research paper thumbnail of DisA Limits RecG Activities at Stalled or Reversed Replication Forks

Cells

The DNA damage checkpoint protein DisA and the branch migration translocase RecG are implicated i... more 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 reve...

Research paper thumbnail of Antitoxin ε Reverses Toxin ζ-Facilitated Ampicillin Dormants

Toxins

Toxin-antitoxin (TA) modules are ubiquitous in bacteria, but their biological importance in stres... more 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...

Research paper thumbnail of Bacillus subtilis RarA Acts as a Positive RecA Accessory Protein

Frontiers in Microbiology

Ubiquitous RarA AAA + ATPases play crucial roles in the cellular response to blocked replication ... more 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 recA, recO, and recF15 cells during unperturbed growth. The rarA gene was epistatic to recO and recF genes in response to H 2 O 2-or MMS-induced DNA damage. Conversely, the inactivation of rarA partially suppressed the HR defect of mutants lacking end-resection (addAB, recJ, recQ, recS) or branch migration (ruvAB, recG, radA) 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 rarA was combined with mutations in genuine RecA accessory genes, RecA accumulation was further reduced in rarA recU and rarA recX double mutant cells, and was blocked in rarA 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.

Research paper thumbnail of Toxin ζ Reduces the ATP and Modulates the Uridine Diphosphate-N-acetylglucosamine Pool

Toxins

Toxin ζ expression triggers a reversible state of dormancy, diminishes the pool of purine nucleot... more 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 E1...

Research paper thumbnail of RecA Regulation by RecU and DprA During Bacillus subtilis Natural Plasmid Transformation

Frontiers in Microbiology

Natural plasmid transformation plays an important role in the dissemination of antibiotic resista... more 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 singlestranded (ss) DNA, whereas RecX inhibits RecA filament formation, leading to net filament disassembly. We show that a null recA (recA) mutation suppresses the plasmid transformation defect of competent recU cells, and that RecU is essential for both chromosomal and plasmid transformation in the recX 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.

Research paper thumbnail of Toxin ζ Triggers a Survival Response to Cope with Stress and Persistence

Frontiers in microbiology, 2017

Bacteria have evolved complex regulatory controls in response to various environmental stresses. ... more Bacteria have evolved complex regulatory controls in response to various environmental stresses. Protein toxins of the ζ superfamily, found in prominent human pathogens, are broadly distributed in nature. We show that ζ is a uridine diphosphate-N-acetylglucosamine (UNAG)-dependent ATPase whose activity is inhibited in vitro by stoichiometric concentrations of ε2 antitoxin. In vivo, transient ζ expression promotes a reversible multi-level response by altering the pool of signaling purine nucleotides, which leads to growth arrest (dormancy), although a small cell subpopulation persists rather than tolerating toxin action. High c-di-AMP levels (absence of phosphodiesterase GdpP) decrease, and low c-di-AMP levels (absence of diadenylate cyclase DisA) increase the rate of ζ persistence. The absence of CodY, a transition regulator from exponential to stationary phase, sensitizes cells to toxin action, and suppresses persisters formed in the ΔdisA context. These changes, which do not affec...

Research paper thumbnail of The N-Terminal Region of the RecU Holliday Junction Resolvase Is Essential for Homologous Recombination

Journal of Molecular Biology, Jun 1, 2009

The RecU Holliday junction (HJ)-resolving enzyme is highly conserved in the Firmicutes phylum of ... more The RecU Holliday junction (HJ)-resolving enzyme is highly conserved in the Firmicutes phylum of bacteria. In Bacillus subtilis, the recU gene has two putative initiation codons, at positions 1 and 33. In rec + cells, only the fulllength RecU polypeptide (206 residues, 23.9 kDa) was detected even after different stress treatments. To address the relevance of the flexible Nterminus, we constructed mutant variants. Experiments in vivo revealed that recUΔ1-32 (which initiates at Met33 and encodes RecUΔ1-32) and recU31 (the conserved Arg31 residue was substituted with alanine to give RecUR31A) are genuine RecU mutants, rendering cells impaired in DNA repair and chromosomal segregation. RecU has three activities: It (i) cleaves HJs, (ii) anneals complementary strands and (iii) modulates RecA activities. RecUR31A binds and cleaves HJ DNA in vitro as efficiently as wild-type RecU, but RuvB•ATPγS•Mg 2+ fails to stimulate the RecUR31A cleavage reaction. In contrast, RecUΔ1-32 forms unstable complexes with DNA and fails to cleave HJs. RecU and its variants are capable of promoting DNA strand annealing and exert a negative effect on deoxy-ATP-dependent RecAmediated DNA strand exchange. This study shows that the flexible Nterminus of RecU is essential for protein activity.

Research paper thumbnail of ParAB Partition Dynamics in Firmicutes: Nucleoid Bound ParA Captures and Tethers ParB-Plasmid Complexes

PLOS ONE, 2015

In Firmicutes, small homodimeric ParA-like (δ 2) and ParB-like (ω 2) proteins, in concert with ci... more In Firmicutes, small homodimeric ParA-like (δ 2) and ParB-like (ω 2) proteins, in concert with cis-acting plasmid-borne parS and the host chromosome, secure stable plasmid inheritance in a growing bacterial population. This study shows that (ω:YFP) 2 binding to parSfacilitates plasmid clustering in the cytosol. (δ:GFP) 2 requires ATP binding but not hydrolysis to localize onto the cell's nucleoid as a fluorescent cloud. The interaction of (δ:CFP) 2 or δ 2 bound to the nucleoid with (ω:YFP) 2 foci facilitates plasmid capture, from a very broad distribution, towards the nucleoid and plasmid pairing. parS-bound ω 2 promotes redistribution of (δ: GFP) 2 , leading to the dynamic release of (δ:GFP) 2 from the nucleoid, in a process favored by ATP hydrolysis and protein-protein interaction. (δD60A:GFP) 2 , which binds but cannot hydrolyze ATP, also forms unstable complexes on the nucleoid. In the presence of ω 2 , (δD60A:GFP) 2 accumulates foci or patched structures on the nucleoid. We propose that (δ: GFP) 2 binding to different nucleoid regions and to ω 2-parS might generate (δ:GFP) 2 gradients that could direct plasmid movement. The iterative pairing and unpairing cycles may tether plasmids equidistantly on the nucleoid to ensure faithful plasmid segregation by a mechanism compatible with the diffusion-ratchet mechanism as proposed from in vitro reconstituted systems.

Research paper thumbnail of Toxin ζ Reversible Induces Dormancy and Reduces the UDP-N-Acetylglucosamine Pool as One of the Protective Responses to Cope with Stress

Toxins, 2014

Toxins of the δ/PezT family, found in the genome of major human pathogens, phosphorylate the pept... more Toxins of the δ/PezT family, found in the genome of major human pathogens, phosphorylate the peptidoglycan precursor uridine diphosphate-N-acetylglucosamine (UNAG) leading to unreactive UNAG-3P. Transient over-expression of a PezT variant impairs cell wall biosynthesis and triggers autolysis in Escherichia coli. Conversely, physiological levels of δ reversibly induce dormancy produce a sub-fraction of membrane-compromised cells, and a minor subpopulation of Bacillus subtilis cells become tolerant of toxin action. We report here that purified δ is a strong UNAG-dependent ATPase, being GTP a lower competitor. In vitro, δ toxin phosphorylates a fraction of UNAG. In vivo, δ-mediated inactivation of UNAG by phosphorylation does not deplete the active UNAG pool, because expression of the toxin enhances the efficacy of genuine cell wall inhibitors (fosfomycin, vancomycin or ampicillin). Transient δ expression together with fosfomycin treatment halt cell proliferation, but ε 2 antitoxin expression facilitates the exit of δ-induced dormancy, suggesting that there is sufficient UNAG for growth. We propose that δ induces diverse cellular responses to cope with stress, being the reduction of the UNAG pool one among them. If the action of δ is not inhibited, e.g., by de novo ε 2 antitoxin synthesis, the toxin markedly enhances the efficacy of antimicrobial treatment without massive autolysis in Firmicutes.

Research paper thumbnail of The Interplay between Different Stability Systems Contributes to Faithful Segregation: Streptococcus pyogenes pSM19035 as a Model

Microbiology Spectrum, 2014

The Streptococcus pyogenes pSM19035 low-copynumber θ-replicating plasmid encodes five segregation... more The Streptococcus pyogenes pSM19035 low-copynumber θ-replicating plasmid encodes five segregation (seg) loci that contribute to plasmid maintenance. These loci map outside of the minimal replicon. The segA locus comprises β 2 recombinase and two six sites, and segC includes segA and also the γ topoisomerase and two ssiA sites. Recombinase β 2 plays a role both in maximizing random segregation by resolving plasmid dimers (segA) and in catalyzing inversion between two inversely oriented six sites. segA, in concert with segC, facilitates replication fork pausing at ssiA sites and overcomes the accumulation of "toxic" replication intermediates. The segB1 locus encodes ω, ε, and ζ genes. The short-lived ε 2 antitoxin and the long-lived ζ toxin form an inactive ζε 2 ζ complex. Free ζ toxin halts cell proliferation upon decay of the ε 2 antitoxin and enhances survival. If ε 2 expression is not recovered, by loss of the plasmid, the toxin raises lethality. The segB2 locus comprises δ and ω genes and six parS sites. Proteins δ 2 and ω 2 , by forming complexes with parS and chromosomal DNA, pair the plasmid copies at the nucleoid, leading to the formation of a dynamic δ 2 gradient that separates the plasmids to ensure roughly equal distribution to daughter cells at cell division. The segD locus, which comprises ω 2 (or ω 2 plus ω2 2) and parS sites, coordinates expression of genes that control copy number, better-than-random segregation, faithful partition, and antibiotic resistance. The interplay of the seg loci and with the rep locus facilitates almost absolute plasmid stability.

Research paper thumbnail of Role of Toxin ζ and Starvation Responses in the Sensitivity to Antimicrobials

PLoS ONE, 2014

A fraction of otherwise antimicrobial-sensitive Bacillus subtilis cells, called persisters, are p... more A fraction of otherwise antimicrobial-sensitive Bacillus subtilis cells, called persisters, are phenotypically tolerant of antimicrobial treatment. We report that, independently of B. subtilis' growth phase, transient f toxin expression induces a dormant state and alters cellular responses so that cells are more sensitive to antimicrobials with different modes of action. This outcome is modulated by fine tuning (p)ppGpp and GTP levels: i) in the presence of low ''dysregulated'' (p)ppGpp levels (as in relA 2 cells) hyper-tolerance to both toxin and antimicrobials was observed; ii) physiological or low (p)ppGpp levels (as in the wild-type, sasA 2 , sasB 2 and relA 2 sasA 2 context) show a normal toxin and antimicrobial tolerance; and iii) lower levels (in relA 2 sasB 2) or absence of (p)ppGpp (in the relA 2 sasA 2 sasB 2 context), in concert with elevated GTP levels, potentiate the efficacy of both toxin and antimicrobial action, rendering tolerance vulnerable to eradication.

Research paper thumbnail of The RecU Holliday junction resolvase acts at early stages of homologous recombination

Nucleic Acids Research, 2008

Homologous recombination is essential for DNA repair and generation of genetic diversity in all o... more Homologous recombination is essential for DNA repair and generation of genetic diversity in all organisms. It occurs through a series of presynaptic steps where the substrate is presented to the recombinase (RecA in bacteria). Then, the recombinase nucleoprotein filament mediates synapsis by first promoting the formation of a D-loop and later of a Holliday junction (HJ) that is subsequently cleaved by the HJ resolvase. The coordination of the synaptic step with the late resolution step is poorly understood. Bacillus subtilis RecU catalyzes resolution of HJs, and biochemical evidence suggests that it might modulate RecA. We report here the isolation and characterization of two mutants of RecU (recU56 and recU71), which promote resolution of HJs, but do not promote RecA modulation. In vitro, the RecU mutant proteins (RecUK56A or RecUR71A) bind and cleave HJs and interact with RuvB. RecU interacts with RecA and inhibits its single-stranded DNA-dependent dATP hydrolysis, but RecUK56A an...

Research paper thumbnail of Molecular anatomy of the Streptococcus pyogenes pSM19035 partition and segrosome complexes

Nucleic Acids Research, 2011

Vancomycin or erythromycin resistance and the stability determinants, du and uef, of Enterococci ... more Vancomycin or erythromycin resistance and the stability determinants, du and uef, of Enterococci and Streptococci plasmids are genetically linked. To unravel the mechanisms that promoted the stable persistence of resistance determinants, the early stages of Streptococcus pyogenes pSM19035 partitioning were biochemically dissected. First, the homodimeric centromere-binding protein, u 2 , bound parS DNA to form a short-lived partition complex 1 (PC1). The interaction of PC1 with homodimeric d [d 2 even in the apo form (Apo-d 2)], significantly stimulated the formation of a long-lived u 2 • parS complex (PC2) without spreading into neighbouring DNA sequences. In the ATP • Mg 2+ bound form, d 2 bound DNA, without sequence specificity, to form a transient dynamic complex (DC). Second, parS bound u 2 interacted with and promoted d 2 redistribution to co-localize with the PC2, leading to transient segrosome complex (SC, parS • u 2 • d 2) formation. Third, d 2 , in the SC, interacted with a second SC and promoted formation of a bridging complex (BC). Finally, increasing u 2 concentrations stimulated the ATPase activity of d 2 and the BC was disassembled. We propose that PC, DC, SC and BC formation were dynamic processes and that the molar u 2 :d 2 ratio and parS DNA control their temporal and spatial assembly during partition of pSM19035 before cell division.

Research paper thumbnail of Bacillus subtilis RecA and its accessory factors, RecF, RecO, RecR and RecX, are required for spore resistance to DNA double-strand break

Nucleic Acids Research, 2014

Bacillus subtilis RecA is important for spore resistance to DNA damage, even though spores contai... more Bacillus subtilis RecA is important for spore resistance to DNA damage, even though spores contain a single non-replicating genome. We report that inactivation of RecA or its accessory factors, RecF, RecO, RecR and RecX, drastically reduce survival of mature dormant spores to ultrahigh vacuum desiccation and ionizing radiation that induce single strand (ss) DNA nicks and double-strand breaks (DSBs). The presence of non-cleavable LexA renders spores less sensitive to DSBs, and spores impaired in DSB recognition or end-processing show sensitivities to X-rays similar to wild-type. In vitro RecA cannot compete with SsbA for nucleation onto ssDNA in the presence of ATP. RecO is sufficient, at least in vitro, to overcome SsbA inhibition and stimulate RecA polymerization on SsbA-coated ssDNA. In the presence of SsbA, RecA slightly affects DNA replication in vitro, but addition of RecO facilitates RecA-mediated inhibition of DNA synthesis. We propose that repairing of the DNA lesions genera...

Research paper thumbnail of The N-Terminal Region of the RecU Holliday Junction Resolvase Is Essential for Homologous Recombination

Journal of Molecular Biology, 2009

The RecU Holliday junction (HJ)-resolving enzyme is highly conserved in the Firmicutes phylum of ... more The RecU Holliday junction (HJ)-resolving enzyme is highly conserved in the Firmicutes phylum of bacteria. In Bacillus subtilis, the recU gene has two putative initiation codons, at positions 1 and 33. In rec + cells, only the fulllength RecU polypeptide (206 residues, 23.9 kDa) was detected even after different stress treatments. To address the relevance of the flexible Nterminus, we constructed mutant variants. Experiments in vivo revealed that recUΔ1-32 (which initiates at Met33 and encodes RecUΔ1-32) and recU31 (the conserved Arg31 residue was substituted with alanine to give RecUR31A) are genuine RecU mutants, rendering cells impaired in DNA repair and chromosomal segregation. RecU has three activities: It (i) cleaves HJs, (ii) anneals complementary strands and (iii) modulates RecA activities. RecUR31A binds and cleaves HJ DNA in vitro as efficiently as wild-type RecU, but RuvB•ATPγS•Mg 2+ fails to stimulate the RecUR31A cleavage reaction. In contrast, RecUΔ1-32 forms unstable complexes with DNA and fails to cleave HJs. RecU and its variants are capable of promoting DNA strand annealing and exert a negative effect on deoxy-ATP-dependent RecAmediated DNA strand exchange. This study shows that the flexible Nterminus of RecU is essential for protein activity.

Research paper thumbnail of Bacillus subtilis RecO Nucleates RecA onto SsbA-coated Single-stranded DNA

Journal of Biological Chemistry, 2008

Research paper thumbnail of Bacillus subtilis RecU protein cleaves Holliday junctions and anneals single-stranded DNA

Proceedings of the …, 2004

Research paper thumbnail of The cell pole: the site of cross talk between the DNA uptake and genetic recombination machinery

Critical Reviews in Biochemistry and Molecular Biology, 2012

Natural transformation is a programmed mechanism characterized by binding of free double-stranded... more Natural transformation is a programmed mechanism characterized by binding of free double-stranded (ds) DNA from the environment to the cell pole in rod-shaped bacteria. In Bacillus subtilis some competence proteins, which process the dsDNA and translocate single-stranded (ss) DNA into the cytosol, recruit a set of recombination proteins mainly to one of the cell poles. A subset of single-stranded binding proteins, working as "guardians", protects ssDNA from degradation and limit the RecA recombinase loading. Then, the "mediators" overcome the inhibitory role of guardians, and recruit RecA onto ssDNA. A RecA•ssDNA filament searches for homology on the chromosome and, in a process that is controlled by "modulators", catalyzes strand invasion with the generation of a displacement loop (D-loop). A D-loop resolvase or "resolver" cleaves this intermediate, limited DNA replication restores missing information and a DNA ligase seals the DNA ends. However, if any step fails, the "rescuers" will repair the broken end to rescue chromosomal transformation. If the ssDNA does not share homology with resident DNA, but it contains information for autonomous replication, guardian and mediator proteins catalyze plasmid establishment after inhibition of RecA. DNA replication and ligation reconstitute the molecule (plasmid transformation). In this review, the interacting network that leads to a cross talk between proteins of the uptake and genetic recombination machinery will be placed into prospective.