PMC

(A) Alleviating interactions between known (pink) or predicted (blue) gene targets and cognate sRNA regulators (brown). (B) Quantitative real-time PCR analysis of target transcript levels in regulator mutants on rich or minimal medium. Values indicate mean fold-change ± standard deviation (error bars) of triplicate biological measurements normalized to a housekeeping gene product (glutathione S-transferase).

(A) Distinct patterns of enrichment in aggravating and alleviating genetic interactions between select envelope bioprocesses in rich and minimal medium. (B) Aggravating genetic interactions (orange edges) and metabolic links (black arrows) between the CA, LPS/ECA and cell wall biosynthetic pathways. (C) Antibiotic sensitivity of select E. coli mutants; bioprocess and gene identity indicated. Phenotypic complementation based on plasmid rescue. (D) Isobologram portraying the enhanced combination potency of amoxicillin (AMOX) and phosphomycin (PMB) against E. coli mutants deficient in cell wall (murA), LPS (lpxA) or CA (wcaB) production. Dotted line indicates additivity; MIC, minimal inhibitory concentration normalized relative to no drug control.

(A) Genetic interaction sub-network (rich medium) involving LPS transport factors. LPS is synthesized at the IM cytoplasmic face, flipped across the bilayer by MsbA, extracted by LptBCFG, and transported to the OM by LptA for final assembly by LptDE. (B) Strain growth in rich liquid medium at 32°C over 24 h. (C) Electron microscopy showing morphological alterations (arrows) in mutant E. coli strains relative to wild-type cells; scale-bar 500 nm. (D) SDS-PAGE immunoblots of marker proteins (I) and silver staining LPS (II). (E) Co-immunoprecipitation of SPA-tagged YhjD with His₆-tagged LPS transport component. (F) LPS-binding by immobilized recombinant YhjD (test) and LptA (positive control) but not LolE (negative control). After extensive washing, bound LPS molecules were visualized by SDS-PAGE and silver staining.

(A) Genetic interaction patterns between secretory and cell division factors. (B) Growth rates (fitness) of secY and ftsK single and double mutants in liquid rich (RM) or minimal medium (MM) at 32°C over 24 h; hypomorphic alleles indicated by asterisks. (C) Pair-wise correlation coefficient profiles of secretory and cell division mutants grown on rich medium or minimal medium. (D) Identification of Sec proteins co-purified from the endogenous affinity tagged Fts protein by tandem mass spectrometry. (E) Fluorescent microscopy of E. coli stains (stained with FM4-64) after growth in minimal or rich medium; scale-bars 2 µm. (F) Quantitative measurements of changes in mutant cell length after growth in rich and minimal medium. Error bars show measurement of standard deviation from three independent experiments.

(A) Alleviating genetic interactions between annotated PG hydrolases and YceG/YebA. (B) Protein architecture (amino acid length indicated); domain structure of YceG unknown. CC, coiled coil; TM, transmembrane helix; SP, signal peptide. (C) Electron microscopy showing impaired membrane invagination and uncleaved PG septa (arrows) in E. coli mutants relative to wild-type cells; scale-bar 500 nm. (D) Cell division defects (arrows), phenotypic complementation (plasmid-based wild-type gene copy) or transgenic rescue. Strains stained with DAPI were visualized using a high content microscopy with differential interference contrast (DIC) and fluorescence optics; scale-bar 2 µm. (E) Differential strain growth with or without ampicillin (AMP, 5 µg/ml). (F) Alleviating double mutant interaction in liquid rich medium at 32°C over 24 h.

(A) Schematic summary of the eSGA gene targets associated with the E. coli outer membrane (OM), inner membrane (IM), periplasm (PP), cytoplasm (CY), regulatory RNA (sRNA), or of uncertain localization. Gene numbers are indicated, with essential genes shown in brackets. (B) Functional distribution of annotated (Group 1), uncertain function (Group 2) and predicted (Group 3) envelope target genes sorted into 20 broadly representative bioprocesses. (C) Schematic summarizing the experimental construction and computational analysis of two E. coli cell-envelope genetic interaction maps. After conjugation and genetic exchange, high density arrays of double-mutants were first selected on rich medium containing Cm and Kan. After outgrowth for 24 hours, surviving colonies were then replica pinned onto minimal M9 medium, to identify condition-dependent genetic interactions. Both sets of plates were digitally imaged and colony sizes quantified to determine gene pairs showing aggravating (synthetic lethal and synthetic sick) and alleviating (buffering) interactions.

(A) Genetic interaction sub-network in rich medium. (B) Aggravating interactions between lptD and yfgH, yceK, and ytfN. (C) Proposed mechanism of drug hypersensitivity (I) modeled on : envelope deficiency results in a porous OM (dashes), thereby allowing vancomycin (VAN) into the periplasm where it blocks PG cross-linking, weakening the cell wall and resulting in cytoplasmic membrane bulges in single mutant strains (II) or catastrophic cell wall failure and lysis in double mutants (III); scale bar equals 2 µm. Quantification (percentage) of bulged or lysed cells following vancomycin treatment according to strain genotype; error bars indicate standard deviation (IV). (D) Growth inhibition of strains treated with vancomycin. Graph shows mean drug disk halo diameter and standard deviation (error bars) in three independent experiments; p-value calculated using Student's t-test. (E) SDS-PAGE immunoblot analyses of marker protein levels in indicated strains during logarithmic growth in rich medium; periplasmic maltose-binding protein (MBP) used as loading control.

(A) Comparison of ratios of alleviating (E-score ≥2) to aggravating (E- score ≤−2) genetic interaction involving essential (E) and non-essential (NE) gene pairs on both growth conditions, p-values were computed using Fisher's exact test. (B) Venn diagram showing the overlap of the genetic interaction pairs derived from this study and the literature. (C) Significance testing of the agreement between the literature and this study (red arrow) compared against expected gene pair frequencies by randomly sampling (blue distribution represents 10,000 null models). (D) Distribution of correlations coefficients between the genetic interaction profiles for gene pairs within the same operon versus randomly drawn gene pairs (panel I). Pair-wise profile correlation coefficient of genes in the Fe-enterobactin “fec” operon in rich medium (panel II) and for LPS 1, 2-glucosyltransferase components of waa operon in minimal medium (panel III). (E) Distribution of genetic interaction profile correlation for gene pairs with correlated co-expression (panel I). Scatter-plot of correlated genetic profiles is shown for cyoD (x-axis) and cyoC (y-axis) in panel II, and for minE (x-axis) and minD (y-axis) in panel III. (F) Distribution of correlation coefficients between the genetic interaction profiles of gene pairs encoding interacting proteins versus randomly drawn gene pairs (panel I), where the p-value was computed using the two-sample Kolmogorov-Smirnov (KS) test. Scatter plot of correlated genetic profiles for mdtI (x-axis) and mdtJ (y-axis) (panel II). Scatter plot of correlated genetic profiles for ftsZ (x-axis) and ftsE (y-axis) (panel III).