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Cited by 1Bacterial Genetics and BiotechnologyMicrotubule and mitosis dynamicsDNA Repair Mechanisms

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Molecular dissection of the chromosome partitioning protein RocS and regulation by phosphorylation

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Demuysere, Margaux, Ducret, Adrien, Grangeasse, Christophe·2024·Journal of Bacteriology
DOI 10.1128/jb.00291-24PMC PMC11500499

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Abstract

Chromosome segregation in bacteria is a critical process ensuring that each daughter cell receives an accurate copy of the genetic material during cell division. Active segregation factors, such as the ParABS system or SMC complexes, are usually essential for this process, but they are surprisingly dispensable in Streptococcus pneumoniae . Rather, chromosome segregation in S. pneumoniae relies on the protein Regulator of Chromosome Segregation (RocS), although the molecular mechanisms involved remain elusive. By combining genetics, in vivo imaging, and biochemical approaches, we dissected the molecular features of RocS involved in chromosome segregation. We investigated the respective functions of the three RocS domains, specifically the C-terminal amphipathic helix (AH), the N-terminal DNA-binding domain (DBD), and the coiled-coil domain (CCD) separating the AH and the DBD. Notably, we found that a single AH is not sufficient for membrane binding and that RocS requires prior oligomerization to interact with the membrane. We further demonstrated that this self-interaction was driven by the N-terminal part of the CCD. On the other hand, we revealed that the C-terminal part of the CCD corresponds to a domain of unknown function (DUF 536) and is defined by three conserved glutamines, which play a crucial role in RocS-mediated chromosome segregation. Finally, we showed that the DBD is phosphorylated by the unique serine-threonine kinase of S. pneumoniae StkP and that mimicking this phosphorylation abrogated RocS binding to DNA. Overall, this study offers new insights into chromosome segregation in Streptococci and paves the way for a deeper understanding of RocS-like proteins in other bacteria. IMPORTANCE: Bacteria have evolved a variety of mechanisms to properly segregate their genetic material during cell division. In this study, we performed a molecular dissection of the chromosome partitioning protein Regulator of Chromosome Segregation (RocS), a pillar element of chromosome segregation in S. pneumoniae that is also generally conserved in the Streptococcaceae family. Our systematic investigation sheds light on the molecular features required for successful pneumococcal chromosome segregation and the regulation of RocS by phosphorylation. In addition, our study also revealed that RocS shares functional domains with the Par protein, involved in an atypical plasmid segregation system. Therefore, we expect that our findings may serve to extend our understanding of RocS and RocS-like proteins while broadening the repertoire of partitioning systems used in bacteria.

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Figures

Fig 1 RocS has a MinD-like AH. (A) Schematic of the RocS protein. The different domains are colored as follows: HTH in red, CC1 in orange, DUF in violet, and AH in yellow. The domain limits are specified in brackets below (B) Spatial localization of a GFP probe fused to 1 or 2 AHs. Upper panel: representative fluorescence images of the S. pneumoniae R6 bgaA::pComX-gfp-(AHRocS)n strains with n = 1 (left) or 2 (right). Lower panel: schematic depiction of the localization of the probes. The single or the double AHs are represented in yellow, the GFP in green, and the inner leaflet of the membrane in gray. (C) Representative images showing the localization of GFP-RocS WT and specified mutants. From top to bottom: GFP channel, overlay GFP and phase-contrast channels. Scale bar, 1 µm. (D) Bar chart representing the percentage of chromosome segregation defects observed in the S. pneumoniae R6 rocS::rocS mutants; (hlpA+) hlpA-mKate2 strain. Bars represent the mean (± SEM) of three independent experiments. The WT was compared separately with the RocS-AH2x or the RocS-AH::TM mutants with an unpaired t-test (P-value 0.0167 and 0.0185, respectively). (E) Growth curves of S. pneumoniae R6 rocS::rocS mutants (mean of three biological replicates).
Fig 1 RocS has a MinD-like AH. (A) Schematic of the RocS protein. The different domains are colored as follows: HTH in red, CC1 in orange, DUF in violet, and AH in yellow. The domain limits are specified in brackets below (B) Spatial localization of a GFP probe fused to 1 or 2 AHs. Upper panel: representative fluorescence images of the S. pneumoniae R6 bgaA::pComX-gfp-(AHRocS)n strains with n = 1 (left) or 2 (right). Lower panel: schematic depiction of the localization of the probes. The single or the double AHs are represented in yellow, the GFP in green, and the inner leaflet of the membrane in gray. (C) Representative images showing the localization of GFP-RocS WT and specified mutants. From top to bottom: GFP channel, overlay GFP and phase-contrast channels. Scale bar, 1 µm. (D) Bar chart representing the percentage of chromosome segregation defects observed in the S. pneumoniae R6 rocS::rocS mutants; (hlpA+) hlpA-mKate2 strain. Bars represent the mean (± SEM) of three independent experiments. The WT was compared separately with the RocS-AH2x or the RocS-AH::TM mutants with an unpaired t-test (P-value 0.0167 and 0.0185, respectively). (E) Growth curves of S. pneumoniae R6 rocS::rocS mutants (mean of three biological replicates).CC BY · PubMed Central
Fig 2 The conserved DUF is a major determinant of RocS function. (A) The self-interaction between RocS, RocS-ΔDUF, and RocS-ΔCC1 was assessed by bacterial two-hybrid assay. A blue coloration indicates a positive interaction as depicted by the positive control (+) and the negative control (-) (B) Coiled-coil prediction by the bioinformatic tool CoCoNat. The sequence of full-length RocS was submitted for analysis. The arrows indicate the bounded residues of the unique predicted CCD. (C) AlphaFold3 model of a RocS dimer. Chain A is depicted as a gray ribbon while chain B is colored according to the corresponding domain, following the same color code as the schematic (Fig. 1A): dark red 1–55: HTH, orange 68–102: CC1, violet 104–146: DUF, and yellow 153–163: AH. The bounded residues of each domain and the three conserved glutamines of the DUF are annotated. The three conserved glutamines are represented as amino acids to visualize their side chain. A magnified view of the DUF at 0°C and 90°C angles enables better visualization of the side chains of the conserved glutamines. (D) Representative images showing the localization of GFP-RocS WT and specified mutants. The GFP channel and the overlay between the GFP and the phase-contrast channels are shown. Scale bar, 1 µm. (E) Bar chart representing the percentage of chromosome segregation defects observed in the S. pneumoniae R6 rocS::rocS mutants; (hlpA+) hlpA-mKate2 strains. Bars represent the mean (± SEM) of three independent experiments. Statistical comparisons between the RocS-ΔCC1 mutant and the RocS-ΔDUF or the RocS-DUFQ3E mutant were performed with an unpaired t-test (P-value 0.0089 and 0.0128, respectively) (F) Growth curves of the WT and specified mutants (mean of three biological replicates).
Fig 2 The conserved DUF is a major determinant of RocS function. (A) The self-interaction between RocS, RocS-ΔDUF, and RocS-ΔCC1 was assessed by bacterial two-hybrid assay. A blue coloration indicates a positive interaction as depicted by the positive control (+) and the negative control (-) (B) Coiled-coil prediction by the bioinformatic tool CoCoNat. The sequence of full-length RocS was submitted for analysis. The arrows indicate the bounded residues of the unique predicted CCD. (C) AlphaFold3 model of a RocS dimer. Chain A is depicted as a gray ribbon while chain B is colored according to the corresponding domain, following the same color code as the schematic (Fig. 1A): dark red 1–55: HTH, orange 68–102: CC1, violet 104–146: DUF, and yellow 153–163: AH. The bounded residues of each domain and the three conserved glutamines of the DUF are annotated. The three conserved glutamines are represented as amino acids to visualize their side chain. A magnified view of the DUF at 0°C and 90°C angles enables better visualization of the side chains of the conserved glutamines. (D) Representative images showing the localization of GFP-RocS WT and specified mutants. The GFP channel and the overlay between the GFP and the phase-contrast channels are shown. Scale bar, 1 µm. (E) Bar chart representing the percentage of chromosome segregation defects observed in the S. pneumoniae R6 rocS::rocS mutants; (hlpA+) hlpA-mKate2 strains. Bars represent the mean (± SEM) of three independent experiments. Statistical comparisons between the RocS-ΔCC1 mutant and the RocS-ΔDUF or the RocS-DUFQ3E mutant were performed with an unpaired t-test (P-value 0.0089 and 0.0128, respectively) (F) Growth curves of the WT and specified mutants (mean of three biological replicates).CC BY · PubMed Central
Fig 3 RocS is phosphorylated on threonine 41. (A) Structure and sequence alignment of the wHTH domains of RocS, Par, and RacA. Left panel: superimposition of the AlphaFold3 model of the wHTH of RocS with the structures of Par (8csh-A) and RacA (5i44-B) after a pairwise alignment using the MatchMaker tool in ChimeraX with default settings. Across all 50 pairs RocS-Par, RMSD between 35 pruned atom pairs was 0.942 angströms. Across all 61 pairs RocS-RacA, RMSD between 35 pruned atom pairs was 1.024 angströms. T41 (from RocS), Y36 (from RacA), and N36 (from Par) are highlighted in pink. Right panel: sequence alignment of the wHTH of RocS with the wHTH of Par and RacA taking into account their secondary structures (32). Sequences are indicated with UniProt identifiers, followed by the protein name. The alignment is colored according to the standard percentage identity color scheme as implemented in Jalview (https://www.jalview.org/). T41 from RocS and the corresponding residues from Par (N36) and RacA (Y36) are highlighted in pink. (B) AlphaFold3-generated model of a RocS dimer in complex with DNA (zoom in on one chain, depicted as a gray ribbon). The DNA-recognition helix and the wing are annotated as α2 and β1-β2, respectively. T41 is highlighted in pink and a putative hydrogen bond between the hydroxyl side chain and the phosphate backbone of DNA is depicted as a blue dashed line. (C) MS/MS spectrum of the triple-charged unlabeled peptide ion at an m/z = 742.6843 corresponding to the mass of the phosphorylated peptide ELPEEDTDKNDKGVTpVVTR from RocS.
Fig 3 RocS is phosphorylated on threonine 41. (A) Structure and sequence alignment of the wHTH domains of RocS, Par, and RacA. Left panel: superimposition of the AlphaFold3 model of the wHTH of RocS with the structures of Par (8csh-A) and RacA (5i44-B) after a pairwise alignment using the MatchMaker tool in ChimeraX with default settings. Across all 50 pairs RocS-Par, RMSD between 35 pruned atom pairs was 0.942 angströms. Across all 61 pairs RocS-RacA, RMSD between 35 pruned atom pairs was 1.024 angströms. T41 (from RocS), Y36 (from RacA), and N36 (from Par) are highlighted in pink. Right panel: sequence alignment of the wHTH of RocS with the wHTH of Par and RacA taking into account their secondary structures (32). Sequences are indicated with UniProt identifiers, followed by the protein name. The alignment is colored according to the standard percentage identity color scheme as implemented in Jalview (https://www.jalview.org/). T41 from RocS and the corresponding residues from Par (N36) and RacA (Y36) are highlighted in pink. (B) AlphaFold3-generated model of a RocS dimer in complex with DNA (zoom in on one chain, depicted as a gray ribbon). The DNA-recognition helix and the wing are annotated as α2 and β1-β2, respectively. T41 is highlighted in pink and a putative hydrogen bond between the hydroxyl side chain and the phosphate backbone of DNA is depicted as a blue dashed line. (C) MS/MS spectrum of the triple-charged unlabeled peptide ion at an m/z = 742.6843 corresponding to the mass of the phosphorylated peptide ELPEEDTDKNDKGVTpVVTR from RocS.CC BY · PubMed Central
Fig 4 RocS phosphorylation and DNA binding and functionality in vivo. (A) Electrophoretic mobility shift assay of RocS-∆AH (WT and point mutants) with a 500 bp ds DNA fragment. From the left to right lane: GeneRuler 1 kb DNA ladder (Thermo Scientific), free DNA, DNA and RocS WT-∆AH, DNA and RocS G15P-∆AH, and DNA and RocS T41D-∆AH. (B) Microscale thermophoresis binding assay of dnaC_Cy5 to increasing concentrations of RocS WT-∆AH (blue) or RocS T41D-∆AH (pink). The variation of the normalized fluorescence (∆FNorm) is plotted against RocS concentration. Measurements are represented by dots (mean of three independent experiments) and the fitted curve by a line. The error bars represent the standard deviation. (C) Co-localization of GFP-RocS WT or T41D-∆AH. Representative fluorescence and phase-contrast images of the S. pneumoniae R6 bgaA::pComX-gfp-RocS WT-∆AH, (hlpA+) hlpA-mKate2 and R6 bgaA::pComX-gfp-RocS T41D-∆AH, (hlpA+) hlpA-mKate2 strains. From left to right, the mCherry and phase-contrast channels overlay, the GFP and phase-contrast channels overlay and the mCherry, the GFP and phase-contrast channels overlay are shown. Scale bar, 1 µm. (D) Representative images of the localization of GFP-RocS WT and specified mutants. The GFP channel and the overlay between the GFP and the phase-contrast channels are shown. White arrows highlight bright foci. Scale bar, 1 µm. (E) Bar chart representing the percentage of chromosome segregation defects observed in the S. pneumoniae R6 rocS::rocS mutants; (hlpA+) hlpA-mKate2 strain. Bars represent the mean (± SEM) of three independent experiments. Statistical comparison between the WT and RocS T41D strain was performed using an unpaired t-test (P-value = 0.0024). (F) Growth curves of the WT and the specified mutants (mean of three biological replicates).
Fig 4 RocS phosphorylation and DNA binding and functionality in vivo. (A) Electrophoretic mobility shift assay of RocS-∆AH (WT and point mutants) with a 500 bp ds DNA fragment. From the left to right lane: GeneRuler 1 kb DNA ladder (Thermo Scientific), free DNA, DNA and RocS WT-∆AH, DNA and RocS G15P-∆AH, and DNA and RocS T41D-∆AH. (B) Microscale thermophoresis binding assay of dnaC_Cy5 to increasing concentrations of RocS WT-∆AH (blue) or RocS T41D-∆AH (pink). The variation of the normalized fluorescence (∆FNorm) is plotted against RocS concentration. Measurements are represented by dots (mean of three independent experiments) and the fitted curve by a line. The error bars represent the standard deviation. (C) Co-localization of GFP-RocS WT or T41D-∆AH. Representative fluorescence and phase-contrast images of the S. pneumoniae R6 bgaA::pComX-gfp-RocS WT-∆AH, (hlpA+) hlpA-mKate2 and R6 bgaA::pComX-gfp-RocS T41D-∆AH, (hlpA+) hlpA-mKate2 strains. From left to right, the mCherry and phase-contrast channels overlay, the GFP and phase-contrast channels overlay and the mCherry, the GFP and phase-contrast channels overlay are shown. Scale bar, 1 µm. (D) Representative images of the localization of GFP-RocS WT and specified mutants. The GFP channel and the overlay between the GFP and the phase-contrast channels are shown. White arrows highlight bright foci. Scale bar, 1 µm. (E) Bar chart representing the percentage of chromosome segregation defects observed in the S. pneumoniae R6 rocS::rocS mutants; (hlpA+) hlpA-mKate2 strain. Bars represent the mean (± SEM) of three independent experiments. Statistical comparison between the WT and RocS T41D strain was performed using an unpaired t-test (P-value = 0.0024). (F) Growth curves of the WT and the specified mutants (mean of three biological replicates).CC BY · PubMed Central

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Metadata

Authors
Demuysere, M., Ducret, A., & Grangeasse, C.
Affiliations
Centre National de la Recherche Scientifique (FR)
Corresponding author
Grangeasse, Christophe
ORCID
Venue
Journal of Bacteriology
Publisher
American Society for Microbiology
Published
2024-10-24
Volume
206
Issue
10
Pages
e0029124-e0029124
ISSN
  • 0021-9193 (ISSN-L)
  • 1098-5530
DOI
10.1128/jb.00291-24
PMC
PMC11500499
OpenAlex
W4402768642
Cited by
1
References
57
Citations by year
2026: 1
FWCI
0.26
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of Bacteriology (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • HAL (submittedVersion, cc-by)
Funders
  • European Commission (H2020, 955626)
  • Centre National de la Recherche Scientifique (UMS344/US8)
  • Université Claude Bernard Lyon 1
  • Fondation Bettencourt Schueller
  • Horizon 2020 Framework Programme (955626)
  • HORIZON EUROPE Excellent Science
  • H2020 Marie Skłodowska-Curie Actions (H2020, 955626)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Microtubule and mitosis dynamics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA Repair Mechanisms
Keywords
ROCS, chromosome segregation, Streptococcus pneumoniae, phosphorylation, amphipathic helix, coiled-coil domain, DNA-binding domain, protein oligomerization, membrane binding, PAR proteins, plasmid partitioning, Streptococcaceae
MeSH terms
Protein Domains, Bacterial Proteins, Chromosomes, Bacterial, Phosphorylation, Streptococcus pneumoniae, Gene Expression Regulation, Bacterial, Chromosome Segregation

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