The periplasmic space cannot be artificially enlarged due to homeostatic regulation 1 maintaining spatial constraints essential for membrane spanning processes and cell 2 viability

The periplasmic space cannot be artificially enlarged due to homeostatic regulation 1 maintaining spatial constraints essential for membrane spanning processes and cell 2 viability. 3 4 Eric Mandela1,+, Christopher J. Stubenrauch1,+, David Ryoo2, Hyea Hwang3,#, Eli J. Cohen4, Von L. Torres1, 5 Pankaj Deo1, Chaille T. Webb1, Cheng Huang5, Ralf B. Schittenhelm5, Morgan Beeby4, JC Gumbart6,*, Trevor 6 Lithgow1,* & Iain D. Hay7,* 7 8 1 Infection & Immunity Program, Biomedicine Discovery Institute and Department of Microbiology, 9 Monash University, Clayton, 3800, Australia 10 2 Interdisciplinary Bioengineering Graduate Program, Georgia Institute of Technology, Atlanta, GA 30332, 11 USA 12 3 School of Materials Science and Engineering, Georgia Institute of Technology, Atlanta, Georgia 3033213 0430, USA 14 4 Department of Life Sciences, Imperial College London, London SW7 2AZ, UK. 15 5 Monash Proteomics & Metabolomics Facility, Department of Biochemistry and Molecular Biology, 16 Biomedicine Discovery Institute, Monash University, Clayton, 3800, Australia 17 6 School of Physics, Georgia Institute of Technology, Atlanta, Georgia 30332-0430, USA 18 7 School of Biological Sciences, The University of Auckland, Auckland 1010, New Zealand 19

INTRODUCTION 42 43 Gram-negative bacteria have a cell envelope composed of two membranes sandwiching between them an 44 aqueous space called the periplasm, in which an essential structural layer of peptidoglycan (PG) resides. The 45 outer membrane is critical to cell growth and these bacteria face challenges to their cell biology in terms of 46 membrane protein assembly and lipid-transport pathways that must traverse the distance from the inner 47 membrane (IM) to the outer membrane (OM) 1 . Recent work investigating the spatial demands for assembly 48 of proteins into the outer membrane has shown precincts of active protein integration into the membrane can 49 deliver new material to the growing outer membrane 2 and that random planar movement from these precincts 50 drives the observed non-uniform distributions of the major proteins of the outer membrane 3, 4 . By contrast to 51 these protein components that diffuse to the outer membrane, elements of the lipid-transport machinery (eg. 52 the Lpt complex) 5,6,7,8,9 span the OM and IM in order to fulfil their function in delivering lipid components 53 to the outer membrane. 54

55
The PG layer is a fundamental aspect of the cell envelope, and it must be dynamically remodeled to allow 56 growth as well as the assembly and transit of trans-envelope structures. PG synthesis and remodeling is a 57 complex process with high levels of redundancy at various steps, involving at least 50 enzymes in E. coli 10 . 58 The Penicillin-binding proteins (PBPs) are the core components responsible for the periplasmic biosynthesis 59 of peptidoglycan. There are multiple PBP complexes including the two core, semi-redundant PBP complexes 60 PBP1a and PBP1b embedded in the IM which are activated by interactions with lipoproteins LpoA and LpoB 61 embedded in the OM. Thus the activation of PG synthesis by these enzymes is spatially regulated, serving as 62 a self-limiting molecular ruler to modulate PG thickness 11 . Cells must possess either a functional PBP1a or 63 PBP1b system for growth 12,13 . 64 4 To understand how trans-envelope processes in E. coli adapt to the presence of an enlarged periplasm, a 77 combination of phenotypic analysis, proteomics, molecular dynamics and a synthetic lethal screen was 78 employed to identify and characterize factors needed to maintain viability in the Lpp +21 strain of E. coli. The 79 genetic screen demanded synthetic growth phenotypes from an array of mutants each lacking a gene that, 80 while non-essential in wild-type E. coli 19 , is essential in the Lpp +21 strain. These genes fall into three functional 81 categories: PG biosynthesis and remodelling, LPS biosynthesis and PG-outer membrane linkage. We show 82 that previously non-essential proteins involved in bridging the gap between the OM and PG become essential 83 in the context of the Lpp +21 strain background. These include previously known PG binding OM proteins 84 (OmpA and Pal) as well as proteins previously not known to play an active role in linking the OM and PG 85 (TolC and YiaD). We observed a thicker more diffuse or heterogeneous PG layer in the Lpp +21 strain and 86 whole cell proteomics revealed that in response to an increased length of Lpp, E. coli increases the levels of a 87 range of cell envelope proteins involved in PG turnover. We discuss the outcomes in terms of how the PG-88 outer membrane linkage functionalizes the periplasm, the evolutionary constrains in place to maintain this 89 functionality, and the specific activity of Lpp in contributing to the load-bearing function of the OM. 90

Resilience and growth of the Lpp +21 strain 94
A phylogenetic assessment of Lpp lengths across diverse bacterial lineages showed a very narrow window of 95 protein size (Fig. 1A), with Lpp being 78 residues in most species of bacteria including E. coli. Lpp lengths 96 of 99 residues or more are at the upper end of the natural range for this protein and, in nature, these longer 97 Lpp proteins are found in the genus Geobacter. The previously described Lpp +21 isoform expressed in E. coli 98 therefore sits near the upper limit in the natural range. We introduced a gene encoding the Lpp +21 isoform into 99 an E. coli background suitable for genetic screens (Fig. S1) and confirmed the size of the protein by SDS-100 PAGE of bacterial cell extracts (Fig. 1B). Enteric bacteria like E. coli have evolved in environments that are 101 not nutrient-rich and are hyper-osmotic with respect to most laboratory growth medium 20, 21 . The Lpp +21 102 isoform had little impact on growth of E. coli. On minimal growth medium, growth rates of the Lpp +21 strain 103 of E. coli were equivalent to the isogenic wild-type E. coli (Fig. 1C). This was likewise true on growth media 104 osmotically balanced with concentrations of sorbitol up to 1.0M (Fig. 1D), and on rich (LB) medium with or 105 without sorbitol (Fig. 1D). 106 107 To establish the extent to which the periplasm had been remodeled in the Lpp +21 strain of E. coli, samples were 108 prepared for cryogenic transmission electron microscopy, a technique in which cells are preserved in a frozen-109 hydrated, near-native state. As expected, the average width of the periplasm was increased in both strains 110 under hyperosmotic conditions when compared to previously reported data where cells were grown in standard 111 laboratory media 18 . As shown in Fig. 1E, the averaged values for the periplasmic width were only slightly 112 greater in the Lpp +21 strain (32-36nm) compared to the isogenic wild-type strain (30-32nm). As evident in 113 individual micrographs, the distance to the center of the PG density from the OM was slightly increased in the 114 Lpp +21 strain (Fig. 1F). The PG morphology was also changed: in the wild-type strain a uniform dark PG layer 115 could be observed in the images, whereas in the Lpp +21 strain a thicker defuse PG layer is present in the images, 116 indicating heterogeneity in both the density and thickness of the PG.  Growth rates for the same strains were measured in rich (LB) growth media with and without sorbitol over 20 hours. (E) The periplasmic width distribution of the indicated strains in hyperosmotic conditions. While PG layer in the wild-type strain is a uniform thin electron dense layer, the PG layer in the Lpp +21 strain is more diffuse and thicker. (F) Cryo EM of cell envelopes in hyperosmotic conditions. Measurements from EM views evaluate the distance between OM and PG in the Lpp +21 strain micrographs. The histogram depicts the frequency with which a given distance is observed between the OM and PG.

Homeostasis in the periplasm 120
To determine the adaptive response to changing the distance constraint between OM and PG, quantitative 121 whole cell proteomics was applied to evaluate the Lpp +21 strain. Triplicate samples of the wild-type and Lpp +21 122 strains were processed for analysis by mass spectrometry and we sought to identify those proteins where the 123 steady-state level increases or decreases three-fold or more (Log2 fold change of ± 1.6) in the Lpp +21 strain 124 ( Fig. 2A; Table S1). The level of the oligopeptide transporter subunits (OppB, OppC, OppD and OppF) are 125 substantially increased in the Lpp +21 strain compared to the wild-type (Table S2). This suggests an increase in 126 PG turnover and an overall increased capacity to recycle PG components, and is consistent with the 127 concomitant increase in AmiC, one of the two major amidases involved in PG remodeling. In addition, 128 proteins implicated in diverse stress-responses (cold shock proteins CspG, CspA, CspI and YdfK, as well as 129 envelope stress protein ZraP and redox stress protein YfcG) were observed at increased steady-state levels in 130 Lpp +21 strain ( Fig. 2A, Table S2). The greatest decreases were seen in the steady state levels of the GatZABCD 131 proteins involved in galactitol phosphotransferase system and DHAP synthesis ( Fig. 2A). The gatABCD genes 132 have been shown to be responsive to factors that change E. coli cell surface tension 22 and Lpp +21 has been 133 reported to significantly decrease cell stiffness 23 . 134 135 A decrease was seen in the steady-state level of the Lpp +21 isoform in the mutant, to approximately one-eighth 136 the level of Lpp in the wild-type strain (Fig. S2). This is consistent with the relative abundance of Lpp and 137 Lpp +21 observed in SDS-PAGE analysis of cell extracts from the two strains (Fig. 1F). However, despite the 138 relative decrease, Lpp +21 remains as a highly abundant component of the OM-PG linkage factors given that 139 Lpp is present at up to 10 6 protein molecules per wild-type cell 1, 24 . 140 141 The mass spectrometry data was processed to allow for an analysis of sub-cellular proteomes 25 ( Fig S3). An 142 initially puzzling observation was that the Lpp +21 strain has a 12% overall reduction of total periplasmic 143 protein compared to wild type (Fig. 2B). This was calculated as the proportion of the summed intensity from 144 identified proteins predicted to reside in the periplasm in the STEPdb: G, E, F2, F3, I annotations 25 ( Fig S3). 145 Lpp +21 strains of E. coli are softer as previously judged by atomic force microscopy 23 , and factors that increase 146 the softness of E. coli also increase outer membrane vesicle (OMV) production 26 . To address whether the 147 measured depletion of periplasmic content reflects an increased production of OMVs, extracts measuring the 148 amount of total protein in the OMV fraction were normalized to OD 600 (Fig 2C, Fig. S3). This confirmed that 149 the presence of Lpp +21 promotes approximately 10-fold more total protein associated with the OMV fraction, 150 reflecting increased OMV production. The overall level of OM proteins associated with the cells was 151 maintained constant ( Fig. S3) but a small OM integrity defect was evident from an increased sensitivity to 152 SDS (Fig. 2D  Lpp +21 can be accommodated in the periplasm, but other factors become essential 156 To directly address the altered phenotype induced by Lpp +21 , we established a robotic synthetic genetic array 157 for factors in E. coli that become essential in order to maintain viability of the Lpp +21 strain ( Fig S4). The 158 screen demands synthetic growth phenotypes from an array of mutants each lacking a gene that, while non-159 essential in wild-type E. coli, become essential in the Lpp +21 strain of E. coli. The endogenous lpp gene was 160 replaced by a gene encoding Lpp +21 in a isogenic library of 3818 E. coli mutants, each of which lacks a non-161 essential gene. Growth on rich medium allowed the rescue of the new library for array into a format suitable 162 for high-throughput screening with a Singer RoToR robotics platform ( Fig S4). Phenotypic analysis was 163 thereafter scored for growth by comparing the growth of the isogenic mutants in the Lpp background ( Fig.  164 3A) with the equivalent mutants in the Lpp +21 background (Fig. 3B). Each of the genes that displayed a 165 noticeable phenotype in these analyses are presented in Table 1. 166 167 The only cytoplasmic factor identified in our screen, YraN is predicted to be a Holliday-junction resolvase 168 related protein, and we therefore speculate that this mutant failed to resolve the merodiploid condition transient 169 in the introduction of the lpp +21 condition to the background strain, making the yraN mutant a technique-170 relevant artefact of the screen. This being the case, only functions performed in the periplasm were recovered 171 as essential to viability for the Lpp +21 strain. 172 173 Most of the components of the LPS biosynthetic machinery are essential genes in E. coli and are thus not 174 represented in the library of non-essential genes. Those few, non-essential genes in the LPS biosynthetic 175 pathway that are in the library, become essential to the Lpp +21 strain (Table 1): the core LPS biosynthesis 176 factors GalU, GmhB and RfaD were shown to be essential in the Lpp +21 strain. The gene encoding the protease 177 YcaL was also detected as essential in the Lpp +21 strain, consistent with its proposed function in LPS 178 biogenesis through quality control of LptD function 27 . 179 180 An essential role for keeping the OM-PG distance

181
Several genes encoding proteins that could play roles in anchoring the PG within the cell envelope were 182 identified as essential in the Lpp +21 background. Independently, none of the major proteins bridging the OM 183 and PG are essential for growth in E. coli 19 and all are therefore represented in the library. In a Lpp +21 184 background the genes encoding the β-barrel protein OmpA and the lipoprotein Pal become essential (Table  185 1). PG-binding domain PF00691 is common to these proteins: appended to a beta-barrel in OmpA, but to a 186 lipoyl anchor in Pal, and is also conserved in other proteins across diverse Gram-negative bacteria ( Fig 3C). 187 In E. coli there are 4 additional proteins containing this PG-binding domain and these were mapped in a 188 sequence similarity network analysis ( Fig 3D). A protein of unknown function, YiaD, is present ( Fig 3D) and 189 it too is essential in a Lpp +21 background (Table 1). We suggest, therefore, that this protein plays a substantive 190 role in OM-PG linkage. The remaining three proteins: MotB, LafU and YfiB, are more divergent to the 191 OmpA/Pal/YiaD cluster. Neither motB, lafU nor yfiB displayed a synthetic phenotype with Lpp +21 , and it has 192 been suggested previously that motB, lafU and yfiB are not expressed at detectable levels under laboratory 193 conditions 28 . 194 195 Detecting genes encoding drug-efflux pumps as important for growth of the Lpp +21 strain was initially 196 surprising. Either the absence of the inner membrane proteins AcrB or the OM component TolC caused a 197 reduction of growth in the Lpp +21 genetic background (Table 1). When antibiotic selection was removed by 198 plating the mutants on medium without chloramphenicol, the synthetic growth defects were observed in the 199 absence of drug selection (Fig. 3D), indicating that this synthetic phenotype is not the result of a decreased 200 drug efflux activity. The trans-envelope AcrAB-TolC multidrug efflux pump has been shown to traverse 201 through the PG and interact directly with PG at several defined sites 29, 30, 31, 32 , and we suggest that this system 202 acts as an additional OM-PG linkage that becomes essential in a Lpp +21 background. Together with the 203 observation that OmpA, Pal, YiaD and TolC are also essential in the Lpp +21 genetic background, these data 204 suggest that functions that maintain local areas of closer contact between the OM and PG are essential for 205 viability. 206 207    OM and patch of PG (Fig. 4). For three of the systems: Lpp only, Lpp with OmpA, and Lpp +21 only, tilting 217 was marginal, with tilt angles of 83±6°, 82±5°, and 79±3°, respectively (all numbers from the last 100 ns of 218 the 200-ns trajectory). These angles are in agreement with previous simulations of Lpp alone (~80°) and 219 slightly larger than those of Lpp with an OmpA monomer (~75°) PG 34, 35 . In initial simulations of Lpp +21 with 220 OmpA, the non-covalent connection between OmpA and PG was quickly disrupted as Lpp +21 extended from 221 its kinked state. Therefore, the simulation was repeated with an enforced OmpA-PG connection. Lpp +21 was 222 observed to both straighten and tilt within the first 100 ns; the tilt angle measured for the last 100 ns was 223 70±5°. 224 225 The distance between the OM inner leaflet phosphorus atoms and the PG sugars was measured in each 226 scenario. In the presence of Lpp, the distances with and without OmpA were similar at 8.3±2.1 nm and 8.1±1.2 227 nm, respectively. This was not true for the other scenarios where the distance for Lpp +21 alone was 11.6±1.3 228 nm, but for Lpp +21 with OmpA, the distance was reduced significantly to 8.7±2.7 nm. Thus, we observe that 229 PG-binding proteins like OmpA can counteract the increased distance imposed by Lpp +21 , inducing it to tilt 230 significantly in accommodation. We also compared our simulations to the distances that were measured by 231 EM (centre of the OM to centre of the PG). In wild-type E. coli (i.e. Lpp+OmpA), the centre-centre distance 232 in the simulations is 10.7 +/-2.2 nm (Fig 4B), similar to the 9.7 -10.8 nm measured in intact cells (Fig. 1F). 233 The centre-centre distance measured in the simulation of Lpp +21 tilted by the presence of OmpA (11.0 +/-2.6 234 nm), fits the observed distances of 10.

239
We observed that over a range of osmotic conditions, and in nutrient-rich or nutrient-poor media, growth rates and mrcB (as well as their OM lipoprotein partners) are essential for viability, indicative of a compromised 298 capability to build the PG-layer. Mutations designed to impact on these interactions lead to transient deposition 299 of "high-density PG" and "multi-layered PG" through dysregulation of the synthetases 11 . The morphology of 300 the PG-layer observed by electron microscopy is suggestive of these high-density and multi-layered PG 301 consistent with transient or local impacts on the OM-PG distance 11 . An essential requirement was also placed 302 on PG-layer remodeling, whereby the PG-binding factor NlpD was found to be essential in Lpp +21 cells: its 303 function is in modulating the activity of the amidase AmiC 46, 47 to remodel PG strands, and AmiC was 304 observed at increased steady-state levels in the Lpp +21 strain. Taken together with the increase in oligopeptide 305 transporter subunits (OppB, OppC, OppD and OppF) in the Lpp +21 strain to recycle PG precursors across the 306 IM, our results suggest a clearance of the malformed PG caused by dysregulation of the PG synthases is a 307 crucial adaptation in the Lpp +21 strain. 308 309

Stiffness, load-bearing and connection of OM-PG 310
The concept of bacterial cell stiffness has emerged as a means to understand the physical parameters that 311 define how readily bacteria can respond to major environmental changes 26,48,49 . Measurements by AFM have 312 revealed a characteristic stiffness in Gram-negative bacterial cells that is contributed by load-bearing outer 313 membrane and its attachment to the underlying PG layer 50 . In E. coli, mutants lacking Lpp, Pal or OmpA are 314 "softer" than wild-type cells 50 , and cells expressing the Lpp +21 isoform are also "softer" than wild-type cells 315 23  done by diluting the saturated culture 1:100 using new media. The cells were then grown to mid-exponential 339 growth phase (OD600 = 0.5-0.6) at 37 °C under shaking. Culture media was supplemented with antibiotics for 340 plasmid selection and maintenance or selection of mutants at the following concentrations: 100 μg/ml 341 ampicillin, 30 μg/ml kanamycin, 34 μg/ml chloramphenicol. 15 g/l agar was added to media before 342 autoclaving when solid media was required (Fig. S4). 343 344

345
The endogenous lpp gene was replaced with the extended lpp +21 gene previously described 17, 18 with minor 346 modifications (Fig. S1). First the replacement was done in the donor E. coli Hfr Cavalli cells using the λ -red 347 recombination system 53 . A gene block was sourced (Integrated DNA Technologies) containing extra 21 amino 348 acid residues (three heptad repeats) inserted between codon 42 and 43 of E. coli Lpp. The gene block also 349 contained 50 bp DNA flanking 5' and 3' ends of the lpp +21 gene. On the 5' end, the extension was homologous 350 to DNA sequence upstream of lpp, while on the 3' end, the extension was homologous to the cat gene. The 351 gene block was combined with the cat gene by Gibson assembly, and the resulting PCR fragment was used to 352 replace lpp. The new Lpp +21 strain was selected by plating on medium containing chloramphenicol. 353 Chromosomal lpp +21 was moved into E. coli BW25113 background by mating with kanamycin-resistant Keio 354 collection strain JW5028, described above, to generate a double mutant 19, 54 (Fig. S4). The mutation was 355 verified by PCR described below (Fig. S1) and sequencing. 356 357

368
Overnight cultured cells, grown in LB without antibiotics, were washed twice in 1 x M9 salts then subcultured 369 in 500 ml M9 minimal media supplemented with 0.5 M sorbitol (1:1000 dilution). The strains were grown to 370 late logarithmic phase without antibiotics, OD600 ≈ 0.9 and spun down to collect culture supernatant. Collected 371 culture supernatant were then processed for OMVs isolation and purification using differential 372 ultracentrifugation technique as discussed previously 55 . OMVs were washed twice in PBS to remove sorbitol 373 then quantified using a bicinchoninic acid assay kit (Thermo Scientific CST#23225 to a 3 x 10 6 target value with maximum injection time of 54 ms. Dynamic exclusion was set to 30 seconds. 396 The 20 most intense multiply charged ions (z ≥ 2) were sequentially isolated and fragmented in the collision 397 cell by higher-energy collisional dissociation (HCD) with a fixed injection time of 54 ms, 15,000 resolution 398 and automatic gain control (AGC) target of 2 x 10 5 . 399 400 20 The raw data files were analyzed using MaxQuant software suite v1.6.5.0 56 against Andromeda search engine 401 57 for protein identification and to obtain their respective label-free quantification (LFQ) values using in-house 402 standard parameters. The proteomics data was analyzed using LFQ-Analyst 58 and the analysis of the data 403 quality analysis is presented in Fig S6. Due to the 21 amino acid insertion in the Lpp +21 isoform, the relative 404 levels of Lpp in the mutant had to be assessed manually. Only the unique peptide (IDQLSSDVQTLNAK)  405 shared between the two isoforms was used to quantify the levels of Lpp and Lpp +21 in the wild-type and mutant 406 strain, respectively (Fig S2). 407 408 To estimate the total relative amount of proteins from the various subcellular compartments, the raw intensities 409 from peptides identified from proteins from different subcellular locations were summed and divided by the 410 total summed intensity from all peptides. Proteome 35% co-membership rpg-35 group 59 and a sequence similarity network was generated with the EFI 418 Enzyme Similarity Tool 60 . This network was visualized with Cytoscape 61 with a similarity score cutoff of 419 30. Each protein is represented by a colored circle node and each similarity match above the similarity score 420 cutoff is represented by an edge between nodes with the length determined by the similarity score. 421 422 Lpp length distribution across bacterial species

423
To determine the amino acid length distribution of Lpp in Gammaproteobacteria (Table S4), amino acid 424 sequences were sourced from the InterPRO database (version 81.0) 62 using the Interpro Family tag -Murein-425 lipoprotein (IPR016367). Filtered Lpp sequences were then concatenated into representative nodes (at least 426 >90% sequence similarity) using the online available amino acid Initiative-Enzyme Similarity Tool (EFI-EST) 427 60 . 428 429 Synthetic genetic interaction array

430
The Lpp +21 isoform was transferred to each of the Keio collection clones by conjugation as described (Fig.  431  S4). First, the Hfr chloramphenicol resistant Lpp +21 strain was arrayed in 384-colony density on LB agar 432 containing chloramphenicol using the Singer rotor HAD (Singer Instruments, United Kingdom). Similarly, 433 the Keio collection arrayed in 384-colony density was pinned on LB agar plates containing kanamycin and 434 incubated overnight at 37 °C. Using the Singer rotor HDA, the Hfr Lpp +21 strain and the Keio collection clones 435 from the 384-colony density were then co-pinned onto LB agar plates and incubated at 37 °C for 16 hours. 436 21 Following conjugation, the colonies were transferred to LB agar with kanamycin (selection 1) at the same 437 colony density and incubated at 37 °C for 16 hours. To select for double mutants (selection 2), colonies from 438 the intermediate selection were pinned on LB agar with both kanamycin and chloramphenicol and incubated 439 at 37 °C for 14 hours. For assessment of synthetic genetic interaction in nutrient-limited media, the double 440 mutants generated were replica pinned in M9 minimal media at the same density and incubated at 37 °C for 441 25-30 hours. Images were acquired using Phenobooth (Singer Instruments, United Kingdom) for analysis. 442 Images were manually screened to cross-reference recipient plate images to the final double antibiotic 443 selection plates images. Candidate synthetic lethal or growth-compromised mutants were then subjected to 444 another round of screening in the same conditions as previously identified (mini-screen) for validation. Four 445 biological replicates were included that were further arrayed in four technical replicates. 446 447 Since the Keio collection yiaD mutant has been identified as containing a potential duplication event 63 , the 448 candidate yiaD synthetic lethal interaction was confirmed through independently constructing a yiaD mutant 449 in the BW25113 strain background (Fig. S5). The lpp +21 variant was subsequently generated in this mutant as 450 described above. As with the Keio yiaD mutant, this strain demonstrated synthetic lethality on M9 media. 451 452 Two colony PCR reactions 53, 63 (Fig. S4) confirmed the identity of all candidate double mutants, using a set 453 of primers flanking the lpp gene, and a set of primers flanking the kanamycin gene (Table S5). 454 455

456
Strains were grown aerobically in M9 minimal media (0.5 M sorbitol) until an OD600 of 0.6 was reached. Cells 457 were collected by spinning at 6000xg for 5 minutes and resuspended to an OD600 of ≈ 12. Cryo-EM, data 458 collection and analysis were performed as described previously 17,18 . 459 460 Generation of simulation systems 461 Initially, two systems were generated: the OM and PG, as previously detailed 49 , with two copies of wild-type 462 Lpp and with two copies of Lpp +21 . For wild-type Lpp, we used the homo trimer from PDB 1EQ7 33 . For 463 Lpp +21 , a monomer was first built using I-TASSER 64 . Next, the trimer of Lpp +21 was built using the wild-type 464 Lpp trimer as a template, further optimized using Targeted Molecular Dynamics (TMD) for 1 ns. For both 465 Lpp and Lpp +21 , the proteins were anchored in the OM via N-terminal acylation while the C-terminus of one 466 copy from each trimer was covalently linked to the PG. The systems generated were prepared for equilibration 467 using the following steps for 1 ns each: 1) minimization for 10,000 steps, 2) melting of lipid tails, 3) restraining 468 only the PG and the protein, and 4) restraining the PG and the protein backbone. Both systems were 469 equilibrated for 200 ns. 470 For each of the two systems (Lpp and Lpp +21 ), a new system was constructed with one Lpp trimer removed 471 and OmpA inserted into the OM. The full-length OmpA structure was taken from Ortiz-Suarez et al. 65 . The 472 PCR confirmation of the lpp +21 mutant strain (Methods). 554 555 Figure S2. Quantitation of Lpp and Lpp +21 isoforms. The sequence of the 21 residues inserted to create the 556 Lpp +21 isoform is also indicated. Mass spectrometry data for Lpp vs Lpp +21 was reanalyzed after extraction 557 from the whole cell proteomic data. Given the different tryptic peptides generated from the two isoforms of 558 Lpp, a shared peptide (red) was used to quantify the relative levels of each Lpp isoform in each of the strains. 559 The graphs document the relative levels of the peptide and show that the presence or absence of sorbitol in 560 the growth medium has no effect on the level of Lpp +21 relative to Lpp. 561 562 Figure S3. subjected to a second round of selection using both antibiotics; (A) depicts images of representative plates 574 generated in each step of the procedure with imaging and manual analysis step, cross-referencing of single 575 gene knock outs and double recombinants, included. (B) depiction of the strains as cartoons generated in each 576 step of the procedure. (C) A representative mini screen of manually selected genes from the main synthetic 577 lethal screen. Sterility controls were included on each mini screen. The mini screen was performed in 384-pin 578 density with each clone arrayed in four biological replicates, each having four technical replicates (blue 579 boxes). Synthetic lethal mutants identified from the mini screen were further verified by PCR to confirm the 580 presence of both gene modifications and rule out partial duplication events. 581 582 Figure S5. Construction and characterization of the validation yiaD mutant. A kanamycin resistance 583 cassette was amplified from pKD4 using primers with overhangs complementary to upstream and downstream 584 27 of yiaD. The PCR fragment was electroporated in BW25113 cells harbouring the λ -red recombineering 585 plasmid (pKD46). Transformants were selected on kanamycin-resistant plates and verified by PCR (methods). 586 Primers flanking the yiaD gene confirm replacement of yiaD with kanamycin cassette and primers amplifying 587 lpp confirm lpp +21 replacement of lpp. The sequence information for all primers used are included in Table  588 S5. 589 590 Figure S6. Proteomics quality control report.   The sequence of the 21 residues inserted to create the Lpp +21 isoform is also indicated. Mass spectrometry data for Lpp vs Lpp +21 was reanalyzed after extraction from the whole cell proteomic data. Given the different tryptic peptides generated from the two isoforms of Lpp, a shared peptide (red) was used to quantify the relative levels of each Lpp isoform in each of the strains. The graphs document the relative levels of the peptide and show that the presence or absence of sorbitol in the growth medium has no effect on the level of Lpp +21 relative to Lpp.  Figure S4. A synthetic lethal screen to determine genes essential to Lpp +21 E. coli. An Hfr donor strain carrying a selectable marker (cat) fused to lpp +21 , replacing the lpp ORF, is mated on agar plates with arrayed Frecipients (384) per plate carrying a selectable marker (kan) replacing other ORF. Upon mating, cells are subjected the first round of selection (intermediate selection) using antibiotic kanamycin and then further subjected to a second round of selection using both antibiotics; (A) depicts images of representative plates generated in each step of the procedure with imaging and manual analysis step, cross-referencing of single gene knock outs and double recombinants, included. (B) depiction of the strains as cartoons generated in each step of the procedure. (C) A representative mini screen of manually selected genes from the main synthetic lethal screen. Sterility controls were included on each mini screen. The mini screen was performed in 384-pin density with each clone arrayed in four biological replicates, each having four technical replicates (blue boxes). Synthetic lethal mutants identified from the mini screen were further verified by PCR to confirm the presence of both gene modifications and rule out partial duplication events. Figure S5. Construction and characterization of the validation yiaD mutant. A kanamycin resistance cassette was amplified from pKD4 using primers with overhangs complementary to upstream and downstream of yiaD. The PCR fragment was electroporated in BW25113 cells harbouring the λ -red recombineering plasmid (pKD46). Transformants were selected on kanamycin-resistant plates and verified by PCR (methods). Primers flanking the yiaD gene confirm replacement of yiaD with kanamycin cassette and primers amplifying lpp confirm lpp +21 replacement of lpp. The sequence information for all primers used are included in Table S5.