Heterogeneous localisation of membrane proteins in Staphylococcus aureus

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.

Organization of Peptidoglycan Synthesis in Nodes and Separate Rings at Different Stages of Cell Division ofStreptococcus pneumoniae

“…9B). Recent notable studies have demonstrated heterogeneous supramolecular localization of membrane proteins generally and at septa of Sau cells, including interacting enzymes that catalyze phospholipid biosynthesis and the MreD regulator of PG synthesis (56,57). This heterogenous localization results in punctate patterns of these proteins at division septa, resembling the localization of Spn PBPs reported here (Fig.…”

“…2 and S2). Polytopic MreD plays a role in supramolecular localization of the phospholipid biosynthesis enzymes, and heterogeneous punctate patterns are lost upon protein overexpression (57). A favored explanation for punctate supramolecular organization is that membrane protein complexes distort local curvature on membranes and thereby perturb diffusion, resulting in distribution patterns for the majority of membrane proteins (57).…”

Recent evolution of a TET-controlled and DPPA3/STELLA-driven pathway of passive DNA demethylation in mammals

“…To quantify this observation, we calculated the coefficient of variation (CV) of nuclear UHRF1-GFP among wt, Dppa3KO, and T12CM ESCs. The CV of a fluorescent signal correlates with its distribution, with low CV values reflecting more homogenous distributions and high CV values corresponding to more heterogeneous distributions 86,87 . Indeed, the pronounced focal accumulation of UHRF1-GFP observed in Dppa3KO and T12CM ESCs corresponded with a highly significant increase in the CV values of nuclear UHRF1-GFP compared with wt ESCs ( Supplementary Fig.…”

SRRF: Universal live-cell super-resolution microscopy

“…1 Hz). Laser-based SRRF microscopy of conventional fluorophores has also been demonstrated in fixed bacteria for multi-colour imaging in B. subtilis ( Vega-Cabrera et al, 2017 ) and z-stack imaging of membrane proteins in S. aureus ( Weihs et al, 2018 ).…”