Stress-Induced Reorganization of the Mycobacterial Membrane Domain

Abstract

Cell elongation occurs primarily at the mycobacterial cell poles, but the molecular mechanisms governing this spatial regulation remain elusive. We recently reported the presence of an intracellular membrane domain (IMD) that was spatially segregated from the conventional plasma membrane in Mycobacterium smegmatis The IMD is enriched in the polar region of actively elongating cells and houses many essential enzymes involved in envelope biosynthesis, suggesting its role in spatially restricted elongation at the cell poles. Here, we examined reorganization of the IMD when the cells are no longer elongating. To monitor the IMD, we used a previously established reporter strain expressing fluorescent IMD markers and grew it to the stationary growth phase or exposed the cells to nutrient starvation. In both cases, the IMD was delocalized from the cell pole and distributed along the sidewall. Importantly, the IMD could still be isolated biochemically by density gradient fractionation, indicating its maintenance as a membrane domain. Chemical and genetic inhibition of peptidoglycan biosynthesis led to the delocalization of the IMD, suggesting the suppression of peptidoglycan biosynthesis as a trigger of spatial IMD rearrangement. Starved cells with a delocalized IMD can resume growth upon nutrient repletion, and polar enrichment of the IMD coincides with the initiation of cell elongation. These data reveal that the IMD is a membrane domain with the unprecedented capability of subcellular repositioning in response to the physiological conditions of the mycobacterial cell.IMPORTANCE Mycobacteria include medically important species, such as the human tuberculosis pathogen Mycobacterium tuberculosis The highly impermeable cell envelope is a hallmark of these microbes, and its biosynthesis is a proven chemotherapeutic target. Despite the accumulating knowledge regarding the biosynthesis of individual envelope components, the regulatory mechanisms behind the coordinated synthesis of the complex cell envelope remain elusive. We previously reported the presence of a metabolically active membrane domain enriched in the elongating poles of actively growing mycobacteria. However, the spatiotemporal dynamics of the membrane domain in response to stress have not been examined. Here, we show that the membrane domain is spatially reorganized when growth is inhibited in the stationary growth phase, under nutrient starvation, or in response to perturbation of peptidoglycan biosynthesis. Our results suggest that mycobacteria have a mechanism to spatiotemporally coordinate the membrane domain in response to metabolic needs under different growth conditions.

Keywords: Mycobacterium; cell envelope; membrane proteins; membranes; peptidoglycan; stress response.

FIG 1

The IMD redistributes during stationary phase. (A) Fluorescence live imaging of the dual IMD marker strain expressing HA-mCherry-GlfT2 (mCh-GlfT2) and Ppm1-mNeonGreen-cMyc (Ppm1-mNG). Cells show reorganization of the IMD during stationary phase. Cell density (the OD₆₀₀) is shown in parentheses to indicate the growth stage. Scale bar, 5 µm. (B) Western blotting detection of IMD markers after sucrose gradient sedimentation of stationary-phase cell lysate (48 h), demonstrating the biochemical isolation of the IMD. IMD markers were HA-mCherry-GlfT2 (anti-HA, 100 kDa), Ppm1-mNeonGreen-cMyc (anti-c-Myc, 59 kDa), and PimB′ (anti-PimB′, 42 kDa). The PM-CW marker was MptA (anti-MptA, 54 kDa). Note that the apparent molecular mass of MptA based on the SDS-PAGE was ~43 kDa (28). (C) Lengths of cells from logarithmic-phase growth (18 h) to stationary-phase growth (38, 63, and 88 h), showing the production of short cells. The black lines indicate averages of 202 cells. (D) Quantification of alkDADA incorporation normalized to cell sizes of stationary-phase cells (OD > 3.0), showing decreased de novo PG synthesis. The black lines indicate averages of 50 cells. *, P < 0.001.

FIG 2

The IMD reorganizes along the sidewall of the cell body during PBST starvation. (A) Fluorescence live imaging of the dual IMD marker strain expressing HA-mCherry-GlfT2 (mCh-GlfT2) and Ppm1-mNeonGreen-cMyc (Ppm1-mNG). Exposure of actively growing cells to PBST led to the redistribution of the IMD. Scale bar, 5 µm. (B) Differences in polar fluorescence enrichment between actively growing (log) and 48-h-starved (PBST) cells. Polar enrichment was calculated as the ratio of mean fluorescence intensities between the polar cap and the sidewall region. The black lines indicate the average of 201 cells. (C) Western blotting detection of IMD proteins from sucrose gradient sedimentation demonstrated the biochemical isolation of the IMD in cells starved in PBST for 30 h. IMD markers were HA-mCherry-GlfT2 (anti-HA, 100 kDa), Ppm1-mNeonGreen-cMyc (anti-c-Myc, 59 kDa), and PimB′ (anti-PimB′, 42 kDa). The PM-CW marker was MptA (anti-MptA, 54 kDa). (D) Quantification of alkDADA incorporation, normalized to cell sizes of cells after 30 h of PBST starvation, showing decreased de novo PG synthesis. The black lines indicate the averages of 50 cells. *, P < 0.001.

FIG 3

Inhibition of PG synthesis by DCS leads to reorganization of the IMD. (A) Fluorescence microscopy images of the dual IMD marker strain expressing HA-mCherry-GlfT2 (mCherry-GlfT2) and Ppm1-mNeonGreen-cMyc (Ppm1-mNG). DCS treatment led to the relocalization of the IMD from polar to sidewall enrichment. Scale bar, 5 µm. (B) The cap-to-sidewall ratio of two marker proteins in cells treated with DCS for 6 h compared with cells before treatment (log), quantitatively demonstrating reorganization from the polar cap to sidewall enrichment. The black lines indicate the averages of 218 cells. (C) Western blotting detection of IMD proteins (Ppm1-mNeonGreen-cMyc, 59 kDa; HA-mCherry-GlfT2, 100 kDa; PimB′, 42 kDa) and the PM-CW protein (MptA, 54 kDa) which were separated by sucrose density gradient sedimentation, illustrating enrichment in IMD fractions after 8 h of DCS treatment. *, P < 0.001.

FIG 4

Genetic inhibition of PG synthesis leads to redistribution of the IMD. (A) Western blot detection of IMD proteins (mTurquoise-GlfT2-FLAG [mTurQ-GlfT2], 100 kDa; PimB′, 42 kDa), and PM-CW protein (MptA, 54 kDa) in the DAP auxotroph (strain mc²1620) expressing a fluorescent IMD marker (mTurQ-GlfT2-FLAG). The IMD fractions (4 to 6) show enrichment of both mTurQ-GlfT2-FLAG and PimB′ when grown with DAP-supplemented medium. Arrowhead, a degradation product which continues to associate with the IMD fraction. (B) Fluorescence microscopy images, demonstrating the relocalization of the IMD from polar to sidewall enrichment over 11 h of DAP deprivation. Scale bar, 5 µm. (C) Quantitative comparison of cells before treatment (log) and those after 10 h of DAP depletion [DAP(−)], demonstrating reorganization of mTruQ-GlfT2-FLAG from polar to sidewall enrichment. The black lines indicate the averages of 204 cells. (D) Western blotting detection of IMD proteins. Cells were starved for DAP for 10 h, and lysate was separated by sucrose density gradient fractionation. Arrow, PimB′. A band below PimB′ of nonspecific binding was occasionally seen under stress conditions. *, P < 0.001.

FIG 5

Polar enrichment of the IMD is restored after starvation and correlates with the initiation of PG synthesis and cell elongation. (A) Fluorescence live imaging of logarithmically growing (log) and starved cells (PBST, 30 h) immediately after cell surface staining with amine-reactive Alexa Fluor 488 fluorescent dye (0 h) or after being grown in fresh medium for 3 h. The single IMD marker strain expressing HA-mCherry-GlfT2 alone was used. Polar regions unstained by the amine-reactive dye indicate the areas of cell envelope growth during the 3-h growth period. Scale bar, 5 µm. (B) Cell envelope elongation after a 3-h recovery period, demonstrating that greater elongation is observed in actively growing cells (log) than in the cells recovering from PBST starvation. The black lines indicate the averages of 204 cells. (C) Kymograph from time-lapse imaging of the dual IMD marker strain expressing HA-mCherry-GlfT2 and Ppm1-mNeonGreen-cMyc and starved in PBST for 6 h and recovered in fresh Middlebrook 7H9 medium, showing recovery of the polar IMD after ~4 h of medium replacement and subsequent cell growth. A representative cell is shown, and others are shown in Fig. S6. The darkest blue (lower right) demarks areas of the graph beyond the length of the cell at that time point. (D) Polar enrichment (cap/sidewall ratio) of the IMD (mCherry-GlfT2) and PG synthesis (alkDADA) increases at the 4-h recovery time point after 6 h of starvation in PBST. The single IMD marker strain expressing HA-mCherry-GlfT2 was used. Representative images are also shown in Fig. S6. The black lines indicate the averages of 201 cells. (E) SIM images of the single IMD marker strain recovering from 6 h of PBST starvation, demonstrating the polar PG synthesis (alkDADA) and the IMD enrichment (mCh-GlfT2) slightly subpolar and adjacent to the PG synthesis after 4 h of recovery. *, P < 0.001; ns, not significant. Scale bar, 5 µm.