Control of a gene transfer agent cluster in Caulobacter crescentus by transcriptional activation and anti-termination.

Gene Transfer Agents (GTAs) are phage-like particles that cannot self-multiply and be infectious. Caulobacter crescentus, a bacterium best known as a model organism to study bacterial cell biology and cell cycle regulation, has recently been demonstrated to produce bona fide GTA particles (CcGTA). Since C. crescentus ultimately die to release GTA particles, the production of GTA particles must be tightly regulated and integrated with the host physiology to prevent a collapse in cell population. Two direct activators of the CcGTA biosynthetic gene cluster, GafY and GafZ, have been identified, however, it is unknown how GafYZ controls transcription or how they coordinate gene expression of the CcGTA gene cluster with other accessory genes elsewhere on the genome for complete CcGTA production. Here, we show that the CcGTA gene cluster is transcriptionally co-activated by GafY, integration host factor (IHF), and by GafZ-mediated transcription anti-termination. We present evidence that GafZ is a transcription anti-terminator that likely forms an anti-termination complex with RNA polymerase, NusA, NusG, and NusE to bypass transcription terminators within the 14 kb CcGTA cluster. Overall, we reveal a two-tier regulation that coordinates the synthesis of GTA particles in C. crescentus.

Widespread prevalence of a methylation-dependent switch to activate an essential DNA damage response in bacteria.

DNA methylation plays central roles in diverse cellular processes, ranging from error-correction during replication to regulation of bacterial defense mechanisms. Nevertheless, certain aberrant methylation modifications can have lethal consequences. The mechanisms by which bacteria detect and respond to such damage remain incompletely understood. Here, we discover a highly conserved but previously uncharacterized transcription factor (Cada2), which orchestrates a methylation-dependent adaptive response in Caulobacter. This response operates independently of the SOS response, governs the expression of genes crucial for direct repair, and is essential for surviving methylation-induced damage. Our molecular investigation of Cada2 reveals a cysteine methylation-dependent posttranslational modification (PTM) and mode of action distinct from its Escherichia coli counterpart, a trait conserved across all bacteria harboring a Cada2-like homolog instead. Extending across the bacterial kingdom, our findings support the notion of divergence and coevolution of adaptive response transcription factors and their corresponding sequence-specific DNA motifs. Despite this diversity, the ubiquitous prevalence of adaptive response regulators underscores the significance of a transcriptional switch, mediated by methylation PTM, in driving a specific and essential bacterial DNA damage response.

Co-opting bacterial viruses for DNA exchange: structure and regulation of gene transfer agents.

Horizontal gene transfer occurs via a range of mechanisms, including transformation, conjugation and bacteriophage transduction. Gene transfer agents (GTAs) are an alternative, less-studied route for interbacterial DNA exchange. Encoded within bacterial or archaeal genomes, GTAs assemble into phage-like particles that selflessly package and transmit host DNA to recipient bacteria. Several unique features distinguish GTAs from canonical phages such as an inability to self-replicate, thus producing non-infectious particles. GTAs are also deeply integrated into the physiology of the host cell and are maintained under tight host-regulatory control. Recent advances in understanding the structure and regulation of GTAs have provided further insights into a DNA transfer mechanism that is proving increasingly widespread across the bacterial tree of life.

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.

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.

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.

Scaling of surface roughness in film deposition with height-dependent step edge barriers.

We perform kinetic Monte Carlo simulations of film growth in simple cubic lattices with solid-on-solid conditions, Ehrlich-Schwoebel (ES) barriers at step edges, and a kinetic barrier related to the hidden off-plane diffusion at multilayer steps. Broad ranges of the diffusion-to-deposition ratio R, detachment probability per lateral neighbor, ε, and monolayer step crossing probability P=exp[-E_{ES}/(k_{B}T)] are studied. Without the ES barrier, four possible scaling regimes are shown as the coverage θ increases: nearly layer-by-layer growth with damped roughness oscillations; kinetic roughening in the Villain-Lai-Das Sarma (VLDS) universality class when the roughness is W∼1 (in lattice units); unstable roughening with mound nucleation and growth, where slopes of logW×logθ plots reach values larger than 0.5; and asymptotic statistical growth with W=θ^{1/2} solely due to the kinetic barrier at multilayer steps. If the ES barrier is present, the layer-by-layer growth crosses over directly to the unstable regime, with no transient VLDS scaling. However, in simulations up to θ=10^{4} (typical of films with a few micrometers), low temperatures (small R, Îµ, or P) may suppress the two or three initial regimes, while high temperatures and P∼1 produce smooth surfaces at all thicknesses. These crossovers help to explain proposals of nonuniversal exponents in previous works. We define a smooth film thickness θ_{c} where W=1 and show that VLDS scaling at that point indicates negligible ES barriers, while rapidly increasing roughness indicates a small ES barrier (E_{ES}∼k_{B}T). θ_{c} scales as ∼exp(const×P^{2/3}) if the other parameters are kept fixed, which represents a high sensitivity on the ES barrier. The analysis of recent experimental data in the light of our results distinguishes cases where E_{ES}/(k_{B}T) is negligible, ∼1, or ≪1.

Mechanistic insight into the repair of C8-linked pyrrolobenzodiazepine monomer-mediated DNA damage.

Pyrrolobenzodiazepines (PBDs) are naturally occurring DNA binding compounds that possess anti-tumor and anti-bacterial activity. Chemical modifications of PBDs can result in improved DNA binding, sequence specificity and enhanced efficacy. More recently, synthetic PBD monomers have shown promise as payloads for antibody drug conjugates and anti-bacterial agents. The precise mechanism of action of these PBD monomers and their role in causing DNA damage remains to be elucidated. Here we characterized the damage-inducing potential of two C8-linked PBD bi-aryl monomers in Caulobacter crescentus and investigated the strategies employed by cells to repair the same. We show that these compounds cause DNA damage and efficiently kill bacteria, in a manner comparable to the extensively used DNA cross-linking agent mitomycin-C (MMC). However, in stark contrast to MMC which employs a mutagenic lesion tolerance pathway, we implicate essential functions for error-free mechanisms in repairing PBD monomer-mediated damage. We find that survival is severely compromised in cells lacking nucleotide excision repair and to a lesser extent, in cells with impaired recombination-based repair. Loss of nucleotide excision repair leads to significant increase in double-strand breaks, underscoring the critical role of this pathway in mediating repair of PBD-induced DNA lesions. Together, our study provides comprehensive insights into how mono-alkylating DNA-targeting therapeutic compounds like PBD monomers challenge cell growth, and identifies the specific mechanisms employed by the cell to counter the same.

Correction: Structural, magnetic and hyperthermia properties and their correlation in cobalt-doped magnetite nanoparticles.

[This corrects the article DOI: 10.1039/D1RA07407E.].

Prophage-like gene transfer agents promote Caulobacter crescentus survival and DNA repair during stationary phase.

Gene transfer agents (GTAs) are prophage-like entities found in many bacterial genomes that cannot propagate themselves and instead package approximately 5 to 15 kbp fragments of the host genome that can then be transferred to related recipient cells. Although suggested to facilitate horizontal gene transfer (HGT) in the wild, no clear physiological role for GTAs has been elucidated. Here, we demonstrate that the α-proteobacterium Caulobacter crescentus produces bona fide GTAs. The production of Caulobacter GTAs is tightly regulated by a newly identified transcription factor, RogA, that represses gafYZ, the direct activators of GTA synthesis. Cells lacking rogA or expressing gafYZ produce GTAs harboring approximately 8.3 kbp fragment of the genome that can, after cell lysis, be transferred into recipient cells. Notably, we find that GTAs promote the survival of Caulobacter in stationary phase and following DNA damage by providing recipient cells a template for homologous recombination-based repair. This function may be broadly conserved in other GTA-producing organisms and explain the prevalence of this unusual HGT mechanism.

Spatial rearrangement of the Streptomyces venezuelae linear chromosome during sporogenic development.

Bacteria of the genus Streptomyces have a linear chromosome, with a core region and two 'arms'. During their complex life cycle, these bacteria develop multi-genomic hyphae that differentiate into chains of exospores that carry a single copy of the genome. Sporulation-associated cell division requires chromosome segregation and compaction. Here, we show that the arms of Streptomyces venezuelae chromosomes are spatially separated at entry to sporulation, but during sporogenic cell division they are closely aligned with the core region. Arm proximity is imposed by segregation protein ParB and condensin SMC. Moreover, the chromosomal terminal regions are organized into distinct domains by the Streptomyces-specific HU-family protein HupS. Thus, as seen in eukaryotes, there is substantial chromosomal remodelling during the Streptomyces life cycle, with the chromosome undergoing rearrangements from an 'open' to a 'closed' conformation.

CTP regulates membrane-binding activity of the nucleoid occlusion protein Noc.

ATP- and GTP-dependent molecular switches are extensively used to control functions of proteins in a wide range of biological processes. However, CTP switches are rarely reported. Here, we report that a nucleoid occlusion protein Noc is a CTPase enzyme whose membrane-binding activity is directly regulated by a CTP switch. In Bacillus subtilis, Noc nucleates on 16 bp NBS sites before associating with neighboring non-specific DNA to form large membrane-associated nucleoprotein complexes to physically occlude assembly of the cell division machinery. By in vitro reconstitution, we show that (1) CTP is required for Noc to form the NBS-dependent nucleoprotein complex, and (2) CTP binding, but not hydrolysis, switches Noc to a membrane-active state. Overall, we suggest that CTP couples membrane-binding activity of Noc to nucleoprotein complex formation to ensure productive recruitment of DNA to the bacterial cell membrane for nucleoid occlusion activity.

Shear dynamics of confined membranes.

We model the nonlinear response of a lubricated contact composed of a two-dimensional lipid membrane immersed in a simple fluid between two parallel flat and porous walls under shear. The nonlinear dynamics of the membrane gives rise to a rich dynamical behavior depending on the shear velocity. In quiescent conditions (i.e., absence of shear), the membrane freezes into a disordered labyrinthine wrinkle pattern. We determine the wavelength of this pattern as a function of the excess area of the membrane for a fairly general form of the confinement potential using a sine-profile ansatz for the wrinkles. In the presence of shear, we find four different regimes depending on the shear rate. Regime I. For small shear, the labyrinthine pattern is still frozen, but exhibits a small drift which is mainly along the shear direction. In this regime, the tangential forces on the walls due to the presence of the membrane increase linearly with the shear rate. Regime II. When the shear rate is increased above a critical value, the membrane rearranges, and wrinkles start to align along the shear direction. This regime is accompanied by a sharp drop of the tangential forces on the wall. The membrane usually reaches a steady-state configuration drifting with a small constant velocity at long times. However, we also rarely observe oscillatory dynamics in this regime. Regime III. For larger shear rates, the wrinkles align strongly along the shear direction, with a set of dislocation defects which assemble in pairs. The tangential forces are then controlled by the number of dislocations, and by the number of wrinkles between the two dislocations within each dislocation pairs. In this dislocation-dominated regime, the tangential forces in the transverse direction most often exceed those in the shear direction. Regime IV. For even larger shear, the membrane organizes into a perfect array of parallel stripes with no defects. The wavelength of the wrinkles is still identical to the wavelength in the absence of shear. In this final regime, the tangential forces due to the membrane vanish. These behaviors give rise to a non-linear rheological behavior of lubricated contacts containing membranes.

Structural, magnetic and hyperthermia properties and their correlation in cobalt-doped magnetite nanoparticles.

Cobalt doped magnetite nanoparticles (Co x Fe3-x O4 NPs) are investigated extensively because of their potential hyperthermia application. However, the complex interrelation among chemical compositions and particle size means their correlation with the magnetic and heating properties is not trivial to predict. Here, we prepared Co x Fe3-x O4 NPs (0 ≤ x ≤ 1) to investigate the effects of cobalt content and particle size on their magnetic and heating properties. A detailed analysis of the structural features indicated the similarity between the crystallite and particle sizes as well as their non-monotonic change with the increase of Co content. Magnetic measurements for the Co x Fe3-x O4 NPs (0 ≤ x ≤ 1) showed that the blocking temperature, the saturation magnetization, the coercivity, and the anisotropy constant followed a similar trend with a maximum at x = 0.7. Moreover, 57Fe Mössbauer spectroscopy adequately explained the magnetic behaviour, the anisotropy constant, and saturation magnetization of low Co content samples. Finally, our study shows that the relaxation loss is a primary contributor to the SAR in Co x Fe3-x O4 NPs with low Co contents as well as their potential application in magnetic hyperthermia.

Diversification of DNA-Binding Specificity by Permissive and Specificity-Switching Mutations in the ParB/Noc Protein Family.

Specific interactions between proteins and DNA are essential to many biological processes. Yet, it remains unclear how the diversification in DNA-binding specificity was brought about, and the mutational paths that led to changes in specificity are unknown. Using a pair of evolutionarily related DNA-binding proteins, each with a different DNA preference (ParB [Partitioning Protein B] and Noc [Nucleoid Occlusion Factor], which both play roles in bacterial chromosome maintenance), we show that specificity is encoded by a set of four residues at the protein-DNA interface. Combining X-ray crystallography and deep mutational scanning of the interface, we suggest that permissive mutations must be introduced before specificity-switching mutations to reprogram specificity and that mutational paths to new specificity do not necessarily involve dual-specificity intermediates. Overall, our results provide insight into the possible evolutionary history of ParB and Noc and, in a broader context, might be useful for understanding the evolution of other classes of DNA-binding proteins.

Bacterial chromosome segregation by the ParABS system.

Proper chromosome segregation during cell division is essential in all domains of life. In the majority of bacterial species, faithful chromosome segregation is mediated by the tripartite ParABS system, consisting of an ATPase protein ParA, a CTPase and DNA-binding protein ParB, and a centromere-like parS site. The parS site is most often located near the origin of replication and is segregated first after chromosome replication. ParB nucleates on parS before binding to adjacent non-specific DNA to form a multimeric nucleoprotein complex. ParA interacts with ParB to drive the higher-order ParB-DNA complex, and hence the replicating chromosomes, to each daughter cell. Here, we review the various models for the formation of the ParABS complex and describe its role in segregating the origin-proximal region of the chromosome. Additionally, we discuss outstanding questions and challenges in understanding bacterial chromosome segregation.

Chromosome Conformation Capture with Deep Sequencing to Study the Roles of the Structural Maintenance of Chromosomes Complex In Vivo.

Recent applications of chromosome conformation capture with deep sequencing (Hi-C and other C techniques) has enabled high-throughput investigations and driven major advances in understanding chromosome organization in bacteria and eukaryotes. C techniques reveal systematically the identities of interacting DNA and the frequency of each interaction in vivo. Beyond a bird's-eye view survey of the global chromosome architecture, C techniques together with genetic perturbation have proven to be powerful in understanding factors that shape chromosome architectures. The structural maintenance of chromosomes (SMC) proteins play major roles in organizing the chromosomes from bacteria to humans, and C techniques have contributed to understanding their mechanism and impact on genome organization in a cellular context. Here, I describe a Hi-C protocol, a variant of C techniques, to construct genome-wide DNA contact maps for bacteria. This protocol is optimized for the gram-negative bacterium Caulobacter crescentus, but it can be readily adapted for any bacterial species of interest.

Adhesion dynamics of confined membranes.

We report on the modeling of the dynamics of confined lipid membranes. We derive a thin film model in the lubrication limit which describes an inextensible liquid membrane with bending rigidity confined between two adhesive walls. The resulting equations share similarities with the Swift-Hohenberg model. However, inextensibility is enforced by a time-dependent nonlocal tension. Depending on the excess membrane area available in the system, three different dynamical regimes, denoted as A, B and C, are found from the numerical solution of the model. In regime A, membranes with small excess area form flat adhesion domains and freeze. Such freezing is interpreted by means of an effective model for curvature-driven domain wall motion. The nonlocal membrane tension tends to a negative value corresponding to the linear stability threshold of flat domain walls in the Swift-Hohenberg equation. In regime B, membranes with intermediate excess areas exhibit endless coarsening with coexistence of flat adhesion domains and wrinkle domains. The tension tends to the nonlinear stability threshold of flat domain walls in the Swift-Hohenberg equation. The fraction of the system covered by the wrinkle phase increases linearly with the excess area in regime B. In regime C, membranes with large excess area are completely covered by a frozen labyrinthine pattern of wrinkles. As the excess area is increased, the tension increases and the wavelength of the wrinkles decreases. For large membrane area, there is a crossover to a regime where the extrema of the wrinkles are in contact with the walls. In all regimes after an initial transient, robust localised structures form, leading to an exact conservation of the number of adhesion domains.

Permissive zones for the centromere-binding protein ParB on the Caulobacter crescentus chromosome.

Proper chromosome segregation is essential in all living organisms. In Caulobacter crescentus, the ParA-ParB-parS system is required for proper chromosome segregation and cell viability. The bacterial centromere-like parS DNA locus is the first to be segregated following chromosome replication. parS is bound by ParB protein, which in turn interacts with ParA to partition the ParB-parS nucleoprotein complex to each daughter cell. Here, we investigated the genome-wide distribution of ParB on the Caulobacter chromosome using a combination of in vivo chromatin immunoprecipitation (ChIP-seq) and in vitro DNA affinity purification with deep sequencing (IDAP-seq). We confirmed two previously identified parS sites and discovered at least three more sites that cluster ∼8 kb from the origin of replication. We showed that Caulobacter ParB nucleates at parS sites and associates non-specifically with ∼10 kb flanking DNA to form a high-order nucleoprotein complex on the left chromosomal arm. Lastly, using transposon mutagenesis coupled with deep sequencing (Tn-seq), we identified a ∼500 kb region surrounding the native parS cluster that is tolerable to the insertion of a second parS cluster without severely affecting cell viability. Our results demonstrate that the genomic distribution of parS sites is highly restricted and is crucial for chromosome segregation in Caulobacter.