Chromosome structure and DNA replication dynamics during the life cycle of the predatory bacterium Bdellovibrio bacteriovorus

Abstract Bdellovibrio bacteriovorus, an obligate predatory Gram-negative bacterium that proliferates inside and kills other Gram-negative bacteria, was discovered more than 60 years ago. However, we have only recently begun to understand the detailed cell biology of this proficient bacterial killer. Bdellovibrio bacteriovorus exhibits a peculiar life cycle and bimodal proliferation, and thus represents an attractive model for studying novel aspects of bacterial cell biology. The life cycle of B. bacteriovorus consists of two phases: a free-living nonreplicative attack phase and an intracellular reproductive phase. During the reproductive phase, B. bacteriovorus grows as an elongated cell and undergoes binary or nonbinary fission, depending on the prey size. In this review, we discuss: (1) how the chromosome structure of B. bacteriovorus is remodeled during its life cycle; (2) how its chromosome replication dynamics depends on the proliferation mode; (3) how the initiation of chromosome replication is controlled during the life cycle, and (4) how chromosome replication is spatiotemporally coordinated with the proliferation program.


Introduction
F rançois J acob stated that "the dream of a bacterium is to become tw o bacteria" (J acob 1965 ).Most bacteria, including wellknown model organisms like Escherichia coli , Bacillus subtilis , and Caulobacter crescentus , use binary division to produce two daughter cells from one parent cell.Ho w ever, some so-called "nonclassical" bacteria use nonbinary fission, which is a more complex cell cycle mode wherein the chromosome replicates into three or more copies and the resulting multinucleoid elongated cell divides into three or more progeny cells.Examples of organisms in this group include Gram-positive bacteria, such as the antibioticpr oducing Streptom yces , and the pr edatory Gr am-negativ e bacterium, Bdellovibrio bacteriovorus , whic h exclusiv el y pr olifer ates inside other Gr am-negativ e bacteria (Fig. 1 ).Recent work demonstrated that B. bacteriovorus can reproduce through nonbinary or binary fission (Pl ąskowska et al. 2023 ).The transition between those two modes is correlated with the size of the prey (see Figs 3  and 4 ): predation on small cells, like Proteus mirabilis , results in the formation of two pr ogen y cells, while predation on larger prey cells results in the formation of three or more progeny cells (Pl ąskowska et al. 2023, Santin et al. 2023 ).
The life cycle of host-dependent (HD) B. bacteriovorus consists of two phases: a free-living nonre plicati ve attack phase and an intr acellular r epr oductiv e phase (for details see Fig. 1 ; for r e vie ws, see Sockett 2009, Rotem et al. 2014, Negus et al. 2017, Caulton and Lovering 2020, Laloux 2020, Lai et al. 2023 ).During the attack phase, the B. bacteriovorus cell is asymmetrically arranged with a flagellum at one cell pole and a pili at the other (the invasive pole).The flagellum facilitates prey-searching movement (Kaplan et al. 2023 ) and the pili are required for the predation (Evans et al. 2007, Kaplan et al. 2023 ).During the r epr oductiv e phase , B .bacteriovorus grows as an elongated cell (called a filament) in the periplasm of its prey.When the host's nutrients are exhausted, the filament, which contains two or more chromosomes, septates into the a ppr opriate e v en or odd number of unigenomic pr ogen y cells.
During the attack phase, the predatory cells are quite small (0.2-0.5 μm wide and 0.5-1.4μm long).Ho w e v er, unlike most other obligatory intracellular bacteria, B. bacteriovorus has not undergone a significant reduction of genome size, and thus possesses a r elativ el y lar ge c hr omosome (3.8 Mbp) (Rendulic et al. 2004 ).
Wild-type B. bacteriovorus is an obligate pr edator, but r ar e sa pr ophytic mutants can grow in the absence of prey and thus act as host-independent (HI) mutants (Figs 1 and 4 ) (Cotter and T homashow 1992a ).T hese HI m utants occur as irr egularl y sha ped filaments (Figs 1 and 4 ) and grow very slowly on rich media (Cotter and Thomashow 1992a , b ).
In this r e vie w, we pr esent the organization of the B. bacteriovorus c hr omosome, explor e how it changes during the life cycle, discuss the c hor eogr a phy of c hr omosome r eplication during the binary and nonbinary pr olifer ation of this pr edatory bacterium, and finall y compar e the r eplication c hor eogr a phy of B. bacteriovorus with those of bacteria that pr olifer ate by either binary or nonbinary modes.
F igure 1. Predatory life c ycle of B .bacteriovorus .T he life cycle of B. bacteriovorus consists of an the attack phase and a the pr olifer ation phase.During the attack phase, a free-swimming, mono-flagellated B. bacteriovorus cell searches for a prey cell and then attaches via its invasive pole to the prey's outer membrane .T he predatory cell then passes through the outer membrane and peptidoglycan layer into the prey's periplasm.Using a plethora of lytic enzymes, it changes the prey's cell shape (Lerner et al. 2012 ) and forms a bdelloplast (dead host cell).From this point, the proliferation phase begins .B .bacteriovorus digests the bdelloplast's cytosolic contents and reuses simple compounds to build its own structures.It grows as a filament, inside which c hr omosome m ultiplication and segr egation occur sim ultaneousl y.Chr omosome r eplication is usuall y initiated at the inv asiv e pole (gr een r eplisome), but initiation may occur at the opposite pole (blue replisome) in filaments dividing into five or more progeny cells (see text for details).When the prey cell's resources are exhausted, the B. bacteriovorus filament sync hr onousl y septates and forms an odd or e v en number of unigenomic pr ogen y cells.Finall y, the matur e daughter cells ar e r eleased fr om the bdelloplast into the envir onment.Rar el y occurring sa pr ophytic v ariants of B. bacteriovorus can exhibit host-independent (HI) growth.regions positioned near the inv asiv e and fla gellated poles, r espectiv el y.B. bacteriovorus under goes either binary fission (in small prey cells) or nonbinary fission (in big prey cells).During the adaptation phase, which follows the invasion of prey, the chromosome is still tightly compacted.At the beginning of the pr olifer ation phase, c hr omosome r eplication is initiated at the inv asiv e pole r egardless of the cell division mode, and the c hr omosome becomes decondensed (blue, solid line).During ongoing replication (dark blue dashed line), one of the duplicated oriC regions is segregated to the former flagellar pole and then the ter region relocates to mid-cell.In both binary and nonbinary dividing cells, Z-ring assembly begins prior to the termination of DNA replication.In a binary-dividing cell, DNA replication is terminated at the mid-cell.In filaments dividing by nonbinary fission, the second round of DNA replication is reinitiated from the chromosome located at the invasive pole; after segregation, one of the newly synthesized oriC copies (the third copy) colocalizes with the first ter in the middle of the filament.The (re)initiation of further rounds of chromosome multiplication also takes place at the inv asiv e pole (see text for details), and the copy number of oriC increases sequentially.At the final stage of replication, the Z-rings are async hr onousl y assembled and the filament is sync hr onousl y divided into an odd or e v en number of pr ogen y cells, eac h of whic h contains a compacted and polarized c hr omosome (ada pted fr om Kalje vi ć et al. 2021, Pl ąskowska et al. 2023 ).

T he c hr omosome of B. bacteriovorus m ust be extremely compacted
The observation that B. bacteriovorus possesses a r elativ el y lar ge c hr omosome r aises the following important questions: (1) Why does an obligate predator that proliferates in other bacteria have so many genes?(2) How is suc h a lar ge c hr omosome compacted in a tiny cell? and (3) How is the c hr omosome structur e sync hr onized with the unusual life cycle of B. bacteriovorus ?
Why does an obligate predator that proliferates in other bacteria have so many genes?B. bacteriovorus HD100 resembles most other bacteria in possessing a single cov alentl y closed c hr omosome.Ho w e v er, this c hr omosome contains many more genes (3584 predicted ORFs; 3.8 Mbp) (Rendulic et al. 2004 ) (Fig. 2 ) than other obligate intracellular parasites, such as Chlamydia (894-1052 ORFs; 1.0-2.0Mbp) (Stephens et al. 1998, Read et al. 2000, Sigalova et al. 2019, Stelzner et al. 2023 ) and Rickettsia (872-1512 ORFs; 1.2-1.3Mpb) (McLeod et al. 2004, Blanc et al. 2007, Gillespie et al. 2008, McGinn and Lamason 2021 ).This likel y r eflects that, compar ed to these other intr acellular pathogens , B .bacteriovorus exhibits a more complex life cycle (see Fig. 1 ) and acts as an obligate predator of other Gramnegative bacteria.T he B .bacteriovorus genome contains many diverse genes that are essential for its predatory beha viors , which include c hemotaxis, attac hment to the pr ey cell, entry into the pr ey's periplasm, ada ptation to the host bacterium and, most importantly, digestion of the bdelloplast content.To feed on its prey, B. bacteriovorus r equir es a wide r epertoir e of enzymes that can degr ade differ ent types of macr omolecules .Indeed, the B .bacteriovorus genome encodes a plethora of hydrolytic enzymes ( ∼300) (Rendulic et al. 2004 ) that degrade DN A, RN A, proteins , lipids , pol ysacc harides , and so on.T he pr oducts of hydr ol ysis ar e r eused for the predator's growth and replication, and up to 80% of de- graded host nucleic acids are incorporated into the DNA of B. bacteriovorus (Matin and Rittenberg 1972 ).B. bacteriovorus cannot endogenously synthesize certain amino acids; instead, these critical components pr esumabl y m ust be obtained from the products of pr ey pr otein hydr ol ysis .T her efor e , B .bacteriovorus possesses a lar ge r epertoir e of tr ansporters (mor e than 100) for tr ansporting amino acids , peptides , amines , and e v en nucleotides, whic h ar e r ar el y tr ansported in bacteria (Bar abote et al. 2007 ).The tr ansporters are also essential for HI m utants gr owing on media rich in amino acids .T he B .bacteriovorus genome further contains many genes encoding hypothetical proteins (1207 ORFs, comprising onethird of the total ORFs) (Rendulic et al. 2004 ).Additional work is needed to assign their biological roles.
In summary, unlike the genomes of other obligate intracellular parasites, that of B. bacteriovorus is sur prisingl y lar ge and contains an enormous number of genes encoding enzymes and transporters, that are indispensable for the particular life cycle of this predatory bacterium.

How is such a large chromosome compacted in a tiny cell?
During the attack phase, no DNA synthesis occurs, most genes ar e downr egulated (Karunker et al. 2013 ), and the predator utilizes ATP mainly to move in search of prey.In this phase of the life cycle, the predator cell can be as small as 0.3 μm wide and 0.8 μm long (Stolp andPetzold 1962 , Laloux 2020 ).Consequently, the c hr omosome must be more than 1500 times shorter than the naked (de void of pr oteins) linear DNA ( ∼1.3 mm).The c hr omosome is so tightly compacted within the small cell that it cannot be penetr ated e v en by small monomeric fluor escent pr oteins (Kalje vi ć et al. 2021 ).As observed for the chromosomes of C. crescentus and Vibrio cholerae , that of B. bacteriovorus is longitudinally arranged, with the origin of c hr omosome r eplication ( oriC ) and the terminus of replication ( ter ) positioned near the invasive and flagellated poles, r espectiv el y (Fig. 3 ) (Kaljevi ć et al. 2021 ).Transmission electron micr oscope and cryoelectr on tomogr a phy r e v ealed that the c hr omosome exhibits a spiral architecture and is organized in two twisted strands along the longitudinal axis of the cell (Butan et al. 2011, Kaplan et al. 2023 ).This spiral organization presumably facilitates  and  Bd3044) that contain putative histone-fold domains (Hocher et al. 2023 ).In vitro studies r e v ealed that, unlike a "classical" histone, the Bd0055 protein does not wrap DNA around itself but instead completely coats linear DN A b y binding end-to-end and thereby forming a nucleohistone filament (Hocher et al. 2023, Hu et al. 2023 ).The Bd0055 gene is highl y expr essed mainl y during the pr olifer ation phase and ranks among the top 6% of genes with the highest expr ession le v el (Hoc her et al. 2023 , The Bdellovibrio viewer: B. bacteriovorus transcriptome 2023 ).Importantly, the Bd0055 and Bd3044 genes are essential throughout the B. bacteriovorus life cycle, as their deletion has been shown to be lethal.Ho w e v er, their functions and impact on DNA organization have yet to be elucidated in vivo .Prior to this work, histones had not been identified in bacteria were commonly believed to be exclusive to eukaryotes and archaea.
Similar to the situation in other bacteria, the global structure of the B. bacteriovorus c hr omosome a ppears to be maintained by con-densins, such as SMC, which spatially and dynamically organizes bacterial c hr omosomes b y extruding DN A into large loops.In the B .bacteriovorus genome , the Bd1158 gene was identified to encode the SMC protein (T he Bdello vibrio viewer: B .bacteriovorus transcriptome 2023 ).In addition to typical bacterial topoisomerases, such as T opA (Bd0964), T op II (Bd2865), and Top IV (Bd0005), the B. bacteriovorus genome encodes topoisomerase VI (Bd2864) (The Bdello vibrio viewer: B .bacteriovorus tr anscriptome 2023 ), whic h is an archaeal-and plant-type topoisomerase (Forterre et al. 2007 ).
T hus , a diverse range of proteins, including unusual ones such as histones and an archaeal/plant topoisomerase, is presumably involved in the chromosomal architecture of B. bacteriovorus .Notabl y, c hr omosomes m ust also be extensiv el y compacted during sporulation in B. subtilis and Streptomyces to fit into the tin y spor e compartment (Khanna et al. 2020, Szafran et al. 2020 ).Ho w e v er, our understanding of c hr omosome or ganization during sporulation also remains incomplete.

How is the chromosome structure synchronized with the unusual life cycle of B. bacteriovorus ?
In the attack phase, the B. bacteriovorus c hr omosome is highly compacted; during the pr olifer ation phase, the DNA must be accessible to proteins involved in essential cellular processes, such as DNA replication, chromosome segregation, and transcription (Fig. 3 ).Unlike the situation in eukaryotic organisms, these pr ocesses occur sim ultaneousl y in bacteria.Consequentl y, the B. bacteriovorus c hr omosome under goes dynamic mor phological changes during proliferation, particularly at the beginning of this phase.After c hr omosome r eplication is initiated, the c hr omosome decondenses and remains in this state until the elongated cell is read y to di vide (Kaljevi ć et al. 2021 ).The decondensation of the c hr omosome has been suggested to be triggered first by replication initiation and further stimulated by the pr ogr ession of r eplication forks (Kaljevi ć et al. 2021 ).Importantly, the length of the elongated cell can exceed 20 times its initial length.Daughter c hr omosomes occupy most of the filament and do not show obvious signs of compaction (Kaljevi ć et al. 2021 ).Before the multiple filament constrictions undergo closure, the chromosomes are reorganized into compact structures to ensure that each tiny daughter cell r eceiv es one copy.This resembles the sporulation of the nonbinary dividing bacterium, Streptomyces , when dozens of chromosomes must be segregated and condensed to ensure the proper formation of unigenomic spores (see Fig. 4 ) (J akimo wicz and van Wezel 2012 ).
Notably, in bacteria, the composition and le v els of NAPs can change during the life cycle, leading to global alterations in the c hr omosome (Amemiya et al. 2021 ).NAPs, a part fr om their arc hitectural functions, also crucially contribute to cellular processes, suc h as DNA r eplication (e.g.HU, IHF, and Fis) and transcription (e.g.H-NS) (Hołówka and Zakrzewska-Czerwi ńska 2020 ).Therefore, it is plausible that, during the B. bacteriovorus life cycle, certain NAPs may not only shape the chromosome architecture but also regulate DNA replication and modulate the transcriptional profile of the cell.
In summary, the B. bacteriovorus c hr omosome under goes dr amatic structural changes as the predatory cell progresses through its life cycle.Further r esearc h is needed to r e v eal the r ele v ant pr oteins and mec hanisms in the c hr omosomal compaction of B. bacteriovorus .The application of techniques such as chromosome conformation ca ptur e (Hi-C) and quantitativ e high-r esolution micr oscopy can pr ovide insights into the thr ee-dimensional struc-ture and life cycle-related changes of the B. bacteriovorus chromosome.

Big and well-fed hosts are the predator's dream home
B. bacteriovorus exhibits an extraordinary ability to adapt to various Gr am-negativ e hosts (Markelov a 2010, Ric hards et al. 2012, Loozen et al. 2015, Shatzkes et al. 2016, Pl ąskowska et al. 2023 ).B. bacteriovorus can pr olifer ate within a prey cell of any size, but the prey cell size is important: In a small prey like P. mirabilis , B. bacteriovorus forms only two daughter cells; in a large prey like exponentiall y gr owing E. coli , it can produce dozens of offspring (Kaljevi ć et al. 2021, Pl ąskowska et al. 2023, Santin et al. 2023 ) (Figs 3 and 4 ).Inter estingl y, while the minim um number of formed daughter cells is obvious (two), the maximum number has not been conclusiv el y determined.Up to 17 offspring were observed (Santin et al. 2023 ) in E. coli , but it remains possible that an e v en lar ger pr ey could yield an e v en gr eater number of daughter cells.Stolp observed 20-30 cells in Aquaspirillum serpens (Stolp 1967, Jurkevitch 2006 ).
The pr olifer ation phase of B. bacteriovorus must be pr ecisel y adjusted to the size and "quality" of the prey cell.Recent studies demonstrated that the duration of B. bacteriovorus proliferation depends on: (1) the size of the prey cell, which determines the number of predator offspring (Pl ąskowska et al. 2023, Santin et al. 2023 ); and (2) the nutritional quality of the prey cell, which affects the rate of predator elongation (Santin et al. 2023 ).It is worth noting that, in contrast to the duration of later cell cycle stages (e.g.cell division), the duration of DN A replication, kno wn as the S-phase, is primarily influenced by the size and nutritional quality of the prey cell (Pl ąskowska et al. 2023, Santin et al. 2023 ).In lar ger pr ey cells, mor e copies of the c hr omosome ar e synthesized, resulting in a longer period of DNA synthesis (see below).In "poorl y nourished" pr ey cells (gr own on minimal medium prior infection), the rate of B. bacteriovorus DNA synthesis is lo w er (and the S-phase is longer) than that in "well-fed" prey cells .T herefore , the spatiotemporal coordination of basic cell cycle processes, particularly that of chromosome replication with the proliferation program of B. bacteriovorus , is necessary for the effective exploitation of resources and space within an inhabited prey cell (Santin et al. 2023 ).
Rar el y, B. bacteriovorus r equir es two independent pr ey cells to complete its life cycle.In this scenario, the undivided filament exits the bdelloplast and enters the second prey cell.After replication is completed, the filament undergoes septation, and the pr ogen y cells leave the second bdelloplast.The noncanonical life cycle may be attributed to a limited assessment of the prey's resources and/or size (Makowski et al. 2019 ).

Choreography of chromosome replication during the reproducti v e phase
DNA replication is a complex and energy-consuming process that r equir es pr ecise contr ol.Acr oss the thr ee domains of life, c hr omosome r eplication is lar gel y r egulated at the initiation step, as a means to pr e v ent unnecessary ener gy loss (Bo y e et al. 2000, Sclafani and Holzen 2007, Zakrzewska-Czerwi ńska et al. 2007 ).In B. bacteriovorus , c hr omosome r eplication occurs exclusiv el y during the intracellular proliferation phase .T hus , the initiation step must be strictly controlled to ensure that DNA replication commences at an appropriate location and time.

(Re)initiation of chromosome replication
The k e y elements involv ed in the initiation of c hr omosome r eplication in B. bacteriovorus , namely the oriC region and the replication initiator pr otein, DnaA, hav e been c har acterized (Mak owski et al. 2016 ).The oriC region of B. bacteriovorus exhibits a typical eubacterial oriC organization comprising DnaA protein binding motifs and an AT-rich DNA unwinding element (DUE) (Makowski et al. 2016 ) (Fig. 2 ).Similar to the situation in other bacteria, binding of the B. bacteriovorus DnaA protein to oriC triggers this region to unwind (Makowski et al. 2016 ) and thereby provide the entry site for a m ultipr otein r eplisome mac hinery that consists of a helicase, an RNA primase, and a DNA pol ymer ase III holoenzyme (Leonard andGrimwade 2015 , Wola ński et al. 2015 ).
The initiation of bacterial c hr omosome r eplication is mainl y regulated by controlling the activity and availability of oriC and/or DnaA.Ho w e v er, r esearc hers hav e not yet identified any regulator of c hr omosome r eplication initiation in B .bacteriovorus .T he activity of B. bacteriovorus DnaA pr esumabl y depends on its nucleotidebound state (ATP versus ADP), as seen in other bacteria (Keyamura et al. 2007 ).RNA-seq analysis (Karunker et al. 2013 ) indicated that dnaA and other genes of the Bdellovibrio transcriptome involved in c hr omosome r eplication ar e expr essed in the pr olifer ation phase but nearly or completely silenced in the attack phase .T his gene silencing may reflect the tight compaction of the c hr omosome during the attack phase, which is likely to block access to the oriC r egion and pr omoters , including those of genes in volv ed in c hr omosome replication.
Chr omosome r eplication does not begin immediately after bdelloplast formation, indicating that B. bacteriovorus r equir es some time to adapt to the prey en vironment (periplasm).T he c hr omosome m ust first under go decompaction to enable various DNA tr ansactions, suc h as DNA r eplication and tr anscription.Real-time micr oscopic observ ation using fluor escent r eporter strains of B. bacteriovorus (Makowski et al. 2019, Pl ąskowska et al. 2023 ) demonstrated that the first replisome appears several dozen minutes ( ∼45 min) after bdelloplast formation.Howe v er, ther e ar e exceptions: when the daughter cell leaves the bdelloplast and immediately attacks the next prey, chromosome replication starts earlier (the r eplisome a ppears ∼23 min after bdelloplast formation).This suggests that certain protein(s) involved in c hr omosome r eplication, particularl y DnaA, ar e not full y degr aded (Mak owski et al. 2019 ).We cannot exclude the possibility that, as seen in C. crescentus , the DnaA protein undergoes specific pr oteol ysis during the tr ansition fr om the gr owth phase to the attack phase.Ho w ever, a specific protease like Lon, which was found in C. crescentus (Jonas et al. 2013 ), has not yet been identified in B. bacteriovorus .
In B. bacteriovorus , the first round of c hr omosome r eplication is initiated at the inv asiv e pole (Figs 1 and 3 ).The assembly of replisomes and the presence of the ParB complex (Kaljevi ć et al. 2021, Pl ąskowska et al. 2023 ) at the cell pole suggest that, similar to the situations in other asymmetric bacterial cells (e.g. C. crescentus and V. cholerae ) (Bowman et al. 2008, Yamaichi et al. 2012 ), the B. bacteriovorus c hr omosome is likel y anc hor ed to the cell pole (presumabl y thr ough the oriC r egion) via one or more pole-localized proteins.It has been postulated that in B. bacteriovorus , the coiledcoil tropom yosin-lik e protein, Di vIVA, together with the filamentforming protein, bactofilin (Laloux 2020, Milner et al. 2020 ), may be involved in maintaining cell polarity.In a picall y extending cells, such as Corynebacterium , Mycobacterium , and Streptomyces , DivIVA is the main component of the polar complex (Flärdh et al. 2012 , Donov an and Br amkamp 2014 ).We cannot exclude the possibility that DivIVA and/or bactofilin are targets for yet unidentified proteins involved in anchoring the B. bacteriovorus chromosome at the cell pole.Ov er all, it r emains unkno wn ho w polarity is ac hie v ed in this predatory bacterium.
Observation of further replication rounds has indicated that r einitiation(s) usuall y occurs at the inv asiv e pole .T hus , the firing of subsequent replication rounds is an asynchronous process in whic h DNA r eplication is sequentiall y r einitiated fr om the c hr omosome located at the inv asiv e pole, r ather than fr om other r eplicated c hr omosomes.In long filaments (fr om whic h fiv e or mor e pr ogen y cells are formed), ho w ever, DN A synthesis may be initiated at the opposite pole in the late stages of c hr omosome m ultiplication (Fig. 1 ).In this case, the former flagellar pole must under go extensiv e r econstruction to become an inv asiv e pole that anchors a daughter chromosome, which can then undergo initiation of DNA synthesis.It is difficult to undertake microscopic observation of later stages of chromosome multiplication in filaments dividing into more than five progeny cells, because the long filaments ar e fr equentl y twisted in the pr ey (Mak owski et al. 2019, Pl ąskowska et al. 2023 ).
In conclusion, the "async hr onous mode" of r eplication r einitiation explains why filaments can contain an odd or e v en number of c hr omosome copies .T he (r e)initiation of DNA r eplication fr om the c hr omosome anc hor ed to the inv asiv e pole pr esumabl y ensur es the pr ecise spatial contr ol of DNA r eplication in B. bacteriovorus , such that the initiation step is a crucial cell cycle c hec kpoint in this bacterium.Under nutritional stress (when the prey's r esources ar e exhausted), it seems r easonable to assume that one or mor e r egulatory mec hanisms) would be activated to pr e v ent the next initiation of DNA replication and control further proliferation of the filament.In C. crescentus , the global transcription factor, CtrA, inhibits replication initiation by binding to the oriC region and additionall y contr ols the expr ession of genes encoding r egulators involved in cell cycle pr ogr ession (Ryan et al. 2002 ).Howe v er, no regulatory mechanism(s) capable of preventing or triggering r eplication (r e)initiation has yet been reported in B. bacteriovorus .Furthermore, it has been postulated that, during the adaptation phase, one or more specific cues reflecting prey quality will modulate c hr omosome r e plication and the subsequent ste ps of B. bacteriovorus pr olifer ation (Rotem et al. 2015 ).Further studies ar e r equired to identify such cues and their targets, which would represent a cell-cycle c hec kpoint that would be expected to be universal across different prey cell types.

Chromosome multiplication
The spatiotempor al c hor eogr a phy of the next step in c hr omosome re plication (i.e.elongation) de pends on the pr olifer ation mode and number of offspring (Fig. 3 ).Bdellovibrio bacteriovorus cells that divide by binary fission provide a simple model for studying chromosome replication dynamics (Fig. 3 ): From observation of B. bacteriovorus dividing into two daughter cells, the rate of DNA synthesis can be determined.Based on the C-period (the time r equir ed to replicate the B. bacteriovorus chromosome; e.g.144 min for cells growing in P. mirabilis ) (Pl ąskowska et al. 2023 ) and genome size (3.8Mbp) (Rendulic et al. 2004 ), the DNA synthesis rate is estimated to be around 220 nucleotides per second (nt/s), which is a ppr oximatel y thr ee to four times slo w er than that of E. coli (600-1000 nt/s) (Fijalkowska et al. 2012 ).This reduced replication rate might reflect the limited availability of nutrients, particularly nucleotides .T he activity of B. bacteriovorus DNA pol ymer ase III is not likely to be the rate-limiting factor, since the subunits of this holoenzyme exhibit high homology with those of E. coli (Makowski et al. 2019 ).
In binary-dividing B. bacteriovorus , after the initiation of DNA r eplication, the r eplication forks split; one fork remains at the inv asiv e pole, while the other migrates to w ar d the opposite pole.
Both forks e v entuall y mov e to the mid-cell, wher e the r eplication is terminated, resulting in the localization of both ter sites in this region of the cell (Fig. 3 ).Meanwhile, one of the ne wl y r eplicated oriC r egions migr ates to w ar d the former fla gellar pole, whic h becomes an inv asiv e pole.After cell division, two daughter cells are formed; each contains a compacted and polarized chromosome with the oriC region located at the invasive pole (Kaljevi ć et al. 2021, Pl ąskowska et al. 2023 ) (Fig. 3 ).
When B. bacteriovorus divides through nonbinary fission, multiple replisomes (three or more) can be observed within a single filament (Figs 1 and 3 ) indicating that the next round of replication is (r e)initiated befor e the pr e vious r ound terminates .T he duration of the S-phase is not dir ectl y pr oportional to the number of pr ogen y cells: in filaments dividing into three or four daughter cells , e .g. the S-phases last ∼ 171 min or 187 min, r espectiv el y (Pl ąsk owska et al. 2023 ).This further confirms the occurrence of ov erla pping r eplication rounds , i.e .multifork replication.The strategy of multifork r eplication not onl y a ppears in fast-gr owing unigenomic bacteria such as E. coli and B. subtilis , but has also been observed in slowgrowing bacteria such as Mycobacterium smegmatis (Trojanowski et al. 2017 ) and multigenomic bacteria like Corynebacterium glutamicum (Böhm et al. 2017 ).
In HI m utants, the pr ocess of c hr omosome r eplication is supposed to be less strictly controlled; HI cells differ in size , shape , and pr esumabl y contain a differ ent number of c hr omosome copies.Ho w e v er, to date, nothing is known about c hr omosome multiplication and septation within the filaments of HI mutants of B. bacteriovorus .

Coordination of chromosome replication with chromosome segregation and septation
The coordination of k e y cell-cycle pr ocesses, suc h as that of c hr omosome replication with segregation and cell division, is crucial for the ability of a bacterial cell to produce viable offspring that contain the full complement of hereditary information.Bacteria hav e e volv ed v arious mec hanisms to ensur e that genetic information is faithfull y tr ansmitted to their offspring.These mechanisms ar e r elativ el y w ell-kno wn in model bacteria such as E. coli , B. subtilis , and C. crescentus , which divide by binary fission (Eswara and Ramamurthi 2017 ).Ho w ever, less is known about the mechanisms that coordinate crucial cell cycle e v ents in organisms dividing by nonbinary fission.
In bacteria, c hr omosome r eplication initiation m ust be closel y coordinated with the segregation of one or mor e ne wl y duplicated oriC regions.In most bacteria, this process is mediated by a ParAB S system consisting of ParA (an ATPase), ParB (binding to the parS sequence), and a centr omer e-like sequence called parS (Kawalek et al. 2023 ).The ParAB S system has been identified in B. bacteriovorus and other nonbinary dividing bacteria, including Streptomyces (Kim et al. 2000 ).In B. bacteriovorus , two parS sequences were r ecentl y identified near the oriC region (Fig. 2 ) and shown to be bound in vitro by the P arB pr otein (Kalje vi ć et al. 2023 ).Micr oscopic observ ations r e v ealed that shortl y after the initiation of the first r eplication r ound, one of the ne wl y synthesized oriC r egions (visible as ParB complexes) is segregated to the former flagellar pole (Fig. 3 ) (Kaljevi ć et al. 2021, Pl ąskowska et al. 2023 ).As chromosome m ultiplication pr ogr esses, the number of P arB segr egation complexes, whic h corr esponds to the number of r eleased daughter cells, gr aduall y incr eases as ne w copies of the oriC r egion ar e synthesized.Befor e septation, c hr omosomes m ust be compacted and uniformly distributed along the filament to ensure that each daughter cell r eceiv es a single copy of the c hr omosome.P arA is pr esumabl y necessary for the proper assembly of ParB complexes and, together with other yet-unidentified proteins, contributes to the precise positioning of the c hr omosome copies.Ho w e v er, the r ole of P arA in c hr omosome segr egation has yet to be elucidated in B. bacteriovorus .Recent studies r e v ealed that the three elements of the ParAB S system experience m ultilayer ed r egulation at the le v els of parA and parB gene tr anscription, P arA and P arB pr otein le v els, and parS accessibility.Mor eov er, the r atio between P arA and P arB (P arA:P arB) fluctuates during the cell cycle and is presumed to be crucial both for the regulation of segrosome formation and for cell cycle pr ogr ession (Kalje vi ć et al. 2023 ).
At the end of the pr olifer ation phase , B .bacteriovorus undergoes m ultiple sync hr onized divisions (Fenton et al. 2010 ) to convert a m ultic hr omosome filament into unigenomic daughter cells.In bacteria, the assembly of the division machinery is orc hestr ated by the FtsZ protein, a bacterial tubulin homolog that polymerizes into a Z-ring at eac h futur e site of cell division (Margolin 2005 , Cameron andMargolin 2023 ).Investigation of the septation pr ocess r e v ealed that two or mor e FtsZ rings ar e formed inside a single elongated cell during predation on large prey.Interestingl y, the assembl y of FtsZ rings was found to be async hr onous, in contrast to their sync hr onous disassembl y.Notabl y, in both binary and nonbinary dividing cells, FtsZ ring assembly begins before DNA synthesis ends.In filaments undergoing nonbinary fission, the multiple FtsZ rings are assembled sequentially (Fig. 3 ) (Pl ąskowska et al. 2023 ).Howe v er, the factors responsible for determining the timing and positioning of Z-rings in B. bacteriovorus remain a mystery.In this bacterium, a replication checkpoint may act to coordinate c hr omosome m ultiplication with the cycle progr ession, suc h as by tuning FtsZ ring placement.In contrast to the situation in B. bacteriovorus , the FtsZ rings of Streptom yces ar e sync hr onousl y assembled (Gr antc har ov a et al. 2005 , J akimo wicz and van Wezel 2012 ) after DNA replication ends (Szafran et al. 2021 ).
Upon replication termination, daughter chromosomes must be physicall y separ ated and tr anslocated befor e filament division is completed.Similar to the situation in other bacteria possessing circular c hr omosomes, dimeric c hr omosomes may form during DNA replication in B. bacteriovorus filaments.This problem is expected to be e v en mor e pr onounced than in other bacteria, as within a single filament, multiple chromosomes are synthesized.
In model organisms such as E. coli or B. subtilis , dimers are resolved by XerC and XerD, two tyr osine r ecombinases that target the 28-nucleotide motif ( dif ) associated with the c hr omosome's re plication termin us .In the B .bacteriovorus genome , the dif site (see Fig. 2 ) and xerCD genes (Bd3064, Bd2300) have also been identified through in silico analysis (Carnoy and Roten 2009 ).Finally, multiple c hr omosomes m ust be tr anslocated a wa y fr om the futur e septum sites to pr e v ent them from becoming trapped or guillotined by a forming septum.In B. bacteriovorus , a DNA translocase called FtsK pr esumabl y coordinates c hr omosome tr anslocation with cell division, as a ftsK gene (Bd0041) was identified in this bacterium (Heidari Tajabadi et al. 2017 ).
Bacteria hav e e volv ed both positiv e and negativ e mec hanisms to spatially and temporally control the polymerization of FtsZ into Z-rings .T he negative regulatory mechanisms include the Min system (in E. coli and B. subtilis ) and nucleoid occlusion protection (in E. coli and B. subtilis ), while the positiv e mec hanisms include the PomXYZ (in Myxococcus xanthus ) and MapZ (in Streptococcus pneumoniae ) proteins (Mahone and Goley 2020 ).Ho w e v er, no suc h system has yet been identified in B. bacteriovorus .It is tempting to speculate that B. bacteriovorus uses one or more of its own mechanisms to pr ecisel y contr ol the tempor al and spatial placement of FtsZ rings.Such a mechanism would prevent the formation of offspring with an extra or missing/guillotined c hr omosome.We cannot exclude the possibility that one or more proteins that interact with ParA (or ParB) and/or contribute to cell division contribute to coordinating c hr omosome r eplication and segr egation with filament septation.In C. crescentus , MipZ has been suggested to coordinate these processes by interacting with ParB at the cell poles and dir ectl y inhibiting the polymerization of FtsZ into Zrings.MipZ forms a gradient within the cell, with its highest concentration at the poles and lo w est at the midcell (Thanbichler and Sha pir o 2006 , Corr ales-Guerr er o et al. 2022 ).

Conclusions and future directions
Although B. bacteriovorus was discov er ed ov er 60 years ago (Stolp andPetzold 1962 , Stolp andStarr 1963 ), this predatory bacterium onl y r elativ el y r ecentl y emer ged as a nov el and intriguing model organism.Due to its peculiar life cycle and bimodal pr olifer ation (Figs 3 and 4 ), B. bacteriovorus r epr esents a noncanonical yet attractive model for studying novel aspects of bacterial cell biology, including c hr omosome or ganization and the c hor eogr a phy of c hr omosome replication.In contrast to model organisms such as E. coli and C. crescentus , but similar to Streptomyces , this bacterium significantl y c hanges its cell size during the life cycle (Fig. 4 ).Consequentl y, the c hr omosomes of B. bacteriovorus m ust be pr ofoundl y r emodeled: they ar e highl y compacted during the attack phase and septation, but become more relaxed during the proliferation phase.A r ecentl y identified histone (Bd0055) in this predatory bacterium appears to be a promising candidate for contributing to the efficient compaction of bacterial c hr omatin (Hoc her et al. 2023 ).It is particularly noteworthy that the mechanism used by this novel bacterial histone to compact DNA differs from that used by eukaryotic histones (as mentioned earlier).It cannot be excluded that this protein, along with one or more other yet unidentified proteins , ma y help orchestrate the remodeling of bacterial chromatin.Further studies are needed to identify the proteins (including regulators) and mechanisms responsible for the extensive changes seen in chromosome organization during the life cycle of B. bacteriovorus .Chromosome decompaction begins upon the initiation of DNA replication (Kaljevi ć et al. 2021 ), suggesting that a c hec kpoint may coordinate c hr omosome or ganization and replication with cell cycle pr ogr ession.
Similarly to the situation in other asymmetric bacteria, such as C. crescentus (Fig. 4 ) and V. cholerae , the oriC region of B. bacteriovorus is located at a cell pole (the inv asiv e pole).In this predatory bacterium, a region near the origin of replication must be anchored at the pole by a yet unknown protein complex.In nonbinary proliferating filaments, each subsequent round of chromosome replication is gener all y initiated fr om the inv asiv e pole-anc hor ed oriC ; the exception to this is found in filaments dividing into five or mor e pr ogen y cells, in whic h the opposite pole can becomes the inv asiv e pole at a late stage of chromosome multiplication.Given this async hr onous mode of replication, the mechanisms responsible for controlling replication initiation are expected to be more intricate than those described for a model organism such as E. coli .Since the DNA replication of B. bacteriovorus starts specificall y fr om the inv asiv e pole-localized c hr omosome, it can be assumed that only the oriC attached to this cell pole is licensed for firing, while other oriC regions remain re plicati ve silent ("dormant").Similarl y, in eukaryotes, onl y licensed origins can initiate replication (Marks et al. 2017 ).T hus , in B .bacteriovorus , the initia-tion of c hr omosome r eplication is both tempor aril y and spatiall y regulated.These unique features of replication initiation choreogr a phy r aise inter esting questions, suc h as: (1) Is ther e an oriC licensing system?(2) Why do potential replication oriC regions remain inactive?(3) What mechanisms control the licensing and dormancy of oriC regions?and (4) How are oriC licensing and DNA r eplication coordinated/r egulated during the pr olifer ation of this predatory bacterium?
Many other interesting questions remain open regarding the cell biology of B. bacteriovorus .For example, we need to understand when and how the predatory cell prevents the next DNA r eplication r ound fr om being trigger ed in adv ance, suc h that all cell cycle e v ents ar e finished befor e the av ailable pr ey r esources ar e completel y exploited.The HI mutant of B. bacteriovorus is also a challenging model, and further studies are needed to delineate how the c hr omosomes of HI m utants ar e or ganized and how these m utants r eplicate.

Figure 2 .
Figure 2. Localization of the origin of c hr omosome r eplication (oriC), the parA and parB genes, and the parS sequence on the B. bacteriovorus c hr omosome .T he directions of triangles within the oriC region represent the orientations of particular DnaA boxes bound by the DnaA protein (Makowski et al. 2016 ).The diamond represents the palindromic parS sequence bound by the ParB protein (Kaljevi ć et al. 2023 ).The distance between the dnaA (encoding the DnaA protein) and bd3908 (encoding a putative rRNA methyltransferase) genes is 6466 bp.Ter and dif indicate the replication termination region and the site that ensures the resolution of chromosome dimers, respectively (Carnoy and Roten 2009 , Kaljevi ć et al. 2021 ).The figure is not drawn to scale.

Figure 3 .
Figure 3. Async hr onous c hor eogr a phy of the initiation of c hr omosome r eplication in B. bacteriovorus dividing by binary or nonbinary fission.During the attack phase, the B. bacteriovorus c hr omosome is highly compacted (y ello w, solid line) and longitudinally arranged, with the oriC (violet) and ter (green)regions positioned near the inv asiv e and fla gellated poles, r espectiv el y.B. bacteriovorus under goes either binary fission (in small prey cells) or nonbinary fission (in big prey cells).During the adaptation phase, which follows the invasion of prey, the chromosome is still tightly compacted.At the beginning of the pr olifer ation phase, c hr omosome r eplication is initiated at the inv asiv e pole r egardless of the cell division mode, and the c hr omosome becomes decondensed (blue, solid line).During ongoing replication (dark blue dashed line), one of the duplicated oriC regions is segregated to the former flagellar pole and then the ter region relocates to mid-cell.In both binary and nonbinary dividing cells, Z-ring assembly begins prior to the termination of DNA replication.In a binary-dividing cell, DNA replication is terminated at the mid-cell.In filaments dividing by nonbinary fission, the second round of DNA replication is reinitiated from the chromosome located at the invasive pole; after segregation, one of the newly synthesized oriC copies (the third copy) colocalizes with the first ter in the middle of the filament.The (re)initiation of further rounds of chromosome multiplication also takes place at the inv asiv e pole (see text for details), and the copy number of oriC increases sequentially.At the final stage of replication, the Z-rings are async hr onousl y assembled and the filament is sync hr onousl y divided into an odd or e v en number of pr ogen y cells, eac h of whic h contains a compacted and polarized c hr omosome (ada pted fr omKalje vi ć et al. 2021 , Pl ąskowska et al. 2023  ).

Figure 4 .
Figure 4. Localization of oriC region(s) in bacteria undergoing binary and/or nonbinary fission (see text for details).