The virulence regulator VirB from<i>Shigella flexneri</i>uses a CTP-dependent switch mechanism to activate gene expression

Jakob, Sissi Brigitte; Steinchen, Wieland; Hanssmann, Juri; Rosum, Julia; Osorio-Valeriano, Manuel; Giammarinaro, Pietro I.; Bange, Gert; Thanbichler, Martin

doi:10.1101/2023.06.01.543266

Cited by 3 publications

(3 citation statements)

References 80 publications

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“…Transcriptional regulation by a DNA-sliding clamp, as observed for KorB-CTP here, is reminiscent of the activation of virulence genes in Shigella flexneri by a VirB-CTP sliding clamp [65][66][67] . However, how a sliding VirB mechanistically counteracts the silencing activity of a nucleoid-associated protein, H-NS, to allow transcription is incompletely understood.…”

Section: Discussion Ctp and Kora Regulate A Korb Functional Switchmentioning

confidence: 99%

“…Such a therapeutic could re-sensitize bacteria to antibiotics they may otherwise be resistant to, while also preventing the spread of multi-drug resistant plasmids. Finally, beyond the classical role of CTP in DNA segregation, the discovery of broader roles for CTP in controlling Noc membrane-binding activity and in gene expression regulation by KorB and VirB suggest that CTP-dependent molecular switches are likely to be more widespread in biology than previously appreciated 7,25,[65][66][67]73 . We speculate that future discovery and characterization of further CTP-binding proteins will ultimately lead to a full appreciation of CTP switches in biology.…”

Section: Final Perspectivesmentioning

confidence: 99%

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Molecular switching of a DNA-sliding clamp to a repressor mediates long-range gene silencing

McLean,

Balaguer-Perez,

Chandanani

et al. 2024

Preprint

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Long-range gene regulation is rare in bacteria and is confined to the classical DNA looping model. Here, we use a combination of biophysical approaches, including X-ray crystallography and single-molecule analysis, to show that long-range gene silencing on the plasmid RK2, a source of multidrug resistance across diverse Gram-negative bacteria, is achieved cooperatively by a DNA-sliding clamp, KorB, and a clamp-locking protein, KorA. We find that KorB is a CTPase clamp that can entrap and slide along DNA to reach distal target promoters. We resolved the tripartite crystal structure of a KorB-KorA-DNA co-complex, revealing that KorA latches KorB into a closed-clamp state. KorA thus stimulates repression by stalling KorB sliding at target promoters to occlude RNA polymerase holoenzymes. Altogether, our findings explain the mechanistic basis for KorB role-switching from a DNA-sliding clamp to a co-repressor, and provide a new paradigm for the long-range regulation of gene expression.

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Section: Discussion Ctp and Kora Regulate A Korb Functional Switchmentioning

confidence: 99%

Section: Final Perspectivesmentioning

confidence: 99%

Molecular switching of a DNA-sliding clamp to a repressor mediates long-range gene silencing

McLean,

Balaguer-Perez,

Chandanani

et al. 2024

Preprint

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“…To investigate whether ParT diffusion leads to its accumulation on parS DNA, we followed the interaction of ParT with parS DNA in real-time using BLI, which allowed us to use a higher concentration of purified ParT than with our optical tweezers setup. We employed a 180-bp dual biotin-labeled parS or non-parS DNA tethered at both ends to a streptavidin-coated probe to form a closed DNA loop (14,19,21,22,32) (Fig. 3E).…”

Section: Part Diffuses and Accumulates On A Pars Dna Loop Independent...mentioning

confidence: 99%

Assembly of a segrosome by a CTP-independent ParB-like protein

Sukhoverkov,

Balaguer-Perez,

Aicart-Ramos

et al. 2024

Preprint

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The ATP- and CTP-dependent ParA-ParB-parS segrosome is a macromolecular complex that segregates chromosomes/plasmids in most bacterial species. CTP binding and hydrolysis enable ParB to slide on DNA and to bridge and condense DNA, thereby dictating the size and dynamics of the tripartite ParABS complex. Several other evolutionarily distinct systems can also segregate DNA, although the full diversity of bacterial DNA partition systems is not yet known. Here, we identify a CTP-independent ParABS system that maintains a conjugative plasmid SCP2 in the filamentous bacterium Streptomyces coelicolor. We demonstrate that an SCP2 ParB-like protein, ParT, loads onto DNA at an 18-bp parS site and diffuses away to the adjacent DNA despite lacking an apparent CTPase domain and detectable NTPase activity. We further show that parS DNA stimulates ParT transition from loading to a diffusing state to accumulate on DNA, and ParT activates the ATPase activity of its cognate partner protein ParA. We also identify numerous structural homologs of ParT, suggesting that CTP-independent diffusion on DNA might be widespread in bacteria despite being previously unappreciated. Overall, our findings uncover a CTP-independent DNA translocation as an alternative and unanticipated mechanism for the assembly of a bacterial DNA segregation complex and suggest that CTP binding and hydrolysis is not a fundamental feature of ParABS-like systems.

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

Antar,

Gruber

2023

Commun Biol

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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.

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The virulence regulator VirB fromShigella flexneriuses a CTP-dependent switch mechanism to activate gene expression

Cited by 3 publications

References 80 publications

Molecular switching of a DNA-sliding clamp to a repressor mediates long-range gene silencing

Molecular switching of a DNA-sliding clamp to a repressor mediates long-range gene silencing

Assembly of a segrosome by a CTP-independent ParB-like protein

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

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