# Sinorhizobium meliloti FcrX coordinates cell cycle and division during free-living growth and symbiosis by a ClpXP-dependent mechanism

Sara Dendene, Shuanghong Xue, Roza Mohammedi, Adam Vieillard, Quentin Nicoud, Odile Valette, Angela Frascella, Anna Bonnardel, Romain Le Bars, Mickaël Bourge, et al.

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Sara Dendene, Shuanghong Xue, Roza Mohammedi, Adam Vieillard, Quentin Nicoud, et al. Sinorhizobium meliloti FcrX coordinates cell cycle and division during free-living growth and symbiosis by a ClpXP-dependent mechanism. Proceedings of the National Academy of Sciences of the United States of America, 2025, 122 (11), pp.e2412367122. 10.1073/pnas.2412367122. hal-05058332

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# Sinorhizobium meliloti FcrX coordinates cell cycle and division during free-living growth and symbiosis by a ClpXP-dependent mechanism

Sara Dendene, Shuanghong Xue, Roza Mohammedi, Adam Vieillard, Quentin Nicoud, Odile Valette, Angela Frascella, Anna Bonnardel, Romain Le Bars, Mickaël Bourge, et al.

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## To cite this version:

Sara Dendene, Shuanghong Xue, Roza Mohammedi, Adam Vieillard, Quentin Nicoud, et al. Sinorhizobium meliloti FcrX coordinates cell cycle and division during free-living growth and symbiosis by a ClpXP-dependent mechanism. Proceedings of the National Academy of Sciences of the United States of America, 2025, 122 (11), 10.1073/pnas.2412367122. hal-05365005

HAL Id: hal-05365005

https://hal.science/view/index/docid/5365005

Submitted on 14 Nov 2025

HAL is a multi-disciplinary open access
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entific research documents, whether they are pub-
lished or not. The documents may come from
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# Sinorhizobium meliloti FcrX coordinates cell cycle and division during free-living growth and symbiosis by a ClpXP-dependent mechanism

Sara Dendeneª, Shuanghong Xueᵇ, Roza Mohammedª, Adam Vieillardª, Quentin Nicoudª, Odile Valetteᵇ, Angela Frascellaᵇ ID, Anna Bonnardelᵇ, Romain Le Barsª, Mickaël Bourgeª, Peter Mergaertª ID, Matteo Brilliᶜ ID, Benoît Alunniª,¹,² ID, and Emanuele G. Biondiª,ª,b,2,3

Affiliations are included on p. 11.

Edited by Giles Oldroyd, University of Cambridge, Cambridge, United Kingdom; received June 26, 2024; accepted December 30, 2024

Sinorhizobium meliloti is a soil bacterium that establishes a nitrogen-fixing symbiosis within root nodules of legumes. In this symbiosis, S. meliloti undergoes a drastic cellular change leading to a terminally differentiated form, called bacteroid, characterized by genome endoreduplication, increased cell size, and high membrane permeability. Bacterial cell cycle (mis)regulation is at the heart of this differentiation process. In free-living cells, the master regulator CtrA ensures the progression of cell cycle by activating cell division (controlled by FtsZ) and inhibiting DNA replication, while on the other hand the so far poorly unknown downregulation of CtrA and FtsZ is essential for bacteroid differentiation. Here, we combine cell biology, biochemistry, and bacterial genetics to understand the functions of FcrX, a factor that controls both CtrA and FtsZ in free-living growth and in symbiosis. Depletion of the essential gene *fcrX* led to abnormally high levels of FtsZ and CtrA and minicell formation. Using multiple complementary techniques, we showed that FcrX may interact with FtsZ and CtrA. Moreover, *fcrX* transcription is directly controlled by CtrA itself and the FcrX protein displays a cell cycle-dependent pattern. We showed further that FcrX also binds the degradosome complex ClpXP and its adaptors CpdR1 and RcdA, and that CtrA degradation efficiency depends on FcrX. We further showed that, despite weak homology with FliJ-like proteins, only FcrX proteins from closely related species are able to complement S. meliloti *fcrX* function. Finally, deregulation of FcrX showed abnormal symbiotic behaviors in plants suggesting a putative role of this factor during bacteroid differentiation.

symbiosis | nitrogen fixation | cell cycle | Sinorhizobium meliloti | Medicago sativa

Sinorhizobium meliloti belongs to the class of Alphaproteobacteria and is known for its dual lifestyle, as a free-living bacterium in the soil and as a symbiotic endophyte within legumes of the genera Medicago, Melilotus, and Trigonella (1). Free-living S. meliloti cells thrive in the soil and divide asymmetrically to produce two physiologically and morphologically different cell types (Fig. 1A): a smaller cell, unable to replicate its DNA, and a larger cell able to replicate its genome once per cell cycle. The small cell has to differentiate first into the large cell type, upon suitable nutrient conditions, before it can undergo genome replication (2).

To ensure a normal progression of the cell cycle, it has to be tightly regulated. In many alphaproteobacterial species, the response regulator CtrA plays a central role in this process by binding to the DNA, mostly activating or inhibiting transcription of more than a hundred target genes, or inhibiting DNA replication by binding to the origin of replication in several species (3, 4). The function of CtrA is best studied in *Caulobacter crescentus*, an aquatic bacterium that divides asymmetrically, like S. meliloti, giving two morphologically and physiologically different daughter cells (5): a stalked cell, able to replicate immediately after division, and a nonreplicative motile cell that is blocked at the G1 phase. CtrA is under strict control, as its levels oscillate during the cell cycle, reaching a maximum in two moments: i) at the G1 phase (motile cell), when it inhibits DNA replication by binding to specific sites that prevent origin binding of DnaA, the initiation factor of DNA replication; and ii) at the end of S-phase/G2, when CtrA also activates the cell division process (6). To permit this pattern, regulation at the transcriptional and posttranslational (by phosphorylation and proteolysis) levels are involved (7, 8). Proteolysis of CtrA has been intensively investigated in *C. crescentus* in which CtrA is under the control of the protease ClpXP and several conserved adapter proteins, such as CpdR and RcdA, and the stability of CtrA significantly increases in mutants lacking the adaptors (9, 10). In S. meliloti, previous studies have suggested that this mechanism is conserved, but differently

## Significance

During the nitrogen-fixing symbiosis between the alphaproteobacterium Sinorhizobium meliloti and the plant *Medicago sativa* (alfalfa), the interplay of molecular mechanisms governing cell cycle and bacteroid differentiation is a remarkable system that has still many details to be discovered. Here, we describe a bacterial cell cycle regulator, named FcrX, that controls two of the main essential players of cell cycle and bacteroid differentiation: the master regulator CtrA and the tubulin-like Z-ring component FtsZ. This essential factor is potentially participating with the degradosome complex driving the proteolysis of those two important regulators. Constitutive expression of FcrX during nodule development shows an increase of plant biomass, opening interesting paths in the amelioration of biological nitrogen fixation for a sustainable agriculture.

The authors declare no competing interest.

This article is a PNAS Direct Submission.

Copyright © 2025 the Author(s). Published by PNAS. This article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).

¹Present address: Institut national de recherche pour l'agriculture, l'alimentation et l'environnement, AgroParisTech, Institut Jean-Pierre Bourgin, Université Paris-Saclay, Versailles 78000, France.

²B.A. and E.G.B. contributed equally to this work.

³To whom correspondence may be addressed. Email: emanuele.biondi@i2bc.paris-saclay.fr.

This article contains supporting information online at https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.2412367122/-/DCSupplemental.

Published March 12, 2025.
<figure><img src="image_4.png" /><figcaption>**Fig. 1.** *fcrX* is an essential gene in *S. meliloti*. (A) Scheme representing the cell cycle progression of *S. meliloti* in free-living condition and in symbiosis with legume plants. The green color refers to CtrA concentration, which is low at the beginning of S-phase and after cell division in large cells. During bacteroid differentiation CtrA is removed in order to induce morphological changes. (B) Transmission contrast (T) and electron microscopy (EM) of a depletion strain of *fcrX* in presence and absence of the inducer (IPTG). In particular, in no IPTG conditions, the depletion of *fcrX* is causing the formation of small cells (red asterisks). (C) Viability test on *S. meliloti* containing empty plasmid and a depletion strain of *fcrX*. Cells of *fcrX* depletion strain or wild type strain carrying an empty plasmid were grown with IPTG and then washed before plating. From left to right, nondiluted to 1/10⁶ diluted cell suspensions were spotted on an agar plate with or without IPTG. (D) EM of a depletion strain of *fcrX* (EM) and overlay of T and epifluorescence microscopy of a depletion strain of *fcrX* labeled with Syto9. (E) Time-lapse microscopy on a depletion strain of *fcrX* (Top) and wild type cells (Bottom). Orange dots represent the mother cells and the blue dots represent the daughter cells. Only daughter cells are able to produce abnormal mini cells. In wild type all daughter cells are dividing normally, although the “small” cells are delayed in their cycle as they must differentiate first into a large cell. Scale bars corresponds to 2 µm.</figcaption></figure>

from *C. crescentus*, this regulation of CtrA plays an essential role as mutants of all degradosome proteins are nonviable or have a strongly reduced fitness (11, 12).

The ortholog of CtrA has been studied in *S. meliloti*, where it has a similar essential function (12, 13). In *S. meliloti*, CtrA indirectly inhibits DNA replication by a yet-unknown process and activates the cell division by repressing the transcription of the *minCDE* system (12), which ultimately inhibits the polymerization of the tubulin-like FtsZ into a Z ring, responsible of cell constriction at the septum (14). The FtsZ protein is composed of a N-terminal core region, containing a GTPase domain, involved in the polymerization activity, and a C-terminal region responsible for the interaction with other actors (15). In *S. meliloti*, FtsZ is present in two copies, FtsZ1 and FtsZ2; however, only FtsZ1 is essential for Z ring formation, while the second copy lacks the C-terminal domain and its deletion is not lethal (16). As is true for many cell cycle actors, FtsZ1 is expressed at the predivisional phase of the cell cycle (17).

In symbiosis with *Medicago* plants, *S. meliloti* colonizes special root organs, called nodules. There, it fixes atmospheric nitrogen into ammonium that is assimilated by the plant; in return, it receives dicarboxylic acids and other nutrients (18). In nodules, after a stage of multiplication within infections threads, *S. meliloti* is internalized inside plant cells and undergoes a drastic cellular change into a terminally differentiated form known as a bacteroid. Bacteroids are characterized by genome endoreduplication, cell enlargement, and high membrane permeability (19). Previous studies have shown an implication of bacterial cell cycle regulation in this differentiation process (11, 12, 20). Indeed, CtrA and FtsZ become undetectable in bacteroids (20, 21) and mutants that overexpress CtrA are characterized by a symbiotic defect (20). Interestingly, the depletion of *ctrA* in *S. meliloti*, similarly to *C. crescentus*, leads to a cell elongation/enlargement and endoreduplication phenotype that is strikingly

similar to that of bacteroids formed in nodules. The mechanism leading to the downregulation of CtrA and FtsZ in bacteroids is not yet known but studies have shown the involvement of plant-produced Nodule-specific Cysteine-Rich (NCR) peptides (22, 23) (Fig. 1A). Legumes, such as *Medicago truncatula*, produce a wide spectrum of NCR peptides (about 600) that are implicated in the disruption of several cellular processes including the cell cycle, thus resulting in the terminal differentiation of *S. meliloti* (1, 19, 22). Indeed, the treatment of a wild type (WT) strain of *S. meliloti* with the NCR247 peptide leads to a downregulation of CtrA and its regulon. Farkas and colleagues also highlighted a potential interaction between this NCR peptide and FtsZ (21). Overall these data strongly suggest that the regulation of bacterial cell cycle and cell division are playing a major role in the symbiosis process (24).

Here, we identified the role of an essential cell cycle regulator, named FcrX, which is associated with the degradosome complex ClpXP. Our experiments focused on the role of FcrX on CtrA and FtsZ1 and FtsZ2, its transcriptional regulation by CtrA, its conservation across the class *Alphaproteobacteria*, and finally, its role during symbiosis.

## Results

**The *fcrX* Gene Is Essential and Controls Cell Cycle in *S. meliloti*.**
The *fcrX* gene (*SMc00655*, 351 bp Open Reading Frame coding for a 116 aa predicted protein, FtsZ CtrA/cell cycle regulator X) is adjacent to *ctrA* in the *S. meliloti* chromosome in a nonoverlapping head-to-head orientation, with a ca. 500 bp long intergenic sequence that controls the transcription of both genes. The *fcrX* gene appears essential in free-living conditions, based on Tn-seq results (25) (*SI Appendix*, Fig. S1A). In order to study the function of *fcrX*, we constructed a depletion strain by deleting the chromosomal copy of *fcrX* and expressing an extra copy of *fcrX* on a plasmid
under the control of an isopropyl β- d-1-thiogalactopyranoside (IPTG)-inducible *lac* promoter (20, 26). This conditional strain of FcrX grew best in a medium supplemented with 100 µM IPTG (*SI Appendix*, Fig. S1B) and upon removal of IPTG (depletion), it showed dramatic growth defects (Fig. 1B and *SI Appendix*, Fig. S1B) as well as a reduced capacity to form colonies (Fig. 1C), confirming that *fcrX* is essential for the viability of *S. meliloti*. To gain more insights into the function(s) of FcrX, we investigated the FcrX-depleted cells by microscopy and flow cytometry (*SI Appendix*, Fig. S2A and B). Interestingly, the absence of FcrX induced a block of DNA replication and the production of DNA-free small cells, referred to as minicells, as revealed by DNA staining by Syto9 and microscopy observation (Fig. 1D). The accumulation of minicells in an FcrX-depleted suspension was confirmed by DNA/membrane double staining with DAPI/Potomac Gold dyes and flow cytometry analysis (*SI Appendix*, Fig. S3, 10.05% of minicells with IPTG against 39.18% of minicells without IPTG). Thus, upon depletion of *fcrX*, an accumulation of cells with only one genome copy and the observation of small cells with lower amount of DNA content was observed. We further quantified by morphometric analysis the extent of minicell formation (*SI Appendix*, Fig. S4A), demonstrating that their accumulation is associated with FcrX depletion.

We monitored cells during the depletion of FcrX by time-lapse microscopy, compared to WT cells, in order to understand the development of minicells at the cellular level. Minicells tended to form only in daughter cells (small cell) while the mother (large cell) remained able to divide efficiently producing daughter cells, for two more additional cycles and then completely stopped dividing (Fig. 1E). This minicell phenotype can be interpreted as the consequence of an imbalanced cell cycle, with an excess of division and a block of DNA replication, suggesting that FcrX may coordinate these two processes in *S. meliloti*. Consistently, the absence of FcrX led to increased levels of CtrA, FtsZ1, and FtsZ2 proteins, as shown by western blot analysis (Fig. 2A), indicating that FcrX indeed negatively controls the accumulation of CtrA and FtsZ1/2. In parallel, we showed that IPTG has no effect on control strains (*SI Appendix*, Fig. S4B). This result strengthens our hypothesis of the implication of FcrX in the regulation of cell cycle and cell division.

**BATCH Shows That FcrX May Interact with CtrA and FtsZ1/2.**
The three dimension (3D) structure prediction of FcrX performed with the Alphafold2 algorithm showed that the FcrX protein may be composed of two alpha helices in a coiled coil configuration (*SI Appendix*, Fig. S5A). The structure did not reveal the presence of a DNA-binding domain, suggesting that its putative mode of action on CtrA and FtsZ1/2 may be by a direct interaction at the protein level. To verify this hypothesis, an affinity column experiment was performed using His6-FcrX (*SI Appendix*, Fig. S6A) loaded nickel columns in order to identify potential FcrX-interacting proteins (*SI Appendix*, Fig. S6B). The presence of FtsZ1/2 and FcrX itself, together with other proteins listed in *SI Appendix*, Table S1, was detected in the affinity column eluate by mass spectrometry (*SI Appendix*, Fig. S6C). The presence of CtrA on the other hand was only detected by western blot (Fig. 2B), as its low abundance makes it hardly detectable by mass spectrometry. In order to confirm this result, we carried out a bacterial two-hybrid (BACTH) experiment using the *Escherichia coli* carrier (Fig. 2C) (27). FcrX, CtrA, and FtsZ1/2 proteins were fused to domains T18 and T25 of the adenylate cyclase from *Bordetella pertussis* in C- and N-terminal orientations and all possible combinations of prey and bait were introduced in the *E. coli* strain HB101. In this system, FcrX interacts with CtrA and with both FtsZ1 and FtsZ2. FtsZ2 lacks the C-terminal domain that is usually implicated in protein-protein interactions and recruitment of FtsZ partners (28). Thus,

the observed interaction between FcrX and FtsZ2 may be explained by either an unusual interaction with the N-terminal FtsZ domain or by the presence of a functional full-length FtsZ homolog in *E. coli* (FtsZ<sub>Ec</sub>), which could promote the formation of a FcrX-FtsZ<sub>Ec</sub>-FtsZ2 ternary complex. We also tested the interaction of FcrX with itself to verify the possible dimerization of the protein, as observed with the affinity column. As shown in the (Fig. 2C) a strong FcrX-FcrX interaction was observed, which is consistent with a putative oligomeric FcrX structure.

**Subcellular Localization of FcrX and Its Putative Interactors.**
To gain more insights about the function of FcrX, we decided to investigate its subcellular localization and whether it colocalizes with its interactors. FtsZ has been shown to have a mid-cell localization while CtrA is potentially polarly localized in *S. meliloti*, as has been shown previously in *C. crescentus* (29, 30). We constructed a strain expressing a C-terminal translational fusion of FcrX with a yellow fluorescent protein (YFP) and further deleted the chromosomal copy of *fcrX* by transduction (*Materials and Methods*). This approach was used in order to avoid all possible competitions of the wild-type FcrX with the tagged version and it confirmed that the fusion protein retained its function. FcrX localized at the septum, as observed by epifluorescence microscopy (with a less frequent localization at the cell pole, presumably after cell division) (Fig. 2D and E). Image analysis in a synchronized cell population confirmed that FcrX shows a localization at the septum in predivisional phase cells, while in all other time points FcrX signal appears diffuse with rare cases in which it forms foci with no defined subcellular localization (Fig. 2E). We wanted to correlate this result with the subcellular localization of the interactors over the cell cycle. Therefore, we constructed a C-terminal translational fusion of FtsZ1 with a cyan fluorescent protein (CFP), which despite the loss of its functionality retained its native localization (29). Using a similar approach, we tagged FtsZ2 with CFP, showing that FtsZ2 also localizes at the septum presumably interacting with FtsZ1, as it does not contain a C-terminal linker necessary for the interaction with the other proteins of the divosome (31). The investigation of the CtrA localization appeared more complex as N- or C-terminal tags strongly stabilized its levels, which is a lethal condition in *S. meliloti* (12). Therefore, we added a sequence coding for the last 15 amino acids of CtrA that constitute the degradation motif, downstream of CFP to ensure the timely degradation of the fusion protein by proteolysis, then deleted the chromosomal copy of *ctrA* by transduction (*Material and Methods*). FtsZ1 and FtsZ2 CFP-tagged proteins showed the expected mid-cell localization while CtrA showed a localization at the cell pole, consistent with FcrX localization (*SI Appendix*, Fig. S7). Together, these results indicate that FcrX may interact with both CtrA and FtsZ1/2 in vivo but at distinct subcellular localizations.

**Transcription of *fcrX* Is Positively Regulated by CtrA.** In order to define and characterize the *fcrX* promoter, we performed a promoter deletion analysis by introducing into otherwise wild-type *S. meliloti* cells, an extra copy of *fcrX* with different putative promoter lengths located on a plasmid downstream of the IPTG-regulated *lacZ* promoter (*SI Appendix*, Fig. S8). Then, the Δ*fcrX*:tetR was transduced from Δ*fcrX*:tetR + *Plac-fcrX* cells into the obtained clones, and selected in the presence or absence of IPTG. All clones should give viable colonies with IPTG while only the constructs containing a functional promoter of *fcrX* should support viability without IPTG (*SI Appendix*, Fig. S8). Constructs with *PfcrX-fcrX* fragments containing at least the region between -1/-287 were viable, while a shorter promoter fragment (-1/-224) was not active, suggesting that the promoter region of *fcrX* resides
<figure><img src="image_5.png" /></figure>

In this figure only localized signals are represented, otherwise the signal must be considered as diffused. Phases of cell cycle could vary among strains carrying other fluorescent fusions, because of the growth speed.

**Fig. 2.** FcrX down-regulates and interacts directly with the master regulator CtrA and the Z ring proteins (FtsZ1, FtsZ2). (A) Western blot (on the Left) using anti-FtsZ, CtrA, FcrX, and GroEL on the *fcrX* depletion strain in comparison with WT and a strain containing the empty vector used in the *fcrX* depletion strain (Empty). For the depletion strain of *fcrX*, a depleted culture was reincubated with IPTG for 2 h (IPTG 2h) or without for 2 h (2h) or 3 h (3h). On the Right, quantification of signals in three replicates. Depletion of 3 h and permissive condition were compared, quantified, and analyzed statistically (asterisk, Student t test, P-value < 0.05). (B) Affinity column using lysate of cellular samples was analyzed by western blot using CtrA, FcrX, and FtsZ antibodies. Percentages represent imidazole concentration used for washes of the columns (see *Materials and Methods* for details). Membrane was stained with Ponceau. Red shows His6-FcrX. Only the column charged with His6-FcrX shows a red signal. “Lys” in “controls” corresponds to lysed cultures used to charge the columns, which, except for *Escherichia coli* strains, clearly contain the proteins of interest. (C) Bacterial Two hybrid (BACTH) testing FcrX interaction with FtsZ1, FtsZ2, CtrA, and itself. The *Upper Right* box shows negative and positive controls provided by the supplier. 25 and 18 are the two subunits of adenylate cyclase of the BACTH (*Materials and Methods*). (D) Functional FcrX-YFP C-terminal fusion observed by transmission (T) and epifluorescence microscopy (EM) in a asynchronous population of *fcrX* deletion genetic background (see text for details). Bar corresponds to 1 µm. Cells showing mid-cell localization are marked with an orange asterisk, while the cell with polar localization is marked with a blue asterisk. (E) YFP-FcrX subcellular localization in a synchronized cell population over a cell cycle (predivisional phase is at 160 min). Analysis performed on >400 cells in each time point.

within the -287 region and that the -224/-287 contains a critical promoter element.

To confirm these results, we used the plasmid pOT1em (32), which contains genes encoding mCherry and Enhanced Green Fluorescent Protein (EGFP) in opposite directions. Several derivatives of the *fcrX* promoter, described in *SI Appendix*, Fig. S8, as well as the full intergenic region between *fcrX* and *ctrA*, were cloned between the ATGs of mCherry and EGFP. Only the full intergenic region between *fcrX* and *ctrA* was able to express both mCherry (*fcrX*) and EGFP (*ctrA*) (Fig. 3A). The expression of the *ctrA* gene is also controlled by the same intergenic region, suggesting that these two genes may share the same transcriptional regulation. Analysis of the region -224/-287 revealed several interesting characteristics. First, the *ctrA*P1 promoter (33) and the predicted P*fcrX* overlap with RNA-seq-determined putative TSSs (transcriptional start site; see *Materials and Methods* for details) separated only by 20/25 bp (*SI Appendix*, Fig. S8). Second, the promoter of *fcrX* contains a CtrA binding box within the 50 bp region upstream the putative TSS (4, 12). These observations suggest that the activation of the *fcrX* promoter may depend on CtrA. In order to test this hypothesis, we mutated the CtrA-binding box by replacing the 5'-TTAA-3' half box with 5'-GCGC-3' in a pOT1em plasmid carrying the full intergenic region between *ctrA* and *fcrX* (Fig. 3A). It was shown that this kind of mutation prevents the fixation of CtrA on the box (34) and therefore affects its transcriptional activity on the downstream gene (Fig. 3A). Microscopy observation showed that the strain containing the mutated CtrA-binding box did not express the mCherry fluorescence, implicating that CtrA may activate *fcrX* transcription by binding to its box. On the other hand, the mutated CtrA binding box did not change the EGFP expression noticeably, excluding the implication of this box in the regulation of *ctrA* transcription (Fig. 3A).

To confirm the positive regulation of *fcrX* expression by CtrA, we tested the steady-state levels of FcrX upon depletion of CtrA by western blot using antibodies directed against FcrX. In the

absence of CtrA, we observed a significant decrease of FcrX (Fig. 3B). Since CtrA is a DNA-binding protein implicated in transcriptional regulation, we also performed a qRT-PCR in the same conditions. Consistently, *fcrX* expression decreased upon CtrA depletion, confirming the previous observation (*SI Appendix*, Fig. S9). These results build up a regulatory model involving a negative feedback loop between CtrA and FcrX.

Cell cycle regulators are known to be dynamically regulated over the cell cycle, consistent with the oscillatory nature of this biological phenomenon. Because FcrX is closely linked to cell cycle regulation, we wanted to check whether FcrX was subject to oscillation. Therefore, synchronization of a wild-type culture of *S. meliloti* was performed as described before (Fig. 3C) (17). Samples were recovered every 30 min over a full cell cycle and the corresponding total RNA was used to perform a qRT-PCR analysis while cell lysates were used for Western blot experiments using antibodies directed against FcrX. Both transcription and translation of FcrX increased at 90 min of the cell cycle, meaning that FcrX is not only a regulator of the cell cycle but it is also subject to cell cycle oscillation. These data are consistent with previous analyses on the ensemble of cell cycle-regulated genes (17).

**FcrX Is Linked to the ClpXP Degradosome Complex.** Based on the presence of ClpP1 in the affinity column (*SI Appendix*, Table S1), and also considering the positive effect of the depletion of *fcrX* on the steady-state levels of CtrA and FtsZ1-2 we hypothesized that FcrX may function as associated factor of the degradosome and/or being degraded by ClpXP. First, in order to consolidate the preliminary observation of FcrX interacting with ClpP1, we tested this interaction in a BACTH experiment (Fig. 4A), also extending this analysis to the other members of the degradosome complex (ClpX, RcdA, and CpdR), as well as including the other copies of ClpP (ClpP2 and ClpP3) (35). Results showed that FcrX is able to give a positive signal with all members of the degradosome suggesting an interaction. It should be noted that we cannot rule
<figure><img src="image_6.png" /><figcaption>**Fig. 3.** FcrX is cell cycle-regulated and its transcription depends on CtrA. (A) Strain of *S. meliloti* containing the intergenic region between *fcrX* and *ctrA* fused with mCherry and EGFP, respectively. *Lower* panels correspond to the same intergenic region mutated in the CtrA box (see text for details). This mutation does not affect the expression of *ctrA* but it completely abolishes the expression of *fcrX*. (B) Transcriptional levels (qRT-PCR) of *fcrX* in different genetic backgrounds. Expression transcriptional levels of *fcrX* normalized with respect to 16S rRNA were tested upon depletion of *fcrX* and *ctrA*. See text for more details. (C) qRT-PCR of *fcrX* (*Upper* part) and Western blot using anti-FcrX antibodies (GroEL as loading control) on a synchronized population of *S. meliloti*. *Bottom* part represents a timeline of cell cycle phases.</figcaption></figure>

out that the interaction is direct, as in *E. coli* a functional ClpXP protein is also present and it may mediate the interaction of FcrX with the other members of the degradosome.

Further, in order to test whether FcrX itself is subject to degradation, we measured the stability of FcrX in WT cells upon treatment with translation-blocking chloramphenicol (Runoff), as previously described (36) (Fig. 4B). This technique allows the evaluation of the half-lives of proteins. Exponential phase cells were treated with chloramphenicol (*Materials and Methods*) and samples at different time points were analyzed by western blot (Fig. 4B). Results showed that FcrX remains stable in comparison with CtrA that has been shown previously to undergo proteolysis by ClpXP (37). The comparison between CtrA and FcrX suggests that the latter may not be proteolyzed by ClpXP during a time scale of a cell cycle, as is CtrA. This leaves, thus, the hypothesis that FcrX is involved in degradation of at least CtrA. Therefore, CtrA stability may be increased upon depletion of FcrX. To test this, we performed a Runoff experiment after depletion of FcrX, followed by western blotting using anti-CtrA antibodies (Fig. 4

C and D). Results showed that upon FcrX depletion, CtrA stability increases, suggesting that the presence of FcrX and its interaction with the ClpXP degradosome are responsible for the regulation of CtrA protein levels. Regarding FtsZ1 and 2, previous results have linked FtsZ to active proteolysis in the closely related bacterium *C. crescentus* (38).

We finally analyzed how the degradosome complex, focusing on ClpX, may show a subcellular localization over the cell cycle that would be consistent with FcrX localization (Fig. 2E). Therefore, we tagged ClpX with CFP and expressed this tagged protein in *S. meliloti* cells. ClpX showed two localizations, at the pole and at the septum, similarly to FcrX (Fig. 4E). We further quantified this observation in a synchronized population by analyzing a large number of cells in different time points of the cell cycle progression. We found that ClpX shows a polar localization over the cell cycle except for the predivisitional stage (as FcrX) in which ClpX (and presumably the whole degradosome) localizes at the septum (Fig. 4F). These data are consistent with a coordinated action of FcrX with the degradosome and suggest that FcrX may be involved in the degradosome activity.
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**Fig. 4.** FcrX associates with the degradosome and controls CtrA stability. (A) BACTH testing FcrX interaction with FtsZ1, FtsZ2, CtrA, and itself. The *Upper Right* box shows negative and positive controls provided by the supplier. 25 and 18 are the two subunits of adenylate cyclase of the BACTH (*Materials and Methods*). (B) Runoff experiment measuring the stability of CtrA (ClpXP-dependent), GroEL (no known proteolysis), and FcrX. The experiment shows that CtrA indeed has a low stability, while GroEL and FcrX remain stable during the time course. (C) Runoff experiment measuring the stability of CtrA in a FcrX depletion background with and without IPTG (presence and absence of FcrX, respectively). The experiment shows that CtrA stability depends on the presence of FcrX. (D) Three replicates of the experiment showed in panel C, in which bands were normalized (based on GroEL) and quantified. (E) ClpX-CFP C-terminal fusion observed by transmission (T) and epifluorescence microscopy (CFP) in a mixed population of *S. meliloti* (see text for details). Bar corresponds to 1 µm. Cells showing mid-cell localization are marked with a blue asterisks, while the cell with polar localization is marked with an orange asterisk. (F) Cell cycle-regulated localization of ClpX-CFP in a synchronized population of *S. meliloti*. More than 1,000 cells were analyzed for each time point (*Materials and Methods*). Phases of cell cycle could vary among strains carrying other fluorescent fusions, because of the growth speed.
**FcrX Is Potentially Involved in the Establishment of the Legume Symbiosis.**
The terminal bacteroid differentiation that *S. meliloti* undergoes during its interaction with *Medicago* plants involves a remodeling of the cell cycle and its regulatory network (11, 12, 20, 23), prompting us to test the involvement of FcrX in the symbiotic process. Indeed, several factors involved in CtrA degradation, such as DivJ and CpdR1, were associated to bacteroid differentiation (11, 20). We tested the role of FcrX by inoculating *Medicago sativa* plants with the FcrX depletion strain and watered the plants with a range of concentrations of IPTG (0, 10, 100, and 1,000 µM, the latter three able to sustain viability in culture), in order to obtain different levels of FcrX expression. At 28 days post inoculation (dpi) we checked nodule colonization using confocal microscopy (Fig. 5A), measured the plant dry weight (Fig. 5B) and assessed bacteroid differentiation by measuring the bacterial DNA content with flow cytometry (SI Appendix, Fig. S10A). Plant weight decreased when FcrX expression was reduced with respect to the condition with the highest IPTG concentration (Fig. 5B). The confocal microscopy images showed a clear nodule colonization defect, with much less plant cells containing bacteroids in the plants watered with 0 µM and 10 µM and compared to the plants watered with 1 mM IPTG, while 100 µM IPTG showed an intermediate level of differentiated cells. These results suggest that expression of FcrX is important for bacteroid differentiation and the establishment of a fully functional symbiosis. Additionally, we measured other parameters reflecting the performance of the symbiosis. First, the number of viable nodule bacteria was measured at 16 and 32 dpi by plating nodule extracts, showing that viability increases with IPTG although we were able to recover viable cells even in the absence of IPTG in the watering (SI Appendix, Fig. S10 B and C). Then, we measured nodule number, weight, and nitrogen fixation activity with the acetylene reduction assay (ARA) (SI Appendix, Fig. S10 D–F) showing that indeed IPTG 1 mM is the best condition for symbiosis. Finally, we counted by cytometry the number of bacteroids in the five conditions tested (X0, X10, X100, X1000, and WT, as analyzed in SI Appendix, Fig. S10) and the relative amounts with respect to X0 (the lowest count, given 1) were, respectively, 1.3, 3.6, 11.9, and 14.4. These results suggests that nitrogen fixation per bacteroid is similar between IPTG 100 and 1,000 mM, but the difference of plant dry weight could be explained by the number of differentiated bacteroids that is ca. three times more in X1000.

Considering our results showing that FcrX inhibits the accumulation of CtrA, FtsZ1, and FtsZ2 potentially through the ClpXP degradosome complex (Fig. 2A), we wondered whether an extra constitutive expression of FcrX, for example from a plasmid, may promote more CtrA and FtsZ1/2 disappearance inside nodule cells and thereby boost the symbiotic process. To do so, we inoculated plants of *M. sativa* with a *S. meliloti* strain containing a second copy of *fcrX* expressed on a pSRK plasmid under the control of the *Plac* promoter, which has a weak transcriptional leakage in *S. meliloti*, in the absence of IPTG (26). This extra copy of FcrX resulted in a significant plant biomass gain at 28 dpi, as compared to the controls (Fig. 5C). In order to understand the nature of this advantage, we also measured other symbiosis parameters, such as bacterial recovery from nodules, nitrogen fixation (ARA), number of nodules, and weight of nodules (SI Appendix, Fig. S11 A–D). No obvious difference emerged from data. Finally, in order to verify whether FcrX is present in fully differentiated bacteroids, we investigated levels of FcrX by using anti-FcrX antibodies. Unlike CtrA and FtsZ1/2 that are absent from mature bacteroids, FcrX remained detectable (Fig. 5D), supporting a role of FcrX during the establishment of bacteroid differentiation and

maintenance. However, its levels are lower than in free-living cells. It should be noted that FcrX is a factor expressed during active cell division, which is completely blocked in differentiated bacteroids. Therefore, we propose that its presence, even at the lower level, may justify an active role of this factor during differentiation, a hypothesis that will need to be further investigated in the future.

**FcrX Is a Conserved Factor in Several Species of the Class Alphaproteobacteria.** We further wanted to investigate the presence and functionality of FcrX in other bacteria. First, the *S. meliloti* FcrX protein sequence was used to search for similar proteins in the Microbes Online database (39). Orthologs of FcrX, defined by BLAST using Bidirectional Best Hit, were found in several rhizobia (*Bradyrhizobium japonicum*, *Rhizobium leguminosarum*), the human pathogen *Brucella abortus* and the phytopathogen *Agrobacterium vitis*. Except for *A. vitis* for which no information is available, previous Tn-seq data on all those species revealed that the putative FcrXs were all essential in the organism of origin (40–42). Those orthologs were tested for their capacity to complement *fcrX* deletion in *S. meliloti*. We also tested a distant homolog of *fcrX* from *C. crescentus*, which codes for the flagellar protein FliJ (43), located in the same genomic context as *fcrX*. Interestingly, the *fcrX-ctrA* synteny is widely preserved among these bacteria suggesting a possible conservation of *fcrX* function (Fig. 6A). In order to test our hypothesis, we constructed *S. meliloti* strains expressing a copy of these orthologs and we deleted by transduction the chromosomal copy of *fcrX*. Results of this experiment showed that only the expression of the orthologs from *R. leguminosarum* and *B. abortus* were able to support *S. meliloti* growth in a *fcrX* deletion background, implying functional conservation of FcrX. A western blotting experiment was carried out confirming that the overexpression of these orthologs is able to down-regulate the accumulation of FtsZ and CtrA (SI Appendix, Fig. S12). On the other hand, the orthologs from *B. japonicum*, *A. vitis*, and the FliJ homolog from *C. crescentus* were not able to complement the *fcrX* deletion. This complementation analysis, although still limited in the number of tested species, revealed that from a functional point of view, each FcrX has acquired specific functions independent from the phylogenetic distance from *S. meliloti*, as for example FcrX from *A. vitis* (a species close to *S. meliloti*) is not able to complement while FcrX from the more distant *B. abortus* does. In order to have a broader view of FcrX conservation, we searched for FcrX/FliJ orthologs/homologs in other alphaproteobacterial species (Fig. 6B and SI Appendix, Table S2). Significantly similar sequences to either the *S. meliloti* FcrX or the *C. crescentus* FliJ, were only found in the sister orders *Caulobacterales* and *Hyphomicrobiales* (syn. *Rhizobiales*). However, the two queries retrieved sequences in complementary sets of species; for example, a search for FcrX in *C. crescentus* retrieved no results (SI Appendix, Table S2). This may be related to a functional diversification of the same ancestral gene gained at the base of the sister orders *Caulobacterales* and *Hyphomicrobiales*, although this is difficult to demonstrate because of the short length and variability of these sequences, even if a phylogenetic tree of all identified homologs seems to suggest an orthology relationship as the topology is largely congruent with a RecG tree. In parallel, we checked the physical distance between *fcrX/fliJ* and *ctrA* genes, finding that the *ctrA* gene is in proximity to *fcrX* orthologs (or *fliJ* in *Caulobacterales*) and is often transcribed from the same intergenic region (Fig. 6 A and B). Finally, we observed that orthologs able to complement the *S. meliloti* *fcrX* deletion belong to a phylogenetic group that spans the families *Brucellaceae-Rhizobiaceae* (the bradyrhizobia are outside of this clade). However, the functional complementation of *fcrX* is not a
<figure><img src="image_15.png" /><figcaption>**Fig. 5.** FcrX is important for symbiosis. (A) Nodules of 42 dpi plants infected by WT (Left row) and the depletion strain of *fcrX*, in different IPTG-watering conditions, were photographed (Upper) and then sectioned and stained with Calcofluor, Syto9, and IP as explained in *Materials and Methods* (Lower panels at high magnification levels). (B) Dry biomass per plant (Left) and aspect (Right) of 42 dpi *M. sativa* infected by WT and the depletion strain of *fcrX* at different IPTG concentrations. Asterisks correspond to significant differences (more than 20 plants for each condition, less than *P* < 0.0001, Kruskal-Wallis test). NI = Noninoculated. (C) Dry biomass per plant (Left) and aspect (Right) of 42 dpi *M. sativa* infected by WT strain containing an empty vector (empty), WT, and a strain expressing an extra copy of *fcrX* (FcrX+). Asterisks correspond to significant differences (More than 20 plants for each condition, less than *P* < 0.0001, Kruskal-Wallis test). No significant difference between WT and Empty vector. NI = Noninoculated. (D) Western blot using FcrX, FtsZ, CtrA, and GroEL antibodies on free-living and bacteroid cells.</figcaption></figure>

conserved feature of specific clades, as for example, FcrX of *A. vitis* does not complement the lack of FcrX in *S. meliloti*. Finally, the functional diversification between FcrX and FliJ is also supported by the fact that, to the best of our investigations, FcrX has no role in the control of motility.

## Discussion

In every organism, important functions of the cell are controlled by key factors (master regulators) coordinating many related elements. This is the case, for example, in the cell cycle of eukaryotes, which is based on cyclins (44), the sporulation process of *Bacillus subtilis*, controlled by Spo0A (45), and the cell cycle regulation in some alphaproteobacteria, such as *C. crescentus*, where it is controlled by the master regulator signal transduction protein CtrA (46). In this work, we identified a key factor of cell cycle and cell division in *S. meliloti* that we named FcrX (FtsZ Cell cycle Regulator X) that in our model controls two key components of cell cycle, CtrA and FtsZ, through the ClpXP degradosome complex.

FcrX is a surprisingly small protein with no homology to other, so far characterized, regulators. We predicted that FcrX is an alpha helix-rich protein that can oligomerize, and shares ancestry with a

previously characterized chaperone involved in flagellum physiology, named FliJ. In *C. crescentus*, this small protein is specifically required for flagellum functioning through the stabilization of another protein FliI, an ATPase involved in the export of flagellar subunits across the membrane using a dedicated type III protein secretion system (43). The analysis of synteny between FcrX orthologs clearly underlined this association between FcrX and FliJ-FliI, as many orthologs of FcrX are still organized in a *fliIJ* operon. Another striking feature of the analysis of the *fcrX* locus in many alphaproteobacteria is its proximity to the *ctrA* gene. As previously hypothesized, CtrA is considered as an ancestral flagellum regulator in many alphaproteobacterial species, including those in which CtrA also plays a role as cell cycle regulator (4, 47). This conserved characteristic may suggest that FcrX evolved from FliJ, extending or changing its targets from flagellar components to the divisome protein FtsZ and the cell cycle regulator CtrA.

Indeed, we showed here that FcrX interacts with CtrA and its function negatively regulates the steady-state levels of CtrA. Conversely, CtrA, in addition to many cell cycle genes (12), also controls *fcrX* transcription in the second half of the DNA replication phase, as clearly shown in Fig. 3 and *SI Appendix*, Fig. S8. From this point of view, CtrA-FcrX forms an essential negative
A

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<figure><img src="image_18.png" /><figcaption>**Fig. 6.** FcrX is conserved among alphaproteobacterial. (A) Typical organization of *fcrX* genomic loci in model alphaproteobacteria. The presence and the relationship between locations of *fcrX* and *ctrA* genes in those alphaproteobacterial species is highlighted with a blue asterisk in Fig. 6B. In *C. crescentus*, the gap between *filB* and *ctrA* is more than 10 kb. (B) Phylogenetic tree of FcrX orthologs-containing species (as described in *Materials and Methods*). The color code of the tree marks the distance between the *fcrX* and *ctrA* genes (values are bp). Species used in Fig. 6A are marked with a blue asterisk, tree based on RecG sequences. (C) Model for the FcrX role in cell cycle regulation with respect to the main functions of cell cycle and CtrA/DivK/ClpXP essential regulators.</figcaption></figure>

feedback loop contributing to the oscillation of CtrA levels during cell cycle (Fig. 6C). The presence of an essential genetic negative feedback loop was also demonstrated in *C. crescentus*, in which DivK and CtrA are the main components of the feedback loop of this species (8). It is tempting to speculate that although the architecture of cell cycle regulation may change between different organisms, CtrA-DivK (linked by a transcriptional relationship) in *C. crescentus* and CtrA-FcrX in *S. meliloti*, the logical principles behind the regulation remain similar. Moreover, it should be highlighted that in *C. crescent*, and presumably in *S. meliloti* as well, phosphorylation plays a crucial role in the activation of CtrA, suggesting that, for example, the activation of FcrX at a phase in which CtrA is also phosphorylated may contribute to a fine-tuning of its activity.

In addition, this CtrA-related crucial regulatory function of FcrX is not its only role, as this master regulator of cell cycle also negatively controls the main component of the divisome, FtsZ (FtsZ1 and FtsZ2 in *S. meliloti*), by protein-protein interaction. However, at this stage it cannot be excluded that FcrX interaction with FtsZ2 (both in BACTH and affinity columns) may involve a ternary complex with FtsZ1 (either from *E. coli* in BACTH or endogenous one in affinity columns) and FcrX, as FtsZ2 lacks a protein interaction domain found in FtsZ1. This dual activity of FcrX makes this factor unique in the knowledge of cell cycle

regulation in *Alphaproteobacteria*. Although cell division has been shown to usually be regulated by CtrA at the transcriptional level in *S. meliloti*, as well as in *C. crescentus* or *B. abortus* (46), here we identified that a negative regulator of cell cycle able to connect directly to both cell cycle regulation and the divisome itself. This dual activity of FcrX is responsible for its severe depletion phenotype, leading to a block of cell cycle producing minicells that contain no DNA, but also keeping a cell with the genome able to produce new minicells. Considering potential applications that need minicells to perform specific functions (48, 49), the depletion of *fcrX* may represent a minicell factory, which may be exploited in the future for biotechnological purposes.

Here, we propose a mechanism of action for FcrX that, by interacting with the degradosome complex ClpXP and its adapters RcdA and CpdR1, may be regulating FtsZ1, FtsZ2, and CtrA levels during cell cycle. The presence of this regulator in several species of *Alphaproteobacteria*, but not in *C. crescentus*, is not surprising as for example the degradation of CtrA has been shown to depend on PopA in *C. crescentus* (9) while this adapter is not present in *S. meliloti* in which there is FcrX.

Another important aspect of FcrX functionality is related to symmetry and bacteroid differentiation, especially in the context of a putative role in degradation of CtrA. It was previously shown that bacteroids in *S. meliloti*, in order to become functionally mature, must
eliminate CtrA and FtsZ, leading to elongated cells with multiple copies of DNA. Moreover, mutants of the inhibition of CtrA, such as *divJ* and *cpdR1*, were associated to a failure in differentiation and symbiosis (references). The finding of a single regulator that is able to negatively control both CtrA and FtsZ suggests that FcrX may play a role during bacteroid differentiation. Indeed, we have shown that FcrX is present in mature bacteroids and its function is required for a correct establishment of symbiosis. Accordingly, a strain constitutively expressing FcrX was able to increase plant biomass with respect to the wild-type situation, suggesting that promoting CtrA and FtsZ downregulation can increase the efficiency of the symbiosis, possibly by predisposing bacteria to terminal differentiation and making this process take place earlier rather than increasing it to a higher level. This will open frontiers of sustainable agriculture by the use of improved bacterial inoculants based on FcrX deregulation, although potential reductions of fitness of such strains may be problematic for the inoculants' survival in the soil. Even more interestingly, FcrX appears as a conserved factor in several rhizobial species further suggesting that this approach of plant growth amelioration may be extended to other agronomically important legumes symbionts.

In conclusion, FcrX is a global factor, related to ClpXP degradosome, controlling two essential key functions of the cell, namely the regulation of cell cycle progression (CtrA) and of cell division (FtsZ) (Fig. 6C). This central position and its integrated role in a negative feedback loop with CtrA suggests that cell physiology may rely on FcrX regulation in order to perform higher levels of coordination of cell cycle. In the future, the investigation should move toward exploring how FcrX is regulated and what is the actual mechanism that links FcrX to ClpX degradosome. FcrX indeed represents a small protein with capacities to interact with very diverse targets that may be a tool or a target for the development of antibiotic therapies.

## Materials and Methods

**Growth Conditions.** The strains used in this study are listed in SI Appendix, Table S3. *S. meliloti* 1021 and *E. coli* strains were grown in Yeast Extract Broth (YEB) medium (0.5% beef extract, 0.1% yeast extract, 0.5% peptone, 0.5% sucrose, 0.04% MgSO₄·7H₂O, pH 7.5) at 30 °C and Lysogeny Broth (LB) medium (1% tryptone, 1% NaCl, 0.5% yeast extract) at 37 °C, respectively. Media were supplemented with appropriate antibiotics: Kanamycin (50 µg/mL), Tetracycline (10 µg/mL), Gentamicin (20 µg/mL) for *E. coli*; Streptomycin (200 µg/mL), Kanamycin (200 µg/mL), Tetracycline (2 µg/mL), Gentamicin (20 µg/mL) for *S. meliloti*. Depletion strains of *S. meliloti* were grown in a medium supplemented with IPTG (100 µM for *fcrX* and 1 mM for *ctrA*); the depletion was accomplished by pelleting the culture, washing the pellet three times using YEB medium, and resuspending it in a medium lacking IPTG at OD<sub>600nm</sub> = 0.3. The synchronization experiment was performed as described previously (17).

**Strain Constructions.** A two-step recombination procedure was used to perform the *fcrX* deletion using the integrative plasmid pNPTS138 as previously described (19). Deletions were verified by PCR using primers flanking the recombination locus (see primers in SI Appendix, Table S4).

To construct the fusion between the protein of interest and the fluorescent proteins (CFP or YFP), the Gateway procedure (Thermo Fisher) was used. First, the gene was amplified by PCR (see primers in SI Appendix, Table S4) and then introduced in the pENTR vector. Then, the vectors were mixed with the destination plasmid carrying the gene coding for the fluorescent protein, to perform the LR reaction as recommended by the manufacturer. The final product was amplified by PCR and cloned in the pSRK vector downstream the *Plac* promoter (24) and electroporated into *S. meliloti* as previously described (50).

To transduce the *fcrX::tetR* deletion, the phage M12 was used (51). To do so, the bacteria were grown in LB containing 2.5 mM CaCl₂ and 2.5 mM MgSO₄ and then mixed with the phage to give a multiplicity of infection of 0.5. The mixture was incubated at room temperature for 45 min and subsequently plated on LB plates with the appropriate antibiotics.

In order to identify the *fcrX* promoter, six different *PfcrX-fcrX* constructions were cloned into the *Plac* inducible plasmid pSRK and then electroporated into wild-type *S. meliloti* cells. All *S. meliloti* clones containing an extra plasmid-encoded copy of *fcrX* with different promoter lengths were transduced using a phage M12 lysate produced from the strain Δ*fcrX*:tetR + *Plac-fcrX* and selected in presence or absence of IPTG. To verify the *fcrX* promoter sequence and its regulation by CtrA, the different constructions were introduced in pOTem1 vector (32) using the RF cloning procedure (52).

*S. meliloti* 1021 (WT) Tn-seq data of the *fcrX* gene during growth in YEB medium was obtained from a previous study (25).

**Nodulation Assays and Analysis.** *M. sativa* cultivar Gabès seeds were scarified with pure sulfuric acid for 8 min. After several washes with distilled water, the seed surface was sterilized with bleach (150 ppm) for 30 min and seeds were washed again. Finally, seeds were soaked overnight under agitation in sterile water and then transferred onto a Kalys agar plate for 1 d at 30 °C in the dark to allow the germination. The seedlings were planted in perlite/sand (2:1 vol/vol) in 1.5 L pots in the greenhouse (24 °C, photoperiod 16 h of light and 8 h of dark, humidity 60%) and were inoculated 7 d after planting with 50 mL per pot of the appropriate bacteria at OD<sub>600nm</sub> = 0.05. Plants were watered every 3 d, alternating tap water and a commercial N-free fertilizer (Plant Prod solution [N-P-K, 0-15-40; Fertil] at 1 g per liter). Plants were harvested at 6 wk post inoculation (42 dpi) to analyze bacteroid colonization and nodule development by confocal microscopy, level of bacteroid differentiation by flow cytometry and plant dry mass measurement (53). ARA has been performed as previously described using only nodule-containing roots (54).

**Electron Microscopy.** Bacteria were prefixed by adding an equal volume of fixative [2% glutaraldehyde in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer 200 mM, pH 7.2) to the culture medium. After 20 min, the medium was replaced by 1% glutaraldehyde in HEPES buffer for at least 1 h at 4 °C. Bacteria were then washed with HEPES buffer, concentrated in 2% agarose (LMP Agarose, Sigma A9414), washed again with HEPES buffer and postfixed in 1% osmium tetroxide (EMS 19150) for 1 h at 4 °C. Samples were washed again in distilled water and treated with 1% uranyl acetate (EMS 22400) for 1 h at 4 °C in the dark. Subsequently, samples were dehydrated in a graded series of acetone and embedded in Epon resin. Ultrathin sections (60 to 90 nm) were cut, stained with uranyl acetate and lead citrate and were analyzed using a Tecnai 200 kV electron microscope (field electron interference). Acquisitions were made with a digital camera (Oneview, Gatan).

**Confocal and Wide-Field Microscopy.** Nodule imaging was performed on an SP8X confocal DMI 6000 CS inverted microscope (Leica) equipped with hybrid and photomultipliers detectors, a 10× dry [Plan Apo + DIC (NA: 0.4, Leica)] and a 63× oil immersion [Plan Apo + DIC (NA: 1.4, Leica)] objectives. For each condition, multiple z-stacks were acquired (excitation: 405 nm; collection of fluorescence: 520 to 580 nm for calcofluor, excitation: 488 nm; collection of fluorescence: 520 to 580 nm for Syto9, and excitation: 561 nm; collection of fluorescence: 520 to 580 nm for Propidium iodide). Stacks were transformed into maximum intensity projections using ImageJ software.

Time-lapse experiments were performed on depleted cells deposited on agarose/YEB (with appropriate antibiotics and inducers) and observed every 10 min (up to 16 h) on a Nikon Eclipse Ti E microscope equipped with a Yokogawa CSU-X1-A1 spinning disk system.

Imaging of fluorescent markers in cells and subcellular localizations.

**qRT-PCR Experiment.** RNA was extracted from bacterial culture samples using Maxwell® 16 LEV miRNA Tissue Kit (Promega). Coding DNA (cDNA) was produced using random hexamers as primers and the GoScriptTM Reverse Transcription kit from Promega. Amplification of 16S ribosome RNA (rRNA) and *fcrX* cDNA was made using the SsoFast EvaGreen Supermix 2 × kit (Bio-Rad, France) on a CFX96 Real-Time System (Bio-Rad) instrument and the results were analyzed by Bio-Rad CFX Maestro version 1.1 software (Bio-Rad). For each sample, biological duplicates were performed. Primers are listed in SI Appendix, Table S4.

**Flow Cytometry.** Cells were fixed by heating 10 min at 70 °C and then stained with DAPI (300 µM) and/or Potomac Gold (1 mM), depending on the experiment. After 10 min of incubation at room temperature, the cells were processed with the Cytoflex S bench-top cytometer (Beckman-Coulter) and the data analyzed with CytExpert 2.5 or Kaluza 2.1 software. DAPI was excited by
a 405-nm solid state laser and emission was collected through a 450/45-nm band pass filter. Potomac Gold was excited by a 561-nm solid state laser and emission was collected through a 610/20-nm band pass filter. A customized parameter was made by making a ratio of Potomac Gold fluorescence intensity on DAPI fluorescence intensity. In presence of minicells, Potomac/DAPI ratio is expected to increase.

**Western Blot Experiments.** The bacterial pellets were prepared and frozen at OD<sub>600nm</sub> = 0.6. Western blot was performed as previously described (19). Anti-GroEL are commercial antibodies against *E. coli* GroEL (Abcam). For anti-FcrX antibodies, *fcrX* was cloned into a pET derivative with N-terminal His6 tag using a Gateway cloning procedure as previously described (55). His6-FcrX was purified on a nickel column and rabbits were injected using a 28 d protocol (19). Purified plasma was then used for western blots. CtrA, FtsZ1-2 rabbit polyclonal and FtsZ2 antibodies were created as previously described (12, 21).

**Protein Interaction Experiments.** For the BACTH experiment (27), the recommendations by the supplier (Euromedex) were applied. To construct recombinant proteins, vectors available from Euromedex were used. These vectors enable the in-frame fusion of the protein subunits of adenylate cyclase from *B. pertussis* (T18, T25) at the C and N terminus. To test protein putative interactions, each appropriate combination of vectors was electroporated into the *E. coli* strain HB101, deleted of the gene coding for the endogenous adenylate cyclase (*cya* strain). Positive control corresponds to the Tol-Pal system from *E. coli* (56).

For biochemical protein-protein interaction analysis, a nickel affinity column was used. Cells of *E. coli* BL21 (D3A) expressing His6-FcrX and *E. coli* BL21 (D3A) with no expression vector were induced 3 h with 100 µM IPTG at 30 °C. Cells were harvested, sonicated as previously described (55) and soluble lysate of both strains was loaded onto prepacked nickel columns. After several washes of extraction buffer (Tris 100 mM, NaCl 500 mM, imidazole 30 mM, pH 7.5), an *S. meliloti* sonicated lysate was loaded on the columns, washed as previously described, followed by elution at increasing concentrations of imidazole (5%, 10%, and 30%), collecting the eluates. Samples were loaded on sodium dodecyl-sulfate polyacrylamide gel electrophoresis gels and analyzed by mass spectrometry (details in *SI Appendix*, Table S1) or western blot using antibodies direct against CtrA and FtsZ1 or FtsZ2 (the same polyclonal antibody is able to detect both copies).

**FcrX Conservation Analysis.** Homologs of RecG, FcrX, and CtrA were identified by first blast (57) hits, with an e-value cutoff of $10^{-4}$. Proteomes for all aliphaproteobacteria considered were downloaded from NCBI. Only complete genomes were considered for the analysis. Genomic distances were calculated by using coordinates in the corresponding "gff" file; the distance dividing two genes was defined as the minimum distance in both directions, i.e., taking the circularity of the genome into account. Ancestral state reconstruction of distances was performed and mapped on trees with function contMap from the R package phytools (58). Alignments were performed with Muscle (59) and refined by hand in AliView (60); maximum likelihood phylogenetic reconstructions were performed with IQTree

1. B. Alunni, B. Gourion, Terminal bacteroid differentiation in the legume-rhizobium symbiosis: Nodule-specific cysteine-rich peptides and beyond. *New Phytol.* **211**, 411-417 (2016).

2. R. Hallez, A.-F. Bellefontaine, J.-J. Letesson, X. De Bolle, Morphological and functional asymmetry in alpha-proteobacteria. *Trends Microbiol.* **12**, 361-365 (2004).

3. M. T. Laub, S. L. Chen, L. Shapiro, H. H. McAdams, Genes directly controlled by CtrA, a master regulator of the Caulobacter cell cycle. *Proc. Natl. Acad. Sci. U.S.A.* **99**, 4632-4637 (2002).

4. M. Brill et al., The diversity and evolution of cell cycle regulation in alpha-proteobacteria: A comparative genomic analysis. *BMC Syst. Biol.* **4**, 52 (2010).

5. V. Hughes, C. Jiang, Y. Brun, Caulobacter crescentus. *Curr. Biol.* **22**, R507-R509 (2012).

6. K. C. Quon, B. Yang, I. J. Domian, L. Shapiro, G. T. Marczynski, Negative control of bacterial DNA replication by a cell cycle regulatory protein that binds at the chromosome origin. *Proc. Natl. Acad. Sci. U.S.A.* **95**, 120-125 (1998).

7. K. R. Ryan, S. Huntwork, L. Shapiro, Recruitment of a cytoplasmic response regulator to the cell pole is linked to its cell cycle-regulated proteolysis. *Proc. Natl. Acad. Sci. U.S.A.* **101**, 7415-7420 (2004).

8. E. G. Biondi et al., Regulation of the bacterial cell cycle by an integrated genetic circuit. *Nature* **444**, 899-904 (2006).

9. K. K. Joshi, M. Bergé, S. K. Radhakrishnan, P. H. Viollier, P. Chien, An adaptor hierarchy regulates proteolysis during a bacterial cell cycle. *Cell* **163**, 419-431 (2015).

10. U. Jenal, The role of proteolysis in the Caulobacter crescentus cell cycle and development. *Res. Microbiol.* **160**, 687-695 (2009).

11. H. Kobayashi, N. J. De Nisco, P. Chien, L. A. Simmons, G. C. Walker, Sinorhizobium meliloti CpdR1 is critical for co-ordinating cell cycle progression and the symbiotic chronic infection. *Mol. Microbiol.* **73**, 586-600 (2009).

(61), with options -nt AUTO -alt1000 -bb 1000, which combines ModelFinder, tree search, ultrafast bootstrap, and SH-aLRT test.

**RNA-seq.** Experiment and analysis were performed as previously described (62). Cultures were harvested at OD<sub>600nm</sub> = 0.6 and frozen in liquid nitrogen as previously described (20). Total RNA was prepared using the RNeasy Mini Kit (Qiagen). Ribosomal RNAs were removed using the Bacterial RiboZero (Illumina) and libraries for MiSeq (V3 cassette) were prepared using the Stranded True Seq RNAseq Kit (Illumina). All fastq files are available at the Sequence Read Archive (SRA) of NCBI (accession PRJNA1223161).

**Alphafold2 Prediction.** A full command-line local installation of Alphafold2 in the I2BC server was used to predict the 3D structure of the FcrX protein. The multiple sequence alignment was constructed using the full databases. All templates downloaded on 19th December 2021, were allowed for structure modeling. Five models were predicted, and all of them achieved a high predicted Local Distance Difference Test (pLDDT) score of approximately 94. We selected the best model determined by an average pLDDT score of 94.95.

**Data, Materials, and Software Availability.** Fastq files data have been deposited in SRA of NCBI (accession number PRJNA1223161 in the NCBI BioProject database, https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1223161) (63). All other data are included in the article and/or *SI Appendix*.

**ACKNOWLEDGMENTS.** S.D. and Q.N. were supported by a PhD fellowship from the Université Paris-Saclay. S.X. benefited from a PhD fellowship from the Chinese Scholarship Council. The present work benefited from the core facilities of Imagerie-Gif (http://www.i2bc.paris-saclay.fr), a member of IBiSA (http://www.ibisa.net), supported by France-Biolmaging (ANR-10-INBS-04-01) and from the support of Saclay Plant Sciences-SPS (ANR-17-EUR-0007). We thank the IMM (Marseille, France) Transcriptomic facility for the RNA preparation and the qRT-PCR experiment; we also thank Artemis Kosta and Hugo le Guenno from the IMM Microscopy platform for Electron Microscopy acquisition and analysis. For plant experiments we thank Amanda Edling De Barros. For the ARA we thank Gautier Bernal and Pascal Ratet (IPS2, Paris-Saclay University). This work was funded by the Agence Nationale de la Recherche, grants no. ANR-17-CE20-0011 and ANR-21-CE20-0040. We thank Amira Boukherissa for ALPHAFOLD analysis and Corine Foucault, Armelle Vigouroux, and Solange Morera for technical help.

Author affiliations: "Commissariat à l'énergie atomique et aux énergies alternatives, CNRS, Institute for Integrative Biology of the Cell, Université Paris-Saclay, Gif-sur-Yvette 91198, France; "CNRS, Laboratoire de Chimie Bactérienne, Institut de Microbiologie de la Méditerranée, Turing Center for Living Systems, Aix-Marseille Université, Marseille 13009, France; and "Department of Biosciences, University of Milan, Milan 20122, Italy

Author contributions: S.D., B.A., and E.G.B. designed research; S.D., S.X., R.M., A.V., Q.N., O.V., A.F., A.B., R.L.B., M. Bourge, M. Brill, B.A., and E.G.B. performed research; S.D., R.L.B., M. Bourge, M. Brill, and E.G.B. analyzed data; and S.D., P.M., M. Brill, B.A., and E.G.B. wrote the paper.

12. F. Pini et al., Cell cycle control by the master regulator CtrA in Sinorhizobium meliloti. *PLoS Genet.* **11**, e1005232 (2015).

13. M. J. Barnett, D. Y. Hung, A. Reisenauer, L. Shapiro, S. R. Long, A homolog of the CtrA cell cycle regulator is present and essential in Sinorhizobium meliloti. *J. Bacteriol.* **183**, 3204-3210 (2001).

14. V. W. Rowlett, W. Margolin, The bacterial Min system. *Curr. Biol.* **23**, R553-R556 (2013).

15. A. Sogues et al., Essential dynamic interdependence of FtsZ and SepF for Z-ring and septum formation in Corynebacterium glutamicum. *Nat. Commun.* **11**, 1641 (2020).

16. W. Margolin, FtsZ and the division of prokaryotic cells and organelles. *Nat. Rev. Mol. Cell Biol.* **6**, 862-871 (2005).

17. N. J. De Nisco, R. P. Abo, C. M. Wu, J. Penterman, G. C. Walker, Global analysis of cell cycle gene expression of the legume symbiont Sinorhizobium meliloti. *Proc. Natl. Acad. Sci. U.S.A.* **111**, 3217-3224 (2014), 10.1073/pnas.1400421111.

18. S. R. Long, Rhizobium-legume nodulation: Life together in the underground. *Cell* **56**, 203-214 (1989).

19. P. Mergaard et al., Eukaryotic control on bacterial cell cycle and differentiation in the Rhizobium-legume symbiosis. *Proc. Natl. Acad. Sci. U.S.A.* **103**, 5230-5235 (2006).

20. F. Pini et al., The DivJ, CbrA and PleC system controls DivK phosphorylation and symbiosis in Sinorhizobium meliloti. *Mol. Microbiol.* **90**, 54-71 (2013).

21. A. Farkas et al., Medicago truncatula symbiotic peptide NCR247 contributes to bacteroid differentiation through multiple mechanisms. *Proc. Natl. Acad. Sci. U.S.A.* **111**, 5183-5188 (2014).

22. W. Van de Velde et al., Plant peptides govern terminal differentiation of bacteria in symbiosis. *Science* **327**, 1122-1126 (2010).
23. J. Penterman *et al.*, Host plant peptides elicit a transcriptional response to control the Sinorhizobium meliloti cell cycle during symbiosis. *Proc. Natl. Acad. Sci. U.S.A.* **111**, 3561–3566 (2014).

24. S. Xue, E. G. Biondi, Coordination of symbiosis and cell cycle functions in Sinorhizobium meliloti. *Biochim. Biophys. Acta Gene Regul. Mech.* **1862**, 691–696 (2019).

25. D. Y. Travin *et al.*, Dual-uptake mode of the antibiotic phazolinic prevents resistance acquisition by gram-negative bacteria. *mBio* **14**, e0021723 (2023).

26. S. R. Khan, J. Gaines, R. M. Roop II, S. K. Farrand, Broad-host-range expression vectors with tightly regulated promoters and their use to examine the influence of TraR and TraM expression on Ti plasmid quorum sensing. *Appl. Environ. Microbiol.* **74**, 5053–5062 (2008).

27. G. Karimova, J. Pidoux, A. Ullmann, D. Ladant, A bacterial two-hybrid system based on a reconstituted signal transduction pathway. *Proc. Natl. Acad. Sci. U.S.A.* **95**, 5752–5756 (1998).

28. J. Xiao, E. D. Goley, Redefining the roles of the FtsZ-ring in bacterial cytokinesis. *Curr. Opin. Microbiol.* **34**, 90–96 (2016).

29. X. Ma *et al.*, Interactions between heterologous FtsA and FtsZ proteins at the FtsZ ring. *J. Bacteriol.* **179**, 6788–6797 (1997).

30. R. T. Wheeler, J. W. Gober, L. Shapiro, Protein localization during the Caulobacter crescentus cell cycle. *Curr. Opin. Microbiol.* **1**, 636–642 (1998).

31. P. J. Buske, P. A. Levin, Extreme C terminus of bacterial cytoskeletal protein FtsZ plays fundamental role in assembly independent of modulatory proteins. *J. Biol. Chem.* **287**, 10945–10957 (2012).

32. T. Meyer *et al.*, Regulation of hydroxycinnamic acid degradation drives agrobacterium fabrum lifestyles. *Mol. Plant Microbe Interact.* **31**, 814–822 (2018).

33. J.-P. Schlüter *et al.*, Global mapping of transcription start sites and promoter motifs in the symbiotic α-proteobacterium Sinorhizobium meliloti 1021. *BMC Genomics* **14**, 156 (2013).

34. A. Reisenauer, K. Quon, L. Shapiro, The CtrA response regulator mediates temporal control of gene expression during the Caulobacter cell cycle. *J. Bacteriol.* **181**, 2430–2439 (1999).

35. A. J. Ogden, J. M. McAleer, M. L. Kahn, Characterization of the Sinorhizobium meliloti HsUV and ClpXP protease systems in free-living and symbiotic states. *J. Bacteriol.* **201**, e00498–18 (2019).

36. D. J. Omnus, M. J. Fink, K. Szwedo, K. Jonas, The Lon protease temporally restricts polar cell differentiation events during the Caulobacter cell cycle. *Elife* **10**, e73875 (2021).

37. K. R. Ryan, E. M. Judd, L. Shapiro, The CtrA response regulator essential for Caulobacter crescentus cell-cycle progression requires a bipartite degradation signal for temporally controlled proteolysis. *J. Mol. Biol.* **324**, 443–455 (2002).

38. B. Williams, N. Bhat, P. Chien, L. Shapiro, ClpXP and ClpAP proteolytic activity on divisome substrates is differentially regulated following the Caulobacter asymmetric cell division. *Mol. Microbiol.* **93**, 853–866 (2014).

39. P. S. Dehal *et al.*, MicrobesOnline: An integrated portal for comparative and functional genomics. *Nucleic Acids Res.* **38**, D396–D400 (2009), 10.1093/nar/gkp919.

40. B. J. Perry, M. S. Akter, C. K. Yost, The use of transposon insertion sequencing to interrogate the core functional genome of the legume symbiont Rhizobium leguminosarum. *Front. Microbiol.* **7**, 1873 (2016).

41. J.-F. Sternon *et al.*, Transposon sequencing of Brucella abortus uncovers essential genes for growth in vitro and inside macrophages. *Infect. Immun.* **86**, e00312–18 (2018).

42. C. Baraquet, W. Dai, J. Mendiola, K. Pechter, C. S. Harwood, Transposon sequencing analysis of Bradyrhizobium diazoefficiens 110spc4. *Sci. Rep.* **11**, 13211 (2021).

43. C. Stephens *et al.*, Identification of the fliI and fliJ components of the Caulobacter flagellar type III protein secretion system. *J. Bacteriol.* **179**, 5355–5365 (1997).

44. Z. Wang, Cell cycle progression and synchronization: An overview. *Methods Mol. Biol.* **2579**, 3–23 (2022).

45. A. T. Kovács, Bacterial differentiation via gradual activation of global regulators. *Curr. Genet.* **62**, 125–128 (2016).

46. K. Poncin, S. Gillet, X. De Bolle, Learning from the master: Targets and functions of the CtrA response regulator in Brucella abortus and other alpha-proteobacteria. *FEMS Microbiol. Rev.* **42**, 500–513 (2018).

47. S. E. Greene, M. Brill, E. G. Biondi, A. Komeili, Analysis of the CtrA pathway in Magnetospirillum reveals an ancestral role in motility in alphaproteobacteria. *J. Bacteriol.* **194**, 2973–2986 (2012).

48. H. Yu *et al.*, Minicells from highly genome reduced Escherichia coli: Cytoplasmic and surface expression of recombinant proteins and incorporation in the minicells. *ACS Synth. Biol.* **10**, 2465–2477 (2021).

49. S.-J. Kim, W. Chang, M.-K. Oh, Escherichia coli minicells with targeted enzymes as bioreactors for producing toxic compounds. *Metab. Eng.* **73**, 214–224 (2022).

50. L. Ferri, A. Gori, E. G. Biondi, A. Mengoni, M. Bazzicalupo, Plasmid electroporation of Sinorhizobium strains: The role of the restriction gene hsdR in type strain Rm1021. *Plasmid* **63**, 128–135 (2010).

51. T. M. Finan *et al.*, General transduction in Rhizobium meliloti. *J. Bacteriol.* **159**, 120–124 (1984).

52. F. van den Ent, J. Löwe, RF cloning: A restriction-free method for inserting target genes into plasmids. *J. Biochem. Biophys. Methods* **67**, 67–74 (2006).

53. Q. Nicoud *et al.*, Sinorhizobium meliloti functions required for resistance to antimicrobial NCR peptides and bacteroid differentiation. *mBio* **12**, e0089521 (2021).

54. T. Kazmierczak *et al.*, Specific host-responsive associations between Medicago truncatula accessions and Sinorhizobium Strains. *Mol. Plant Microbe Interact.* **30**, 399–409 (2017).

55. J. M. Skerker, M. S. Prasol, B. S. Perchuk, E. G. Biondi, M. T. Laub, Two-component signal transduction pathways regulating growth and cell cycle progression in a bacterium: A system-level analysis. *PLoS Biol.* **3**, e334 (2005).

56. J. A. Wojdyla *et al.*, Structure and function of the Escherichia coli Tol-Pal stator protein TolR. *J. Biol. Chem.* **290**, 26675–26687 (2015).

57. C. Camacho *et al.*, BLAST+: Architecture and applications. *BMC Bioinformatics* **10**, 421 (2009).

58. L. J. Revell, phytools: An R package for phylogenetic comparative biology (and other things). *Methods Ecol. Evol.* **3**, 217–223 (2012).

59. R. C. Edgar, MUSCLE: A multiple sequence alignment method with reduced time and space complexity. *BMC Bioinformatics* **5**, 113 (2004).

60. A. Larsson, AliView: A fast and lightweight alignment viewer and editor for large datasets. *Bioinformatics* **30**, 3276–3278 (2014).

61. L.-T. Nguyen, H. A. Schmidt, A. von Haeseler, B. Q. Minh, IQ-TREE: A fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. *Mol. Biol. Evol.* **32**, 268–274 (2015).

62. W. Beroual *et al.*, The noncoding RNA CcnA modulates the master cell cycle regulators CtrA and GcrA in Caulobacter crescentus. *PLoS Biol.* **20**, e3001528 (2022).

63. E. G. Biondi, Transcriptome of Sinorhizobium meliloti 1021 in rich medium. NCBI BioProject. https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1223161. Deposited 13 February 2025.
## Supporting Information for

*Sinorhizobium meliloti* FcrX coordinates cell cycle and division during free-living growth and symbiosis by a ClpXP-dependent mechanism

Sara Dendene, Shuanghong Xue, Roza Mohammed, Adam Vieillard, Quentin Nicoud, Odile Valette, Angela Frascella, Anna Bonnardel, Romain Le Bars, Mickaël Bourge, Peter Mergaert, Matteo Brilli, Benoît Alunni

Emanuele G. Biondi

Email: emanuele.biondi@i2bc.paris-saclay.fr

### This PDF file includes:

- Figures S1 to S12

- Tables S1 to S4
<figure><img src="image_19.png" /></figure>

<figure><img src="image_20.png" /><figcaption>**Fig. S1. Data supporting *fcrX* essentiality in *Sinorhizobium meliloti*.</figcaption></figure>

(A) Schematics of the *fcrX-ctrA* locus with Tn-seq insertion frequency when cells are grown in rich medium (YEB). Data come from di Cenzo et al., 2022.

(B) Growth curves of S. meliloti Δ*fcrX*-Plac-*fcrX* strain cultivated in rich medium (YEB) with different IPTG concentrations, as reported in the figure. Wild type strain, which grows as a strain containing an empty plasmid, was shown as positive control.
<figure><img src="image_21.png" /></figure>

<figure><img src="image_22.png" /><figcaption>**Fig. S2.** Cells depleted of FcrX are minicells.</figcaption></figure>

(A) Transmission contrast microscopy of DfcrX-Plac-fcrX with (left) and without IPTG (right). Bars correspond to 10µm.

(B) Image analysis of DfcrX-Plac-fcrX with (grey) and without IPTG (red) (see Material and Methods for details).
<figure><img src="image_23.png" /><figcaption>**Fig. S3.** Cells depleted of FcrZ are empty of DNA.
Flow cytometry using Potomac (membrane quantity) and DAPI (DNA content) in DfcrX+Plac-fcrX with (red) and without IPTG (green). WT cells (blue) were used to determine threshold of Potomac/DAPI ratio. A minimum of 50.000 events were measured per condition. Cells schemes on top illustrate what DNA proportion compared to membrane mass is theoretically expected.</figcaption></figure>
<figure><img src="image_24.png" /><figcaption>Fig. S4. Cells depleted of FcrX are minicells.</figcaption></figure>

A) Flow cytometry analysis using Potomac (membrane quantity) and DAPI (DNA content) in DfcrX+Plac-fcrX with (left) and without IPTG (right). Upper panels represent DNA content of events. Side-Scatter (SSC) represents size/complexity of events. Gated events from the middle panels (minicells) were represented in red in the lower panels. Threshold of minicells gating was determined by taking all events with DNA content inferior to 1C. Density color code in dot plots is represented by a gradient from blue (low events) to red (high events). A minimum of 50.000 events were measured.

B) Western blot using FcrX, CtrA, FtsZ and GroEL antibodies using controls grown with 1 mM IPTG and without. Results were quantified showing that there was no difference between samples. This experiment shows that IPTG has no effect in wild-type conditions on the levels of proteins analyzed in this work. No significant difference was found among samples.
<figure><img src="image_25.png" /><figcaption>**Fig. S5.** Predicted structure of FcrX.</figcaption></figure>

Structure of FcrX (from two different angles) was predicted using AlphaFold2. Darker blue corresponds to higher prediction confidence. The predicted local distance difference test (pLDDT) score (0-100) is a per-residue confidence score, with values greater than 90 indicating high confidence, and values below 50 indicating low confidence.
<figure><img src="image_26.png" /></figure>

<table><caption>Fig. S6. His6-FcrX interacting proteins purified by affinity on a nickel column.</caption><thead><tr><th>Description</th><th>Score Sequest HT</th></tr></thead><tbody><tr><td>Cyclic beta-(1,2)-glucan synthase NdvB</td><td>83,56</td></tr><tr><td>ATPase subunit beta</td><td>67,00</td></tr><tr><td>ATPase subunit alpha</td><td>42,00</td></tr><tr><td>Nitrous-oxide reductase</td><td>30,00</td></tr><tr><td>ATPase subunit alpha</td><td>27,00</td></tr><tr><td>ATPase subunit b 1</td><td>27,00</td></tr><tr><td>ATPase subunit delta</td><td>26,00</td></tr><tr><td>Glutaminase</td><td>23,00</td></tr><tr><td>Endopeptidase Clp 1 Smc01903</td><td>23,00</td></tr><tr><td>Membrane protein YidC</td><td>21,00</td></tr><tr><td>RNA polymerase sigma factor RpoD</td><td>19,00</td></tr><tr><td>ATPase subunit alpha</td><td>16,00</td></tr><tr><td>Flagellin B</td><td>15,00</td></tr><tr><td>Protein RecA</td><td>14,00</td></tr><tr><td>Endopeptidase Clp 1 Smc01903</td><td>10,00</td></tr><tr><td>Cell division protein FtsZ 1</td><td>9,00</td></tr><tr><td>Nitrogen fixation protein FixI</td><td>9,00</td></tr><tr><td>Nitrogen fixation protein FixH</td><td>5,00</td></tr><tr><td>Cell division protein FtsZ 1</td><td>4,76</td></tr><tr><td>Nitrogen fixation protein FixG</td><td>4,00</td></tr></tbody></table>

(A) Purified His6-FcrX (see Material and Methods) as used for the affinity column and antibodies preparation.

(B) SDS-PAGE gel of affinity column experiments, performed as described in Materials and Methods. His6-FcrX (left) precipitated proteins and a negative control (right) were eluted from the column using 30% imidazole buffer and loaded on gel or analyzed by mass spectrometry.

(C) Summary of results of mass spectrometry (as described in Materials and methods and showed in Table S1). The peptide identification score (sequest ht) is represented in the second column (>2).
<figure><img src="image_27.png" /><figcaption>**Fig. S7.** Subcellular localization of FtsZ1, FtsZ2 and CtrA. C-terminal translational fusions of FtsZ1, FtsZ2 and CtrA with CFP were constructed as described in the text. Scale bar corresponds to 2 mm.</figcaption></figure>
<figure><img src="image_28.png" /><figcaption>**Fig. S8.** Determination of *fcrX* promoter by deletion analysis.</figcaption></figure>

Several complementation regions were constructed with different length of the putative *fcrX*
promoter, adding also a Plac promoter (inducible by IPTG). A transduction lysate containing the
*fcrX* gene locus replaced by the tetracycline resistance cassette was transduced in the different S.
*meliloti* backgrounds containing the different *fcrX* constructs (results of the transduction on the right
table with and without IPTG). See text for more details. At the bottom, an RNAseq profile
corresponding to the promoter region shows the consistency of RNAseq profile (see materials and
methods) with the genetics presented here.
A

<figure><img src="image_29.png" /></figure>

B

<figure><img src="image_30.png" /><figcaption>**Fig. S9.** FcrX protein levels of FcrX upon *fcrX* or *ctrA* depletion.</figcaption></figure>

(A) Western blot using anti-FcrX (and anti-GroEL for loading control) antibodies using FcrX depletion and CtrA depletion samples. First lane is purified His6-FcrX. M = Marker (sizes are reported).

(B) Quantification of FcrX band intensity from the Western blot results as shown in panel A (three replicates). Only the difference between + and the depletion conditions in the CtrA depletion strain background is represented, although FcrX is also reduced significantly in the FcrX depletion background.
<figure><img src="image_31.png" /><figcaption>**Fig S10.** *fcrX* expression level affects the symbiosis between *S. meliloti* and *M. sativa*.</figcaption></figure>

WT = Wild type; X = FcrX depletion strains (*DfcrX* + *Plac-fcrX* at different concentration of IPTG (0, 10, 100, 1000 mM). T-

(A) DNA content of bacteroids extracted from 32 dpi nodules determined by flow cytometry as shown in Figure 5A. Heat-killed cells were incubated with propidium iodide (PI) for 20 minutes in order to label the DNA.

(B) Viable cells recovered from single nodules at 16 dpi.

(C) Viable cells recovered from single nodules at 32 dpi.

(D) Number of nodules per plant at 32 dpi.

(E) Nodule dry weight per plant at 32 dpi.

(F) Nitrogen fixation per plant measured by acetylene reduction at 32 dpi. Mean of X0, X10, X100, X1000, respectively, 1.52 (SD 0.95), 3.46 (SD = 0.96), 9.67 (SD = 1.957), 18.58 (SD = 3.24).
<figure><img src="image_32.png" /></figure>

<figure><img src="image_33.png" /></figure>

<figure><img src="image_34.png" /></figure>

<figure><img src="image_35.png" /><figcaption>Fig. S11. Plant analysis as in Figure 5C.</figcaption></figure>

(A) Viable bacterial cells (cfu) recovered per nodule at 32 dpi. No statistical difference has been detected.

(B) Nitrogenase activity per plant measured by acetylene reduction assay at 32 dpi. No statistical difference has been detected.

(C) Number of nodules per plant at 32 dpi. No statistical difference has been detected.

(D) Nodule dry weight per plant at 32 dpi. No statistical difference has been detected.
<figure><img src="image_36.png" /><figcaption>**Fig. S12.** CtrA and FtsZ1 and FtsZ2 steady state levels in *S. meliloti* *fcrX* depletion strains complemented by *S. meliloti*, *B. abortus* and *R. leguminosarum* *fcrXs*. Western blots were performed as described in Materials and Methods.</figcaption></figure>
<table><caption>Table S1. Mass spectrometry data of affinity column of figure S6B.</caption><thead><tr><th colspan="5">Below details of the analysis:</th></tr></thead><tbody><tr><td>Reduction</td><td></td><td></td><td></td><td></td></tr><tr><td>Akylation</td><td></td><td></td><td></td><td></td></tr><tr><td>Digestion</td><td></td><td>Trypsine/LysC</td><td>Primega</td><td></td></tr><tr><td>Q-Exactive</td><td></td><td></td><td></td><td></td></tr><tr><td>RSLC</td><td></td><td>90mins</td><td></td><td></td></tr><tr><td>MS/MS</td><td></td><td>Top10</td><td></td><td></td></tr><tr><td></td><td></td><td>90min</td><td></td><td></td></tr><tr><td></td><td></td><td>QC Helix</td><td></td><td></td></tr><tr><td></td><td></td><td>RV</td><td></td><td></td></tr><tr><td></td><td></td><td>FFM150</td><td></td><td></td></tr><tr><td>Modification :</td><td></td><td>Carbamidomethyl Cystein</td><td></td><td></td></tr><tr><td></td><td></td><td>Oxidation Methionine</td><td></td><td></td></tr><tr><td colspan="5">Filtres: Master Protein, Min 2 Peptides per Protein & High Confidence Peptides</td></tr><tr><td colspan="5">Confidence table:</td></tr><tr><td></td><td>Z =1</td><td>Z=2</td><td>Z=3</td><td>Z=4</td></tr><tr><td>High</td><td>Xcorr &gt;1.2</td><td>Xcorr &gt;1.9</td><td>Xcorr &gt;2.3</td><td>Xcorr &gt;2.6</td></tr><tr><td>Medium</td><td>1.2 &gt; Xcorr &gt; 0.7</td><td>1.9 &gt; Xcorr &gt; 0.8</td><td>2.3 &gt; Xcorr &gt; 1</td><td>2.6 &gt; Xcorr &gt; 1.2</td></tr><tr><td>Low</td><td>Xcorr &lt; 0.7</td><td>Xcorr &lt; 0.8</td><td>Xcorr &lt; 1</td><td>Xcorr &lt; 1.2</td></tr><tr><td colspan="5">Consensus Step Workflow</td></tr><tr><td colspan="5">Result name: 20180418_02_036_EM_BD6</td></tr><tr><td colspan="5">Result file: Z:\CHIHIRO\036_EM036EM\20180418_02_036_EM_BD6.pdResult</td></tr><tr><td colspan="5">Description: Result filtered for high confident peptides</td></tr><tr><td colspan="5">Workflow based on template: CWP. Basic. RequestHT. bestPSMscore</td></tr><tr><td colspan="5">Creation date: 4/24/2018 9:55:04 AM</td></tr><tr><td colspan="5">Created with Discoverer version: 2.1.0.81</td></tr><tr><td colspan="5">The pipeline tree:</td></tr><tr><td colspan="5">1.(0) MSF Files</td></tr><tr><td colspan="5">1.(1) PSM Grouper</td></tr><tr><td colspan="5">1.(2) Peptide Validator</td></tr><tr><td colspan="5">1.(3) Peptide and Protein Filter</td></tr><tr><td colspan="5">1.(4) Protein Scorer</td></tr><tr><td colspan="5">1.(5) Protein Grouping</td></tr><tr><td colspan="5">Post-processing nodes:</td></tr><tr><td colspan="5">1.(7) Display Filter</td></tr><tr><td colspan="5">Processing node 0: MSF Files</td></tr><tr><td colspan="5">1. Spectrum Storage Settings:</td></tr><tr><td colspan="5">- Spectra to Store: Identified or Quantified</td></tr><tr><td colspan="5">2. Merging of Identified Peptide and Proteins:</td></tr><tr><td colspan="5">- Merge Mode: Globally by Search Engine Type</td></tr><tr><td colspan="5">- File Limit for Automatic Merge: 10</td></tr><tr><td colspan="5">3. FASTA Title Line Display:</td></tr><tr><td colspan="5">- Reported FASTA Title Lines: Best match</td></tr><tr><td colspan="5">- Title Line Rule: standard</td></tr><tr><td colspan="5">4. PSM Filters:</td></tr><tr><td colspan="5">- Maximum Delta Cn: 0.05</td></tr><tr><td colspan="5">- Maximum Rank: 0</td></tr><tr><td colspan="5">- Maximum Delta Mass: 0 ppm</td></tr><tr><td colspan="5">Hidden Parameters:</td></tr><tr><td colspan="5">- MSF File(s): Z:\CHIHIRO\036_EM036EM\20180418_02_036_EM_BD6.msf</td></tr><tr><td colspan="5">Processing node 1: PSM Grouper</td></tr><tr><td colspan="5">1. Peptide Group Modifications:</td></tr><tr><td colspan="5">- Site Probability Threshold: 75</td></tr><tr><td colspan="5">2. Display Options:</td></tr><tr><td colspan="5">- Modification Sites Shown: Best Position</td></tr><tr><td colspan="5">Processing node 2: Peptide Validator</td></tr><tr><td colspan="5">1. General Validation Settings:</td></tr><tr><td colspan="5">- Validation Mode: Only PSM level FDR Calculation based on score</td></tr><tr><td colspan="5">- Target FDR (Strict) for PSMs: 0.01</td></tr><tr><td colspan="5">- Target FDR (Relaxed) for PSMs: 0.05</td></tr><tr><td colspan="5">- Target FDR (Strict) for Peptides: 0.01</td></tr><tr><td colspan="5">- Target FDR (Relaxed) for Peptides: 0.05</td></tr><tr><td colspan="5">2. Specific Validator Settings:</td></tr><tr><td colspan="5">- Validation Based on: q-Value</td></tr><tr><td colspan="5">- Use Concatenated FDR Calculation for PSM Level FDR Calculation Based on Score: False</td></tr><tr><td colspan="5">- Reset Confidences for Nodes without Decoy Search (Fixed score thresholds): False</td></tr><tr><td colspan="5">Processing node 3: Peptide and Protein Filter</td></tr><tr><td colspan="5">1. Peptide Filters:</td></tr><tr><td colspan="5">- Peptide Confidence At Least: High</td></tr><tr><td colspan="5">- Keep Lower Confident PSMs: False</td></tr><tr><td colspan="5">- Minimum Peptide Length: 6</td></tr><tr><td colspan="5">- Remove Peptides Without Protein Reference: False</td></tr><tr><td colspan="5">2. Protein Filters:</td></tr><tr><td colspan="5">- Minimum Number of Peptide Sequences: 2</td></tr><tr><td colspan="5">- Count Only Rank 1 Peptides: False</td></tr><tr><td colspan="5">- Count Peptides Only for Top Scored Protein: False</td></tr><tr><td colspan="5">Processing node 4: Protein Scorer</td></tr><tr><td colspan="5">No parameters</td></tr><tr><td colspan="5">Processing node 5: Protein Grouping</td></tr><tr><td colspan="5">1. Protein Grouping:</td></tr><tr><td colspan="5">- Apply strict parimony principle: True</td></tr></tbody></table>
<table><thead><tr><th colspan="12">Identified proteins (245-80 kDa)</th></tr><tr><th>Accession</th><th>Description</th><th>Score Sequest HT</th><th>Coverage</th><th># Peptides</th><th>#PSMs</th><th>#Unique Pept</th><th># Protein Gro</th><th># AAs</th><th>MW [kDa]</th><th>calc. pl</th></tr></thead><tbody><tr><td>17380408</td><td>Cyclic beta-(1,2)-glucan synthase NdvB</td><td>83,56</td><td>14,00</td><td>32,00</td><td>33,00</td><td>32,00</td><td>1,00</td><td>2832,00</td><td>315,55</td><td>6,11</td></tr><tr><td>81633813</td><td>ATP synthase subunit alpha</td><td>27,00</td><td>28,00</td><td>11,00</td><td>11,00</td><td>11,00</td><td>1,00</td><td>509,00</td><td>54,65</td><td>6,74</td></tr><tr><td>21362573</td><td>Glutaminase</td><td>23,00</td><td>31,00</td><td>7,00</td><td>9,00</td><td>7,00</td><td>1,00</td><td>315,00</td><td>33,97</td><td>7,65</td></tr><tr><td>132237</td><td>Protein RecA</td><td>14,00</td><td>16,00</td><td>5,00</td><td>6,00</td><td>5,00</td><td>1,00</td><td>348,00</td><td>37,40</td><td>5,33</td></tr><tr><td>232113</td><td>Cell division protein FtsZ 1</td><td>9,00</td><td>8,00</td><td>4,00</td><td>4,00</td><td>4,00</td><td>1,00</td><td>590,00</td><td>62,94</td><td>5,17</td></tr><tr><td>120199</td><td>Nitrogen fixation protein FixI</td><td>8,00</td><td>6,00</td><td>3,00</td><td>3,00</td><td>3,00</td><td>1,00</td><td>757,00</td><td>79,51</td><td>7,03</td></tr><tr><td>120197</td><td>Nitrogen fixation protein FixG</td><td>4,00</td><td>5,00</td><td>2,00</td><td>2,00</td><td>2,00</td><td>1,00</td><td>524,00</td><td>58,27</td><td>8,82</td></tr></tbody></table>

<table><thead><tr><th colspan="12">Identified proteins (80-45 kDa)</th></tr><tr><th>Accession</th><th>Description</th><th>Score Sequest</th><th>Coverage</th><th># Peptides</th><th>#PSMs</th><th>#Unique Pept</th><th># Protein Gro</th><th># AAs</th><th>MW [kDa]</th><th>calc. pl</th></tr></thead><tbody><tr><td>2842625</td><td>Nitrous-oxide reductase</td><td>30,00</td><td>18,00</td><td>9,00</td><td>10,00</td><td>9,00</td><td>1,00</td><td>639,00</td><td>70,71</td><td>5,43</td></tr><tr><td>38503127</td><td>Membrane protein insertase YidC</td><td>21,00</td><td>16,00</td><td>7,00</td><td>8,00</td><td>7,00</td><td>1,00</td><td>594,00</td><td>66,06</td><td>7,06</td></tr><tr><td>7388096</td><td>RNA polymerase sigma factor RpoD=</td><td>19,00</td><td>12,00</td><td>7,00</td><td>7,00</td><td>7,00</td><td>1,00</td><td>684,00</td><td>77,13</td><td>5,07</td></tr><tr><td>21362573</td><td>Glutaminase</td><td>19,00</td><td>25,00</td><td>6,00</td><td>7,00</td><td>6,00</td><td>1,00</td><td>315,00</td><td>33,97</td><td>7,65</td></tr><tr><td>18202306</td><td>ATP-dependent Clp 1 Smc01903</td><td>10,00</td><td>14,00</td><td>2,00</td><td>3,00</td><td>2,00</td><td>1,00</td><td>208,00</td><td>23,22</td><td>6,05</td></tr><tr><td>120199</td><td>Nitrogen fixation protein FixI</td><td>9,00</td><td>7,00</td><td>4,00</td><td>4,00</td><td>4,00</td><td>1,00</td><td>757,00</td><td>79,51</td><td>7,03</td></tr><tr><td>81633813</td><td>ATP synthase subunit alpha</td><td>5,00</td><td>5,00</td><td>2,00</td><td>2,00</td><td>2,00</td><td>1,00</td><td>509,00</td><td>54,65</td><td>6,74</td></tr></tbody></table>

<table><thead><tr><th colspan="12">Identified proteins (45-25 kDa)</th></tr><tr><th>Accession</th><th>Description</th><th>Score Sequest</th><th>Coverage</th><th># Peptides</th><th>#PSMs</th><th>#Unique Pept</th><th># Protein Gro</th><th># AAs</th><th>MW [kDa]</th><th>calc. pl</th></tr></thead><tbody><tr><td>81633814</td><td>ATP synthase subunit beta</td><td>45,00</td><td>44,00</td><td>14,00</td><td>16,00</td><td>14,00</td><td>1,00</td><td>504,00</td><td>53,25</td><td>5,49</td></tr><tr><td>81633813</td><td>ATP synthase subunit alpha</td><td>42,00</td><td>25,00</td><td>11,00</td><td>14,00</td><td>11,00</td><td>1,00</td><td>509,00</td><td>54,65</td><td>6,74</td></tr><tr><td>17366440</td><td>Flagellin B</td><td>15,00</td><td>15,99</td><td>4,00</td><td>5,00</td><td>4,00</td><td>1,00</td><td>394,00</td><td>40,72</td><td>4,40</td></tr><tr><td>21362573</td><td>Glutaminase</td><td>7,00</td><td>12,00</td><td>3,00</td><td>3,00</td><td>3,00</td><td>1,00</td><td>315,00</td><td>33,97</td><td>7,65</td></tr><tr><td>232113</td><td>Cell division protein FtsZ 1</td><td>4,76</td><td>3,00</td><td>2,00</td><td>2,00</td><td>2,00</td><td>1,00</td><td>590,00</td><td>62,94</td><td>5,17</td></tr></tbody></table>

<table><thead><tr><th colspan="12">Identified proteins (25-17 kDa)</th></tr><tr><th>Accession</th><th>Description</th><th>Score Sequest</th><th>Coverage</th><th># Peptides</th><th>#PSMs</th><th>#Unique Pept</th><th># Protein Gro</th><th># AAs</th><th>MW [kDa]</th><th>calc. pl</th></tr></thead><tbody><tr><td>81633814</td><td>ATP synthase subunit beta</td><td>67,00</td><td>51,00</td><td>16,00</td><td>23,00</td><td>16,00</td><td>1,00</td><td>504,00</td><td>53,25</td><td>5,49</td></tr><tr><td>18202306</td><td>ATP-dependent Clp 1 Smc01903</td><td>23,00</td><td>22,00</td><td>4,00</td><td>6,00</td><td>4,00</td><td>1,00</td><td>208,00</td><td>23,22</td><td>6,05</td></tr><tr><td>81633813</td><td>ATP synthase subunit alpha</td><td>16,00</td><td>15,00</td><td>6,00</td><td>6,00</td><td>6,00</td><td>1,00</td><td>509,00</td><td>54,65</td><td>6,74</td></tr></tbody></table>
<table><caption>Table S3. Strains used in this work.</caption><thead><tr><th>Description</th><th>Antibiotics</th><th>Source</th><th>Name of the strain</th></tr></thead><tbody><tr><td>Sinorhizobium meliloti 1021</td><td>Sm (200μg/ml)</td><td>Biondi lab</td><td>EB446</td></tr><tr><td>Sinorhizobium meliloti 1021 ΔcrrA::tet pSRK-Km::crrA</td><td>Sm (200μg/ml) + Km (20μg/ml) + Tc (10μg/ml) + 1mM IPTG</td><td>Pini et al., 2015</td><td>EB 249</td></tr><tr><td>Sinorhizobium meliloti 1021 ΔfcrX::tet pSRK-Gm::fcrX</td><td>Sm (200μg/ml) + Gm (20μg/ml) + Tc (10μg/ml) + 100μM IPTG</td><td></td><td>EB 1889</td></tr><tr><td>Escherichia coli ΔHT101 PUT18C::fcrX + PKNT25::crrA</td><td>Amp (100μg/ml) + Km (50μg/ml) + Xgal (40μg/ml) + 500μM IPTG</td><td></td><td>EB 2778</td></tr><tr><td>Escherichia coli ΔHT101 PUT18C::fcrX + PKNT25::crrA</td><td>Amp (100μg/ml) + Km (50μg/ml) + Xgal (40μg/ml) + 500μM IPTG</td><td></td><td>EB 2779</td></tr><tr><td>Escherichia coli ΔHT101 PUT18C::fcrX + PKNT25::crrA</td><td>Amp (100μg/ml) + Km (50μg/ml) + Xgal (40μg/ml) + 500μM IPTG</td><td></td><td>EB 2780</td></tr><tr><td>Escherichia coli ΔHT101 PUT18C::fcrX + PKNT25::crrA</td><td>Amp (100μg/ml) + Km (50μg/ml) + Xgal (40μg/ml) + 500μM IPTG</td><td></td><td>EB 2589</td></tr><tr><td>Escherichia coli ΔHT101 PKT25::fcrX + PUT18C::fcrX</td><td>Amp (100μg/ml) + Km (50μg/ml) + Xgal (40μg/ml) + 500μM IPTG</td><td></td><td>EB 2586</td></tr><tr><td>Escherichia coli ΔHT101 PKT25::fcrX + PUT18C::fcrX</td><td>Amp (100μg/ml) + Km (50μg/ml) + Xgal (40μg/ml) + 500μM IPTG</td><td></td><td>EB 2781</td></tr><tr><td>Escherichia coli ΔHT101 PKT25::fcrX + PUT18C::fcrX</td><td>Amp (100μg/ml) + Km (50μg/ml) + Xgal (40μg/ml) + 500μM IPTG</td><td></td><td>EB 2581</td></tr><tr><td>Escherichia coli ΔHT101 PKT25::fcrX + PUT18C::fcrX</td><td>Amp (100μg/ml) + Km (50μg/ml) + Xgal (40μg/ml) + 500μM IPTG</td><td></td><td>EB 2582</td></tr><tr><td>Escherichia coli ΔHT101 PKT25::fcrX + PKNT25::ftsZ2</td><td>Amp (100μg/ml) + Km (50μg/ml) + Xgal (40μg/ml) + 500μM IPTG</td><td></td><td>EB 2782</td></tr><tr><td>Escherichia coli ΔHT101 PKT25::fcrX + PKNT25::ftsZ2</td><td>Amp (100μg/ml) + Km (50μg/ml) + Xgal (40μg/ml) + 500μM IPTG</td><td></td><td>EB 2783</td></tr><tr><td>Escherichia coli ΔHT101 PKT25::fcrX + PKNT25::ftsZ2</td><td>Amp (100μg/ml) + Km (50μg/ml) + Xgal (40μg/ml) + 500μM IPTG</td><td></td><td>EB 2784</td></tr><tr><td>Escherichia coli ΔHT101 PKT25::fcrX + PKNT25::ftsZ2</td><td>Amp (100μg/ml) + Km (50μg/ml) + Xgal (40μg/ml) + 500μM IPTG</td><td></td><td>EB 2783</td></tr><tr><td>Escherichia coli ΔHT101 PKT25::fcrX + PKNT25::ftsZ2</td><td>Amp (100μg/ml) + Km (50μg/ml) + Xgal (40μg/ml) + 500μM IPTG</td><td></td><td>EB 2787</td></tr><tr><td>Escherichia coli ΔHT101 PKT25::fcrX + PKNT25::ftsZ2</td><td>Amp (100μg/ml) + Km (50μg/ml) + Xgal (40μg/ml) + 500μM IPTG</td><td></td><td>EB 2584</td></tr><tr><td>Escherichia coli ΔHT101 PKT25::fcrX + PKNT25::ftsZ2</td><td>Amp (100μg/ml) + Km (50μg/ml) + Xgal (40μg/ml) + 500μM IPTG</td><td></td><td>EB 2583</td></tr><tr><td>Escherichia coli ΔHT101 PKT25::digS + PUT18C::ftsZ2</td><td>Amp (100μg/ml) + Km (50μg/ml) + Xgal (40μg/ml) + 500μM IPTG</td><td></td><td>EB 2782</td></tr><tr><td>Escherichia coli ΔHT101 PKT25::digS + PUT18C::ftsZ2</td><td>Amp (100μg/ml) + Km (50μg/ml) + Xgal (40μg/ml) + 500μM IPTG</td><td></td><td>EB 3394</td></tr><tr><td>Escherichia coli ΔHT101 PKT25::fcrX + PKNT25::ftsZ1</td><td>Amp (100μg/ml) + Km (50μg/ml) + Xgal (40μg/ml) + 500μM IPTG</td><td></td><td>EB 3395</td></tr><tr><td>Escherichia coli ΔHT101 PKT25::fcrX + PKNT25::ftsZ1</td><td>Amp (100μg/ml) + Km (50μg/ml) + Xgal (40μg/ml) + 500μM IPTG</td><td></td><td>EB 3396</td></tr><tr><td>Escherichia coli ΔHT101 PKT25::fcrX + PKNT25::ftsZ1</td><td>Amp (100μg/ml) + Km (50μg/ml) + Xgal (40μg/ml) + 500μM IPTG</td><td></td><td>EB 3397</td></tr><tr><td>Escherichia coli ΔHT101 PKT25::fcrX + PKNT25::ftsZ1</td><td>Amp (100μg/ml) + Km (50μg/ml) + Xgal (40μg/ml) + 500μM IPTG</td><td></td><td>EB 3398</td></tr><tr><td>Escherichia coli ΔHT101 PKT25::fcrX + PKNT25::ftsZ1</td><td>Amp (100μg/ml) + Km (50μg/ml) + Xgal (40μg/ml) + 500μM IPTG</td><td></td><td>EB 3399</td></tr><tr><td>Escherichia coli ΔHT101 PKT25::fcrX + PKNT25::ftsZ1</td><td>Amp (100μg/ml) + Km (50μg/ml) + Xgal (40μg/ml) + 500μM IPTG</td><td></td><td>EB 3400</td></tr><tr><td>Escherichia coli ΔHT101 PKNT25::fcrX + PKNT25::ftsZ1</td><td>Amp (100μg/ml) + Km (50μg/ml) + Xgal (40μg/ml) + 500μM IPTG</td><td></td><td>EB 3401</td></tr><tr><td>Escherichia coli ΔHT101 PKNT25::fcrX + PKNT25::fcrX</td><td>Amp (100μg/ml) + Km (50μg/ml) + Xgal (40μg/ml) + 500μM IPTG</td><td></td><td>EB 2659</td></tr><tr><td>Escherichia coli ΔHT101 PKNT25::fcrX + PKNT25::fcrX</td><td>Amp (100μg/ml) + Km (50μg/ml) + Xgal (40μg/ml) + 500μM IPTG</td><td></td><td>EB 2658</td></tr><tr><td>Escherichia coli ΔHT101 PKNT25::fcrX + PKNT25::fcrX</td><td>Amp (100μg/ml) + Km (50μg/ml) + Xgal (40μg/ml) + 500μM IPTG</td><td></td><td>EB 2660</td></tr><tr><td>Escherichia coli ΔHT101 PKNT25::fcrX + PKNT25::fcrX</td><td>Amp (100μg/ml) + Km (50μg/ml) + Xgal (40μg/ml) + 500μM IPTG</td><td></td><td>EB 2661</td></tr><tr><td>Sinorhizobium meliloti 1021 ΔfcrX::tet pSRK Plac yfp::fcrX</td><td>Sm (200μg/ml) + Gm (20μg/ml)</td><td></td><td>EB 3064</td></tr><tr><td>Sinorhizobium meliloti 1021 ΔfcrX::tet pSRK Plac yfp</td><td>Sm (200μg/ml) + Gm (20μg/ml)</td><td></td><td>EB 3017</td></tr><tr><td>Sinorhizobium meliloti 1021 pSRK Plac ftsZ1::cfp</td><td>Sm (200μg/ml) + Gm (20μg/ml)</td><td></td><td>EB 3076</td></tr><tr><td>Sinorhizobium meliloti 1021 pSRK Plac ftsZ2::cfp</td><td>Sm (200μg/ml) + Gm (20μg/ml)</td><td></td><td>EB 3110</td></tr><tr><td>Sinorhizobium meliloti 1021 ΔcrrA::tet pSRK Plac crrA::cfp15aa</td><td>Sm (200μg/ml) + Gm (20μg/ml)</td><td></td><td>EB 3233</td></tr><tr><td>Sinorhizobium meliloti 1021 pSRK Plac clpX::cfp</td><td>Sm (200μg/ml) + Gm (20μg/ml)</td><td></td><td>EB 3327</td></tr><tr><td>Sinorhizobium meliloti 1021 ΔfcrX::tet pSRK prom(1) fcrX</td><td>Sm (200μg/ml) + Tc (2μg/ml) + Gm (20μg/ml) + 100μM IPTG</td><td></td><td>EB 3071</td></tr><tr><td>Sinorhizobium meliloti 1021 ΔfcrX::tet pSRK prom(2) fcrX</td><td>Sm (200μg/ml) + Tc (2μg/ml) + Gm (20μg/ml) + 100μM IPTG</td><td></td><td>EB 3072</td></tr><tr><td>Sinorhizobium meliloti 1021 ΔfcrX::tet pSRK prom(3) fcrX</td><td>Sm (200μg/ml) + Tc (2μg/ml) + Gm (20μg/ml) + 100μM IPTG</td><td></td><td>EB 3073</td></tr><tr><td>Sinorhizobium meliloti 1021 ΔfcrX::tet pSRK prom(5) fcrX</td><td>Sm (200μg/ml) + Tc (2μg/ml) + Gm (20μg/ml) + 100μM IPTG</td><td></td><td>EB 3074</td></tr><tr><td>Sinorhizobium meliloti 1021 ΔfcrX::tet pSRK prom(7) fcrX</td><td>Sm (200μg/ml) + Tc (2μg/ml) + Gm (20μg/ml) + 100μM IPTG</td><td></td><td>EB 3075</td></tr><tr><td>Sinorhizobium meliloti 1021 pOTIem pfcX(1)</td><td>Sm (200μg/ml) + Gm (20μg/ml)</td><td></td><td>EB 3065</td></tr><tr><td>Sinorhizobium meliloti 1021 pOTIem pfcX(3)</td><td>Sm (200μg/ml) + Gm (20μg/ml)</td><td></td><td>EB 3066</td></tr><tr><td>Sinorhizobium meliloti 1021 pOTIem pfcX(4)</td><td>Sm (200μg/ml) + Gm (20μg/ml)</td><td></td><td>EB 3067</td></tr><tr><td>Sinorhizobium meliloti 1021 pOTIem pfcX(5)</td><td>Sm (200μg/ml) + Gm (20μg/ml)</td><td></td><td>EB 3068</td></tr><tr><td>Sinorhizobium meliloti 1021 pOTIem pfcX(5) mutated CrrA box</td><td>Sm (200μg/ml) + Gm (20μg/ml)</td><td></td><td>EB 3113</td></tr><tr><td>Sinorhizobium meliloti 1021 pOTIem pfcX(7)</td><td>Sm (200μg/ml) + Gm (20μg/ml)</td><td></td><td>EB 3069</td></tr><tr><td>Sinorhizobium meliloti 1021 pOTIem pfcXfull</td><td>Sm (200μg/ml) + Gm (20μg/ml)</td><td></td><td>EB 3070</td></tr><tr><td>Sinorhizobium meliloti 1021 pOTIem pfcXfull mutated CrrA box</td><td>Sm (200μg/ml) + Gm (20μg/ml)</td><td></td><td>EB 3119</td></tr><tr><td>Sinorhizobium meliloti pSRK::fcrX</td><td>Sm (200μg/ml) + Gm (20μg/ml)</td><td></td><td>EB 1658</td></tr><tr><td>Sinorhizobium meliloti 1021 pOTIem pfcX(3)</td><td>Sm (200μg/ml) + Gm (20μg/ml)</td><td></td><td>EB 3066</td></tr><tr><td>Sinorhizobium meliloti 1021 pOTIem pfcX(4)</td><td>Sm (200μg/ml) + Gm (20μg/ml)</td><td></td><td>EB 3067</td></tr><tr><td>Sinorhizobium meliloti 1021 pOTIem pfcX(5)</td><td>Sm (200μg/ml) + Gm (20μg/ml)</td><td></td><td>EB 3068</td></tr><tr><td>Sinorhizobium meliloti 1021 pOTIem pfcX(7)</td><td>Sm (200μg/ml) + Gm (20μg/ml)</td><td></td><td>EB 3069</td></tr><tr><td>Sinorhizobium meliloti 1021 pOTIem pfcXfull</td><td>Sm (200μg/ml) + Gm (20μg/ml)</td><td></td><td>EB 3070</td></tr><tr><td>Sinorhizobium meliloti 1021 pSRK::Gm</td><td>Sm (200μg/ml) + Gm (20μg/ml)</td><td></td><td>EB 350</td></tr><tr><td>Sinorhizobium meliloti 1021 pSRK::Km</td><td>Sm (200μg/ml) + Km (100μg/ml)</td><td></td><td>EB 261</td></tr><tr><td>Sinorhizobium meliloti 1021 pSRK blr2202 (fcrX homolog from Bradyrhizobium japonicum)</td><td>Sm (200μg/ml) + Gm (20μg/ml)</td><td></td><td>EB 3185</td></tr><tr><td>Sinorhizobium meliloti 1021 pSRK flu (ATG) (Caulobacter crescentus)</td><td>Sm (200μg/ml) + Gm (20μg/ml)</td><td></td><td>EB 3151</td></tr><tr><td>Sinorhizobium meliloti 1021 pSRK avi_3526 (fcrX homolog from A, vitis)</td><td>Sm (200μg/ml) + Gm (20μg/ml)</td><td></td><td>EB 3189</td></tr><tr><td>Sinorhizobium meliloti 1021 ΔcrrX::tet pSRK-RL3537 (fcrX homolog from R leguminosarum)</td><td>Sm (200μg/ml) + Gm (20μg/ml) + Tc (2μg/ml) + 100μM IPTG</td><td></td><td>EB 3191</td></tr><tr><td>Sinorhizobium meliloti 1021 ΔfcrX::tet pSRK bab1-1615 (fcrX homolog from Brucella militensis biovar Abortus)</td><td>Sm (200μg/ml) + Gm (20μg/ml) + Tc (2μg/ml) + 100μM IPTG</td><td></td><td>EB 3153</td></tr></tbody></table>
<table><caption>Table S4. Primers used in this work.</caption><thead><tr><th>Primer's name</th><th>Sequence (5'-3')</th></tr></thead><tbody><tr><td>fcrx forward</td><td>GGCATATGAAAGCACGTGAGAGCCTAGT</td></tr><tr><td>fcrx reverse</td><td>GGGGATCCAAGTTAGAGCGCCCTTTGTG</td></tr><tr><td>SMC00655_F1 (Apal)</td><td>GGGGGCCCAATCGGTGTTTTGACCTTCG</td></tr><tr><td>SMC00655_R2 (EcoRI)</td><td>GGAATTCGACTAGGCTCTCACGTGCT</td></tr><tr><td>SMC00655_F3 (EcoRI)</td><td>GGAATTCGCTCGAGGAGCGGGATAC</td></tr><tr><td>SMC00655_R4 (SphI)</td><td>GGGCATGCATTCTCTCCTGGCAATCC</td></tr><tr><td>ctrA forward</td><td>GGCATATGCGGGTTCTACTGATCGAAG</td></tr><tr><td>ctrA 15AA reverse</td><td>GGGGATCCTCAGGCGGTTTCCAGGTAGTCGCTGCCTTCCGGCTCGCGCAGCACCTTGTACAGCTCGTCCATGCCG</td></tr><tr><td>ftsZ1 forward</td><td>GGCATATGGCCATCAACTTGCAGAAGC</td></tr><tr><td>ftsZ1 reverse</td><td>GCTCGACTGGCGGCGCAGG</td></tr><tr><td>ftsZ2 forward</td><td>GGCATATGACGGAATACAAGAAGCCG</td></tr><tr><td>ftsZ2 reverse</td><td>CTGCAGCGTACGCGAAGGAAC</td></tr><tr><td>fcrx forward for BACTH</td><td>TCTAGAGATGAAAGCACGTGAGAGCCTAGT</td></tr><tr><td>fcrx reverse for BACTH</td><td>TACCCGGGGGCGCGCAGCGCGCACCCG</td></tr><tr><td>ftsZ1 forward for BACTH</td><td>GGTCTAGAGATGGCCATCAACTTGCAGAAGC</td></tr><tr><td>ftsZ1 reverse for BACTH</td><td>TACCCGGGGCTCGACTGGCGGCGCAGG</td></tr><tr><td>ftsZ2 forward for BACTH</td><td>TCTAGAGATGACGGAATACAAGAAGCCG</td></tr><tr><td>ftsZ2 reverse for BACTH</td><td>TACCCGGGGCTGCAGCGTACGCGAAGGAAC</td></tr><tr><td>ctrA forward for BACTH</td><td>TCTAGAGATGCGGGTTCTACTGATCG</td></tr><tr><td>ctrA reverse for BACTH</td><td>GGTACCCGGCGGTTTCCAGGTAGTCG</td></tr><tr><td>yfp/CFP reverse</td><td>GGGGATCCTTACTTGTACAGCTCGTCCATGCCG</td></tr><tr><td>pfcrX-53 pb fcrX forward</td><td>GGCATATGTATCCCGCTCGGGAACAGTA</td></tr><tr><td>pfcrX-130 pb fcrX forward</td><td>GGCATATGCGAACCGGCTTCTCTAGTGG</td></tr><tr><td>pfcrX-180 pb fcrX forward</td><td>GGCATATGTTTTGTTTCCGGCTCGGTTG</td></tr><tr><td>pfcrX-227 pb fcrX forward</td><td>GGCATATGGGTTACCAGCGGTAGGCATT</td></tr><tr><td>pfcrX-289 pb fcrX forward</td><td>GGCATATGGCATCAGCATTTCACGGAGCC</td></tr><tr><td>pfcrX-371 pb fcrX forward</td><td>GGCATATGCGACTCGTAAATGCGCGAAA</td></tr><tr><td>fcrx forward for qRT-PCR</td><td>GTCGGGTATTTCGACCCTT</td></tr><tr><td>fcrx reverse for qRT-PCR</td><td>CATCTTCAGCTCGCGAATGG</td></tr><tr><td>16S RNA forward for qRT-PCR</td><td>AAAGGCGACGATCCATAGCTG</td></tr><tr><td>16S RNA reverse for qRT-PCR</td><td>AACCCTAGGGCCTTCATCACT</td></tr><tr><td>megaprimer pfcrX-53 pb fcrX forward</td><td>GGGTACCAAGCTTTATGCTTCTAGTGGTATCCCGCTCGGGAACAGTA</td></tr><tr><td>megaprimer pfcrX-180 pb fcrX forward</td><td>GGGTACCAAGCTTTATGCTTCTAGTGGTTTTGTTTCCGGCTCGGTTG</td></tr><tr><td>megaprimer pfcrX-227 pb fcrX forward</td><td>GGGTACCAAGCTTTATGCTTCTAGTGGGGTTACCAGCGGTAGGCATT</td></tr><tr><td>megaprimer pfcrX-289 pb fcrX forward</td><td>GGGTACCAAGCTTTATGCTTCTAGTGGGCATCAGCATTTACGGAGCC</td></tr><tr><td>megaprimer pfcrX- full pb fcrX forward</td><td>CTCGCCCTTGCTCACCATAGTCTTCCCTTTTCCGCCG</td></tr><tr><td>megaprimer pfcrX- full pb fcrX reverse</td><td>CCTCGCCCTTGGAAACCATACGCAATACTCCCGTGGATAC</td></tr><tr><td>megaprimer pfcrX-289 pb fcrX forward MUT</td><td>ATCAGCATTTACGGAGCCGCGCATCATCGCACCTATGATT</td></tr><tr><td>megaprimer pfcrX- full pb fcrX forward MUT</td><td>AATCATAGGTGCGATGATGCGCGGCTCCGTAAATGCTGAT</td></tr><tr><td>RL3537 (fcrX) forward</td><td>GGCATATGCGTATGAAGTCGCGAGAAAG</td></tr><tr><td>RL3537 (fcrX) reverse</td><td>GGGGATCCTACATGGCTTCTCAGTGCG</td></tr><tr><td>bab1-1615 (fcrX) forward</td><td>GGCATATGAAGCCACGTGAAAGCCTC</td></tr><tr><td>bab1-1615 (fcrX) reverse</td><td>GGGGATCCTTCAGTTGCCAGGATGCACT</td></tr><tr><td>avi_3526 (fcrX) forward</td><td>GGCATATGTCAAAACAGAGTGACGGCG</td></tr><tr><td>avi_3526 (fcrX) reverse</td><td>GGGGATCCGGCCAGGCAATGAAACCATC</td></tr><tr><td>fijJ forward</td><td>GGCATGTGACCAAGTGGGCCGCC</td></tr><tr><td>fijJ reverse</td><td>GGGGATCCGCTGGCTCTGCACCTCAC</td></tr></tbody></table>