Combined Study of Gene Expression and Chromosome Three-Dimensional Structure in Escherichia coli During Growth Process.

The chromosome structure of different bacteria has its unique organization pattern, which plays an important role in maintaining the spatial location relationship between genes and regulating gene expression. Conversely, transcription also plays a global role in regulating the three-dimensional structure of bacterial chromosomes. Therefore, we combine RNA-Seq and Hi-C technology to explore the relationship between chromosome structure changes and transcriptional regulation in E. coli at different growth stages. Transcriptome analysis indicates that E. coli synthesizes many ribosomes and peptidoglycan in the exponential phase. In contrast, E. coli undergoes more transcriptional regulation and catabolism during the stationary phase, reflecting its adaptability to changes in environmental conditions during growth. Analyzing the Hi-C data shows that E. coli has a higher frequency of global chromosomal interaction in the exponential phase and more defined chromosomal interaction domains (CIDs). Still, the long-distance interactions at the replication termination region are lower than in the stationary phase. Combining transcriptome and Hi-C data analysis, we conclude that highly expressed genes are more likely to be distributed in CID boundary regions during the exponential phase. At the same time, most high-expression genes distributed in the CID boundary regions are ribosomal gene clusters, forming clearer CID boundaries during the exponential phase. The three-dimensional structure of chromosome and expression pattern is altered during the growth of E. coli from the exponential phase to the stationary phase, clarifying the synergy between the two regulatory aspects.

Direct observation of a crescent-shape chromosome in expanded Bacillus subtilis cells.

Bacterial chromosomes are folded into tightly regulated three-dimensional structures to ensure proper transcription, replication, and segregation of the genetic information. Direct visualization of chromosomal shape within bacterial cells is hampered by cell-wall confinement and the optical diffraction limit. Here, we combine cell-shape manipulation strategies, high-resolution fluorescence microscopy techniques, and genetic engineering to visualize the shape of unconfined bacterial chromosome in real-time in live Bacillus subtilis cells that are expanded in volume. We show that the chromosomes predominantly exhibit crescent shapes with a non-uniform DNA density that is increased near the origin of replication (oriC). Additionally, we localized ParB and BsSMC proteins - the key drivers of chromosomal organization - along the contour of the crescent chromosome, showing the highest density near oriC. Opening of the BsSMC ring complex disrupted the crescent chromosome shape and instead yielded a torus shape. These findings help to understand the threedimensional organization of the chromosome and the main protein complexes that underlie its structure.

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.

Bacterial nucleoid is a riddle wrapped in a mystery inside an enigma.

Bacterial chromosome, the nucleoid, is traditionally modeled as a rosette of DNA mega-loops, organized around proteinaceous central scaffold by nucleoid-associated proteins (NAPs), and mixed with the cytoplasm by transcription and translation. Electron microscopy of fixed cells confirms dispersal of the cloud-like nucleoid within the ribosome-filled cytoplasm. Here, I discuss evidence that the nucleoid in live cells forms DNA phase separate from riboprotein phase, the "riboid." I argue that the nucleoid-riboid interphase, where DNA interacts with NAPs, transcribing RNA polymerases, nascent transcripts, and ssRNA chaperones, forms the transcription zone. An active part of phase separation, transcription zone enforces segregation of the centrally positioned information phase (the nucleoid) from the surrounding action phase (the riboid), where translation happens, protein accumulates, and metabolism occurs. I speculate that HU NAP mostly tiles up the nucleoid periphery-facilitating DNA mobility but also supporting transcription in the interphase. Besides extruding plectonemically supercoiled DNA mega-loops, condensins could compact them into solenoids of uniform rings, while HU could support rigidity and rotation of these DNA rings. The two-phase cytoplasm arrangement allows the bacterial cell to organize the central dogma activities, where (from the cell center to its periphery) DNA replicates and segregates, DNA is transcribed, nascent mRNA is handed over to ribosomes, mRNA is translated into proteins, and finally, the used mRNA is recycled into nucleotides at the inner membrane. The resulting information-action conveyor, with one activity naturally leading to the next one, explains the efficiency of prokaryotic cell design-even though its main intracellular transportation mode is free diffusion.

Chromosome structure modeling tools and their evaluation in bacteria.

The three-dimensional (3D) structure of bacterial chromosomes is crucial for understanding chromosome function. With the growing availability of high-throughput chromosome conformation capture (3C/Hi-C) data, the 3D structure reconstruction algorithms have become powerful tools to study bacterial chromosome structure and function. It is highly desired to have a recommendation on the chromosome structure reconstruction tools to facilitate the prokaryotic 3D genomics. In this work, we review existing chromosome 3D structure reconstruction algorithms and classify them based on their underlying computational models into two categories: constraint-based modeling and thermodynamics-based modeling. We briefly compare these algorithms utilizing 3C/Hi-C datasets and fluorescence microscopy data obtained from Escherichia coli and Caulobacter crescentus, as well as simulated datasets. We discuss current challenges in the 3D reconstruction algorithms for bacterial chromosomes, primarily focusing on software usability. Finally, we briefly prospect future research directions for bacterial chromosome structure reconstruction algorithms.

Transcription-induced domains form the elementary constraining building blocks of bacterial chromosomes.

Transcription generates local topological and mechanical constraints on the DNA fiber, leading to the generation of supercoiled chromosome domains in bacteria. However, the global impact of transcription on chromosome organization remains elusive, as the scale of genes and operons in bacteria remains well below the resolution of chromosomal contact maps generated using Hi-C (~5-10 kb). Here we combined sub-kb Hi-C contact maps and chromosome engineering to visualize individual transcriptional units. We show that transcriptional units form discrete three-dimensional transcription-induced domains that impose mechanical and topological constraints on their neighboring sequences at larger scales, modifying their localization and dynamics. These results show that transcriptional domains constitute primary building blocks of bacterial chromosome folding and locally impose structural and dynamic constraints.

Transcription-replication interactions reveal bacterial genome regulation.

Organisms determine the transcription rates of thousands of genes through a few modes of regulation that recur across the genome1. In bacteria, the relationship between the regulatory architecture of a gene and its expression is well understood for individual model gene circuits2,3. However, a broader perspective of these dynamics at the genome scale is lacking, in part because bacterial transcriptomics has hitherto captured only a static snapshot of expression averaged across millions of cells4. As a result, the full diversity of gene expression dynamics and their relation to regulatory architecture remains unknown. Here we present a novel genome-wide classification of regulatory modes based on the transcriptional response of each gene to its own replication, which we term the transcription-replication interaction profile (TRIP). Analysing single-bacterium RNA-sequencing data, we found that the response to the universal perturbation of chromosomal replication integrates biological regulatory factors with biophysical molecular events on the chromosome to reveal the local regulatory context of a gene. Whereas the TRIPs of many genes conform to a gene dosage-dependent pattern, others diverge in distinct ways, and this is shaped by factors such as intra-operon position and repression state. By revealing the underlying mechanistic drivers of gene expression heterogeneity, this work provides a quantitative, biophysical framework for modelling replication-dependent expression dynamics.

Understanding the Impact of Temperate Bacteriophages on Their Lysogens Through Transcriptomics.

Temperate phages are found integrated as prophages in the majority of bacterial genomes. Some prophages are cryptic and fixed in the bacterial chromosome, but others are active and can be triggered into a replicative form either spontaneously or by exposure to inducing factors. Prophages are commonly associated with the ability to confer toxin production or other virulence-associated traits on their host cell. More recent studies have shown they can play a much bigger role in altering the physiology of their hosts. The technique described here has enabled us to investigate how prophages affect gene expression in the opportunistic bacterium Pseudomonas aeruginosa. In this work, the growth of the wild-type P. aeruginosa strain PAO1 was compared with that of isogenic lysogens carrying different combinations of prophages from the Liverpool Epidemic Strain (LES) LESB58. In a lysogen culture, a proportion of bacterial cells will be supporting lytic bacteriophage replication (spontaneous induction) with a high level of expression per cell of late phage genes, such as those associated with the assembly of phage particles, thus masking the low-level gene expression associated with lysogen-restricted gene expression. The impact of spontaneous induction can thus obscure prophage gene expression across a lysogen population. Growth profiling experiments were used to identify spontaneous induction, which was minimal during the early exponential growth phase. This study reports how to prepare sample cultures during the early exponential growth phase and how to set up adequate controls despite low cell numbers. These protocols ensure the reliable and reproducible comparison of wild-type and lysogenic bacteria under various conditions, thus improving the transcriptomic profiling of prophage genomes and aiding in the identification of previously unrecognized prophage functions.

Dual-Fluorescence Chromosome-Located Labeling System for Accurate In Vivo Single-Cell Gene Expression Analysis in Pseudomonas syringae.

Epigenetic regulation as a means for bacterial adaptation is receiving increasing interest in the last decade. Significant efforts have been directed towards understanding the mechanisms giving raise to phenotypic heterogeneity within bacterial populations and its adaptive relevance. Phenotypic heterogeneity mostly refers to phenotypic variation not linked to genetic differences nor to environmental stimuli. Recent findings on the relevance of phenotypic heterogeneity on some bacterial complex traits are causing a shift from traditional assays where bacterial phenotypes are defined by averaging population-level data, to single-cell analysis that focus on bacterial individual behavior within the population. Fluorescent labeling is a key asset for single-cell gene expression analysis using flow cytometry, fluorescence microscopy, and/or microfluidics.We previously described the generation of chromosome-located transcriptional gene fusions to fluorescent reporter genes using the model bacterial plant pathogen Pseudomonas syringae. These fusions allow researchers to follow variation in expression of the gene(s) of interest, without affecting gene function. In this report, we improve the analytic power of the method by combining such transcriptional fusions with constitutively expressed compatible fluorescent reporter genes integrated in a second, neutral locus of the bacterial chromosome. Constitutively expressed fluorescent reporters allow for the detection of all bacteria comprising a heterogeneous population, regardless of the level of expression of the concurrently monitored gene of interest, thus avoiding the traditional use of stains often incompatible with samples from complex contexts such as the leaf.

Modeling the compaction of bacterial chromosomes by biomolecular crowding and the cross-linking protein H-NS.

Cells orchestrate the action of various molecules toward organizing their chromosomes. Using a coarse-grained computational model, we study the compaction of bacterial chromosomes by the cross-linking protein H-NS and cellular crowders. In this work, H-NS, modeled as a mobile "binder," can bind to a chromosome-like polymer with a characteristic binding energy. The simulation results reported here clarify the relative role of biomolecular crowding and H-NS in condensing a bacterial chromosome in a quantitative manner. In particular, they shed light on the nature and degree of crowder and H-NS synergetics: while the presence of crowders enhances H-NS binding to a chromosome-like polymer, the presence of H-NS makes crowding effects more efficient, suggesting two-way synergetics in chain compaction. Also, the results show how crowding effects promote clustering of bound H-NS. For a sufficiently large concentration of H-NS, the cluster size increases with the volume fraction of crowders.

Tight coupling of cell width to nucleoid structure in Escherichia coli.

Cell dimensions of rod-shaped bacteria such as Escherichia coli are connected to mass growth and chromosome replication. During their interdivision cycle (τ min), cells enlarge by elongation only, but at faster growth in richer media, they are also wider. Changes in width W upon nutritional shift-up (shortening τ) occur during the division process. The elusive signal directing the mechanism for W determination is likely related to the tightly linked duplications of the nucleoid (DNA) and the sacculus (peptidoglycan), the only two structures (macromolecules) existing in a single copy that are coupled, temporally and spatially. Six known parameters related to the nucleoid structure and replication are reasonable candidates to convey such a signal, all simple functions of the key number of replication positions n(=C/τ), the ratio between the rates of growth (τ-1) and of replication (C-1). The current analysis of available literature-recorded data discovered that, of these, nucleoid complexity NC[=(2n-1)/(n×ln2)] is by far the most likely parameter affecting cell width W. The exceedingly high correlations found between these two seemingly unrelated measures (NC and W) indicate that coupling between them is of major importance to the species' survival. As an exciting corollary, to the best of our knowledge, a new, indirect approach to estimate DNA replication rate is revealed. Potential involvement of DNA topoisomerases in W determination is also proposed and discussed.

Development of a Data-Driven Integrative Model of a Bacterial Chromosome.

The chromosome of archetypal bacteria E. coli is known for a complex topology with a 4.6 × 106 base pairs (bp) long sequence of nucleotides packed within a micrometer-sized cellular confinement. The inherent organization underlying this chromosome eludes general consensus due to the lack of a high-resolution picture of its conformation. Here we present our development of an integrative model of E. coli at a 500 bp resolution (https://github.com/JMLab-tifrh/ecoli_finer), which optimally combines a set of multiresolution genome-wide experimentally measured data within a framework of polymer based architecture. In particular the model is informed with an intragenome contact probability map at 5000 bp resolution derived via the Hi-C experiment and RNA-sequencing data at 500 bp resolution. Via dynamical simulations, this data-driven polymer based model generates an appropriate conformational ensemble commensurate with chromosome architectures that E. coli adopts. As a key hallmark of the E. coli chromosome the model spontaneously self-organizes into a set of nonoverlapping macrodomains and suitably locates plectonemic loops near the cell membrane. As novel extensions, it predicts a contact probability map simulated at a higher resolution than precedent experiments and can demonstrate segregation of chromosomes in a partially replicating cell. Finally, the modular nature of the model helps us devise control simulations to quantify the individual role of key features in hierarchical organization of the bacterial chromosome.

Massive integration of large gene libraries in the chromosome of Escherichia coli.

Large gene libraries are frequently created in Escherichia coli plasmids, which can induce cell toxicity and expression instability due to the high gene dosage. To address these limitations, gene libraries can be integrated in a single copy into the bacterial chromosome. Here, we describe an efficient system for the massive integration (MAIN) of large gene libraries in the E. coli chromosome that generates in-frame gene fusions that are expressed stably. MAIN uses a thermosensitive integrative plasmid that is linearized in vivo to promote extensive integration of the gene library via homologous recombination. Positive and negative selections efficiently remove bacteria lacking gene integration in the target site. We tested MAIN with a library of 107 VHH genes that encode nanobodies (Nbs). The integration of VHH genes into a custom target locus of the E. coli chromosome enabled stable expression and surface display of the Nbs. Next-generation DNA sequencing confirmed that MAIN preserved the diversity of the gene library after integration. Finally, we screened the integrated library to select Nbs that bind a specific antigen using magnetic and fluorescence-activated cell sorting. This allowed us to identify Nbs binding the epidermal growth factor receptor that were not previously isolated in a similar screening of a multicopy plasmid library. Our results demonstrate that MAIN enables large gene library integration into the E. coli chromosome, creating stably expressed in-frame fusions for functional screening.

Connecting the dots: key insights on ParB for chromosome segregation from single-molecule studies.

Bacterial cells require DNA segregation machinery to properly distribute a genome to both daughter cells upon division. The most common system involved in chromosome and plasmid segregation in bacteria is the ParABS system. A core protein of this system - partition protein B (ParB) - regulates chromosome organization and chromosome segregation during the bacterial cell cycle. Over the past decades, research has greatly advanced our knowledge of the ParABS system. However, many intricate details of the mechanism of ParB proteins were only recently uncovered using in vitro single-molecule techniques. These approaches allowed the exploration of ParB proteins in precisely controlled environments, free from the complexities of the cellular milieu. This review covers the early developments of this field but emphasizes recent advances in our knowledge of the mechanistic understanding of ParB proteins as revealed by in vitro single-molecule methods. Furthermore, we provide an outlook on future endeavors in investigating ParB, ParB-like proteins, and their interaction partners.

Apparent simplicity and emergent robustness in the control of the Escherichia coli cell cycle.

To examine how bacteria achieve robust cell proliferation across diverse conditions, we developed a method that quantifies 77 cell morphological, cell cycle, and growth phenotypes of a fluorescently labeled Escherichia coli strain and >800 gene deletion derivatives under multiple nutrient conditions. This approach revealed extensive phenotypic plasticity and deviating mutant phenotypes were often nutrient dependent. From this broad phenotypic landscape emerged simple and robust unifying rules (laws) that connect DNA replication initiation, nucleoid segregation, FtsZ ring formation, and cell constriction to specific aspects of cell size (volume, length, or added length) at the population level. Furthermore, completion of cell division followed the initiation of cell constriction after a constant time delay across strains and nutrient conditions, identifying cell constriction as a key control point for cell size determination. Our work provides a population-level description of the governing principles by which E. coli integrates cell cycle processes and growth rate with cell size to achieve its robust proliferative capability. A record of this paper's transparent peer review process is included in the supplemental information.

The bacterial replication origin BUS promotes nucleobase capture.

Genome duplication is essential for the proliferation of cellular life and this process is generally initiated by dedicated replication proteins at chromosome origins. In bacteria, DNA replication is initiated by the ubiquitous DnaA protein, which assembles into an oligomeric complex at the chromosome origin (oriC) that engages both double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) to promote DNA duplex opening. However, the mechanism of DnaA specifically opening a replication origin was unknown. Here we show that Bacillus subtilis DnaAATP assembles into a continuous oligomer at the site of DNA melting, extending from a dsDNA anchor to engage a single DNA strand. Within this complex, two nucleobases of each ssDNA binding motif (DnaA-trio) are captured within a dinucleotide binding pocket created by adjacent DnaA proteins. These results provide a molecular basis for DnaA specifically engaging the conserved sequence elements within the bacterial chromosome origin basal unwinding system (BUS).

Mid-cell migration of the chromosomal terminus is coupled to origin segregation in Escherichia coli.

Bacterial chromosomes are dynamically and spatially organised within cells. In slow-growing Escherichia coli, the chromosomal terminus is initially located at the new pole and must therefore migrate to midcell during replication to reproduce the same pattern in the daughter cells. Here, we use high-throughput time-lapse microscopy to quantify this transition, its timing and its relationship to chromosome segregation. We find that terminus centralisation is a rapid discrete event that occurs ~25 min after initial separation of duplicated origins and ~50 min before the onset of bulk nucleoid segregation but with substantial variation between cells. Despite this variation, its movement is tightly coincident with the completion of origin segregation, even in the absence of its linkage to the divisome, suggesting a coupling between these two events. Indeed, we find that terminus centralisation does not occur if origin segregation away from mid-cell is disrupted, which results in daughter cells having an inverted chromosome organisation. Overall, our study quantifies the choreography of origin-terminus positioning and identifies an unexplored connection between these loci, furthering our understanding of chromosome segregation in this bacterium.

To let go or not to let go: how ParA can impact the release of the chromosomal anchoring in Caulobacter crescentus.

Chromosomal maintenance is vital for the survival of bacteria. In Caulobacter crescentus, chromosome replication initiates at ori and segregation is delayed until the nearby centromere-like region parS is replicated. Our understanding of how this sequence of events is regulated remains limited. The segregation of parS has been shown to involve multiple steps including polar release from anchoring protein PopZ, slow movement and fast ParA-dependent movement to the opposite cell pole. In this study, we demonstrate that ParA's competing attractions from PopZ and from DNA are critical for segregation of parS. Interfering with this balance of attractions-by expressing a variant ParA-R195E unable to bind DNA and thus favoring interactions exclusively between ParA-PopZ-results in cell death. Our data revealed that ParA-R195E's sole interactions with PopZ obstruct PopZ's ability to release the polar anchoring of parS, resulting in cells with multiple parS loci fixed at one cell pole. We show that the inability to separate and segregate multiple parS loci from the pole is specifically dependent on the interaction between ParA and PopZ. Collectively, our results reveal that the initial steps in chromosome segregation are highly regulated.

Dynamic ParB-DNA interactions initiate and maintain a partition condensate for bacterial chromosome segregation.

In most bacteria, chromosome segregation is driven by the ParABS system where the CTPase protein ParB loads at the parS site to trigger the formation of a large partition complex. Here, we present in vitro studies of the partition complex for Bacillus subtilis ParB, using single-molecule fluorescence microscopy and AFM imaging to show that transient ParB-ParB bridges are essential for forming DNA condensates. Molecular Dynamics simulations confirm that condensation occurs abruptly at a critical concentration of ParB and show that multimerization is a prerequisite for forming the partition complex. Magnetic tweezer force spectroscopy on mutant ParB proteins demonstrates that CTP hydrolysis at the N-terminal domain is essential for DNA condensation. Finally, we show that transcribing RNA polymerases can steadily traverse the ParB-DNA partition complex. These findings uncover how ParB forms a stable yet dynamic partition complex for chromosome segregation that induces DNA condensation and segregation while enabling replication and transcription.

Cell cycle-dependent organization of a bacterial centromere through multi-layered regulation of the ParABS system.

The accurate distribution of genetic material is crucial for all organisms. In most bacteria, chromosome segregation is achieved by the ParABS system, in which the ParB-bound parS sequence is actively partitioned by ParA. While this system is highly conserved, its adaptation in organisms with unique lifestyles and its regulation between developmental stages remain largely unexplored. Bdellovibrio bacteriovorus is a predatory bacterium proliferating through polyploid replication and non-binary division inside other bacteria. Our study reveals the subcellular dynamics and multi-layered regulation of the ParABS system, coupled to the cell cycle of B. bacteriovorus. We found that ParA:ParB ratios fluctuate between predation stages, their balance being critical for cell cycle progression. Moreover, the parS chromosomal context in non-replicative cells, combined with ParB depletion at cell division, critically contribute to the unique cell cycle-dependent organization of the centromere in this bacterium, highlighting new levels of complexity in chromosome segregation and cell cycle control.