Permissive zones for the centromere-binding protein ParB on the Caulobacter crescentus chromosome

Tran, Ngat T.; Stevenson, Clare E. M.; Som, Nicolle F.; Thanapipatsiri, Anyarat; Jalal, Adam S. B.; Le, Tung B. K.

doi:10.1093/nar/gkx1192

Cited by 46 publications

(61 citation statements)

References 45 publications

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“…2B). The highly conserved arginine-rich patch (G 101 ERRWR), crucial for Caulobacter ParB spreading (10), resides on helix α2 ( Fig. 1).…”

Section: Resultsmentioning

confidence: 99%

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A CTP-dependent gating mechanism enables ParB spreading on DNA

Jalal

Tran

Stevenson

et al. 2019

Preprint

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The tripartite ParA-ParB-parS complex ensures faithful chromosome segregation in the majority of bacterial species. ParB nucleates on a centromere-like parS site and spreads to neighboring DNA to form a network of protein-DNA complexes. This nucleoprotein network interacts with ParA to partition the parS locus, hence the chromosome to each daughter cell. Here, we determine the cocrystal structure of a C-terminal domain truncated ParB-parS complex from Caulobacter crescentus, and show that its N-terminal domain adopts alternate conformations. The multiple conformations of the N-terminal domain might facilitate the spreading of ParB on the chromosome. Next, using ChIPseq we show that ParBs from different bacterial species exhibit variation in their intrinsic capability for spreading, and that the N-terminal domain is a determinant of this variability. Finally, we show that the C-terminal domain of Caulobacter ParB possesses no or weak non-specific DNA-binding activity. Engineered ParB variants with enhanced non-specific DNA-binding activity condense DNA in vitro but do not spread further than wild-type in vivo. Taken all together, our results emphasize the role of the N-terminal domain in ParB spreading and faithful chromosome segregation in Caulobacter crescentus. Proper chromosome segregation is essential in all domains of life. In two-thirds of known bacterial species, faithful chromosome segregation is mediated by the conserved ParA-ParB-parS system (1-10). This tripartite complex consists of a Walker-box ATPase ParA, a centromere-binding protein ParB, and a centromere-like DNA sequence parS. The parS site is the first DNA locus to be segregated after chromosome replication (2,5,11,12). ParB, a DNA-binding protein, nucleates on parS before binding to adjacent non-specific DNA to form a network of protein-DNA complexes. This nucleoprotein network interacts with ParA to partition the chromosome to each daughter cell. In Caulobacter crescentus, ParA forms a protein gradient emanating from the opposite pole of the cell to the ParB-parS complex (13-15). The DNA-bound ParB complexes stimulate the ATPase activity of ParA, causing the ParA gradient to retract, bringing the ParB-DNA complex to the opposite pole of the cell in a retreating gradient of ParA (15)(16)(17)(18)(19). In Caulobacter crescentus, ParA and ParB are essential for chromosome segregation and cell viability (3,13). In other bacterial species, engineered strains lacking ParB are still viable but have elevated numbers of anucleate cells due to defects in chromosome segregation (2,7,9,12,16,(20)(21)(22).The binding of multiple ParB molecules onto non-specific DNA after nucleation at parS (i.e. spreading) is a crucial event; bacterial cells harboring a nucleation-competent but spreadingdefective parB allele are impaired in plasmid/chromosome segregation (23-25). Spreading was first discovered for the F-plasmid-encoded SopB protein and the P1 plasmid-encoded ParB protein (26,27), and was subsequently found to be a general feature of many plasmid and chromo...

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“…2B). The highly conserved arginine-rich patch (G 101 ERRWR), crucial for Caulobacter ParB spreading (10), resides on helix α2 ( Fig. 1).…”

Section: Resultsmentioning

confidence: 99%

“…Fixed cells were incubated at RT for 30 minutes, then quenched with 0.125 M glycine for 15 minutes at RT. All subsequent steps were performed exactly as described in Tran et al (2018) (10). A detailed protocol was described in the Supplementary Materials and Methods.…”

Section: Chromatin Immunoprecipitation With Deep Sequencing (Chip-seq)mentioning

confidence: 99%

A CTP-dependent gating mechanism enables ParB spreading on DNA

Jalal

Tran

Stevenson

et al. 2019

Preprint

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“…Engineered strains harboring a nucleation-competent but spreading-defective mutant of parB are either unviable 10 or have elevated number of anucleate cells 4,7,8,15,[31][32][33][34] . Despite the importance of spreading for proper chromosome segregation, the mechanism by which a few parS-bound ParB can recruit hundreds more ParB molecules to the vicinity of parS to assemble a high molecular-weight nucleoprotein complex is not fully understood.Since the first report in 1995 35 , ParB spreading has been observed in vivo by chromatin immunoprecipitation in multiple bacterial species 12,[15][16][17]19,36 . The nucleation of ParB on parS has also been demonstrated in vitro 4,10,16,17,20,[37][38][39] , however parS-dependent ParB spreading has resisted biochemical reconstitution [17][18][19]40,41 .…”

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confidence: 99%

“…Since the first report in 1995 35 , ParB spreading has been observed in vivo by chromatin immunoprecipitation in multiple bacterial species 12,[15][16][17]19,36 . The nucleation of ParB on parS has also been demonstrated in vitro 4,10,16,17,20,[37][38][39] , however parS-dependent ParB spreading has resisted biochemical reconstitution [17][18][19]40,41 .…”

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ParB spreading on DNA requires cytidine triphosphatein vitro

Jalal

Tran

2019

Preprint

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In all living organisms, it is essential to transmit genetic information faithfully to the next generation. The SMC-ParAB-parS system is widely employed for chromosome segregation in bacteria. A DNA-binding protein ParB nucleates on parS sites and must associate with neighboring DNA, a process known as spreading, to enable efficient chromosome segregation. Despite its importance, how the initial few ParB molecules nucleating at parS sites recruit hundreds of further ParB to spread is not fully understood. Here, we reconstitute a parS-dependent ParB spreading event using purified proteins from Caulobacter crescentus and show that CTP is required for spreading. We further show that ParB spreading requires a closed DNA substrate, and a DNA-binding transcriptional regulator can act as a roadblock to attenuate spreading unidirectionally in vitro. Our biochemical reconstitutions recapitulate many observed in vivo properties of ParB and opens up avenues to investigate the interactions between ParB-parS with ParA and SMC.Faithful chromosome segregation is essential in all domains of life if daughter cells are each to inherit the full set of genetic information. The SMC-ParAB-parS complex is widely employed for chromosome segregation in bacteria 1-13 . The centromere parS is the first DNA locus to be segregated following chromosome replication 8,9,14,15 . ParB specifically nucleates on parS before spreading outwards to the flanking DNA and bridges/cages DNA together to form a nucleoprotein network in vivo [16][17][18][19][20][21][22][23] . This nucleoprotein complex recruits SMC to disentangle and organize replicated DNA 3,11,13,24,25 . ParB-parS also interacts with an ATPase ParA to power the segregation of replicated chromosomes [26][27][28][29][30] . Engineered strains harboring a nucleation-competent but spreading-defective mutant of parB are either unviable 10 or have elevated number of anucleate cells 4,7,8,15,[31][32][33][34] . Despite the importance of spreading for proper chromosome segregation, the mechanism by which a few parS-bound ParB can recruit hundreds more ParB molecules to the vicinity of parS to assemble a high molecular-weight nucleoprotein complex is not fully understood.Since the first report in 1995 35 , ParB spreading has been observed in vivo by chromatin immunoprecipitation in multiple bacterial species 12,[15][16][17]19,36 . The nucleation of ParB on parS has also been demonstrated in vitro 4,10,16,17,20,[37][38][39] , however parS-dependent ParB spreading has resisted biochemical reconstitution [17][18][19]40,41 . Unsuccessful attempts to reconstitute spreading in vitro suggests that additional factors might be missing. Recently, works by Soh et al (2019) and Osorio-Valeriano et al (2019) on Bacillus subtilis and Myxococcus xanthus ParB, respectively, showed that ParB binds and hydrolyzes cytidine triphosphate (CTP) to cytidine diphosphate (CDP), and that CTP modulates the binding affinity of ParB to parS 42,43 . A co-crystal structure of Bacillus ParB with CDP and that of a Myxococcus ParB-li...

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“…Asymmetric cell division enables the development of different cell types throughout the organism to specialize by partitioning recognizes and cooperatively binds the cognate sequence, parS. A combination of homodimerization and nonspecific DNA-protein interactions leads to the formation of a nucleoprotein complex spreading for several kilobases around the parS site 34,35 . The exact mechanism of the ParB nucleation around parS is still largely unclear, but one model suggests a "nucleation and caging" mechanism in which a core of tightly bound ParB dimers forms around parS 36 .…”

Section: Introductionmentioning

confidence: 99%

Synthetic pluripotent bacterial stem cells

Molinari

Shis

Chappell

et al. 2018

Preprint

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We describe a synthetic genetic circuit for controlling asymmetric cell division in E. coli in which a progenitor cell creates a differentiated daughter cell while retaining its original phenotype.Specifically, we engineered an inducible system that can bind and segregate plasmid DNA to a single position in the cell. Upon cell division, co-localized plasmids are kept by one and only one of the daughter cells. The other daughter cell receives no plasmid DNA and is hence irreversibly differentiated from its sibling. In this way, we achieved asymmetric cell division through asymmetric plasmid partitioning. We then used this system to achieve physical separation of genetically distinct cells by tying motility to differentiation. Finally, we characterized an orthogonal inducible circuit that enables the simultaneous asymmetric partitioning of two plasmid species, resulting in cells that have four distinct differentiated states. These results point the way towards engineering multicellular systems from prokaryotic hosts. biochemical or physical tasks throughout the organism. In multicellular eukaryotes, cellular differentiation is generally achieved through complex regulatory networks that utilize transcriptional regulation, post-transcriptional modifications, and chromatin remodeling 19-21 . To differentiate asymmetrically, a progenitor cell (such as a stem cell) senses chemical cues in the environment to alter the transcriptional landscape in the daughter cell. This transcriptional rearrangement of the daughter cell is complete enough that de-differentiation is rare and reproducible in the lab only through the very specific and simultaneous induction of many genes 22 .Several attempts have been made to create synthetic bacteria that have multiple stable transcriptional profiles 3,23 . The first of these was the corepressive toggle switch in E. coli designed by Gardner and colleagues 3 . However, the corepressive toggle does not create irreversibly differentiated cell types. It is sensitive to intrinsic and extrinsic sources of gene regulatory noise 3,24-27 , and can only transiently maintain one state before stochastically switching back to the other.Others have used recombinases 28 to alter DNA sequences permanently, or controllably alter the copy numbers of plasmids to affect gene regulation and cell differentiation 29,30 .Here, we repurposed two key elements of the chromosome partitioning system (par) of Caulobacter crescentus 31 to create asymmetric cell division and irreversible differentiation in E. coli. The par system is common in prokaryotes and is principally responsible for partitioning low copy number plasmids or chromosomes upon cell division 32 . The par systems generally rely on the interaction of three elements: a centromere-like cis-acting sequence, parS, present on the plasmid/chromosome; a centromere-binding trans-acting protein, ParB; and an NTPase, usually called ParA 33 . The initial step is the formation of the partition complex, in which all the copies of the plasmid or chromosomal DNA are gat...

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Permissive zones for the centromere-binding protein ParB on the Caulobacter crescentus chromosome

Cited by 46 publications

References 45 publications

A CTP-dependent gating mechanism enables ParB spreading on DNA

A CTP-dependent gating mechanism enables ParB spreading on DNA

ParB spreading on DNA requires cytidine triphosphatein vitro

Synthetic pluripotent bacterial stem cells

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