The C-Terminal Domain of the Bacterial SSB Protein Acts as a DNA Maintenance Hub at Active Chromosome Replication Forks

We have investigated in vivo the role of the carboxy-terminal domain of the Bacillus subtilis Single-Stranded DNA Binding protein (SSBCter) as a recruitment platform at active chromosomal forks for many proteins of the genome maintenance machineries. We probed this SSBCter interactome using GFP fusions and by Tap-tag and biochemical analysis. It includes at least 12 proteins. The interactome was previously shown to include PriA, RecG, and RecQ and extended in this study by addition of DnaE, SbcC, RarA, RecJ, RecO, XseA, Ung, YpbB, and YrrC. Targeting of YpbB to active forks appears to depend on RecS, a RecQ paralogue, with which it forms a stable complex. Most of these SSB partners are conserved in bacteria, while others, such as the essential DNA polymerase DnaE, YrrC, and the YpbB/RecS complex, appear to be specific to B. subtilis. SSBCter deletion has a moderate impact on B. subtilis cell growth. However, it markedly affects the efficiency of repair of damaged genomic DNA and arrested replication forks. ssbΔCter mutant cells appear deficient in RecA loading on ssDNA, explaining their inefficiency in triggering the SOS response upon exposure to genotoxic agents. Together, our findings show that the bacterial SSBCter acts as a DNA maintenance hub at active chromosomal forks that secures their propagation along the genome

The lactococcal abortive infection protein AbiP is membrane-anchored and binds nucleic acids

AbstractAbiP, a lactococcal abortive phage infection system, has previously been shown to arrest phage bIL66M1 DNA replication around 10 min after infection and to inhibit the switch off of phage early transcripts. We report here the functional characterization and implication in the abortive infection phenotype of two domains identified in the AbiP sequence. We show that AbiP is a protein anchored to the membrane by an N-terminal membrane-spanning domain. Our results further suggest that membrane localization may be required for the anti-phage activity of AbiP. The remainder of the protein, which contains a putative nucleic acid binding domain, is shown to be located on the cytosolic side. Purified AbiP is shown to bind nucleic acids with an approximately 10-fold preference for RNA relative to ssDNA. AbiP interaction with both ssDNA and RNA molecules occurs in a sequence-independent manner. We have analyzed the effect of substitutions of aromatic and basic residues on the surface of the putative binding fold. In vitro and in vivo studies of these AbiP derivatives indicate that the previously reported effects on phage development might be dependent on the nucleic acid binding activity displayed by the membrane-bound protein

A distinct single-stranded DNA-binding protein encoded by the Lactococcus lactis bacteriophage bIL67

Single-stranded binding proteins (SSBs) are found to participate in various processes of DNA metabolism in all known organisms. We describe here a SSB protein encoded by the Lactococcus lactis phage bIL67 orf14 gene. It is the first noted attempt at characterizing a SSB protein from a lactococcal phage. The purified Orf14bIL67 binds unspecifically to ssDNA with the same high affinity as the canonical Bacillus subtilis SSB. Electrophoretic mobility-shift assays performed with mutagenized Orf14bIL67 protein derivatives suggest that ssDNA-binding occurs via a putative OB-fold structure predicted by three-dimensional modeling. The native Orf14bIL67 forms homotetramers as determined by gel filtration studies. These results allow distinguishing the first lactococcal phage protein with single-strand binding affinity, which defines a novel cluster of phage SSBs proteins. The possible role of Orf14bIL67 in phage multiplication cycle is also discussed

Assembly mechanism and cryoEM structure of RecA recombination nucleofilaments from Streptococcus pneumoniae

Abstract RecA-mediated Homologous Recombination (HR) is a key mechanism for genome maintenance and plasticity in bacteria. It proceeds through RecA assembly into a dynamic filament on ssDNA, the presynaptic filament, which mediates DNA homology search and ordered DNA strand exchange. Here, we combined structural, single molecule and biochemical approaches to characterize the ATP-dependent assembly mechanism of the presynaptic filament of RecA from Streptococcus pneumoniae ( Sp RecA), in comparison to the Escherichia coli RecA ( Ec RecA) paradigm. Ec RecA polymerization on ssDNA is assisted by the Single-Stranded DNA Binding (SSB) protein, which unwinds ssDNA secondary structures that block Ec RecA nucleofilament growth. We report that neither of the two paralogous pneumococcal SSBs could assist Sp RecA polymerization on ssDNA. Instead, we found that the conserved RadA helicase promotes this Sp RecA nucleofilamentation in an ATP-dependent manner. This allowed us to solve the atomic structure of such a long native Sp RecA nucleopolymer by cryoEM stabilized with ATPγS. It was found to be equivalent to the crystal structure of the Ec RecA filament with a marked difference in how RecA mediates nucleotide orientation in the stretched ssDNA. Then, our results show that Sp RecA and Ec RecA HR activities are different, in correlation with their distinct ATP-dependent ssDNA binding modes

Role of the Single-Stranded DNA–Binding Protein SsbB in Pneumococcal Transformation: Maintenance of a Reservoir for Genetic Plasticity

Bacteria encode a single-stranded DNA (ssDNA) binding protein (SSB) crucial for genome maintenance. In Bacillus subtilis and Streptococcus pneumoniae, an alternative SSB, SsbB, is expressed uniquely during competence for genetic transformation, but its precise role has been disappointingly obscure. Here, we report our investigations involving comparison of a null mutant (ssbB−) and a C-ter truncation (ssbBΔ7) of SsbB of S. pneumoniae, the latter constructed because SSBs' acidic tail has emerged as a key site for interactions with partner proteins. We provide evidence that SsbB directly protects internalized ssDNA. We show that SsbB is highly abundant, potentially allowing the binding of ∼1.15 Mb ssDNA (half a genome equivalent); that it participates in the processing of ssDNA into recombinants; and that, at high DNA concentration, it is of crucial importance for chromosomal transformation whilst antagonizing plasmid transformation. While the latter observation explains a long-standing observation that plasmid transformation is very inefficient in S. pneumoniae (compared to chromosomal transformation), the former supports our previous suggestion that SsbB creates a reservoir of ssDNA, allowing successive recombination cycles. SsbBΔ7 fulfils the reservoir function, suggesting that SsbB C-ter is not necessary for processing protein(s) to access stored ssDNA. We propose that the evolutionary raison d'être of SsbB and its abundance is maintenance of this reservoir, which contributes to the genetic plasticity of S. pneumoniae by increasing the likelihood of multiple transformation events in the same cell

Etude moléculaire de la protéine SSB de Bacillus subtilis (comparaison fonctionnelle avec son paralogue YwpH et mise en évidence de son interaction avec l'hélicase PriA du re-démarrage de la réplication)

Les protéines SSB sont essentielles à certains mécanismes cellulaires faisant intervenir l'ADN simple brin (ADNsb), tels que réplication, recombinaison ou réparation. Elles protègent et structurent l'ADNsb, rendant son utilisation efficace par différentes machineries qui y sont spécifiquement recrutées. Elles agissent aussi comme des connecteurs entre l'ADNsb et des partenaires protéiques avec lesquels elles interagissent. Il est récemment apparu que plusieurs protéines de type SSB pouvaient être présentes dans un même organisme, ce qui pose la question des fonctions exercées par chacune d'elles. En prenant comme modèle d'étude la bactérie Bacillus subtilis, nous avons entrepris la caractérisation comparée des deux protéines paralogues SSB et YwpH. YwpH a 80% de similarité avec le domaine de liaison à l'ADNsb de SSB, mais est dépourvue du domaine d'interaction protéine-protéine. Elle se lie à l'ADNsb et stimule certaines enzymes qui l'utilisent, avec néanmoins une efficacité inférieure à celle de SSB. Elle n'est pas essentielle et n'est détectée qu'en milieu minimum en phase stationnaire de croissance. Bien que sa fonction cellulaire ne soit pas encore élucidée, sa caractérisation biochimique a permis d'augmenter les connaissances sur cette protéine et d'avancer des hypothèses quant à son rôle. En parallèle, nous avons mis en évidence une nouvelle interaction physique et fonctionnelle entre SSB et PriA, protéine primaire du re-démarrage de la réplication. Nos résultats révèlent une implication majeure de SSB dans ce mécanisme particulier et un modèle dans lequel SSB permettrait le recrutement et l'ancrage de PriA à proximité de la fourche de réplication est proposé.SSB proteins are essential for cellular mechanisms using single stranded DNA (ssDNA), such as replication, recombination or DNA repair. They protect and structure the ssDNA, rendering it effective to be used by various proteins specifically recruited on it. In addition to that, SSB act as connectors between ssDNA and their interacting partners. Recently, it has been revealed that several SSB paralogs could be present within the same organism, raising the question of the functions exerted by each one of them. By using the Gram+ bacterium Bacillus subtilis as a model system, we undertook a compared characterization of the two paralogs SSB and YwpH. YwpH display 80% similarity to ihe ssDNA interacting domain of SSB but it lacks the protein-protein interacting domain of SSB. It is able to bind to single stranded nucleic acids and to stimulate certain enzymes that use ssDNA as substrate, although with a lower efficiency than SSB. YwpH is not essential for bacterial growth and it is only detected during stationary phase in minimum medium. Even though the cellular function of YwpH remains to be elucidated, its biochemical characterization made possible to increase our knowledge of this protein, and to suggest several hypothesis for its role. Besides, we have identified and characterized anew physical and functional interaction between B. subtilis SSB and PriA, the primary determinant of the replication restart. Our results have revealed a major implication of SSB in this particular mechanism. A model in which SSB would allow the recruitment and the anchoring of PriA near the replication fork is suggested.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Etude moléculaire de la protéine SSB de Bacillus subtilis (comparaison fonctionnelle avec son paralogue YwpH et mise en évidence de son interaction avec l'hélicase PriA du re-démarrage de la réplication)

Les protéines SSB sont essentielles à certains mécanismes cellulaires faisant intervenir l'ADN simple brin (ADNsb), tels que réplication, recombinaison ou réparation. Elles protègent et structurent l'ADNsb, rendant son utilisation efficace par différentes machineries qui y sont spécifiquement recrutées. Elles agissent aussi comme des connecteurs entre l'ADNsb et des partenaires protéiques avec lesquels elles interagissent. Il est récemment apparu que plusieurs protéines de type SSB pouvaient être présentes dans un même organisme, ce qui pose la question des fonctions exercées par chacune d'elles. En prenant comme modèle d'étude la bactérie Bacillus subtilis, nous avons entrepris la caractérisation comparée des deux protéines paralogues SSB et YwpH. YwpH a 80% de similarité avec le domaine de liaison à l'ADNsb de SSB, mais est dépourvue du domaine d'interaction protéine-protéine. Elle se lie à l'ADNsb et stimule certaines enzymes qui l'utilisent, avec néanmoins une efficacité inférieure à celle de SSB. Elle n'est pas essentielle et n'est détectée qu'en milieu minimum en phase stationnaire de croissance. Bien que sa fonction cellulaire ne soit pas encore élucidée, sa caractérisation biochimique a permis d'augmenter les connaissances sur cette protéine et d'avancer des hypothèses quant à son rôle. En parallèle, nous avons mis en évidence une nouvelle interaction physique et fonctionnelle entre SSB et PriA, protéine primaire du re-démarrage de la réplication. Nos résultats révèlent une implication majeure de SSB dans ce mécanisme particulier et un modèle dans lequel SSB permettrait le recrutement et l'ancrage de PriA à proximité de la fourche de réplication est proposé.SSB proteins are essential for cellular mechanisms using single stranded DNA (ssDNA), such as replication, recombination or DNA repair. They protect and structure the ssDNA, rendering it effective to be used by various proteins specifically recruited on it. In addition to that, SSB act as connectors between ssDNA and their interacting partners. Recently, it has been revealed that several SSB paralogs could be present within the same organism, raising the question of the functions exerted by each one of them. By using the Gram+ bacterium Bacillus subtilis as a model system, we undertook a compared characterization of the two paralogs SSB and YwpH. YwpH display 80% similarity to ihe ssDNA interacting domain of SSB but it lacks the protein-protein interacting domain of SSB. It is able to bind to single stranded nucleic acids and to stimulate certain enzymes that use ssDNA as substrate, although with a lower efficiency than SSB. YwpH is not essential for bacterial growth and it is only detected during stationary phase in minimum medium. Even though the cellular function of YwpH remains to be elucidated, its biochemical characterization made possible to increase our knowledge of this protein, and to suggest several hypothesis for its role. Besides, we have identified and characterized anew physical and functional interaction between B. subtilis SSB and PriA, the primary determinant of the replication restart. Our results have revealed a major implication of SSB in this particular mechanism. A model in which SSB would allow the recruitment and the anchoring of PriA near the replication fork is suggested.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Etude fonctionnelle du rôle de la protéine SSB dans le maintien de l intégrité du génome de la bactérie bacillus subtilis

Les processus du métabolisme de l ADN produisent de l ADN simple brin (ADN sb). La fonction essentielle des protéines SSB (Single Stranded DNA Binding protein) est de se polymériser sur l ADN simple brin, afin de le structurer et le protéger. Le domaine C-terminal (Cter) des SSB bactériennes interagit physiquement et fonctionnellement avec plusieurs protéines de la dynamique du génome. Nous avons montré chez Bacillus subtilis que le Cter de SSB joue un ro le de plateforme de rétention de 12 protéines intervenant dans les processus de réplication, recombinaison, réparation et re-démarrage de la réplication de l ADN. Cette plateforme est localisée aux fourches de réplication actives du chromosome, qu elle peut surveiller et ainsi anticiper d éventuels arrêts de réplication. L interactome de SSB est impliqué dans la viabilité des cellules en condition de croissance normale ou stressée par des agents génotoxiques. La dissection fonctionnelle in vivo du Cter de SSB par mutagénèse a été entreprise pour déterminer les résidus essentiels à l interaction entre SSB et ses multiples partenaires. En fonction de la mutation, une perte partielle du partenariat est observée modulant les fonctions cellulaires du Cter de SSB. La viabilité de mutants de délétion du Cter de SSB semble dépendante de motifs internes découverts dans le Cter de SSB. Dans leur ensemble, ces résultats confortent le rôle central de SSB dans la mise en place des multiples voies de secours des fourches arrêtées et des lésions induites sur l ADN. Le Cter de SSB semble essentiel à la croissance cellulaire via sa propriété d interactions avec plusieurs protéines spécifiques de la dynamique de l ADN.Single stranded DNA (ssDNA) is produced during DNA metabolism. Essential function of SSB protein (Single Stranded DNA Binding protein) consists of binding on ssDNA in order to structure and protect it. Bacterial SSB C-terminal domain (C-ter) interacts physically and functionally with several DNA dynamic s proteins. We showed that Bacillus subtilis SSB Cter acts as a recruitment platform for 12 proteins involved in DNA replication, recombination, repair and replication restart. This platform is localized at active chromosomal replication forks and is able to survey them and anticipate accidental arrest. SSB s interactome is implied in cell viability in normal or genotoxic stressed growth conditions. In vivo SSB Cter functional dissection by directed mutagenesis was used to determine essentials residues for SSB interactions with multiple proteins. Partial loss of partnership is observed depending on SSB mutation and results in variation of SSB Cter cellular functions. Internal sequences found in SSB Cter seem responsible of SSB Cter deletion mutant viability. Together, these results arguing a central role for SSB in coordination of multiple pathway acting in DNA damage repair and replication fork restart. SSB Cter seems essential to cell viability by its capacity to interact with many plurals and specifics DNA dynamic s proteins.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Direct Visualization of Horizontal Gene Transfer by Transformation in Live Pneumococcal Cells Using Microfluidics

Natural genetic transformation is a programmed mechanism of horizontal gene transfer in bacteria. It requires the development of competence, a specialized physiological state during which proteins involved in DNA uptake and chromosomal integration are produced. In Streptococcus pneumoniae, competence is transient. It is controlled by a secreted peptide pheromone, the competence-stimulating peptide (CSP) that triggers the sequential transcription of two sets of genes termed early and late competence genes, respectively. Here, we used a microfluidic system with fluorescence microscopy to monitor pneumococcal competence development and transformation, in live cells at the single cell level. We present the conditions to grow this microaerophilic bacterium under continuous flow, with a similar doubling time as in batch liquid culture. We show that perfusion of CSP in the microfluidic chamber results in the same reduction of the growth rate of individual cells as observed in competent pneumococcal cultures. We also describe newly designed fluorescent reporters to distinguish the expression of competence genes with temporally distinct expression profiles. Finally, we exploit the microfluidic technology to inject both CSP and transforming DNA in the microfluidic channels and perform near real time-tracking of transformation in live cells. We show that this approach is well suited to investigating the onset of pneumococcal competence together with the appearance and the fate of transformants in individual cells