Evidence that Listeria innocua modulates its membrane's stored curvature elastic stress, but not fluidity, through the cell cycle

Abstract

This paper reports that the abundances of endogenous cardiolipin and phosphatidylethanolamine halve during elongation of the Gram-positive bacterium Listeria innocua. The lyotropic phase behaviour of model lipid systems that describe these modulations in lipid composition indicate that the average stored curvature elastic stress of the membrane is reduced on elongation of the cell, while the fluidity appears to be maintained. These findings suggest that phospholipid metabolism is linked to the cell cycle and that changes in membrane composition can facilitate passage to the succeding stage of the cell cycle. This therefore suggests a means by which bacteria can manage the physical properties of their membranes through the cell cycle.

Figure 1

Identification of growth phases and image analysis of cultures. Panel A, Growth curve for L. innocua NCTC 11288 grown in TSB at 37 °C (n = 3 independent experimental measurements per point). The lag phase lasted for approximately 150 min (------); exponential phase (untreated cultures) began at about 150 min and by 400 min the cells were in the stationary phase. In cultures treated with RIF, stationary phase was induced immediately although some growth was still detected for ~120 min. Samples in panels B and C (untreated controls) were collected after 240 and 360 min and sample in panel C was collected 240 min after RIF administration (at 480 min). Panels B–D show light and fluorescence micrographs of L. innocua; for fluorescent imaging samples were stained with Nile red (1000 × magnification; scale bar = 1 µm). A graph of the distribution of cell lengths is shown for each sample with Gaussian distribution (red line). Data represents 180–200 cells from at least three images from four independent cultivations. The Box plots show medians, 50% of data in the range (box), non-outliers range (whiskers) and outliers (circles and stars). Sample B is representative of cells in exponential phase growth (240 min after inoculation) with the majority of cells appearing to be in the B period (>80% synchrony; mean = 1.34 µm, ± 0.29 µm). Sample C is representative of cells at the end of the exponential phase (360 min after inoculation) and represents the B period of older cultures (>80% synchrony; mean = 1.22 µm, ±0.26 µm). Sample D is representative of cells arrested at the C/D boundary by RIF (80-90% synchrony; mean = 1.92 µm, ±0.44 µm). Only 6.7% of cells in the C/D boundary samples are as short or shorter than those seen to predominate in the untreated samples (t-test, p =  < 0.01).

Figure 2

Profiling of the phospholipids in L. innocua NCTC 11288 at different stages of the cell cycle. Panel A shows representative ³¹P NMR spectra of the lipid fraction collected from B period cultures and cultures at the boundary of the C and D periods. The relative area of the integrations of the appropriate resonance(s) were taken as a fraction of the total integrations for each spectrum and used to generate the fraction size given for each sample. The shift of phosphate mono-ester-containing lipids such as PA, is pH dependent. PE exhibits several resonances due to concentration and pH-dependent solvent interactions^, . Panel B shows the abundance of lipids in the B period and at the C/D period boundary from cells collected in the exponential phase. Asterisks mark the head groups (CL, PE) for which the difference in abundance is considerable (when comparing standard deviations, n = 5 independent samples profiled using quantitative ³¹P NMR). CL, cardiolipin; lyso-PA, lyso-phosphatidic acid; lyso-PG, lyso-phosphatidylglycerol; lysyl-CL, lysyl-cardiolipin; lysyl-PG, lysyl-phosphatidylglycerol; PA, phosphatidic acid, PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PS, phosphatidylserine.

Figure 3

Stacked spectra showing the phase behaviour of two-lipid systems as a function of lipid head group composition. (A), DOPG/TOCL mixtures at 273 K; (B), DOPE/DOPG at 273 K; (C), DOPE/DOPG at 310 K; (D), DOPE/TOCL scan at 273 K. The 36:64 and 18:82 mixtures of DOPE:DOPG represent the ratio of these two lipids in the B period and at the C/D boundary, respectively. DOPG, dioleoylphosphatidylglycerol; TOCL, tetraoleoylcardiolipin; DOPE, phosphatidylethanolamine. Full temperature scans of each lipid mixture can be found in Figs S8–10.