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

Margaux Demuysere, Adrien Ducret, Christophe Grangeasse

► To cite this version:

Margaux Demuysere, Adrien Ducret, Christophe Grangeasse. Molecular dissection of the chromosome partitioning protein RocS and regulation by phosphorylation. Journal of Bacteriology, 2024, 206 (10), pp.00291-24. 10.1128/jb.00291-24. hal-05004085

HAL Id: hal-05004085

https://hal.science/hal-05004085v1

Submitted on 3 Dec 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
# Molecular dissection of the chromosome partitioning protein RocS and regulation by phosphorylation

Margaux Demuysere,¹ Adrien Ducret,¹ Christophe Grangeasse¹

**AUTHOR AFFILIATION** See affiliation list on p. 15.

**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.

**KEYWORDS** chromosome segregation, *Streptococcus pneumoniae*, protein phosphorylation, bacterial cell cycle, DNA binding

Chromosome inheritance is crucial in all domains of life. Unlike eukaryotes, bacteria do not rely on a spindle-like mitotic apparatus to organize chromosome segregation. Instead, they combine passive and active mechanisms to concomitantly replicate, segregate, and transcribe their genetic material in synchronization with cell division (1).

**Editor** Patricia A. Champion, University of Notre Dame, Notre Dame, Indiana, USA

Address correspondence to Christophe Grangeasse, christophe.grangeasse@cnrs.fr.

Margaux Demuysere and Adrien Ducret contributed equally to this article. Author order was determined alphabetically

The authors declare no conflict of interest.

See the funding table on p. 15.

**Received** 25 July 2024

**Accepted** 10 September 2024

**Published** 24 September 2024

Copyright © 2024 Demuysere et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
Most of our understanding of bacterial chromosome segregation comes from observa-
tions in the model bacteria *Bacillus subtilis*, *Escherichia coli*, and *Caulobacter crescentus*
(2). Although these studies have pointed toward shared mechanisms, they have also
highlighted divergences, such as the relative importance of conserved mechanisms
depending on the studied organism (3), as well as unique strategies to cope with various
constraints specific to their physiology, mode of growth and environmental niches (4, 5).

One example of this duality is the broadly distributed ParABS system, responsible for the early segregation of the origin region through the combination of a CTPase (ParB) and an ATPase (ParA) activities (6). *C. crescentus*, in particular, relies on this system to orchestrate chromosome segregation (7). However, the system is absent in *E. coli* (8), while *B. subtilis* uses it mainly during sporulation (9). The versatility of the ParABS system is also illustrated by its plasmid-encoded version, which assists in the segregation of low-copy plasmids to ensure their maintenance (8). Another ubiquitous factor for chromosome segregation is the Structural Maintenance of Chromosome (SMC) complex, SMC-ScpAB, or its MukBEF and MksBEF counterparts (3). These complexes are proposed to participate in chromosome segregation *via* an ATP-driven loop-extrusion mechanism (2). Again, their importance varies, from having little impact on chromosome segregation in *C. crescentus* to being essential in *B. subtilis* and *E. coli* when grown in a nutrient-rich medium (3).

Over the past decade, *Streptococcus pneumoniae* has emerged as a model organism for deciphering the molecular mechanisms that control the bacterial cell cycle (10). In particular, these studies offered crucial insights into the mechanisms of cell division and morphogenesis, pioneering the discovery that protein phosphorylation is a fundamental regulatory mechanism in the pneumococcal cell cycle (11–14). However, a fine understanding of chromosome segregation in this bacterium remains largely elusive. The first studies reported the presence of an incomplete ParABS system, in which the ParA protein was lacking (10, 15). Furthermore, this system, as well as the SMC complex, appeared only moderately involved in chromosome segregation, as their single and dual deletion only generated a low percentage of anucleate cells (15, 16). This suggested the existence of an additional player, which was identified recently as the Regulator of Chromosome Segregation (RocS) protein (17). Indeed, the deletion of *rocS* is extremely deleterious for chromosome segregation and is synthetic lethal with the deletion of *parB* and *smc*, thus establishing it as the main driver of chromosome segregation in *S. pneumoniae* (17). RocS is a protein bound to the membrane and the nucleoid *via* its C-terminal AH and its N-terminal DNA-binding domain (DBD), respectively (Fig. 1A). Both domains are essential for RocS localization and function in chromosome segregation (17). However, the molecular mechanisms involved remain elusive.

In this work, we performed a mutational dissection of RocS to reveal the key features required for chromosome segregation. In particular, we demonstrate that the CCD of RocS, which separates the AH and the DBD, can be divided into two subdomains, each serving distinct functions. Furthermore, we revealed that a domain of unknown function (DUF 536) plays a major role in the RocS-assisted chromosome segregation. Finally, we show that the DBD is phosphorylated by StkP, thus negatively modulating DNA binding and subsequent chromosome segregation. Overall, this study provides new insights into chromosome segregation in Streptococci and paves the way for a better understanding of RocS-related proteins in other bacteria.

RESULTS

The amphipathic helix of RocS requires multivalency to associate with the
membrane

It was previously demonstrated that RocS harbors a C-terminal AH. This AH mediates the interaction of RocS with the membrane, which is crucial for its localization and function (17). To gain insights into the binding properties of the AH, we fused it to the C-terminus of mGFP and expressed the GFP-AH<sub>RocS</sub> fusion ectopically under the control of an inducible promoter. We observed the fluorescence distribution within these cells using
<figure><img src="image_2.png" /><figcaption>**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-(AH<sub>RocS</sub>)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-AH<sub>2x</sub> 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).</figcaption></figure>

phase-contrast and fluorescence microscopy. The signal of GFP-AH<sub>RocS</sub> was diffuse in the cytoplasm, indicating that a single AH was not sufficient to mediate a stable association with the membrane (Fig. 1B). We have reported previously that the AH of RocS shares a homologous sequence with the AH of MinD from *E. coli* (17) (Fig. S1). This protein is known to interact with the membrane *via* its AH upon ATP-dependent dimerization (18, 19). Therefore, we tested whether duplicating the AH of RocS would be sufficient to target the GFP to the membrane. In contrast to the monovalent construct GFP-AH<sub>RocS</sub> and consistent with previous results obtained with the AH of MinD (19), the signal of GFP-(AH<sub>RocS</sub>)₂ was mainly localized at the cell periphery (Fig. 1B). This indicates that this fusion binds to the membrane of *S. pneumoniae* and suggests that RocS requires at least dimerization to associate with the membrane.

To investigate the dynamics of membrane association and its role in RocS function, we assessed the localization of GFP-tagged RocS harboring a duplicated AH (GFP-RocS-AH<sub>2x</sub>) expressed at the native chromosomal locus. Throughout the study, we confirmed the expression and stability of all our constructs with a polyclonal anti-GFP antibody (Fig. S2). The localization of GFP-RocS-AH<sub>2x</sub> was similar to GFP-RocS WT, that is, they formed bright foci close to the membrane (Fig. 1C). We also examined chromosome segregation using the merodiploid HlpA-mKate2 fluorescent reporter to visualize the nucleoid and quantify segregation defects (see Material and Methods, Fig. S3) (16). Cells expressing RocS-AH<sub>2x</sub> exhibited limited segregation defects (Fig. 1D, segregation defects RocS WT: 0.92%, *N* = 1,220 vs RocS-AH<sub>2x</sub>: 1.66%, *N* = 1,730—*P*-value = 0.0167) and cell growth was not impacted (Fig. 1E). A similar phenotype was observed when we substituted the AH of RocS for the transmembrane domain of MapZ (TM-Ex<sub>1</sub>), a transmembrane protein unrelated to chromosome segregation (20–22). In particular, GFP-RocS-AH::TM displayed a WT-like localization (Fig. 1C) and its untagged version supported normal growth (Fig. 1E). Chromosome segregation was also slightly impacted compared to the WT (Fig.
1D, segregation defects RocS WT: 0.92%, N = 1,220 vs RocS-AH::TM: 3.13%, N = 1,168—P-value = 0.0185). Finally, we compared both mutants to a strain expressing a version of RocS devoid of AH (RocS-ΔAH) which has been previously characterized (17). As expected, this mutant was considerably more impacted, with growth and chromosome segregation defects comparable to the markerless deletion mutant ΔrocS (Fig. 1D and E, segregation defects RocS-ΔAH: 22.16%, N = 1,193 vs ΔrocS: 24.73%, N = 1,077—P-value = 0.3067). In summary, RocS requires prior oligomerization to interact with the membrane and this interaction is required for the RocS function. In addition, altering the nature of the membrane anchor does not drastically impair RocS function.

## The coiled-coil domain drives RocS oligomerization

Since RocS needs to oligomerize to interact with the membrane, we investigated its ability to self-interact using a bacterial adenylate cyclase-based two-hybrid (BACTH) assay. Specifically, the N-terminus of RocS and derivatives was fused to either the T18 or the T25 fragment of the catalytic domain of adenylate cyclase (CyaA) from *Bordetella pertussis* (23). As anticipated, we detected a strong interaction between full-length CyaA-RocS hybrids after 48 h of incubation (Fig. 2A).

We next sought to identify the determinants of this self-interaction. According to two *in silico* coiled-coil structure prediction tools (CoCoNat (24) and CoCoPRED (25)), the C-terminal AH of RocS is immediately preceded by a CCD spanning approximately the entire length of the helix joining the AH to the DBD (Fig. 2B; Fig. S4). The CCD is predicted to assemble into a parallel dimer (Fig. 2B; Fig. S4). In addition, the analysis of the sequence indicates that the CCD contains a conserved domain of unknown function DUF 536 (Pfam PF04394, InterPro IPR007489, see below), recently renamed RocS-like_C and herein referred to as DUF. The CCD was thus divided into two subdomains, the CC₁ from residues D68-E102 and the DUF from residues D104-D146 (Fig. 2C), which were examined separately to decipher their potential contribution to the oligomerization of RocS. Removing CC₁ completely abrogated the interaction between the CyaA fusions (Fig. 2A). In line with this observation, GFP-RocS-ΔCC₁ displayed a signal dispersed in the cytoplasm and no longer formed distinct foci, nor was enriched at the membrane (Fig. 2D). In addition, a strain expressing RocS-ΔCC₁ was characterized by severe growth and chromosome segregation defects comparable to a ΔrocS mutant (Fig. 2E and F), thus confirming that self-interaction is crucial for the function of RocS. By contrast, we detected a strong interaction between CyaA-RocS-ΔDUF derivatives (Fig. 2A), indicating that this domain is not required for homotypic interaction. However, GFP-RocS-ΔDUF exhibited a signal similar to the ΔCC₁ fusion, that is, cytosolic (Fig. 2D), revealing that this domain is still essential for the interaction of RocS with the membrane. Surprisingly, the expression of the corresponding untagged truncated protein resulted in intermediate growth and chromosome segregation defects (Fig. 2E and F). Indeed, while the deletion of RocS or the ablation of the CC₁ domain generated around 24% of cells with an abnormal chromosome content (Fig. 2E, segregation defects ΔrocS: 24.73%, N = 1,077 vs RocS-ΔCC₁: 22.97%, N = 1,318—P-value = 0.3564), the absence of the DUF only resulted in 16% of cells with a chromosome segregation defect (Fig. 2E, segregation defects RocS-ΔDUF: 15.69%, N = 1,915 vs ΔrocS: P-value = 0.0068; vs RocS-ΔCC₁: P-value = 0.0089). Note that all the truncated versions of RocS were stable and expressed at similar levels (Fig. S2). Collectively, these results highlight two distinct modules within the CCD, the CC1 and the DUF, each contributing differently to the function of RocS. While the CC₁ is required for the homodimerization and subsequent membrane association of RocS, the DUF likely serves another function that is crucial for RocS-mediated chromosome segregation.

## Three conserved glutamines underpin the function of the DUF 536

To build upon our observations regarding the DUF, we analyzed its sequence conservation. According to Pfam (26), the DUF536 domain is defined by the presence of three highly conserved glutamines (Fig. 2C; Fig. S5). To test whether these residues contribute
<figure><img src="image_3.png" /><figcaption>**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).</figcaption></figure>

to the function of RocS, we mutated them into alanine ($DUF_{Q3A}$) or glutamate ($DUF_{Q3E}$) and investigated the localization of their cognate GFP fusion. First, we observed that the signal displayed by $GFP-RocS_{Q3E}$ was cytosolic, reminiscent of the localization of GFP-RocS-ΔDUF (Fig. 2D). Furthermore, the RocS-DUF$_{Q3E}$ mutant phenocopied the RocS-ΔDUF mutant, presenting similar growth and segregation defects (Fig. 2E and F, segregation defects RocS-DUF$_{Q3E}$: 16.93%, *N* = 1,172 vs RocS-ΔDUF: 15.69%, *N* = 1,915—*P*-value = 0.4532). Unexpectedly, we observed a completely different behavior when the glutamines were mutated into alanines. Indeed, $GFP-RocS-DUF_{Q3A}$ formed bright static foci, characteristic of the localization of the WT fusion (Fig. 2D). However, the expression of RocS-DUF$_{Q3A}$ did not result in a WT-like phenotype. On the contrary, growth was even more affected than for a RocS-DUF$_{Q3E}$ mutant and the segregation defects were also very severe (Fig. 2E and F, segregation defects RocS-DUF$_{Q3A}$: 20.10%, *N* = 1,002 vs RocS-DUF$_{Q3E}$: *P*-value = 0.1551). Note that all the mutated versions of RocS were stable and expressed at similar levels (Fig. S2). In summary, these results establish the conserved
glutamines as major determinants of the function of the DUF. Moreover, they reveal the
previously overlooked importance of this domain for RocS function.

## The DNA-binding domain of RocS is phosphorylated by StkP

We have previously shown that the first 55 amino acids of RocS are predicted to form
a helix-turn-helix (HTH) domain, a DNA-recognition motif in which two α-helices are
separated by a sharp turn (17, 27). An AlphaFold3 prediction further suggests that the
HTH of RocS could be followed by an additional β-strand hairpin (wing), characteristic
of the winged HTH (wHTH) subfamily (Fig. 2C, 3A, and B) (27). Interestingly, a DALI
search of the wHTH prediction model of RocS revealed that this domain shares structural
homology with the *Staphylococcus aureus* plasmid partitioning protein Par (28) and
the *Bacillus subtilis* chromosome segregation protein RacA (4, 29) (Fig. 3A), which both
harbor a MerR-type wHTH domain (30, 31). According to the structure of the correspond-
ing protein-DNA complexes, the helix α2 is inserted in the major groove while the wing
primarily contacts the phosphate backbone and possibly the minor groove (30, 31). This
interaction is also suggested by the AlphaFold3 modeling of a RocS dimer in a complex
with a double-stranded (ds) DNA fragment (Fig. 3B).

Interestingly, threonine 41 (T41), located within the wing (Fig. 3A and B), was recently found to be phosphorylated when cells were grown in the presence of ampicillin (14). This antibiotic is known to increase the activity of the unique Serine-Threonine kinase StkP in S. pneumoniae (12). To assess whether T41 is also phosphorylated by StkP in normal growth conditions, we immunoprecipitated a GFP-RocS fusion expressed from the native promoter at the chromosomal locus in a WT or a ΔstkP genetic background and performed a phosphoproteomic analysis following a phosphopeptide enrichment procedure (see Materials and Methods). We detected that T41 was phosphorylated in WT cells but not in the strain ΔstkP (Fig. 3C), thus confirming that RocS is a bona fide substrate of StkP in normal growth conditions.

## Modulation of DNA binding by phosphorylation affects chromosome segregation

The structural alignment of the wHTH domains from RocS and RacA shows that T41 corresponds to Y36 in RacA (Fig. 3A), an amino acid previously implicated in the interaction of RacA with the phosphate backbone of DNA (30). Therefore, we hypothesized that phosphorylation might affect its interaction with DNA. To explore this idea, we overproduced and purified a phosphomimetic (T41D) version of RocS from *E. coli* (Fig. S6). As previously described (17), this construct did not contain the AH and was labeled with a 6xHistidine tag, which was subsequently removed. The binding of the purified proteins to DNA was then assayed by an electrophoretic mobility shift assay (EMSA). RocS WT-ΔAH and RocS G15P-ΔAH (Fig. 4A), previously shown to respectively retain or lose their DNA-binding ability (17), were respectively used as positive and negative controls. We observed that DNA incubated with RocS T41D-ΔAH migrated freely, suggesting that this mutant was unable to interact stably with DNA (Fig. 4A). This observation was confirmed by measuring the binding affinities of RocS-WT and RocS-T41D using microscale thermophoresis (MST). Indeed, the affinity of RocS-T41D for DNA was considerably decreased with an affinity constant (K_D) above 500 µM, more than 10 times higher than the WT (K_D = 34 µM) (Fig. 4B). This indicates that T41 is directly involved in the interaction with DNA and further suggests that the phosphorylation of T41 could disrupt this interaction.

We also examined the nucleoid-binding properties of RocS T41D in *cellulo*. For this purpose, we constructed a fusion of GFP with a truncated version of RocS, in which the AH domain was removed, and expressed ectopically under the control of an inducible promoter. As observed previously when expressed from the native chromosomal locus (17), GFP-RocS-ΔAH co-localized with the nucleoid when expressed from an inducible promoter (Fig. 4C). By contrast, its phosphomimetic counterpart GFP-RocS-T41D-ΔAH no longer co-localized with the nucleoid and displayed a uniform signal inside the
<figure><img src="image_4.png" /></figure>

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.

cytoplasm (Fig. 4C). Taken together, these results show that mimicking phosphorylation impairs DNA binding. This further suggests that phosphorylation negatively modulates the ability of RocS to bind DNA.

Finally, we interrogated the functionality of the phosphomimetic mutant *in vivo* by looking at its subcellular localization and its effect on chromosome segregation and growth. As shown in Fig. 4D, the GFP-RocS T41D fusion did not form the bright foci characteristic of the wild type (17). Instead, it displayed a membrane-enriched localization with transient and local fluorescent enrichments, similar to a truncated mutant lacking the wHTH (GFP-RocS-ΔwHTH, Fig. 4D). Importantly, the GFP-RocS T41D fusion was stable and expressed at similar levels than the wild type (Fig. S2). As self-interaction and subsequent membrane association are not affected in both cases (Fig. S4D and S7), this signifies that RocS does not form higher-order assemblies in the absence of the DBD or when it is phosphorylated. Yet, RocS T41D and RocS-ΔwHTH mutants exhibited a strikingly different phenotype. While the expression of RocS-ΔwHTH resulted in a level of chromosome segregation defects comparable to a ΔrocS mutant (Fig. 4E, segregation defects RocS-ΔwHTH: 22.57%, N = 1,395 vs ΔrocS: 24.88%, N = 1,511—P-value = 0.5416), the expression of RocS T41D generated two times less chromosome segregation defects (Fig. 3F, segregation defects RocS T41D: 9.37%, N = 1,733). Moreover, the phosphomimetic mutant presented normal growth, which contrasted with the strong growth defect
<figure><img src="image_5.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_10.png" /></figure>

FIG 4 RocS phosphorylation and DNA binding and functionality in vivo. (A) Electrophoretic mobility shift assay of RocS-ΔAH (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-ΔAH, DNA and RocS G15P-ΔAH, and DNA and RocS T41D-ΔAH. (B) Microscale thermophoresis binding assay of dnaC_Cy5 to increasing concentrations of RocS WT-ΔAH (blue) or RocS T41D-ΔAH (pink). The variation of the normalized fluorescence (ΔFNorm) 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-ΔAH. Representative fluorescence and phase-contrast images of the S. pneumoniae R6 bgaA::pComX-gfp-RocS WT-ΔAH, (hlpA+) hlpA-mKate2 and R6 bgaA::pComX-gfp-RocS T41D-ΔAH, (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).

observed in both RocS-ΔwHTH and ΔrocS mutants (Fig. 4F). In conclusion, interfering
with the phospho-regulation of RocS is less deleterious than the removal of its DBD.
DISCUSSION

Chromosome segregation in bacteria is a critical process that ensures the faithful partitioning of the genetic material to each daughter cell. Although *S. pneumoniae* encodes for the well-characterized proteins ParB and SMC, which are critical for chromosome segregation in other bacteria (3, 6), these two proteins appear to have a minor contribution (15, 16). In addition, the pneumococcus lacks ParA, the P-loop ATPase that directs segregation to opposite sides of the cell (10, 15). In this context, the discovery of RocS as the major contributor to chromosome segregation in *S. pneumoniae* revealed that the molecular mechanisms governing chromosome segregation are significantly different from those at play in other bacteria (17). In this study, we have dissected the molecular features of RocS to shed light on this alternative partition mechanism and understand the relative contribution of each domain of RocS to chromosome segregation.

Our results show that RocS is organized into three major functional domains: a C-terminal AH acting as a membrane anchor, a central CCD mediating multimerization, and an N-terminal wHTH domain-binding DNA. Although previously classified as a MarR-type wHTH domain (17), advances in secondary structure prediction (33) reveal that the DBD is more likely to have a MerR-type wHTH fold (Fig. 3A and B). In particular, the RocS wHTH shows strong structural similarity to two distant members of the MerR-type wHTH family: the *B. subtilis* chromosome segregation protein RacA (29, 30) and the *S. aureus* plasmid partitioning protein Par (28, 31) (Fig. 3A). Both proteins contain three domains: an N-terminal wHTH domain, a central coiled-coil region, involved in oligomerization and possibly protein-protein interactions, and a C-terminal disordered domain (30, 31). RacA contributes to DNA segregation during sporulation through the binding of repetitive centromere-like sequences on the nucleoid and the interaction with the polar anchor DivIVA (4, 29, 30, 34). Therefore, the RacA protein can be considered as a sub-cellular positioning system that tethers the nucleoid to the pole. However, the biased localization of RocS at the cell equators (17) argues against the implication of DivIVA or an alternative polar anchor in RocS-dependent DNA segregation. On the other hand, Par is a single protein partition system involved in the segregation of the low-copy number plasmid pSK1 (28). Interestingly, the architecture of Par shares an additional similarity with RocS, as it is also predicted to contain the DUF 536. Moreover, an alignment of both sequences reveals that the C-terminal amino acid stretch of Par is practically identical to the AH of RocS and MinD (Fig. S1).

The conservation of the DUF-AH module in Par (Fig. S1) suggests that it could also bind to the membrane and share a common DNA partition mechanism with RocS. While more work is required to identify such a mechanism, it can narrow down previously suggested scenarios (31). By analogy with the partitioning systems of eukaryotic extrachromosomal elements (35), it has been proposed that the Par-dependent plasmid segregation system could bind both specific sequence repeats on the plasmid and degenerated sequence repeats on the chromosome, thus using the nucleoid as a vehicle (31). Therefore, the Par protein could be considered as a sub-cellular positioning system that tethers plasmids to the nucleoid. However, this hitchhiking model cannot be applied to RocS and direct evidence for this mechanism in Par is still lacking. Alternatively, we propose that RocS, and possibly Par, could be considered as a sub-cellular positioning system that tethers the targeted DNA to the membrane. However, this still poses the question of how a single DNA-binding protein can drive segregation by membrane tethering.

The absence of an energy-driven partitioning system might indicate a predominant role in entropy-driven segregation. Entropic forces have previously been proposed to drive bacterial chromosome segregation by favoring the natural separation of replicated DNA molecules, thereby increasing the overall disorder within the cell (36, 37). However, purely entropic forces are generally insufficient for bacterial chromosome segregation and could even hinder chromosome segregation if precise physical conditions are not met (38). In this context, the proteins previously identified as segregation factors could
help create conditions that promote entropy-driven segregation, ensuring that each daughter cell receives one copy of the chromosome. For instance, a biophysical model suggests that ParB and SMC alter the effective topology of the chromosome, thereby redirecting and amplifying entropic forces to enable accurate chromosome segregation (38). Consistently, by tethering the nucleoid to the membrane, RocS could alter the physical state of the chromosome to create the right conditions for entropy-directed segregation. As previously mentioned, the single or double deletion of *parB* and/or *smc* only generates a small percentage of anucleate cells in *S. pneumoniae*. However, the deletion of either gene is synthetic lethal when combined with the deletion of *rocS* (17). Altogether, this suggests that ParB, SMC, and RocS may have additive and possibly synergetic actions on chromosome segregation by altering the topology of the chromosome.

An alternative way to drive chromosome segregation would be through the intervention of another protein. As mentioned before, RocS and Par are predicted to contain the same domain of unknown function (DUF 536). This DUF domain is not required for RocS homodimerization (Fig. 2A) but is crucial for its function in chromosome segregation (Fig. 2E). Moreover, when we mutated three conserved glutamines of the DUF domain into alanines, we observed a strong effect on chromosome segregation while the localization of RocS remained unaffected (Fig. 2D and E). Our findings show that the formation of bright foci near the membrane, typical of the WT localization, relies on RocS ability to dimerize, associate with the membrane (Fig. 2D), and interact with DNA (Fig. 4D). Therefore, the DUF domain, especially through its three conserved glutamines, likely plays an additional crucial role in RocS-mediated chromosome segregation. While we cannot rule out that these conserved glutamines may stabilize interactions at the dimer or oligomer interface, they might also be involved in interactions with an additional partner. Interestingly, a sequence alignment of all proteins predicted to contain the DUF indicates that most proteins also harbor a putative C-terminal MinD-like AH (Fig. S8). Therefore, it is tempting to speculate that the DUF 536 and the AH form a functional unit, which could work in tandem with a yet-to-be-identified partner to form a complete partitioning system. Future work is definitely needed to address this question.

Finally, we showed that the N-terminal wHTH domain, which mediates DNA binding, is phosphorylated by StkP on T41 (Fig. 3C). Specifically, we revealed that a phosphomimetic mutation (T41D) affects the interaction of RocS with DNA (Fig. 4A, B, and C). Protein phosphorylation has been previously suggested to regulate chromosome segregation in mycobacteria (39) and possibly *B. subtilis* (40). On the other hand, Serine-Threonine phosphorylation by StkP in *S. pneumoniae* is generally considered a central regulator of the pneumococcal cell elongation and division, in particular by modulating cell elongation, constriction, and final separation (20, 41–43). Therefore, our study identifies an additional layer of regulation controlled by StkP, related to chromosome segregation. In sum, StkP would not only be dedicated to the control of cell division but would act as a comprehensive coordinator of several, if not all, processes required to achieve a successful cell cycle. This hypothesis is supported by the characterization of other potential substrates of StkP and/or its cognate phosphatase.phpP that are involved in DNA replication and repair such as MutL, SsB, DnaD, and PolA (14, 44). Future studies focusing on the dynamics of RocS phosphorylation during the cell cycle, also in relation to the phosphorylation of the other substrates, should clarify the role of RocS phosphorylation and subsequent perturbation of its association with DNA during the cell cycle.
# MATERIALS AND METHODS

## Strains and growth conditions

Strains of *S. pneumoniae* were grown in C + Y or THY medium at 37°C. For growth on a plate, THY agar supplemented with 3% horse blood was used. The expression of fluorescent probes under the control of the pComX promoter was achieved by the addition of 1 µM of the inducer ComS (45) 30 min prior to imaging or for the whole culture duration for crude extracts preparation. Growth was monitored with a Tecan SUNRISE microtiter plate reader set up with a 550-nm filter. Cells were first grown up to OD<sub>550</sub> = 0.3 in C + Y medium. They were then diluted at OD<sub>550</sub> = 0.0001 and transferred in a Nunc Edge 96-well, non-treated, flat-bottom MicroplateGreen plate, whose lid was preventively treated with a 0.05% Triton-X100 and 20% ethanol mixture to avoid condensation. Growth was monitored at 37°C with OD measurements taken every 10 min after brief shaking. Throughout the manuscript, the growth curves represent the mean of three biological replicates. *E. coli* XL1-Blue, *E. coli* BL21 star (DE3), and *E. coli* BTH101 strains were used for cloning, recombinant protein expression, and BACTH, respectively. All were grown in LB medium supplemented with appropriate chemicals and antibiotics.

## Allelic replacement mutagenesis

The construction the strains used in this study is detailed in Table S1. The oligonucleotides used for cloning purposes are listed in Table S2. Strains of *S. pneumoniae* were derived from R800 rpsL1 (resistant to streptomycin) and transformed with PCR products of interest. Briefly, bacteria were cultivated in C + Y medium (pH 6.8) at 37°C without agitation, and competence was induced at around OD<sub>550</sub> = 0.1 by the addition of the synthetic competence stimulating peptide 1 (CSP1) (46) at 100 ng/10<sup>8</sup> cells. DNA was then mixed with competent cells and incubated at 37°C for 30 min before plating dilutions of the transformed bacteria mixed in THY-agar supplemented with 3% of defibrinated horse blood. After 2 h of incubation at 37°C, an additional THY-agar layer supplemented with the appropriate antibiotic (streptomycin 200 µg/mL, kanamycin 250 µg/mL, or chloramphenicol 4.5 µg/mL) was poured and the cells were left to incubate overnight. Gene replacement or deletion was achieved by homologous recombination following a two-step selection process (47). First, the gene was replaced by a *kanR-rpsL* cassette restoring sensitivity to streptomycin while conferring resistance to kanamycin. Colonies were selected on plates supplemented with kanamycin (0.25 mg/mL). The second step consisted of the cassette's removal and selection on THY-agar plates supplemented with streptomycin (0.2 mg/mL). All strains were verified by colony PCR, antibiotic sensitivity testing, and locus sequencing.

The merodiploid copy of *hlpA* fused to *mKate2* was inserted along with a chloramphenicol resistance marker as previously described (16) and colonies were selected on THY-agar plates supplemented with chloramphenicol (4.5 µg/mL).

## Plasmids construction

The construction of the plasmids used in this study is detailed in Table S1. The oligonucleotides used for cloning purposes are listed in Table S2. BACTH plasmids carrying the full-length RocS were constructed as follows: *rocS* was PCR-amplified from the *S. pneumoniae* R6 strain and inserted in the pUT18C and pkT25 vectors digested XbaI and Acc65I restriction enzymes (New England Biolabs). The derivatives deletion mutants were PCR amplified using designated primers and the resulting linear plasmids were re-circularized using Gibson assembly (New England Biolabs). Expression plasmids carrying mutated versions of RocS were PCR-amplified from a pt7-7 vector carrying *rocS-ΔAH-6His* (17) and circularized using Gibson assembly (New England Biolabs). Plasmids were verified by sequencing and stored in the *E. coli* XL1B strain.
Microscopy techniques and image analyses

The cells were grown up to OD<sub>550</sub> = 0.1–0.2, concentrated 10 times by centrifugation and resuspension, and 1 µL was spotted onto a 1% agarose/C + Y pad. Visualization was performed using a Nikon TiE microscope fitted with an Orca-CMOS Flash4 V2 camera with a 100 × 1.45 objective. Images were collected using NIS-Elements (Nikon) and were analyzed with ImageJ (http://rsb.info.nih.gov/ij/) and the MicrobeJ plugin (48). For the quantification of the chromosome segregation defects, cells were manually classified in the MicrobeJ editor according to their HlpA-mKate2 profile. Specifically, cells were considered as missegregated when exhibiting (i) no HlpA-mKate2 signal, (ii) a cytosolic HlpA-mKate signal, and (iii) an asymmetrical HlpA-mKate2 distribution between two dividing cells (Fig. S3). Statistical analyses were performed on biological triplicates, using unpaired Student's t-test with MicrobeJ, and GraphPad Prism 10 software (www.graphpad.com). Bonferroni correction was used to correct for multiple comparisons.

Preparation of *S. pneumoniae* crude extracts and immunoblot analysis

The strains were grown in C + Y medium up to OD<sub>550</sub> = 0.3–0.5, pelleted and resuspended in TE buffer (10 mM Tris/HCl, pH 8.0, 1 mM EDTA) supplemented with a cocktail of protease inhibitors [CLAPA (Chymostatine 1 µg/mL, Leupeptine 1 µg/mL, Antipaine 1 µg/mL, Pepstatine 1 µg/mL, Aprotinine 5 µg/mL, Sigma-Aldrich) and 1 µg/mL cOmplete, EDTA-free, Roche). Cells were sonicated (Branson 450) with 20 pulses at 3/30 followed by two times 20 pulses at 4/40. The protein concentration was determined by the Bradford method using the Pierce protein assay reagent. 20 µg of total protein extract was loaded onto an SDS-PAGE gel and analyzed by western blotting. A polyclonal anti-gfp primary antibody (Amsbio, TP401) diluted in 1% BSA-TBST (1:10,000) was used in combination with a goat anti-rabbit secondary antibody coupled with horseradish peroxidase (BioRad, 170–6515) diluted in 1% BSA-TBST (1:5,000). The signal was revealed using a SuperSignal West Pico PLUS Chemiluminescent Substrate kit (Thermo Scientific, 34580) according to the manufacturer's instructions.

Bacterial adenylate cyclase two-hybrid assay

The BACTH assay was performed with the BACTH system kit (Euromedex) according to the manufacturer's protocol (49). Briefly, N-terminal fusions of RocS full-length or truncation mutants with T18 or T25 were co-transformed and re-streaked on an LB-agar plate supplemented with ampicillin 100 µg/mL, kanamycin 50 µg/mL, isopropyl-β-D-thiogalactopyranoside (IPTG) 0.5 mM, and X-gal 100 µg/mL. Plates were grown at room temperature and photos were taken at 24, 48, and 72 h to monitor the color shift of the colonies.

Protein production and purification

Recombinant plasmids overexpressing RocS-ΔAH-6His and its variants were transformed *E. coli* BL21 STAR. Cells were grown in 2 L of LB medium supplemented with ampicillin (100 µg/mL) up to OD<sub>600</sub> = 0.4–0.6 before inducing protein expression with 0.5 mM of isopropyl β-D-1-thiogalactopyranoside (IPTG) and further incubation at 25°C with shaking overnight.

Cells were collected by centrifugation and resuspended in buffer A (25 mM Tris/HCl, pH 7.5, 1 M NaCl, 10% glycerol) supplemented with a cocktail of protease inhibitors (CLAPA), 6 µg/mL of DNase I (Roche), 6 µg/mL of RNase A (Roche), and 10 µg/mL of lysozyme (Sigma-Aldrich). After three passages through a Continuous Flow Cell Disruptor (Constant Systems Ltd) at 1.5 kbar, membranes were further solubilized by the addition of 1% Triton (Euromedex) for 30 min. The lysate was clarified via centrifugation (14,000 × g, 30 min, 4°C) and the supernatant was diluted with buffer A1 (25 mM Tris/HCl, pH 7.5; 10% glycerol) to lower the concentration of NaCl to 500 mM (or 300 mM for RocS G15P-ΔAH-6His) before loading onto a 5 mL HisTrap column (Cytiva). The column was washed with 10% of buffer B (25 mM Tris/HCl, 500 mM NaCl, 300 mM imidazole, and
10% glycerol) before being eluted following a linear gradient. Peak fractions were pooled and dialyzed overnight at 4°C in the presence of Tobacco Etch Virus (TEV) protease (0.025 mg per mg of protein), 1 mM DTT, and 0.5 mM EDTA against buffer A2 (25 mM Tris/HCl, 300 mM NaCl, 10% glycerol) supplemented with 1 mM DTT and 0.5 mM EDTA. The next day, the cleaved sample was loaded onto a 5 mL HisTrap column (Cytiva). The flow-through was collected, concentrated, and injected onto a HiLoad 16/600 Superdex 200 pg size exclusion chromatography column (Cytiva) equilibrated in buffer C (25 mM Tris/HCl, pH 7.5, 150 mM NaCl, 5% glycerol). Peak fractions were pooled, concentrated up to 10–20 mg/mL, and flash-frozen in liquid nitrogen for subsequent storage at –70°C.

## Immunoprecipitation and mass spectrometry analysis

S. pneumoniae strains carrying a chromosomally encoded GFP-tagged RocS in a WT or Δ*stkP* background were grown at 37°C in 500 mL of THY broth until OD~550~ = 0.4. Cells were pelleted and washed with buffer A (20 mM Tris/HCl pH 7.5, 200 mM NaCl). The pellet was resuspended in buffer B (100 mM Tris/HCl, 150 mM NaCl, 5 mM MgCl~2~) supplemented with a cocktail of protease inhibitors (CLAPA and 1 µg/mL cOmplete, EDTA-free, Roche) and phosphatase inhibitors (Phosphatase Inhibitor Cocktail 2, Sigma-Aldrich), 6 µg/mL of DNase I (Roche), 6 µg/mL of RNase A (Roche), 50 U of mutanolysine (Sigma-Aldrich), and 8 mg/mL of lysozyme (Sigma-Aldrich). The cells were then sonicated (Branson 450) at 4°C for 4 × 1 min at 4/40. Lysates were further incubated with 1% Triton-X100 (Euromedex) for 1 h at 4°C, then cleared by centrifugation for 30 min at 14,000 × g. The lysates were incubated with 40 µL of GFP-TRAP agarose resin (Chromotek) for 2 h at 4°C on a rotating wheel. After collection by centrifugation, the beads were washed three times with buffer C (10 mM Tris/HCl, pH 7.5, 150 mM NaCl, 500 µM EDTA) containing protease and phosphatase inhibitors. Finally, GFP-RocS was eluted with 30 µL of Laemmli 2× and, heating at 95°C for 10 min. The eluted samples were run onto an SDS-PAGE gel 12.5% and stained with Coomassie.

## Nano LC-MS/MS analysis of purified GFP-RocS

The protein sample in gel was excised from SDS-PAGE and subjected to manual in-gel reduction, alkylation, and digestion. The gel band was reduced with 10 mM DTT in 100 mM NH~4~HCO~3~ (Sigma Aldrich) for 1 h at 57°C and alkylated for 1 h in the dark with 55 mM iodoacetamide in 100 mM NH~4~HCO~3~ (Sigma Aldrich), washed in 25 mM NH~4~HCO~3~, dehydrated with acetonitrile, and dried in a speed-vac. Then the gel pieces were rehydrated with 40 µL trypsin solution, 12.5 ng/µL in 50 mM NH~4~HCO~3~ (trypsin porcine, PROMEGA) for 1 h on ice, and incubated in 50 mM NH~4~HCO~3~ overnight at 37°C. The peptides were extracted twice with 50 µL of acetonitrile/water/formic acid-45/45/10 vol/vol/vol followed by a final extraction with 50 µL of acetonitrile/formic acid (FA)-95/05 vol/vol. Peptides were dried in a speed-vac before nano LC-MS/MS analysis and then suspended in 0.1% HCOOH.

The sample was analyzed using an Ultimate 3000 nano-RSLC (Thermo Scientific, San Jose California) coupled online with a Q Exactive HF mass spectrometer via a nano-elec-trospray ionization source (Thermo Scientific, San Jose California).

5 µL of peptide mixtures was loaded on a PepMap NEO C18 trap-column (300 µm ID × 5 mm, 5 µm, Thermo Fisher Scientific) for 3.0 min at 20 µL/min with 2% ACN, 0.05% TFA in H~2~O, and then separated on a C18 Acclaim Pepmap100 nano-column, 50 cm × 75 mm i.d, 2 mm, 100 Å (Thermo Scientific) with a 40 min linear gradient from 8.8% to 50% buffer B (A: 0.1% FA in H~2~O, B: 0.1% FA in ACN), from 50 to 90% of B in 0.5 min, hold for 4 min, and returned to the initial conditions in 0.5 min for 15 min. The total duration was set to 60 min at a flow rate of 300 nL/min. The oven temperature was kept constant at 40°C.

The sample was analyzed with the TOP20 HCD method: MS data were acquired in a data-dependent strategy selecting the fragmentation events based on the 20 most abundant precursor ions in the survey scan (375–1,600 Th). The resolution of the survey scan was 60,000 at m/z 200 Th. The ion target value for the survey scans in the Orbitrap and the MS² mode were set to 3E6 and 1E5, respectively, and the maximum injection
time was set to 60 ms for both scan modes. Parameters for acquiring HCD MS/MS spectra were as follows: resolution 15,000 at m/z 200 Th, collision energy = 27, and an isolation width of 2 m/z. The precursors with an unknown charge state or a charge state of 1 were excluded. Peptides selected for MS/MS acquisition were then placed on an exclusion list for 20 s using the dynamic exclusion mode to limit duplicates.

## Data analysis

Proteins were identified by database searching using Sequest HT and MS Amanda with Proteome Discoverer 2.5 software (Thermo Scientific) against the RocS sequence and uniprot *S. pneumoniae* R6 database. Precursor mass tolerance was set at 10 ppm, and fragment mass tolerance was set at 0.02 Da, and up to two missed cleavages were allowed. Oxidation (M), acetylation (Protein N-terminus), and Phosphorylation (S, T, Y) were set as variable modification, and Carbamidomethylation (C) as fixed modification. Trypsin was selected as full and two miss-cleavage were allowed. False discovery rate for peptides and proteins was set at 1%. phosphopeptides identified in high confidence were checked manually.

## Electrophoretic mobility shift assay

EMSAs were performed in 6% native polyacrylamide gel. The DNA fragment was amplified on the genome of the *S. pneumoniae* R6 strain *via* PCR using primers CTC GGGACTTTTTTAAGTATGCCC and CCAACTGCTGGACGAGCTGC TAAG. Purified RocS-ΔAH (WT or point mutants) was mixed with 100 ng of DNA fragment (1,000:1 ratio) in a final volume of 20 µL. The binding buffer contained 25 mM Tris/HCl pH 7.5, 100 mM NaCl, and 5% glycerol. The reaction was incubated for 30 min at room temperature. The polyacrylamide gels were pre-run at 4°C at 60 V in 0.5x Tris-Borate-EDTA buffer prior to the loading of the samples. The complexes were then resolved by migration at 4°C for 4 h at 60V. DNA was visualized by bromure ethidium staining and imaging on a UV-transilluminator.

## Microscale thermophoresis

The affinity of RocS WT and mutants for DNA was measured using MST with a Monolith NT.115 Series instrument (NanoTemper Technologies). The DNA probe labeled with Cyanin 5 in 5' (dnaC_Cy5) was reconstituted by annealing a 50 bp forward primer (Cy5-GTCAACCAAGCTTACGAAGCGTCACAACCAGCTGATGAAATTATTGCTCA) labeled with Cyanine 5 at its 5'-extremity and its unlabeled complementary strand (TGAGCAA TAATT TCATCAGCTGGTTGTGACGCTTCGTAAGCTTGGTTGAC) at 95°C for 10 min with subsequent progressive cooling at room temperature. A mix of 10 nM DNA probe (1:1 vol/vol) with increasing concentrations of RocS (from 0.0061 to 200 µM) was loaded into standard Monolith NT.115 capillaries and MST was measured at 25°C in MST buffer (25 mM Tris/HCl pH 7.5, 100 mM NaCl, 5% glycerol) supplemented with 0.025% of Tween-20. The measurements were performed in triplicates and the analysis was carried out with the Monolith Software. The affinity constant (K_D) was derived from the fluorescence change measured in the function of RocS concentration. The data are represented here as the variation of the normalized fluorescence in the function of RocS concentration (three independent experiments). The normalized fluorescence corresponds to the ratio between fluorescence after thermophoresis and initial fluorescence values.

## Structures and AlphaFold model analysis

We generated a model of RocS structure as a dimer bound to dsDNA with AlphaFold3 (Fig. 2C, 3A, and B) (50). We searched for structural homologs of the wHTH domain (residues 1–55) in the protein bank database using the Dali server (http://ekhidna.bio-center.helsinki.fi/dali) (51). The structures of Par (8csh-A), RacA (5i44-B), and the
AlphaFold model of RocS (ranked_0) were aligned based on their secondary structures using the MatchMaker tool from ChimeraX (https://www.rbvi.ucsf.edu/chimerax) (52).

## Sequence alignments and analysis

The 11 C-terminal amino acids (aa) of MinD, RocS, and Par were aligned with MAFFT (53). The wTHH sequences from RocS (aa 1–55), Par (aa 1–51), and RacA (aa 1–50) were aligned according to their secondary structures [structures of Par (8csh-A) and RacA (5i44-B) and the AlphaFold model of RocS (ranked_0) were entered as queries] using Promals3D (http://prodata.swmed.edu/promals3d) (32). The sequences of DUF 536-containing proteins were retrieved from InterPro and aligned with MAFFT (53). All alignments were visualized using Jalview (http://www.jalview.org) (54). AmphipaSeek was used to predict amphipathic helix from the C-terminal sequence of DUF53-containing proteins (55). The circular phylogenetic tree was generated with annotations using iTOL (56).

## ACKNOWLEDGMENTS

We would like to thank Céline Freton for expert technical assistance with microscale thermophoresis experiments, Alexis Michon for his help in data processing, and members of the Grangeasse lab for fruitful discussions. We acknowledge the contribution of the Protein Science plateform of the "Structure Féderative de Recherche" Biosciences Gerland-Lyon-Sud (UMS344/US8), in particular, Adeline Page and Frédéric Delolme for mass spectrometry analysis and Virginie Gueguen-Chaignon for technical advice. In addition, we would like to thank Linda Doubravová and Bohumil Kubeša who kindly shared that RocS was also found phosphorylated in their phosphoproteomics data.

This project has received funding from the European Union's Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement N°955626. It was also supported by grants from the Centre National de la Recherche Scientifique, Université Claude Bernard Lyon 1, and the Bettencourt Schueller Foundation.

## AUTHOR AFFILIATION

¹Molecular Microbiology and Structural Biochemistry, Université de Lyon, CNRS, Lyon, France

## AUTHOR ORCIDs

Margaux Demuysere http://orcid.org/0009-0003-1954-0613

Adrien Ducret http://orcid.org/0000-0002-9076-405X

Christophe Grangeasse http://orcid.org/0000-0002-5484-4589

## FUNDING

<table><thead><tr><td>Funder</td><td>Grant(s)</td><td>Author(s)</td></tr></thead><tbody><tr><td>EC | H2020 | PRIORITY 'Excellent science' | H2020 Marie Skłodowska-Curie Actions (MSCA)</td><td>955626</td><td>Christophe Grangeasse</td></tr><tr><td>Centre National de la Recherche Scientifique (CNRS)</td><td></td><td>Christophe Grangeasse</td></tr><tr><td>Université Claude Bernard Lyon 1 (UCBL)</td><td></td><td>Christophe Grangeasse</td></tr><tr><td>Fondation Bettencourt Schueller (Bettencourt Schueller Foundation)</td><td></td><td>Christophe Grangeasse</td></tr></tbody></table>

## AUTHOR CONTRIBUTIONS

Margaux Demuysere, Conceptualization, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – original draft, Writing – review and editing |
Adrien Ducret, Conceptualization, Formal analysis, Investigation, Methodology, Software, Supervision, Validation, Writing – original draft, Writing – review and editing | Christophe Grangeasse, Conceptualization, Formal analysis, Funding acquisition, Investigation, Project administration, Resources, Supervision, Validation, Visualization, Writing – original draft, Writing – review and editing

DATA AVAILABILITY

All strains, plasmids, and raw data generated in the course of this work are available from
the author upon request.

ADDITIONAL FILES

The following material is available online.

Supplemental Material

Supplemental material (JB00291-24-s0001.pdf). Fig. S1 to S8; Tables S1 and S2.

REFERENCES

1. Reyes-Lamothe R, Sherratt DJ. 2019. The bacterial cell cycle, chromosome inheritance and cell growth. Nat Rev Microbiol 17:467–478. https://doi.org/10.1038/s41579-019-0212-7

2. Gogou C, Japaridze A, Dekker C. 2021. Mechanisms for chromosome segregation in bacteria. Front Microbiol 12:685687. https://doi.org/10.3389/fmicb.2021.685687

3. Nolivos S, Sherratt D. 2014. The bacterial chromosome: architecture and action of bacterial SMC and SMC-like complexes. FEMS Microbiol Rev 38:380–392. https://doi.org/10.1111/1574-6976.12045

4. Ben-Yehuda S, Fujita M, Liu XS, Gorbatyuk B, Skoko D, Yan J, Marko JF, Liu JS, Eichenberger P, Rudner DZ, Losick R. 2005. Defining a centromere-like element in *Bacillus subtilis* by identifying the binding sites for the chromosome-anchoring protein RacA. Mol Cell 17:773–782. https://doi.org/10.1016/j.molcel.2005.02.023

5. Kawalek A, Wawrzyniak P, Bartosik AA, Jagura-Burdzy G. 2020. Rules and exceptions: the role of chromosomal ParB in DNA segregation and other cellular processes. Microorganisms 8:0. https://doi.org/10.3390/microorganisms8010105

6. Jalal ASB, Le TBK. 2020. Bacterial chromosome segregation by the ParABS system. Open Biol 10:200097. https://doi.org/10.1098/rsob.200097

7. Mohl DA, Easter J, Gober JW. 2001. The chromosome partitioning protein, ParB, is required for cytokinesis in *Caulobacter crescentus*. Mol Microbiol 42:741–755. https://doi.org/10.1046/j.1365-2958.2001.02643.x

8. Gerdes K, Møller-Jensen J, Bugge Jensen R. 2000. Plasmid and chromosome partitioning: surprises from phylogeny. Mol Microbiol 37:455–466. https://doi.org/10.1046/j.1365-2958.2000.01975.x

9. Ireton K, Gunther NW, Grossman AD. 1994. spoOJ is required for normal chromosome segregation as well as the initiation of sporulation in *Bacillus subtilis*. J Bacteriol 176:5320–5329. https://doi.org/10.1128/jb.176.17.5320-5329.1994

10. Pinho MG, Kjos M, Veening J-W. 2013. How to get (a)round: mechanisms controlling growth and division of coccoid bacteria. Nat Rev Microbiol 11:601–614. https://doi.org/10.1038/nrmicro3088

11. Fleurie A, Cluzel C, Guiral S, Freton C, Galisson F, Zanella-Cleon I, Di Guilmi A-M, Grangeasse C. 2012. Mutational dissection of the S/T-kinase StkP reveals crucial roles in cell division of *Streptococcus pneumoniae*. Mol Microbiol 83:746–758. https://doi.org/10.1111/j.1365-2958.2011.07962.x

12. Beilharz K, Nováková L, Fadda D, Branny P. Massidda O, Veening J-W. 2012. Control of cell division in *Streptococcus pneumoniae* by the conserved Ser/Thr protein kinase StkP. Proc Natl Acad Sci U S A 109:E905–13. https://doi.org/10.1073/pnas.1119172109

13. Grangeasse C. 2016. Rewiring the pneumococcal cell cycle with serine/threonine- and tyrosine-kinases. Trends Microbiol 24:713–724. https://doi.org/10.1016/j.tim.2016.04.004

14. Ulrych A, Fabrik I, Kupčík R, Vajrychová M, Doubravová L, Branny P. 2021. Cell wall stress stimulates the activity of the protein kinase StkP of *Streptococcus pneumoniae*, leading to multiple phosphorylation. J Mol Biol 433:167319. https://doi.org/10.1016/j.jmb.2021.167319

15. Minnen A, Attaiech L, Thon M, Gruber S, Veening J-W. 2011. SMC is recruited to oriC by ParB and promotes chromosome segregation in *Streptococcus pneumoniae*. Mol Microbiol 81:676–688. https://doi.org/10.1111/j.1365-2958.2011.07722.x

16. Kjos M, Veening J-W. 2014. Tracking of chromosome dynamics in live *Streptococcus pneumoniae* reveals that transcription promotes chromosome segregation. Mol Microbiol 91:1088–1105. https://doi.org/10.1111/mmi.12517

17. Mercy C, Ducret A, Slager J, Lavergne J-P, Freton C, Nagarajan SN, Garcia PS, Noirot-Gros M-F, Dubarry N, Nourikyan J, Veening J-W, Grangeasse C. 2019. RocS drives chromosome segregation and nucleoid protection in *Streptococcus pneumoniae*. Nat Microbiol 4:1661–1670. https://doi.org/10.1038/s41564-019-0472-z

18. Hu Z, Lutkenhaus J. 2003. A conserved sequence at the C-terminus of MinD is required for binding to the membrane and targeting MinC to the septum. Mol Microbiol 47:345–355. https://doi.org/10.1046/j.1365-2958.2003.03321.x

19. Szeto TH, Rowland SL, Habrukowich CL, King GF. 2003. The MinD membrane targeting sequence is a transplantable lipid-binding helix. J Biol Chem 278:40050–40056. https://doi.org/10.1074/jbc.M306876200

20. Fleurie A, Lesterlin C, Manuse S, Zhao C, Cluzel C, Lavergne J-P, Franz-Wachtel M, Macek B, Combet C, Kuru E, VanNieuwenhze MS, Brun YV, Sherratt D, Grangeasse C. 2014. MapZ marks the division sites and positions FtsZ rings in *Streptococcus pneumoniae*. Nature New Biol 516:259–262. https://doi.org/10.1038/nature13966

21. Holečková N, Doubravová L, Massidda O, Molle V, Buriánková K, Benada O, Kofroňová O, Ulrych A, Branny P. 2014. LocZ is a new cell division protein involved in proper septum placement in *Streptococcus pneumoniae*. MBio 6:e01700–14. https://doi.org/10.1128/mBio.01700-14

22. van Raaphorst R, Kjos M, Veening J-W. 2017. Chromosome segregation drives division site selection in *Streptococcus pneumoniae*. Proc Natl Acad Sci U S A 114:E5959–E5968. https://doi.org/10.1073/pnas.1620608114

23. Ladant D, Ullmann A. 1999. Bordatella pertussis adenylate cyclase: a toxin with multiple talents. Trends Microbiol 7:172–176. https://doi.org/10.1016/s0966-842x(99)01468-7

24. Madeo G, Savojardo C, Manfredi M, Martelli PL, Casadio R. 2023. CoCoNat: a novel method based on deep learning for coiled-coil prediction. Bioinformatics 39:btad495. https://doi.org/10.1093/bioinformatics/btad495

25. Feng S-H, Xia C-Q, Shen H-B. 2022. CoCoPRED: coiled-coil protein structural feature prediction from amino acid sequence using deep
neural networks. Bioinformatics 38:720-729. https://doi.org/10.1093/
bioinformatics/btbab744

26. Mistry J, Chuguransky S, Williams L, Qureshi M, Salazar GA, Sonnhammer ELL, Tosatto SCE, Paladin L, Raj S, Richardson LJ, Finn RD, Bateman A. 2021. Pfam: the protein families database in 2021. Nucleic Acids Res 49:D412–D419. https://doi.org/10.1093/nar/gkaa913

27. Aravind L, Anantharaman V, Balaji S, Babu MM, Iyer LM. 2005. The many faces of the helix-turn-helix domain: transcription regulation and beyond. FEMS Microbiol Rev 29:231-262. https://doi.org/10.1016/j.femsre.2004.12.008

28. Simpson AE, Skurray RA, Firth N. 2003. A single gene on the staphylococcal multiresistance plasmid pSK1 encodes a novel partitioning system. J Bacteriol 185:2143-2152. https://doi.org/10.1128/JB.185.7.2143-2152. 2003

29. Ben-Yehuda S, Rudner DZ, Losick R. 2003. RacA, a bacterial protein that anchors chromosomes to the cell poles. Science 299:532-536. https://doi.org/10.1126/science.1079914

30. Schumacher MA, Lee J, Zeng W. 2016. Molecular insights into DNA binding and anchoring by the *Bacillus subtilis* sporulation kinetochore-like RacA protein. Nucleic Acids Res 44:5438-5449. https://doi.org/10.1093/nar/gkw248

31. Chan HY, Jensen SO, LeBard RJ, Figgett WA, Lai E, Simpson AE, Brzoska AJ, Davies DS, Connolly AM, Cordwell SJ, Travis BA, Salinas R, Skurray RA, Firth N, Schumacher MA. 2022. Molecular analysis of pSK1 par: a novel plasmid partitioning system encoded by staphylococcal multiresistance plasmids. J Mol Biol 434:167770. https://doi.org/10.1016/j.jmb.2022.167770

32. Pei J, Grishin NV. 2014. PROMALS3D: multiple protein sequence alignment enhanced with evolutionary and three-dimensional structural information. Methods Mol Biol 1079:263-271. https://doi.org/10.1007/978-1-62703-646-7_17

33. Jumper J, Evans R, Pritzel A, Green T, Figurnov M, Ronneberger O, Tunyasuvunakool K, Bates R, Židek A, Potapenko A, et al. 2021. Highly accurate protein structure prediction with AlphaFold. Nature New Biol 596:583-589. https://doi.org/10.1038/s41586-021-03819-2

34. Wu LJ, Errington J. 2003. RacA and the Soj-SpoOJ system combine to effect polar chromosome segregation in sporulating *Bacillus subtilis*. Mol Microbiol 49:1463-1475. https://doi.org/10.1046/j.1365-2958.2003.03643.x

35. Sau S, Ghosh SK, Liu Y-T, Ma C-H, Jayaram M. 2019. Hitchhiking on chromosomes: a persistence strategy shared by diverse selfish DNA elements. Plasmid 102:19-28. https://doi.org/10.1016/j.plasmid.2019.01.004

36. Jun S, Mulder B. 2006. Entropy-driven spatial organization of highly confined polymers: lessons for the bacterial chromosome. Proc Natl Acad Sci U S A 103:12388-12393. https://doi.org/10.1073/pnas.0605305103

37. Jun S, Wright A. 2010. Entropy as the driver of chromosome segregation. Nat Rev Microbiol 8:600-607. https://doi.org/10.1038/nrmicro2391

38. Harju J, van Teeseling MCF, Broedersz CP. 2024. Loop-extruders alter bacterial chromosome topology to direct entropic forces for segregation. Nat Commun 15:4618. https://doi.org/10.1038/s41467-024-49039-w

39. Baronian G, Ginda K, Berry L, Cohen-Gonsaud M, Zakrzewska-Czerwińska J, Jakimowicz D, Molle V. 2015. Phosphorylation of *Mycobacterium tuberculosis* ParB participates in regulating the ParABS chromosome segregation system. PLoS One 10:e0119907. https://doi.org/10.1371/journal.pone.0119907

40. Shi L, Pigeonneau N, Ravikumar V, Dobrinic P, Macek B, Franjevic D, Noirot-Gros M-F, Mijakovic I. 2014. Cross-phosphorylation of bacterial serine/threonine and tyrosine protein kinases on key regulatory residues. Front Microbiol 5:495. https://doi.org/10.3389/fmicb.2014.00495

41. Fleurie A, Manuse S, Zhao C, Campo N, Cluzel C, Lavergne J-P, Freton C, Combet C, Guiral S, Soufi B, Macek B, Kuru E, VanNieuwenhze MS, Brun YV, Di Guilmi A-M, Claverys J-P, Galinier A, Grangeasse C. 2014. Interplay of the serine/threonine-kinase StkP and the paralogs DivIVA and GpsB in pneumococcal cell elongation and division. PLoS Genet 10:e1004275. https://doi.org/10.1371/journal.pgen.1004275

42. Stamsås GA, Straume D, Ruud Winther A, Kjos M, Frantzen CA, Hävarstein LS. 2017. Identification of EloR (Spr1851) as a regulator of cell elongation in *Streptococcus pneumoniae*. Mol Microbiol 105:954-967. https://doi.org/10.1111/mmi.13748

43. Martínez-Caballero S, Freton C, Molina R, Bartual SG, Gueguen-Chaignon V, Mercy C, Gago F, Mahasenan KV, Muñoz IG, Lee M, Hesek D, Mobashery S, Hermoso JA, Grangeasse C. 2023. Molecular basis of the final step of cell division in *Streptococcus pneumoniae*. Cell Rep 42:112756. https://doi.org/10.1016/j.celrep.2023.112756

44. Hirschfeld C, Gómez-Mejia A, Bartel J, Hentschker C, Rohde M, Maaß S, Hammerschmidt S, Becher D. 2019. Proteomic investigation uncovers potential targets and target sites of pneumococcal serine-threonine kinase StkP and phosphatase PHP. Front Microbiol 10:3101. https://doi.org/10.3389/fmicb.2019.03101

45. Berg KH, Biørnstad TJ, Straume D, Hävarstein LS. 2011. Peptide-regulated gene depletion system developed for use in *Streptococcus pneumoniae*. J Bacteriol 193:5207-5215. https://doi.org/10.1128/JB.05170-11

46. Martin B, Prudhomme M, Alloing G, Granadel C, Claverys J-P. 2000. Cross-regulation of competence pheromone production and export in the early control of transformation in *Streptococcus pneumoniae*. Mol Microbiol 38:867-878. https://doi.org/10.1046/j.1365-2958.2000.02187.x

47. Sung CK, Li H, Claverys JP, Morrison DA. 2001. An rpsL cassette, janus, for gene replacement through negative selection in *Streptococcus pneumoniae*. Appl Environ Microbiol 67:5190-5196. https://doi.org/10.1128/AEM.67.11.5190-5196.2001

48. Ducret A, Quardokus EM, Brun YV. 2016. MicrobeJ, a tool for high throughput bacterial cell detection and quantitative analysis. Nat Microbiol 1:16077. https://doi.org/10.1038/nmicrobiol.2016.77

49. Karimova G, Pidoux J, Ullmann A, Ladant D. 1998. A bacterial two-hybrid system based on a reconstituted signal transduction pathway. Proc Natl Acad Sci USA 95:5752-5756. https://doi.org/10.1073/pnas.95.10.5752

50. Abramson J, Adler J, Dunger J, Evans R, Green T, Pritzel A, Ronneberger O, Willmore L, Ballard AJ, Bambrick J, et al. 2024. Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature New Biol 630:493-500. https://doi.org/10.1038/s41586-024-07487-w

51. Holm L. 2020. DALI and the persistence of protein shape. Protein Sci 29:128-140. https://doi.org/10.1002/pro.3749

52. Pettersen EF, Goddard TD, Huang CC, Meng EC, Couch GS, Croll TI, Morris JH, Ferrin TE. 2021. UCSF ChimeraX: structure visualization for researchers, educators, and developers. Protein Sci 30:70-82. https://doi.org/10.1002/pro.3943

53. Katoh K, Standley DM. 2013. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol 30:772-780. https://doi.org/10.1093/molbev/mst010

54. Waterhouse AM, Procter JB, Martin DMA, Clamp M, Barton GJ. 2009. Jalview version 2-a multiple sequence alignment editor and analysis workbench. Bioinformatics 25:1189-1191. https://doi.org/10.1093/bioinformatics/btp033

55. Sapay N, Guermeur Y, Deléage G. 2006. Prediction of amphipathic in-plane membrane anchors in monotopic proteins using a SVM classifier. BMC Bioinformatics 7:255. https://doi.org/10.1186/1471-2105-7-255

56. Letunic I, Bork P. 2024. Interactive Tree of Life (iTOL) v6: recent updates to the phylogenetic tree display and annotation tool. Nucleic Acids Res 52:W78-W82. https://doi.org/10.1093/nar/gkae268