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.

Prophage induction can facilitate the in vitro dispersal of multicellular Streptomyces structures.

Streptomyces are renowned for their prolific production of specialized metabolites with applications in medicine and agriculture. These multicellular bacteria present a sophisticated developmental cycle and play a key role in soil ecology. Little is known about the impact of Streptomyces phage on bacterial physiology. In this study, we investigated the conditions governing the expression and production of "Samy", a prophage found in Streptomyces ambofaciens ATCC 23877. This siphoprophage is produced simultaneously with the activation of other mobile genetic elements. Remarkably, the presence and production of Samy increases bacterial dispersal under in vitro stress conditions. Altogether, this study unveiled a new property of a bacteriophage infection in the context of multicellular aggregate dynamics.

Dynamics of the Streptomyces chromosome: chance and necessity.

Streptomyces are prolific producers of specialized metabolites with applications in medicine and agriculture. Remarkably, these bacteria possess a large linear chromosome that is genetically compartmentalized: core genes are grouped in the central part, while the ends are populated by poorly conserved genes including antibiotic biosynthetic gene clusters. The genome is highly unstable and exhibits distinct evolutionary rates along the chromosome. Recent chromosome conformation capture (3C) and comparative genomics studies have shed new light on the interplay between genome dynamics in space and time. Here, we review insights that illustrate how the balance between chance (random genome variations) and necessity (structural and functional constraints) may have led to the emergence of spatial structuring of the Streptomyces chromosome.

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.

Principles of bacterial genome organization, a conformational point of view.

Bacterial chromosomes are large molecules that need to be highly compacted to fit inside the cells. Chromosome compaction must facilitate and maintain key biological processes such as gene expression and DNA transactions (replication, recombination, repair, and segregation). Chromosome and chromatin 3D-organization in bacteria has been a puzzle for decades. Chromosome conformation capture coupled to deep sequencing (Hi-C) in combination with other "omics" approaches has allowed dissection of the structural layers that shape bacterial chromosome organization, from DNA topology to global chromosome architecture. Here we review the latest findings using Hi-C and discuss the main features of bacterial genome folding.

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.

Aminoglycoside resistance 16S rRNA methyltransferases block endogenous methylation, affect translation efficiency and fitness of the host.

In Gram-negative bacteria, acquired 16S rRNA methyltransferases ArmA and NpmA confer high-level resistance to all clinically useful aminoglycosides by modifying, respectively, G1405 and A1408 in the A-site. These enzymes must coexist with several endogenous methyltransferases that are essential for fine-tuning of the decoding center, such as RsmH and RsmI in Escherichia coli, which methylate C1402 and RsmF C1407. The resistance methyltransferases have a contrasting distribution--ArmA has spread worldwide, whereas a single clinical isolate producing NpmA has been reported. The rate of dissemination of resistance depends on the fitness cost associated with its expression. We have compared ArmA and NpmA in isogenic Escherichia coli harboring the corresponding structural genes and their inactive point mutants cloned under the control of their native constitutive promoter in the stable plasmid pGB2. Growth rate determination and competition experiments showed that ArmA had a fitness cost due to methylation of G1405, whereas NpmA conferred only a slight disadvantage to the host due to production of the enzyme. MALDI MS indicated that ArmA impeded one of the methylations at C1402 by RsmI, and not at C1407 as previously proposed, whereas NpmA blocked the activity of RsmF at C1407. A dual luciferase assay showed that methylation at G1405 and A1408 and lack of methylation at C1407 affect translation accuracy. These results indicate that resistance methyltransferases impair endogenous methylation with different consequences on cell fitness.

Molecular anatomy of the Streptococcus pyogenes pSM19035 partition and segrosome complexes.

Vancomycin or erythromycin resistance and the stability determinants, δω and ωεζ, of Enterococci and Streptococci plasmids are genetically linked. To unravel the mechanisms that promoted the stable persistence of resistance determinants, the early stages of Streptococcus pyogenes pSM19035 partitioning were biochemically dissected. First, the homodimeric centromere-binding protein, ω2, bound parS DNA to form a short-lived partition complex 1 (PC1). The interaction of PC1 with homodimeric δ [δ2 even in the apo form (Apo-δ2)], significantly stimulated the formation of a long-lived ω2·parS complex (PC2) without spreading into neighbouring DNA sequences. In the ATP·Mg2+ bound form, δ2 bound DNA, without sequence specificity, to form a transient dynamic complex (DC). Second, parS bound ω2 interacted with and promoted δ2 redistribution to co-localize with the PC2, leading to transient segrosome complex (SC, parS·ω2·Î´2) formation. Third, δ2, in the SC, interacted with a second SC and promoted formation of a bridging complex (BC). Finally, increasing ω2 concentrations stimulated the ATPase activity of δ2 and the BC was disassembled. We propose that PC, DC, SC and BC formation were dynamic processes and that the molar ω2:δ2 ratio and parS DNA control their temporal and spatial assembly during partition of pSM19035 before cell division.

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.

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.

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.

A toxin-antitoxin module as a target for antimicrobial development.

The emergence and spread of pathogenic bacteria that have become resistant to multiple antibiotics through lateral gene transfer have created the need of novel antimicrobials. Toxin-antitoxin (TA) modules, which have been implicated in plasmid maintenance and stress management, are ubiquitous among plasmids from vancomycin or methicillin resistant bacteria. In the Streptococcus pyogenes pSM19035-encoded TA loci, the labile epsilon antitoxin binds to free zeta toxin and neutralizes it. When the zeta toxin is freed from the epsilon antitoxin, it induces a reversible state of growth arrest with a drastic reduction on the rate of replication, transcription and translation. However, upon prolonged zeta toxin action, the cells can no longer be rescued from their stasis state. A compound that disrupts the epsilon.zeta interaction can be considered as an attractive antimicrobial agent. Gene epsilon was fused to luc (Luc-epsilon antitoxin) and zeta to the gfp gene (zeta-GFP). Luc-epsilon or epsilon antitoxin neutralizes the toxic effect of the zeta or zeta-GFP toxin. In the absence of the antitoxin, free zeta or zeta-GFP triggers a reversible loss of cell proliferation, but the zetaK46A-GFP variant fails to block growth. Bioluminescence resonance energy transfer (BRET) assay was developed for high-throughput screening (HTS). To develop the proper controls, molecular dynamics studies were used to predict that the Asp18 and/or Glu22 residues might be relevant for epsilon.zeta interaction. Luc-epsilon efficiently transfers the excited energy to the fluorescent acceptor molecule (zeta-GFP or zetaK46A-GFP) and rendered high bioluminescence BRET signals. The exchange of Asp18 to Ala from zeta (D18A) affects Luc-epsilon.zetaD18A K46A-GFP interaction. In this study, we validate the hypothesis that it is possible to disrupt a TA module and offer a novel and unexploited targets to fight against antibiotic-resistant strains.

Plasmid pSM19035, a model to study stable maintenance in Firmicutes.

pSM19035 is a low-copy-number theta-replicating plasmid, which belongs to the Inc18 family. Plasmids of this family, which show a modular organization, are functional in evolutionarily diverse bacterial species of the Firmicutes Phylum. This review summarizes our understanding, accumulated during the last 20 years, on the genetics, biochemistry, cytology and physiology of the five pSM19035 segregation (seg) loci, which map outside of the minimal replicon. The segA locus plays a role both in maximizing plasmid random segregation, and in avoiding replication fork collapses in those plasmids with long inverted repeated regions. The segB1 locus, which acts as the ultimate determinant of plasmid maintenance, encodes a short-lived epsilon(2) antitoxin protein and a long-lived zeta toxin protein, which form a complex that neutralizes zeta toxicity. The cells that do not receive a copy of the plasmid halt their proliferation upon decay of the epsilon(2) antitoxin. The segB2 locus, which encodes two trans-acting, ParA- and ParB-like proteins and six cis-acting parS centromeres, actively ensures equal or roughly equal distribution of plasmid copies to daughter cells. The segC locus includes functions that promote the shift from the use of DNA polymerase I to the replicase (PolC-PolE DNA polymerases). The segD locus, which encodes a trans-acting transcriptional repressor, omega(2), and six cis-acting cognate sites, coordinates the expression of genes that control copy number, better-than-random segregation and partition, and assures the proper balance of these different functions. Working in concert the five different loci achieve almost absolute plasmid maintenance with a minimal growth penalty.

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.

Toxin ζ Triggers a Survival Response to Cope with Stress and Persistence.

Bacteria have evolved complex regulatory controls in response to various environmental stresses. Protein toxins of the ζ superfamily, found in prominent human pathogens, are broadly distributed in nature. We show that ζ is a uridine diphosphate-N-acetylglucosamine (UNAG)-dependent ATPase whose activity is inhibited in vitro by stoichiometric concentrations of ε2 antitoxin. In vivo, transient ζ expression promotes a reversible multi-level response by altering the pool of signaling purine nucleotides, which leads to growth arrest (dormancy), although a small cell subpopulation persists rather than tolerating toxin action. High c-di-AMP levels (absence of phosphodiesterase GdpP) decrease, and low c-di-AMP levels (absence of diadenylate cyclase DisA) increase the rate of ζ persistence. The absence of CodY, a transition regulator from exponential to stationary phase, sensitizes cells to toxin action, and suppresses persisters formed in the ΔdisA context. These changes, which do not affect the levels of stochastic ampicillin (Amp) persistence, sensitize cells to toxin and Amp action. Our findings provide an explanation for the connection between ζ-mediated growth arrest (with alterations in the GTP and c-di-AMP pools) and persistence formation.

ParAB Partition Dynamics in Firmicutes: Nucleoid Bound ParA Captures and Tethers ParB-Plasmid Complexes.

In Firmicutes, small homodimeric ParA-like (δ2) and ParB-like (ω2) proteins, in concert with cis-acting plasmid-borne parS and the host chromosome, secure stable plasmid inheritance in a growing bacterial population. This study shows that (ω:YFP)2 binding to parS facilitates plasmid clustering in the cytosol. (δ:GFP)2 requires ATP binding but not hydrolysis to localize onto the cell's nucleoid as a fluorescent cloud. The interaction of (δ:CFP)2 or δ2 bound to the nucleoid with (ω:YFP)2 foci facilitates plasmid capture, from a very broad distribution, towards the nucleoid and plasmid pairing. parS-bound ω2 promotes redistribution of (δ:GFP)2, leading to the dynamic release of (δ:GFP)2 from the nucleoid, in a process favored by ATP hydrolysis and protein-protein interaction. (δD60A:GFP)2, which binds but cannot hydrolyze ATP, also forms unstable complexes on the nucleoid. In the presence of ω2, (δD60A:GFP)2 accumulates foci or patched structures on the nucleoid. We propose that (δ:GFP)2 binding to different nucleoid regions and to ω2-parS might generate (δ:GFP)2 gradients that could direct plasmid movement. The iterative pairing and unpairing cycles may tether plasmids equidistantly on the nucleoid to ensure faithful plasmid segregation by a mechanism compatible with the diffusion-ratchet mechanism as proposed from in vitro reconstituted systems.

Characterization of diacetylenic liposomes as carriers for oral vaccines.

In order to evaluate liposomes as vehicle for oral vaccines the characterization and stability of polymerized and non-polymerized liposomes were examined. Mixtures of 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3 phosphocholine) (DC8,9PC) with saturated 1,2-dimiristoyl-sn-glycero-3-phosphocholine in molar ratio 1:1 were used. Saturated and non-saturated lipids were combined to give a chemically modified membrane by UV polymerization derived from DC8,9PC. Characterization was carried out by electronic microscopy, differential scanning calorimetry (DSC) and by hydrophobicity factor (HF). The stability towards the digestive tract (including saliva): acidic solutions, bile and pancreatin are compared to buffer pH 7.4, measuring the release of Glucose-6-phosphate or bovine plasma albumin entrapment. The polymerized liposomes showed further augmentation of the HF and the size. DSC showed phase separation and lower Tt if compared to data obtained for DC8,9PC. The HF, as main factor is discussed in relation to in vitro stability, suggesting that polymerized and non-polymerized liposomes would serve effectively as an oral delivery vehicle.

Role of toxin ζ and starvation responses in the sensitivity to antimicrobials.

A fraction of otherwise antimicrobial-sensitive Bacillus subtilis cells, called persisters, are phenotypically tolerant of antimicrobial treatment. We report that, independently of B. subtilis' growth phase, transient ζ toxin expression induces a dormant state and alters cellular responses so that cells are more sensitive to antimicrobials with different modes of action. This outcome is modulated by fine tuning (p)ppGpp and GTP levels: i) in the presence of low "dysregulated" (p)ppGpp levels (as in relA (-) cells) hyper-tolerance to both toxin and antimicrobials was observed; ii) physiological or low (p)ppGpp levels (as in the wild-type, sasA (-), sasB (-) and relA (-) sasA (-) context) show a normal toxin and antimicrobial tolerance; and iii) lower levels (in relA (-) sasB (-)) or absence of (p)ppGpp (in the relA (-) sasA (-) sasB (-) context), in concert with elevated GTP levels, potentiate the efficacy of both toxin and antimicrobial action, rendering tolerance vulnerable to eradication.

The ζ toxin induces a set of protective responses and dormancy.

The ζε module consists of a labile antitoxin protein, ε, which in dimer form (ε(2)) interferes with the action of the long-living monomeric ζ phosphotransferase toxin through protein complex formation. Toxin ζ, which inhibits cell wall biosynthesis and may be bactericide in nature, at or near physiological concentrations induces reversible cessation of Bacillus subtilis proliferation (protective dormancy) by targeting essential metabolic functions followed by propidium iodide (PI) staining in a fraction (20-30%) of the population and selects a subpopulation of cells that exhibit non-inheritable tolerance (1-5×10(-5)). Early after induction ζ toxin alters the expression of ∼78 genes, with the up-regulation of relA among them. RelA contributes to enforce toxin-induced dormancy. At later times, free active ζ decreases synthesis of macromolecules and releases intracellular K(+). We propose that ζ toxin induces reversible protective dormancy and permeation to PI, and expression of ε(2) antitoxin reverses these effects. At later times, toxin expression is followed by death of a small fraction (∼10%) of PI stained cells that exited earlier or did not enter into the dormant state. Recovery from stress leads to de novo synthesis of ε(2) antitoxin, which blocks ATP binding by ζ toxin, thereby inhibiting its phosphotransferase activity.