Journal: bioRxiv

Article Title: Peptidoglycan turnover promotes active transport of protein through the bacterial cell wall

doi: 10.1101/2025.09.12.675941

Figure Lengend Snippet: (A) Illustration of the Gram-positive cell envelope. (B) Micrographs of E. coli (left) and B. subtilis (right) expressing cytosolic GFP and mNeonGreen, respectively, before and after detergent-mediated lysis. E. coli data from reference . ( C ) Illustration of phospholipase-based assay to measure cell-wall permeability. ( D ) Cytometer forward scatter vs fluorescence of cells labeled with propidium iodide and treated with 2 μg/mL phospholipase. ( E ) Fluorescence versus time of cells labeled with propidium iodide and treated with 2 μg/mL phospholipase at t =0. ( F ) Mean percent survival versus time. Cells were treated with 2 μg/mL phospholipase at t = 45 seconds. Permeabilized cells were incubated in PBS for 30 minutes prior to the experiment. Error bars indicate +/- 1 s.d. across N = 6, 11, 3 technical replicates for no-enzyme control, phospholipase treatment, and phospholipase after permeabilization, respectively. ( G ) Mean time it takes for 50% of the cells to die, t 1/2 , for phospholipase treated cells and phospholipase-treated permeabilized cells. Error bars indicate +/- 1 s.d. across N = 6, 11 technical replicates (black dots). p -value is for a Student’s two-sided t -test.

Article Snippet: During phosphate buffer saline (PBS) incubation, the growth medium in the flow cell was exchanged with PBS (no divalent cation supplements) + 2 mM IPTG using the ONIX system.

Techniques: Expressing, Lysis, Permeability, Cytometry, Fluorescence, Labeling, Incubation, Control

Journal: bioRxiv

Article Title: Peptidoglycan turnover promotes active transport of protein through the bacterial cell wall

doi: 10.1101/2025.09.12.675941

Figure Lengend Snippet: ( A ) Diagram of the two modes of peptidoglycan synthesis. The Rod complex (blue) synthesizes peptidoglycan oriented along the circumference of the cell. PBP1 (red, encoded by ponA ) synthesizes peptidoglycan isotropically. Inset: a diagram of the inner face of the cell wall with oriented Rod complex-synthesized strands (blue), non-oriented PBP1-synthesized strands (red), peptidoglycan cross-links (light blue) and wall teichoic acids (black). ( B ) Distribution of protein masses for secreted proteins in B. subtilis 168. Data obtained from the UniProt database after filtering for proteins with the ‘secreted’ label and without the ‘membrane’ label in the subcellular localization field . ( C ) Mean percent survival of cells treated with varying phospholipase concentrations over time. Phospholipase was added at t = 45 seconds, as indicated by the dashed line. Each experiment was performed once. (D) Mean percent survival of PBS permeabilized cells over time. Cells were incubated in PBS for 0, 15, 30, or 60 minutes prior to the experiment. Cells were treated with 2 μg/mL phospholipase at t = 45 seconds. Data are presented as mean ± standard deviation (SD). Shown here for comparison, the untreated and 30-minute permeabilized data from . N = 11, 2, 3, 3, 1 replicates. (E) t 1/2 (time it takes to reach 50% survival) versus time spent incubating in PBS. Colored dots represent the t 1/2 values for individual experiments. The black dots indicate mean t 1/2 values. The error bars are the standard deviation. Statistical significance calculated using Student’s t -test.

Article Snippet: During phosphate buffer saline (PBS) incubation, the growth medium in the flow cell was exchanged with PBS (no divalent cation supplements) + 2 mM IPTG using the ONIX system.

Techniques: Synthesized, Membrane, Incubation, Standard Deviation, Comparison

Journal: bioRxiv

Article Title: Peptidoglycan turnover promotes active transport of protein through the bacterial cell wall

doi: 10.1101/2025.09.12.675941

Figure Lengend Snippet: (A) Charged surface representations of mono-ubiqutin (mUbq), phospholipase (PLA2), and mNeonGreen (mNG). (B) Micrographs of B. subtilis before and 30 minutes after detergent-mediated lysis. (C) Single-cell normalized mNeonGreen fluorescence versus time from cells upon detergent treatment in microfluidic perfusion chambers (blue lines). The black line is the mean of the cell traces. The black dashed line indicates the time point at which detergent is perfused. The red dashed line indicates the time at which detergent is perfused out. n = 108 cells across N = 3 technical replicates. ( D ) The probability distribution for the lag time mNeonGreen efflux upon cell lysis ( t lag ). Mean (circle) and standard deviation (error bars) shown. ( E ) Mean normalized single-cell fluorescence versus time for PBS-incubated cells upon lysis. n = 85 and 49 cells for N = 1 experiment for each duration of PBS treatment. ( F ) Mean normalized single cell mass versus time for cells upon detergent treatment on an agarose pad. Error bars indicate +/- 1 s.d. from n = 14 cells across N = 1 experiment. ( G ) Normalized single-cell fluorescence versus time upon detergent treatment for cells expressing the mono-ubiquitin probe (blue lines). The black line the mean of n = 117 cells across N = 3 technical replicates. Inset: mean fluorescence from 0-10 minutes after lysis with detergent wash (blue) and without wash (pink). n = 170 cells across N = 3 technical replicates.

Article Snippet: During phosphate buffer saline (PBS) incubation, the growth medium in the flow cell was exchanged with PBS (no divalent cation supplements) + 2 mM IPTG using the ONIX system.

Techniques: Lysis, Fluorescence, Standard Deviation, Incubation, Expressing, Ubiquitin Proteomics

Journal: bioRxiv

Article Title: Peptidoglycan turnover promotes active transport of protein through the bacterial cell wall

doi: 10.1101/2025.09.12.675941

Figure Lengend Snippet: ( A ) Normalized fluorescence traces of the PBS-incubated cells shown in . The black line indicates the mean normalized trace. The shaded region indicates the standard error of the mean. The first dashed black line indicates detergent perfusion. The second dashed black line indicates the wash. (B) Fold change in OD600 of E.coli and B. subtilis after resuspension in detergent (LB + 5% N-lauroylsarcosine). N = 3 replicates.

Article Snippet: During phosphate buffer saline (PBS) incubation, the growth medium in the flow cell was exchanged with PBS (no divalent cation supplements) + 2 mM IPTG using the ONIX system.

Techniques: Fluorescence, Incubation

Journal: bioRxiv

Article Title: Peptidoglycan turnover promotes active transport of protein through the bacterial cell wall

doi: 10.1101/2025.09.12.675941

Figure Lengend Snippet: ( A ) Normalized mNeonGreen fluorescence over time from wild-type (gray, from ) and Δ lytC Δ lytD (+Δ slrR ) (green) cells after detergent treatment. n = 31 cells across N = 1 experiment. (B) Diagram of normalized fluorescence traces. Comparing the gray (i) trace to the black trace, the shorter lag indicates larger initial pore size but the same slope ( m ) indicates the same rate of hydrolysis. Comparing the light gray (ii) trace to the black trace, the shorter lag and steeper slope arise from a faster hydrolysis rate. ( C ) (left) Lag time before mNeonGreen efflux after lysis versus efflux rate for untreated wild-type cells shown in . (right) Larger initial pore sizes at the time of lysis correspond to lower lag times for a given efflux rate. ( D ) Mean percent survival over time for wild-type cells treated with 60 mM sodium azide (light blue) one minute prior to the addition of 2 μg/mL phospholipase. Permeabilized (perm.) cells (pink) were incubated in PBS for 30 minutes prior to the experiment. N = 2 and 3 technical replicates for the sodium azide-treated and permeabilized sodium azide-treated traces. Wild-type + phospholipase data from . ( E ) Mean time that it takes for 50% of the cells die), t 1/2 . The error bars indicate +/- 1 s.d. p -values from Student’s two-sided t -test, ns: p > 0.05. ( F ) Mean normalized single-cell fluorescence versus time after lysis for cells expressing the mono-ubiquitin probe un-treated (bue, from ) and treated with 60 mM sodium azide (light blue). Confidence intervals indicate +/- 1 s.e.m. n = 132 cells across N = 3 technical replicates for sodium azide-treated cells. Inset: probability distribution for the fluorescence values at t = 15 minutes for untreated (blue) and sodium-azide treated (light blue) cells. p -values calculated using Student’s t -test. ( G ) Lag before efflux versus mNeonGreen efflux rate for untreated (gray, from ) and sodium azide-treated (light blue) cells. Cells were treated with LB + 60 mM sodium azide for 15 minutes prior to membrane lysis. n = 138 cells across N = 3 technical replicates for sodium azide-treated cells. p -values calculated using Student’s t -test. ( H ) Fraction of fluorescent D-amino acids retained in cell wall for untreated (blue) and sodium azide-treated (60 mM, light blue) cells. Error bars indicate +/- 1 s.d. N = 2, 2 technical replicates.

Article Snippet: During phosphate buffer saline (PBS) incubation, the growth medium in the flow cell was exchanged with PBS (no divalent cation supplements) + 2 mM IPTG using the ONIX system.

Techniques: Fluorescence, Pore Size, Lysis, Incubation, Expressing, Ubiquitin Proteomics, Membrane