Biology subjects

Antar, H.

Publications and source records attributed to Antar, H..

4 recordsLinked to original sources

bioRxiv2023

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.

biophysics↗

bioRxiv2023

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. It is encoded by the large invasion plasmid, pINV, and is thought to counteract the transcriptional silencing mediated by the nucleoid structuring protein, H-NS. Mutations in virB lead to loss of virulence. Studies suggest 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. We show that VirB slowly but detectably hydrolyses CTP, which is mildly stimulated by the virS targeting sequences found on pINV. CTP and DNA binding promote VirB clamp closure. Curiously, DNA stimulation of clamp closure appears efficient even without virS sequences. These findings suggest that VirB acts as a CTP-dependent DNA clamp and may indicate that so far elusive factors might prevent offsite DNA clamping in vivo.

microbiology↗

bioRxiv2021

ParS-independent recruitment of the bacterial chromosome-partitioning protein ParB

The ParABS system plays an essential role in prokaryotic chromosome segregation. After loading at the parS site on the genome, ParB proteins rapidly redistribute to distances of ~15 kb away from the loading site. It has remained puzzling how this large-distance spreading can occur along DNA that is loaded with hundreds of proteins. Using single-molecule in vitro visualization, we here show that, unexpectedly, ParB can load onto DNA independently and distantly of parS, whereby loaded ParB molecules are themselves able to recruit additional ParB proteins from bulk. Strikingly, this recruitment can occur in-cis but also in-trans whereby, 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 also show that cooperative ParB-ParB recruitment enhances spreading. ParS-independent recruitment explains how ParB can cover substantial genomic distance during chromosome segregation which is vital for the bacterial cell cycle.

biophysics↗

bioRxiv2021

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

Three-component ParABS systems are widely distributed factors for plasmid partitioning and chromosome segregation in bacteria. ParB protein acts as an adaptor between the 16 bp centromeric parS DNA sequences and the DNA segregation ATPase ParA. It accumulates at high concentrations at and near a parS site by assembling a partition complex. ParB dimers form a DNA sliding clamp whose closure at parS requires CTP binding. The mechanism underlying ParB loading and the role of CTP hydrolysis however remain unclear. We show that CTP hydrolysis is dispensable for Smc recruitment to parS sites in Bacillus subtilis but is essential for chromosome segregation by ParABS in the absence of Smc. Our results suggest that CTP hydrolysis contributes to partition complex assembly via two mechanisms. It recycles off-target ParB clamps to allow for new attempts at parS targeting and it limits the extent of spreading from parS by promoting DNA unloading. We also propose a model for how parS DNA catalyzes ParB clamp closure involving a steric clash between ParB protomers binding to opposing parS half sites.

biochemistry↗