Biology subjects

Kaljevic, J.

Publications and source records attributed to Kaljevic, J..

7 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

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↗

bioRxiv2023

Distinct dynamics and proximity networks of hub proteins at the prey-invading cell pole in a predatory bacterium

In bacteria, cell poles function as subcellular compartments where proteins localize during specific lifecycle stages, orchestrated by polar "hub" proteins. Whereas most described bacteria inherit an "old" pole from the mother cell and a "new" pole from cell division, polarizing cells at birth, non-binary division poses challenges for establishing cell polarity, particularly for daughter cells inheriting only new poles. We investigated polarity dynamics in the obligate predatory bacterium Bdellovibrio bacteriovorus, proliferating through filamentous growth followed by non-binary division within prey bacteria. Monitoring the subcellular localization of two proteins known as polar hubs in other species, RomR and DivIVA, revealed RomR as an early polarity marker in B. bacteriovorus. RomR already marks the future anterior poles of the progeny during the predators growth phase, in a define time window closely following the onset of divisome assembly and the end of chromosome segregation. In contrast to RomRs stable unipolar localization in the progeny, DivIVA exhibits a dynamic pole-to-pole localization. This behaviour changes shortly before division of the elongated predator cell, where DivIVA accumulates at all septa and both poles. In vivo protein interaction networks for DivIVA and RomR, mapped through endogenous miniTurbo-based proximity labeling, further underscore their distinct roles in cell polarization and the importance of the anterior "invasive" cell pole in prey-predator interactions. Our work emphasizes the strict spatiotemporal coordination of cellular processes underlying B. bacteriovorus proliferation, offering insights into the subcellular organization of bacteria with filamentous growth and non-binary division.

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

Modulation of prey size reveals adaptability and robustness in the cell cycle of an intracellular predator

Despite the remarkable diversity of bacterial lifestyles, the sophisticated regulatory networks underlying bacterial replication have only been investigated in a limited number of model species so far. In bacteria that do not rely on canonical binary division for proliferation, the coordination of major cellular processes is still mysterious. Moreover, bacterial growth and division remain largely unexplored within spatially confined niches where nutrients are limited. This includes the lifecycle of the model endobiotic predatory bacterium Bdellovibrio bacteriovorus, which grows by filamentation within its host or prey cells and produces a variable number of daughter cells. Here, we examined how the size of the micro-compartment in which predators replicate (i.e., the prey bacterium) impacts their cell cycle progression at the single-cell level. Using Escherichia coli with genetically encoded size differences, we show that the duration of the predator cell cycle scales with prey size. As a result, the size of the prey determines the number of predator offspring, through a relationship that is maintained across prey species. Strikingly, the nutritional quality of the prey cell determined the specific growth rate of predators regardless of prey size, reminiscent of the effect of medium composition on the growth of other bacteria. Tuning the predatory cell cycle by modulating prey dimensions also allowed us to reveal invariable temporal connections between key cellular processes. Altogether, our data uncover adaptability and robustness shaping the enclosed replication of B. bacteriovorus. Consequently, predators optimally exploit the finite prey resources and space while ensuring a strict cell cycle progression. This study opens the way to explore diverse cell cycle control strategies by extending their characterization to intracellular lifestyles, beyond canonical models and growth conditions.

microbiology↗

bioRxiv2020

Chromosome dynamics and spatial organization during the non-binary cell cycle of a predatory bacterium

In bacteria, the dynamics of chromosome replication and segregation are tightly coordinated with cell cycle progression, and largely rely on specific spatiotemporal arrangement of the chromosome. Whereas these key processes are mostly investigated in species that divide by binary fission, they remain mysterious in bacteria producing larger number of descendants. Here, we establish the predatory bacterium Bdellovibrio bacteriovorus as a model to investigate the non-binary processing of a circular chromosome. Our data reveal its extreme compaction in a dense polarized nucleoid. We also show that a first binary-like cycle of replication and asymmetric segregation is followed by multiple asynchronous rounds of replication and progressive ParABS-dependent partitioning, uncoupled from cell division. Surprisingly, ParB localization at the centromere is cell-cycle regulated. Altogether, our findings support a model of complex chromosome choreography, leading to the generation of variable numbers of offspring, highlighting the adaptation of conserved mechanisms to achieve non-binary reproduction in bacteria.

microbiology↗