Chromosome Dynamics in Bacteria: Triggering Replication at the Opposite Location and Segregation in the Opposite Direction

ABSTRACT Maintaining the integrity of the genome is essential to cell survival. In the bacterium Caulobacter crescentus, the single circular chromosome exhibits a specific orientation in the cell, with the replication origin (ori) residing at the pole of the cell bearing a stalk. Upon initiation of replication, the duplicated centromere-like region parS and ori move rapidly to the opposite pole where parS is captured by a microdomain hosting a unique set of proteins that contribute to the identity of progeny cells. Many questions remain as to how this organization is maintained. In this study, we constructed strains of Caulobacter in which ori and the parS centromere can be induced to move to the opposite cell pole in the absence of chromosome replication, allowing us to ask whether once these chromosomal foci were positioned at the wrong pole, replication initiation and chromosome segregation can proceed in the opposite orientation. Our data reveal that DnaA can initiate replication and ParA can orchestrate segregation from either cell pole. The cell reconstructs the organization of its ParA gradient in the opposite orientation to segregate one replicated centromere from the new pole toward the stalked pole (i.e., opposite direction), while displaying no detectable viability defects. Thus, the unique polar microdomains exhibit remarkable flexibility in serving as a platform for directional chromosome segregation along the long axis of the cell. IMPORTANCE Bacteria can accomplish surprising levels of organization in the absence of membrane organelles by constructing subcellular asymmetric protein gradients. These gradients are composed of regulators that can either trigger or inhibit cell cycle events from distinct cell poles. In Caulobacter crescentus, the onset of chromosome replication and segregation from the stalked pole are regulated by asymmetric protein gradients. We show that the activators of chromosome replication and segregation are not restricted to the stalked pole and that their organization and directionality can be flipped in orientation. Our results also indicate that the subcellular location of key chromosomal loci play important roles in the establishment of the asymmetric organization of cell cycle regulators.

Download Full-text

Choreography of the Mycobacterium Replication Machinery during the Cell Cycle

mBio ◽

10.1128/mbio.02125-14 ◽

2015 ◽

Vol 6 (1) ◽

Cited By ~ 39

Author(s):

Damian Trojanowski ◽

Katarzyna Ginda ◽

Monika Pióro ◽

Joanna Hołówka ◽

Partycja Skut ◽

...

Keyword(s):

Cell Cycle ◽

Chromosome Segregation ◽

Mycobacterium Smegmatis ◽

Chromosome Replication ◽

Specific Cell ◽

Replication Machinery ◽

Content Type ◽

Chromosome Dynamics ◽

Mycobacterial Cell ◽

Slow Growing

ABSTRACT It has recently been demonstrated that bacterial chromosomes are highly organized, with specific positioning of the replication initiation region. Moreover, the positioning of the replication machinery (replisome) has been shown to be variable and dependent on species-specific cell cycle features. Here, we analyzed replisome positions in Mycobacterium smegmatis, a slow-growing bacterium that exhibits characteristic asymmetric polar cell extension. Time-lapse fluorescence microscopy analyses revealed that the replisome is slightly off-center in mycobacterial cells, a feature that is likely correlated with the asymmetric growth of Mycobacterium cell poles. Estimates of the timing of chromosome replication in relation to the cell cycle, as well as cell division and chromosome segregation events, revealed that chromosomal origin-of-replication (oriC) regions segregate soon after the start of replication. Moreover, our data demonstrate that organization of the chromosome by ParB determines the replisome choreography. IMPORTANCE Despite significant progress in elucidating the basic processes of bacterial chromosome replication and segregation, understanding of chromosome dynamics during the mycobacterial cell cycle remains incomplete. Here, we provide in vivo experimental evidence that replisomes in Mycobacterium smegmatis are highly dynamic, frequently splitting into two distinct replication forks. However, unlike in Escherichia coli, the forks do not segregate toward opposite cell poles but remain in relatively close proximity. In addition, we show that replication cycles do not overlap. Finally, our data suggest that ParB participates in the positioning of newly born replisomes in M. smegmatis cells. The present results broaden our understanding of chromosome segregation in slow-growing bacteria. In view of the complexity of the mycobacterial cell cycle, especially for pathogenic representatives of the genus, understanding the mechanisms and factors that affect chromosome dynamics will facilitate the identification of novel antimicrobial factors.

Download Full-text

Chromosome dynamics in bacteria: triggering replication at opposite location and segregation in opposite direction

10.1101/521781 ◽

2019 ◽

Author(s):

Ady B. Meléndez ◽

Inoka P. Menikpurage ◽

Paola E. Mera

Keyword(s):

Opposite Direction ◽

Caulobacter Crescentus ◽

Chromosome Replication ◽

Replication Initiation ◽

Opposite Orientation ◽

Dividing Cells ◽

Bacterial Replication ◽

High Flexibility ◽

Replication Initiator ◽

Chromosomal Loci

ABSTRACTThe accurate onset of chromosome replication and segregation are fundamental for the survival of the cell. In bacteria, regulation of chromosome replication lies primarily at the initiation step. The bacterial replication initiator DnaA recognizes the origin of replication (ori) and opens this double stranded site allowing for the assembly of the DNA replication machinery. Following the onset of replication initiation, the partitioning protein ParA triggers the onset of chromosome segregation by direct interactions with ParB-bound to the centromere. The subcellular organization of ori and centromere are maintained after the completion of each cell cycle. It remains unclear what triggers the onset of these key chromosome regulators DnaA and ParA. One potential scenario is that the microenvironment of where the onset of replication and segregation take place hosts the regulators that trigger the activity of DnaA and ParA. In order to address this, we analyzed whether the activity of DnaA and ParA are restricted to only one site within the cell. In non-dividing cells of the alpha proteobacterium Caulobacter crescentus, ori and centromere are found near the stalked pole. To test DnaA’s ability to initiate replication away from the stalked pole, we engineered a strain where movement of ori was induced in the absence of chromosome replication. Our data show that DnaA can initiate replication of the chromosome independently of the subcellular localization of ori. Furthermore, we discovered that the partitioning protein ParA was functional and could segregate the replicated centromere in the opposite direction from the new pole toward the stalked pole. We showed that the organization of the ParA gradient can be completely reconstructed in the opposite orientation by rearranging the location of the centromere. Our data reveal the high flexibility of the machineries that trigger the onset of chromosome replication and segregation in bacteria. Our work also provides insights into the coordination between replication and segregation with the cellular organization of specific chromosomal loci.

Download Full-text

Multilayered control of chromosome replication in Caulobacter crescentus

Biochemical Society Transactions ◽

10.1042/bst20180460 ◽

2019 ◽

Vol 47 (1) ◽

pp. 187-196 ◽

Cited By ~ 3

Author(s):

Antonio Frandi ◽

Justine Collier

Keyword(s):

Cell Cycle ◽

Dna Replication ◽

Caulobacter Crescentus ◽

Response Regulator ◽

Classical Model ◽

Optimal Growth ◽

Chromosome Replication ◽

S Phase ◽

Circular Chromosome ◽

Swarmer Cell

Abstract The environmental Alphaproteobacterium Caulobacter crescentus is a classical model to study the regulation of the bacterial cell cycle. It divides asymmetrically, giving a stalked cell that immediately enters S phase and a swarmer cell that stays in the G1 phase until it differentiates into a stalked cell. Its genome consists in a single circular chromosome whose replication is tightly regulated so that it happens only in stalked cells and only once per cell cycle. Imbalances in chromosomal copy numbers are the most often highly deleterious, if not lethal. This review highlights recent discoveries on pathways that control chromosome replication when Caulobacter is exposed to optimal or less optimal growth conditions. Most of these pathways target two proteins that bind directly onto the chromosomal origin: the highly conserved DnaA initiator of DNA replication and the CtrA response regulator that is found in most Alphaproteobacteria. The concerted inactivation and proteolysis of CtrA during the swarmer-to-stalked cell transition license cells to enter S phase, while a replisome-associated Regulated Inactivation and proteolysis of DnaA (RIDA) process ensures that initiation starts only once per cell cycle. When Caulobacter is stressed, it turns on control systems that delay the G1-to-S phase transition or the elongation of DNA replication, most probably increasing its fitness and adaptation capacities.

Download Full-text

Novel Divisome-Associated Protein Spatially Coupling the Z-Ring with the Chromosomal Replication Terminus in Caulobacter crescentus

mBio ◽

10.1128/mbio.00487-20 ◽

2020 ◽

Vol 11 (2) ◽

Author(s):

Shogo Ozaki ◽

Urs Jenal ◽

Tsutomu Katayama

Keyword(s):

Cell Cycle ◽

Cell Division ◽

Caulobacter Crescentus ◽

Enteric Bacteria ◽

Chromosome Replication ◽

Chromosomal Replication ◽

Content Type ◽

Physical Linkage ◽

Z Ring ◽

Replication Terminus

ABSTRACT Cell division requires proper spatial coordination with the chromosome, which undergoes dynamic changes during chromosome replication and segregation. FtsZ is a bacterial cytoskeletal protein that assembles into the Z-ring, providing a platform to build the cell division apparatus. In the model bacterium Caulobacter crescentus, the cellular localization of the Z-ring is controlled during the cell cycle in a chromosome replication-coupled manner. Although dynamic localization of the Z-ring at midcell is driven primarily by the replication origin-associated FtsZ inhibitor MipZ, the mechanism ensuring accurate positioning of the Z-ring remains unclear. In this study, we showed that the Z-ring colocalizes with the replication terminus region, located opposite the origin, throughout most of the C. crescentus cell cycle. Spatial organization of the two is mediated by ZapT, a previously uncharacterized protein that interacts with the terminus region and associates with ZapA and ZauP, both of which are part of the incipient division apparatus. While the Z-ring and the terminus region coincided with the presence of ZapT, colocalization of the two was perturbed in cells lacking zapT, which is accompanied by delayed midcellular positioning of the Z-ring. Moreover, cells overexpressing ZapT showed compromised positioning of the Z-ring and MipZ. These findings underscore the important role of ZapT in controlling cell division processes. We propose that ZapT acts as a molecular bridge that physically links the terminus region to the Z-ring, thereby ensuring accurate site selection for the Z-ring. Because ZapT is conserved in proteobacteria, these findings may define a general mechanism coordinating cell division with chromosome organization. IMPORTANCE Growing bacteria require careful tuning of cell division processes with dynamic organization of replicating chromosomes. In enteric bacteria, ZapA associates with the cytoskeletal Z-ring and establishes a physical linkage to the chromosomal replication terminus through its interaction with ZapB-MatP-DNA complexes. However, because ZapB and MatP are found only in enteric bacteria, it remains unclear how the Z-ring and the terminus are coordinated in the vast majority of bacteria. Here, we provide evidence that a novel conserved protein, termed ZapT, mediates colocalization of the Z-ring with the terminus in Caulobacter crescentus, a model organism that is phylogenetically distant from enteric bacteria. Given that ZapT facilitates cell division processes in C. crescentus, this study highlights the universal importance of the physical linkage between the Z-ring and the terminus in maintaining cell integrity.

Download Full-text

Scaffold-scaffold interactions regulate cell polarity in a bacterium

10.1101/2020.08.21.262345 ◽

2020 ◽

Author(s):

Wei Zhao ◽

Samuel W. Duvall ◽

Kimberly A. Kowallis ◽

Chao Zhang ◽

Dylan T. Tomares ◽

...

Keyword(s):

Cell Cycle ◽

Cell Polarity ◽

Chromosome Segregation ◽

Time Course ◽

Asymmetric Cell Division ◽

Caulobacter Crescentus ◽

Unknown Mechanism ◽

Cell Pole ◽

Signaling Complex ◽

Client Proteins

AbstractThe localization of two biochemically distinct signaling hubs at opposite cell poles provides the foundation for asymmetric cell division in Caulobacter crescentus. Here we identify an interaction between the scaffolds PodJ and PopZ that regulates the assembly of the new cell pole signaling complex. Time-course imaging of a mCherry-sfGFP-PopZ fluorescent timer throughout the cell cycle revealed that existing PopZ resides at the old cell pole while newly translated PopZ accumulates at the new cell pole. Our studies suggest that interactions between PodJ and PopZ promotes the sequestration of older PopZ and robust accumulation of newl PopZ at the new cell pole. Elimination of the PodJ-PopZ interaction impacts PopZ client proteins, leading to chromosome segregation defects in one-third of cells. Additionally, this PopZ-PodJ interaction is crucial for anchoring PodJ and preventing PodJ extracellular loss at the old cell pole through unknown mechanism. Therefore, segregation of PopZ protein at the old pole and recruitment of newly translated PopZ at the new pole via the PodJ scaffold ensures stringent inheritance and maintenance of the polarity axis within dividing C. crescentus cells.

Download Full-text

Genomic Adaptations to the Loss of a Conserved Bacterial DNA Methyltransferase

mBio ◽

10.1128/mbio.00952-15 ◽

2015 ◽

Vol 6 (4) ◽

Cited By ~ 10

Author(s):

Diego Gonzalez ◽

Justine Collier

Keyword(s):

Cell Cycle ◽

Cell Division ◽

Experimental Evolution ◽

Dna Methyltransferase ◽

Metabolic Regulation ◽

Caulobacter Crescentus ◽

Point Mutations ◽

Rich Medium ◽

Content Type ◽

Evolution Approach

ABSTRACTCcrM is an orphan DNA methyltransferase nearly universally conserved in a vast group ofAlphaproteobacteria.InCaulobacter crescentus, it controls the expression of key genes involved in the regulation of the cell cycle and cell division. Here, we demonstrate, using an experimental evolution approach, thatC. crescentuscan significantly compensate, through easily accessible genetic changes like point mutations, the severe loss in fitness due to the absence of CcrM, quickly improving its growth rate and cell morphology in rich medium. By analyzing the compensatory mutations genome-wide in 12 clones sampled from independent ΔccrMpopulations evolved for ~300 generations, we demonstrated that each of the twelve clones carried at least one mutation that potentially stimulatedftsZexpression, suggesting that the low intracellular levels of FtsZ are the major burden of ΔccrMmutants. In addition, we demonstrate that the phosphoenolpyruvate-carbohydrate phosphotransfer system (PTS) actually modulatesftsZandmipZtranscription, uncovering a previously unsuspected link between metabolic regulation and cell division inAlphaproteobacteria. We present evidence that point mutations found in genes encoding proteins of the PTS provide the strongest fitness advantage to ΔccrMcells cultivated in rich medium despite being disadvantageous in minimal medium. This environmental sign epistasis might prevent such mutations from getting fixed under changing natural conditions, adding a plausible explanation for the broad conservation of CcrM.IMPORTANCEIn bacteria, DNA methylation has a variety of functions, including the control of DNA replication and/or gene expression. The cell cycle-regulated DNA methyltransferase CcrM modulates the transcription of many genes and is critical for fitness inCaulobacter crescentus. Here, we used an original experimental evolution approach to determine which of its many targets make CcrM so important physiologically. We show that populations lacking CcrM evolve quickly, accumulating an excess of mutations affecting, directly or indirectly, the expression of theftsZcell division gene. This finding suggests that the most critical function of CcrM inC. crescentusis to promote cell division by enhancing FtsZ intracellular levels. During this work, we also discovered an unexpected link between metabolic regulation and cell division that might extend to otherAlphaproteobacteria.

Download Full-text

Surface Sensing Stimulates Cellular Differentiation in Caulobacter crescentus

10.1101/844324 ◽

2019 ◽

Cited By ~ 1

Author(s):

Rhett A. Snyder ◽

Courtney K. Ellison ◽

Geoffrey B. Severin ◽

Christopher M. Waters ◽

Yves V. Brun

Keyword(s):

Cell Cycle ◽

Cell Differentiation ◽

Cellular Differentiation ◽

Cell Cycle Progression ◽

Caulobacter Crescentus ◽

Chromosome Replication ◽

Fundamental Aspect ◽

Cycle Progression ◽

Surface Sensing ◽

Surface Contact

AbstractCellular differentiation is a fundamental strategy used by cells to generate specialized functions at specific stages of development. The bacterium C. crescentus employs a specialized dimorphic life cycle consisting of two differentiated cell types. How environmental cues, including mechanical inputs such as contact with a surface, regulate this cell cycle remain unclear. Here, we find that surface sensing by the physical perturbation of retracting extracellular pilus filaments accelerates cell cycle progression and cellular differentiation. We show that physical obstruction of dynamic pilus activity by chemical perturbation or by a mutation in the outer membrane pilus pore protein, CpaC, stimulates early initiation of chromosome replication. In addition, we find that surface contact stimulates cell cycle progression by demonstrating that surface-stimulated cells initiate early chromosome replication to the same extent as planktonic cells with obstructed pilus activity. Finally, we show that obstruction of pilus retraction stimulates the synthesis of the cell cycle regulator, cyclic diguanylate monophosphate (c-di-GMP) through changes in the activity and localization of two key regulatory histidine kinases that control cell fate and differentiation. Together, these results demonstrate that surface contact and mechanosensing by alterations in pilus activity stimulate C. crescentus to bypass its developmentally programmed temporal delay in cell differentiation to more quickly adapt to a surface-associated lifestyle.SignificanceCells from all domains of life sense and respond to mechanical cues [1–3]. In eukaryotes, mechanical signals such as adhesion and surface stiffness are important for regulating fundamental processes including cell differentiation during embryonic development [4]. While mechanobiology is abundantly studied in eukaryotes, the role of mechanical influences on prokaryotic biology remains under-investigated. Here, we demonstrate that mechanosensing mediated through obstruction of the dynamic extension and retraction of tight adherence (tad) pili stimulates cell differentiation and cell cycle progression in the dimorphic α-proteobacterium Caulobacter crescentus. Our results demonstrate an important intersection between mechanical stimuli and the regulation of a fundamental aspect of cell biology.

Download Full-text

Regulation of Bacterial Cell Cycle Progression by Redundant Phosphatases

Journal of Bacteriology ◽

10.1128/jb.00345-20 ◽

2020 ◽

Vol 202 (17) ◽

Author(s):

Jérôme Coppine ◽

Andreas Kaczmarczyk ◽

Kenny Petit ◽

Thomas Brochier ◽

Urs Jenal ◽

...

Keyword(s):

Cell Cycle ◽

Cell Cycle Progression ◽

Environmental Changes ◽

Caulobacter Crescentus ◽

Response Regulator ◽

Model Organism ◽

Replication Initiation ◽

Cycle Progression ◽

Content Type ◽

Two Component

ABSTRACT In the model organism Caulobacter crescentus, a network of two-component systems involving the response regulators CtrA, DivK, and PleD coordinates cell cycle progression with differentiation. Active phosphorylated CtrA prevents chromosome replication in G1 cells while simultaneously regulating expression of genes required for morphogenesis and development. At the G1-S transition, phosphorylated DivK (DivK∼P) and PleD (PleD∼P) accumulate to indirectly inactivate CtrA, which triggers DNA replication initiation and concomitant cellular differentiation. The phosphatase PleC plays a pivotal role in this developmental program by keeping DivK and PleD phosphorylation levels low during G1, thereby preventing premature CtrA inactivation. Here, we describe CckN as a second phosphatase akin to PleC that dephosphorylates DivK∼P and PleD∼P in G1 cells. However, in contrast to PleC, no kinase activity was detected with CckN. The effects of CckN inactivation are largely masked by PleC but become evident when PleC and DivJ, the major kinase for DivK and PleD, are absent. Accordingly, mild overexpression of cckN restores most phenotypic defects of a pleC null mutant. We also show that CckN and PleC are proteolytically degraded in a ClpXP-dependent way before the onset of the S phase. Surprisingly, known ClpX adaptors are dispensable for PleC and CckN proteolysis, raising the possibility that as yet unidentified proteolytic adaptors are required for the degradation of both phosphatases. Since cckN expression is induced in stationary phase, depending on the stress alarmone (p)ppGpp, we propose that CckN acts as an auxiliary factor responding to environmental stimuli to modulate CtrA activity under suboptimal conditions. IMPORTANCE Two-component signal transduction systems are widely used by bacteria to adequately respond to environmental changes by adjusting cellular parameters, including the cell cycle. In Caulobacter crescentus, PleC acts as a phosphatase that indirectly protects the response regulator CtrA from premature inactivation during the G1 phase of the cell cycle. Here, we provide genetic and biochemical evidence that PleC is seconded by another phosphatase, CckN. The activity of PleC and CckN phosphatases is restricted to the G1 phase since both proteins are degraded by ClpXP protease before the G1-S transition. Degradation is independent of any known proteolytic adaptors and relies, in the case of CckN, on an unsuspected N-terminal degron. Our work illustrates a typical example of redundant functions between two-component proteins.

Download Full-text

Segregation but Not Replication of the Pseudomonas aeruginosa Chromosome Terminates at Dif

mBio ◽

10.1128/mbio.01088-18 ◽

2018 ◽

Vol 9 (5) ◽

Cited By ~ 6

Author(s):

Bijit K. Bhowmik ◽

April L. Clevenger ◽

Hang Zhao ◽

Valentin V. Rybenkov

Keyword(s):

Pseudomonas Aeruginosa ◽

Chromosome Segregation ◽

Large Scale ◽

Critical Role ◽

Genetic Material ◽

Chromosome Replication ◽

Content Type ◽

Chromosome Partitioning ◽

Bacterial Chromosomes ◽

Large Domains

ABSTRACT Coordination between chromosome replication and segregation is essential for equal partitioning of genetic material between daughter cells. In bacteria, this is achieved through the proximity of the origin of replication, oriC, and the chromosome partitioning site, parS. We report here that in Pseudomonas aeruginosa, segregation but not replication is also controlled at the terminus region of the chromosome. Using the fluorescent repressor operator system (FROS), we investigated chromosome segregation in P. aeruginosa strain PAO1-UW, wherein the chromosome dimer resolution site, dif, is asymmetrically positioned relative to oriC. In these cells, segregation proceeded sequentially along the two chromosomal arms and terminated at dif. In contrast, chromosome replication terminated elsewhere, opposite from oriC. We further found two large domains on the longer arm of the chromosome, wherein DNA segregated simultaneously. Notably, GC-skew, which reflects a bias in nucleotide usage between the leading and lagging strands of the chromosome, switches polarity at the dif locus but not necessarily at the terminus of replication. These data demonstrate that termination of chromosome replication and segregation can be physically separated without adverse effects on bacterial fitness. They also reveal the critical role of the dif region in defining the global layout of the chromosome and the progression of chromosome segregation and suggest that chromosome packing adapts to its subcellular layout. IMPORTANCE Segregation of genetic information is a central event in cellular life. In bacteria, chromosome segregation occurs concurrently with replication, sequentially along the two arms from oriC to dif. How the two processes are coordinated is unknown. We explored here chromosome segregation in an opportunistic human pathogen, Pseudomonas aeruginosa, using its strain with markedly unequal chromosomal arms. We found that replication and segregation diverge in this strain and terminate at very different locations, whereas the longer chromosomal arm folds into large domains to align itself with the shorter arm. The significance of this research is in establishing that segregation and replication of bacterial chromosomes are largely uncoupled from each other and that the large-scale structure of the chromosome adapts to its subcellular layout.

Download Full-text

Caulobacterrequires a dedicated mechanism to initiate chromosome segregation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0807448105 ◽

2008 ◽

Vol 105 (40) ◽

pp. 15435-15440 ◽

Cited By ~ 100

Author(s):

Esteban Toro ◽

Sun-Hae Hong ◽

Harley H. McAdams ◽

Lucy Shapiro

Keyword(s):

Chromosome Segregation ◽

Caulobacter Crescentus ◽

Time Lapse ◽

Replication Initiation ◽

Circular Chromosome ◽

Chromosomal Inversions ◽

Initiation Of Replication ◽

Time Lapse Imaging ◽

Single Circular Chromosome

Chromosome segregation in bacteria is rapid and directed, but the mechanisms responsible for this movement are still unclear. We show thatCaulobacter crescentusmakes use of and requires a dedicated mechanism to initiate chromosome segregation.Caulobacterhas a single circular chromosome whose origin of replication is positioned at one cell pole. Upon initiation of replication, an 8-kb region of the chromosome containing both the origin andparSmoves rapidly to the opposite pole. This movement requires the highly conservedParABSlocus that is essential inCaulobacter.We use chromosomal inversions andin vivotime-lapse imaging to show thatparSis theCaulobactersite of force exertion, independent of its position in the chromosome. WhenparSis moved farther from the origin, the cell waits forparSto be replicated before segregation can begin. Also, a mutation in the ATPase domain of ParA halts segregation without affecting replication initiation. Chromosome segregation inCaulobactercannot occur unless a dedicatedparSguiding mechanism initiates movement.

Download Full-text