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. 2018 Feb 26;8(1):3657.
doi: 10.1038/s41598-018-21750-x.

Heterogeneous localisation of membrane proteins in Staphylococcus aureus

Felix Weihs  1 , Katarzyna Wacnik  1 , Robert D Turner  1 , Siân Culley  2   3 , Ricardo Henriques  2   3 , Simon J Foster  4
Affiliations

Heterogeneous localisation of membrane proteins in Staphylococcus aureus

Felix Weihs et al. Sci Rep. .

Abstract

The bacterial cytoplasmic membrane is the interface between the cell and its environment, with multiple membrane proteins serving its many functions. However, how these proteins are organised to permit optimal physiological processes is largely unknown. Based on our initial findings that 2 phospholipid biosynthetic enzymes (PlsY and CdsA) localise heterogeneously in the membrane of the bacterium Staphylococcus aureus, we have analysed the localisation of other key membrane proteins. A range of protein fusions were constructed and used in conjunction with quantitative image analysis. Enzymes involved in phospholipid biosynthesis as well as the lipid raft marker FloT exhibited a heterogeneous localisation pattern. However, the secretion associated SecY protein, was more homogeneously distributed in the membrane. A FRET-based system also identified novel colocalisation between phospholipid biosynthesis enzymes and the respiratory protein CydB revealing a likely larger network of partners. PlsY localisation was found to be dose dependent but not to be affected by membrane lipid composition. Disruption of the activity of the essential cell division organiser FtsZ, using the inhibitor PC190723 led to loss of PlsY localisation, revealing a link to cell division and a possible role for FtsZ in functions not strictly associated with septum formation.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
The localisation of PlsY-GFP is dose-dependent. (a) Growth curves (left Y-axis) of S. aureus SH1000 FW6 (IPTG-inducible plsY-gfp expression) and fluorescence (right Y-axis) of whole culture samples. 50 or 500 µM IPTG were added to cultures at an OD600 ~ 1 and samples were analysed at 30 min intervals. A.U. fluorescence for 50 (formula image) and 500 (formula image) µM IPTG induction. OD600 for 50 (formula image) and 500 (formula image) µM IPTG induction. (b) Fluorescence images (convolved and deconvolved) of S. aureus SH1000 FW6 with different expression levels of plsY-gfp as seen in (a). Scale bars represent 1 µm. (c) CV-factor calculation of deconvolved fluorescence images of PlsY-GFP (FW6). Significance values were calculated using a two-tailed unpaired student t-test. ****P < 0.0001; ***P < 0.001.
Figure 2
Figure 2
Phospholipid synthesis enzymes are distributed heterogeneously in the membrane of S. aureus. (a–c) Phase contrast and fluorescence images (deconvolved) of S. aureus expressing plsY-eyfp (FW1), pgsA-eyfp (FW2) and cls2-eyfp (FW5) under their native promoter. Cells were counterstained with the fluorescent D-amino acid HADA for 5 min, which is incorporated into the cell wall indicating the cell-cycle stage. (d) Fluorescence images (deconvolved) of Cls2-eYFP in S. aureus SH1000 FW5 at the upper and lower end of the cell focus showing the localisation of Cls2-eYFP on different three-dimensional levels. The cartoon schematically illustrates the distribution of Cls2-eYFP at the base of the septum. All scale bars represent 1 µm.
Figure 3
Figure 3
Membrane proteins in S. aureus show different localisation profiles. (a) Phase contrast and fluorescence images (deconvolved) of S. aureus SH1000 expressing floT-eyfp (FW8) or secY-gfp (JGL231) under their native promoter. Cells were counterstained with HADA for 5 min. (b) CV-factor calculation of deconvolved images of PlsY-GFP (JGL232), FloT-eYFP (FW8) and SecY-GFP (JGL231). Significance values were calculated against PlsY-GFP using a two-tailed unpaired student t-test. ***P < 0.001. (c) Colocalisation studies of PlsY-GFP with a range of membrane proteins translationally fused to mCherry in S. aureus RN4220 (strains FW14-FW20). Fusions were expressed from an IPTG-inducible plasmid and fluorescence images were deconvolved. White arrows indicate matching foci of fluorescence signals while red arrows show non-matching signal foci. All scale bars represent 1 µm.
Figure 4
Figure 4
Phospholipid synthesis enzymes, MreD and CydB interact with PlsY. (a) FRET efficiencies calculated using a donor photo bleaching FRET system. All investigated strains expressed plsY-gfp together with a protein of interest translationally fused to mCherry (strains FW14-FW20). Significance values were calculated using a two-tailed unpaired student t-test. **P < 0.01. The interaction analyses of PlsY with MreD, CdsA, SecY and MscL were shown previously. (b) FRET efficiencies of protein interactions on a subcellular level of PlsY with MreD, CydB or SecY. Non-dividing and dividing cells were analysed. In addition, dividing cells were further dissected into the septum and periphery. In the case of interactions with SecY some negative FRET efficiency values were calculated. We do not, of course, claim a true negative FRET efficiency, simply that the donor fluorophore bleached more rapidly in these experiments. It was necessary to include these results for completeness.
Figure 5
Figure 5
Role of the cell wall in membrane protein localization. (a) Phase contrast and fluorescence images (deconvolved) of E. coli C43(DE3) with episomal IPTG-induced expression of mreD-eyfp (E. coli C43(DE3) mreD-eyfp). (b) Phase contrast and fluorescence images (deconvolved) of spheroplasts of E. coli C43(DE3) mreD-eyfp. (c) CV-factor calculation of deconvolved images of MreD-eYFP in E. coli C43(DE3) mreD-eyfp rods and spheroplasts. (d) Fluorescence images (deconvolved) of protoplasted and native cells of S. aureus SH1000 JGL232 (plsY-gfp). White arrows indicate septal PlsY-GFP localisation, which is not seen in protoplasts. (e) CV-factor calculation of deconvolved images of PlsY-GFP in native cells and protoplasts of S. aureus SH1000 JGL232 (plsY-gfp) (based on 10 cells for each group). All significance values were calculated using a two-tailed unpaired student t-test. ***P < 0.001. All scale bars represent 1 µm.
Figure 6
Figure 6
Inhibition of FtsZ disrupts the localisation pattern of PlsY. (a) Fluorescence images (deconvolved) of: FW21, S. aureus SH1000 plsY-eyfp in CL-deficient background (∆cls1/2); FW22, S. aureus SH1000 plsY-eyfp in LPG-deficient background (∆mprF): FW23, S. aureus SH1000 plsY-eyfp in WTA-deficient background (∆tarO); JGL232 (S. aureus SH1000 plsY-eyfp) treated with the FtsZ inhibitor PC190723 and the squalene-synthase inhibitor zaragozic acid. Scale bars represent 1 µm. (b) CV-factor calculation of deconvolved images of all investigated groups. Significance values against the untreated group were calculated using a two-tailed unpaired student t-test. ****P < 0.0001; *P < 0.05.
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