Centromere binding specificity in assembly of the F plasmid partition complex

The segregation of plasmid F of Escherichia coli is highly reliable. The Sop partition locus, responsible for this stable maintenance, is composed of two genes, sopA and sopB and a centromere, sopC, consisting of 12 direct repeats of 43 bp. Each repeat carries a 16-bp inverted repeat motif to which SopB binds to form a nucleoprotein assembly called the partition complex. A database search for sequences closely related to sopC revealed unexpected features that appeared highly conserved. We have investigated the requirements for specific SopB–sopC interactions using a surface plasmon resonance imaging technique. We show that (i) only 10 repeats interact specifically with SopB, (ii) no base outside the 16-bp sopC sites is involved in binding specificity, whereas five bases present in each arm are required for interactions, and (iii) the A-C central bases contribute to binding efficiency by conforming to a need for a purine–pyrimidine dinucleotide. We have refined the SopB–sopC binding pattern by electro-mobility shift assay and found that all 16 bp are necessary for optimal SopB binding. These data and the model we propose, define the basis of the high binding specificity of F partition complex assembly, without which, dispersal of SopB over DNA would result in defective segregation

Modeling supercoiled DNA interacting with an anchored cluster of proteins: towards a quantitative estimation of chromosomal DNA supercoiling

We investigate the measurement of DNA supercoiling density ($\sigma$) along chromosomes using interaction frequencies between DNA and DNA-anchored clusters of proteins. Specifically, we show how the physics of DNA supercoiling leads, in bacteria, to the quantitative modeling of binding properties of ParB proteins around their centromere-like site, {\it parS}. Using this framework, we provide an upper bound for $\sigma$ in the {\it Escherichia coli} chromosome, consistent with plasmid values, and offer a proof of concept for a high accuracy measurement. To reach these conclusions, we revisit the problem of the formation of ParB clusters. We predict, in particular, that they result from a non-equilibrium, stationary balance between an influx of produced proteins and an outflux of excess proteins, i.e., they behave like liquid-like protein condensates with unconventional ``leaky'' boundaries.Comment: 5 pages + Supplementary Info (including 3 figures

Novel Insights into the Bovine Polled Phenotype and Horn Ontogenesis in Bovidae

Despite massive research efforts, the molecular etiology of bovine polledness and the developmental pathways involved in horn ontogenesis are still poorly understood. In a recent article, we provided evidence for the existence of at least two different alleles at the Polled locus and identified candidate mutations for each of them. None of these mutations was located in known coding or regulatory regions, thus adding to the complexity of understanding the molecular basis of polledness. We confirm previous results here and exhaustively identify the causative mutation for the Celtic allele (PC) and four candidate mutations for the Friesian allele (PF). We describe a previously unreported eyelash-and-eyelid phenotype associated with regular polledness, and present unique histological and gene expression data on bovine horn bud differentiation in fetuses affected by three different horn defect syndromes, as well as in wild-type controls. We propose the ectopic expression of a lincRNA in PC/p horn buds as a probable cause of horn bud agenesis. In addition, we provide evidence for an involvement of OLIG2, FOXL2 and RXFP2 in horn bud differentiation, and draw a first link between bovine, ovine and caprine Polled loci. Our results represent a first and important step in understanding the genetic pathways and key process involved in horn bud differentiation in Bovidae

Plasmid Localization and Partition in Enterobacteriaceae

International audienc

Ndd, the Bacteriophage T4 Protein That Disrupts the Escherichia coli Nucleoid, Has a DNA Binding Activity

Early in a bacteriophage T4 infection, the phage ndd gene causes the rapid destruction of the structure of the Escherichia coli nucleoid. Even at very low levels, the Ndd protein is extremely toxic to cells. In uninfected E. coli, overexpression of the cloned ndd gene induces disruption of the nucleoid that is indistinguishable from that observed after T4 infection. A preliminary characterization of this protein indicates that it has a double-stranded DNA binding activity with a preference for bacterial DNA rather than phage T4 DNA. The targets of Ndd action may be the chromosomal sequences that determine the structure of the nucleoid

Probing the ATP-binding site of P1 ParA: partition and repression have different requirements for ATP binding and hydrolysis

The ParA family of proteins is involved in partition of a variety of plasmid and bacterial chromosomes. P1 ParA plays two roles in partition: it acts as a repressor of the par operon and has an undefined yet indispensable role in P1 plasmid localization. We constructed seven mutations in three putative ATP-binding motifs of ParA. Three classes of phenotypes resulted, each represented by mutations in more than one motif. Three mutations created ‘super-repressors’, in which repressor activity was much stronger than in wild-type ParA, while the remainder damaged repressor activity. All mutations eliminated partition activities, but two showed a plasmid stability defect that was worse than that of a null mutation. Four mutant ParAs, two super-repressors and two weak repressors, were analyzed biochemically, and all exhibited damaged ATPase activity. The super-repressors bound site-specifically to the par operator sequence, and this activity was strongly stimulated by ATP and ADP. These results support the proposal that ATP binding is essential but hydrolysis is inhibitory for ParA’s repressor activity and suggest that ATP hydrolysis is essential for plasmid localization

La Ségrégation du plasmide F d'Escherichia coli (régulation de l'activité ATPase de la protéine moteur de partition SopA)

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

Spatial control over near-critical-point operation ensures fidelity of ParABS-mediated bacterial genome segregation

In bacteria, most low-copy-number plasmid and chromosomally encoded partition systems belong to the tripartite ParAB S partition machinery. Despite the importance in genetic inheritance, the mechanisms of ParAB S -mediated genome partition are not well understood. Combining theory and experiment, we provided evidences that the ParAB S system – partitioning via the ParA gradient-based Brownian ratcheting – operates near a critical point in vivo . This near-critical-point operation adapts the segregation distance of replicated plasmids to the half-length of the elongating nucleoid, ensuring both cell halves to inherit one copy of the plasmids. Further, we demonstrated that the plasmid localizes the cytoplasmic ParA to buffer the partition fidelity against the large cell-to-cell fluctuations in ParA level. Thus, the spatial control over the near-critical-point operation not only ensures both sensitive adaption and robust execution of partitioning, but sheds light on the fundamental question in cell biology: How do cells faithfully measure cellular-scale distance by only using molecular-scale interactions

Three ParA dimers cooperatively assemble on type Ia partition promoters

ABSTRACT Accurate DNA segregation is essential for faithful inheritance of genetic material. In bacteria, this process is mainly ensured by a partition system (Par) composed of two proteins, ParA and ParB, and a centromere site. The auto-regulation of Par operon expression is important for efficient partitioning, and is primarily mediated by ParA for type Ia plasmid partition systems. For the plasmid F, four ParA F monomers were proposed to bind to four repeated sequences in the promoter region. By contrast, using quantitative surface plasmon resonance, we showed that three ParA F dimers bind to this region. We uncovered that one perfect inverted repeat (IR) motif, consisting of two hexamer sequences spaced by 28-bp, constitutes the primary ParA F DNA binding site. A similar but degenerated motif overlaps the former. ParA F binding to these motifs is well supported by biochemical and modeling analyses. In addition, molecular dynamics simulations predict that the winged-HTH domain displays high flexibility, which may favor the cooperative ParA binding to the promoter region. We propose that three ParA F dimers bind cooperatively to overlapping motifs thus covering the promoter region. A similar organization is found on both closely related and distant plasmid partition systems, suggesting that such promoter organization for auto-regulated Par operons is widespread and may have evolved from a common ancestor

Three ParA Dimers Cooperatively Assemble on Type Ia Partition Promoters

International audienceAccurate DNA segregation is essential for faithful inheritance of genetic material. In bacteria, this process is mainly ensured by partition systems composed of two proteins, ParA and ParB, and a centromere site. Auto-regulation of Par operon expression is important for efficient partitioning and is primarily mediated by ParA for type Ia plasmid partition systems. For the F-plasmid, four ParAF monomers were proposed to bind to four repeated sequences in the promoter region. By contrast, using quantitative surface-plasmon-resonance, we showed that three ParAF dimers bind to this region. We uncovered that one perfect inverted repeat (IR) motif, consisting of two hexamer sequences spaced by 28-bp, constitutes the primary ParAF DNA binding site. A similar but degenerated motif overlaps the former. ParAF binding to these motifs is well supported by biochemical and modeling analyses. Molecular dynamics simulations predict that the winged-HTH domain displays high flexibility, which may favor the cooperative ParA binding to the promoter. We propose that three ParAF dimers bind cooperatively to overlapping motifs, thus covering the promoter region. A similar organization is found on closely related and distant plasmid partition systems, suggesting that such promoter organization for auto-regulated Par operons is widespread and may have evolved from a common ancestor