2006. with peptides in early logarithmic growth phase. A minimum of 50 cells were examined per condition. (B) Unstained membrane areas are not representative of damaged membranes. SIM photos of bSS82 expressing intracellular GFP and stained with Nile reddish are demonstrated. Arrows show unstained membrane domains in cells retaining intracellular GFP, showing that unstained membrane domains are not representative of damaged membranes. Cells were cultivated until early exponential growth phase and treated with 1 MIC of tyrocidines A and C, respectively. Photos were taken after 10 min of treatment. Pub, 2 m. (C) Quantification of fluid membrane patches. 168 was cultivated until early exponential growth phase and treated with 1 MIC of tyrocidines A and C, respectively. Membranes were stained with FM5-95. A minimum of 300 cells were examined per condition. (D) Formation of fluid membrane patches is definitely self-employed of protein and lipid synthesis. 168 was treated with inhibitory concentrations of either chloramphenicol (100 g/ml) or triclosan (2.5 g/ml) for 10 min to inhibit synthesis of proteins or lipids, respectively. Cells were then treated with peptides for an additional 10 min. Membranes were stained with FM5-95. Arrows show membrane patches. Download FIG?S3, TIF file, 1.6 MB. Copyright ? 2018 Wenzel et al. This content is distributed under the terms of the Creative Commons Attribution 4.0 International license. FIG?S4. Fluid lipid clustering does not require MreB. (A) Lipid patches are self-employed of MreC (strain 3481), the membrane-anchoring protein of MreB. (B) Lipid patches are self-employed of MreB. MreB organizes RIFs in mutant (strain 4277). Naturally happening RIFs are indicated by packed arrows; fluid lipid accumulations (clustered RIFs) are indicated by open arrows. Bars, 2 m. All experiments were performed in early exponential growth phase. Download FIG?S4, TIF file, 0.6 MB. Copyright ? 2018 Wenzel et al. This content is distributed under the terms of the Creative Commons Attribution Rabbit Polyclonal to NRIP2 4.0 International license. TEXT?S1. MreB is not required to establish RIFs. Download Text S1, DOCX file, 0.0 MB. Copyright ? 2018 Wenzel et al. This content is distributed Walrycin B under the terms of the Creative Commons Attribution 4.0 International license. FIG?S5. Formation of RIFs depends on the growth phase and not on the presence of MreB. (A) Growth of 168 (crazy type [WT], green) and 4277 (4277 (50). At this time point, the WT has an OD of approximately 0.25 (exponential phase) while the triple mutant has an OD of approximately 0.15 (lag phase). Time points at which microscopically visible RIFs were observed are designated with an asterisk. (B) DiIC12 microscopy of 168 (WT) and 4277 (mutant compared to the 168 WT and mutant. The mutant develops slightly faster than the strain, providing an explanation of why RIFs were observed with the strain but not with the strain in our earlier study (50). Cells were cultivated at 30C in LB supplemented with 20 mM MgCl2. Download FIG?S6, TIF file, 0.1 MB. Copyright ? 2018 Wenzel et al. This content is distributed under the terms of the Creative Commons Attribution 4.0 International license. FIG?S7. 31P solid-state NMR spectra of model bilayers. POPG-POPE (A) (at 3:1) or POPE (B) bilayers were mechanically oriented with the normal parallel to the magnetic field direction and equilibrated at 93% relative moisture in the absence and presence of 2% tyrocidine A, 2% tyrocidine C, and 2% gramicidin S in the indicated temps. In the absence of peptide, the 31P spectra exhibited a predominant maximum at 30 ppm (310 K), indicative of a good alignment of the phospholipid head groups. This value shifted to 35 ppm below the phase transition temperature of the lipid combination (290 K). At intermediate temps, two 31P NMR peaks are distinguishable, suggesting the coexistence of liquid crystalline and gel-phase lipid domains. The presence of tyrocidine A Walrycin B at a lipid-to-peptide percentage of 50 was found to have minimal effects within the bilayer orientation with only a small increase in the nonoriented spectral intensities extending up to ?11 ppm. These are indicative of small rearrangements at Walrycin B the level of the phospholipid head groups or due to loss of orientational order of lipid membrane domains. The transition to the gel phase was lowered by 5 to 10 K, suggesting the peptide stabilizes the liquid crystalline phase. Whereas the spectral collection boarding is similar in the presence of tyrocidine C and gramicidin S, the effect of these peptides within the phase transition temp was less pronounced. The 31P solid-state NMR signals that arise from your liquid crystalline and gel phase were then integrated and compared.