Biology subjects

Antar, H.

Publications and source records attributed to Antar, H..

7 recordsLinked to original sources

bioRxiv2025

A LexA-like repressor and global H-NS-like regulators enable the fine-tuning of R-tailocin expression in environmental Pseudomonas

Bacteria rely on an arsenal of weapons to challenge their opponents in highly competitive environments. To specifically counter closely related bacteria, specialized weapons with a narrow activity spectrum are deployed, particularly contractile phage tail-like particles or R-tailocins. Their production leads to the lysis of the producing cells, indicating that their expression must be carefully orchestrated so that only a small percentage of cells produce R-tailocins for the benefit of the entire population. In this study, we set out to better understand how the production of these phage tail-like weapons is regulated in environmental pseudomonads using the competitive plant root colonizer and environmental model Pseudomonas protegens CHA0. Using an RNA sequencing (RNA-seq) approach, we found that genes involved in DNA repair, particularly the SOS response program, are upregulated following exposure of the pseudomonad to the DNA-damaging agents mitomycin C and hydrogen peroxide, while genes involved in cell division and primary metabolism are downregulated. The R-tailocin and prophage gene clusters were also upregulated in response to these DNA damaging agents. By combining reverse genetics, transcriptional reporters and chromatin immunoprecipitation sequencing (ChIP-seq), we show that the R-tailocin locus-specific LexA-like regulator PrtR1 represses R-tailocin gene expression by binding directly to the promoter region of the cluster, while the histone-like nucleoid structuring (H-NS) proteins MvaT and MvaV act as master regulators that indirectly regulate R-tailocin cluster expression. Our results suggest that at least these three regulators operate in concert to ensure tight control of R-tailocin expression and cell lytic release in environmental Pseudomonas strains. Author SummaryTo face their opponents in highly competitive environments, bacteria rely on an arsenal of weapons. To specifically target closely related bacterial strains, specialized weapons with a narrow activity spectrum are produced, notably contractile phage tail-like particles such as R-tailocins. Since production of these particles leads to the lysis and death of the producing cell, their expression must be tightly regulated. Here, we set out to better understand how the expression of these phage tail-like weapons is regulated in environmental bacteria using the competitive plant root colonizer and environmental model Pseudomonas protegens CHA0. We found that following DNA-damaging stress, which activates DNA repair systems and downregulates primary metabolism, the expression of gene clusters encoding viral-like particles such as the R-tailocins is upregulated. We identified cluster-specific and global regulators that are either directly or indirectly involved in the control of R-tailocin cluster expression. Our results contribute to a better understanding of the regulatory mechanisms of viral-like particle production in environmental Pseudomonas bacteria.

microbiology↗

bioRxiv2024

Orchestrated long-distance gene activation by a ParB-like BisD-CTP DNA clamp in low-frequency transfer competence development in Pseudomonas putida

Integrative conjugative elements (ICEs) are mobile DNA that remain integrated within the host bacterial genome until they activate their excision and transfer to recipient cells via conjugation. ICE transfer is initiated in a small subpopulation of cells that undergo a hierarchical gene expression cascade leading to transfer competence formation. In this study, we demonstrate that transfer competence formation of the ICEclc element in stationary phase Pseudomonas putida cells is regulated by the coordinated activity of a two-component transcriptional activator, BisC and BisD. Chromatin immunoprecipitation of tagged BisC or BisD followed by high throughput sequencing showed that both proteins accumulate at similar sites around ICEclc transfer competence promoters in P. putida cells. Genetic dissection and single cell microscopy showed that BisD is a dual domain protein, with a C-terminal gene activator domain and an N-terminal ParB-like domain, forming dimeric DNA clamps that self-load at distant sites and reach target promoters after extensive one-dimensional DNA sliding. Expressed mCherry-BisD in P. putida ICEclc cells form discrete fluorescent foci, dependent on parS-like sequences on the ICE. This focus formation is similar as what is seen with canonical ParB proteins accumulating on chromosomal DNA, but in case of BisD corresponds to both chromosomal and excised ICE-molecules. Sliding of BisD over [~]50 kb of ICE-DNA is asymmetric and follows the direction of ICE gene transcription. This may help to establish a temporal order of activation of transfer competence formation, optimizing ICE transfer. Given that ICEclc is activated in stationary phase cells, we hypothesize that BisD is not involved in segregating excised ICE-DNA among daughter cells, but rather in actively directing ICE-DNA molecules towards the (multiple) conjugative systems that are produced in transfer competent cells. BisD thus serves as a twin function protein, integrating gene activation and DNA segregation functions.

microbiology↗

bioRxiv2024

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, gyrases, 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.

biophysics↗

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↗