David Ranava

Yiying Yang

Luis Orenday-Tapia

François Rousset

Catherine Turlan

Lun Cui

Violette Morales

Cyril Moulin

Gladys Munoz

Jérôme Rech

Anne Caumont-Sarcos

Cécile Albenne

David Bikard

Raffaele Ieva
email: raffaele.ieva@univ-tlse3.fr

Outer membrane lipoprotein DolP interacts with the BAM complex and promotes fitness during envelope stress response

In Gram-negative bacteria, coordinated remodelling of the outer membrane (OM) and the peptidoglycan is crucial for envelope integrity. Envelope stress caused by unfolded OM proteins (OMPs) activates sigmaE (s E ) in Enterobacteria. s E upregulates OMP biogenesis factors, including the b-barrel assembly machinery (BAM) that catalyzes OMP-folding. Elevated s E activity, however, can be detrimental for OM integrity. Here we report that DolP (YraP), a s Eupregulated OM lipoprotein important for envelope integrity, is a novel interactor of BAM and we demonstrate that OM-assembled BamA is a critical determinant of the BAM-DolP complex. Mid-cell recruitment of DolP had been previously associated to activation of septal peptidoglycan remodelling during cell division, but its role during envelope stress was unknown. We now show that DolP promotes cell fitness upon stress-induced activation of s E and opposes a detrimental effect caused by the overaccumulation of BAM in the OM. During envelope stress, DolP loses its association with the mid-cell, thus suggesting a possible link between envelope stress caused by impaired OMP biogenesis and the regulation of a late step of cell division.

Introduction

The outer membrane (OM) of Gram-negative bacteria forms a protective barrier against harmful compounds, including several antimicrobials. This envelope structure surrounds the inner membrane and the periplasm that contains the peptidoglycan, a net-like structure made of glycan chains and interconnecting peptides. During cell division, the multi-layered envelope structure is remodelled by the divisome machinery [START_REF] Den Blaauwen | The divisome at 25: the road ahead[END_REF] . At a late step of division, septal peptidoglycan synthesized by the divisome undergoes splitting, initiating the formation of the new poles of adjacent daughter cells. Finally, remodelling of the OM barrier completes formation of the new poles in the cell offspring. The mechanisms by which cells coordinate OM remodelling with peptidoglycan splitting, preserving the permeability barrier of this protective membrane, are ill-defined [START_REF] Egan | Regulation of peptidoglycan synthesis and remodelling[END_REF] .

Integral outer membrane proteins (OMPs) are crucial to maintain the OM permeability barrier. OMPs fold into amphipathic b-barrel structures that span the OM and carry out a variety of tasks. Porins are OMPs that facilitate the diffusion of small metabolites. Other OMPs function as cofactor transporters, secretory channels, or machineries for the assembly of proteins and lipopolysaccharide (LPS) [START_REF] Calmettes | Structural Aspects of Bacterial Outer Membrane Protein Assembly[END_REF][START_REF] Nikaido | Molecular basis of bacterial outer membrane permeability revisited[END_REF] , a structural component of the external OM leaflet that prevents the diffusion of noxious chemicals [START_REF] Nikaido | Molecular basis of bacterial outer membrane permeability revisited[END_REF] . The b-barrel assembly machinery (BAM) is a multi-subunit complex that mediates the folding and membrane insertion of OMPs transiting through the periplasm [START_REF] Ranava | Bacterial machineries for the assembly of membrane-embedded beta-barrel proteins[END_REF][START_REF] Schiffrin | Outer membrane protein folding from an energy landscape perspective[END_REF] . The essential and evolutionarily conserved BamA insertase subunit is an OMP consisting of an amino (N)-terminal periplasmic domain made of polypeptide transport-associated (POTRA or P) motifs and a carboxy (C)terminal 16-stranded b-barrel membrane domain that catalyzes OMP biogenesis [START_REF] Webb | Evolution of the beta-barrel assembly machinery[END_REF][START_REF] Voulhoux | Role of a highly conserved bacterial protein in outer membrane protein assembly[END_REF] .

The flexible pairing of b-strands 1 and 16 of the BamA b-barrel controls a lateral gate connecting the interior of the barrel towards the surrounding lipid bilayer [START_REF] Noinaj | Structural insight into the biogenesis of beta-barrel membrane proteins[END_REF][START_REF] Iadanza | Lateral opening in the intact beta-barrel assembly machinery captured by cryo-EM[END_REF][START_REF] Gu | Structural basis of outer membrane protein insertion by the BAM complex[END_REF][START_REF] Bakelar | The structure of the beta-barrel assembly machinery complex[END_REF] .

Conformational dynamics of the BamA b-barrel region proximal to the lateral gate is thought to locally increase the entropy of the surrounding lipid bilayer [START_REF] Noinaj | Structural insight into the biogenesis of beta-barrel membrane proteins[END_REF][START_REF] Doerner | Extreme Dynamics in the BamA beta-Barrel Seam[END_REF][START_REF] Gessmann | Outer membrane beta-barrel protein folding is physically controlled by periplasmic lipid head groups and BamA[END_REF] and assist the insertion of nascent OMPs into the OM [START_REF] Gu | Structural basis of outer membrane protein insertion by the BAM complex[END_REF][START_REF] Doyle | Bacterial outer membrane proteins assemble via asymmetric interactions with the BamA beta-barrel[END_REF][START_REF] Tomasek | Structure of a nascent membrane protein as it folds on the BAM complex[END_REF] . The N-terminal periplasmic portion of BamA from the enterobacterium Escherichia coli contains five POTRA motifs that serve as a scaffold for four lipoproteins, BamBCDE, which assist BamA during OMP biogenesis [START_REF] Sklar | Lipoprotein SmpA is a component of the YaeT complex that assembles outer membrane proteins in Escherichia coli[END_REF][START_REF] Kim | Structure and function of an essential component of the outer membrane protein assembly machine[END_REF][START_REF] Wu | Identification of a multicomponent complex required for outer membrane biogenesis in Escherichia coli[END_REF] . The N-terminal POTRA motif is also the docking site of the periplasmic chaperone SurA [START_REF] Bennion | Dissection of beta-barrel outer membrane protein assembly pathways through characterizing BamA POTRA 1 mutants of Escherichia coli[END_REF] . Together with the chaperones Skp and DegP, SurA contributes to monitor unfolded OMPs transported into the periplasm by the inner membrane general secretory (Sec) pathway [START_REF] Rizzitello | Genetic evidence for parallel pathways of chaperone activity in the periplasm of Escherichia coli[END_REF][START_REF] Crane | The Sec System: Protein Export in Escherichia coli[END_REF] .

Defective OMP assembly causes periplasmic accumulation of unfolded protein transport intermediates. This envelope stress is signalled across the inner membrane to induce the sigmaE (s E )-mediated transcriptional response [START_REF] Walsh | OMP peptide signals initiate the envelope-stress response by activating DegS protease via relief of inhibition mediated by its PDZ domain[END_REF] . In the absence of stress, s E is sequestered by the inner membrane-spanning RseA factor.

By-products of misfolded OMP turnover activate degradation of RseA, liberating s E [START_REF] Ades | Regulation by destruction: design of the sigmaE envelope stress response[END_REF] . The s E response copes with stress i) by upregulating genes involved in OMP biogenesis, such as the bam genes [START_REF] Rhodius | Conserved and variable functions of the sigmaE stress response in related genomes[END_REF] , and ii) by lowering the OMP biogenesis burden via a post-transcriptional mechanism [START_REF] Guillier | Modulating the outer membrane with small RNAs[END_REF] . Whereas s E is essential [START_REF] De Las Penas | SigmaE is an essential sigma factor in Escherichia coli[END_REF] , a tight control of cytosolic s E availability is necessary for optimal cell fitness and to prevent a potentially detrimental effect on the envelope [START_REF] Missiakas | Modulation of the Escherichia coli sigmaE (RpoE) heat-shock transcriptionfactor activity by the RseA, RseB and RseC proteins[END_REF][START_REF] De Las Penas | The sigmaE-mediated response to extracytoplasmic stress in Escherichia coli is transduced by RseA and RseB, two negative regulators of sigmaE[END_REF][START_REF] Nicoloff | Appropriate Regulation of the sigma(E)-Dependent Envelope Stress Response Is Necessary To Maintain Cell Envelope Integrity and Stationary-Phase Survival in Escherichia coli[END_REF][START_REF] Mitchell | Envelope stress responses: balancing damage repair and toxicity[END_REF] . Remarkably, the functions of a number of genes upregulated by s E remains unknown. Among those, yraP encodes a ~20 kDa OM-anchored lipoprotein largely conserved in g and b proteobacteria 32- 36 . YraP is crucial for OM integrity [START_REF] Onufryk | Characterization of six lipoproteins in the sigmaE regulon[END_REF] and pathogenicity [START_REF] Morris | YraP Contributes to Cell Envelope Integrity and Virulence of Salmonella enterica Serovar Typhimurium[END_REF] . A recent study showed that YraP localizes at the mid-cell during a late step of cell division, where it contributes to the regulation of septal peptidoglycan splitting by an unknown mechanism [START_REF] Tsang | NlpD links cell wall remodeling and outer membrane invagination during cytokinesis in Escherichia coli[END_REF] . These observations do not explain why YraP is upregulated in response to s E activation and how YraP helps coping with envelope stress.

During cell division, envelope stress caused by defective OMP biogenesis would place an additional burden on the already-complicated envelope reshaping process faced by cells. It remains unknown whether stress caused by unfolded OMPs influences envelope remodelling at the forming poles of two adjacent daughter cells. Here we present a functional investigation of yraP prompted by a genome-wide synthetic-defect screen. We demonstrate genetic interactions of yraP with bam genes and we provide evidence that YraP can associate with the BAM complex via an interaction with the BamA subunit. YraP is not critical for OMP assembly but promotes fitness upon activation of the s E response. Upon envelope stress or when BAM overaccumulates in the OM, YraP loses its association with the mid-cell, thus suggesting a possible link between the envelope stress response and septal peptidoglycan hydrolysis during a late step of cell division. Hence, we propose to rename YraP as DolP (division-and OM stress-associated lipoprotein).

Results

A genome-wide synthetic-defect screen identifies dolP genetic interactions

The mutant allele DdolP::kan [START_REF] Baba | Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection[END_REF] was introduced into E. coli BW25113 by P1 transduction. The resulting DdolP strain grew normally on LB medium (Figs. S1A andS1B), but was highly susceptible to vancomycin (Figs. 1A andS1B). This antibiotic is normally excluded from the OM of wild-type cells but inhibits growth of cells lacking OMP biogenesis factors such as skp and surA (Fig. S1B). The expression of C-terminally fused DolP protein variants in place of its wild-type form restored vancomycin resistance (Fig. 1A and Fig. S1C). This result supports the notion that DolP is important for envelope integrity [START_REF] Tsang | NlpD links cell wall remodeling and outer membrane invagination during cytokinesis in Escherichia coli[END_REF][START_REF] Onufryk | Characterization of six lipoproteins in the sigmaE regulon[END_REF][START_REF] Seib | The meningococcal vaccine antigen GNA2091 is an analogue of YraP and plays key roles in outer membrane stability and virulence[END_REF][START_REF] Bos | Involvement of Neisseria meningitidis lipoprotein GNA2091 in the assembly of a subset of outer membrane proteins[END_REF] . However, the role of DolP during envelope stress remains poorly understood.

A previous study showed that lack of DolP causes a severe growth defect in a surA deletion strain, however the cause of this synthetic phenotype has remained unclear [START_REF] Onufryk | Characterization of six lipoproteins in the sigmaE regulon[END_REF] . To gain insights into the role of DolP, we subjected DdolP cells to a genome-wide synthetic-defect screen exploiting a Clustered Regularly Interspersed Short Palindromic Repeat interference (CRISPRi) approach. Targeting of the catalytically inactive dCas9 nuclease by gene-specific single guides RNAs (sgRNAs) enables gene repression (Fig. 1B) [START_REF] Cui | A CRISPRi screen in E. coli reveals sequence-specific toxicity of dCas9[END_REF] . The EcoWG1 sgRNA library targeting the entire genome of E. coli MG1655 [START_REF] Calvo-Villamanan | On-target activity predictions enable improved CRISPR-dCas9 screens in bacteria[END_REF] was introduced into isogenic DdolP or dolP + MG1655-derivative strains. The fitness of each knockdown was then compared in these backgrounds by deep-sequencing of the sgRNA library after ~17 growth generations. The outputs obtained from two independent tests were highly reproducible (Fig. S2A). A strong fitness defect in the DdolP strain was caused by the targeting of envC (Fig. 1C, Fig. S2B and Tables S1 andS2), followed by the targeting of ftsX and ftsE (Fig. 1C, Tables S1 andS2). A validation growth test showed that the synthetic fitness defect observed for DdolP cells was caused by dCas9-dependent silencing of ftsX and envC (Fig. 1D, panels 6 and 7). The ABC transporter-like complex FtsE/FtsX has multiple roles in organizing the cell divisome, including the recruitment of periplasmic EnvC, a LytM domain-containing factor required for the activation of amidases that hydrolyse septal peptidoglycan [START_REF] Pichoff | Roles of FtsEX in cell division[END_REF] .

This peptidoglycan remodelling reaction is mediated by two sets of highly controlled and partially redundant amidases, AmiA/AmiB and AmiC [START_REF] Heidrich | Involvement of N-acetylmuramyl-L-alanine amidases in cell separation and antibiotic-induced autolysis of Escherichia coli[END_REF][START_REF] Uehara | LytM-domain factors are required for daughter cell separation and rapid ampicillin-induced lysis in Escherichia coli[END_REF] . Whereas AmiA and AmiB are activated at the inner membrane/peptidoglycan interface by the coordinated action of FtsE/FtsX and EnvC, activation of AmiC requires the OManchored LytM domain-containing lipoprotein NlpD [START_REF] Yang | An ATP-binding cassette transporter-like complex governs cell-wall hydrolysis at the bacterial cytokinetic ring[END_REF][START_REF] Uehara | Daughter cell separation is controlled by cytokinetic ring-activated cell wall hydrolysis[END_REF] . Under laboratory conditions, the activity of only one of these two pathways is sufficient for septal peptidoglycan splitting, whereas inhibition of both pathways leads to formation of chains of partially divided cells, i.e. cells that have begun to divide but are blocked at the step of septal peptidoglycan splitting [START_REF] Uehara | Daughter cell separation is controlled by cytokinetic ring-activated cell wall hydrolysis[END_REF] . A recent report showed that dolP is necessary to complete septal peptidoglycan splitting and to promote cell separation when the AmiA/AmiB pathway is inactive, somehow linking DolP to AmiC activation [START_REF] Tsang | NlpD links cell wall remodeling and outer membrane invagination during cytokinesis in Escherichia coli[END_REF] . Thus, the reduced fitness caused by silencing of envC, ftsE or ftsX in DdolP cells (Fig. 1C) may be explained by impaired cell separation when both the AmiA/AmiB and the AmiC pathways are not active. In keeping with this notion, amiA itself was found among the negative fitness hits of the CRISPRi screen (Fig. 1C). amiB was not a hit (Table S2), probably because AmiA is sufficient to split septal peptidoglycan in the absence of other amidases [START_REF] Chung | Rapid beta-lactam-induced lysis requires successful assembly of the cell division machinery[END_REF] .

Most importantly, the CRISPRi approach identified novel dolP-genetic interactions that had a score similar to that obtained for amiA (Fig. 1C). These included an interaction with rseA, encoding the inner membrane s E -sequestering factor, as well as with bamD, encoding an essential subunit of the BAM complex [START_REF] Onufryk | Characterization of six lipoproteins in the sigmaE regulon[END_REF][START_REF] Malinverni | YfiO stabilizes the YaeT complex and is essential for outer membrane protein assembly in Escherichia coli[END_REF] . In accordance with the screen output, a serial dilution assay confirmed that silencing of bamD (as well as bamE, encoding a stoichiometric interactor of BamD) causes a fitness defect in cells lacking DolP (Fig. 1D, panels 8 and 9). In addition, the interaction of dolP with rseA was confirmed in the genetic background of a BW25113 strain (Fig. 1E). Further genes, involved in OMP biogenesis and more generally in protein secretion, had a lower interaction score (Table S2 and Fig. S2C) and are highlighted in Fig. 1C. These comprise bamA, the OMP chaperoneencoding gene surA, as well as the genes encoding the Sec ancillary complex SecDF-YajC that contributes to efficient secretion of proteins including OMPs [START_REF] Crane | The Sec System: Protein Export in Escherichia coli[END_REF] and that was shown to interact with the BAM complex [START_REF] Carlson | Profiling the Escherichia coli membrane protein interactome captured in Peptidisc libraries[END_REF][START_REF] Alvira | Inter-membrane association of the Sec and BAM translocons for bacterial outer-membrane biogenesis[END_REF] . Collectively, the results of the CRISPRi screen indicate that the function of DolP is particularly critical for cell fitness upon inactivation of septal peptidoglycan hydrolysis by AmiA, as well as when the assembly of proteins in the OM is impaired.

DolP improves cell fitness when the OM undergoes stress

At a first glance, the newly identified genetic interaction between dolP and bamD (Figs. 1C and1D) points to a possible role of DolP in OMP biogenesis.

However, the overall protein profile of the crude envelope fraction was not affected by the deletion of dolP (Fig. S3A, lanes 1-4). OMPs, such as the very abundant OmpA and OmpC [START_REF] Li | Quantifying absolute protein synthesis rates reveals principles underlying allocation of cellular resources[END_REF] , can be recognized by their characteristic heat-modifiable migration patterns when separated by SDS-PAGE [START_REF] Nakamura | Effects of heating in dodecyl sulfate solution on the conformation and electrophoretic mobility of isolated major outer membrane proteins from Escherichia coli K-12[END_REF] (Fig. S3 A-D). The levels of OmpA (Fig. S3B), LamB (Fig. S3C) and OmpC (Fig. S3D) were not affected in DdolP samples. Similarly, the assembly kinetics of an autotransporter OMP was not impaired (Fig. S3E). Importantly, the levels of proteins encoded by s E -upregulated genes, such as BamA and BamE, were not increased in DdolP cells (Fig. 2A), indicating a wild-type-like s E activity in this strain. The envelope protein profiles were not affected also when dolP was deleted in cells lacking one of the OMP periplasmic chaperones Skp or DegP (Fig. S3A, lanes 5-12), suggesting that DolP does not play a function redundant with that of these periplasmic factors. Taken together, these observations suggest that DolP is not crucial for efficient OMP biogenesis. The genetic interaction of dolP with bamD and surA (Fig. 1C) may also suggest that DolP is required for optimal survival when the OM undergoes modifications caused by activation of the s E response [START_REF] Onufryk | Characterization of six lipoproteins in the sigmaE regulon[END_REF][START_REF] Malinverni | YfiO stabilizes the YaeT complex and is essential for outer membrane protein assembly in Escherichia coli[END_REF][START_REF] Rouviere | a periplasmic protein with peptidyl-prolyl isomerase activity, participates in the assembly of outer membrane porins[END_REF] . This hypothesis is supported by the observation that s E activation caused by rseA silencing or deletion worsens the fitness of DdolP cells (Figs. 1C and1E).

To further test the importance of DolP under envelope stress conditions, we deleted dolP in a strain lacking bamB, which was identified with a lower genetic score by the CRISPRi approach (Table S2). In DbamB cells, the s E response is partially activated [START_REF] Wu | Identification of a multicomponent complex required for outer membrane biogenesis in Escherichia coli[END_REF][START_REF] Charlson | Differential effects of yfgL mutation on Escherichia coli outer membrane proteins and lipopolysaccharide[END_REF] , causing the upregulation of bam genes (Fig. 2B). A strain carrying the simultaneous deletion of dolP and bamB was viable but growthdefective. Normal growth was restored by ectopic expression of a C-terminally polyhistidine tagged DolP protein variant (Fig. 2C). The DbamB envelope protein profile presented a marked reduction of the major heat-modifiable OMPs, OmpA and OmpC, however the concomitant lack of DolP did not enhance this OMP defect (Fig. S3A, lanes 13-18). Notably, phase-contrast analysis of the same DdolP DbamB strain revealed a number of cells with altered morphology (Fig. 2D). This result suggests that the reduced fitness of DdolP DbamB cells cannot be ascribed to an exacerbation of the OMP biogenesis defect already caused by lack of BamB, pointing to a different role of DolP under envelope stress conditions.

DolP promotes growth in cells with increased levels of BAM complex.

The s E response leads to alteration of the OM protein content, with an increase of the level of the BAM complex and a reduction of numerous other proteins, including the most abundant OMPs and some lipoproteins [START_REF] Rhodius | Conserved and variable functions of the sigmaE stress response in related genomes[END_REF][START_REF] Guo | a new σE-dependent sRNA, combats envelope stress by repressing synthesis of Lpp, the major outer membrane lipoprotein[END_REF][START_REF] Gogol | Small RNAs endow a transcriptional activator with essential repressor functions for single-tier control of a global stress regulon[END_REF] . To gain further insights into the role of DolP during activation of the s E response, we explored the effect of prolonged overproduction of BAM. To this end, the genes encoding wild-type BamABCD and a C-terminally polyhistidine-tagged BamE protein variant were ectopically expressed via the isopropylthiogalactoside (IPTG)inducible trc promoter (P trc ) as a transcriptional unit, adapting a previously established method [START_REF] Roman-Hernandez | Reconstitution of bacterial autotransporter assembly using purified components[END_REF] . With 400 µM IPTG, the concentration of BAM complex that accumulated in the membrane fraction was roughly similar to the concentration of the major OMPs OmpA or OmpC (Fig. 3A, lane 2). Importantly, we noticed that prolonged BAM overproduction caused a partial detrimental effect in the wild-type BW25113 strain (Fig. 3B). The excess of BamA was responsible for impaired growth, as the excess of different subsets of BAM subunits that did not include BamA or an excess of OmpA obtained using a similar overproduction plasmid (see also the subsequent description of Fig. S7B) had no detectable effects in our growth tests (Fig. 3B). The detrimental effect of BAM overproduction was caused by the overaccumulation of BamA in the OM, as the overproduction of an assemblydefective BamA variant, BamA DP1 , that lacks the N-terminal POTRA1 motif and largely accumulates in the periplasm [START_REF] Bennion | Dissection of beta-barrel outer membrane protein assembly pathways through characterizing BamA POTRA 1 mutants of Escherichia coli[END_REF] , did not impair growth to the same extent (Figs. S4A-C). Even a slight increment of BAM, due to the leaky transcriptional activity of the trc promoter (Fig. 3A, lane 1) caused a mild but noticeable deterioration of the OM permeability barrier to vancomycin (Fig. 3C), suggesting that a small increment in BAM levels can enhance the permeability of the OM.

Strikingly, in the absence of vancomycin, we noticed that the detrimental effect caused by IPTG-induced OM overaccumulation of BAM was more severe in a DdolP strain (Fig. 3D). This difference was particularly evident with 200 µM IPTG, which had only a minor inhibitory effect on the growth of wild-type cells but strongly impaired the growth of a DdolP strain. Similar to dolP, skp is upregulated by s E and its deletion causes sensitivity to vancomycin (Fig. S1B). In contrast to DdolP, a Dskp strain harbouring the same BAM overproduction plasmid could grow as the wildtype reference (Fig. 3D), suggesting a specific effect of DolP in supporting growth when BAM is overproduced.

DolP interacts with BamA assembled in the OM

The observed interaction between dolP and bam genes prompted us to investigate whether DolP physically interacts with the BAM complex. To this end, a construct encoding C-terminally protein A-tagged DolP was ectopically expressed in DdolP cells. The envelope of cells expressing DolP ProtA was solubilized using digitonin as main mild-detergent component prior to IgG-affinity chromatography (Fig. 4A, coomassie staining). Site-specific enzymatic cleavage of an amino acid linker between DolP and the protein A tag was used for protein elution. Notably, BamA, BamC, BamD and BamE were immunodetected in the elution fraction of protein A-tagged DolP (Fig. 4A, lane 3). In contrast, the inner membrane protein CyoA and cytosolic RpoB were not detected. Next, BAM ProtA (consisting of wild-type BamABCD and a C-terminally protein A-tagged BamE protein variant) was ectopically overproduced to isolate the BAM complex via IgG-affinity purification (Fig. 4B, Coomassie staining). Importantly, in addition to the BamE bait and other subunits of the BAM complex, DolP was also immunodetected in the elution fraction (Fig. 4B, lane 3). Other proteins of the bacterial envelope (Skp, OmpA, and F 1 b of the F 1 F O ATP synthase) or cytosolic RpoB were not detected (Fig. 4B). Taken together, our native pull-down analysis indicates that DolP and BAM have affinity for each other.

To explore whether the central BAM subunit, BamA, is a critical determinant of the BAM-DolP interaction, we performed Ni-affinity purification using the solubilized envelope fraction obtained from cells overproducing BamA and Cterminally polyhistidine-tagged DolP. Under these conditions, BamA was efficiently co-eluted together with DolP His , demonstrating that BamA and DolP can interact even in the absence of stoichiometric amounts of the BAM lipoproteins (Fig. 4C, coomassie staining). To assess if the interaction of DolP and BAM takes place at the OM, DolP His was overproduced together with the assembly-defective form BamA DP1 . When expressed together with DolP His , assembly-defective BamA DP1 was highly depleted in the corresponding eluate (Fig. 4C, lane 7), even though BamA DP1 was only marginally reduced in the crude envelope fraction with respect to wild-type BamA (Fig. 4C, lane 3). In contrast to BamA DP1 , the BamA DP2 variant, which is efficiently assembled into the OM (Fig. S4), was co-eluted to a similar extent as wild-type BamA (Fig. 4C, lane 8). This result indicates that DolP has affinity for OMassembled BamA.

In seeking a detergent that would interfere with the interaction of BAM and DolP, and allow their purification as separate components, we solubilized the envelope fraction with increasing amounts of n-dodecyl b-D-maltoside (DDM), a detergent previously used to isolate the native BAM complex [START_REF] Roman-Hernandez | Reconstitution of bacterial autotransporter assembly using purified components[END_REF] . At concentrations of DDM between 0.3% (w/v) and 1% (w/v), we observed a drastic reduction in the amounts of BAM subunits that were co-eluted with DolP His (Fig. 4D), indicating that the BAM-DolP interaction is sensitive to DDM. We thus used 1% (w/v) DDM to extract and purify His-tagged DolP or His-tagged BAM as separate components (Fig. 4E). When analyzed by blue native-PAGE and immunoblotting, purified DolP gave rise to a diffused signal at around 450 kDa (Fig. 4F, lane 1), suggesting a dynamic multimeric organization of this protein. Purified BAM migrated as expected at 250 kDa (Fig. 4F, lane 7). When roughly equimolar amounts of purified BAM and DolP were pre-incubated in the presence of a low DDM concentration and subsequently resolved by blue native-PAGE, a complex with an apparent molecular weight higher than that of the BAM complex was detected with both anti-BamA and anti-DolP specific antibodies (Fig. 4F, lanes 3 to 6). Taken together our results demonstrate that the BAM complex and DolP interact to form a larger complex and that OM-assembled BamA is a critical determinant of this interaction. Only a portion of BAM and DolP associate to form the BAM-DolP complex, indicating that the interaction is substoichiometric.

BamA overaccumulation in the OM reduces DolP mid-cell localization

In light of our observation that DolP interacts with BAM, we asked whether the envelope localization patterns of DolP and BAM are reciprocally linked. First, we monitored the effect of DolP expression on the localization of the chromosomally encoded BamD mCherry subunit of the BAM complex. This protein generated a fluorescence signal throughout the envelope that was not affected by the lack or the overproduction of DolP (Fig. S5). Next, we checked the effect of BAM overaccumulation in the OM on DolP localization. DolP associates with the OM and accumulates at mid-cell during a late step of cell division [START_REF] Tsang | NlpD links cell wall remodeling and outer membrane invagination during cytokinesis in Escherichia coli[END_REF] . To monitor the localization of DolP, we used a strain harbouring a chromosomal dolP-gfp fusion (Fig. 1A). The localization of the DolP GFP fusion protein (Fig. S6A) was analyzed concomitantly with other two chromosomally-encoded markers of the division septum, ZipA mCherry or NlpD mCherry . ZipA is involved in an early step of divisome assembly and accumulates at division sites before, as well as, during envelope constriction (Fig. S6B) [START_REF] Hale | Direct binding of FtsZ to ZipA, an essential component of the septal ring structure that mediates cell division in E. coli[END_REF] . Instead, NlpD is a late marker of cell division involved in the activation of AmiC and accumulates at septa that are already undergoing constriction (Fig. S6C) [START_REF] Uehara | LytM-domain factors are required for daughter cell separation and rapid ampicillin-induced lysis in Escherichia coli[END_REF][START_REF] Uehara | Daughter cell separation is controlled by cytokinetic ring-activated cell wall hydrolysis[END_REF] . DolP GFP accumulated at mid-cell sites where the envelope appeared invaginated, showing a localization pattern similar to that of NlpD mCherry (Fig. S6C) [START_REF] Tsang | NlpD links cell wall remodeling and outer membrane invagination during cytokinesis in Escherichia coli[END_REF] . We investigated the effect of short-lived (1 hour) BAM overproduction on DolP GFP localization. Strikingly, we found that BAM overproduction depleted DolP GFP from mid-cell sites (Figs. 5A, center, and S8C). In contrast, no obvious effects on cell division nor on mid-cell recruitment of ZipA mCherry and NlpD mCherry was observed (Fig. S7A). The overproduction of BamA alone was sufficient to alter the distribution of the DolP GFP fluorescence signal in constricting cells, reducing its intensity at constriction sites and enhancing it at decentred positions along the cell axis (Fig. 5B, right plot). In contrast, the overproduction of only the four BAM lipoproteins (Fig. 5A, right) as well as the overproduction of OmpA (Fig. S7B) had no obvious effects on DolP mid-cell localization.

As BAM catalyzes OMP assembly, we asked whether this activity interferes with DolP mid-cell localization. To address this question, we made use of an inactive BamA mutant form (Fig. S8A) harbouring a polyhistidine peptide extension at its C-terminal b-strand [START_REF] Hartmann | Sequence-Specific Solution NMR Assignments of the beta-Barrel Insertase BamA to Monitor Its Conformational Ensemble at the Atomic Level[END_REF] . Similar to the overaccumulation of the BAM complex or BamA, the overaccumulation of BamA His interfered with DolP mid-cell localization (Figs. S8B and S8C), without affecting ZipA mCherry and NlpD mCherry (Fig. S7C), indicating that the cellular localization of DolP does not depend on the OMPassembly activity of BamA. In contrast, the ability of BamA to assemble into the OM was a critical determinant of the observed septal depletion of DolP. In fact, the periplasm-accumulating BamA DP1/His variant (Fig. S8D) did not impair DolP GFP midcell localization (Fig. S8B, center), whereas the OM-overaccumulating BamA DP2/His did (Figs. S8B, right, S8C, and S8D). Taken together, these results suggest that the overaccumulation of BamA in the OM interferes with the recruitment of DolP at midcell sites.

DolP mid-cell localization is impaired under envelope stress conditions

As DolP is critical for fitness under envelope stress conditions, we asked whether the localization of DolP GFP would be affected in mutants undergoing envelope stress. To this end, we analyzed the localization of DolP GFP in strains lacking either the OMP chaperone SurA or the lipoprotein BamB. Both DsurA and DbamB strains are defective in OMP biogenesis and produce higher levels of BAM complex due to activation of the s E response [START_REF] Wu | Identification of a multicomponent complex required for outer membrane biogenesis in Escherichia coli[END_REF][START_REF] Rouviere | a periplasmic protein with peptidyl-prolyl isomerase activity, participates in the assembly of outer membrane porins[END_REF][START_REF] Charlson | Differential effects of yfgL mutation on Escherichia coli outer membrane proteins and lipopolysaccharide[END_REF][START_REF] Vertommen | Characterization of the role of the Escherichia coli periplasmic chaperone SurA using differential proteomics[END_REF] . Importantly, the frequency of mid-cell labelling by DolP GFP was reduced in DsurA cells both in minimal (Fig. S9A) and LB (Fig. 5C) culture media. In contrast, lack of SurA did not affect septal recruitment of the late cell division marker NlpD (Fig. S9B). The analysis of the fluorescence plot profiles of constricted cells clearly showed a marked reduction of the DolP GFP signal at mid-cell sites and higher fluorescence levels in decentred positions along the cell axis (Fig. 5C, right plot). As for the DsurA strain, DolP GFP accumulated at the mid-cell with a lower frequency when bamB was deleted (Fig. S9C). Together, these results indicate that the depletion of DolP at mid-cell sites occurs during envelope stress-induced activation of the s E response.

Discussion

Despite its critical role in maintaining OM integrity, the reason why DolP is upregulated by s E has remained unclear [START_REF] Morris | YraP Contributes to Cell Envelope Integrity and Virulence of Salmonella enterica Serovar Typhimurium[END_REF][START_REF] Tsang | NlpD links cell wall remodeling and outer membrane invagination during cytokinesis in Escherichia coli[END_REF][START_REF] Onufryk | Characterization of six lipoproteins in the sigmaE regulon[END_REF] . Here we demonstrate that DolP interacts with the BAM complex and is crucial for fitness in cells with elevated s E activity and when BAM overaccumulates in the OM. Our results have important implications for the understanding of the mechanisms that coordinate OM and peptidoglycan remodelling and help preserving envelope integrity.

A previous study [START_REF] Onufryk | Characterization of six lipoproteins in the sigmaE regulon[END_REF] has shown that the concomitant deletion of dolP and surA generates a severe synthetic growth defect. However, the role of DolP during envelope stress and in OMP biogenesis had remained unclear [START_REF] Onufryk | Characterization of six lipoproteins in the sigmaE regulon[END_REF][START_REF] Bos | Involvement of Neisseria meningitidis lipoprotein GNA2091 in the assembly of a subset of outer membrane proteins[END_REF] . Here we demonstrate that DolP interacts with the BamA subunits of the BAM complex. DolP, however, is not a stoichiometric component of the BAM complex and thus this interaction is probably transient or has a lower affinity compared to the interactions between BamA and the lipoproteins of the BAM complex. Several pieces of evidence presented in this study suggest that DolP is not strictly required for OMP assembly into the OM. In addition, we show that the deletion of dolP does not cause envelope stress and activation of the s E response. Increased s E activity has been reported to alleviate growth defects caused by the absence or inactivation of OMP biogenesis factors [START_REF] Konovalova | A Suppressor Mutation That Creates a Faster and More Robust sigmaE Envelope Stress Response[END_REF][START_REF] Kern | Suppressor Mutations in degS Overcome the Acute Temperature-Sensitive Phenotype of DeltadegP and DeltadegP Deltatol-pal Mutants of Escherichia coli[END_REF][START_REF] Hart | Fine-Tuning of sigma(E) Activation Suppresses Multiple Assembly-Defective Mutations in Escherichia coli[END_REF][START_REF] Leiser | Reversal of the ΔdegP phenotypes by a novel rpoE allele of Escherichia coli[END_REF] . On the contrary, activation of s E caused a growth defect in a DdolP strain. This result suggests that, under envelope stress conditions, DolP plays a fitness role that is not directly related to OMP biogenesis. More specifically, we have obtained evidence that DolP opposes a detrimental effect that can derive from the overaccumulation of BAM complex in the OM.

Further studies are warranted to determine the molecular bases of how DolP promotes fitness during activation of the s E response. It is worth noting, however, that DolP consists of two BON (bacterial OsmY and nodulation) domains, folding motifs that have been proposed to interact with hydrophobic ligands such as lipids [START_REF] Yeats | The BON domain: a putative membrane-binding domain[END_REF] . Phospholipid-binding by DolP has been reported in a recent study [START_REF] Bryant | Structure-function analyses of dual-BON domain protein DolP identifies phospholipid binding as a new mechanism for protein localisation[END_REF] . A key aspect of our results hints at a possible role of the lipid-binding function of DolP in improving the fitness of cells that undergo envelope stress. We have shown that DolP interacts with OM-assembled BamA, a subunit of the BAM complex that is predicted to interfere with the organization of the surrounding lipid bilayer and to generate an energetically favourable environment for the insertion of nascent OMPs in the OM [START_REF] Fleming | A combined kinetic push and thermodynamic pull as driving forces for outer membrane protein sorting and folding in bacteria[END_REF][START_REF] Horne | Role of the lipid bilayer in outer membrane protein folding in Gram-negative bacteria[END_REF] . BamA-mediated membrane destabilization was shown by molecular dynamics simulations, as well as by reconstituting BamA into proteoliposomes [START_REF] Noinaj | Structural insight into the biogenesis of beta-barrel membrane proteins[END_REF][START_REF] Sinnige | Solid-state NMR studies of full-length BamA in lipid bilayers suggest limited overall POTRA mobility[END_REF] .

Here, we have provided evidence that the progressive overaccumulation of BAM in the OM has an increasingly detrimental effect, enhancing the OM permeability at low increments and impairing growth at higher levels. Our observation that DolP is needed for optimal growth when the BAM complex overaccumulates in the OM is compatible with a role of DolP in membrane stabilization at BAM sites. It remains to be seen how DolP docks to BamA and whether it influences the organization of BAM sites and of the surrounding lipid bilayer.

Another key finding of our study is that DolP septal localization is sensitive to envelope stress conditions. During envelope stress, the OM undergoes a significant alteration of its protein composition. Activation of the s E response triggers activation of genes encoding OMP biogenesis factors, but also the posttranscriptional downregulation of many OMPs including porins and OmpA [START_REF] Rhodius | Conserved and variable functions of the sigmaE stress response in related genomes[END_REF] . OMPs are largely arranged in clusters [START_REF] Rassam | Supramolecular assemblies underpin turnover of outer membrane proteins in bacteria[END_REF][START_REF] Gunasinghe | The WD40 Protein BamB Mediates Coupling of BAM Complexes into Assembly Precincts in the Bacterial Outer Membrane[END_REF][START_REF] Jarosławski | High-resolution architecture of the outer membrane of the Gram-negative bacteria Roseobacter denitrificans[END_REF] embedded by highly organized LPS molecules in the external leaflet of the OM [START_REF] Nikaido | Molecular basis of bacterial outer membrane permeability revisited[END_REF] and the rigidity of their b-barrel structures contributes to the mechanical stiffness of the OM [START_REF] Lessen | Building Blocks of the Outer Membrane: Calculating a General Elastic Energy Model for β-Barrel Membrane Proteins[END_REF] . Posttranscriptional downregulation targets of s E activation also include the lipoproteins Pal and Lpp [START_REF] Guo | a new σE-dependent sRNA, combats envelope stress by repressing synthesis of Lpp, the major outer membrane lipoprotein[END_REF][START_REF] Gogol | Small RNAs endow a transcriptional activator with essential repressor functions for single-tier control of a global stress regulon[END_REF] , which are critical for OM integrity [START_REF] Cascales | Pal lipoprotein of Escherichia coli plays a major role in outer membrane integrity[END_REF][START_REF] Asmar | the Braun lipoprotein, turns 50-major achievements and remaining issues[END_REF] . Whereas we have shown that BAM overaccumulation in the OM influences DolP localization, it is possible that other modifications of the OM during envelope stress contribute to impair the recruitment of DolP at constriction sites.

Distinct biogenesis and surveillance pathways are required to maintain the protective function of the multi-layered envelope of Gram-negative bacteria [START_REF] Egan | Regulation of peptidoglycan synthesis and remodelling[END_REF] . Our CRISPRi analysis shows that DolP promotes efficient cell growth when the AmiA pathway of septal peptidoglycan splitting is impaired. This result is in line with the conclusions of a previous study implicating DolP in the regulation of NlpD-mediated activation of AmiC [START_REF] Tsang | NlpD links cell wall remodeling and outer membrane invagination during cytokinesis in Escherichia coli[END_REF] . Cells lacking both NlpD and AmiC have reduced OM integrity [START_REF] Tsang | NlpD links cell wall remodeling and outer membrane invagination during cytokinesis in Escherichia coli[END_REF] , which may contribute to explain the vancomycin sensitivity of DdolP cells. It remains poorly understood whether envelope stress caused by the accumulation of unfolded OMPs influences peptidoglycan remodelling. Intriguingly, our observation that mid-cell localization of DolP is reduced in surA and bamB deletion strains points to a possible role of DolP in linking envelope stress to septal peptidoglycan hydrolysis. Reduced levels of DolP at mid-cell sites, and thus impaired AmiC activation [START_REF] Tsang | NlpD links cell wall remodeling and outer membrane invagination during cytokinesis in Escherichia coli[END_REF] , could play an important role in coping with envelope stress, for instance by regulating the window of time available to restore normal OMP biogenesis prior to completing the formation of the new poles in the cell offspring.

Taken together, our results reveal an unprecedented link between activation of the envelope stress response and a late step of cell division. The fitness role of DolP during envelope stress emerges as an exploitable target in the development of new antibacterial therapies.

Materials and Methods

Bacterial strains and growth conditions

All E. coli strains used in this study are listed in Table S3. Strains newly generated for this study derive from BW25113 [D(araD-araB)567 D(rhaD-rhaB)568 DlacZ4787 (::rrnB-3) hsdR514 rph-1] [START_REF] Grenier | Complete Genome Sequence of Escherichia coli BW25113[END_REF] or MG1655 (F -λ -ilvG -rfb-50 rph-1) [START_REF] Blattner | The complete genome sequence of Escherichia coli K-12[END_REF] .

Deletions of dolP, rseA, surA, bamB, degP, or skp in a BW25113 strain were achieved by P1 transduction of the DdolP::kan R , DrseA::kan R , DsurA::kan R , DbamB::kan R , DdegP::kan R or Dskp::kan R alleles, respectively, obtained from the corresponding Keio collection strains [START_REF] Baba | Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection[END_REF] . BW25113 derivative strains harbouring chromosomal fusions of constructs encoding superfolder GFP downstream of dolP or mCherry downstream of nlpD, zipA and bamD were obtained by l-red recombination as previously described [START_REF] Datsenko | One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products[END_REF] . Briefly, a kanamycin-resistance cassette was amplified from plasmid pKD4 using oligonucleotides carrying extensions of approximately 50 nucleotides homologous to regions immediately upstream or downstream the stop codon of the interested genes. DpnI-digested and purified PCR products were electroporated into the BW25113 or derivative strains. Recombinant clones were selected at 37°C on LB agar plates containing kanamycin. When necessary, the kan R cassette inserted into a mutated locus (gene deletion or fusion) was removed upon transformation with the heat-curable plasmid pCP20 [START_REF] Datsenko | One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products[END_REF] . The MG1655 derivative strain LC-E75, harbouring a dCas9-encoding construct under the control of the P tet promoter, has been described [START_REF] Cui | A CRISPRi screen in E. coli reveals sequence-specific toxicity of dCas9[END_REF] . Cells were cultured in home-made lysogeny broth (LB) medium (1% (w/v) tryptone, 0.5% (w/v) yeast extract, 5 mg/ml (NaCl), commercially available Miller LB Broth (Sigma) or M9 minimal medium containing M9 salts (33.7 mM Na 2 HPO 4 , 22 mM KH 2 PO 4 , 8.55 mM NaCl, 9.35 mM NH 4 Cl) and supplemented with 0.2% w/v glycerol and all the amino acids, or all amino acids except methionine and cysteine in the case of [START_REF] Seib | The meningococcal vaccine antigen GNA2091 is an analogue of YraP and plays key roles in outer membrane stability and virulence[END_REF] S pulsechase labeling. Antibiotics were used at the following concentrations: ampicillin 100 µg/ml, kanamycin 50 µg/ml, vancomycin 60 µg/ml. For spot tests, cells were cultured to mid-log phase, washed with M9 salts and serially diluted in ice-cold M9 salts prior to spotting on agar plates.

Plasmid construction

All plasmids used in data figures are listed in Table S4. Plasmids for the ectopic expression of BAM subunits, DolP, or OmpA are derived from a pTrc99a vector. The plasmid pBAM His (pJH114), which harbours a P trc promoter followed by the bamABCDE open reading frames and an octahistidine tag fused downstream of bamE, was described [START_REF] Roman-Hernandez | Reconstitution of bacterial autotransporter assembly using purified components[END_REF] . The region of pBAM His comprising the segment that spans from the bamA start codon to the bamE stop codon was deleted by site-directed mutagenesis, generating pCtrl. Plasmids pBamA His was generated by restrictionfree cloning, inserting the bamA ORF without its stop codon downstream of the P trc promoter and upstream of the octahistidine encoding region in pCtrl. pBamA His was subjected to site directed mutagenesis to generate pBamA, encoding wild-type, non-tagged BamA. The dolP ORF amplified from the BW25113 genomic DNA was used to replace the bamABCDE ORFs in pJH114 by restriction-free cloning, generating pDolP His . The plasmid pBamA-DolP His was generated by restriction-free cloning of the bamA ORF between the P trc promoter and dolP in pDolP His . Sitedirected mutagenesis was conducted on pBAM His (pJH114) to obtain pBamACDE His , pBamABDE His , pBamBCDE His , pBamCDE His . A sequence encoding the tobacco etch virus protease cleavage site (TEV site) followed by a tandem Protein-A tag was amplified from pYM10 [START_REF] Knop | Epitope tagging of yeast genes using a PCR-based strategy: more tags and improved practical routines[END_REF]

CRISPRi screen and data analysis

Strain LC-E75 (dolP + ) and its DdolP derivative were transformed with the EcoWG1 library which contains 5 guides per gene as previously described [START_REF] Calvo-Villamanan | On-target activity predictions enable improved CRISPR-dCas9 screens in bacteria[END_REF] . After culturing pooled transformant cells in LB at 37°C to early exponential phase (optical density at 600 nm [OD 600 ] = 0.2), a sample was withdrawn for plasmid isolation (t start ). Subsequently, cultures were supplemented with 1 µM anhydrotetracycline (aTc) to induce dCas9 expression and further incubated at 37°C. When cultures reached an OD 600 of 2 they were diluted 1:100 into LB supplemented with 1 µM aTc and incubated at the same temperature until an OD 600 of 2. This step was repeated one more time prior to withdrawing a sample for isolation of plasmid DNA (t end ).

Sequencing indexes were used to assign reads to each sample. Illumina sequencing samples were prepared and analyzed as previously described [START_REF] Cui | A CRISPRi screen in E. coli reveals sequence-specific toxicity of dCas9[END_REF] .

Briefly, a two-step PCR was performed with Phusion polymerase (Thermo Scientific) using indexed primers. The first PCR adds the first index and the second PCR adds the second index and flow-cell attachment sequences. Pooled PCR products were gel-purified. Sequencing was performed on a NextSeq550 machine (Illumina). The total number of reads obtained for each sample was used to normalize raw reads by sample size. Replicates were pooled to increase depth before another normalization by sample size. Guides with less than 100 normalized read counts in initial time points were discarded. For each screen, sgRNA fitness was calculated as the log2-transformed ratio of normalized reads counts between the final and the initial time point:

𝑙𝑜𝑔2𝐹𝐶 = 𝑙𝑜𝑔2 𝑁𝑜𝑟𝑚𝑎𝑙𝑖𝑧𝑒𝑑 𝑟𝑒𝑎𝑑𝑠 23456 + 1 𝑁𝑜𝑟𝑚𝑎𝑙𝑖𝑧𝑒𝑑 𝑟𝑒𝑎𝑑𝑠 3439356 + 1
For each sample, log2FC values were centered by subtracting the median log2FC of non-targeting control guides. We then calculated for each sgRNA the difference of log2FC value between the DdolP screen and the dolP + screen. Guides were ranked from the lowest negative values (negative fitness effect in DdolP compared to dolP + ) to the highest positive values (positive fitness effect in DdolP compared to dolP + ) and the significance of the interaction between dolP and each gene was evaluated by performing a minimum hypergeometric (mHG) test on the ranked list for each gene using the mHG R package (v. 1.1) [START_REF] Mcleay | Motif Enrichment Analysis: a unified framework and an evaluation on ChIP data[END_REF] . False-discovery rate (FDR) was used to correct p-values for multiple testing. For each gene, the median difference of log2FC between DdolP and dolP + screens was used as a measure of the genetic interaction.

Cell fractionation

To prepare whole-cell lysates, cells were cultured to early exponential phase (OD 600 = 0.2-0. ). The total protein content was denatured at 98°C for 5 min prior SDS-PAGE analysis.

To perform lysozyme/EDTA lysis, cells were cultured to early exponential phase, collected by centrifugation, resuspended in 33 mM Tris-HCl pH 8 to an OD 600 of 1.

The cell suspension was then supplemented with 0.1 mg/ml lysozyme (Sigma), 2 mM EDTA and incubated on ice for 20 minutes to induce lysis. After addition of 10 mM MgSO 4, the membrane fraction was collected by centrifugation at 16000x g. The supernatant was further centrifuged at 100000x g to remove any residual membrane fraction, which was discarded. The obtained soluble fraction was subjected to protein precipitation by adding 10% (w/v) trichloroacetic acid (TCA).

TCA precipitates were solubilized in Laemmli Sample Buffer (BioRad) prior to SDS-PAGE analysis.

The crude envelope fractions directly analyzes by SDS-PAGE or used for native affinity purification of affinity tagged BAM complex or DolP were prepared from cells that were cultured in LB until early exponential phase and, where indicated, supplemented with 400 µM IPTG for 1 hour to induce ectopic expression.

Cells were collected by centrifugation at 6000x g at 4°C, resuspended in 20 mM

Tris-HCl pH 8, and mechanically disrupted using a Cell Disruptor (Constant Systems LTD) set to 0.82 kPa. The obtained cell lysate fractions were clarified by centrifugation at 6000x g and 4°C. The supernatant was then subjected to ultracentrifugation at 100000x g at 4°C to collect the envelope fraction.

Isolation of native protein complexes by IgG-or nickel-affinity chromatography

The envelope fraction was resuspended at a concentration of approximately 10 mg/ml in solubilization buffer ( To facilitate extraction of membrane proteins, samples were subjected to mild agitation for 1 hour at 4°C. Insoluble material was removed by centrifugation at 16000x g at 4°C. To perform IgG affinity purification, membrane-extracted proteins were incubated for 1.5 hours at 4°C with purified human IgG (Sigma) that had been previously coupled to CNBr-activated Sepharose beads (GE Healthcare). After extensive washes of the resin with solubilization buffer containing 0.3% (w/v) digitonin and 0.03% (w/v) DDM, bound proteins were eluted by incubation with

AcTEV protease (ThermoFisher) overnight at 4°C under mild agitation. To perform nickel (Ni)-affinity purification, membrane-extracted proteins were supplemented with 20 mM imidazole and incubated with Protino Ni-NTA agarose beads (Macherey-Nagel) for 1 hour at 4°C. After extensive washes of the resin with solubilization buffer supplemented with 50 mM imidazole, 0.3% (w/v) digitonin, 0.03% (w/v) DDM, and the EDTA-free protease inhibitor cocktail (Roche), bound proteins were eluted using the same buffer supplemented with 500 mM imidazole.

In vitro reconstitution of the BAM-DolP interaction and BN-PAGE analysis

Envelope fractions were obtained from cells carrying pBAM His or pDolP His and cultured until early exponential phase in LB medium at 37°C and subsequently supplemented with 400 µM IPTG for 1.5 hours to induce the expression of the BAM complex genes or dolP.

Pulse-chase in vivo protein labeling

Pulse-chase labeling of cellular proteins with radioactive [START_REF] Seib | The meningococcal vaccine antigen GNA2091 is an analogue of YraP and plays key roles in outer membrane stability and virulence[END_REF] S methionine and cysteine was conducted as previously described [START_REF] Ieva | Interaction of an autotransporter passenger domain with BamA during its translocation across the bacterial outer membrane[END_REF] . Briefly, overnight cultures were washed and freshly diluted into M9 medium to OD 600 = 0.03 and incubated at 37 °C.

When the cultures reached OD 600 = 0.3, ectopic expression of EspP was induced by adding 200 μM IPTG for 30 min. [START_REF] Seib | The meningococcal vaccine antigen GNA2091 is an analogue of YraP and plays key roles in outer membrane stability and virulence[END_REF] S labelling was performed by supplementing cultures with a mixture of [ 35 S]-methionine and [ 35 S]-cysteine followed after 30 seconds by the addition of an excess of cold methionine and cysteine. Samples were withdrawn at different chase times, immediately mixed with an equal volume of ice and subjected to TCA precipitation, prior to immunoprecipitation.

Antibodies

Proteins separated by SDS-PAGE or blue native-PAGE and blotted onto PVDF membranes were immunodecorated using epitope-specific rabbit polyclonal antisera with the following exceptions. The F 1 b subunit of the ATP F 1 F O synthase was detected using a rabbit polyclonal antiserum raised against an epitope of the homologous protein of Saccharomyces cerevisiae (Atp2). RpoB was detected using a mouse monoclonal antibody (NeoClone Biotechnology). The secondary immunodecoration was conducted using anti-rabbit or anti-mouse peroxidaseconjugated antibodies produced in goat (Sigma). EspP immunoprecipitation was conducted using a rabbit polyclonal antiserum specific for a C-terminal epitope of EspP.

Epifluorescence microscopy and analysis

Overnight cultures of E. coli BW25113 and its derivative strains were diluted into fresh M9 medium containing 0.2% glycerol or LB medium and grown at 30°C to OD 600 = 0.2-0. fold-change (log2FC) between t end and t start calculated for each sgRNAs (Fig. S2B) was grouped by gene target, and their median (Table S1) was used to derive fitness 1%, 0.1% (lane 1), 0.8%, 0.3% (lane 2), 0.3%, 0.8% (lane 3), 0.1%, 1% (lane 4).

His-tagged DolP was purified by Ni-affinity chromatography. In all cases, proteins were eluted in the presence of 0.3% (w/v) digitonin and 0.03% (w/v) DDM. Load 0.2%; Elution 100%. (C) Gene Ontology analysis of the genes with a significant synthetic interaction with dolP. Using FDR<0.05 as a threshold, 27 genes were selected for Gene Ontology (GO) analysis for cellular component (upper panel) or biological process (bottom panel) [START_REF] Ashburner | Gene ontology: tool for the unification of biology. The Gene Ontology Consortium[END_REF] . Both analyses show an overrepresentation of genes associated with cell envelope and membrane function. Table S1. Log2FC values of sgRNAs in the WT or ΔdolP screen.

See source data file (Table S1) Table S2. Genetic interaction scores.

See source data file (Table S2)

Contrast

DolP GFP

  and fused by restriction-free cloning with the last codon of the bamE gene in pBAM His to generate pBAM ProtA . A stop codon was introduced downstream of the bamE last codon to generate pBAM. Plasmids encoding the DP1 and DP2 BamA variant were obtained by site-directed mutagenesis deleting the portion of bamA ORFs corresponding to residues E22-K89 or P92-G172, respectively. The TEV site and the tandem Protein-A construct amplified from pYM10 were inserted by restriction-free cloning downstream of the dolP last codon in pDolP His , generating pDolP ProtA . pDolP was derived from pDolP ProtA using site-directed mutagenesis to introduce a stop codon immediately downstream of the dolP ORF. The ompA ORF was amplified from the BW25113 genomic DNA and inserted by restriction-free cloning between P trc and the His-tag encoding construct of pCtrl to generate pOmpA His . The sgRNAs plasmids are derived from psgRNAcos[START_REF] Cui | A CRISPRi screen in E. coli reveals sequence-specific toxicity of dCas9[END_REF] . To generate sgRNA-encoding plasmids the DNA sequences AGCTGCACCTGCTGCGAATA (bamD sgRNA, plasmid pCAT187), sgRNA, plasmid pCAT191), and CTGAGCCGCCGACCGATTTA (ftsX sgRNA, pCAT193) were inserted into a BsaI site of the psgRNAcos.

  20 mM Tris-HCl pH 7.4, 100 mM NaCl, 0.1 mM EDTA, 2 mM PMSF) supplemented with EDTA-free protease inhibitor cocktail (Roche), and 1.1% (w/v) of a mild detergent component corresponding to digitonin (Merck) and n-dodecyl b-D-maltoside (DDM, Merck) as indicated in Figure Legends.

3 .

 3 When indicated, cultures were supplemented with 400 µM of IPTG to induce ectopic expression of plasmid-borne genes for 1 hour prior to collecting samples for microscopy analysis. Culture volumes of 0.6 µl were deposited directly onto slides coated with 1% (w/v) agarose in a phosphate-buffered saline solution and visualized by epifluorescence microscopy. Cells were imaged at 30°C using an Eclipse TI-E/B Nikon wide field epifluorescence inverted microscope with a phase contrast objective (Plan APO LBDA 100X oil NA1.4 Phase) and a Semrock filter mCherry (Ex: 562BP24; DM: 593; Em: 641BP75) or FITC (Ex: 482BP35; DM: 506; Em: 536BP40). Images were acquired using a CDD OrcaR2 (Hamamatsu) camera with illumination at 100% from a HG Intensilight source and with an exposure time of 1-3 seconds, or using a Neo 5.5 sCMOS (Andor) camera with illumination at 60% from a LED SPECTRA X source (Lumencor) with an exposure time of 2 seconds. Nis-Elements AR software (Nikon) was used for image capture. Image analysis was conducted using the Fiji and ImageJ software. The fraction of cells with DolP GPF signals at mid-cell sites was estimated using the Fiji Cell Counter plugin. Collective profiles of fluorescence distribution versus the relative position along the cell axis were generated using the Coli-Inspector macro run in ImageJ within the plugin ObjectJ 80 , selecting only cells with a constriction (80% of cell diameter) as qualified objects. experiments; D.R., Y.Y., L.O., F.R., L.C., V.M., C.M., G.M., A.C.-S, C.T., J.R. and C.A. performed the experiments and analyzed the data together with R.I. and D.B.; A.C.-S, D.R., Y.Y., L.O. and F.R. prepared the figures; all authors discussed the project and the experimental results, and commented on the manuscript.

Fig. 1 .

 1 Fig. 1. Genome-wide screen of dolP interactions.

  gene scores. Right: Volcano plot of the dolP genetic interaction (GI) scores. The xaxis shows a genetic interaction score calculated for each gene based on the minimum hypergeometric (mHG) test conducted on the ranked difference of sgRNA-specific log2FC values between the DdolP and the dolP + screens. The yaxis shows the log10 of the false discovery rate (FDR) of the test. The dashed line shows FDR = 0.05. In both panels, genes highlighted in orange have FDR < 0.05 and GI>1 whereas genes highlighted in red have FDR < 0.05 and GI>2. (D and E) Validation of the genetic interactions determined in (C). (D) LC-E75 (dolP + ) or its DdolP derivative strain expressing sgRNAs that target the indicated genes were serially diluted and spotted on LB agar lacking or supplemented with aTc to induce expression of dcas9, as indicated. (E) BW25113 derivative cells deleted of rseA or both rseA and dolP were transformed with an empty vector (pCtrl) or a plasmid encoding DolP (pDolP His ). Ectopic expression of DolP His was driven by the leaky transcriptional activity of P trc in the absence of IPTG. (D and E) 10-fold serial dilutions of the indicated transformants were spotted on LB agar.

Fig. 2 .Fig. 3 .

 23 Fig. 2. DolP promotes fitness in cells that undergo envelope stress.

Fig. 4 .

 4 Fig. 4. DolP associates with the BAM complex via an interaction with BamA.

(

  E) The envelope fraction of BW25113 cells overproducing DolP His or the BAM complex containing C-terminally His-tagged BamE was subjected to protein extraction with 1% (w/v) DDM and Ni-affinity purification and gel filtration chromatographies. The elution fractions were analyzed by SDS-PAGE and coomassie staining. The double asterisk indicates a contaminant protein in the elution of DolP.(F) Roughly equimolar quantities of purified His-tagged BAM complex and DolP were incubated alone for 1 hour at 4°C (lanes 1, 2 and 7), or together for 1 hour at 4°C (lanes 3 and 6) or for 30 min at 25°C (lanes 4 and 5), prior to blue native-PAGE and immunoblotting using the indicated antisera.

Fig. 5 .

 5 Fig. 5. BamA overaccumulation in the OM and envelope stress interfere with mid-cell localization of DolP.

Fig. S1 .

 S1 Fig. S1. The deletion of dolP severely impairs growth in the presence of vancomycin.

Fig. S2 .

 S2 Fig. S2. Reproducibility of the CRISPRi screens and ontology analysis of gene hits.

Fig. S3 .

 S3 Fig. S3. DolP is not crucial for OMP biogenesis.

Fig. S4 .

 S4 Fig. S4. The detrimental effect of BAM overproduction is caused by the overaccumulation of BAM in the OM

Fig. S5 .

 S5 Fig. S5. Effect of lack or overproduction of DolP on BAM localization.

Fig. S6 .

 S6 Fig. S6. DolP accumulates at mid-cell sites during a late step of cell division.

Fig. S7 .

 S7 Fig. S7. Overproduction of BAM does not influence septal recruitment of NlpD and ZipA.

Fig. S8 .

 S8 Fig. S8. BamA overaccumulation in the OM impairs septal DolP localization.

Fig. S9 .

 S9 Fig. S9. Envelope stress influences DolP localization

  Figure 1.

  Figure 2.

  Figure 5.

  Figure S3.

  Figure S5.

  3) in LB medium at 37°C and collected. Where indicated, IPTG was

	added 60 min prior to cell collection. Cells were pelleted by centrifugation, washed
	once with M9 salt and lysed with Laemmli Sample Buffer (BioRad) (69 mM Tris-HCl,
	pH 6.8, 11.1% [v/v] glycerol, 1.1% [w/v] lithium dodecyl sulfate [LDS], 0.005% [w/v]

bromophenol blue, supplemented with 357 mM b-mercaptoethanol and 2 mM phenylmethylsulfonyl fluoride

[PMSF]

  The envelope fractions were solubilized and purified by Ni-

	Purifier 10 (GE Healthcare) at 4°C. The column containing bound proteins was
	washed with equilibration buffer supplemented with 50 mM imidazole. Proteins were
	eluted in equilibration buffer, applying a gradient of imidazole from 50 mM to 500
	mM and further separated by gel filtration using an HiLoad 16/600 Superdex 200
	(GE Healthcare) in equilibration buffer. Eluted proteins were concentrated using an
	ultrafiltration membrane with a 10 kDa molecular weight cutoff (Vivaspin 6,
	Sartorius). To reconstitute the BAM-DolP complex in vitro, equimolar concentrations
	of purified BAM and DolP were used. Purified proteins were mixed in equilibration
	buffer for 1 hour at 4°C or for 30 min at 25°C as indicated in Figure Legends. The
	reaction was further diluted 1:4 times in ice-cold blue native buffer (20 mM Tris-HCl
	pH 7.4, 50 mM NaCl, 0.1 mM EDTA, 1% [w/v] digitonin, 10% w/v glycerol) and ice
	cold blue native loading buffer (5% coomassie brilliant blue G-250, 100 mM Bis-
	affinity and size exclusion chromatography, adapting a previously published
	protocol. Briefly, after membrane solubilization with 50 mM Tris-HCl pH 8.0, 150
	mM NaCl, and 1% (w/v) DDM, and removal of insoluble material by
	ultracentrifugation at 100000x g, 4°C, soluble proteins were loaded onto a Ni-

column (HisTrap FF Crude, GE Healthcare) pre-equilibrated with 50 mM Tris-HCl pH 8.0, 150 mM NaCl, and 0.03% (w/v) DDM (equilibration buffer), using an ÄKTA Tris-HCl, pH 7.0, 500 mM 6-aminocaproic acid) prior to loading onto home-made 5-13% blue native polyacrylamide gradient gels. Resolved protein complexes were blotted onto a PVDF membrane and immunolabeled. Where non-relevant gel lanes were removed, a white space was used to separate contiguous parts of the same gel.

Table S3 : List of strains Name Genotype and relevant features Source

 S3

	1005	Table S4: List of plasmids pBamBCDE His Ectopic expression of BamB, BamC,	This study
			BamD and BamE subunits; BamE is C-
	1006	Name	Relevant features terminally His-tagged	Source
		pCtrl pBamCDE His	Reference empty vector for ectopic Ectopic expression of BamC, BamD and	This study This study
		BW25113 pBAM	D(araD-araB)567 D(rhaD-rhaB)568 DlacZ4787(::rrnB-3) hsdR514 rph-1 (wild-type reference) BamE subunits; BamE is C-terminally protein expression Ectopic expression of all 5 BAM subunits This study Grenier et al., 2014 74 His-tagged
		DrseA DdolP DbamB (pJH114) pBAM His pBamA	BW25113 rseA::kan BW25113 dolP::kan BW25113 bamB::kan BamE is C-terminally His-tagged Ectopic expression of all 5 BAM subunits; Ectopic expression of wild-type BamA	This study This study Hernandez et This study Roman This study
		DbamB DdolP	BW25113 DbamB dolP::kan	This study al., 2014 55
		DdolP DrseA Dskp pBAM ProtA pBamA His DdolP Dskp DdegP pBamA DP1/His	BW25113 DdolP rseA::kan BW25113 skp::kan Ectopic expression of all 5 BAM subunits; Ectopic expression of C-terminally His-BamE is C-terminally Protein A-tagged; a tagged BamA BW25113 DdolP skp::kan BW25113 degP::kan TEV site amino acid linker is positioned Ectopic expression of a C-terminally His-	This study This study This study This study This study This study This study
		DdolP DdegP dolP-gfp DsurA pBAM DP1/ProtA pBamA DP2/His	BW25113 DdolP degP::kan BW25113 dolP-gfp tagged BamA variant lacking POTRA1 immediately upstream of the tag BW25113 surA::kan Ectopic expression of all 5 BAM subunits; Ectopic expression of a C-terminally His-	This study This study This study This study
		DsurA dolP-gfp	BW25113 surA::kan dolP-gfp BamA harbours the deletion of POTRA1 tagged BamA variant lacking POTRA2	This study
		DbamB dolP-gfp BW25113 bamB::kan dolP-gfp and BamE is C-terminally Protein A-	This study
		DdolP bamD-mCherry	BW25113 DdolP bamD-mCherry tagged; a TEV site amino acid linker is	This study
		bamD-mCherry	BW25113 bamD-mCherry positioned immediately upstream of the	This study
		dolP-gfp nlpD-mCherry	BW25113 dolP-gfp nlpD-mCherry tag	This study
		DsurA dolP-gfp pBAM DP2/ProtA	BW25113 dolP-gfp nlpD-mCherry surA::kan Ectopic expression of all 5 BAM subunits;	This study This study
		nlpD-mCherry dolP-gfp	BW25113 dolP-gfp zipA-mCherry BamA harbours the deletion of POTRA2	This study
		zipA-mCherry	and BamE is C-terminally Protein A-
		JCM166	MC4100 ara r/-D(λatt-lom)::bla P BAD yaeT araC tagged; a TEV site amino acid linker is DyaeT (BamA depletion strain) positioned immediately upstream of the	Wu et al., 2005 19
		LC-E75 pBamABDE His	F -λ -ilvG -rfb-50 rph-1 186attB::P tet -dcas9, lattB::mCherry tag Ectopic expression of BamA, BamB,	Cui et al., 2018 38 This study
		LC-E75 DdolP	LC-E75 dolP::kan BamD and BamE subunits; BamE is C-	This study
			terminally His-tagged
		pBamACDE His	Ectopic expression of BamA, BamC,	This study
			BamD and BamE subunits; BamE is C-
			terminally His-tagged

Acknowledgments

We thank Harris Bernstein (NIDDK/NIH, Bethesda, MD) for providing key reagents and for comments on the manuscript. We thank Tanneke Den Blaauwen (University of Amsterdam) and Nathalie Dautin (IBPC/CNRS, Paris) for discussion.

We thank the Light Imaging Toulouse CBI (LITC) platform for assistance with and maintenance of the microscopy instrumentation. Financial support was provided by: the Fondation pour la Recherche Médicale to D.R.; the Chinese Scholarship Council to Y.Y.; the Ecole Normale Supérieure to F.R.; the European Research Council (Europe Union's Horizon 2020 research and innovation program, grant agreement No 677823), the French governmental Investissement d'Avenir program and the Laboratoire d'Excellence "Integrative Biology of Emerging Infectious Diseases" (ANR-10-LABX-62-IBEID) to D.B.; the CNRS ATIP-Avenir program to R.I.