Candidate undecaprenyl phosphate translocases enable conditional microbial fitness and pathogenesis

The mechanisms that enable adaptation of peptidoglycan, the structural unit of the bacterial cell wall, to shifting extracellular conditions such as pH remain largely unknown. Here, we identify a DUF368-containing membrane protein in the cholera pathogen Vibrio cholerae that is critical for pathogenesis and alkaline fitness. V. cholerae and Staphylococcus aureus lacking their cognate DUF368-containing protein have pH-dependent cell wall defects consistent with surface accumulation of undecaprenyl phosphate (C55-P), an essential lipid carrier for the biogenesis of peptidoglycan and other key bacterial cell surface polymers. In both species, DUF368-containing proteins exhibit synthetic genetic interactions with putative transporters from the DedA family, suggesting these proteins represent complementary long-sought C55-P translocases that enable envelope maintenance functions critical for microbial fitness within and outside the host. One-Sentence Summary DUF368-containing and DedA-family proteins are undecaprenyl phosphate transporter candidates and are required for bacterial alkaline fitness and pathogenesis.

The phenotypes of DUF368-deficient S. aureus and V. cholerae strongly suggest DUF368 133 domains are involved in C55-P recycling, likely as C55-P transporters. 134 Analyses of spontaneous suppressors of Δvca0040 V. cholerae, which have distinctive 135 colony morphology ( fig. S6A) revealed that VCA0040's requirement for V. cholerae fitness 136 is controlled by [Na + ] in addition to pH. Suppressor colonies lacked stationary phase cell 137 shape defects and whole-genome sequencing revealed four suppressor genes, three of 138 which (secD1, secF1, and ppiD) function in Sec-mediated protein secretion ( fig. S6B). 139 The fourth, yfgO, is a protein of unknown function from the AI-2E transporter family, 140 members of which have recently been implicated in PG biosynthesis and Na + /H + antiport 141 (19,20). Deletions of each gene rescued Δvca0040's shape defects, even though alkaline pH and Na + both contribute to the environmental control of the defect of DUF368-154 deficient V. cholerae. The Na + requirement appears to be species-dependent, as ΔSAOUHSC_00846 S. aureus, although alkaline-sensitive, were not similarly rescued by 156 Na + depletion (Fig. 2C). 157 We next carried out a synthetic transposon screen to define vca0040's genetic network. 158 Many of the identified synthetic sick/lethal interactions were related to cell envelope 159 homeostasis (Fig. 3B,table S7). vca0040's most striking interaction was with ΔSAOUHSC_00846, we found frequent promoter region (and no coding region) 171 mutations in two DedA proteins (SAOUHSC_00901 and SAOUHSC_02816) (Fig. 3E). 172 Incorporating these mutations into an IPTG-inducible system rescued SAOUHSC_00846 173 even without induction, suggesting the isolated mutations increase expression of DedA-174 family proteins and compensate for the loss of SAOUHSC_00846, as in V. cholerae (Fig. 175 3F). The conserved synthetic interaction between DUF368 and DedA proteins suggests 176 that DedA family members are also required for C55-P transport, consistent with recent 177 reports that eukaryotic DedA proteins may bind lipids, and bacterial DedA mutants show 178 decreases in LPS modifications that require C55 carriers (23)(24)(25). V. cholerae lacking both yghB and vca0040 were viable at acidic pH, suggesting at least one other V. 180 cholerae protein can carry out C55-P translocation ( fig. S7C). DedA proteins may be more 181 active in different conditions than DUF368 proteins, since opposite from the Δvca0040 182 mutant, a ΔyghB mutant was not able to grow without Na + , suggesting an SMF-183 independent (i.e., PMF-dependent) function ( fig. S7D). Consistent with the idea of 184 conditional C55-P translocase activity, the outer membrane-permeable lptD4213 E. coli 185 strain (26), which has only DedA paralogs and not DUF368, had heightened sensitivity to 186 amphomycin and tunicamycin at alkaline pH (table S4). 187 We used an infant rabbit model, which mimics severe human cholera (27) idea, freshly grown rod-shaped WT or Δvca0040 V. cholerae were incubated in cell-free 205 CF samples. Δvca0040, but not WT, cells became spherical when exposed to CF,206 suggesting that VCA0040 function is critical for maintenance of V. cholerae shape in the 207 in vivo milieu (Fig. 4G). An infection defect was not detected for ΔSAOUHSC_00846 S. 208 aureus in a murine IV infection/organ abscess model, where the pathogen is unlikely to 209 encounter alkaline environments ( fig. S9). 210 We propose that DUF368-containing and DedA family proteins are C55-P translocases 211 that provide functional redundancy in this crucial step of envelope maintenance. The 212 apparent environmental specificity of DUF368-containing and DedA proteins (and 213 potentially other proteins such as BacA/UppP) could support C55-P flux in diverse 214 microbial niches (Fig. 4H  Biol. 6, 106-16 (2011  Acknowledgements: 376 We thank members of the Waldor lab for helpful comments and discussions during the 377 study. We also thank the Thermo Fisher Scientific Center for Multiplexed Proteomics at

Materials and Methods
Note: Parts of this section are adapted from the thesis of B.S (29).

Bacterial strains, media and growth conditions
All V. cholerae strains used in this study are derivatives of HaitiWT, a spontaneous streptomycin-resistant variant of a clinical isolate from the Haiti cholera epidemic (30). All S. aureus strains used in this study are derivatives of HG003 (itself a derivative of NCTC8325). Strain and plasmid information is listed in Table S10. Genome annotation of HaitiWT and a batch BLAST dictionary assigning putative VC locus tags (the conventional V. cholerae gene naming system) to HaitiWT loci is given in Table S11. All constructed strains were verified by Sanger sequencing of the targeted locus (Genewiz, USA).
For routine culture, V. cholerae were grown in non-buffered LB at 37°C and S. aureus were grown in non-buffered TSB at 37°C. Cultures for spent supernatant analysis in V. cholerae were obtained by subculturing 37°C-grown overnight cultures 1:1000 in fresh media and growing for 24 hours at 30°C. To collect spent supernatants, cultures were centrifuged for 10 minutes at 5000 rcf at 4°C. Supernatants were transferred to a new tube, re-centrifuged, and sterile filtered with 0.22 µm syringe filters into new tubes. Supernatants were stored at 4°C for future use. Cultures used for pH quantification were centrifuged for 10 minutes at 5000 rcf before measurement. All pH measurements, including for media and buffer formulation, were performed with a pH meter (Thermo ORION, USA) freshly calibrated to two appropriate pH standards from 4, 7, and 10.

Bioinformatic methods
Information on the loci, genomes, and accession numbers for genes and proteins from this study is available in Table S10.

Phylogenetic analysis
To identify sequences with annotated DUF368 domains, Annotree (31) was used with the Pfam identifier PF04018 at a cutoff e-value of 1x10 -30 . The results from this search are included in Table S1. Phyla with "_X" names were manually collated into a single phylum for ease of analysis. In a related effort, to identify precise homologues of vca0040, we used a HMMER search with the VCA0040 sequence from WT V. cholerae (Table S2). To analyze the relative distributions of PF04018, PF09335 (DedA), and PF02673 (BacA), we used Annotree at a cutoff e-value of 1x10 -15 . Differential species lists with all possible combinations of conservation were generated with a custom Venn diagram generator (https://bioinformatics.psb.ugent.be/webtools/Venn/) and are provided in Table S9.
Structural modeling Structural prediction of DUF368 and DedA-family proteins in this study was performed with AlphaFold2 on the CoLabFold publicly accessible interface (32). Sequences were modeled as monomers using mmseqs2 for multiple sequence alignment. Structures were ranked by pLDDT and the top-ranked structures were visualized with ChimeraX (UCSF, USA).

Cloning, vectors, and strain construction V. cholerae
Cloning of expression vectors and deletion plasmids was performed by standard isothermal assembly techniques with 25 bp overhangs on each fragment (HiFi DNA Assembly Kit, NEB, USA). V. cholerae mutants were generated by allelic exchange as previously described with the suicide vector pCVD442 bearing 500-700 bp upstream and downstream homology arms of the targeted locus (33). Either SM10λpir or MFDλpir E. coli (a DAP auxotroph) were used as the donor strain with identical conjugation conditions apart from DAP supplementation. Single crossover transconjugants were isolated by selective plating on LB + Sm/Cb and passaged in 10% sucrose overnight at RT to select for double crossover events. Cells were plated on LB + Sm, re-patched onto LB + Sm and LB + Sm/Cb, and Sm R Cb S colonies screened by colony PCR for the successful deletion. For deletions involving vca0040, vca0040 was always deleted last and at least 2 clones per strain were stocked and verified for the correct phenotype to guard against spontaneous suppressor formation. For native vca0040 expression from the ectopic chromosomal site, the full coding sequence along with 350 bp upstream was cloned to include the native promoter and inserted at a known neutral genomic location in V. cholerae (34). For expression of SAOUHSC_00846 from the vca0040 locus in Δvca0040 V. cholerae, we used allelic exchange to replace the vca0040 coding sequence with a SAOUHSC_00846 sequence codon-optimized for V. cholerae. For depletion of yghB from Δvca0040 V. cholerae, we used pAM299, a suicide vector which replaces the native allele of targeted gene with an arabinose-inducible copy (35). For overexpression studies, we used pBAD18 for plasmid-based arabinose induction (36).
S. aureus S. aureus SAOUHSC_00846 knockout was generated with a one-step allelic exchange using pTarKO plasmid bearing 1000 bp upstream and downstream homology arms of the targeted locus flanking a kanamycin cassette as previously described (37). Briefly, assembled pTarKO plasmid was electroporated into electrocompetent S. aureus RN4220 tarOoff and selected for double crossovers on TSB with kanamycin, neomycin and targocil. The kanamycin insertion in SAOUHSC_00846 was then transduced to S. aureus HG003 with phage phi85. For overexpression studies, we used pLOW for plasmid-based IPTG induction (38). Assembled plasmids were electroporated into RN4220 WT and transduced to S. aureus HG003 ∆SAOUHSC_00846 with phage phi85.
V. parahaemolyticus V. parahaemolyticus RIMD2210633 mutants were constructed with a similar allelic exchange system to V. cholerae as previously described, with the suicide vector pDM4 (28).

Growth assays
Growth curves in liquid medium For automated OD600 measurements, 1 mL of a saturated 37°C LB overnight V. cholerae culture was washed once with 1 mL fresh media (specific to the experiment) and resuspended in 1mL fresh media. Resuspended bacteria were diluted to a starting dilution of 1:4000 by performing a 1:100 dilution into 1 mL fresh media and a subsequent 1:40 dilution into 195 µL of specific media aliquoted into sterile 96 well plates (Corning, USA). At least 3 technical replicates per strain and media condition were run per plate along with at least 3 blank media wells. Growth curves were performed in a BioTek Epoch2 spectrophotometer with shaking and OD600 readings were taken every 10 minutes for 20-24 hours. Data for each condition were averaged across technical and biological replicates corrected against the baseline blank OD600 values.
Overexpression vector plate dilution assays on solid medium V. cholerae strains were grown overnight at 37°C in LB with addition of 50 µg/mL carbenicillin for strains with pBAD18. The cultures were diluted 1:100 into 5 mL of LB (supplemented with 50 µg/mL carbenicillin and 0.2% arabinose for strains with pBAD18) and grown at 37 °C to an OD600 of ~0.7. Cells were normalized to an OD600 of 0.1 and then serially 10-fold diluted six times. 5 µL of the dilution series were plated on LB agar 100 mM Tris pH 9 (with 0.2% arabinose, 0.2% glucose, or no sugar added) and incubated at 30°C for 18-24 hours. S. aureus strains were grown overnight at 37 °C in TSB with addition of 10 µg/mL erythromycin for strains with pLOW. The cultures were diluted 1:100 into 5 mL of TSB (supplemented with 10 µg/mL erythromycin and 1 mM IPTG for strains with pLOW) and grown at 37 °C to an OD600 of ~0.7. Cells were normalized to an OD600 of 0.1 and then serially 10-fold diluted six times. 5 µL of the dilution series were plated on TSB agar 100 mM Tris pH 9 (with or without 1 mM IPTG) and incubated at 37°C for 18-24 hours.

Live and single timepoint phase-contrast microscopy
For single timepoint imaging of live cells, samples from the indicated cultures were concentrated as necessary and immobilized on 0.8% agarose pads in sterile PBS on glass slides (Gene Frames, Thermo, USA) and dried before coverslip placement. For time-lapse imaging of live cells, samples were spotted on 0.8% agarose pads in sterile LB before imaging in a temperature-controlled chamber. Cells were imaged with a Nikon Eclipse Ti microscope equipped with an Andor NeoZyla camera and a 100× oil phase 3 1.4-numerical-aperture (NA) objective. Image analysis was performed with ImageJ. Images in manuscript figures are representative of at least 10 fields from the same sample (>200 cells) and multiple independent replicate cultures.

Sphere formation and incubation assays
To induce sphere formation, V. cholerae 37°C overnight cultures (where Δvca0040 cells are still largely rod shaped) were back-diluted 1:100 into the indicated supernatant or fresh media and grown for 4 hours shaking at 200 rpm at 30°C. Then, cells were imaged as described above. For the D/L-Ala treatment assay, overnight cultures were expanded in fresh LB for 90 minutes prior to spike-in of the amino acid for 1 hour.

Minimal inhibitory concentration assays V. cholerae
To quantify MICs for various antimicrobial agents, 37°C overnight cultures of V. cholerae were diluted 1:100,000 in fresh media. Diluted cultures were used to inoculate 96-well plates containing twelve 2-fold dilutions of the indicated agent in LB medium at a ratio of 50 µL culture: 50 µL medium. Four technical replicates were performed per strain per dilution. Plates were incubated for 24 hours at 37°C and MIC values were read as the first dilution where no turbidity was observed. For repeat assays, MICs were performed with independent overnight cultures.

S. aureus and E. coli
37˚C overnight cultures of S. aureus or E. coli were diluted to OD600 = 0.01 and then further diluted 1:100 in fresh TSB media. Diluted cultures were added to 96-well plates containing 2-fold dilutions of the indicated agent in TSB medium or TSB medium 5 mM Tris pH 8.5 at a ratio of 75 µL culture: 75 µL media. Plates were incubated for 24 hours at 30°C (37°C for E. coli) with shaking. MIC values were read off as the first dilution where no turbidity was observed.

Peptidoglycan characterization
Preparation of V. cholerae and S. aureus sacculi For V. cholerae, strains were grown overnight at 37°C in LB + 200 µg/mL streptomycin. For each sample, 500 µL of overnight culture were collected and centrifuged at 5000 rcf for 5 min, washed once with 500 µL of the corresponding media, and resuspended in 500 µL of the corresponding media. This culture was then added to 50 mL of M63 media (pH 7 with 100 mM NaCl, pH 8 with 100 mM NaCl or pH 8 with 0 mM NaCl) supplemented with 2% glucose and 200 µg/mL streptomycin and incubated at 30°C for approximately 6 hours until the OD600 reached ~0.5. Bacteria cells were collected by centrifugation at 6000 rcf for 10 minutes at 4°C and resuspended in 1.5 mL of 1 x PBS. The resuspension was added dropwise into 1.5 mL of boiling 5% SDS solution. The mixture was boiled for 1 hour and stirred for 2 more hours after the heat was turned off. For S. aureus, strains were grown overnight at 37°C in 3 mL Tryptic Soy Broth (TSB). 500 µL of the overnight culture was added to 100 mL of TSB without pH adjustment or TSB + 100 mM bicine pH 8.5 and incubated at 37°C for ~2 hours until the OD600 reached 0.5. The bacterial cells were collected by centrifugation at 5000 rcf for 10 min at 4°C and resuspended in 1.5 mL of 1 x PBS. Resuspended samples were boiled as for V. cholerae.
Total PG and crosslinking quantification from sacculi samples Peptidoglycan was extracted from boiled samples as described previously for Gramnegative and Gram-positive organisms (39,40). Once boiled, cell wall material was pelleted by ultracentrifugation and washed with water. Clean sacculi were digested with muramidase (100 μg/ml) and soluble muropeptides reduced using 0.5 M sodium borate pH 9.5 and 10 mg/mL sodium borohydride. The pH of the samples was then adjusted to 3.5 with phosphoric acid. UPLC analyses were performed on a Waters-UPLC system equipped with an ACQUITY UPLC BEH C18 Column, 130Å, 1.7 μm, 2.1 mm × 150 mm (Waters Corporation, USA) and identified at Abs. 204 nm. Muropeptides were separated using a linear gradient from buffer A (phosphate buffer 50 mM pH 4.35) to buffer B (phosphate buffer 50 mM pH 4.95 methanol 15% (v/v)). Identification of individual peaks was assigned by comparison of the retention times and profiles to validated chromatograms. The relative amount of each muropeptide was calculated by dividing the peak area of a muropeptide by the total area of the chromatogram. The abundance of PG (total PG) was assessed by normalizing the total area of the chromatogram to the OD600. The degree of cross-linking was calculated as described previously (41).
Intracellular UDP-M5 quantification For V. cholerae, strains were grown overnight at 37°C in LB + 200 µg/mL streptomycin. For each sample, 500 µL of overnight culture were collected and centrifuged at 5000 rcf for 5 min, washed once with 500 µL of the corresponding media, and resuspended in 500 µL of the corresponding media. 80 µL of this resuspension was then added to 8 mL of M63 media (pH 7 with 100 mM NaCl, pH 8 with 100 mM NaCl or pH 8 with 0 mM NaCl) supplemented with 2% glucose and 200 µg/mL streptomycin and incubated at 30°C for approximately 6 hours until the OD600 reached ~0.5. Bacteria cells were collected by centrifugation at 5000 rcf for 10 minutes at 4°C, washed twice with 1 mL of ice cold 0.9% NaCl, and resuspended in 200 µL of milliQ water. The resuspension was boiled for 30 min, centrifuged at 20,000 rcf for 15 min, and the supernatant was filtered and analyzed. For S. aureus, strains were grown overnight at 37°C in 3 mL Tryptic Soy Broth (TSB). 80 µL of the overnight culture was added to 8 mL of TSB without pH adjustment or TSB + 100 mM bicine pH 8.5 and incubated at 37°C for ~2 hours until the OD600 reached ~0.5. Cultures were then processed as for V. cholerae. Quantification of soluble UDP-M5 muropeptide levels by LC-MS of filtered supernatants was performed as previously described (42). Detection and characterization of soluble muropeptides by LC-MS was performed on an UPLC system interfaced with a Xevo G2/XS Q-TOF mass spectrometer (Waters Corporation) using previously reported conditions (42). UDP-M5 levels were quantified by integrating peak areas from extracted ion chromatograms (EICs) of the corresponding m/z value.

Amphomycin-FL (ampho-FL) synthesis and staining
To synthesize ampho-FL, 5 mg of amphomycin (Cayman Chemical, USA) was dissolved in 200 µL of dimethyl formamide (DMF) and combined with 7 µL of triethylamine. Separately, 3 mg of fluorescein-C5,6-NHS (ThermoFisher, USA) was dissolved in 200 µL of DMF and then added to the amphomycin solution. The reaction mixture was stirred at room temperature in the dark for 24 hours. The solution was diluted in one equal volume of DMSO and purified by reverse-phase HPLC (Agilent 1260 Infinity) using a C18 stationary phase column (Luna 5 µM C18(2) 100 Å, 250 x 10 mm). HPLC conditions were as follows: Phase A: water (0.1% formic acid); Phase B: acetonitrile (0.1% formic acid). Phase B: 0-2 min, 50%; 2-15 min, linear gradient 50%-100%, 15-17 min, 100%. The wavelength of the detector was set at 254 nm. The flow rate was 4.7 mL/min. The mixture of conjugated fluorescein-C5,6 products eluted at 9 min. HPLC eluates were collected in a 50 mL round-bottom flask, concentrated by rotatory evaporation, transferred with DMSO to a tared microcentrifuge tube and lyophilized, resulting in 1.7 mg of ampho-FL (M+1 = 1649.23). For ampho-FL labeling, overnight cultures of S. aureus HG003 WT or ∆SAOUHSC_00846 were diluted 1:100 in fresh TSB media + 100 mM bicine pH 8.5. The new cultures were grown to OD600 = 0.5. 1 mL of the culture was centrifuged at 1000 rcf for 5 min. The pellet was resuspended in 500 µL of 1 x Tris-buffered saline (TBS) pH 9. 98 µL of the mixture was transferred to a new tube and added with 2 µL of 10 mg/mL ampho-FL conjugate in DMSO. The mixture was incubated in the dark at room temperature for 10 minutes, washed three times with 500 µL of 1 x TBS pH 9, and resuspended in 100 µL of 1 x TBS pH 9. 10 µL bacteria was then added to a 0.8% agarose pad in TBS pH 9 and imaged as described above. Bacterial cells were imaged using DIC at a 30 ms exposure and ampho-FL signal was captured with the FITC filter at a 1 second exposure. Fluorescence intensity of ampho-FL was quantified using ImageJ. FITC signal profiles were generated by drawing bisecting lines through dividing cells. The FITC signal was recorded at the background to the left of the cells (A), at the left wall peak (B), at the middle walls (C), at the right wall peak (D), and at the background to the right of the cells (E). The average signal was calculated by (B+C+D)/4-(A+E)/2.

Transposon-insertion sequencing (TIS)
Generation of transposon libraries in the Δvca0040 and ΔyghB backgrounds was performed as previously described for HaitiWT V. cholerae (10). Strains were conjugated to SM10λpir E. coli bearing the donor transposon vector pSC189. Due to the moderate conjugation defect of Δvca0040, we used a 5x concentration of the conjugation reactions compared to the other libraries. Reactions were plated on 245 mm 2 LB+Sm/Kn agar plates to isolate V. cholerae transconjugants and incubated overnight at 30°C. Two independent Δvca0040 and ΔyghB libraries were generated, consisting of approximately 200,000 colonies each and stocked at an OD600 ~10 in LB + 25% glycerol. For synthetic TIS analyses, a frozen aliquot of each library was thawed and used for genomic DNA extraction with the Wizard kit (Promega, USA). DNA libraries were prepared in an identical manner to previous TIS experiments from our group (ref) and sequenced on an in-house MiSeq platform (lllumina, USA). Reads were trimmed, mapped, and processed as previously described with the Con-ARTIST TIS analysis pipeline (13). To identify synthetic interaction loci, we used WT as the "input" library and Δvca0040 as the "output" library during analysis.

RNAseq
For transcriptomic analyses, triplicate WT and Δvca0040 V. cholerae 37°C overnight cultures were back-diluted 1:100 into fresh LB medium and grown at 30°C for 8 hours with shaking. At 8 hours, samples were checked by microscopy to ensure onset of sphere formation in the mutant samples, at which point 2 mL of each culture was spun down (5 minutes at 5000 rcf at RT). RNA was extracted with Trizol reagent (Sigma-Aldrich, USA) per the manufacturer's instructions. Isolated RNA was then DNase-treated and reisolated with ethanol precipitation according to a standard protocol. RNA samples were quality checked with a Bioanalyzer to confirm RIN values > 6. Library preparation and sequencing was performed by the Microbial Genome Sequencing Center (Pittsburgh, USA). RNAseq analysis was performed largely as described (43). Reads were mapped to the V. cholerae KW3 genome (NCBI assembly GCA_001318185.1) with Bowtie2 (Galaxy) and a read matrix was generated with featureCounts (44). Read matrices were then analyzed with the default DESeq2 pipeline in RStudio to identify differentially expressed genes. Data shrinkage was performed with ashr (45).

Multiplexed tandem mass tag (TMT) proteomics
For proteomic analyses, triplicate WT and ΔsecDF1 V. cholerae 37°C overnight cultures were grown as described for RNAseq. At 8 hours of growth, whole cell pellet (WCP) samples were prepared by centrifuging 1 mL of cells for 5 minutes at 5000 rcf at RT, washing once in fresh LB, and flash frozen and kept at -80°C. MS analysis was performed by the Thermo-Fisher Center for Multiplexed Proteomics (TCMP) at Harvard Medical School according to standard protocols. WCP samples were subjected to a total proteomics workflow with fractionation. Pelleted cells were first lysed in 8 M urea, 200 mM EPPS and 1% SDS with phosphatase and protease inhibitors. Then, samples were reduced with DTT and alkylated with iodoacetamide. Alkylated proteins were precipitated with methanol/chloroform and resuspended in 200 mM EPPS pH 8 and digested sequentially with 1:50 LysC and 1:100 trypsin. Peptides were labeled with TMT16 reagents, pooled, and fractionated by a basic reverse phase (bRP) protocol into 12 fractions. Fractions were then dried, cleaned on a C18-packed stage tip, and eluted into an MS sample vial for analysis. Samples were resuspended in 5% ACN/5% formic acid and analyzed by LC-MS3 on an Orbitrap Lumos mass spectrometer. Peptides were detected (MS1) and quantified (MS2) in the Orbitrap, and sequenced (MS2) in the ion trap. MS2 spectra were searched with the COMET algorithm against the V. cholerae KW3 proteome, its reversed complement, and known contaminants. Spectral matches were filtered to a 1% false discovery rate using the target-decoy strategy combined with linear discriminant analysis. Proteins were quantified from peptides with a summed signal to noise threshold of >150 and isolation specificity of >0.5.

Spontaneous suppressor isolation and sequencing V. cholerae
To ensure independent isolation of spontaneous suppressors, the Δvca0040 strain was re-derived 11 times from different conjugation reactions. From each colony PCR-and cell shape defect-verified Δvca0040 clone, colonies with mutant morphology (small and completely blue) on LB+Sm/X-gal plates were re-streaked onto new LB+Sm/X-gal plates and grown for 36-48 hours at 37°C. This process was repeated once. From the tertiary plates, a single colony with WT morphology (large with a white halo) was re-streaked to confirm a stable suppressor phenotype. Suppressors were checked by microscopy of 30°C overnight cultures to confirm cell shape defect reversion and verified by colony PCR to confirm the absence of vca0040 from their genome. Validated suppressors were grown overnight, and genomic DNA was extracted as described above. Library preparation and sequencing were either outsourced to the Microbial Genome Sequencing Center (Pittsburgh, USA) or performed in-house. For in-house whole genome sequencing, gDNA was tagmented and barcoded with the Nextera XT library preparation kit (Illumina, USA), quality checked by BioAnalyzer and sequenced on a MiSeq platform to at least 20-50x depth of the V. cholerae genome (~4 Mbp). Genome assembly and variant identification was performed with CLC Genomics Workbench 12 (Qiagen, Germany). Variants were filtered against an assembled HaitiWT isolate re-sequenced with the same workflow. Mutations were assigned as suppressors if they were present in >90% of reads and did not occur in a known poorly mapping or highly varying region such as a tRNA locus. For the secondary screen in secDF2-Δvca0040 bacteria, the Δvca0040 deletion was rederived three independent times in the ΔsecD2 or ΔsecF2 background. Suppressors were isolated identically to those in the parental Δvca0040 background.

S. aureus
Multiple 2 mL cultures of S. aureus HG003 ∆SAOUHSC_00846 were grown in TSB overnight at 37°C. 190 µL from each independent overnight culture was then separately plated on TSA 100 mM Tris pH 9 plates (where the mutant is entirely inhibited for growth) and grown at 37°C for 24 hours. Suppressors were confirmed by re-streaking on TSA 100 mM Tris pH 9 plates. Validated suppressors were grown overnight, and genomic DNA was extracted as described above. Library preparation, sequencing, and variant identification was performed as described above for V. cholerae.

Infant rabbit V. cholerae infections and cecal fluid imaging
Infant rabbit oral infections with V. cholerae were performed as described previously (10). Briefly, 2-3-day-old New Zealand White rabbits (Charles River Laboratories) co-housed with their dams were orally gavaged with 10 9 CFU V. cholerae in a 500 μL gavage volume. Inocula were prepared by centrifuging a late exponential phase culture (OD600 0.6-0.8) V. cholerae and resuspending in 2.5% sodium bicarbonate. For varying the pH of the inocula, sodium bicarbonate was pH-adjusted to pH 9 or 7 with NaOH or HCl immediately before resuspension. Infected kits were returned to their dam and monitored for 16-20 hours post-infection, when they were sacrificed by isoflurane inhalation and intracardiac injection of 20 mEq potassium chloride. Small intestinal segments and the cecum were isolated by dissection, homogenized by bead beading in PBS, and dilutions were plated on appropriate agar plates for colony enumeration. Plates were counted after an overnight incubation at 30°C. During dissection of the cecum, a 28 G needle was used to extract crude cecal fluid (CF). A sample of crude CF was taken for CFU plating and imaging, and the remainder was centrifuged at 21000 rcf for 2 minutes. The pellet was discarded and supernatants were frozen at -20°C for downstream analyses. For CF incubation assays, overnight cultures of V. cholerae were back-diluted 1:100 in CF and grown for 4 hours at 30°C shaking before imaging.

Mouse S. aureus intravenous infection
S. aureus HG003 (WT) and KanR-marked ΔSAOUHSC_00846 were grown overnight at 37°C. Cultures were mixed at a 1:1 ratio, combined 1:1 with 50% glycerol, and stored at frozen at -80°C in several aliquots. For infections, an aliquot was thawed and diluted in PBS to a density of ~3x10 7 CFU/ml. 100 μl was injected into the tail vein of 8-9-week-old female Swiss Webster mice. Mice were monitored daily and at days 2 and 5 postinfection, the heart, lungs, right kidney, liver, and spleen were collected. Organs were homogenized with stainless steel beads in PBS and plated on TSA and TSA + kanamycin (TSAK) and incubated overnight to enumerate total ΔSAOUHSC_00846 CFU, respectively.

Animal use statement
All animal work in this study was performed in accordance with the NIH Guide on Use of and Care for Laboratory Animals and with the approval of the Brigham and Women's Hospital IACUC (Protocol #2016N000334 for infant rabbits and #2016N000416 for mice).

Statistics
Statistical tests used and replicate information are indicated in the figure legends and relevant methods sections. Statistical analyses were performed in Prism (Graphpad, USA).

Supplementary Text
Variable annotation of coding sequences in the reference V. cholerae genome During our study, which mainly used a contemporary pandemic strain of V. cholerae (HaitiWT or KW3, GCA_001318185.1), we noted several annotation inconsistencies with genes ostensibly conserved in the reference shotgun assembly (GCA_000006745.1) of N16961 V. cholerae. We found that compared to newer assemblies of N16961 (GCA_003063785.1 and GCA_900205735.1), the original assembly has ~50 more ORFs annotated as pseudogenes due to frameshifts. Although these ORFs are still annotated, these loci lack protein accessions and are not included in coding sequence tables. Three of these were directly related to our study: 1) lacZ, a widely-used reporter gene (VC2338), 2) secF2 (VCA0692), and 3) yghB (VCA0534). lacZ is known to be functional in N16961 and has been used repeatedly as a reporter locus. Re-sequencing of our laboratory N16961 stock and examination of newer N16961 assemblies confirmed that secF2 and yghB are in fact intact in this strain and lack the predicted frameshift. Notably, the "pseudogene" annotation of secF2 led to its usage as a safe-harbor locus for genome editing in V. cholerae in a recent report (46), but caution should be used for this method. The newer, long-read hybrid assemblies of N16961 V. cholerae may circumvent these issues and improved sequence and annotation curation will benefit future studies (47).
Phylogenetic conservation of DUF368 and DedA proteins       In the absence of a competitive defect, the TSAK counts should be 50% of those on the TSA plates (and thus are very close together on the log10plotted graph).

Fig. S11. Essentiality and genetic suppression of a DUF368 protein in V. parahaemolyticus.
The V. parahaemolyticus DUF368 protein, VPA1624, was depleted with the pAM299 system and is clearly required for growth even on neutral LB plates. VPA1624 apparently genetically interacts with the V. parahaemolyticus secDF1 and yfgO loci, as overexpression of these suppressors originally identified in V. cholerae rescues the VPA1624-depleted strain.

Supplementary Tables
All supplementary tables are included as additional spreadsheets with the submission. Table S1. Bacterial and archaeal clades with PF04018-containing proteins. Annotree was used to plot and identify species with an annotated DUF368 domain. Each tab of the spreadsheet is a different level of bacterial classification (Phylum>Order>Family>Genus>Species). Archaeal clades are combined on the tab "Archaea". Table S2. Homologues of VCA0040 in bacteria. HMMER was used to identify homologous sequences to VCA0040 in the Uniprot database. Each species is listed once.   For the top 40 up-regulated genes in Δvca0040, manual curation of subcellular localization was performed with SignalP and pSortb. Table S6. Multiplexed comparative proteomics of whole-cell WT and ΔsecDF1 V. cholerae. Fold change was calculated by dividing the average normalized relative abundance of proteins in ΔsecDF1 by that of WT. Table S7. Synthetic transposon-insertion screening in Δvca0040 V. cholerae. Note that intergenic regions (IG_) and regions with <5 informative sites (possible transposon insertion sites) were excluded from analysis. Hits on the second tab were thresholded at an inverse p-value >100 before sorting by mean log2 fold change. Table S8. Synthetic transposon-insertion screening in ΔyghB V. cholerae. Note that intergenic regions (IG_) and regions with <5 informative sites (possible transposon insertion sites) were excluded from analysis. Hits were thresholded at an inverse p-value >100 and log2FC less or greater than 1 (2-fold change). Table S9. Conservation of PF04018, PF09335, and PF02763 in bacterial species. Spreadsheet was generated by Annotree queries for all possible combinations of the three protein families. The "Master" sheet lists all species-level organisms with the indicated combination of domains. The "Model Species" sheet lists selected microbes of particular interest and can be used to look up any given species in the reference table.
Note that species of interest should be manually confirmed for presence or absence by BLAST to guard against misannotation or non-annotation issues.

Table S10. Strains and vectors and genomic information used in this study.
Table S11. BLAST dictionary for assignment of putative VC names to HaitiWT V. cholerae loci. All coding sequences from HaitiWT were used as queries in a batch BLAST of the N16961 V. cholerae proteome. The top hit by e-value was selected and VC name automatically assigned. However, as batch BLAST outputs a hit regardless of confidence, manual curation is still required for specific loci when using this dictionary.