DNA supercoiling enhances DNA condensation by ParB proteins.

The ParABS system plays a critical role in bacterial chromosome segregation. The key component of this system, ParB, loads and spreads along DNA to form a local protein-DNA condensate known as a partition complex. As bacterial chromosomes are heavily supercoiled due to the continuous action of RNA polymerases, topoisomerases and nucleoid-associated proteins, it is important to study the impact of DNA supercoiling on the ParB-DNA partition complex formation. Here, we use an in-vitro single-molecule assay to visualize ParB on supercoiled DNA. Unlike most DNA-binding proteins, individual ParB proteins are found to not pin plectonemes on supercoiled DNA, but freely diffuse along supercoiled DNA. We find that DNA supercoiling enhances ParB-DNA condensation, which initiates at lower ParB concentrations than on DNA that is torsionally relaxed. ParB proteins induce a DNA-protein condensate that strikingly absorbs all supercoiling writhe. Our findings provide mechanistic insights that have important implications for our understanding of bacterial chromosome organization and segregation.

Dynamic ParB-DNA interactions initiate and maintain a partition condensate for bacterial chromosome segregation.

In most bacteria, chromosome segregation is driven by the ParABS system where the CTPase protein ParB loads at the parS site to trigger the formation of a large partition complex. Here, we present in vitro studies of the partition complex for Bacillus subtilis ParB, using single-molecule fluorescence microscopy and AFM imaging to show that transient ParB-ParB bridges are essential for forming DNA condensates. Molecular Dynamics simulations confirm that condensation occurs abruptly at a critical concentration of ParB and show that multimerization is a prerequisite for forming the partition complex. Magnetic tweezer force spectroscopy on mutant ParB proteins demonstrates that CTP hydrolysis at the N-terminal domain is essential for DNA condensation. Finally, we show that transcribing RNA polymerases can steadily traverse the ParB-DNA partition complex. These findings uncover how ParB forms a stable yet dynamic partition complex for chromosome segregation that induces DNA condensation and segregation while enabling replication and transcription.

Direct observation of a crescent-shape chromosome in expanded Bacillus subtilis cells.

Bacterial chromosomes are folded into tightly regulated three-dimensional structures to ensure proper transcription, replication, and segregation of the genetic information. Direct visualization of chromosomal shape within bacterial cells is hampered by cell-wall confinement and the optical diffraction limit. Here, we combine cell-shape manipulation strategies, high-resolution fluorescence microscopy techniques, and genetic engineering to visualize the shape of unconfined bacterial chromosome in real-time in live Bacillus subtilis cells that are expanded in volume. We show that the chromosomes predominantly exhibit crescent shapes with a non-uniform DNA density that is increased near the origin of replication (oriC). Additionally, we localized ParB and BsSMC proteins - the key drivers of chromosomal organization - along the contour of the crescent chromosome, showing the highest density near oriC. Opening of the BsSMC ring complex disrupted the crescent chromosome shape and instead yielded a torus shape. These findings help to understand the threedimensional organization of the chromosome and the main protein complexes that underlie its structure.

VirB, a transcriptional activator of virulence in Shigella flexneri, uses CTP as a cofactor.

VirB is a transcriptional activator of virulence in the gram-negative bacterium Shigella flexneri encoded by the large invasion plasmid, pINV. It counteracts the transcriptional silencing by the nucleoid structuring protein, H-NS. Mutations in virB lead to loss of virulence. Studies suggested that VirB binds to specific DNA sequences, remodels the H-NS nucleoprotein complexes, and changes DNA supercoiling. VirB belongs to the superfamily of ParB proteins which are involved in plasmid and chromosome partitioning often as part of a ParABS system. Like ParB, VirB forms discrete foci in Shigella flexneri cells harbouring pINV. Our results reveal that purified preparations of VirB specifically bind the ribonucleotide CTP and slowly but detectably hydrolyse it with mild stimulation by the virS targeting sequences found on pINV. We show that formation of VirB foci in cells requires a virS site and CTP binding residues in VirB. Curiously, DNA stimulation of clamp closure appears efficient even without a virS sequence in vitro. Specificity for entrapment of virS DNA is however evident at elevated salt concentrations. These findings suggest that VirB acts as a CTP-dependent DNA clamp and indicate that the cellular microenvironment contributes to the accumulation of VirB specifically at virS sites.

ParB proteins can bypass DNA-bound roadblocks via dimer-dimer recruitment.

The ParABS system is essential for prokaryotic chromosome segregation. After loading at parS on the genome, ParB (partition protein B) proteins rapidly redistribute to distances of ~15 kilobases from the loading site. It has remained puzzling how this large-distance spreading can occur along DNA loaded with hundreds of proteins. Using in vitro single-molecule fluorescence imaging, we show that ParB from Bacillus subtilis can load onto DNA distantly of parS, as loaded ParB molecules themselves are found to be able to recruit additional ParB proteins from bulk. Notably, this recruitment can occur in cis but also in trans, where, at low tensions within the DNA, newly recruited ParB can bypass roadblocks as it gets loaded to spatially proximal but genomically distant DNA regions. The data are supported by molecular dynamics simulations, which show that cooperative ParB-ParB recruitment can enhance spreading. ParS-independent recruitment explains how ParB can cover substantial genomic distance during chromosome segregation, which is vital for the bacterial cell cycle.

Relief of ParB autoinhibition by parS DNA catalysis and recycling of ParB by CTP hydrolysis promote bacterial centromere assembly.

Three-component ParABS systems are widely distributed factors for plasmid partitioning and chromosome segregation in bacteria. ParB acts as adaptor protein between the 16­base pair centromeric parS DNA sequences and the DNA segregation proteins ParA and Smc (structural maintenance of chromosomes). Upon cytidine triphosphate (CTP) and parS DNA binding, ParB dimers form DNA clamps that spread onto parS-flanking DNA by sliding, thus assembling the so-called partition complex. We show here that CTP hydrolysis is essential for efficient chromosome segregation by ParABS but largely dispensable for Smc recruitment. Our results suggest that CTP hydrolysis contributes to partition complex assembly via two mechanisms. It promotes ParB unloading from DNA to limit the extent of ParB spreading, and it recycles off-target ParB clamps to allow for parS retargeting, together superconcentrating ParB near parS. We also propose a model for clamp closure involving a steric clash when binding ParB protomers to opposing parS half sites.