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Nature Microbiology (2023 )Cite this article R-CHNH2-COOH

How bacteria link their growth rate to external nutrient conditions is unknown. To investigate how Bacillus subtilis cells alter the rate at which they expand their cell walls as they grow, we compared single-cell growth rates of cells grown under agar pads with the density of moving MreB filaments under a variety of growth conditions. MreB filament density increases proportionally with growth rate. We show that both MreB filament density and growth rate depend on the abundance of Lipid II and murAA, the first gene in the biosynthetic pathway creating the cell wall precursor Lipid II. Lipid II is sensed by the serine/threonine kinase PrkC, which phosphorylates RodZ and other proteins. We show that phosphorylated RodZ increases MreB filament density, which in turn increases cell growth rate. We also show that increasing the activity of this pathway in nutrient-poor media results in cells that elongate faster than wild-type cells, which means that B. subtilis contains spare ‘growth capacity’. We conclude that PrkC functions as a cellular rheostat, enabling fine-tuning of cell growth rates in response to Lipid II in different nutrient conditions.

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Data generated and analysed during this study are either presented in the paper or in the supplementary materials. Mass proteomic data are available via ProteomeXchange with identifier PXD039295. Source data are provided with this paper.

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We thank R. Carballido-Lopez, C. Billaudeau and A. Chastanet for helping explore the MreB velocity discrepancy; P. Levin, E. Libby, P. Stoddard and A. Florez for helpful advice and discussions; A. Mollo, S. Rowe and D. Kahne’s lab for biotin-d -Lys and S. aureus PBP4; and L. Lavis for JF dyes. This work was funded by National Institutes of Health Grants DP2AI117923-01 to E.G., as well as support from the Volkswagen Foundation. This work was supported by the NSF-Simons Center for Mathematical and Statistical Analysis of Biology at Harvard (award number 1764269) and the Harvard Quantitative Biology Initiative.

Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA, USA

Yingjie Sun, Sylvia Hürlimann & Ethan Garner

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E.G. and Y.S. conceived the project and wrote the paper. Y.S. and S.H. conducted all experiments.

The authors declare no competing interests.

Nature Microbiology thanks the anonymous reviewers for their contribution to the peer review of this work.

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a, Density of directionally moving MreB-mNeonGreen (bYS09) filaments in cells growing in different media determined from TIRF-SIM imaging. b, Velocity of single molecules of MreB-HaloTag (bYS40), HaloTag-Pbp2a (bYS201), and mNeonGreen-MreC (bYS170) are constant across different growth media - MreB-HaloTag and HaloTag-Pbp2a were labeled with 50 pM of HaloTag-JF549 for 30 min. See also Supplementary Movie 2, 3. c, Velocity of fully labeled MreB-mNeonGreen filaments remains mostly constant across growth rates - Cells expressing MreB-mNeonGreen(bYS09) were grown in different media, and MreB velocity determined by single-molecule tracking and MSD analysis. d, Velocity of MreB-mNeonGreen filaments (bYS09) in cells growing in different media determined by TIRF-SIM imaging. e, MreB velocity remains constant during shifts from poor to rich media - MreB-mNeonGreen (bYS09) cells were grown in S750 glycerol for 6 hs, washed in CH, then placed under a pad made of CH media immediately before imaging with TIRF. Plotted is the growth rate and velocity of directionally moving MreB filaments at each time point. f, Comparison of MreB velocity determined by each group analyzing the MreB-HaloTag (bYS40) data in Extended Data Fig. 1b. The MreB velocity was determined by single-molecule tracking and MSD analysis. g, Comparison of velocities determined by each group analyzing the MreB-mNeonGreen (bYS09) data from the growth shift in Fig. 1d. h, Comparison of velocities determined by each group independently growing and imaging cells containing MreB-msfGFP (bMD159) in rich and minimal media. Carballido-Lopez’s group measured the velocity of MreB-msfGFP in LB and S media. We measured the velocity of MreB-msfGFP in LB, CH, and S750 glycerol. While both groups still disagree, as each continues to attain self-consistant velocities across experiments, both groups remain committed to working together to resolve this discrepancy in the near future. i, Comparison of MreB velocity of MreB-mNeonGreen (bYS09) cells harvested at different OD600 values. j, MreB velocity of MreB-mNeonGreen (bYS09) over time with cells in MatTek dishes vs cells on a CH agar pad sandwiched between two coverslips. The MreB velocity of cells in MatTek dishes was fairly constant over 90 minutes, however the MreB velocity of cells on pads between two coverslips decreases after 20–30 minutes. N values are available in Supplementary Table 2. Boxes indicate 25–75 percentiles, whiskers indicate 10–90 percentiles, the midline indicates median, and + indicates the mean. Error bars and envelopes represent standard deviation. P-values were calculated using one-way ANOVA with multiple comparisons.

a, The abundance of Rod complex components in WT (PY79) cells grown in different media. Abundances are taken from the proteomic mass spectrometry datasets published in Dion et al.13. These datasets included two biological replicates. Each protein abundance is normalized to the mean abundance of that protein in WT cells grown in CH media. Note that membrane proteins, like RodA, were not detected in every replicate. Dotted line represents protein abundances in CH. b, The abundance of Mur enzymes in WT (PY79) cells grown in different media. Abundances are taken from the proteomic mass spectrometry datasets published in Dion et al.13. These datasets included two biological replicates. Each protein abundance is normalized to the mean abundance of that protein in WT cells grown in CH media. Dotted line represents protein abundances in CH. c, The relative abundance of MurAA determined by (Left axis) proteomic mass spectrometry in WT (PY79) and Pxyl-murAA (bYS365) cells growing in glycerol at different xylose concentrations, and (Right axis) the relative intensity of WT murAA-mNeonGreen (bYS397) and Pxyl-murAA-mNeonGreen (bYS1087) cells growing in glycerol at different xylose concentrations. Protein abundances are normalized to WT cells grown in S750 glycerol. The proteomic mass spectrometry dataset included 2–3 biological replicates. Note some error bars are too small to resolve. d, Western blot showing accumulation of Lipid II in WT (PY79) cells grown in S750 glycerol in the presence of cefotaxime (0.25 μg/ml). Fosfomycin (100 μg/ml) depletes Lipid II from cells. The experiment was repeated twice with similar results. e, Western blot showing accumulation of Lipid II in WT (PY79) cells grown in S750 glycerol in the presence of moenomycin (2.5 μg/ml). The experiment was repeated twice with similar results. f, Western blot showing Lipid II levels increases as murAA induction levels in Pxyl-murAA (bYS365). The experiment was repeated twice with similar results. g, MreB velocity remains constant with murAA induction. Cells containing xylose-inducible MurAA and MreB-mNeonGreen (bYS497) were grown in S750 glycerol with a range of MurAA inductions. The velocity of MreB filaments was determined by particle tracking. h, mNeonGreen-MreC velocity under different inductions of Pxyl-murAA (bYS499) grown in S750 glycerol. i, Sub-MIC treatment of moenomycin (2.5 μg/ml) and cefotaxime (0.25 μg/ml) in both CH (left) and S750 glycerol (right) can reduce the growth rate of WT (bYS09) cells without reducing MreB filament density. d, Western blot showing accumulation of Lipid II in WT (PY79) cells grown in S750 glycerol in the presence of cefotaxime (0.25 μg/ml). Fosfomycin (100 μg/ml) depletes Lipid II from cells. e, Western blot showing accumulation of Lipid II in WT (PY79) cells grown in S750 glycerol in the presence of moenomycin (2.5 μg/ml). f, Western blot showing Lipid II levels increases as murAA induction level increases in Pxyl-murAA (bYS365). g, MreB velocity remains constant with murAA induction. Cells containing xylose-inducible MurAA and MreB-mNeonGreen (bYS497) were grown in S750 glycerol with a range of MurAA inductions. The velocity of MreB filaments was determined by particle tracking. h, mNeonGreen-MreC velocity during different inductions of Pxyl-murAA (bYS499) grown in S750 glycerol. i, Sub-MIC treatment of moenomycin (2.5 μg/ml) and cefotaxime (0.25 μg/ml) in both CH (left) and S750 glycerol(right) can reduce the growth rate of WT (bYS09) cells without reducing the MreB filament density. Dashed lines represent the mean of WT values. All constructs are at the native locus unless otherwise indicated. N values are available in Supplementary Table 2. Boxes indicate 25–75 percentiles, whiskers indicate 10–90 percentiles, the midline indicates median, and + indicates the mean. Error bars represent standard deviation.

a, The relative abundance of PrpC and PrkC in WT (PY79) cells grown in different media. Abundances are from the mass spectrometry datasets in Dion et al.13. These datasets included two biological replicates. Each protein abundance is normalized to the mean abundance of that protein in WT cells grown in CH. PrkC is not detected in every replicate. Dotted line represents protein abundances in CH. b, Growth rates of ΔprkC (bYS542), ΔprpC (bYS543), ectopic prkC overexpression (bYS539 with 30 mM xylose), and ectopic prpC overexpression (bYS545 with 30 mM xylose) in cells grown in S750 maltose. c, Growth rates of WT (PY79), ΔprkC (bYS542), and ectopic prkC overexpression (bYS539 with 30 mM xylose) in S750 glycerol, S750 maltose, S750 glucose, CH and LB. d, The relative abundance of YrzL, YpiB, and ClpC/P/X protease in WT (PY79) grown in different media. The abundances are from the mass spectrometry datasets in Dion et al.13. These datasets included two biological replicates. Each protein abundance is normalized to the mean abundance of that protein in WT cells grown in CH. Dotted line represents protein abundances in CH. YrzL and YpiB are not detected in every replicate. Dashed lines represent the mean of WT values. All constructs are at the native locus unless otherwise indicated. N values are available in Supplementary Table 2. Boxes indicate 25–75 percentiles, whiskers indicate 10–90 percentiles, the midline indicates median, and + indicates the mean. P-values were calculated using one-way ANOVA with multiple comparisons.

a and b, PhosTag SDS-PAGE was used to quantitate the fractional phosphorylation of HaloTag-RodZ (labeled with JF549 HaloTag during steady state growth). Separated bands were quantitated with a gel imager. a, PhosTag SDS-PAGE gel of HaloTag-RodZ fusions. Top: SDS-PAGE supplemented with PhosTag causes a band shift with HaloTag-RodZ (bYS163), but not in RodZ(S85A) (bYS165) or ΔprkC (bYS646) cells. Bottom: Regular SDS PAGE gel showing no band shift is observed. The experiment was repeated twice with similar results. b, Top: PhosTag SDS-PAGE of HaloTag-RodZ in cells ectopically overexpressing prkC (bYS643, 30 mM xylose), ectopically overexpressing prpC (bYS644, 30 mM xylose), and under different levels of murAA (bYS642) induction. Bottom: regular SDS PAGE gel, showing no second band is observed. The experiment was repeated twice with similar results. c, Spot plating assay of PY79, ΔprkC (bYS542), and ΔprpC (bYS543) cells on LB agar plates containing 25 μg/ml fosfomycin. The ability of the cells to grow on plates was examined after a 12-hour incubation at 37 °C. d-i, Growth of different strains in a shaking plate reader at 37 °C, where the indicated concentrations of antibiotics were added at time 0. Envelopes represent standard deviation. Each condition was repeated at least 3 times. Note the envelopes on some of the curves are too small to resolve. d, Bulk growth of WT (PY79) cells in CH and S750 glycerol with different concentrations of fosfomycin. e-f, Bulk growth of WT(PY79) and ΔprkC (bYS542) cells grown in CH or S750 glycerol the indicated concentrations of fosfomycin. g-i, ΔprkC (bYS542) shows growth defect relative to WT(PY79) after exposure to sub-MIC levels of antibiotics. Bulk growth curves of WT (PY79) and ΔprkC (bYS542) cells in the presence of sub-MIC (g) moenomycin (5 μg/ml), (h) cefotaxime (0.25 and 0.5 μg/ml), and (i) methilinam (2.5 μg/ml). Dashed lines represent the mean of WT values. All constructs are at the native locus unless otherwise indicated. N values are available in Supplementary Table 2. Envelopes represent standard deviation.

a, Nutrient downshifts of WT (PY79) and ΔprkC (bYS542). Cells were assayed as they were shifted from growth in CH to growth in S750 glycerol. b, c, Cell width decreases (b), and cell length increases (c) with increasing growth rate in the growth accelerated mutants. WT (bME07), RodZ(S85E) (bSKH389), overexpressing murAA (bSKH55) with 30 mM xylose, or ectopically overexpressing prkC (bSKH387) with 30 mM xylose. Unless otherwise indicated, all strains were assayed in S750 glycerol. d, Cell volume remains constant in growth accelerated mutants- Volume for each cell is calculated as (π/6) D2+ (π/4) D2 (L-D), where D is the cell width and L is the cell length. e, The interdivision time of cells decreases as the accelerated cells grow faster – Interdivision time was measured by measuring the time between Z ring constrictions in mNeonGreen-pbp2B in S750 glycerol: WT cells (bME07), cells containing RodZ(S85E) (bSKH389), cells overexpressing murAA (bSKH55) with 30 mM xylose, or cells ectopically overexpressing prkC (bSKH387) with 30 mM xylose. Dashed lines represent the mean of WT values. All constructs are at the native locus unless otherwise indicated. N values are available in Supplementary Table 2. Error bars and envelopes represent standard deviation.

Supplementary Discussions 1 and 2, Notes, Tables 1–3 and References.

Imaging of MreB filaments in different media. TIRF-SIM imaging of MreB-mNeonGreen (bYS09) cells grown in different media and imaged with TIRF-SIM microscopy at 1 s intervals using 100 ms exposures.

Single-molecule imaging of MreB-HaloTag sparsely labelled with JF549. MreB-HaloTag (bYS40) cells were grown in different media, incubated with 50 pM of HaloTag-JF549 ligand in that same media for 30 min, then washed and prepared as indicated in methods. Cells were imaged at 0.5 s intervals using TIRFM with 0.5 s exposures.

Imaging of mNeonGreen-MreC in different media. TIRF imaging of cells expressing mNeonGreen-MreC at the native locus (bYS170) grown in different media. Cells were imaged at 1 s intervals using TIRFM with 1 s exposures.

Imaging of MreB filaments during nutrient upshifts. TIRF-SIM imaging of MreB-mNeonGreen (bYS09) during a nutrient upshift from S750 maltose to CH starting at time 0. Cells were imaged at 1 s intervals with TIRF-SIM using 100 ms exposures.

Imaging of MreB filaments during different levels of murAA induction and murAA depletion. TIRF-SIM imaging of (1) MreB-mNeonGreen (bYS09), (2) MreB-mNeonGreen, Pxyl-murAA (bYS497) with different levels of induction indicated at the bottom of the panel and (3) a MurAA depletion. For the MurAA depletion, bYS497 was first grown in CH with 0.5 mM xylose, washed two times with CH, then resuspended in fresh CH media without xylose and grown for 4 h. All cells were imaged with TIRF at 1 s intervals using 300 ms exposures.

TIRF imaging shows no directional motions of single molecules of MraY, MurG, MurJ or Amj. All cells were grown in CH and imaged at 1 s intervals with TIRF using 300 ms exposures. Column 1: imaging of Phyperspank-HaloTag-MurG (bYS993) induced with 100 uM IPTG. Column 2: imaging of Pxyl-mNeonGreen-MraY (bYS453) induced with 1 mM xylose. Column 3: imaging of Pxyl-mNeonGreen-MurJ (bYS609) induced with 1 mM xylose. Column 4: imaging of Pxyl-mNeonGreen-Amj (bYS611) induced with 1 mM xylose.

Imaging of MreB filaments during MurJ and Amj depletions. Cells were grown in CH or S750 glycerol and imaged at 1 s intervals with TIRF using 300 ms exposures. Column 1: MreB-mNeonGreen (bYS09) growing in CH or S750 glycerol. Column 2: MreB-mNeonGreen, Pxyl-MurJ, Pxyl-Amj (bYS670) cells induced with 0.5 mM xylose growing in CH or S750 glycerol.

PrkC-mediated acceleration of cell growth. Phase-contrast time lapses comparing the growth of WT (PY79) cells grown in S750 glycerol to cells growing in the same media where PrkC was ectopically overexpressed (bYS539) with 30 mM xylose. Cells were imaged using phase contrast at 2 min intervals with 300 ms exposures.

TIRF imaging of HaloTag-PrkC and HaloTag-RodZ. The first series shows HaloTag-PrkC (bYS765) growing in CH and S750 glycerol. HaloTag-PrkC (bYS765) was incubated with 1 uM HaloTag-JF549 ligand for 30 min, washed and then prepared as detailed in methods. The second series shows HaloTag-RodZ (bYS163) growing in CH and S750 glycerol. HaloTag-RodZ (bYS163) was incubated with 1 uM HaloTag-JF549 ligand for 30 min, washed, then imaged. Cells were imaged using TIRFM at 1 s intervals with 0.5 s exposures.

TIRF imaging of MreB with RodZ phosphomutants. MreB-mNeonGreen (bYS09), MreB-mNeonGreen with RodZ(S85A) (bYS748) and MreB-mNeonGreen with RodZ(S85E) (bYS749) cells were grown in S750 maltose and S750 glycerol. All cells were imaged at 1 s intervals using TIRFM with 300 ms exposures.

Source data for Extended Data Fig. 1.

Source data for Extended Data Fig. 2.

Source data for Extended Data Fig. 3.

Source data for Extended Data Fig. 4.

Source data for Extended Data Fig. 5.

Unprocessed regular SDS–PAGE, PhosTag gels and western blots gels for lipid II detection.

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Sun, Y., Hürlimann, S. & Garner, E. Growth rate is modulated by monitoring cell wall precursors in Bacillus subtilis. Nat Microbiol (2023). https://doi.org/10.1038/s41564-023-01329-7

DOI: https://doi.org/10.1038/s41564-023-01329-7

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