Activity of MukBEF for chromosome management in E. coli and its inhibition by MatP.

Structural maintenance of chromosomes (SMC) complexes share conserved structures and serve a common role in maintaining chromosome architecture. In the bacterium Escherichia coli, the SMC complex MukBEF is necessary for rapid growth and the accurate segregation and positioning of the chromosome, although the specific molecular mechanisms involved are still unknown. Here, we used a number of in vivo assays to reveal how MukBEF controls chromosome conformation and how the MatP/matS system prevents MukBEF activity. Our results indicate that the loading of MukBEF occurs preferentially on newly replicated DNA, at multiple loci on the chromosome where it can promote long-range contacts in cis even though MukBEF can promote long-range contacts in the absence of replication. Using Hi-C and ChIP-seq analyses in strains with rearranged chromosomes, the prevention of MukBEF activity increases with the number of matS sites and this effect likely results from the unloading of MukBEF by MatP. Altogether, our results reveal how MukBEF operates to control chromosome folding and segregation in E. coli.

Ribosomal RNA operons define a central functional compartment in the Streptomyces chromosome.

Streptomyces are prolific producers of specialized metabolites with applications in medicine and agriculture. These bacteria possess a large linear chromosome genetically compartmentalized: core genes are grouped in the central part, while terminal regions are populated by poorly conserved genes. In exponentially growing cells, chromosome conformation capture unveiled sharp boundaries formed by ribosomal RNA (rrn) operons that segment the chromosome into multiple domains. Here we further explore the link between the genetic distribution of rrn operons and Streptomyces genetic compartmentalization. A large panel of genomes of species representative of the genus diversity revealed that rrn operons and core genes form a central skeleton, the former being identifiable from their core gene environment. We implemented a new nomenclature for Streptomyces genomes and trace their rrn-based evolutionary history. Remarkably, rrn operons are close to pericentric inversions. Moreover, the central compartment delimited by rrn operons has a very dense, nearly invariant core gene content. Finally, this compartment harbors genes with the highest expression levels, regardless of gene persistence and distance to the origin of replication. Our results highlight that rrn operons are structural boundaries of a central functional compartment prone to transcription in Streptomyces.

Computational Tools for the Multiscale Analysis of Hi-C Data in Bacterial Chromosomes.

Just as in eukaryotes, high-throughput chromosome conformation capture (Hi-C) data have revealed nested organizations of bacterial chromosomes into overlapping interaction domains. In this chapter, we present a multiscale analysis framework aiming at capturing and quantifying these properties. These include both standard tools (e.g., contact laws) and novel ones such as an index that allows identifying loci involved in domain formation independently of the structuring scale at play. Our objective is twofold. On the one hand, we aim at providing a full, understandable Python/Jupyter-based code which can be used by both computer scientists and biologists with no advanced computational background. On the other hand, we discuss statistical issues inherent to Hi-C data analysis, focusing more particularly on how to properly assess the statistical significance of results. As a pedagogical example, we analyze data produced in Pseudomonas aeruginosa, a model pathogenetic bacterium. All files (codes and input data) can be found on a GitHub repository. We have also embedded the files into a Binder package so that the full analysis can be run on any machine through Internet.

Dynamics of the compartmentalized Streptomyces chromosome during metabolic differentiation.

Bacteria of the genus Streptomyces are prolific producers of specialized metabolites, including antibiotics. The linear chromosome includes a central region harboring core genes, as well as extremities enriched in specialized metabolite biosynthetic gene clusters. Here, we show that chromosome structure in Streptomyces ambofaciens correlates with genetic compartmentalization during exponential phase. Conserved, large and highly transcribed genes form boundaries that segment the central part of the chromosome into domains, whereas the terminal ends tend to be transcriptionally quiescent compartments with different structural features. The onset of metabolic differentiation is accompanied by a rearrangement of chromosome architecture, from a rather 'open' to a 'closed' conformation, in which highly expressed specialized metabolite biosynthetic genes form new boundaries. Thus, our results indicate that the linear chromosome of S. ambofaciens is partitioned into structurally distinct entities, suggesting a link between chromosome folding, gene expression and genome evolution.

Multiscale Dynamic Structuring of Bacterial Chromosomes.

Since the nucleoid was isolated from bacteria in the 1970s, two fundamental questions emerged and are still in the spotlight: how bacteria organize their chromosomes to fit inside the cell and how nucleoid organization enables essential biological processes. During the last decades, knowledge of bacterial chromosome organization has advanced considerably, and today, such chromosomes are considered to be highly organized and dynamic structures that are shaped by multiple factors in a multiscale manner. Here we review not only the classical well-known factors involved in chromosome organization but also novel components that have recently been shown to dynamically shape the 3D structuring of the bacterial genome. We focus on the different functional elements that control short-range organization and describe how they collaborate in the establishment of the higher-order folding and disposition of the chromosome. Recent advances have opened new avenues for a deeper understanding of the principles and mechanisms of chromosome organization in bacteria.

A low Smc flux avoids collisions and facilitates chromosome organization in Bacillus subtilis.

SMC complexes are widely conserved ATP-powered DNA-loop-extrusion motors indispensable for organizing and faithfully segregating chromosomes. How SMC complexes translocate along DNA for loop extrusion and what happens when two complexes meet on the same DNA molecule is largely unknown. Revealing the origins and the consequences of SMC encounters is crucial for understanding the folding process not only of bacterial, but also of eukaryotic chromosomes. Here, we uncover several factors that influence bacterial chromosome organization by modulating the probability of such clashes. These factors include the number, the strength, and the distribution of Smc loading sites, the residency time on the chromosome, the translocation rate, and the cellular abundance of Smc complexes. By studying various mutants, we show that these parameters are fine-tuned to reduce the frequency of encounters between Smc complexes, presumably as a risk mitigation strategy. Mild perturbations hamper chromosome organization by causing Smc collisions, implying that the cellular capacity to resolve them is limited. Altogether, we identify mechanisms that help to avoid Smc collisions and their resolution by Smc traversal or other potentially risky molecular transactions.

Distinct Activities of Bacterial Condensins for Chromosome Management in Pseudomonas aeruginosa.

Three types of structurally related structural maintenance of chromosomes (SMC) complexes, referred to as condensins, have been identified in bacteria. Smc-ScpAB is present in most bacteria, whereas MukBEF is found in enterobacteria and MksBEF is scattered over the phylogenic tree. The contributions of these condensins to chromosome management were characterized in Pseudomonas aeruginosa, which carries both Smc-ScpAB and MksBEF. In this bacterium, SMC-ScpAB controls chromosome disposition by juxtaposing chromosome arms. In contrast, MksBEF is critical for chromosome segregation in the absence of the main segregation system, and it affects the higher-order architecture of the chromosome by promoting DNA contacts in the megabase range. Strikingly, our results reveal a prevalence of Smc-ScpAB over MksBEF involving a coordination of their activities with chromosome replication. They also show that E. coli MukBEF can substitute for MksBEF in P. aeruginosa while prevailing over Smc-ScpAB. Our results reveal a hierarchy between activities of bacterial condensins on the same chromosome.

Conformational Studies of Bacterial Chromosomes by High-Throughput Sequencing Methods.

The development of next-generation sequencing technologies has allowed the application of different methods dedicated to the study of DNA-protein interactions and chromosome conformation to entire bacterial genome. By combining these approaches, the role of various parameters and factors involved in gene expression and chromosome organization can be disclosed at the molecular level over the full genome. Here we describe two methods that profoundly revolutionized our vision of DNA-protein interactions and spatial organization of chromosomes. Chromosome conformation capture (3C) coupled to deep sequencing (3C-seq) enables studies of the genome-wide chromosome folding and its control by different parameters and structural factors. Chromatin immunoprecipitation (ChIP) followed by high-throughput DNA sequencing (ChIP-seq) revealed the extent and regulation of DNA-protein interactions in vivo and highlight the role of structural factors in the control of chromosome organization. In this chapter, we describe a detailed protocol of 3C-seq and ChIP-seq experiments that, when combined, allows the spatial study of the chromosome and the factors that promote specific folding. Data processing and analysis for both experiments are also discussed.

Multiscale Structuring of the E. coli Chromosome by Nucleoid-Associated and Condensin Proteins.

As in eukaryotes, bacterial genomes are not randomly folded. Bacterial genetic information is generally carried on a circular chromosome with a single origin of replication from which two replication forks proceed bidirectionally toward the opposite terminus region. Here, we investigate the higher-order architecture of the Escherichia coli genome, showing its partition into two structurally distinct entities by a complex and intertwined network of contacts: the replication terminus (ter) region and the rest of the chromosome. Outside of ter, the condensin MukBEF and the ubiquitous nucleoid-associated protein (NAP) HU promote DNA contacts in the megabase range. Within ter, the MatP protein prevents MukBEF activity, and contacts are restricted to ∼280 kb, creating a domain with distinct structural properties. We also show how other NAPs contribute to nucleoid organization, such as H-NS, which restricts short-range interactions. Combined, these results reveal the contributions of major evolutionarily conserved proteins in a bacterial chromosome organization.

Long range chromosome organization in Escherichia coli: The position of the replication origin defines the non-structured regions and the Right and Left macrodomains.

The Escherichia coli chromosome is organized into four macrodomains (Ori, Ter, Right and Left) and two non-structured regions. This organization influences the segregation of sister chromatids, the mobility of chromosomal DNA, and the cellular localization of the chromosome. The organization of the Ter and Ori macrodomains relies on two specific systems, MatP/matS for the Ter domain and MaoP/maoS for the Ori domain, respectively. Here by constructing strains with chromosome rearrangements to reshuffle the distribution of chromosomal segments, we reveal that the difference between the non-structured regions and the Right and Left lateral macrodomains relies on their position on the chromosome. A change in the genetic location of oriC generated either by an inversion within the Ori macrodomain or by the insertion of a second oriC modifies the position of Right and Left macrodomains, as the chromosome region the closest to oriC are always non-structured while the regions further away behave as macrodomain regardless of their DNA sequence. Using fluorescent microscopy we estimated that loci belonging to a non-structured region are significantly closer to the Ori MD than loci belonging to a lateral MD. Altogether, our results suggest that the origin of replication plays a prominent role in chromosome organization in E. coli, as it determines structuring and localization of macrodomains in growing cell.

Regional Control of Chromosome Segregation in Pseudomonas aeruginosa.

Chromosome segregation in bacteria occurs concomitantly with DNA replication, and the duplicated regions containing the replication origin oriC are generally the first to separate and migrate to their final specific location inside the cell. In numerous bacterial species, a three-component partition machinery called the ParABS system is crucial for chromosome segregation. This is the case in the gammaproteobacterium Pseudomonas aeruginosa, where impairing the ParABS system is very detrimental for growth, as it increases the generation time and leads to the formation of anucleate cells and to oriC mispositioning inside the cell. In this study, we investigate in vivo the ParABS system in P. aeruginosa. Using chromatin immuno-precipitation coupled with high throughput sequencing, we show that ParB binds to four parS site located within 15 kb of oriC in vivo, and that this binding promotes the formation of a high order nucleoprotein complex. We show that one parS site is enough to prevent anucleate cell formation, therefore for correct chromosome segregation. By displacing the parS site from its native position on the chromosome, we demonstrate that parS is the first chromosomal locus to be separated upon DNA replication, which indicates that it is the site of force exertion of the segregation process. We identify a region of approximatively 650 kb surrounding oriC in which the parS site must be positioned for chromosome segregation to proceed correctly, and we called it "competence zone" of the parS site. Mutant strains that have undergone specific genetic rearrangements allow us to propose that the distance between oriC and parS defines this "competence zone". Implications for the control of chromosome segregation in P. aeruginosa are discussed.

The MaoP/maoS Site-Specific System Organizes the Ori Region of the E. coli Chromosome into a Macrodomain.

The Ori region of bacterial genomes is segregated early in the replication cycle of bacterial chromosomes. Consequently, Ori region positioning plays a pivotal role in chromosome dynamics. The Ori region of the E. coli chromosome is organized as a macrodomain with specific properties concerning DNA mobility, segregation of loci and long distance DNA interactions. Here, by using strains with chromosome rearrangements and DNA mobility as a read-out, we have identified the MaoP/maoS system responsible for constraining DNA mobility in the Ori region and limiting long distance DNA interactions with other regions of the chromosome. MaoP belongs to a group of proteins conserved in the Enterobacteria that coevolved with Dam methylase including SeqA, MukBEF and MatP that are all involved in the control of chromosome conformation and segregation. Analysis of DNA rings excised from the chromosome demonstrated that the single maoS site is required in cis on the chromosome to exert its effect while MaoP can act both in cis and in trans. The position of markers in the Ori region was affected by inactivating maoP. However, the MaoP/maoS system was not sufficient for positioning the Ori region at the »-¾ regions of the cell. We also demonstrate that the replication and the resulting expansion of bulk DNA are localized centrally in the cell. Implications of these results for chromosome positioning and segregation in E. coli are discussed.

Polymer modeling of the E. coli genome reveals the involvement of locus positioning and macrodomain structuring for the control of chromosome conformation and segregation.

The mechanisms that control chromosome conformation and segregation in bacteria have not yet been elucidated. In Escherichia coli, the mere presence of an active process remains an open question. Here, we investigate the conformation and segregation pattern of the E. coli genome by performing numerical simulations on a polymer model of the chromosome. We analyze the roles of the intrinsic structuring of chromosomes and the forced localization of specific loci, which are observed in vivo. Specifically, we examine the segregation pattern of a chromosome that is divided into four structured macrodomains (MDs) and two non-structured regions. We find that strong osmotic-like organizational forces, which stem from the differential condensation levels of the chromosome regions, dictate the cellular disposition of the chromosome. Strikingly, the comparison of our in silico results with fluorescent imaging of the chromosome choreography in vivo reveals that in the presence of MDs the targeting of the origin and terminus regions to specific positions are sufficient to generate a segregation pattern that is indistinguishable from experimentally observed patterns.

Chromosomal organization and segregation in Pseudomonas aeruginosa.

The study of chromosomal organization and segregation in a handful of bacteria has revealed surprising variety in the mechanisms mediating such fundamental processes. In this study, we further emphasized this diversity by revealing an original organization of the Pseudomonas aeruginosa chromosome. We analyzed the localization of 20 chromosomal markers and several components of the replication machinery in this important opportunistic γ-proteobacteria pathogen. This technique allowed us to show that the 6.3 Mb unique circular chromosome of P. aeruginosa is globally oriented from the old pole of the cell to the division plane/new pole along the oriC-dif axis. The replication machinery is positioned at mid-cell, and the chromosomal loci from oriC to dif are moved sequentially to mid-cell prior to replication. The two chromosomal copies are subsequently segregated at their final subcellular destination in the two halves of the cell. We identified two regions in which markers localize at similar positions, suggesting a bias in the distribution of chromosomal regions in the cell. The first region encompasses 1.4 Mb surrounding oriC, where loci are positioned around the 0.2/0.8 relative cell length upon segregation. The second region contains at least 800 kb surrounding dif, where loci show an extensive colocalization step following replication. We also showed that disrupting the ParABS system is very detrimental in P. aeruginosa. Possible mechanisms responsible for the coordinated chromosomal segregation process and for the presence of large distinctive regions are discussed.

Molecular basis for a protein-mediated DNA-bridging mechanism that functions in condensation of the E. coli chromosome.

The E. coli chromosome is condensed into insulated regions termed macrodomains (MDs), which are essential for genomic packaging. How chromosomal MDs are specifically organized and compacted is unknown. Here, we report studies revealing the molecular basis for Terminus-containing (Ter) chromosome condensation by the Ter-specific factor MatP. MatP contains a tripartite fold with a four-helix bundle DNA-binding motif, ribbon-helix-helix and C-terminal coiled-coil. Strikingly, MatP-matS structures show that the MatP coiled-coils form bridged tetramers that flexibly link distant matS sites. Atomic force microscopy and electron microscopy studies demonstrate that MatP alone loops DNA. Mutation of key coiled-coil residues destroys looping and causes a loss of Ter condensation in vivo. Thus, these data reveal the molecular basis for a protein-mediated DNA-bridging mechanism that mediates condensation of a large chromosomal domain in enterobacteria.

Sister chromatid interactions in bacteria revealed by a site-specific recombination assay.

The process of Sister Chromosome Cohesion (SCC), which holds together sister chromatids upon replication, is essential for chromosome segregation and DNA repair in eukaryotic cells. Although cohesion at the molecular level has never been described in E. coli, previous studies have reported that sister sequences remain co-localized for a period after their replication. Here, we have developed a new genetic recombination assay that probes the ability of newly replicated chromosome loci to interact physically. We show that Sister Chromatid Interaction (SCI) occurs exclusively within a limited time frame after replication. Importantly, we could differentiate sister cohesion and co-localization since factors such as MatP and MukB that reduced the co-localization of markers had no effect on molecular cohesion. The frequency of sister chromatid interactions were modulated by the activity of Topo-IV, revealing that DNA topology modulates cohesion at the molecular scale in bacteria.

A MatP-divisome interaction coordinates chromosome segregation with cell division in E. coli.

Initiation of chromosome segregation in bacteria is achieved by proteins acting near the origin of replication. Here, we report that the precise choreography of the terminus region of the Escherichia coli chromosome is also tightly controlled. The segregation of the terminus (Ter) macrodomain (MD) involves the structuring factor MatP. We characterized that migration of the Ter MD from the new pole to mid-cell and its subsequent persistent localization at mid-cell relies on several processes. First, the replication of the Ter DNA is concomitant with its recruitment from the new pole to mid-cell in a sequential order correlated with the position on the genetic map. Second, using a strain carrying a linear chromosome with the Ter MD split in two parts, we show that replisomes are repositioned at mid-cell when replication of the Ter occurs. Third, we demonstrate that anchoring the Ter MD at mid-cell depends on the specific interaction of MatP with the division apparatus-associated protein ZapB. Our results reveal how segregation of the Ter MD is integrated in the cell-cycle control.

Long-range chromosome organization in E. coli: a site-specific system isolates the Ter macrodomain.

The organization of the Escherichia coli chromosome into a ring composed of four macrodomains and two less-structured regions influences the segregation of sister chromatids and the mobility of chromosomal DNA. The structuring of the terminus region (Ter) into a macrodomain relies on the interaction of the protein MatP with a 13-bp target called matS repeated 23 times in the 800-kb-long domain. Here, by using a new method that allows the transposition of any chromosomal segment at a defined position on the genetic map, we reveal a site-specific system that restricts to the Ter region a constraining process that reduces DNA mobility and delays loci segregation. Remarkably, the constraining process is regulated during the cell cycle and occurs only when the Ter MD is associated with the division machinery at mid-cell. The change of DNA properties does not rely on the presence of a trans-acting mechanism but rather involves a cis-effect acting at a long distance from the Ter region. Two specific 12-bp sequences located in the flanking Left and Right macrodomains and a newly identified protein designated YfbV conserved with MatP through evolution are required to impede the spreading of the constraining process to the rest of the chromosome. Our results unravel a site-specific system required to restrict to the Ter region the consequences of anchoring the Ter MD to the division machinery.