Biology subjects

Le, T. B. K.

Publications and source records attributed to Le, T. B. K..

At least 19 recordsLinked to original sources

bioRxiv2026

Repurposing a chromosome segregation ParB-CTPase fold into an ATPase toxin for contact-dependent growth inhibition in plant and animal pathogens

Bacterial competition drives the evolution of antibacterial mechanisms, yet how new activities arise remains poorly understood. A major route to innovation is the reuse of pre-existing genetic systems, whereby conserved protein modules are repurposed in new biological contexts to generate new capabilities. Here, we show that the ParB-CTPase fold, a conserved nucleotide-binding module best known for its role in chromosome segregation, can be functionally repurposed as an antibacterial toxin. We identify ToxB, a ParB-like domain embedded within the polymorphic toxin region of contact-dependent inhibition systems and show that it functions as a potent antibacterial effector. Structural and biochemical analyses reveal that ToxB retains the core architecture of the ParB-CTPase fold but lacks DNA-binding capability and preferentially binds ATP. This shift in nucleotide specificity underpins a distinct mode of action, in which ATP binding and hydrolysis trigger rapid nucleoid compaction, chromosome segregation defects, oxidative stress, cell chaining, and ultimately cell lysis. ToxB also exhibits toxic activity in plant cells, suggesting that it targets conserved cellular processes. Together, these findings provide direct experimental evidence that the ParB-NTPase fold is biologically versatile and can be repurposed for biological roles fundamentally distinct from its ancestral function in DNA segregation.

microbiology↗

bioRxiv2025

Stable inheritance of the Streptomyces linear plasmid SCP1 by dual ParABS partition systems

Low-copy-number plasmids often rely on dedicated maintenance mechanisms, such as partitioning systems, to ensure stable inheritance across generations. These partition systems actively segregate sister plasmid copies during cell division and are classified by the NTPase types they encode. While the distribution and organization of partition system types are well characterized in Enterobacteriaceae plasmids, their functions and diversity across broader bacterial taxa remain poorly understood. Here, we analyze a large and diverse plasmid database to examine the distribution of partition system types and find that plasmids encoding multiple partition systems are more common than previously recognized. Notably, many plasmids encode multiple partition systems of the same type, an organization that has not been previously investigated. To further investigate, we employ the Streptomyces linear plasmid SCP1, which encodes two type I ATP- and CTP-dependent parABS partition systems, as a model. Sequence analysis shows that both SCP1-encoded ParBs harbor less conserved CTPase domains than their chromosomal counterparts, suggesting they might diverge from canonical ParB functions. However, using chromatin immunoprecipitation with deep sequencing, biochemical assays, and targeted mutagenesis, we demonstrate that both proteins are bona fide ParB CTPase proteins: they recognize distinct parS sites on SCP1, bind and hydrolyze CTP, and slide to accumulate on DNA. Despite both systems being functional, only parABS1, but not parABS2, is crucial for SCP1 maintenance under standard laboratory conditions. Altogether, these findings provide the first functional characterization of dual ParB-CTPase partition systems coexisting on a single plasmid, advancing our understanding of plasmid maintenance in Streptomyces, and reveal new aspects of the diversity and distribution of plasmid partition systems in bacteria.

microbiology↗

bioRxiv2025

Versatile NTP recognition and domain fusions expand the functional repertoire of the ParB-CTPase fold beyond chromosome segregation

Nucleotide triphosphate (NTP)-dependent molecular switches regulate essential cellular processes by cycling between active and inactive states through nucleotide binding and hydrolysis. These mechanisms were long thought to rely exclusively on ATPase or GTPase proteins, until the discovery of CTPase activity in the bacterial chromosome segregation protein ParB. In the ParABS system, CTP binding enables ParB accumulation around the centromere-like parS DNA sites to activate the ATPase ParA, thereby facilitating chromosome partitioning to daughter cells. CTP hydrolysis then releases ParB from DNA for recycling. This discovery uncovered a new regulatory principle, but the broader diversity of proteins employing a CTPase mechanism remains unclear. Here, we conduct a large-scale survey of proteins harboring the ParB-CTPase fold across bacteria, archaea, bacteriophages, and eukaryotes. While many ParB-like proteins follow the canonical ParABS organization with ParA partners, we also identify numerous orphan homologs encoded outside of the parAB operon, frequently linked to mobile genetic elements that may have driven their rapid diversification. The ParB-CTPase folds in these divergent proteins are often fused to lineage-specific domains with diverse predicted biological activities. We further demonstrate that while many homologs retain CTP-binding, others instead bind ATP or GTP, revealing a broader spectrum of nucleotide specificities than previously appreciated. Our findings establish the ParB-CTPase fold as a widely distributed and evolutionarily versatile NTP-binding module, repeatedly co-opted through domain fusion and shifts in nucleotide specificity to enable functions far beyond the classical ParABS-mediated DNA segregation.

microbiology↗

bioRxiv2025

Dynamics and interplay of gene expression and chromosome organization across a predatory lifecycle

The obligate predatory bacterium Bdellovibrio bacteriovorus alternates between a motile attack phase and a growth phase inside another bacterium, during which it undergoes multiple rounds of DNA replication followed by non-binary division and progeny release. How its chromosome is dynamically reorganized across these contrasting states remains unclear. Here, we used synchronized predatory infections combined with time-resolved RNA-seq, Hi-C, ChIP-seq, and quantitative microscopy to map transcriptional activity and chromosome architecture across seven key stages of the predatory lifecycle. We uncover a dramatic shift in nucleoid organization - from an ultra-compact, transcriptionally restricted state in attack-phase cells to a progressively decompacted, ori-centered conformation during intracellular replication. Notably, distinct transcriptional and chromosome folding states not only mark the canonical attack and growth phases, but also critical transitions such as prey invasion, replication onset, and predator division. Temporal gene expression profiles reflect the coordinated regulation of core cellular functions across the predatory cycle. Furthermore, spatial localization of RNA polymerase, ribosomes, DNA-binding proteins, and freely diffusing proteins highlight the tight, cell cycle-dependent coupling between nucleoid compaction, chromosome accessibility, and transcriptional activity. Altogether, our findings reveal the intricate dynamics and coordination of genome folding and gene expression in a complex bacterial cell cycle driven by prey-predator interactions.

microbiology↗

bioRxiv2025

A bacterial CARD-NLR immune system controls the release of gene transfer agents

Bacteria have evolved a wide array of immune systems to detect and defend against external threats including mobile genetic elements (MGEs) such as bacteriophages, plasmids, and transposons. MGEs are often selfish, exploiting their bacterial hosts to propagate, however they can also provide adaptive advantages through horizontal gene transfer. Gene transfer agents (GTAs), which are non-infectious domesticated prophages, represent a unique class of beneficial MGEs that facilitate bacterial gene transfer. Despite their domestication, GTAs retain phage-like features, including the requirement for host cell lysis to release particles, that may inadvertently trigger host immunity. How GTAs might avoid, subvert, or possibly adopt host immune systems to complete their life stages is poorly understood. Here, we identify a tripartite system, LypABC, that is essential for GTA-mediated cell lysis in Caulobacter crescentus. LypABC resembles caspase recruitment domain-nucleotide-binding leucine-rich repeat (CARD-NLR) anti-phage defence systems that mediate abortive infection wherein infected cells die to prevent phage proliferation, thereby protecting the overall bacterial population. LypABC-deficient cells produce host DNA-packed GTA particles and eventually die but cannot lyse to release GTA particles. Moreover, overproduction of LypABC is highly toxic to both GTA-producing and non-producing cells, highlighting the need for strict regulation. We find that such regulation is achieved transcriptionally by a repressor, RogB, which binds the promoters of lypABC and of essential GTA activator genes, thus coupling GTA activation and host cell lysis. While traditionally considered antagonistic towards MGEs, our findings here suggest that immunity components are versatile and can be adapted to support MGEs.

microbiology↗

bioRxiv2025

Coupling chromosome organization to genome segregation in Archaea

Chromosome segregation is a fundamental process in all life forms and requires coordination with genome organization, replication and cell division. The mechanism that mediates chromosome segregation in archaea remains enigmatic, despite the centre-stage role assumed by these organisms in the discourse about the origin of eukaryotes. Previously, we identified two proteins, SegA and SegB, which form a minimalist chromosome partition machine in Sulfolobales. Here we uncover patterns and mechanisms that the SegAB system employs to link chromosome organization to genome segregation. Deletion of the genes causes growth and chromosome partition defects. SegB binds to multiple sites scattered across the chromosome, but mainly localised close to the segAB locus in most archaeal genera. The sites are predominantly intragenic and enriched in one of the two compartments into which the chromosome folds. We show that SegB coalesces into multiple foci through the nucleoid, exhibiting a biased localisation towards the cell periphery, which hints at potential tethers to the cell membrane. The short-range DNA compaction activity of SegB and long-range looping of distant sites point to a significant role for SegB in chromosome condensation that in turn enables genome segregation. Collectively, our data put forward SegAB as important players in bridging chromosome organization to genome segregation in archaea.

microbiology↗

bioRxiv2024

Assembly of a segrosome by a CTP-independent ParB-like protein

The ATP- and CTP-dependent ParA-ParB-parS segrosome is a macromolecular complex that segregates chromosomes/plasmids in most bacterial species. CTP binding and hydrolysis enable ParB to slide on DNA and to bridge and condense DNA, thereby dictating the size and dynamics of the tripartite ParABS complex. Several other evolutionarily distinct systems can also segregate DNA, although the full diversity of bacterial DNA partition systems is not yet known. Here, we identify a CTP-independent ParABS system that maintains a conjugative plasmid SCP2 in the filamentous bacterium Streptomyces coelicolor. We demonstrate that an SCP2 ParB-like protein, ParT, loads onto DNA at an 18-bp parS site and diffuses away to the adjacent DNA despite lacking an apparent CTPase domain and detectable NTPase activity. We further show that parS DNA stimulates ParT transition from loading to a diffusing state to accumulate on DNA, and ParT activates the ATPase activity of its cognate partner protein ParA. We also identify numerous structural homologs of ParT, suggesting that CTP-independent diffusion on DNA might be widespread in bacteria despite being previously unappreciated. Overall, our findings uncover a CTP-independent DNA translocation as an alternative and unanticipated mechanism for the assembly of a bacterial DNA segregation complex and suggest that CTP binding and hydrolysis is not a fundamental feature of ParABS-like systems.

microbiology↗

bioRxiv2024

Molecular switching of a DNA-sliding clamp to a repressor mediates long-range gene silencing

Long-range gene regulation is rare in bacteria and is confined to the classical DNA looping model. Here, we use a combination of biophysical approaches, including X-ray crystallography and single-molecule analysis, to show that long-range gene silencing on the plasmid RK2, a source of multidrug resistance across diverse Gram-negative bacteria, is achieved cooperatively by a DNA-sliding clamp, KorB, and a clamp-locking protein, KorA. We find that KorB is a CTPase clamp that can entrap and slide along DNA to reach distal target promoters. We resolved the tripartite crystal structure of a KorB-KorA-DNA co-complex, revealing that KorA latches KorB into a closed-clamp state. KorA thus stimulates repression by stalling KorB sliding at target promoters to occlude RNA polymerase holoenzymes. Altogether, our findings explain the mechanistic basis for KorB role-switching from a DNA-sliding clamp to a co-repressor, and provide a new paradigm for the long-range regulation of gene expression.

microbiology↗

bioRxiv2023

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

Gene Transfer Agents (GTAs) are domesticated prophages that cannot self-multiply and be infectious but might have been co-opted to perform biological functions for the host bacteria. 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). Two direct activators of the CcGTA biosynthetic gene cluster, GafY and GafZ, have been identified, however, it is unknown how GafYZ controls transcription mechanistically 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 novel 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.

microbiology↗

bioRxiv2023

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 centromere parS 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 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. Author summaryThe precise distribution of genetic material to the progeny is essential for all living organisms, and the ParABS system is critical for this process in bacteria. Our study provides novel insights into the regulation of this system in a predatory bacterium that exhibits a non-canonical cell cycle, in which the chromosome is copied and segregated multiple times during growth inside a prey bacterium. Our work reveals the subcellular dynamics and multi-level regulation of the ParABS system in this bacterium, which results in the unique cell-cycle dependent assembly of the segregation complex at the chromosomal centromere. Our findings provide a deeper understanding of the adaptation of the ParABS system across bacterial species and developmental stages.

microbiology↗

bioRxiv2022

Requirement for specific bacterial genome maintenance pathways in repair of C8-linked pyrrolobenzodiazepine (PBD) bi-aryl 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 antibacterial 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.

microbiology↗

bioRxiv2022

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

In Bacillus subtilis, a ParB-like nucleoid occlusion protein (Noc) binds specifically to Noc-binding sites (NBS) around the chromosome to help coordinate chromosome segregation and cell division. Noc does so by binding to cytidine triphosphate (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 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 re-engagement of the NTD and DBD from the same ParB/Noc subunit.

microbiology↗

bioRxiv2021

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 (i) CTP is required for Noc to form the NBS-dependent nucleoprotein complex, and (ii) 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.

microbiology↗

bioRxiv2021

Chemical synthesis and purification of a non-hydrolyzable CTP analog CTPγS

Slowly- or non-hydrolyzable analogs of ATP and GTP, for example adenosine 5-({gamma}-thio)triphosphate (ATP{gamma}S) and guanosine 5-({gamma}-thio)triphosphate (GTP{gamma}S), have been widely employed to probe the roles of ATP and GTP in biosystems, and these compounds are readily available from commercial sources. In contrast, cytosine 5-({gamma}-thio)triphosphate (CTP{gamma}S) is not widely available commercially. The recent discovery of ParB as the founding member of a new class of CTPase enzyme (Osorio-Valeriano et al., 2019; Soh et al., 2019) and the possibility of multiple other undiscovered CTPases necessitate the use of the CTP{gamma}S to elucidate the roles of CTP hydrolysis in these systems. Here, we adapt a procedure for the synthesis of modified phosphoanhydrides (Hofer et al., 2015) to chemically synthesize and purify a milligram-scale of CTP{gamma}S.

biochemistry↗

bioRxiv2020

Spatial rearrangement of the Streptomyces venezuelae linear chromosome during sporogenic development

Depending on the species, bacteria organize their chromosomes with either spatially separated or closely juxtaposed replichores. However, in contrast to eukaryotes, significant changes in bacterial chromosome conformation during the cell cycle have not been demonstrated to date. Streptomyces are unique among bacteria due to their linear chromosomes and complex life cycle. These bacteria develop multigenomic hyphae that differentiate into chains of unigenomic exospores. Only during sporulation-associated cell division, chromosomes are segregated and compacted. In this study, we show that at entry to sporulation, arms of S. venezuelae chromosomes are spatially separated, but they are closely aligned within the core region during sporogenic cell division. Arm juxtaposition is imposed by the segregation protein ParB and condensin SMC. Moreover, we disclose that the chromosomal terminal regions are organized into domains by the Streptomyces-specific protein - HupS. Thus, we demonstrate chromosomal rearrangement from open to close conformation during Streptomyces life cycle.

microbiology↗

bioRxiv2019

ParB spreading on DNA requires cytidine triphosphate in vitro

In all living organisms, it is essential to transmit genetic information faithfully to the next generation. The SMC-ParAB-parS system is widely employed for chromosome segregation in bacteria. A DNA-binding protein ParB nucleates on parS sites and must associate with neighboring DNA, a process known as spreading, to enable efficient chromosome segregation. Despite its importance, how the initial few ParB molecules nucleating at parS sites recruit hundreds of further ParB to spread is not fully understood. Here, we reconstitute a parS-dependent ParB spreading event using purified proteins from Caulobacter crescentus and show that CTP is required for spreading. We further show that ParB spreading requires a closed DNA substrate, and a DNA-binding transcriptional regulator can act as a roadblock to attenuate spreading unidirectionally in vitro. Our biochemical reconstitutions recapitulate many observed in vivo properties of ParB and opens up avenues to investigate the interactions between ParB-parS with ParA and SMC.

microbiology↗

bioRxiv2019

Structural and biochemical analyses of Caulobacter crescentus ParB reveal the role of its N-terminal domain in chromosome segregation

Proper chromosome segregation is essential in all living organisms. The ParA-ParB-parS system is widely employed for chromosome segregation in bacteria. Previously, we showed that Caulobacter crescentus ParB requires cytidine triphosphate to escape the nucleation site parS and spread by sliding to the neighboring DNA 1. Here, we provide the structural basis for this transition from nucleation to spreading by solving co-crystal structures of a C-terminal domain truncated C. crescentus ParB with parS and with a CTP analog. Nucleating ParB is an open clamp, in which parS is captured at the DNA-binding domain (the DNA-gate). Upon binding CTP, the N-terminal domain (NTD) self-dimerizes to close the NTD-gate of the clamp. The DNA-gate also closes, thus driving parS into a compartment between the DNA-gate and the C-terminal domain. CTP hydrolysis and/or the release of hydrolytic products are likely associated with re-opening of the gates to release DNA and to recycle ParB. Overall, we suggest a CTP-operated gating mechanism that regulates ParB nucleation, spreading, and recycling.

microbiology↗

bioRxiv2019

Evolving a new protein-DNA interface via sequential introduction of permissive and specificity-switching mutations

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 what were the mutational paths that led to changes in specificity. Using a pair of evolutionarily related DNA-binding proteins, each with a different DNA preference (ParB and Noc: both having 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 a 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 in understanding the evolution of other classes of DNA-binding proteins.

microbiology↗