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.


Introduction
The precise segregation of genetic material during cellular division is fundamental across all domains of life.In eukaryotes, sister chromosomes are tethered to the spindle during mitosis via kinetochores, which are protein complexes that assemble at the chromosomal centromeres.The kinetochores serve as attachment points for the spindle to pull chromosomes apart.Bacteria exploit a conceptually analogous strategy to partition copies of their chromosomal DNA into future daughter cells [1].
In most bacterial lineages, chromosome segregation is achieved by the active partitioning of the duplicated chromosomal origins (oriC) via the ParABS system [2,3].The leading player in this system, the DNA-binding CTPase ParB, loads onto the chromosome by binding to parS sites -short palindromic sequences, usually found near the oriC [2,[4][5][6][7][8].Binding of ParB-CTP on parS allows ParB to spread away and cover adjacent DNA, leading to the accumulation of ParB on a bacterial centromere-like region and the formation of a higher-order nucleoprotein structure known as the partitioning complex [9][10][11][12][13][14], reminiscent of the eukaryotic kinetochore.CTP hydrolysis favors the opening of the ParB clamp and its removal from the DNA, replenishing the pool available for binding on one or more parS sites [11,14,15].Upon initiation of chromosome replication at oriC, a second partitioning complex assembles on the duplicated centromere, which will be segregated by the ParABS system.Briefly, ParB�parS interacts with ParA, a protein that dimerizes in its ATP-bound form and associates non-specifically with the DNA, therefore coating the entire nucleoid [16,17].The partitioning complex stimulates the ATPase activity of ParA ATP , displacing it from the oriC-proximal edge of the DNAbound "cloud" and generating a ParA ATP gradient towards the opposite cell pole.Repeated ParA-ParB interactions drive the movement of the centromere towards the highest ParA ATP concentration and across the cell, followed by the rest of the sister chromosome [3,[18][19][20][21].
In several organisms, this process is enhanced by specific landmark polar proteins that either anchor the chromosomal oriC [22][23][24][25] or sequester the released ParA ADP monomers to maintain the ParA ATP gradient [26][27][28].These mechanisms play a role in ensuring the unidirectionality of chromosome segregation and establishing polarity in future daughter cells [29].Bacteria also evolved diverse strategies to precisely couple the positioning and timing of cell constriction with the segregation of either oriC [30] or the chromosomal terminus (ter) [31,32].Consequently, segregation of bacterial chromosomes is tightly coordinated in time and space with cell cycle progression [33].While ParABS was demonstrated to be essential for survival in a few bacterial species [34][35][36][37], its inactivation or depletion usually leads to pleiotropic phenotypes emanating from impaired oriC segregation, including aberrant chromosome numbers but also cell cycle progression and cell division defects [3].
ParABS systems translocate chromosomes in a variety of organisms with vastly different lifestyles, cell cycles, and differentiation programs.Despite significant advances in understanding the subcellular dynamics, biochemistry, and interactions of the ParABS components during chromosome segregation [3,38], the adaptation of this highly conserved system across species and between developmental stages remains unclear.Only a few investigations have examined the regulatory mechanisms tuning the ParABS system [39].For example, studies have highlighted the importance of balancing the ParA and ParB levels to ensure correct cell cycle progression in Caulobacter crescentus [34], the ParA (Soj)-dependent transcriptional modulation of the parAB operon in Bacillus subtilis [40] and the developmental regulation of parA and parB expression that couples the segregation of the linear chromosome with the mycelial lifestyle of Streptomyces coelicolor [41].Except for the latter, most available data on ParABS-dependent chromosome partitioning come from bacteria that proliferate classically through vegetative growth and binary division (one mother cell generating two daughter cells).However, many species possessing a ParABS system rely on relatively complex cell cycles involving non-binary division events, generating larger and sometimes variable numbers of progeny from a polyploid mother cell (i.e., Cyanobacteria, Bdellovibrionata, and Actinobacteria) [42][43][44].The exploration of how the ParABS system partitions numerous copies of the chromosome during the intricate cell cycle of these organisms is still limited.
The obligate predatory bacterium Bdellovibrio bacteriovorus proliferates through filamentation and non-binary division inside the envelope of other diderm bacteria.Its cell cycle comprises two main phases determined by its presence outside or inside a prey (Fig 1A) [45,46].The first phase, called attack phase (AP), corresponds to a G1 cell cycle stage during which B. bacteriovorus do not replicate their single circular chromosome as they search for prey [47].Upon contact, predators attach to the prey surface and invade their periplasm, i.e., the space located between the inner and outer membranes [48,49].Upon a G1-S transition, B. bacteriovorus starts the second stage of its cell cycle called the growth phase (GP), during which the cell elongates and copies its DNA multiple times.A first round of chromosome replication and segregation, which starts from the oriC localized at one cell pole [47], is followed by the asynchronous firing of additional replication rounds using the oriC of multiple chromosome copies as a template [47,50].The newly synthesized chromosomal origins segregate progressively [47], unlike in Streptomyces, which also grows as polyploid filaments but uncouples DNA replication during vegetative growth from the ParABS-dependent segregation of multiple chromosome copies at sporulation.The filamentous and polyploid mother B. bacteriovorus cell eventually divides by synchronous constriction at multiple locations along the cell body, releasing a variable, odd, or even number of offspring [47,[50][51][52].The extent of the GP and the number of predator daughter cells is determined by the size of the prey [53], which remains a closed nest as it is being digested by B. bacteriovorus, until newborn predators break the prey open to resume their predatory cycle [54].
In contrast to all other ParB homologs involved in chromosome segregation that always localize at the parS site [7,35,37,[55][56][57][58][59][60][61][62][63][64][65], ParB in B. bacteriovorus (ParB Bb ) does not mark the centromere throughout the cell cycle.Our previous report showed that fluorescently tagged ParB Bb , produced at native or constitutive levels, accumulates near oriC only after the initiation of chromosome replication, while the protein displays a diffuse localization in the predator progeny [47].The constitutive production of ParB Bb disturbs the progressive segregation of chromosomal copies, resulting in newborn B. bacteriovorus with aberrant cell length, oriC numbers, and nucleoid size.Thus, although ParB is critical for proper oriC partitioning and cell cycle progression in B. bacteriovorus, the formation of a ParB�parS higher order structure at the centromere-like region is prevented during the non-proliferative stage of the predatory cycle [47].The underlying mechanism behind this cell cycle-dependent on-off behaviour of ParB Bb is unknown.Furthermore, the subcellular dynamics of ParA have not been explored in bacteria that segregate multiple pairs of sister chromosomes during their vegetative cell cycle.An in-depth view of the ParABS system throughout the lifecycle of B. bacteriovorus is crucial to better understand how it operates in species with non-binary proliferation.With its intricate chromosome dynamics and unique centromere organization during the cell cycle, B. bacteriovorus represents a compelling model to uncover new levels of regulation in the highly conserved ParABS system.
Here we provide key insights into the ParABS system in B. bacteriovorus, by monitoring the subcellular localization, expression, and protein levels of ParA Bb and ParB Bb during all stages of the predatory cell cycle.Moreover, we characterize ParB Bb CTPase and parS binding activities.Our data reveal multiple regulation layers in the ParABS system of this bacterium.We show that ParA Bb and ParB Bb protein levels fluctuate differently during the cell cycle despite being expressed from the same operon, their fine balance being critical for proper cell cycle

The ParABS system is critical for survival and exhibits specific localization patterns in B. bacteriovorus
The B. bacteriovorus genome encodes homologs of ParA and ParB and harbors two parS sites near the chromosomal origin, indicating the presence of a ParABS system for chromosome segregation (Fig 1B).All our attempts to individually delete parA Bb and parB Bb were unsuccessful (86 and 90 screened recombinants, respectively, see Methods), underscoring that the ParABS system is likely essential in this organism under the tested conditions.We have shown previously that ParB Bb labeled with the fluorescent protein mCherry localizes as foci that mark the multiple copies of chromosomal oriC during their partitioning in the growth phase (GP) [47], consistent with the conserved role of ParB in chromosome segregation.However, the localization of ParB Bb appeared to be cell-cycle regulated in B. bacteriovorus [47], whereas all ParB proteins described in other species permanently cluster at the centromere [7,35,37,[55][56][57][58][59][60][61][62][63][64][65].Using a ParB Bb -msfGFP fusion produced as a single copy from the native chromosomal locus, we confirmed that ParB Bb forms foci only during the growth phase of the predator cell cycle (Fig 1C pole, which we were able to distinguish using the invasive cell pole marker RomR-mCherry [47,66]

A fine-tuned balance of parA Bb and parB Bb expression underlies proper chromosome segregation and cell cycle progression in B. bacteriovorus
Remarkably, the ParB Bb -associated fluorescence seemed to drop at the end of the cell cycle, unlike ParA Bb -msfGFP (see the last time points in Fig 1C , 1F).Thus, our data suggest that these key players of the ParABS system undergo an unknown regulation resulting in distinct changes in protein abundance throughout the predatory cell cycle.To delve deeper into the cell-cycle control of the ParABS system in B. bacteriovorus, we first investigated the regulation of its expression.RT-PCR shows that parA Bb and parB Bb are part of the same operon (Figs 2A and S2A).Furthermore, their expression is cell-cycle regulated as the corresponding mRNA was detected mainly in GP and not in AP (Figs 2A and S2A), in agreement with previous reports [69,70].
Consistent with the significance of the cell cycle-dependent parAB Bb expression in B. bacteriovorus, we previously found that the constitutive expression of parB Bb (parB Bb ++ ) negatively impacts cell cycle progression and chromosome segregation [47] [71,72], this is the first evidence of cell-cycle-dependent changes in the relative levels of ParA and ParB proteins.

The formation of ParB Bb �parS complexes is inhibited in non-replicative predator cells
In addition to the sudden decrease of ParB Bb protein levels at the end of the GP, the subcellular localization of ParB Bb fluorescent fusions also changed, shifting from clear foci to diffuse localization (Figs 1C and 3B).However, both events could not be easily distinguished due to their occurrence within a relatively short time window and the low fluorescence of the natively produced ParB Bb fusion at that stage.Therefore, we sought to uncouple the ability of ParB  [47].Furthermore, these cells displayed the same phenotypes observed when the untagged ParB Bb is constitutively produced (i.e., morphological and chromosome segregation defects; S3H, S2B Figs), further supporting the relevance of these ParB Bb fusions.Thus, a third level of control is applied to ParB Bb in B. bacteriovorus to prevent its accumulation at the centromere, regardless of its protein level.

ParB Bb specifically accumulates on B. bacteriovorus parS in a CTPdependent manner
The formation of partitioning complexes in other species requires both the initial binding and the spreading of ParB on the DNA [8,10,11,13,14,73].To investigate ParB Bb 's ability to perform these two critical functions, we assessed its capacity to bind and spread from parS and to hydrolyze CTP in vitro.The two parS sequences in the proximity of the B. bacteriovorus chromosomal oriC (parS Bb ) are identical and closely resemble the parS consensus (S4A Fig) [2].In a biolayer interferometry assay, purified ParB Bb bound a linear DNA substrate carrying parS Bb , and the addition of CTP triggered ParB Bb sliding and falling off (marked by the higher Kd in the presence of CTP;

The parS Bb chromosomal context prevents ParB Bb accumulation in nonreplicative cells
Having established that ParB Bb activities are not per se drastically different than previously characterized ParB homologs, we investigated the possibility that in AP cells, the chromosomal

Discussion
Altogether, our study provides novel insights into the complex regulation of the conserved ParABS system in a bacterium that segregates multiple copies of its chromosome during its non-binary proliferation.We followed the subcellular dynamics, expression, and protein levels of the key players in that system, the proteins ParA and ParB, throughout the predatory cell cycle of B. bacteriovorus.Additionally, we assessed the ability of ParB Bb to form nucleoprotein complexes on parS sites in vitro and in vivo.We discovered that the ParABS system is coupled to cell cycle progression via three levels of regulation that modulate (i

Intricate subcellular dynamics of ParA Bb and ParB Bb during the replicative stage of the cell cycle
We investigated the localization of ParA Bb in B. bacteriovorus during the predatory cell cycle using a functional, natively produced fluorescent fusion.The subcellular localization of ParA has been observed in Streptomyces species, which also feature a polyploid growth stage [74,75].While the chromosome is copied multiple times during vegetative growth in these bacteria, the ParABS system only acts later in a distinct developmental stage to separate nucleoids in future spores [76].Thus, our data provide the first visualization of the ParA protein in a bacterium in which segregating multiple chromosome copies is an integral part of the cell cycle, rather than a conditional process as in Streptomyces.In non-replicative cells, ParA Bb is uniformly distributed on the nucleoid, in contrast with the ParA gradient observed in other species in which oriC is localized at one cell pole [3].The absence of a ParB Bb �parS nucleoprotein complex at the centromere-like region (see below) might explain this non-polarized ParA Bb pattern in AP B. bacteriovorus cells.Furthermore, our observations of ParA Bb and ParB Bb during chromosome replication and partitioning in the filamentous predator cell revealed several dynamic ParA Bb "clouds" between segregating ParB Bb foci, demonstrating that the ParABS system segregates multiple chromosomal copies simultaneously in the B. bacteriovorus cell.
In contrast to binary-dividing species like C. crescentus, the partitioning complexes do not move from one pole to another across the entire predator cell.Instead, ParB Bb foci adopt a clear "beads-on-a-string" pattern ( [47] and this study), reminiscent of the ParABS-mediated positioning of low-copy plasmids at non-polar locations [77].It is still unclear how the segregation of each chromosome is spatially confined within the filamenting B. bacteriovorus cell body (which is filled with other replicating and segregating copies of the chromosome) to achieve this regular distribution [47].We found that perturbations in the ParABS system, such as alterations of the ParA Bb :ParB Bb balance, prevent this exquisite arrangement of newly synthesized centromeres.In other bacteria, specific hub proteins at the cell poles polarize the choreography of ParABS-dependent segregation [29], but no homologs of these proteins were identified in B. bacteriovorus.Future research will determine whether this bacterium uses polar landmarks or other mechanisms to spatially organize chromosome partitioning along the mother cell.

Coupling centromere organization to cell cycle progression
Our previous work uncovered a striking difference between the ParB protein in B. bacteriovorus and those characterized in other species.Whereas ParB homologs always localize at the parS site [7,35,37,[55][56][57][58][59][60][61][62][63][64][65], ParB Bb is unable to accumulate at the chromosomal centromere in non-replicative cells, independently of its protein levels [47].This study offers unambiguous support to this finding by showing that (i) the unique on-off localization pattern of ParB Bb is independent of the fluorescent tag, and (ii) the protein fusions used to monitor ParB Bb subcellular localization and abundance represent reliable reporters of the native protein during the entire B. bacteriovorus cell cycle.Furthermore, we identified ParB Bb as a bona-fide CTPase like several other ParB homologs [10,13,73,78], which strongly suggests that ParB Bb accumulation at the centromere results from its binding on parS and CTP-dependent sliding on adjacent DNA, while subsequent CTP hydrolysis unloads the protein.
Remarkably, we found that the absence of ParB Bb �parS complex depends on the chromosomal context of parS, as ParB Bb formed a focus in B. bacteriovorus AP cells when parS was present on a plasmid.This result indicates that fluctuations in the cellular concentration of CTP, if any, are not responsible for the cell cycle-dependent localization of ParB Bb .It also hints that ParB Bb has the capacity to bind and spread from parS in the absence of ongoing chromosome replication, even though the first ParB Bb focus systematically appears after DNA replication initiation in GP cells [47].Moreover, our data reveal that the direct chromosomal context of parS sites has a major role in preventing ParB Bb complex formation, as ParB Bb lost its ability to accumulate on the plasmidic parS when this sequence was flanked by short DNA stretches adjacent to parS1 or parS2 on the chromosome.This intriguing effect was only observed in B. bacteriovorus, and not in E. coli cells.Therefore, we propose that the local topology of the DNA in the immediate vicinity of the parS sequences, possibly induced by B. bacteriovorusspecific proteins during the GP-AP transition, might interfere with parS binding and/or the spreading of ParB Bb .Identifying the specific inhibition that prevents ParB Bb from accumulating at the centromere in a cell-cycle and chromosomal context-dependent manner will certainly reveal additional complexity in bacterial centromere organization.

Cell cycle-dependent fluctuations of the ParA and ParB protein balance
The ParA Bb :ParB Bb balance appears to be crucial for B. bacteriovorus cell cycle progression, as constitutive production of ParA Bb or ParB Bb throughout the cell cycle led to cellular dysfunction, unless both proteins are overproduced.It is likely that the excess of either protein interferes with their fine-tuned interplay during the chromosome segregation process, resulting in the observed phenotypes.The compensatory effect of the double ParA Bb and ParB Bb overproduction is consistent with this idea.
Intriguingly, our results also hint that ParA Bb and ParB Bb protein levels are differentially controlled in the non-replicative stage of the B. bacteriovorus cell cycle and suggest that ParB Bb is specifically degraded in a cell cycle-dependent manner.Indeed, although the co-expression of parA Bb and parB Bb is turned off during AP, protein concentration is not expected to change between the mother cell and the newborn cells upon cell division.Yet, ParB Bb protein levels dropped within minutes at the GP-AP transition, becoming undetectable in the AP, unlike ParA Bb .Whereas there has been no report of a mechanism regulating ParB degradation in other bacteria, a recent study identified ParB as a candidate substrate of the conserved Lon protease in C. crescentus [79].Future investigation will be needed to reveal the potential proteolytic mechanism that depletes ParB Bb at the end of the B. bacteriovorus growth phase.
While it is clear that ParB Bb levels are tightly regulated during the B. bacteriovorus cell cycle, the physiological role of the disappearance of ParB Bb at the GP-AP transition remains to be determined.We hypothesize that in addition to the importance of proper ParA Bb and ParB Bb levels during growth to ensure accurate chromosome segregation, B. bacteriovorus benefits from clearing the cell from ParB Bb in the AP.This idea is rationalized by the incapacity of ParB Bb to cluster at the chromosomal oriC during that stage, which would result in the entire ParB Bb pool being free in the cytoplasm in the absence of degradation.We propose that such excess of cytosolic ParB Bb in AP cells introduces unknown deleterious effects, since we observed that the ParB Bb overproduction phenotype was largely alleviated when the protein could form a parS-bound cluster on a plasmid in AP cells.
Although studies in Streptomyces coelicolor have shown developmental stage-specific modulation of ParA and ParB protein levels resulting from the differential control of parAB transcription [41], to the best of our knowledge, this is the first report of fluctuating ParA:ParB protein ratios across the lifecycle of a bacterium.Altogether, our study sheds new light on the intricate dynamics and adaptation of the conserved ParABS system in bacteria.Our work hints that investigating the ParABS system in organisms with distinctive lifestyles will unveil new levels of complexity in centromere organization, chromosome segregation, and cell cycle control.

Strains
The strains and plasmids used in this study are listed in S1 Table, along with their respective construction methods (S2 Table ).Standard molecular cloning techniques were employed, and DNA assembly was conducted using the NEBuilder HiFi mix from New England Biolabs.All oligos used in the study can be found in S3 Table .The B. bacteriovorus strains were produced from the wild-type HD100 strain, while the E. coli strains employed as prey were generated from MG1655.Microscopy, conjugation, bacterial two-hybrid, and POLAR assay were conducted utilizing E. coli MG1655, S17-λpir, BL21, BTH101, and TOP10, CC118 λpir, TB28, respectively.All plasmids were introduced into B. bacteriovorus through mating, following the methodology described below.Scarless allelic replacements into the HD100 chromosome were achieved via a two-step recombination approach using a pK18mobsacB-derived suicide vector.The overexpression experiments were performed using the low-copy plasmid pTNV215 (RSF1010 replicon) as a backbone [47].Chromosomal modifications were screened by PCR and verified by Sanger DNA sequencing, following the protocol detailed in [47].

Routine culturing of B. bacteriovorus and E. coli
E. coli strains were grown in LB medium.For imaging experiments, overnight starter cultures from single colonies were diluted at least 1:500 in fresh medium until exponential phase.B. bacteriovorus strains were grown as described in DNB medium (Dilute Nutrient Broth, Becton, Dickinson, and Company) supplemented with 2 mM CaCl 2 and 3 mM MgCl 2 salts in the presence of E. coli prey at 30˚C with continuous shaking [80].When appropriate, antibioticresistant E. coli strains were used as prey for overnight culturing of the corresponding antibiotic-resistant B. bacteriovorus.When required, kanamycin, gentamycin, ampicillin, chloramphenicol, or tetracycline was added in liquid and solid media at 50 μg/ml, 10 μg/ml, 50 μg/ml, 15 μg/ml, and 7.5 μg/ml respectively.

Plasmid conjugation by mating
Mating was carried out between the E. coli S17-λpir donor strain carrying the plasmid to be conjugated and the B. bacteriovorus receiver strain, following the protocol detailed in [47].The exponentially growing E. coli donor strains were harvested and washed twice in DNB medium before resuspending in DNB salts at a 1:10 ratio of the initial volume.This donor suspension was combined with an equal volume of fresh overnight lysate of a receiver HD100 strain.The mating mixture was then incubated for a minimum of 4 hours at 30˚C with shaking before being plated on a selective medium using the double-layer technique [80].Single plaques were isolated, and transconjugants were validated by microscopy (when applicable), PCR, and sequencing.

RT-PCR
A predator: prey mixture (using a 1:1 volume ratio, which minimizes the excess of free attack phase cells) was prepared and incubated for several hours with shaking at 30˚C.RNA samples were collected at different times during synchronous predation to track the expression of parA Bb (bd3906), parAB Bb (bd3905-bd3906), parB Bb (bd3905) and dnaK (bd1298) throughout the predatory cycle of B. bacteriovorus on E. coli prey.A NucleoSpin RNA kit (MACHEREY-NAGEL) or SV Total RNA Isolation System (Promega) was used to isolate RNA, following the manufacturer's instructions.An additional TURBO DNase (Invitrogen) treatment was performed for 1 hour at 37˚C following manufacturer's instructions.RNA quality was assessed by measuring the 260/280 nm and 260/230 nm absorbance ratios.Reverse transcription (RT) and PCR were carried out utilizing the QIAGEN OneStep RT-PCR kit with the following thermocycling parameters: 50˚C for 30 minutes, 94˚C for 15 minutes, followed by 30 cycles of 94˚C for 1 minute, 50˚C for 1 minute, and 72˚C for 1 minute, with a final step of 72˚C for 10 minutes.

Bacterial-two hybrid assay (BACTH)
Interaction between protein pairs was assessed by the adenylate cyclase-based bacterial twohybrid technique, as detailed in [81].Briefly, the proteins of interest were merged with the isolated T18 and T25 catalytic domains of the Bordetella pertussis adenylate cyclase.The two plasmids producing the fusion proteins were introduced into the BTH101 reporter strain, and cotransformants were incubated on a selective medium overnight at 30˚C.A single colony from each co-transformation was inoculated into 400 μl LB medium supplemented with ampicillin (200 μg/ml), kanamycin (50 μg/ml), and IPTG (0.5 mM).After incubation overnight at 30˚C, 5 μl of each culture was placed onto LB plates supplemented with ampicillin, kanamycin, IPTG (using the abovementioned concentrations), and X-gal (40 ng/μl) and incubated at 30˚C.An interaction assay with pKT25-Zip and pUT18C-Zip, two Zip protein domains, was included as a positive control.The experiments were conducted in triplicate, and representative results are presented.

PopZ-linked apical recruitment (POLAR) assay
Protein-protein interaction between ParA Bb and ParB Bb was assessed using the POLAR assay, similarly as in [68].Briefly, proteins of interest were fused to msfGFP-H3H4 or mScarlet to generate the bait and prey protein fusions, respectively.Bait-and prey-encoding plasmids, cloned in E.coli TOP10 and CC118 λpir, respectively, were introduced in electrocompetent E. coli TB28 cells containing the temperature-sensitive helper plasmid pAH69.Selection of cotransformants and loss of pAH69 were allowed by overnight incubation on LB plates supplemented with chloramphenicol and tetracycline at 37˚C.An overnight starter was prepared by inoculating LB medium supplemented with appropriate antibiotics with a single colony and grown at 37˚C.A 6 ml fresh co-culture was prepared in LB medium with antibiotics using a 1:100 dilution and incubated at 37˚C.Once OD 600 reached 0.2, cells were collected by centrifugation (5000 rpm for 2 min) and resuspended in 6 ml 1x M9 minimal medium with salts supplemented with 0.2% Casaminoacids.The 6 ml culture was then split into two 3 ml cultures, one supplemented with 100 μM IPTG for prey protein induction only and the other with both 100 μM IPTG and 0.2% arabinose to induce the expression of the prey protein, PopZ Cc , and the bait protein.Snapshots were performed following a 2 h induction at 37˚C.

Live-cell imaging
B. bacteriovorus were grown overnight with the appropriate E. coli prey, and antibiotics were used when needed.Subsequently, they were grown on wild-type MG1655 for at least one generation without antibiotics before the beginning of the imaging experiment.For snapshots of fresh AP B. bacteriovorus, the cells were deposited on 1.2% agarose pads prepared with DNBsalt media.For the time-lapse imaging of synchronous predation cycles, MG1655 E. coli cells were grown in 2TYE medium to the exponential phase (OD 600 = 0.4-0.6),harvested at 5000 x g at room temperature for 5 minutes, washed twice, and resuspended in DNB medium.E. coli and B. bacteriovorus were then combined in a 1:3 to 1:5 volume ratio to allow the majority of prey cells to be infected.This setup ensured infection of all E. coli cells, which was optimal for time-lapse imaging.The remaining free Bdellovibrio cells did not pose any issue in that setup as they neither proliferate nor can re-infect existing bdelloplasts.In all synchronous predation imaging experiments, the prey-predator mixing step was indicated as time 0. The cells were either directly deposited on DNB-agarose pads for imaging or left shaking at 30˚C before imaging for the indicated durations.In time-lapse experiments, the same fields of view on the pad were imaged at regular intervals, with the enclosure temperature set to 27˚C.When appropriate, prior to imaging, cells were incubated for 5 minutes with DAPI (Life Technologies) at a final concentration of 5 μg/ml for nucleoid staining experiments.Regarding snapshots of E. coli strains, overnight cultures were diluted at least 1:500 and allowed to grow to the exponential phase before being deposited on 1.2% agarose pads prepared with PBS buffer supplemented with 0.2% glucose, 0.2% casamino acids, 1 μg/ml thiamine, 2 mM MgSO 4 , and 0.1 mM CaCl2), except for POLAR strains which were diluted 1:100 and imaged on 1.2% agarose pads prepared with 1x M9 minimal medium with salts following induction as described above.

Image processing
To prepare figures, images were processed using FIJI [82], with contrast and brightness settings being kept consistent for all regions of interest in each figure unless stated otherwise.Denoising (Denoise.ai,Nikon) was applied to all phase contrast and fluorescence channels for Fig 1C , 1F, 1G to improve the display of time-lapse images captured with low exposure (necessary to preserve cell viability).Figures were constructed and labeled using Adobe Illustrator.B. bacteriovorus outlines were obtained with Oufti [83] for AP cells, or drawn manually in Adobe Illustrator for GP cells within bdelloplasts.

Protein overexpression and purification
Expression and purification of ParB Bb with a C-terminal His-tag were conducted as follows.E. coli BL21(DE3) cells containing pET21, which expresses C-terminal His-tagged ParB Bb , were cultivated at 37˚C in autoinducing media supplemented with ampicillin (200 μg/ml).Following a 5-hour induction, cells were harvested by centrifugation and suspended in 35 ml lysis buffer (100 mM Tris-HCl [pH8], 300 mM NaCl, 5% glycerol (v/v), supplemented with a protease inhibitor cocktail (Complete, Roche)).The suspended cells were stored at 20˚C.The frozen cells were thawed on ice and lysed using three passages through a French pressure cell at 1500 psi.The suspension was then centrifuged for 15 minutes at 40 000 x g at 4˚C, and the supernatant was filtered through 0.45 μm filters before loading onto a 1 ml HisTrap column (GE Healthcare) pre-equilibrated with buffer A (100 mM Tris-HCl [pH8], 300 mM NaCl, and 5% [v/v] glycerol).The protein was eluted from the column using an increasing imidazole gradient (15-300 mM) in the same buffer.As a final purification step, size-exclusion chromatography was performed using a HiLoad 16/60 Superdex 75 column (GE Healthcare) with buffer B (100 mM Tris-HCl [pH8], 300 mM NaCl, and 5% [v/v] glycerol).The ParB Bb -containing fractions were pooled and analyzed for purity using SDS-PAGE.Glycerol was added to the ParB Bb fractions to a final volume of 10% (v/v), followed by 10 mM EDTA and 1 mM DTT.The purified ParB Bb was aliquoted, snap-frozen in liquid nitrogen, and stored at -80˚C.

Construction of DNA substrates for BLI assays
All DNA constructs were designed using VectorNTI (ThermoFisher) and synthesized chemically (gBlocks dsDNA fragments, IDT).To produce a linear biotinylated 40 bp DNA substrate, complementary oligos (with and without biotin) were heated at 98˚C for 5 min before being left to cool down to room temperature overnight to form 50 mM double-stranded DNA.A 180 bp DNA construct was created with M13F and M13R homologous regions at each end.To produce a dual biotin-labeled DNA substrate, PCR reactions were carried out using a 2x GoTaq PCR master mix (Promega), biotin-labeled M13F and biotin-labeled M13R primers, and gBlocks fragments as a template.PCR products were separated by electrophoresis and then purified from the gel.

Measurement of protein-DNA interaction by bio-layer interferometry (BLI)
Bio-layer interferometry experiments were performed using a BLItz system equipped with Dip-and-Read Streptavidin Biosensors (Molecular Devices) as previously described [15].The streptavidin biosensor was first equilibrated in a low-salt binding buffer (100 mM Tris-HCl (pH 8), 100 mM NaCl, 1 mM MgCl2, and 0.005% Tween 20) for at least 10 min before each experiment.Biotinylated double-stranded DNA (dsDNA) was then immobilized onto the surface of the biosensor through a cycle of baseline (30 s), association (120 s), and dissociation (120 s).During the association phase, different concentrations of ParB Bb dimers, with or without NTPs at varying concentrations, were added to the binding buffer and allowed to associate with the immobilized DNA for 120 s.Finally, the sensor was transferred into a protein-free binding buffer to monitor the dissociation kinetics for 120 s.The sensor was recycled by dipping in a high-salt buffer (100 mM Tris-HCl (pH 8), 1000 mM NaCl, 1 mM MgCl 2 , and 0.005% Tween 20) for 5 min to remove bound ParB Bb .All sensorgrams were recorded and analyzed using the BLItz analysis software (BLItz Pro version 1.2, Molecular Devices) and replotted using DataGraph for presentation.The data were fitted using a one-site-specific binding model in GraphPad Prism Version 5.04 to calculate the binding affinity constant Kd.All experiments were conducted at least in triplicate, and a representative sensorgram is presented in each figure.

DNA preparation for EnzCheck phosphate assay
A 20 bp palindromic single-stranded DNA fragment (parS Bb : GGATGTTCCACGTGGAAC ATCC or parS Cc : GGATGTTTCACGTGAAACATCC; 100 mM in 1 mM Tris-HCl pH 8.0, 5 mM NaCl buffer) was heated at 98˚C for 5 min before being left to cool down to room temperature overnight to form 50 μM double-stranded DNA.The parS Bb and parS Cc sequences are underlined.

Measurement of NTPase activity by EnzCheck phosphate assay
The EnzCheck Phosphate Assay Kit from Thermo Fisher was used to measure NTP hydrolysis.A reaction buffer containing 1 mM NTP and 1 μM of ParB Bb was analyzed in a Biotek EON plate reader at 25˚C 15 hours with readings taken every minute.The reaction buffer consisted of 740 μl of ultrapure water, 50 μl of a 20x customized reaction buffer (100 mM Tris pH 8.0, 2 M NaCl, and 20 mM MgCl 2 ), 200 μl of MESG substrate solution, and 10 μL of purine nucleoside phosphorylase (1 unit).Control reactions were performed with buffer only, buffer plus protein, or buffer plus NTP only.The plates were shaken continuously at 280 rpm for 15 hours at 25˚C.Each assay was performed at least in triplicate.Data analysis was done using Data-Graph, and the NTPase rates were calculated by fitting a linear regression in DataGraph.

Western blot analysis
For Western blot analysis, the AP samples were prepared following the method used in [47], starting from 1.5 ml cleared predation lysates.For the growth phase samples, a 1:1 volume ratio of E. coli prey to predator mixture was established, minimizing the excess of attack phase cells.The presence of uninfected, free E. coli cells did not interfere with downstream Western blot analysis as antibodies recognized proteins present only in B. bacteriovorus, and normalization by total protein estimates was not done between AP and GP samples (see below).Samples were collected at indicated time intervals for the time course analysis.NuPage Bis-Tris SDS precast polyacrylamide gels (Invitrogen) were used to load the samples and were run at 190 V for 50 minutes in the NuPAGE MOPS SDS running buffer.The assessment of equal total protein loading was carried out using PonceauS staining.The resulting stained membrane was imaged using an Image Quant LAS 500 camera (GE Healthcare).Standard Western blotting procedures were followed using primary antibodies against ParB Bb (rabbit serum obtained after injecting purified ParB Bb -6xhis protein; CER Group, Marloie, Belgium), GFP (mouse monoclonal JL-8 antibody, Takara), ParA Cc (rabbit polyclonal antibody, Le lab) and mCherry (polyclonal antibody, Thermo Fisher, product # PA5-34974).Secondary antibodies were goat anti-mouse IgG-peroxidase antibody (Sigma) for JL-8 and goat anti-rabbit IgG-peroxidase antibody (Sigma) for mCherry, ParA Cc and ParB Bb .Detection of antibody binding was performed by visualizing chemiluminescence from the reaction of horseradish peroxidase with luminol, imaged with an Image Quant LAS 500 camera (GE Healthcare).The quantification of band intensities was done separately for the AP and GP samples using the Image-QuantTL software.Normalization of band intensities to obtain relative protein levels was done by the total PonceauS intensity in the corresponding lane, used as loading and transfer control.Means and standard deviations were calculated and plotted using GraphPad Prism.Image processing was done with ImageJ.The assembly and annotation of figures were performed using Adobe Illustrator.Replicates of corresponding experiments presented in

Cell, nucleoid, and spot detection from microscopy images
The automated cell detection tool Oufti [83] was used to detect outlines of AP B. bacteriovorus cells, uninfected E. coli cells, or entire bdelloplasts with subpixel precision from phase contrast images.Fluorescence signals were added to cell meshes after background subtraction.Oufti was also used to detect diffraction-limited fluorescent foci and nucleoids with subpixel precision from fluorescence images, using the spotDetection and objectDetection modules.The detected spots and objects were added to the corresponding cell in the Oufti cell lists, including features related to coordinates, morphology, and intensity.The same optimized nucleoid detection parameters were used to ensure consistency and comparisons as done previously [47].Parameters for spot detection were optimized for each dataset or control set of images.

Quantitative image analysis from cell meshes
Fluorescence-related analysis, nucleoids, spots-related information, and other properties of individual cells based on microscopy images were extracted from Oufti cellLists data and plotted using custom codes in MATLAB (Mathworks), described below.

Fluorescence profiles
The custom Matlab script MeanIntProfile.m was used to obtain mean relative fluorescence profiles.Briefly, the fluorescence profile of each cell (corresponding to the array of fluorescence intensity per cell segment provided by the relevant signal field in the Oufti cellList) was first normalized by the corresponding array of steparea values (corresponding to the area of each segment of the cell), then divided by their sum to obtain relative fluorescence values for each cell (to account for potential concentration differences between cells).When needed, arrays of relative fluorescence were oriented based on the position of the maximal fluorescence intensity of the indicated signal in each cell half.Cell length vectors were normalized from 0 to 1, and the corresponding relative fluorescence profiles from individual cells were interpolated to a fixed-dimension vector and concatenated before averaging.

Fluorescence intensity analysis of time-lapse experiments
Bdelloplasts outlines were detected in the first frame of the time-lapse using Oufti and copied to all following frames using MATLAB.The resulting time-lapse cell list was reused in Oufti after background subtraction to add the fluorescence signal(s) to the bdelloplasts.The mean fluorescence profiles per cell over time were computed using a custom Matlab script (Mean-FluoIntPerCell_TimeLapse), which extracts and plots the total fluorescence per cell (here bdelloplast) divided by the area of the corresponding cell (bdelloplast) over time.

Kymographs and demographs
Demographs of relative fluorescence intensity in cells sorted by length were plotted as in [47,84].When needed, arrays of relative fluorescence intensity values were oriented based on the position of the maximal fluorescence intensity of the indicated signal in each cell half.Kymographs were obtained using the built-in kymograph function in Oufti [83].

Nucleoid size measurement
To measure nucleoid size, we used the objectDetection module in Oufti [83].We considered the nucleoid area as a proxy for nucleoid size, based on previous work [85], which demonstrated that variations do not influence nucleoid area measurements in DAPI signal intensity.The nucleoid area was obtained from the nucleoid area field in the Oufti cell lists and exported to MATLAB for all cells with a single nucleoid.The nucleoid area values were then converted to μm 2 and used to generate violin plots of nucleoid area distributions using R [86].

Statistical analyses
The sample sizes and the number of repeats are included in the figure legends.Means, standard deviations, and coefficients of variation (CV) were calculated in GraphPad Prism, MATLAB (Mathworks), R, or Microsoft Excel.

Data, resources, and software availability
Matlab and R codes and datasets have been deposited at Zenodo (doi: 10.5281/zenodo.8325025).

Fig 1 .
Fig 1. Subcellular localization of the ParABS system during the B. bacteriovorus cell cycle.(A) Schematics of the Bdellovibrio bacteriovorus cell cycle.Numbers indicate key steps in the cycle: 1. Freely-swimming attack phase (AP) cells, 2. Attachment of B. bacteriovorus to its prey, 3. B. bacteriovorus resides in the periplasm of the prey, which is now called bdelloplast, 4. Filamentous growth and consumption of prey content, 5. Pre-divisional state, 6.The non-binary division of the mother cell generates an odd or even number of daughter cells, which mature before 7. escaping the prey remnants and resuming the cell cycle.Attack phase (AP) and growth phase (GP) represent the non-replicative and

,
S1 Video).Conversely, the ParB Bb -msfGFP fluorescence signal was weak and diffuse in the cytoplasm in non-replicating cells, i.e., in the free attack-phase (AP) cells(Fig  1D)  or during the time window corresponding to the G1-S transition upon prey invasion[47] (Fig1C).To obtain more insights into the functioning of the ParABS system during the cell cycle of B. bacteriovorus, we constructed a functional ParA Bb -msfGFP fusion (S1A Fig) produced natively as a single copy and monitored its subcellular localization.We found that ParA Bb -msfGFP co-localized with the nucleoid in non-replicative cells (Fig1E), in agreement with the non-specific DNA binding of ParA proteins[16,17].Unlike in other species with a polarly localized oriC, ParA Bb -msfGFP localization was not visibly biased towards a particular cell replicative stages of the cell cycle, respectively.(B) Chromosomal context of the parABS system in the WT B. bacteriovorus genome.The parAB operon is shown in blue.bd3908 encodes a putative rRNA methyltransferase; bd3907 encodes an unknown protein; parA and parB are bd3906 and bd3905, respectively; bd3904 encodes a bactofilin homolog.The genomic distance between chromosomal ori and both parS sites is indicated.(C) Cell-cycle dependent localization of ParB Bb .B. bacteriovorus strain parB Bb ::parB Bb -msfgfp (GL1654) was mixed with E. coli prey and imagined in time-lapse after 60 min with 8-min intervals.Overlays of phase contrast and fluorescence images of selected time points are shown; multiple well-separated ParB Bb -msfGFP foci appear over time; in this example, ParB Bb -msfGFP foci disappear at time point 664 min.The time-lapse is shown in S1 Video.(D) Endogenous ParB Bb does not form an oriC-bound focus during AP.Representative phase contrast and fluorescence images of the parB Bb ::parB Bb -msfgfp strain (GL1654) for representative AP cells; histogram representing the percentage of cells with zero, one, or two ParB Bb -msfGFP foci in the same strain.(E) ParA Bb is nucleoid bound.Left to right: representative phase contrast and fluorescence images of AP cells of the parA Bb ::parA Bb -msfgfp strain (GL2134) stained with DAPI; demographs of the corresponding fluorescent signals in the same cells sorted by length and oriented based on signal intensity; heatmaps represent relative fluorescence intensities; mean pole-to-pole profiles of relative fluorescence intensity of the corresponding signals in the same cells.(F) Endogenous ParA Bb moves dynamically along the growing predator filament.From left to right: B. bacteriovorus strain parA Bb ::parA Bb -msfgfp (GL2134) was mixed with prey and imaged in time-lapse after 80 min with 5-min intervals; left: overlay of phase contrast and fluorescence images, time points are shown in min; right: kymograph of the ParA Bb -msfGFP signal along the cell length for the same cell until time point 445 min.The time-lapse is shown in S2 Video.(G) The dynamic interplay between ParB Bb and ParA Bb .B. bacteriovorus strain parA Bb :: parA Bb -msfgfp parB Bb ::parB Bb -mcherry (GL2154) was mixed with prey and imagined in time-lapse after 75 min with 8-min intervals.Top: phase contrast; bottom: overlay of ParB Bb -mCherry and ParA Bb -msfGFP signals.Arrowheads point to a ParB Bb focus on the edge of a ParA Bb -msfGFP cloud.Bottom: pole-to-pole profiles of relative fluorescence intensity of the corresponding fusions in the same cells; arrowheads point to ParB Bb foci at the edge of ParA Bb -msfGFP clouds.The time-lapse is shown in S3 Video.Scale bars are 1 μm.n indicate the number of cells analyzed in a representative experiment.B. bacteriovorus and bdelloplasts outlines in panels C, F, G were drawn manually based on the phase contrast images.All experiments were performed at least twice.See also S1 Fig. https://doi.org/10.1371/journal.pgen.1010951.g001 (S1B Fig).During the B. bacteriovorus proliferative phase inside the prey, ParA Bb -msfGFP displayed a dynamic localization pattern reminiscent of the DNA-bound ParA "cloud" characterized in other organisms (Fig 1F, S2 Video), which pulls apart duplicated ParB�parS partitioning complexes via repeated ParA-ParB interactions [19,67].In addition, we detected an interaction between ParA Bb and ParB Bb in a bacterial two-hybrid assay (S1C Fig) and in a POLAR recruitment assay (S1D-S1F Fig) [68], suggesting a similar interplay between these proteins in B. bacteriovorus.However, in contrast to bacteria that segregate only two copies of their chromosome, several ParA Bb -msfGFP accumulations were present at the same time and moved dynamically along the filamentous predator cell, shifting from one subcellular region to another (Fig 1F, S2 Video).Using a strain carrying both fluorescently labeled ParA Bb and ParB Bb (S1G Fig), we observed that ParB Bb -mCherry foci were mainly located at the edges of ParA Bb -msfGFP clouds (Fig 1G, arrowheads, S3 Video).This complex localization pattern is consistent with the idea that the B. bacteriovorus ParABS system is adapted to drive the simultaneous or sequential segregation of multiple chromosome copies.

The protein levels of
ParA Bb and ParB Bb vary differently during the predatory cell cycle Since ParA Bb and ParB Bb fluorescence profiles hinted at distinct relative protein abundance despite being expressed from the same transcript, we sought to obtain detailed insights into ParA Bb and ParB Bb protein levels during the cell cycle.Western blot using an antibody against ParB Bb shows that the endogenous (untagged) ParB Bb protein levels vary during the cell cycle, being almost unnoticeable in AP but present in higher amounts in GP (Figs 3A, S3A and S6A), mirroring the fluorescence signal of the natively produced ParB Bb -msfGFP fusion (Fig 1C).Accordingly, single-cell analysis of the ParB Bb -msfGFP fluorescence intensity profile over time indicates that ParB Bb proteins accumulate during cell growth but rapidly drop when B. bacteriovorus divides into multiple daughter cells (Fig 3B, magenta, S3B Fig).Notably, the levels of natively produced ParB Bb -msfGFP or ParB Bb -mCherry protein fusions, detected with the same anti-ParB Bb antibody, fully reflected the endogenous untagged ParB Bb profile during a synchronized B. bacteriovorus cell cycle (Figs 3A, S3C and S6A), confirming that these fusions are reliable reporters of the native ParB Bb protein.Unlike ParB Bb , ParA Bb -msfGFP was detected throughout the cell cycle, including in AP (Figs 3C-3D, S3D and S6B-S6C), consistent with the corresponding fluorescence signal (Fig 1F).ParA Bb -msfGFP fluorescence intensity remained high during the last cell cycle stages, including cell division (Fig 3B, green, S3E Fig).These results show that, besides cell-cycle regulated expression, the ParABS system in B. bacteriovorus is subjected to an additional level of control that results in distinct ParA Bb and ParB Bb protein levels at different cell cycle stages.While in vitro studies showed that the ParA:ParB balance is important for efficient partitioning

Fig 2 .Fig 3 .
Fig 2. Biphasic expression of the parAB Bb operon during the B. bacteriovorus cell cycle is critical for proper chromosome segregation.(A) Biphasic expression of the parAB Bb operon.Top: schematic representation of the B. bacteriovorus parAB and dnaK (housekeeping gene) genomic context; graphical illustrations depict the cell cycle stages corresponding to the indicated time points.The fragments corresponding to the parA-parB junction or inside the dnaK gene amplified by RT-PCR are indicated below (AB, 400 bp; K, 400 bp).RNA samples were isolated from wild-type B. bacteriovorus in AP, 1 h, and 3 h after mixing with prey.Bottom: agarose gel analysis of the indicated fragments amplified by PCR from cDNA (top panel), genomic DNA (middle panel, positive control), and total RNA (bottom panel, negative control, ensuring RT-PCR bands result from the cDNA and not from potential DNA contamination in the RNA sample).Molecular weight markers (in kb) are indicated on the left.(B) Balanced parA Bb :parB Bb expression is essential for proper chromosome partitioning.Left: phase contrast and fluorescence images of representative AP cells stained with DAPI.The schematics illustrate the presence of a plasmid allowing the constitutive expression (from the PnptII promoter) of the indicated gene(s) parB Bb (GL1261), parA Bb (GL1460), or both (GL1004) in an otherwise WT background.Cells with abnormal nucleoid areas are observed when parA Bb (parA Bb ++ ) or parB Bb (parB Bb ++ ) is overexpressed, but to a lower extent when both are overexpressed (parAB Bb + Bb to form foci (due to parS binding and spreading on adjacent DNA) from ParB Bb protein levels by constitutively expressing parB Bb -msfgfp.In these cells, the fluorescence signal (S3F Fig) and protein levels (S3G Fig) of ParB Bb -msfGFP in AP were stronger than upon expression from the native locus.Still, overproduced ParB Bb -msfGFP was diffuse in AP cells (Fig 3E), consistent with our previous results with ParB Bb -mCherry Fig 4A), like other characterized ParB homologs [11,13-15,73].Moreover, ParB Bb accumulated on a closed DNA loop containing parS Bb in a CTP-dependent manner, reflecting the capacity of ParB Bb to bind parS Bb and escape onto neighboring DNA (Fig 4B).This accumulation was specific to the cognate parS Bb , as no binding was observed when we used a scrambled parS sequence (nonS; Fig 4B) or parS from Caulobacter crescentus (parS Cc ; Fig 4B).Finally, measurements of inorganic phosphate release showed that ParB Bb hydrolyses CTP but no other NTPs in the presence of parS Bb (S4B Fig).Thus, the B. bacteriovorus ParB protein is a bona-fide parS-binding CTPase.Consistent with those in vitro data, ParB Bb specifically accumulated on parS Bb in vivo in E. coli (which lacks an endogenous 3h, 4h, and 5h after mixing with prey (GP), and AP.All samples were run on the same gel; for enhanced figure clarity, sample positions have been rearranged, as indicated by the dashed line.Graphical illustration of the cell cycle stages corresponding to the indicated time points.Arrowhead points to ParA Bb -msfGFP (detected also in the AP).Ponceau staining served as a loading control and quantification from full lanes (displayed in S6B Fig) was used to normalize band intensities.Quantifications are shown as the means of independent triplicates.Error bars represent standard deviations.All replicates are shown in S6B Fig. (D) Top: Western blots of whole-cell protein extracts from AP cells of B. bacteriovorus WT, parB Bb ::parB Bb -msfgfp (ParB Bb, GL1654) and parA Bb ::parA Bb -msfgfp (ParA Bb , GL2134) strains were probed with an anti-msfGFP antibody.Only ParA Bb -msfGFP protein is detected in AP.Bottom: quantifications are shown as the means of independent triplicates, normalized as in A and C. Error bars represent standard deviations.All replicates and Ponceau staining are shown in S6C Fig. (E) Overproduction of ParB Bb -msfGFP does not lead to the parS-bound focus in AP.Left: representative phase contrast and fluorescence images of AP cells of a WT B. bacteriovorus strain constitutively producing ParB Bb -msfGFP (GL1003).Right: histogram representing the percentage of cells with zero, one, or two ParB Bb -msfGFP foci in the same strain; n indicates the number of cells analyzed in a representative experiment.Scale bars are 1 μm.All experiments were performed at least twice.See also S3 Fig. https://doi.org/10.1371/journal.pgen.1010951.g003ParABS), used as a heterologous system.Indeed, ParB Bb -msfGFP formed foci when produced in E. coli, only in the presence of a plasmid carrying parS Bb and not when the plasmid carried the non-cognate parS Cc (Figs 4C and S4C).Interestingly, ParB from Caulobacter crescentus (ParB Cc ) also failed to form a focus and was diffuse in the cytoplasm when ectopically produced in AP B. bacteriovorus cells (Fig 4D), even though ParB Cc can bind parS Bb in vitro (S4D Fig) and in E. coli (S4E Fig).Therefore, the absence of ParB Bb �parS Bb complex in non-replicative B. bacteriovorus cells is unlikely to result from an unusual functionality of the ParB Bb protein itself.

Fig 4 .
Fig 4. ParB Bb is a CTPase with a specific affinity for its cognate parS Bb .(A) CTP reduces the binding of ParB Bb on parS Bb on a linear substrate.Left: schematics of the Streptavidin (SA)-coated probe and the linear 40-bp DNA substrate containing the cognate parS Bb sequence.Right: ParB Bb titration curves in the presence or absence of CTP obtained from biolayer interferometry (BLI) analysis (see B and Material and Methods).All reactions contained 1 mM CTP (when appropriate), 0.5 μM 40 bp parS Bb DNA, and an increasing concentration of ParB Bb .The BLI experiment was done in triplicates; data were fitted to calculate the binding affinity constant Kd (nM); Kd = 134 ± 23 nM for the 40 bp parS Bb , and Kd = 5500 ± 397 nM for the 40 bp parS Bb + 1mM CTP.In this assay, ParB Bb spreading displaces it from the short linear parS fragments by pushing it into the solution.The higher Kd constant (lower parS affinity) in the presence of CTP thus indicates CTP-induced spreading of ParB Bb .(B) ParB Bb accumulation at parS Bb is specific and CTP-dependent.Left: schematics of the SA-coated probe and a closed DNA substrate with a parS sequence, allowing ParB accumulation through parS binding and CTP binding-dependent spreading.CTP hydrolysis removes ParB from the DNA.Right: BLI analysis of the interaction between 1 μM ParB Bb -6xhis in the presence or absence of 1 mM CTP and a 180 bp DNA substrate containing the cognate parS Bb (blue), scrambled nonS (black) or non-cognate parS Cc (grey) sequence.The BLI probe was dipped into a bufferonly solution (base), then into a premix of protein +/-CTP (association), and finally returned to a buffer-only solution (dissociation).Each BLI experiment was done in triplicate, and a representative sensorgram is presented.(C) ParB Bb protein can bind to its cognate parS sequence in E. coli.Representative phase contrast and fluorescence images of E. coli carrying a plasmid with parS Bb and allowing constitutive production of ParB Bb -msfGFP (GL1669; plasmid schematics on the left).The scale bar is 2 μm.See Fig 5D for histogram representing the percentage of cells with zero, one, or two ParB Bb -msfGFP foci in the same strain.(D) A constitutively produced fusion of Caulobacter crescentus ParB (ParB Cc ) to msfGFP does not form foci in AP B. bacteriovorus cells.Left: representative phase contrast and fluorescence images of AP cells of a WT strain constitutively producing ParB Cc -msfGFP from a plasmid (GL2108; schematics on the left).The fluorescence signal shows the partial nucleoid exclusion pattern of freely diffusing cytosolic proteins, characteristic of AP B. bacteriovorus cells 1 .Right: histogram of the percentage of cells with zero, one, or two ParB Cc -msfGFP foci in the same strain; n indicates the number of cells analyzed in a representative experiment.The scale bar is 1 μm.See also S4 Fig. https://doi.org/10.1371/journal.pgen.1010951.g004 parA Bb and parB Bb gene expression, (ii) ParA Bb and ParB Bb protein levels, and (iii) chromosomal parS accessibility during the non-proliferative and replicative stages of the predator lifecycle (Fig 6).Furthermore, our data indicate that the combination of these layers of cell cycle-dependent control contributes to clearing the B. bacteriovorus cell and the centromere from ParB Bb during the non-proliferative stage, which appears crucial for cell cycle progression (see below).

Fig 5 .
Fig 5.The genomic context of parS Bb plays a role in ParB Bb localization.(A) Changing the native context of the parS Bb enables ParB Bb binding.Left: representative phase contrast and fluorescence images of AP cells of WT B. bacteriovorus strain constitutively producing ParB Bb -msfGFP from a plasmid carrying a copy of the parS Bb sequence (GL1751).Right: histogram representing the percentage of cells with zero, one, or two ParB Bb -msfGFP foci in the same strain.(B-C) Short chromosomal regions around each parS Bb site are enough to prevent ParB Bb from clustering on the centromere.Top: schematic representation of the parAB operon in B. bacteriovorus and the upstream parS sequences; chromosomal regions around parS Bb sequences ("extended parS") cloned in the plasmids are indicated; plasmid schematics are shown on the right.Bottom: representative phase contrast and fluorescence images of AP cells of WT strain constitutively producing ParB Bb -msfGFP from a plasmid carrying parS 1extended (B, GL1749) or parS 2extended (C, GL1925); histogram representing the percentage of cells with zero, one, or two ParB Bb -msfGFP foci in the same strain.Scale bars are 1 μm.(D) The genomic context of the parS Bb prevents ParB Bb focus formation only in B. bacteriovorus.Top: representative phase contrast and fluorescence images of E. coli strains carrying plasmids as in Figs 3D, 5A-5C, constitutively producing ParB Bb -msfGFP in the absence of parS (GL1661), or in the presence of parS Bb (GL1669), parS 1extended (GL1737) or parS 2extended (GL1899), respectively.Schematics illustrate the parB Bb -msfgfp expression plasmid with or without (extended) parS sequences.Bottom: histogram representing the percentage of cells with zero, one, or two ParB Bb -msfGFP foci in the same strains.The scale bar is 1 μm.n indicates the number of cells analyzed in a representative experiment; all experiments were performed at least twice.See also S5 Fig. https://doi.org/10.1371/journal.pgen.1010951.g005

Fig 6 .
Fig 6.Model for coupling cell cycle progression and centromere organization through multi-layered regulation of the ParABS system in B. bacteriovorus.Schematic representation of the subcellular localization of ParA Bb (green) and ParB Bb (magenta) throughout the predatory cell cycle in B. bacteriovorus.The three levels of ParABS regulation are i) transcription of the parAB operon, ii) ParA Bb and ParB Bb protein levels, and iii) accessibility of the chromosomal parS site.In growing cells (1), the parAB operon transcription is "on", ParB Bb and ParA Bb levels are high, and parS is accessible, enabling ParB Bb to form nucleoprotein complexes on the centromere.The ParABS system rearranges concomitantly with non-binary division (2), transitioning towards an "off" state.During the attack phase (3), ParA Bb levels remain high, but ParB Bb levels are low due to potential protein degradation at the end of the growth phase, combined with the absence of new transcription of the parAB operon.The parS chromosomal context is incompatible with the formation of a ParB Bb complex at the centromere during this stage of the cell cycle.https://doi.org/10.1371/journal.pgen.1010951.g006 Fig 3A, 3C-3D are provided in S6A-S6C Fig, respectively.

S1Fig.
ParA Bb subcellular localization and interaction with ParB Bb .Related to Fig 1. (A) The endogenous ParA Bb -msfGFP fusion is functional.Violin plots of cell length, cell area, and nucleoid area distributions in parA Bb ::parA Bb -msfgfp (GL2134) and WT B. bacteriovorus strains, measured from cells in Fig 1E.The lines indicate the 25, 50, and 75 percent quantiles from bottom to top.Mean, standard deviation and coefficient of variation (CV) values are shown on top of the corresponding plot.n indicate the number of cells analyzed in a representative experiment.(B) ParA Bb is not oriented towards any cell pole in AP cells.Left to right: representative phase contrast and fluorescence images of AP cells of parA Bb ::parA Bb -msfgfp romR::romR-mcherry strain (GL2155); demographs of the corresponding fluorescent signals in the same cells sorted by length and oriented based on RomR-mCherry signal intensity; heatmaps represent relative fluorescence intensities; mean pole-to-pole profiles of relative fluorescence intensity of the corresponding fusions in the same cells.Scale bar is 1 μm.(C) ParA Bb and ParB Bb interact in a bacterial two-hybrid assay.BTH101 reporter cells producing the indicated proteins fused to the T18 or T25 adenylate cyclase domain were spotted on X-gal agar plates supplemented with IPTG.Interaction between two proteins results in blue colony color.The Zip-Zip interaction serves as a positive control.(D) Schematic representation of the POLAR assay.POLAR takes advantage of PopZ from C. crescentus (grey), which spontaneously forms clusters at the cell poles or septa when produced in E. coli.The unlabeled PopZ is produced along with an msfGFP-H3H4 fusion to the bait protein of choice (here ParB Bb ), depicted in magenta; H3H4 is a PopZ self-interacting domain (shown in blue) that is sufficient to draw the bait protein to PopZ clusters.The prey protein (ParA Bb ) is depicted in green.The prey protein is recruited to the PopZ-bait cluster if prey and bait proteins interact (upper half of the cell), whereas a lack of interaction leaves the localization of the prey protein unmodified by the bait protein (lower part of the cell).(E) ParA Bb does not interact with msfGFP-H3H4 (control).Representative fluorescence images of E. coli cells expressing ParA Bb -mScarlet in the absence (left) or presence (right) of msfGFP-H3H4 without bait fusion.ParA Bb -mScarlet (expressed from GL2295) displays a nucleoid-bound pattern and is not recruited to the polar PopZ cluster via msfGFP-H3H4; pole-to-pole profiles of relative fluorescence intensity of the corresponding fusions in the one cell.(F) ParA Bb and ParB Bb interact in the POLAR assay.Same as in (E) for cells producing ParA Bb -mScarlet as prey (green) in the absence (left) or presence (right) of ParB Bb -msfGFP as bait fused to msfGFP-H3H4 (magenta).ParA Bb partially colocalizes with the ParB Bb -msfGFP-H3H4 cluster (expressed from GL2294) visible as fluorescent foci (arrowheads); pole-to-pole profiles of relative fluorescence intensity of the corresponding fusions in the same cell.Scale bar is 2 μm.(G) A strain in which both native ParA Bb and ParB Bb are labeled displays morphology and chromosome aspect ratio similar to wildtype.Violin plots of cell length, cell area, and nucleoid area distributions in parA Bb ::parA Bbmsfgfp parB Bb ::parB Bb -mcherry (GL2154) and WT B. bacteriovorus strains, measured from cells in Fig 1G.The lines indicate the 25, 50, and 75 percent quantiles from bottom to top.Mean and standard deviation values are shown on top of the corresponding plot.n indicate the number of cells analyzed in a representative experiment.All experiments were performed at least twice.(TIF) S2 Fig. Biphasic expression of the parA Bb and parB Bb genes and proper balancing of parAB Bb expression contributes to progressive ori segregation.Related to Fig 2. (A) Expression of the parA Bb and parB Bb genes, individually, follow the same biphasic pattern as the fragment covering both genes.RT-PCR experiment as in Fig 2A using primer pairs hybridizing within the parA Bb (left) or the parB Bb gene (right).(B) Overexpression of parA Bb leads to chromosome segregation defects.B. bacteriovorus strain parB Bb ::parB Bb -mcherry / pTNV215-parA Bb (GL2129) was mixed with prey and imaged in time-lapse after 120 min with 8-min intervals.Left: phase contrast and fluorescence images of selected time points; arrowhead points to an altered ParB Bb -mCherry behavior (patches instead of well-separated foci) during the cell cycle.(C) Overexpression of parB Bb or parA Bb leads to pronounced phenotypes, largely rescued by overexpression of both.Violin plots of cell length, cell area, and nucleoid area distributions in WT / pTNV215-parB Bb (parB Bb ++ , GL1261); WT / pTNV215-parA Bb (parA Bb ++ , GL1460); WT / pTNV215-parB Bb -parA Bb (parAB Bb ++ , GL1004) and WT B. bacteriovorus from cells in Fig 2B.The lines indicate the 25, 50, and 75 percent quantiles from bottom to top.Mean, standard deviation and coefficient of variation (CV) values are shown on top of the corresponding plot.n indicate the number of cells analyzed in a representative experiment.(D) Overexpression of both parA Bb and parB Bb has no obvious effect on ori segregation.B. bacteriovorus strain WT / pTNV215-parB Bb -mcherry stop -RBS-parA Bb -msfgfp (GL1004) was mixed with prey and imaged in time-lapse after 75 min with 8-min intervals.Left: phase contrast and fluorescence images of selected time points; arrowheads point to well-distributed ParB Bb -mCherry foci (which mark chromosomal ori) during the cell cycle; ParA Bb -msfGFP not shown for simplicity.All experiments were performed at least twice.Scale bars are 1 μm.(TIF) S3 Fig.The levels of ParB Bb and ParA Bb vary differently during the cell cycle, and ParB Bb is unable to form foci when overproduced.Related to Fig 3. (A) The anti-ParB Bb antibody detects overproduced (right) but not native levels (right) of untagged ParB Bb in AP; control experiment for Fig 3A.Western blots of whole-cell protein extracts from AP cells of WT B. bacteriovorus and a strain constitutively producing the untagged ParB Bb (GL1261) were probed with an anti-ParB Bb antibody represented in the schematics on the left.(B) Same as in Fig 3B for the strain natively producing ParB Bb -msfGFP (GL1654).(C) The protein levels of natively produced ParB Bb -msfGFP or ParB Bb -mCherry detected with the same anti-ParB Bb antibody compare with the endogenous untagged ParB Bb profile during a synchronized B. bacteriovorus cell cycle.Western blots of whole-cell protein extracts from B. bacteriovorus strains parB Bb ::parB Bb -msfgfp (GL1654) and parB Bb ::parB Bb -mcherry (GL906) were probed with an anti-ParB Bb antibody, as represented in the schematics.Protein samples are isolated at time points throughout the predatory cell cycle: AP, 1h, 2h, 3h, and 4h after mixing with prey.Arrowhead indicates detected ParB Bb protein during the growth phase.Ponceau staining of the same membranes (where bands were most visible, ~30-50 kDa) is illustrated below each blot as a loading control.Molecular weight markers (kDa) are shown on the side.(D) ParA Bb protein is detected in the AP.Western blots of whole-cell protein extracts from Caulobacter crescentus (positive control), E. coli MG1655 (negative control), and WT B. bacteriovorus (in triplicates), were probed with an anti-ParA Cc antibody, as represented in the schematics on the left.Untagged ParA Bb (~29 kDa) is detected in the AP in all three samples.Ponceau staining of the same membranes (where bands were most visible, ~30-50 kDa) is illustrated below each blot as a loading control.Molecular weight markers (kDa) are shown on the side.(E) Same as in Fig 3B for the strain natively producing ParA Bb -msfGFP (GL2134).(F) Mean msfGFP fluorescence measured for AP cells of WT, parB Bb ::parB Bb -msfgfp (parB Bb -msfgfp, GL1654) and WT / pTNV215-parB Bb -msfgfp (parB Bb -msfgfp ++ , GL1003).n indicate the number of cells analyzed in a representative experiment; mean values are represented.Error bars indicate standard deviations.(G) Western blots of whole-cell protein extracts from AP cells of WT B. bacteriovorus parB Bb ::parB Bb -msfgfp (parB Bb -msfgfp, GL1654, left) and WT / pTNV215-parB Bb -msfgfp (parB Bb -msfgfp ++ , GL1003, right) strains were probed with an anti-msfGFP antibody, represented in the schematics on the left.Arrowhead points to the ParB Bb -msfGFP protein detected in AP when overproduced.(H) Overproduction of ParB Bb -msfGFP leads to morphological and chromosome segregation defects.Violin plots of cell length and nucleoid area distributions for the cells in Fig 3E and WT B. bacteriovorus.The lines indicate the 25, 50, and 75 percent quantiles from bottom to top.Mean, standard deviation and coefficient of variation (CV) values are shown on top of the corresponding plot.n indicate the number of cells analyzed in a representative experiment.All experiments were performed at least twice.(TIF) S4 Fig. Investigation of the ParB Bb features near the CTP binding pocket and their impact on parS Bb recognition.Related to Fig 4. (A) parS Bb sequence matches with the consensus.Top: parS sequence from B. bacteriovorus.Bottom: a sequence logo generated by WebLogo 3.0 [1], using parS sequence alignments from [5] as input.(B) ParB Bb is a CTPase.Continuous monitoring of the released inorganic phosphate (Pi) by recording the absorbance at 360 nm overtime at 25˚C.The NTP hydrolysis of ParB Bb was also monitored in the presence of ATP, GTP, or UTP, with a 22 bp parS Bb DNA duplex.(C) ParB Bb does not cluster without parS or on non-cognate parS.Left: representative phase contrast and fluorescence images of MG1655 E. coli strain constitutively producing ParB Bb -msfGFP from a plasmid carrying no parS sequence (GL1661) or the C. crescentus parS (parS Cc ; GL2024).Right: histograms representing the percentage of cells with zero, one, or two ParB Bb -msfGFP foci in the same strains.The scale bar is 2 μm; schematics illustrate parB Bb -msfgfp expression plasmids.(D) ParB from C. crescentus (ParB Cc ) is more promiscuous to parS binding than ParB Bb .BLI analysis of the interaction between 1 μM ParB Cc and a 40 bp cognate parS Cc (grey) or a non-cognate parS Bb (blue).ParB Cc binds both parS sequences.(E) ParB Cc can bind to its cognate parS Cc and a noncognate parS Bb in E. coli.Left: representative phase contrast and fluorescence images of MG1655 E. coli strain constitutively producing ParB Cc -msfGFP from a plasmid carrying parS Cc (left, GL2025) or parS Bb (right, GL2026); fluorescent foci are observed in both cases.

mimicking the parB Bb ++ phenotype (Figs 2B and S2B-S2C). Remarkably, simultaneous constitutive expres- sion of both parA Bb and parB Bb (from the same promoter) decreased the severity of the single overexpression phenotypes (Figs 2B and S2B-S2D). Thus, our data show that the ParABS sys
. Altering the native parA Bb expression similarly impaired cell cycle progression and ori partitioning,