Phenotypic profiling of mutants lacking Bam-associated proteins

Heat map of fitness scores for the E. coli BW25113 parent strain and ΔbamB, ΔbamC, ΔbamE, ΔsurA, Δskp, or ΔdegP mutants grown in various stress conditions. Colony size measurements for each condition were normalised by comparing to colony size of that strain when grown on LB medium at 37°C. Growth of each strain on LB is set to 1 and each condition normalised to this. Fitness scores above 1 represent better growth than the LB condition as measured by colony size and scores below 1 indicate smaller colony size than the LB condition. Some conditions are abbreviated due to space restrictions: EMIC - 1-Ethyl-3-methylimidazolium chloride, EDTA - Ethylenediaminetetraacetic acid, SDS – Sodium dodecyl sulfate.

Genetic interaction analysis of Bam-associated genes by TraDIS

TraDIS libraries were constructed in the E. coli BW25113 parent strain and ΔbamB, ΔbamC, ΔbamE, ΔsurA, Δskp, or ΔdegP mutants to identify genes that become essential in each knockout strain compared to the parent library (conditionally essential). A. Venn diagrams showing conditionally essential genes identified within the ΔbamB, ΔbamC and ΔbamE TraDIS libraries or the ΔsurA, Δskp and ΔdegP libraries. B. Analysis of KEGG categories enriched in conditionally essential gene lists in the ΔbamB, ΔbamE, ΔsurA and ΔdegP TraDIS libraries, represented as bubble plots. No data is shown for the ΔbamC and Δskp conditionally essential gene lists as there was no significant enrichment of any KEGG categories.

Incorporation of heptose into LPS is essential in ΔbamB, ΔsurA and ΔdegP

A. Transposon insertions in the genes gmhA, gmhB, hldE, waaD, waaC, and waaF in the parent, ΔbamB, ΔsurA and ΔdegP TraDIS libraries. Transposon cut-off is set to 20. Essential genes are represented as red arrows. B. Schematic of the heptose biosynthesis pathway and LPS structure in E. coli K-12 BW25113 with gene labels for LPS biosynthesis enzymes indicated next to the linkage they form or component they ligate. Enzymes identified as conditionally-essential are labelled in red text. C. Membrane fluidity was measured in ΔgmhA, ΔgmhB, ΔhldE, ΔwaaD, ΔwaaC, ΔwaaF, ΔwaaG, ΔwaaP and ΔwaaY mutants and compared to the parent strain by using pyrenedeconoic acid fluorescence. D. To determine the extent of Bam complex activity, successful folding and insertion of OmpT into the outer membrane was measured in the ΔgmhA, ΔgmhB, ΔhldE, ΔwaaD, ΔwaaC, ΔwaaF, ΔwaaG, ΔwaaP, ΔwaaY and ΔompT mutants and compared to the parent strain. For panels C and D, experiments were performed in biological and technical triplicate with standard deviation represented by error bars. Two sample t-tests were used to assess statistical significance of differences from the WT strain with *** indicating p-values of <0.001, * indicating p-values of <0.05, and NS as not significant (p-value ≥ 0.05).

Synthesis of ECA is essential in the absence of the chaperone SurA

A. Transposon insertions in genes of the ECA biosynthesis pathway in the parent or ΔsurA mutant TraDIS libraries. Transposon cut-off is set to 100. Essential genes are represented as red arrows. B. Schematic representation of the ECA biosynthesis pathway with the names of proteins labelled next to the reaction for which they are responsible. Conditionally essential proteins are labelled in red text. C. Transposon insertions in the waaL gene in either the parent or ΔsurA mutant TraDIS libraries. Transposon cut-off is set to 100. D. Analysis of phospholipid content in the parent or ΔsurA mutant with further mutations to disrupt synthesis of the major anionic phospholipids. The ΔpgsA and ΔsurAΔpgsA strains are also ΔlppΔrcsF. Phospholipid samples were separated and visualised by TLC using a solvent mixture of methanol/ chloroform/ water with a ratio of 2:2:1.8 before being visualised by phosphomolybdic acid and charring.

meso-DAP containing peptidoglycan is essential in the absence of Bam accessory lipoproteins

A. Transposon insertions in the genes dapF and lysA in the parent or ΔbamB, ΔbamC, ΔbamE mutant TraDIS libraries. Transposon cut-off is set to 20. Essential genes are represented as red arrows. B. Schematic representation of the L-lysine biosynthesis pathway with the names of proteins labelled next to the reaction for which they are responsible. Conditionally essential proteins are labelled in red text. A schematic structure of E. coli peptidoglycan is shown with the site of meso-DAP incorporation shown. C. Efficiency of plating assay showing survival of the ΔdapF, ΔbamB, ΔbamC, ΔbamE, ΔdapFΔbamB, ΔdapFΔbamC, or ΔdapFΔbamE mutants grown on LB with or without 1 mM meso-DAP or 10 % sucrose. Cells were grown overnight in LB supplemented with 1 mM mesoDAP before being normalised to OD600 = 1.00 and serially diluted 1:10 before 2 µl was spotted on agar plates and incubated at 37°C overnight.

Loss of BamB leads to changes in DNA replication control

A. Schematic representing DNA replication control in E. coli. DiaA stimulates initiation at the origin of replication, oriC, by DnaA. SeqA prevents premature reinitiation of replication. Hda acts together with the β-sliding clamp to hydrolyse ATP-bound DnaA by regulatory inactivation of DnaA (RIDA). This can also occur through datA-dependent DnaA-ATP hydrolysis (DDAH).

B. Transposon insertions in the genes hda, seqA and diaA in the parent or ΔbamB mutant TraDIS libraries. Conditionally-essential genes are represented as red arrows and genes that increase fitness are in green. C. Efficiency of plating assay showing survival of the parent dCas9 expressing strain LC-E18 (WT) or ΔbamB mutant derivatives carrying the pSGRNA plasmid, which encodes CRISPRi guide RNA targeting either seqA or hda. Cells were grown on LB with or without 40 nM anhydro-tetracycline (aTc) to induce guide RNA expression. D. The E. coli BW25113 parent strain, single, or double ΔbamB and ΔdiaA mutants were spotted on LB agar in 384-well format, in triplicate, and grown overnight at 37°C before being imaged. Fitness was calculated based on colony size and fitness ratios generated relative to the WT parent. E. Flow cytometry of E. coli LC-E18 cells grown in LB followed by replication run-out assay. Forward scatter and side scatter are plotted with WT cell data indicated in blue and ΔbamB data points in red. Fluorescence is plotted and represents chromosomal content for each cell with chromosome numbers for each peak marked

Conservation analysis of Bam-associated proteins in Gram-negative bacteria

Conservation of the BamB, BamC, BamE, SurA, DegP and Skp proteins among key Gram-negative bacterial species represented by a presence/absence map plotted onto a phylogenetic tree generated with iTOL [76]. Gene count in each species is represented by coloured boxes.

Correlation of phenotypic profiles

Heatmap of Pearson correlation coefficients for each pair of strain phenotypic profiles. Correlation of fitness ratios for each strain was assessed by calculating the Pearson correlation coefficient for averaged fitness scores across replicates using Python before being plotted as a heatmap. Black squares indicate a p-value ≥0.05 meaning the correlation coefficient achieved was not statistically significant.

Reproducibility across TraDIS library sequencing runs

Correlogram of insertion indexes between individual sequencing runs for each TraDIS library. The colour and size of the bubbles correspond to the strength of the Pearson correlation coefficient between the indices for the same genes across the runs. The blue colour represents positive correlation.

Transposon library construction metrics

TraDIS library construction metrics showing the percentage of sequencing reads that matched the transposon tag and that mapped to the chromosome. Total sequencing length as a function of total unique insertion sites, the total number of unique insertion sites for each sample, total number of sequencing reads, and the number of reads that mapped are also shown.

Circular plots of transposon insertion density in TraDIS libraries.

Circular plot of transposon insertion sites in the parent BW25113 (WT), ΔdegP, Δskp, ΔsurA, ΔbamE, ΔbamC, ΔbamB TraDIS libraries, listed from innermost tracks outwards, generated using DNAPlotter. The outermost track marks the BW25113 genome in base pairs with the inner grey bar tracks corresponding to sense and antisense CDS.

Examples of known synthetic lethal interactions

Transposon insertions in the genes degP, skp, bamB or bamE in the WT parent strain, ΔbamB, or ΔsurA mutant TraDIS libraries. Conditionally-essential genes are represented as red arrows, non-essential genes are represented by grey arrows. Transposon cut-off is set to 50.

Transposon insertions in genes required for heptose biosynthesis in the ΔbamC, ΔbamE and Δskp TraDIS libraries

Transposon insertions in the genes gmhA, gmhB, hldE, waaD, waaC, and waaF in the parent, ΔbamC, ΔbamE and Δskp TraDIS libraries. Transposon cut-off is set to 20. Essential genes are represented as red arrows and non-essential genes are represented by grey arrows.

Transposon insertions in the waaG and waaP genes in the WT, ΔbamB, ΔdegP and ΔsurA TraDIS libraries

Transposon insertions in the genes waaB, waaS, waaP, waaG and waaQ in the parent, ΔbamB, ΔdegP and ΔsurA TraDIS libraries. Transposon cut-off is set to 20. Genes are represented by grey arrows.

Replication run-out assay standards

Flow cytometry of E. coli MG1655 cells grown in media supporting different growth rates followed by replication run-out assay. Cells were grown in AB minimal medium supplemented with either 0.4% acetate, 0.2% glucose, or 0.2% glucose + 0.5% casamino acids, or LB supplemented 0.2% glucose at 37°C with aeration before being treated with rifampicin, cephalexin and stained with Sytox Green. Fluorescence is plotted and represents chromosomal content for each cell with chromosome numbers for each peak marked. An overlay of the separate data panels is presented.

Reproducibility of replication run-out assays for the parent strain

Flow cytometry of E. coli LCE-18 cells grown in LB supplemented with 0.2% glucose at 37°C with aeration before being treated with rifampicin, cephalexin and stained with Sytox Green. Fluorescence is plotted and represents chromosomal content for each cell with chromosome numbers for each peak marked. Each experiment was repeated on 4 separate occasions and an overlay of the separate data panels is presented.

Reproducibility of replication run-out assays for the ΔbamB strain

Flow cytometry of E. coli LCE-18 ΔbamB cells grown in LB supplemented with 0.2% glucose at 37°C with aeration before being treated with rifampicin, cephalexin and stained with Sytox Green. Fluorescence is plotted and represents chromosomal content for each cell with chromosome numbers for each peak marked. Each experiment was repeated on 4 separate occasions and an overlay of the separate data panels is presented.

Replication run-out assays for the parent, ΔbamB, ΔbamC, ΔbamE and Δskp strains

Flow cytometry of E. coli LCE-18 WT parent, ΔbamB, ΔbamC, ΔbamE or Δskp cells grown in LB supplemented with 0.2% glucose at 37°C with aeration before being treated with rifampicin, cephalexin and stained with Sytox Green. Fluorescence is plotted and represents chromosomal content for each cell with chromosome numbers for each peak marked. An overlay of the separate data panels is presented.