Effects of membrane lipid composition on the organization and signalling properties of bacterial chemoreceptors
The bacterial cytoplasmic membrane is one of the most dynamic cellular structures, functioning mainly as selective permeability barrier. The aim of this thesis was to investigate effects of lipid bilayer composition on several bacterial cell properties like growth, chemosensing and motility. I mainl...
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|The bacterial cytoplasmic membrane is one of the most dynamic cellular structures, functioning mainly as selective permeability barrier. The aim of this thesis was to investigate effects of lipid bilayer composition on several bacterial cell properties like growth, chemosensing and motility. I mainly focused on the effect of cardiolipin (CL), a minor component of the bacterial cytoplasmic membrane, synthesised by three enzymes (ClsA, YbhO, YmdC) in Escherichia coli. While individual deletions of these synthase genes affected growth and partly cell morphology, the triple knockout ΔclsA ΔybhO ΔymdC grew comparably to the parental strain. Surprisingly, cardiolipin deficiency caused no or only minor growth defects under various osmotic and antibiotic stress conditions, which indicates that cardiolipin is expendable for growth of E. coli. Survival of motile microorganisms does not only depend on growth but also on their ability to sense and control their directed motility in chemical gradients. Therefore, E. coli utilizes methyl-accepting chemotaxis proteins, which are clustering at the cell poles and laterally along the cell body. We focused on the influence of membrane- dependent interactions between the receptors and the phospholipids. With this we could show that the membrane plays not only a considerable role in chemoreceptor functionality but also in clustering. We showed the reduced assembly of Tar clusters in a strain with cardiolipin deficiency, independent of temperature and growth phase. Clustering in a cardiolipin-deficient background could be restored by modification of the transmembrane domain of Tar. To investigate whether the decrease in clustering could be the consequence of reduced protein mobility, hindering the assembly process in combination with hydrophobic mismatch, we utilized FRAP microscopy that allows measurements of fluorescent protein recovery after photobleaching. We performed FRAP measurements for the major E. coli receptors, Tar and Tsr. We observed that the diffusion of Tar is significantly decelerated in the cardiolipin-deficient strain at a narrow temperature range around 18°C. At higher and lower temperature, the difference in Tar recovery in the WT and cardiolipin-deficient strain is negligible, indicating a sudden drop of Tar mobility in a critical temperature range in the cardiolipin-deficient strain. Solubilizing the membrane using benzyl alcohol could restore the effect. Additionally, the effect could also be abolished by extension of the Tar transmembrane domain. For Tsr we could not detect any membrane- dependent effect on receptor diffusion. We assume that the cardiolipin- dependent increase of Tar receptor mobility in a certain temperature range is due to protein-membrane interactions depending inter alia on the length of transmembrane domains. The connections between the cardiolipin mediated enhancement in receptor clustering and the cardiolipin-dependent increase of receptor mobility remains to be explored. Additionally, we could show, that cardiolipin is influencing the motility system of E. coli. With raising temperatures, the motility of a cardiolipin-deficient Tar-only strain increases, in contrast to the parental strain. This could be confirmed by examining the expression of the motility genes fliC and fliA. Furthermore, we examined the effect of membrane alterations on E. coli, by overexpressing various phospholipid synthases, as well as the effect of changing membrane composition in B. subtilis.