In vivo assembly of bacterial partition condensates on circular supercoiled and linear DNA.

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.

Identification of gene targets that potentiate the action of rifampicin on Mycobacterium bovis BCG.

Tuberculosis (TB) caused by bacteria of the Mycobacterium tuberculosis complex remains one of the most important infectious diseases of mankind. Rifampicin is a first line drug used in multi-drug treatment of TB, however, the necessary duration of treatment with these drugs is long and development of resistance is an increasing impediment to treatment programmes. As a result, there is a requirement for research and development of new TB drugs, which can form the basis of new drug combinations, either due to their own anti-mycobacterial activity or by augmenting the activity of existing drugs such as rifampicin. This study describes a TnSeq analysis to identify mutants with enhanced sensitivity to sub-minimum inhibitory concentrations (MIC) of rifampicin. The rifampicin-sensitive mutants were disrupted in genes of a variety of functions and the majority fitted into three thematic groups: firstly, genes that were involved in DNA/RNA metabolism, secondly, genes involved in sensing and regulating mycobacterial cellular systems, and thirdly, genes involved in the synthesis and maintenance of the cell wall. Selection at two concentrations of rifampicin (1/250 and 1/62 MIC) demonstrated a dose response for mutants with statistically significant sensitivity to rifampicin. The dataset reveals mechanisms of how mycobacteria are innately tolerant to and initiate an adaptive response to rifampicin; providing putative targets for the development of adjunctive therapies that potentiate the action of rifampicin.

Visualization of the Association of Dimeric Protein Complexes on Specific Enhancers in the Salivary Gland Nuclei of Drosophila Larva.

Transcription factors (TFs) regulate gene expression by recognizing specific target enhancers in the genome. The DNA-binding and regulatory activity of TFs depend on the presence of additional protein partners, leading to the formation of versatile and dynamic multimeric protein complexes. Visualizing these protein-protein interactions (PPIs) in the nucleus is key for decrypting the molecular cues underlying TF specificity in vivo. Over the last few years, Bimolecular Fluorescence Complementation (BiFC) has been developed in several model systems and applied in the analysis of different types of PPIs. In particular, BiFC has been applied when analyzing PPIs with hundreds of TFs in the nucleus of live Drosophila embryos. However, the visualization of PPIs at the level of specific target enhancers or genomic regions of interest awaits the advent of DNA-labelling methods that can be coupled with BiFC. Here, we present a novel experimental strategy that we have called BiFOR and that is based on the coupling of BiFC with the bacterial ANCHOR DNA-labelling system. We demonstrate that BiFOR enables the precise quantification of the enrichment of specific dimeric protein complexes on target enhancers in Drosophila salivary gland nuclei. Given its versatility and sensitivity, BiFOR could be applied more widely to other tissues during Drosophila development. Our work sets up the experimental basis for future applications of this strategy.

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.

Species Discrimination within the Metarhizium PARB Clade: Ribosomal Intergenic Spacer (rIGS)-Based Diagnostic PCR and Single Marker Taxonomy.

(1) Background: The entomopathogenic fungus Metarhizium anisopliae sensu lato forms a species complex, comprising a tight cluster made up of four species, namely M. anisopliae sensu stricto, M. pinghaense, M. robertsii and M. brunneum. Unambiguous species delineation within this "PARB clade" that enables both the taxonomic assignment of new isolates and the identification of potentially new species is highly solicited. (2) Methods: Species-discriminating primer pairs targeting the ribosomal intergenic spacer (rIGS) sequence were designed and a diagnostic PCR protocol established. A partial rIGS sequence, referred to as rIGS-ID800, was introduced as a molecular taxonomic marker for PARB species delineation. (3) Results: PARB species from a validation strain set not implied in primer design were clearly discriminated using the diagnostic PCR protocol developed. Using rIGS-ID800 as a single sequence taxonomic marker gave rise to a higher resolution and statistically better supported delineation of PARB clade species. (4) Conclusions: Reliable species discrimination within the Metarhizium PARB clade is possible through both sequencing-independent diagnostic PCR and sequencing-dependent single marker comparison, both based on the rIGS marker.

A framework to validate fluorescently labeled DNA-binding proteins for single-molecule experiments.

Due to the enhanced labeling capability of maleimide-based fluorescent probes, lysine-cysteine-lysine (KCK) tags are frequently added to proteins for visualization. In this study, we employed an in vitro single-molecule DNA flow-stretching assay as a sensitive way to assess the impact of the KCK tag on the property of DNA-binding proteins. Using Bacillus subtilis ParB as an example, we show that, although no noticeable changes were detected by in vivo fluorescence imaging and chromatin immunoprecipitation (ChIP) assays, the KCK tag substantially altered ParB's DNA compaction rates and its response to nucleotide binding and to the presence of the specific sequence (parS) on the DNA. While it is typically assumed that short peptide tags minimally perturb protein function, our results urge researchers to carefully validate the use of tags for protein labeling. Our comprehensive analysis can be expanded and used as a guide to assess the impacts of other tags on DNA-binding proteins in single-molecule assays.

VirB, a key transcriptional regulator of Shigella virulence, requires a CTP ligand for its regulatory activities.

IMPORTANCE: Shigella species cause bacillary dysentery, the second leading cause of diarrheal deaths worldwide. There is a pressing need to identify novel molecular drug targets. Shigella virulence phenotypes are controlled by the transcriptional regulator, VirB. We show that VirB belongs to a fast-evolving, plasmid-borne clade of the ParB superfamily, which has diverged from versions with a distinct cellular role-DNA partitioning. We report that, like classic members of the ParB family, VirB binds a highly unusual ligand, CTP. Mutants predicted to be defective in CTP binding are compromised in a variety of virulence attributes controlled by VirB, likely because these mutants cannot engage DNA. This study (i) reveals that VirB binds CTP, (ii) provides a link between VirB-CTP interactions and Shigella virulence phenotypes, (iii) provides new insight into VirB-CTP-DNA interactions, and (iv) broadens our understanding of the ParB superfamily, a group of bacterial proteins that play critical roles in many bacteria.

Robust ParB Binding to Half-parS Sites in Pseudomonas aeruginosa-A Mechanism for Retaining ParB on the Nucleoid?

Chromosome segregation in Pseudomonas aeruginosa is assisted by the tripartite ParAB-parS system, composed of an ATPase (ParA), a DNA-binding protein (ParB) and its target parS sequence(s). ParB forms a nucleoprotein complex around four parSs (parS1-parS4) that overlaps oriC and facilitates relocation of newly synthesized ori domains inside the cells by ParA. Remarkably, ParB of P. aeruginosa also binds to numerous heptanucleotides (half-parSs) scattered in the genome. Here, using chromatin immunoprecipitation-sequencing (ChIP-seq), we analyzed patterns of ParB genome occupancy in cells growing under conditions of coupling or uncoupling between replication and cell division processes. Interestingly, a dissipation of ParB-parS complexes and a shift of ParB to half-parSs were observed during the transition from the exponential to stationary phase of growth on rich medium, suggesting the role of half-parSs in retaining ParB on the nucleoid within non-dividing P. aeruginosa cells. The ChIP-seq analysis of strains expressing ParB variants unable to dislocate from parSs showed that the ParB spreading ability is not required for ParB binding to half-parSs. Finally, a P. aeruginosa strain with mutated 25 half-parSs of the highest affinity towards ParB was constructed and analyzed. It showed altered ParB coverage of the oriC region and moderate changes in gene expression. Overall, this study characterizes a novel aspect of conserved bacterial chromosome segregation machinery.

CTP switches in ParABS-mediated bacterial chromosome segregation and beyond.

Segregation of genetic material is a fundamental process in biology. In many bacterial species, segregation of chromosomes and low-copy plasmids is facilitated by the tripartite ParA-ParB-parS system. This system consists of a centromeric parS DNA site and interacting proteins ParA and ParB that are capable of hydrolyzing adenosine triphosphate and cytidine triphosphate (CTP), respectively. ParB first binds to parS before associating with adjacent DNA regions to spread outward from parS. These ParB-DNA complexes bind to ParA and, through repetitive cycles of ParA-ParB binding and unbinding, move the DNA cargo to each daughter cell. The recent discovery that ParB binds and hydrolyzes CTP as it cycles on and off the bacterial chromosome has dramatically changed our understanding of the molecular mechanism used by the ParABS system. Beyond bacterial chromosome segregation, CTP-dependent molecular switches are likely to be more widespread in biology than previously appreciated and represent an opportunity for new and unexpected avenues for future research and application.

The CTP-binding domain is disengaged from the DNA-binding domain in a cocrystal structure of Bacillus subtilis Noc-DNA complex.

In Bacillus subtilis, a ParB-like nucleoid occlusion protein (Noc) binds specifically to Noc-binding sites (NBSs) on the chromosome to help coordinate chromosome segregation and cell division. Noc does so by binding to CTP to form large membrane-associated nucleoprotein complexes to physically inhibit the assembly of the cell division machinery. The site-specific binding of Noc to NBS DNA is a prerequisite for CTP-binding and the subsequent formation of a membrane-active DNA-entrapped protein complex. Here, we solve the structure of a C-terminally truncated B. subtilis Noc bound to NBS DNA to reveal the conformation of Noc at this crucial step. Our structure reveals the disengagement between the N-terminal CTP-binding domain and the NBS-binding domain of each DNA-bound Noc subunit; this is driven, in part, by the swapping of helices 4 and 5 at the interface of the two domains. Site-specific crosslinking data suggest that this conformation of Noc-NBS exists in solution. Overall, our results lend support to the recent proposal that parS/NBS binding catalyzes CTP binding and DNA entrapment by preventing the reengagement of the CTP-binding domain and the DNA-binding domain from the same ParB/Noc subunit.

Visualizing the Genome: Experimental Approaches for Live-Cell Chromatin Imaging.

Over the years, our vision of the genome has changed from a linear molecule to that of a complex 3D structure that follows specific patterns and possesses a hierarchical organization. Currently, genomics is becoming "four-dimensional": our attention is increasingly focused on the study of chromatin dynamics over time, in the fourth dimension. Recent methods for visualizing the movements of chromatin loci in living cells by targeting fluorescent proteins can be divided into two groups. The first group requires the insertion of a special sequence into the locus of interest, to which proteins that recognize the sequence are recruited (e.g., FROS and ParB-INT methods). In the methods of the second approach, "programmed" proteins are targeted to the locus of interest (i.e., systems based on CRISPR/Cas, TALE, and zinc finger proteins). In the present review, we discuss these approaches, examine their strengths and weaknesses, and identify the key scientific problems that can be studied using these methods.

Characterization of a pathway-specific activator of edeine biosynthesis and improved edeine production by its overexpression in Brevibacillus brevis.

Edeines are a group of non-ribosomal antibacterial peptides produced by Brevibacillus brevis. Due to the significant antibacterial properties of edeines, increasing edeine yield is of great interest in biomedical research. Herein, we identified that EdeB, a member of the ParB protein family, significantly improved edeine production in B. brevis. First, overexpression of edeB in B. brevis X23 increased edeine production by 92.27%. Second, in vitro bacteriostasis experiment showed that edeB-deletion mutant exhibited less antibacterial activity. Third, RT-qPCR assay demonstrated that the expression of edeA, edeQ, and edeK, which are key components of the edeine biosynthesis pathway, in edeB-deletion mutant X23(ΔedeB) was significantly lower than that in wild-type B. brevis strain X23. Finally, electrophoretic mobility shift assay (EMSA) showed that EdeB directly bound to the promoter region of the edeine biosynthetic gene cluster (ede BGC), suggesting that EdeB improves edeine production through interaction with ede BGC in B. brevis.

Regulation of DNA replication initiation by ParA is independent of parS location in Bacillus subtilis.

Replication and segregation of the genetic information is necessary for a cell to proliferate. In Bacillus subtilis, the Par system (ParA/Soj, ParB/Spo0J and parS) is required for segregation of the chromosome origin (oriC) region and for proper control of DNA replication initiation. ParB binds parS sites clustered near the origin of replication and assembles into sliding clamps that interact with ParA to drive origin segregation through a diffusion-ratchet mechanism. As part of this dynamic process, ParB stimulates ParA ATPase activity to trigger its switch from an ATP-bound dimer to an ADP-bound monomer. In addition to its conserved role in DNA segregation, ParA is also a regulator of the master DNA replication initiation protein DnaA. We hypothesized that in B. subtilis the location of the Par system proximal to oriC would be necessary for ParA to properly regulate DnaA. To test this model, we constructed a range of genetically modified strains with altered numbers and locations of parS sites, many of which perturbed chromosome origin segregation as expected. Contrary to our hypothesis, the results show that regulation of DNA replication initiation by ParA is maintained when a parS site is separated from oriC. Because a single parS site is sufficient for proper control of ParA, the results are consistent with a model where ParA is efficiently regulated by ParB sliding clamps following loading at parS.

A joint-ParB interface promotes Smc DNA recruitment.

Chromosomes readily unlink and segregate to daughter cells during cell division, highlighting a remarkable ability of cells to organize long DNA molecules. SMC complexes promote DNA organization by loop extrusion. In most bacteria, chromosome folding initiates at dedicated start sites marked by the ParB/parS partition complexes. Whether SMC complexes recognize a specific DNA structure in the partition complex or a protein component is unclear. By replacing genes in Bacillus subtilis with orthologous sequences from Streptococcus pneumoniae, we show that the three subunits of the bacterial Smc complex together with the ParB protein form a functional module that can organize and segregate foreign chromosomes. Using chimeric proteins and chemical cross-linking, we find that ParB directly binds the Smc subunit. We map an interface to the Smc joint and the ParB CTP-binding domain. Structure prediction indicates how the ParB clamp presents DNA to the Smc complex, presumably to initiate DNA loop extrusion.

Genus-Specific Interactions of Bacterial Chromosome Segregation Machinery Are Critical for Their Function.

Most bacteria use the ParABS system to segregate their newly replicated chromosomes. The two protein components of this system from various bacterial species share their biochemical properties: ParB is a CTPase that binds specific centromere-like parS sequences to assemble a nucleoprotein complex, while the ParA ATPase forms a dimer that binds DNA non-specifically and interacts with ParB complexes. The ParA-ParB interaction incites the movement of ParB complexes toward the opposite cell poles. However, apart from their function in chromosome segregation, both ParAB may engage in genus-specific interactions with other protein partners. One such example is the polar-growth controlling protein DivIVA in Actinomycetota, which binds ParA in Mycobacteria while interacts with ParB in Corynebacteria. Here, we used heterologous hosts to investigate whether the interactions between DivIVA and ParA or ParB are maintained across phylogenic classes. Specifically, we examined interactions of proteins from four bacterial species, two belonging to the Gram positive Actinomycetota phylum and two belonging to the Gram-negative Pseudomonadota. We show that while the interactions between ParA and ParB are preserved for closely related orthologs, the interactions with polarly localised protein partners are not conferred by orthologous ParABs. Moreover, we demonstrate that heterologous ParA cannot substitute for endogenous ParA, despite their high sequence similarity. Therefore, we conclude that ParA orthologs are fine-tuned to interact with their partners, especially their interactions with polarly localised proteins are adjusted to particular bacterial species demands.

Catching a Walker in the Act-DNA Partitioning by ParA Family of Proteins.

Partitioning the replicated genetic material is a crucial process in the cell cycle program of any life form. In bacteria, many plasmids utilize cytoskeletal proteins that include ParM and TubZ, the ancestors of the eukaryotic actin and tubulin, respectively, to segregate the plasmids into the daughter cells. Another distinct class of cytoskeletal proteins, known as the Walker A type Cytoskeletal ATPases (WACA), is unique to Bacteria and Archaea. ParA, a WACA family protein, is involved in DNA partitioning and is more widespread. A centromere-like sequence parS, in the DNA is bound by ParB, an adaptor protein with CTPase activity to form the segregation complex. The ParA ATPase, interacts with the segregation complex and partitions the DNA into the daughter cells. Furthermore, the Walker A motif-containing ParA superfamily of proteins is associated with a diverse set of functions ranging from DNA segregation to cell division, cell polarity, chemotaxis cluster assembly, cellulose biosynthesis and carboxysome maintenance. Unifying principles underlying the varied range of cellular roles in which the ParA superfamily of proteins function are outlined. Here, we provide an overview of the recent findings on the structure and function of the ParB adaptor protein and review the current models and mechanisms by which the ParA family of proteins function in the partitioning of the replicated DNA into the newly born daughter cells.

Centromere Interactions Promote the Maintenance of the Multipartite Genome in Agrobacterium tumefaciens.

Many pathogens or symbionts of animals and plants contain multiple replicons, a configuration called a multipartite genome. Multipartite genomes enable those species to replicate their genomes faster and better adapt to new niches. Despite their prevalence, the mechanisms by which multipartite genomes are stably maintained are poorly understood. Agrobacterium tumefaciens is a plant pathogen that contains four replicons: a circular chromosome (Ch1), a linear chromosome (Ch2), and two large plasmids. Recent work indicates that their replication origins are clustered at the cell poles in a manner that depends on their ParB family centromeric proteins: ParB1 for Ch1 and individual RepB paralogs for Ch2 and the plasmids. However, understanding of these interactions and how they contribute to genome maintenance is limited. By combining genome-wide chromosome conformation capture (Hi-C) assays, chromatin-immunoprecipitation sequencing (ChIP-seq), and live cell fluorescence microscopy, we provide evidence here that centromeric clustering is mediated by interactions between these centromeric proteins. We further show that the disruption of centromere clustering results in the loss of replicons. Our data establish the role of centromeric clustering in multipartite genome stability. IMPORTANCE About 10% of sequenced bacteria have multiple replicons, also known as multipartite genomes. How these multipartite genomes are maintained is still poorly understood. Here, we use Agrobacterium tumefaciens as a model and show that the replication origins of the four replicons are clustered through direct interactions between the centromeric proteins; disruption of origin clustering leads to the loss of replicons. Thus, our study provided evidence that centromeric clustering is important for maintaining multipartite genomes.

Fluorescent labeling of genomic loci in Drosophila imaginal discs with heterologous DNA-binding proteins.

Using the Drosophila melanogaster Hox gene Ultrabithorax (Ubx) as an example, we demonstrate the use of three heterologous DNA-binding protein systems-LacI/LacO, ParB1/ParS1, and ParB2/ParS2-to label genomic loci in imaginal discs with the insertion of a small DNA tag. We compare each system, considering the impact of labeling in genomic regions (1) inside versus outside of a transcribed gene body and (2) with varying chromatin accessibility. We demonstrate the value of this system by interrogating the relationship between gene expression level and enhancer-promoter distance, as well as inter-allelic distance at the Ubx locus. We find that the distance between an essential intronic cis-regulatory element, anterobithorax (abx), and the promoter does not vary with expression level. In contrast, inter-allelic distance correlates with Ubx expression level.

Subcellular Dynamics of a Conserved Bacterial Polar Scaffold Protein.

In order to survive, bacterial cells rely on precise spatiotemporal organization and coordination of essential processes such as cell growth, chromosome segregation, and cell division. Given the general lack of organelles, most bacteria are forced to depend on alternative localization mechanisms, such as, for example, geometrical cues. DivIVA proteins are widely distributed in mainly Gram-positive bacteria and were shown to bind the membrane, typically in regions of strong negative curvature, such as the cell poles and division septa. Here, they have been shown to be involved in a multitude of processes: from apical cell growth and chromosome segregation in actinobacteria to sporulation and inhibition of division re-initiation in firmicutes. Structural analyses revealed that DivIVA proteins can form oligomeric assemblies that constitute a scaffold for recruitment of other proteins. However, it remained unclear whether interaction with partner proteins influences DivIVA dynamics. Using structured illumination microscopy (SIM), single-particle tracking (SPT) microscopy, and fluorescent recovery after photobleaching (FRAP) experiments, we show that DivIVA from Corynebacterium glutamicum is mobilized by its binding partner ParB. In contrast, we show that the interaction between Bacillus subtilis DivIVA and its partner protein MinJ reduces DivIVA mobility. Furthermore, we show that the loss of the rod-shape leads to an increase in DivIVA dynamics in both organisms. Taken together, our study reveals the modulation of the polar scaffold protein by protein interactors and cell morphology. We reason that this leads to a very simple, yet robust way for actinobacteria to maintain polar growth and their rod-shape. In B. subtilis, however, the DivIVA protein is tailored towards a more dynamic function that allows quick relocalization from poles to septa upon division.

Conformation and dynamic interactions of the multipartite genome in Agrobacterium tumefaciens.

Bacterial species from diverse phyla contain multiple replicons, yet how these multipartite genomes are organized and segregated during the cell cycle remains poorly understood. Agrobacterium tumefaciens has a 2.8-Mb circular chromosome (Ch1), a 2.1-Mb linear chromosome (Ch2), and two large plasmids (pAt and pTi). We used this alpha proteobacterium as a model to investigate the global organization and temporal segregation of a multipartite genome. Using chromosome conformation capture assays, we demonstrate that both the circular and the linear chromosomes, but neither of the plasmids, have their left and right arms juxtaposed from their origins to their termini, generating interarm interactions that require the broadly conserved structural maintenance of chromosomes complex. Moreover, our study revealed two types of interreplicon interactions: "ori-ori clustering" in which the replication origins of all four replicons interact, and "Ch1-Ch2 alignment" in which the arms of Ch1 and Ch2 interact linearly along their lengths. We show that the centromeric proteins (ParB1 for Ch1 and RepBCh2 for Ch2) are required for both types of interreplicon contacts. Finally, using fluorescence microscopy, we validated the clustering of the origins and observed their frequent colocalization during segregation. Altogether, our findings provide a high-resolution view of the conformation of a multipartite genome. We hypothesize that intercentromeric contacts promote the organization and maintenance of diverse replicons.