Influence of cardiolipin on the chemosensing of E. coli

To examine the effect of cardiolipin on protein interactions upon stimulation with an attractant or repellent in E. coli, we used a stimulus-dependent FRET assay.

As described before (see Material and Methods, 3.12.2), the Förster (fluorescence) Resonance Energy Transfer (FRET) assay relies on a phosphorylation-dependency of the interaction between the response regulator CheY fused to the yellow fluorescent protein (CheY-YFP) and its phosphatase CheZ fused to the cyan fluorescent protein (CheZ-CFP) (166, 268).

That enables the analysis of the intracellular pathway response towards certain chemotactic stimuli. More accurately, this assay (illustrated in Supplementary Figure S1) can be used to monitor the activity of the kinase CheA upon stimulation. We determined the amplitudes and dose-response curves by

91 continuous stimulation of the cells with increasing serial dilutions of the compound of interest, with concentrations ranging from below the limit of response to those exceeding the maximum FRET response. From obtained dose-response curves, the EC50 value was calculated corresponding to the ligand concentration triggering the half-maximum response. This value can be utilised to precisely analyse the sensitivity of certain strains (CL +/-) under different conditions (e.g.

temperature). For all experiments concentrations of 0.01 μM to 100 μM for methyl-aspartate (MeAsp), 0.01 μM to 3 mM serin, and 0.03 μM to 100 μM for NiCl2 were used. For pH-dependent experiments, a range from pH 6 to 8.3 was used. The experiments were carried out at 22°C and later also at 18°C, due to obtained protein diffusion results in FRAP experiments (see 4.1.5). Results are summed up in Figure 16, A-G.

We could show that at 22°C experimental temperature, the cardiolipin deletion mutant expressing Tar as only chemoreceptor is responding differently in the FRET assay compared to WT-Tar (Figure 16, A). The response curve of the cardiolipin knockout strain seems not to be monotonic but biphasic. The response amplitude of the mutant is smaller than the response amplitude of the Tar+ WT strain at attractant concentrations higher than 3 μM MeAsp. For lower concentrations, the mutant strain lacking cardiolipin (CL- Tar+) is significantly more sensitive. For attractant levels, lower than 0.1 μM MeAsp the WT-Tar is not responding whereas the CL- Tar+ is still sensing. The Tar+ strain has an EC50 of 2 μM MeAsp, and the CL- deletion Tar+ mutant exhibits an EC50 of 0.6 μM MeAsp. The time the cells need to adapt to certain different attractant (MeAsp) concentrations are shown in Figure 16, C. For example, for an attractant concentration of 1 μM MeAsp the Tar+ strain needs around 400 seconds to adapt, whereas the CL- Tar+ mutant is adapting in roughly 200 seconds. The differences in adaptation time might result from different response intensities. This can be seen in Figure 16, D, where the adaptation time is plotted against the relative response of the Tar receptor towards MeAsp. At smaller response values until approximately 0.6, the cardiolipin knockout mutant adapts faster than the WT with unmodified membrane composition, whereas at relative response values higher than 0.6 up to 0.9 the WT adapts faster. At a relative Tar response of 1, the WT needs twice as long to adapt than the cardiolipin deficient mutant. However,

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further experiments regarding the adaptation modalities would be necessary to obtain more precise insights into the fluctuation adaptation behaviour of the cardiolipin knockout strain. So far our results indicate a slightly better sensitivity of Tar in the cardiolipin deficient strain, especially at lower attractant concentration. As we could detect a difference in attractant sensing of the Tar receptor, we also tested the influence of cardiolipin on the repellent sensing using NiCl2 (Figure 16, E) as ligand. Initial experiments show a continuously smaller response amplitude of the cardiolipin lacking mutant compared to the WT. The WT is sensing the repellent in a low nanomolar range, whereas the mutant starts sensing in a range higher than 1μM. The EC50 values are 1.3 μM for the WT and 2.7 μM for the mutant. To get further insights into the effect of cardiolipin on the chemosensing behaviour of Tar, we carried out further experiments, examining the receptor response towards step-wise changes of the external pH (Figure 16, F). Generally, the pH-taxis of E. coli used to obviate acidic or basic external conditions likely depends on the opposing pH sensing behaviour of the two main chemoreceptors Tar and Tsr (3, 4). Previous observations reported an attractant response of Tar for more acidic pH values lower than seven and a repellent response for basic pH levels higher than 7 (4-6). Throughout the attractant as well as the repellent side of our FRET experiment, the amplitudes of the cardiolipin deficient strain are smaller. However, these preliminary results do not seem to be conclusive when comparing to the results with attractant (MeAsp) and repellent (NiCl2).

After detecting the influence of cardiolipin on the Tar receptor, we were additionally testing the influence of cardiolipin on the second main receptor in E.

coli, Tsr. With our experiments, we could show that for Tsr no significant difference in attractant response is detectable (figure 16, G). The EC50 values are here 30 μM serin for Tsr+ and 27 μM serin for the CL null Tsr+ mutant. These results are leading to the idea that cardiolipin has an influence on the Tar receptor but not on the Tsr receptor of E. coli.

As described earlier, the chemoreceptors constitute an essential part of the bacterial chemotaxis system and are located in the cytoplasmic membrane. It is known that lipid bilayers, such as the cytoplasmic membrane, are

temperature-93 dependent regarding their fluidity and phase transition (7). Corresponding results to the temperature dependency of the interplay between the cell membrane fluidity and the chemoreceptors diffusion are shown later (Results, 4.1.5). In this context, we repeated the Tar+ FRET experiments at a slightly lower experimental temperature of 18°C, to investigate the influence of this temperature shift on the sensing abilities of the receptor. The results are shown in Figure 16, B. In contrast to the results at 22°C, here the curve of the CL- strain curve is continuously below the WT one. This means the response amplitudes are generously smaller compared to the cardiolipin containing WT. Additionally, the cardiolipin-deficient strain is less sensitive at lower concentrations of MeAsp. At 18°C, the Tar receptor expressed in the CL- strain is not sensing MeAsp concentrations lower than 0.1 μM, whereas the WT starts sensing already at this concentration level.

The EC50 of the WT Tar+ strain is 0.32 μM, for the CL- Tar+ strains its 0.67 μM MeAsp. Comparing the two temperatures, some differences in Tar response can be detected. For WT as well as for the CL- strain at 18°C, the response amplitude is in the maximum, at 50% of the response amplitude at 22°C. Additionally, at 22°C, it seems the CL- strain is with regards to the Tar response more sensitive at lower MeAsp concentration. However, at 18°C, the cardiolipin deficient strain is throughout the response curve less sensitive and showing lower amplitudes, compared to the corresponding WT.

With our experiments, we could show that the attractant sensing of Tar can be affected by membrane composition and the external temperature additionally influencing the membrane fluidity. A temperature shift leads to a difference in response amplitudes and also the sensitivity changes.

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Figure 16. Chemotaxis in E. coli. FRET amplitude response curves of E. coli WT (grey) and E.

coli ∆clsAybhOymdC (green) expressing Tar as only chemoreceptor at 22°C (A) and 18°C (B).

Each point represents the values of response amplitudes, measured from the buffer-baseline, for at least three independent experiments. Error bars indicate the standard error. Preliminary adaptation curves of E. coli WT (grey) and E. coli ∆clsAybhOymdC (green) to MeAsp(C). Plotted adaptation time against amplitudes for E. coli WT (grey) and E. coli ∆clsAybhOymdC (green), one biological replicate (D). Measures response amplitudes of E. coli WT (grey) and E. coli

∆clsAybhOymdC (green) to NiCl2, one biological replicate (E). Measures response amplitudes of E. coli WT (grey) and E. coli ∆clsAybhOymdC (green) towards pH, one biological replicate for the WT, three biological replicates for CL-. Error bars indicate standard error (F). Dose response curve of E. coli WT (grey) and E. coli ∆clsAybhOymdC (green) expressing Tsr from plasmid towards serine (G). Each point represents the mean kinase activity, normalized by the baseline in buffer, from three biological replicas for the WT and one replicate CL-, error bars indicating the standard error.