RESUMEN
In bacteria, faithful DNA segregation of chromosomes and plasmids is mainly mediated by ParABS systems. These systems, consisting of a ParA ATPase, a DNA binding ParB CTPase, and centromere sites parS, orchestrate the separation of newly replicated DNA copies and their intracellular positioning. Accurate segregation relies on the assembly of a high-molecular-weight complex, comprising a few hundreds of ParB dimers nucleated from parS sites. This complex assembles in a multi-step process and exhibits dynamic liquid-droplet properties. Despite various proposed models, the complete mechanism for partition complex assembly remains elusive. This study investigates the impact of DNA supercoiling on ParB DNA binding profiles in vivo, using the ParABS system of the plasmid F. We found that variations in DNA supercoiling does not significantly affect any steps in the assembly of the partition complex. Furthermore, physical modeling, leveraging ChIP-seq data from linear plasmids F, suggests that ParB sliding is restricted to approximately 2 Kbp from parS, highlighting the necessity for additional mechanisms beyond ParB sliding over DNA for concentrating ParB into condensates nucleated at parS. Finally, explicit simulations of a polymer coated with bound ParB suggest a dominant role for ParB-ParB interactions in DNA compaction within ParB condensates.
RESUMEN
Plasmid families harbor different maintenances functions, depending on their size and copy number. Low copy number plasmids rely on active partition systems, organizing a partition complex at specific centromere sites that is actively positioned using NTPase proteins. Some low copy number plasmids lack an active partition system, but carry atypical intracellular positioning systems using a single protein that binds to the centromere site but without an associated NTPase. These systems have been studied in the case of the Escherichia coli R388 and of the Staphylococcus aureus pSK1 plasmids. Here we review these two systems, which appear to be unrelated but share common features, such as their distribution on plasmids of medium size and copy number, certain activities of their centromere-binding proteins, StbA and Par, respectively, as well as their mode of action, which may involve dynamic interactions with the nucleoid-packed chromosome of their hosts.
Asunto(s)
Variaciones en el Número de Copia de ADN , Nucleósido-Trifosfatasa , Humanos , Plásmidos/genética , Nucleósido-Trifosfatasa/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Bacterianas/genética , Segregación CromosómicaRESUMEN
DNA segregation ensures that cell offspring receive at least one copy of each DNA molecule, or replicon, after their replication. This important cellular process includes different phases leading to the physical separation of the replicons and their movement toward the future daughter cells. Here, we review these phases and processes in enterobacteria with emphasis on the molecular mechanisms at play and their controls.
Asunto(s)
Cromosomas Bacterianos , Enterobacteriaceae , Enterobacteriaceae/genética , Cromosomas Bacterianos/genética , ADN , Replicón , Replicación del ADNRESUMEN
The mycobacterial cell envelope consists of a typical plasma membrane, surrounded by a complex cell wall and a lipid-rich outer membrane. The biogenesis of this multilayer structure is a tightly regulated process requiring the coordinated synthesis and assembly of all its constituents. Mycobacteria grow by polar extension and recent studies showed that cell envelope incorporation of mycolic acids, the major constituent of the cell wall and outer membrane, is coordinated with peptidoglycan biosynthesis at the cell poles. However, there is no information regarding the dynamics of incorporation of other families of outer membrane lipids during cell elongation and division. Here, we establish that the translocation of non-essential trehalose polyphleates (TPP) occurs at different subcellular locations than that of the essential mycolic acids. Using fluorescence microscopy approaches, we investigated the subcellular localization of MmpL3 and MmpL10, respectively involved in the export of mycolic acids and TPP, in growing cells and their colocalization with Wag31, a protein playing a critical role in regulating peptidoglycan biosynthesis in mycobacteria. We found that MmpL3, like Wag31, displays polar localization and preferential accumulation at the old pole whereas MmpL10 is more homogenously distributed in the plasma membrane and slightly accumulates at the new pole. These results led us to propose a model in which insertion of TPP and mycolic acids into the mycomembrane is spatially uncoupled.
Asunto(s)
Mycobacterium tuberculosis , Mycobacterium , Trehalosa/metabolismo , Ácidos Micólicos/metabolismo , Peptidoglicano/metabolismo , Proteínas Bacterianas/metabolismo , Membrana Celular/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Pared Celular/metabolismo , Mycobacterium/metabolismo , Mycobacterium tuberculosis/metabolismoRESUMEN
The human pathogen Mycobacterium tuberculosis requires a P1B-ATPase metal exporter, CtpC (Rv3270), for resistance to zinc poisoning. Here, we show that zinc resistance also depends on a chaperone-like protein, PacL1 (Rv3269). PacL1 contains a transmembrane domain, a cytoplasmic region with glutamine/alanine repeats and a C-terminal metal-binding motif (MBM). PacL1 binds Zn2+, but the MBM is required only at high zinc concentrations. PacL1 co-localizes with CtpC in dynamic foci in the mycobacterial plasma membrane, and the two proteins form high molecular weight complexes. Foci formation does not require flotillin nor the PacL1 MBM. However, deletion of the PacL1 Glu/Ala repeats leads to loss of CtpC and sensitivity to zinc. Genes pacL1 and ctpC appear to be in the same operon, and homologous gene pairs are found in the genomes of other bacteria. Furthermore, PacL1 colocalizes and functions redundantly with other PacL orthologs in M. tuberculosis. Overall, our results indicate that PacL proteins may act as scaffolds that assemble P-ATPase-containing metal efflux platforms mediating bacterial resistance to metal poisoning.
Asunto(s)
Adenosina Trifosfatasas , Mycobacterium tuberculosis , Adenosina Trifosfatasas/metabolismo , Transporte Biológico , Humanos , Metales/metabolismo , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Zinc/metabolismoRESUMEN
Low-copy-number plasmids require sophisticated genetic devices to achieve efficient segregation of plasmid copies during cell division. Plasmid R388 uses a unique segregation mechanism, based on StbA, a small multifunctional protein. StbA is the key protein in a segregation system not involving a plasmid-encoded NTPase partner, it regulates the expression of several plasmid operons, and it is the main regulator of plasmid conjugation. The mechanisms by which StbA, together with the centromere-like sequence stbS, achieves segregation, is largely uncharacterized. To better understand the molecular basis of R388 segregation, we determined the crystal structure of the conserved N-terminal domain of StbA to 1.9 Å resolution. It folds into an HTH DNA-binding domain, structurally related to that of the PadR subfamily II of transcriptional regulators. StbA is organized in two domains. Its N-terminal domain carries the specific stbS DNA binding activity. A truncated version of StbA, deleted of its C-terminal domain, displays only partial activities in vivo, indicating that the non-conserved C-terminal domain is required for efficient segregation and subcellular plasmid positioning. The structure of StbA DNA-binding domain also provides some insight into how StbA monomers cooperate to repress transcription by binding to the stbDR and to form the segregation complex with stbS.
Asunto(s)
Proteínas Bacterianas , Segregación Cromosómica , Nucleósido-Trifosfatasa , Plásmidos , Proteínas Bacterianas/química , ADN/química , ADN/metabolismo , Nucleósido-Trifosfatasa/química , Nucleósido-Trifosfatasa/metabolismo , Operón , Plásmidos/genética , Dominios ProteicosRESUMEN
Accurate 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.
Asunto(s)
Centrómero/metabolismo , Cromosomas Bacterianos/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Sitios de Unión , Cromosomas Bacterianos/genética , ADN Primasa/metabolismo , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Escherichia coli/metabolismo , Simulación de Dinámica Molecular , Operón/genética , Plásmidos/genética , Regiones Promotoras Genéticas/genética , Dominios Proteicos , Multimerización de ProteínaRESUMEN
In bacteria, most low-copy-number plasmid and chromosomally encoded partition systems belong to the tripartite ParABS partition machinery. Despite the importance in genetic inheritance, the mechanisms of ParABS-mediated genome partition are not well understood. Combining theory and experiment, we provided evidence that the ParABS system-DNA partitioning in vivo via the ParA-gradient-based Brownian ratcheting-operates near a transition point in parameter space (i.e., a critical point), across which the system displays qualitatively different motile behaviors. 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. The spatial control over the near-critical-point operation not only ensures both sensitive adaptation and robust execution of partitioning but also sheds light on the fundamental question in cell biology: how do cells faithfully measure cellular-scale distance by only using molecular-scale interactions?
Asunto(s)
Proteínas Bacterianas , ADN , Proteínas Bacterianas/genética , ADN/genética , ADN Bacteriano/genética , Plásmidos/genéticaRESUMEN
ParABS, the most widespread bacterial DNA segregation system, is composed of a centromeric sequence, parS, and two proteins, the ParA ATPase and the ParB DNA binding proteins. Hundreds of ParB proteins assemble dynamically to form nucleoprotein parS-anchored complexes that serve as substrates for ParA molecules to catalyze positioning and segregation events. The exact nature of this ParBS complex has remained elusive, what we address here by revisiting the Stochastic Binding model (SBM) introduced to explain the non-specific binding profile of ParB in the vicinity of parS. In the SBM, DNA loops stochastically bring loci inside a sharp cluster of ParB. However, previous SBM versions did not include the negative supercoiling of bacterial DNA, leading to use unphysically small DNA persistences to explain the ParB binding profiles. In addition, recent super-resolution microscopy experiments have revealed a ParB cluster that is significantly smaller than previous estimations and suggest that it results from a liquid-liquid like phase separation. Here, by simulating the folding of long (≥ 30 kb) supercoiled DNA molecules calibrated with realistic DNA parameters and by considering different possibilities for the physics of the ParB cluster assembly, we show that the SBM can quantitatively explain the ChIP-seq ParB binding profiles without any fitting parameter, aside from the supercoiling density of DNA, which, remarkably, is in accord with independent measurements. We also predict that ParB assembly results from a non-equilibrium, stationary balance between an influx of produced proteins and an outflux of excess proteins, i.e., ParB clusters behave like liquid-like protein condensates with unconventional "leaky" boundaries.
Asunto(s)
Proteínas Bacterianas/química , Centrómero/química , Segregación Cromosómica , ADN Bacteriano/química , ADN Superhelicoidal/química , Modelos Biológicos , Nucleoproteínas/química , Unión Proteica , Procesos EstocásticosRESUMEN
Bacterial ParB partitioning proteins involved in chromosomes and low-copy-number plasmid segregation are cytosine triphosphate (CTP)-dependent molecular switches. CTP-binding converts ParB dimers to DNA clamps, allowing unidimensional diffusion along the DNA. This sliding property has been proposed to explain the ParB spreading over large distances from parS centromere sites where ParB is specifically loaded. We modeled such a "clamping and sliding" mechanism as a typical reaction-diffusion system, compared it to the F plasmid ParB DNA binding pattern, and found that it can account neither for the long range of ParB binding to DNA nor for the rapid assembly kinetics observed in vivo after parS duplication. Also, it predicts a strong effect on the F plasmid ParB binding pattern from the presence of a roadblock that is not observed in ChIP-sequencing (ChIP-seq). We conclude that although "clamping and sliding" can occur at short distances from parS, another mechanism must apply for ParB recruitment at larger genomic distances.
RESUMEN
Multidrug resistance (MDR) often results from the acquisition of mobile genetic elements (MGEs) that encode MDR gene(s), such as conjugative plasmids. The spread of MDR plasmids is founded on their ability of horizontal transference, as well as their faithful inheritance in progeny cells. Here, we investigated the genetic factors involved in the prevalence of the IncI conjugative plasmid pESBL, which was isolated from the Escherichia coli O104:H4 outbreak strain in Germany in 2011. Using transposon-insertion sequencing, we identified the pESBL partitioning locus (par). Genetic, biochemical and microscopic approaches allowed pESBL to be characterized as a new member of the Type Ib partitioning system. Inactivation of par caused mis-segregation of pESBL followed by post-segregational killing (PSK), resulting in a great fitness disadvantage but apparent plasmid stability in the population of viable cells. We constructed a variety of pESBL derivatives with different combinations of mutations in par, conjugational transfer (oriT) and pnd toxin-antitoxin (TA) genes. Only the triple mutant exhibited plasmid-free cells in viable cell populations. Time-lapse tracking of plasmid dynamics in microfluidics indicated that inactivation of pnd improved the survival of plasmid-free cells and allowed oriT-dependent re-acquisition of the plasmid. Altogether, the three factors-active partitioning, toxin-antitoxin and conjugational transfer-are all involved in the prevalence of pESBL in the E. coli population.
Asunto(s)
Conjugación Genética , Infecciones por Escherichia coli/transmisión , Escherichia coli O104/genética , Proteínas de Escherichia coli/genética , Transferencia de Gen Horizontal , Plásmidos/genética , Resistencia betalactámica/genética , Antibacterianos/farmacología , Infecciones por Escherichia coli/microbiología , Humanos , Sistemas Toxina-Antitoxina/genéticaRESUMEN
Liquid-liquid phase-separated (LLPS) states are key to compartmentalizing components in the absence of membranes; however, it is unclear whether LLPS condensates are actively and specifically organized in the subcellular space and by which mechanisms. Here, we address this question by focusing on the ParABS DNA segregation system, composed of a centromeric-like sequence (parS), a DNA-binding protein (ParB), and a motor (ParA). We show that parS and ParB associate to form nanometer-sized, round condensates. ParB molecules diffuse rapidly within the nucleoid volume but display confined motions when trapped inside ParB condensates. Single ParB molecules are able to rapidly diffuse between different condensates, and nucleation is strongly favored by parS. Notably, the ParA motor is required to prevent the fusion of ParB condensates. These results describe a novel active mechanism that splits, segregates, and localizes non-canonical LLPS condensates in the subcellular space.
Asunto(s)
Adenosina Trifosfato/fisiología , Fenómenos Fisiológicos Bacterianos , Proteínas de Escherichia coli/fisiología , Transición de Fase , ADN Primasa/fisiología , ADN Bacteriano , Microscopía/métodos , Nanopartículas , Imagen Individual de Molécula/métodosRESUMEN
The ParB-parS partition complexes that bacterial replicons use to ensure their faithful inheritance also find employment in visualization of DNA loci, as less intrusive alternatives to fluorescent repressor-operator systems. The ability of ParB molecules to interact via their N-terminal domains and to bind to non-specific DNA enables expansion of the initial complex to a size both functional in partition and, via fusion to fluorescent peptides, visible by light microscopy. We have investigated whether it is possible to dispense with the need to insert parS in the genomic locus of interest, by determining whether ParB fused to proteins that bind specifically to natural DNA sequences can still assemble visible complexes. In yeast cells, coproduction of fusions of ParB to a fluorescent peptide and to a TALE protein targeting an endogenous sequence did not yield visible foci; nor did any of several variants of these components. In E.coli, coproduction of fusions of SopB (F plasmid ParB) to fluorescent peptide, and to dCas9 together with specific guide RNAs, likewise yielded no foci. The result of coproducing analogous fusions of SopB proteins with distinct binding specificities was also negative. Our observations imply that in order to assemble higher order partition complexes, ParB proteins need specific activation through binding to their cognate parS sites.
Asunto(s)
Proteínas Bacterianas/metabolismo , Centrómero/química , Centrómero/metabolismo , ADN Bacteriano/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Secuencia de Bases , Sitios de Unión , Proteína 9 Asociada a CRISPR , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Plásmidos/genética , Unión Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Simportadores/genética , Simportadores/metabolismoRESUMEN
Transposons are found in a wide variety of forms throughout the prokaryotic world where they actively contribute to the adaptive strategies of bacterial communities and hence, to the continuous emergence of new multiresistant pathogens. Contrasting with their biological and societal impact, only a few bacterial transposons have been the subject of detailed molecular studies. In this chapter, we propose a set of reliable biochemical methods as a primary route for studying new transposition mechanisms. These methods include (a) a straightforward approach termed "thermal shift induction" to produce the transposase in a soluble and properly folded configuration prior to its purification, (b) an adaptation of classical electrophoretic mobility shift assays (EMSA) combined to fluorescently labeled DNA substrates to determine the DNA content of different complexes assembled by the transposase, and (c) a highly sensitive "in-gel" DNA footprinting assay to further characterize these complexes at the base pair resolution level. A combination of these approaches was recently applied to decipher the molecular organization of key intermediates in the Tn3-family transposition pathway, a mechanism that has long been refractory to biochemical studies.
Asunto(s)
Bacterias/genética , Bacterias/metabolismo , Elementos Transponibles de ADN , Transposasas/metabolismo , Fenómenos Fisiológicos Bacterianos , Proteínas de Unión al ADN/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Expresión Génica , Sustancias Macromoleculares/metabolismo , Unión Proteica , Coloración y Etiquetado , Temperatura , Transposasas/genéticaRESUMEN
Plasmids are ubiquitous in the microbial world and have been identified in almost all species of bacteria that have been examined. Their localization inside the bacterial cell has been examined for about two decades; typically, they are not randomly distributed, and their positioning depends on copy number and their mode of segregation. Low-copy-number plasmids promote their own stable inheritance in their bacterial hosts by encoding active partition systems, which ensure that copies are positioned in both halves of a dividing cell. High-copy plasmids rely on passive diffusion of some copies, but many remain clustered together in the nucleoid-free regions of the cell. Here we review plasmid localization and partition (Par) systems, with particular emphasis on plasmids from Enterobacteriaceae and on recent results describing the in vivo localization properties and molecular mechanisms of each system. Partition systems also cause plasmid incompatibility such that distinct plasmids (with different replicons) with the same Par system cannot be stably maintained in the same cells. We discuss how partition-mediated incompatibility is a consequence of the partition mechanism.
Asunto(s)
Enterobacteriaceae/genética , Plásmidos/genética , Proteínas Bacterianas/genética , Enterobacteriaceae/metabolismo , ReplicónRESUMEN
Chromosome and plasmid segregation in bacteria are mostly driven by ParABS systems. These DNA partitioning machineries rely on large nucleoprotein complexes assembled on centromere sites (parS). However, the mechanism of how a few parS-bound ParB proteins nucleate the formation of highly concentrated ParB clusters remains unclear despite several proposed physico-mathematical models. We discriminated between these different models by varying some key parameters in vivo using the F plasmid partition system. We found that "Nucleation & caging" is the only coherent model recapitulating in vivo data. We also showed that the stochastic self-assembly of partition complexes (i) is a robust mechanism, (ii) does not directly involve ParA ATPase, (iii) results in a dynamic structure of discrete size independent of ParB concentration, and (iv) is not perturbed by active transcription but is by protein complexes. We refined the "Nucleation & caging" model and successfully applied it to the chromosomally encoded Par system of Vibrio cholerae, indicating that this stochastic self-assembly mechanism is widely conserved from plasmids to chromosomes.
Asunto(s)
Proteínas Bacterianas/metabolismo , Cromosomas Bacterianos/fisiología , Plásmidos/fisiología , Vibrio cholerae/metabolismo , Segregación Cromosómica , Cromosomas Bacterianos/genética , Modelos Teóricos , Plásmidos/genética , Procesos Estocásticos , Biología de Sistemas/métodos , Vibrio cholerae/fisiologíaRESUMEN
Chromatin immunoprecipitation (ChIP) coupled with next-generation sequencing (NGS) is widely used for studying the nucleoprotein components that are involved in the various cellular processes required for shaping the bacterial nucleoid. This methodology, termed ChIP-sequencing (ChIP-seq), enables the identification of the DNA targets of DNA binding proteins across genome-wide maps. Here, we describe the steps necessary to obtain short, specific, high-quality immunoprecipitated DNA prior to DNA library construction for NGS and high-resolution ChIP-seq data.
Asunto(s)
Inmunoprecipitación de Cromatina/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Análisis de Secuencia de ADN/métodos , Proteínas Bacterianas/metabolismo , ADN/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Biblioteca de Genes , Nucleoproteínas/metabolismoRESUMEN
Bacterial centromeres-also called parS, are cis-acting DNA sequences which, together with the proteins ParA and ParB, are involved in the segregation of chromosomes and plasmids. The specific binding of ParB to parS nucleates the assembly of a large ParB/DNA complex from which ParA-the motor protein, segregates the sister replicons. Closely related families of partition systems, called Bsr, were identified on the chromosomes and large plasmids of the multi-chromosomal bacterium Burkholderia cenocepacia and other species from the order Burkholeriales. The centromeres of the Bsr partition families are 16 bp palindromes, displaying similar base compositions, notably a central CG dinucleotide. Despite centromeres bind the cognate ParB with a narrow specificity, weak ParB-parS non cognate interactions were nevertheless detected between few Bsr partition systems of replicons not belonging to the same genome. These observations suggested that Bsr partition systems could have a common ancestry but that evolution mostly erased the possibilities of cross-reactions between them, in particular to prevent replicon incompatibility. To detect novel similarities between Bsr partition systems, we have analyzed the binding of six Bsr parS sequences and a wide collection of modified derivatives, to their cognate ParB. The study was carried out by Surface Plasmon Resonance imaging (SPRi) mulitplex analysis enabling a systematic survey of each nucleotide position within the centromere. We found that in each parS some positions could be changed while maintaining binding to ParB. Each centromere displays its own pattern of changes, but some positions are shared more or less widely. In addition from these changes we could speculate evolutionary links between these centromeres.
Asunto(s)
Proteínas Bacterianas/genética , Burkholderia cepacia/genética , Centrómero , Cromosomas Bacterianos , Plásmidos , Resonancia por Plasmón de Superficie/métodosRESUMEN
Precise and rapid DNA segregation is required for proper inheritance of genetic material. In most bacteria and archaea, this process is assured by a broadly conserved mitotic-like apparatus in which a NTPase (ParA) displaces the partition complex. Competing observations and models imply starkly different 3D localization patterns of the components of the partition machinery during segregation. Here we use super-resolution microscopies to localize in 3D each component of the segregation apparatus with respect to the bacterial chromosome. We show that Par proteins locate within the nucleoid volume and reveal that proper volumetric localization and segregation of partition complexes requires ATPase and DNA-binding activities of ParA. Finally, we find that the localization patterns of the different components of the partition system highly correlate with dense chromosomal regions. We propose a new mechanism in which the nucleoid provides a scaffold to guide the proper segregation of partition complexes.