Article Figures & Data
Figures
- FIG 1
Cell mass increase and cell wall incorporation continue during EP insufficiency. (A to J) Overnight cultures of the Δ6 endo (ΔshyABC Δvc1537 ΔtagE1,2 PIPTG::shyA) strain grown in the presence of IPTG (200 μM) were washed twice and diluted 100-fold into growth medium with IPTG (ShyA +) or without IPTG (ShyA -). At the indicated time points, optical density (OD600) (A), dry mass (mg/liter) (B), and viable cell counts (CFU/ml) (C) were measured. Data are means of at least three biological replicates, and error bars represent standard deviations. (D to G) Cells were imaged at 3 h, and representative cells are shown in panel D. ImageJ was used to measure cell area (E), cell length (F), and cell width (G). Raw data points are shown, and error bars represent standard deviations. Asterisks denote statistical difference relative to ShyA + via a Mann-Whitney test (****, P < 0.0001). (H) Δ6 endo strain was grown in the presence of HADA (100 μM) for 3 h, washed twice, and then imaged. All bars, 5 μm. (I) After 2 h of growth, relative PG content of Δ6 endo strain (normalized to OD600) was measured via UPLC analysis (see Materials and Methods for details). Bar graphs show data normalized to Shy A+. Error bars represent standard deviations of three biological replicates, and asterisks denote statistical difference via unpaired t test (**, P < 0.01). (J) After 3 h of growth, cells were pelleted and resuspended in 20 mM NaCl (osmotic shock treatment) for 5 min. Shock treatment was stopped by adding PBS to 180 mM. Percent survival is CFU/ml after treatment divided by CFU/ml before treatment. Raw data points of three biological replicates are shown. Asterisks denote statistical difference via unpaired t test (****, P < 0.0001).
- FIG 2
Cell mass increase and PG incorporation during EP insufficiency relies on aPBP activity. Δ6 endo grown overnight in IPTG (200 μM) was washed twice and diluted 100-fold into fresh medium with inducer (ShyA+) (A) or without inducer (B) (ShyA -) (B). After 2 h of growth, either vehicle (0.1% DMSO, shown in black), the aPBP inhibitor moenomycin (aPBP-, 10 μg ml−1, 8× MIC, shown in red), the MreB inhibitor MP265 (MreB-, 300 μM, 15× MIC, shown in blue), or the PBP2 inhibitor amdinocillin (PBP2-, 10 μg ml−1, 20× MIC, shown in blue) were added. At the indicated time points, OD600 was measured; data are averages of three biological replicates, and error bars represent standard deviations. (C) At 2 h after drug treatment, 120 ml of cells was harvested and dried on a heat block (65°C) until the mass steadied (∼24 h). Dry mass values were transformed into milligrams per liter. Data are averages of three biological replicates, and error bars represent standard deviations. Asterisks denote statistical difference via paired multiple t tests (****, P < 0.0001). n.s., not significant. (D) Representative cell morphologies 2 h after drug addition are shown. (E) Cell area (square micrometers) and (F) cell length (micrometer) were measured in ImageJ. Raw data points are shown. Asterisks denote statistical difference via Kruskal-Wallis test (**, P < 0.01; *, P < 0.05). (G) An overnight culture of the Δ6 endo ΔldtA ΔldtB strain was diluted into medium without inducer. After 2 h of ShyA depletion, HADA (100 μM) was added for another 1 h. Cells were then washed twice and imaged. Antibiotics moenomycin (aPBP -), MP265 (MreB -), and amdinocillin (PBP2 -) were added for 1 h after the 2-h initial depletion, followed by 1-h addition of HADA. All bars, 5 μm. (H) At 2 h after drug treatment, relative PG content of Δ6 endo strain relative to OD600 was measured via UPLC analysis (see Materials and Methods for details). Data are normalized to the ShyA+ DMSO sample. Error bars represent standard deviations of three biological replicates. Asterisks denote statistical difference via unpaired t tests (****, P < 0.0001; *, P < 0.05).
- FIG 3
MreB movement continues during EP insufficiency. Δ6 endo (A to D) or Δ8 endo (E to G) strain expressing an mreBmsfGFPsw fusion from its native chromosomal locus was diluted from an overnight culture grown in the presence of IPTG into growth medium without inducer (ShyA -). After 3 h, cells were imaged using epifluorescence microscopy (A to D) or TIRF (E to G). MreB movement was analyzed using Fiji (TrackMate). (A) A representative single moving MreB focus track (red circle) is shown (frames are 2.5 s apart). (B to D) Representative kymographs of MreB foci are shown for cells grown in the presence of the MreB inhibitor MP265 (MreB -) (B), in the presence of inducer (ShyA +) (C), or in the absence of inducer (ShyA -) (D). (E to G) TIRF was used to assess MreB focus velocity (E), mean cluster size (F), and mean cluster number (G). Raw data points are shown, and error bars represent standard deviations. Asterisks denote statistical difference via Mann-Whitney test (**, P < 0.01; ****, P < 0.0001).
- FIG 4
Cross-species complementation of Δ6 endo phenotypes with an EP from Neisseria gonorrhoeae. (A and B) Δ6 endo strain was transformed with (arabinose-inducible) pBAD33 expressing an N. gonorrhoeae EP (MepMNgo). Derivatives of MepMNgo include an N-terminal DsbA signal sequence (ss), domain 1 truncation (ΔDom1), or active site mutation (H373A). (A) Cells were washed and spot-plated on medium containing either no inducer, IPTG (200 μM) (chromosomal ShyA +), or arabinose (0.2%) (pBAD33-encoded MepM +). Plates were incubated at 37°C for 24 h and then imaged. (B) Cells were diluted 100-fold and grown without inducer, with IPTG (ShyA +), or with arabinose (MepM +) for 3 h and then imaged. Bars, 5 μm.
- FIG 5
Summary diagram of the consequences of EP insufficiency in V. cholerae. Characteristics of EP-insufficient (- EP) V. cholerae in the presence or absence of the Rod system or aPBP inhibitors.
Supplemental Material
MOVIE S1
Time-lapse microscopy of Δ6 endo mutant recovering from shyA depletion. shyA was depleted from the Δ6 endo mutant for 3 h in LB. The ShyA-depleted cells were spotted onto an LB agarose pad (0.8% [wt/vol] agarose) containing 500 μM IPTG to induce shyA expression. Cells were incubated at 37°C, and images were taken at 10-min intervals for 8 h. Time-lapse frames were stabilized using ImageJ plugin “StackReg.” Movie S1, AVI file, 0.8 MB
Copyright © 2021 Murphy et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
FIG S1
ShyA levels after depletion in a Δ6 endopeptidase mutant and its effect on growth, survival, and PG composition. (A) Wild-type, Δ6 endo, and ΔshyA strains were grown overnight in LB broth containing inducer (IPTG, 200 μM) and then washed three times with fresh LB. Cells were then diluted 100-fold into 150 ml prewarmed LB medium without inducer for ShyA depletion. Samples were collected at indicated time points. For Western blotting, cell extracts (adjusted to same protein concentration) were separated on 10% SDS-PAGE gels and subjected to Western blot analysis using ShyA polyclonal antibody. (B) ShyA band intensities were quantified (ImageJ), and the intensity value of the nonspecific background band detected in the ΔshyA mutant was subtracted. Residual ShyA protein levels were normalized to nondepleted ShyA at 0 h (100%). Data are averages of three biological replicates, and error bars represent standard deviations. (C and D) Δ6 endo cells grown for 2 h in the presence (ShyA +) or absence (ShyA -) of inducer were harvested for PG content analysis (see Materials and Methods). (C) PG chromatograms and (D) a table showing the relative molar abundance (as a percentage) of monomers, dimers, and trimers are shown. Standard deviations are shown. Crosslink percentage is the proportion of crosslinked peptide side chains (calculated based on dimer and trimer content) and anhydro muropeptides (with a residue of (1-6 anhydro) N-acetyl muramic acid, abbreviated “Anh”) are the terminal subunits of the sugar chains and hence used to calculate the chain length. Values are means of three biological replicates. Percent change was calculated relative to the IPTG-treated sample, and P values were generated using a multiple-comparison t test (****, P < 0.0001; ***, P < 0.001, ** P < 0.01, * < 0.05). (E and F) WT, N16961 Δ6 endo, and E7946 Δ9 endo (mreB::mreBmsfGFPsw) were treated as described in the legend to Fig. 1. At the indicated time points, OD600 (E) was measured via spectrophotometry, and cells were diluted serially onto LB IPTG (200 μM) plates to determine colony-forming units (CFU) per milliliter (F). Note that data for WT and Δ6 endo strains are reproduced from Fig. 1A and B. Data are averages of three biological replicates; error bars represent standard deviations. Download FIG S1, TIF file, 0.4 MB.
Copyright © 2021 Murphy et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
FIG S2
Mass increase during EP insufficiency requires aPBPs, whereas the Rod system influences cell morphology. Δ6 endo strain was grown overnight in IPTG (200 μM), washed twice, and diluted 100-fold into fresh medium containing IPTG (ShyA +) (A) or not containing IPTG (ShyA -) (B). After 2 h of EP depletion (dashed vertical line), either no antibiotic (DMSO, 0.1%, shown in black), the aPBP inhibitor moenomycin (aPBP -, 10 μg ml−1, 8× MIC, shown in red), the MreB inhibitor MP265 (MreB -, 300 μM, 15× MIC, shown in blue), or the PBP2 inhibitor mecillinam (PBP2-, 10 μg ml−1, 20× MIC, shown in blue) were added. At the indicated time points, cells were diluted serially onto LB IPTG (200 μM) plates to determine viable colony-forming units (CFU) per ml (B). Data are averages of three biological replicates; error bars represent standard deviations. (C to E) At 2 h post-drug treatment (+ 2 h), cells were harvested and spotted on a 0.8% agarose pad containing PBS for phase contrast microscopy. ImageJ was used to measure cell area (C), length (D), and width (E) of individual cells (n > 200). P values were generated using a Kruskal-Wallis multiple-comparison test (****, P < 0.0001; ***, P < 0.001; ** P < 0.01; *, P < 0.05). Note that ShyA - data are the same as shown in Fig. 2. (F to H) Cells were grown as described above for panels A and B but treated with DMSO (1%) or the aPBP (pbp1b) inhibitor cefsulodin (1 mg ml−1, shown in red). Cells were imaged at 2 h post-drug treatment on a 0.8% agarose pad; representative images are shown (E). All scale bars, 5 μM. At the indicated time points, optical density (OD600) (G) and viable cell counts (CFU/ml) (H) were measured. Data are means of at least three biological replicates; error bars represent standard deviations. Download FIG S2, TIF file, 0.4 MB.
Copyright © 2021 Murphy et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
MOVIE S2
mreBmsfGFP movement in Δ6 endopeptidase after 3 h of EP depletion. Cells were treated as described in the legend to Fig. 3. Following depletion, cells were transferred to an agarose pad lacking IPTG and imaged by time-lapse microscopy; images were taken every 2.5 s at 100-ms exposure time. Download Movie S2, AVI file, 0.6 MB.
Copyright © 2021 Murphy et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
FIG S3
Growth rate, mean square displacement analysis, and cluster analysis of the mreBmsfGFP strain. (A) Wild-type E7946 and mreBmsfGFP-containing derivative were grown overnight in LB. Cells were diluted 1,000-fold into fresh medium, and 200 μl of each was loaded into a 100-well plate. Growth of each culture was monitored by optical density at 600 nm (OD600) in a Bioscreen C plate reader (Growth Curves America). (B to D) Δ6 endo mreBmsfGFPsw strain was grown with (ShyA +) or without (ShyA -) IPTG and imaged using TIRF microscopy. (B) Example MSD curves for two regions of interest (ROIs) for both the ShyA+ and ShyA- condition. (C) Alpha values and percentages of MreBmsfGFP patches exhibiting directed motion. (D) Representative images of mreBmsfGFPsw clusters in Δ6 endo ShyA+ and ShyA- strains used for analysis. Scale bars, 5 μm. Download FIG S3, TIF file, 0.4 MB.
Copyright © 2021 Murphy et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
FIG S4
ShyA and orthologous MepM amino acid sequence alignment. (A) Amino acid sequences of V. cholerae endopeptidase ShyA (VCA0079) and orthologs from N. gonorrhoeae (NGO1686) and E. coli (b1856) were aligned in Clustal Omega (74). Numbers indicate the amino acid position relative to the start codon, and alignment gaps are denoted with dashes. Symbols indicate the similarity of aligned residues: identical (*), strong similarity (:), and weak similarity (.). (B) Summary of BLAST alignments of b1656 and NGO1686 amino acid sequences to ShyA. Download FIG S4, TIF file, 0.4 MB.
Copyright © 2021 Murphy et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
FIG S5
Cross-species complementation of Δ6 endo strain with orthologous EPs. (A and B) Δ6 endo strain was transformed with (arabinose-inducible) pBAD33 expressing an E. coli EP (MepMEco). Derivatives of MepMEco include an N-terminal DsbA signal sequence (ss), domain 1 truncation (ΔDom1). (A) Strains were diluted and spot-plated on medium containing either no inducer, IPTG (200 μM) (ShyA +), or arabinose (0.2%) (MepM +). Plates were incubated at 37°C for 24 h and then imaged. (B) Alternatively, these strains were diluted into fresh medium containing either no inducer, IPTG, or arabinose (ara) and grown for 3 h and then imaged on a 0.8% agarose pad. Scale bar, 5 μm. (C) Δ6 endo strain carrying pBAD33 expressing the indicated constructs (“pBAD” column) were diluted into fresh medium containing either no inducer, IPTG, or arabinose (ara) and grown for 3 h (“experiment” column). Cells were then spot-plated on medium containing either IPTG (200 μM) (ShyA expressed), arabinose (0.2%) (MepM expressed) or no inducer. Plates were incubated at 37 ˚C for 24 h and then imaged. Download FIG S5, TIF file, 0.6 MB.
Copyright © 2021 Murphy et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
TABLE S1
Summary of strains and oligonucleotides used in this study. Download Table S1, XLSX file, 0.01 MB.
Copyright © 2021 Murphy et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
MOVIE S3
mreBmsfGFP movement in Δ6 endopeptidase expressing shyA. Time-lapse movie showing movement of MreBmsfGFPsw particles. Images were taken every 5 s at 100-ms exposure time. The movie is minimally processed (ImageJ’s walking average function). Download Movie S3, AVI file, 0.1 MB.
Copyright © 2021 Murphy et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
