An Stomatin, Prohibitin, Flotillin, and HflK/C-Domain Protein Required to Link the Phage-Shock Protein to the Membrane in Bacillus subtilis

Abstract

Membrane surveillance and repair is of utmost importance to maintain cellular integrity and allow cellular life. Several systems detect cell envelope stress caused by antimicrobial compounds and abiotic stresses such as solvents, pH-changes and temperature in bacteria. Proteins containing an Stomatin, Prohibitin, Flotillin, and HflK/C (SPFH)-domain, including bacterial flotillins have been shown to be involved in membrane protection and membrane fluidity regulation. Here, we characterize a bacterial SPFH-domain protein, YdjI that is part of a stress induced complex in Bacillus subtilis. We show that YdjI is required to localize the ESCRT-III homolog PspA to the membrane with the help of two membrane integral proteins, YdjG/H. In contrast to classical flotillins, YdjI resides in fluid membrane regions and does not enrich in detergent resistant membrane fractions. However, similarly to FloA and FloT from B. subtilis, deletion of YdjI decreases membrane fluidity. Our data reveal a hardwired connection between phage shock response and SPFH proteins.

Keywords: Bacillus subtilis; SPFH-domain proteins; YdjI; cell envelope stress response; flotillins; phage-shock protein.

FIGURE 1

(A) Schematic overview of the pspA-ydj-operon. σ^W-dependent promotor PM8J2-64 starts transcription at 671,209(+); transcription starts with pspA, continuing with ydjG, ydjH and subsequently ydjI. (B) SPFH-proteins in B. subtilis and their domains. (C) Proteins of the ydj-operon and their protein-domains.

FIGURE 2

YdjI and PspA intracellular localization. Morphology of the exponentially growing strains labeled with Nile Red in unstressed (upper panel) and in alkali stress conditions (15 mM NaOH), lower panel. (A) YdjI-mNeonGreen and (B) PspA-GFP. Scale bars: 3 μm.

FIGURE 3

Co-localization analysis of PspA and YdjI proteins. (A) Micrographs showing exponentially growing cells of expressing YdjI-mCherry2 and PspA-GFP in alkali stress condition (15 mM NaOH). Arrows indicate co-localized foci. Scale bar: 4 μm. (B) Scattered dot plots of Pearson’s correlation (R values) assessed by Coloc-2 plugin from FIJI. Red to green (YdjI:PspA) and green to red (PspA:YdjI) co-localization analysis are shown. Error bars represent mean and standard deviation of values calculated for 24 cells. (C) Foci population composition in percentage of cells (n = 1650). (D) Ratio of total number of foci in each channel per total of cells (n = 1650).

FIGURE 4

YdjI localizes into fluid membrane regions and does not co-localize with FloT. (A) YdjI co-localizes with fluid membrane regions as indicated by co-localization with Dil-C12 staining (arrows). (B) Localization of YdjI and FloT are independent. Micrograph show no localization of FloT and YdjI in a double tagged strain floT-mNeonGreen; ydjI-mcherry. Cells are in exponentially growing phase under unstressed or alkali stress conditions (15 mM NaOH). Scale bars: 3 μm. (C) Pearson’s co-localization analysis (n > 20) and fluorescence intensity correlation graphs (right panel) are shown for the YdjI and Dil-C12 localization. The graph displays a pixel by pixel intensity correlation between Dil-C12 and YdjI-mNeonGreen fluorescence. (D) Pearson’s co-localization analysis (n > 20) and fluorescence intensity correlation graphs are shown for the YdjI-mCherry and FloT-mNeonGreen localization. The graph on the right displays a pixel by pixel intensity correlation between FloT-mNeonGreen and YdjI-mCherry fluorescence. (E) Localization of YdjI in the detergent soluble membrane fraction (DSM). DSM/DRM extraction using strains expressing YdjI-mCherry (ASB049) and FloT-mCherry (ASB044) and subsequent immunoblotting using anti-mCherry antibodies is shown.

FIGURE 5

YdjI is required for PspA membrane anchoring and complex formation. (A) PspA-GFP foci are shown in unstressed and alkali stress conditions. (B) Deletion of ydjI delocalize PspA and the cells show no PspA foci in both conditions. Scale bars: 4 μm (C) ydji-mNeonGreen is shown under unstressed or stressed conditions (15 mM NaOH). (D) Deletion of pspA leads to YdjI overexpression. YdjI foci are still formed (arrows). Scale bars: 3 μm.

FIGURE 6

YdjG and YdjH serve as membrane anchor for PspA-YdjI protein complex and seem to have a role in cell-shape maintenance. Deletion of (A) ydjG and (B) ydjH delocalize YdjI foci formation under both unstressed and alkali stress conditions. The same is shown for PspA delocalization upon deletion of (C) ydjG and (D) ydjH. Arrows indicate a strong cell-shape defect upon deletion of either YdjG or YdjH, under stress conditions. Scale bars: 3 μm. (E) Interactions of proteins encoded in the pspA-ydjGHI operon via Bacterial-2-Hybrid. Blue colonies indicate protein–protein interaction, while in white colonies the target proteins do not interact. The positive and negative controls are marked by a plus minus, respectively. In pUT18C and pKT25 plasmids the T18 or T25 fragment of the adenylate cyclase is N-terminally fused to the protein of interest. pUT18 and pKNT25 encode for a C-terminal fusion to the protein of interest. The diagram represents a summary of the B2H analysis. The size of the arrow indicates the intensity of the blue color. Dashed lines indicate weaker protein–protein interactions.

FIGURE 7

MreB is not required for YdjI and PspA foci formation. (A) YdjI localization in unstressed and alkaline stressed conditions. (B) PspA localization in unstressed and alkaline stressed conditions. (C) YdjI localization in a mreB mutant background. Note that in stressed and unstressed conditions YdjI foci are seen. (D) Upon deletion of the cytoskeletal protein mreB, PspA still localizes in membrane associated foci. Foci numbers in strains lacking mreB are reduced (see also Table 1). (E) Generalized polarization analysis after Laurdan staining of WT 168, MreB, and YdjI mutant strain are shown (n > 100); deletion of mreB causes increase in membrane fluidity, whereas YdjI deletion increases membrane rigidity. Scale bars 4 μm.