The Main (Glyco) Phospholipid (MPL) of Thermoplasma Acidophilum
Total Page：16
File Type：pdf, Size：1020Kb
International Journal of Molecular Sciences Review The Main (Glyco) Phospholipid (MPL) of Thermoplasma acidophilum Hans-Joachim Freisleben 1,2 1 Goethe-Universität, Gustav-Embden-Zentrum, Laboratory of Microbiological Chemistry, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany; [email protected] 2 Universitas Indonesia, Medical Research Unit, Faculty of Medicine, Jalan Salemba Raya 6, Jakarta 10430, Indonesia Received: 19 September 2019; Accepted: 18 October 2019; Published: 21 October 2019 Abstract: The main phospholipid (MPL) of Thermoplasma acidophilum DSM 1728 was isolated, purified and physico-chemically characterized by differential scanning calorimetry (DSC)/differential thermal analysis (DTA) for its thermotropic behavior, alone and in mixtures with other lipids, cholesterol, hydrophobic peptides and pore-forming ionophores. Model membranes from MPL were investigated; black lipid membrane, Langmuir-Blodgett monolayer, and liposomes. Laboratory results were compared to computer simulation. MPL forms stable and resistant liposomes with highly proton-impermeable membrane and mixes at certain degree with common bilayer-forming lipids. Monomeric bacteriorhodopsin and ATP synthase from Micrococcus luteus were co-reconstituted and light-driven ATP synthesis measured. This review reports about almost four decades of research on Thermoplasma membrane and its MPL as well as transfer of this research to Thermoplasma species recently isolated from Indonesian volcanoes. Keywords: Thermoplasma acidophilum; Thermoplasma volcanium; tetraether lipid; main phospholipid; black lipid membrane; Langmuir-Blodgett monolayer; liposomes; proton permeability; bacteriorhodopsin reconstitution; light-driven ATP synthesis 1. Introduction The scientific history of Thermoplasma species started in 1970, when Darland and co-workers isolated a “thermophilic, acidophilic mycoplasm” from a self-heating coal refuse pile [1]. Thermoplasma acidophilum generated its own micro-environment of sulfuric acid at pH 1–2 and 55–56 ◦C by oxidative degradation of pyrite-containing material and was grouped into the genus Mycoplasma because it lacks a cell wall. Hence, the cytoplasmic membrane must be sufficiently resistant towards the extreme environmental milieu by its unique membrane components, mainly tetraether lipid (TEL) [2]. It took some more years, until Archaebacteria were introduced [3], the phylogeny of the mycoplasmas re-analyzed [4], and Thermoplasma acidophilum re-grouped into the Archaeal order of Thermoplasmatales, family Thermoplasmataceae, where it was first the only species, later accompanied by T. volcanium [5]. Physico-chemical characterization of the membrane lipids of Thermoplasma (as reported here) was mainly performed at the Goethe-University Frankfurt am Main with a laboratory stem DSM 1728 Göttingen, Germany. This research was transferred to Indonesia [6–8], since Thermoplasma and other Archaeal species had been found in Indonesian volcanoes [9]. Meanwhile, Archaea have been isolated and cultured from there [10,11] and research will be continued from wild type species, until Indonesian laboratory stems will have been established. Int. J. Mol. Sci. 2019, 20, 5217; doi:10.3390/ijms20205217 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2019, 20, 5217 2 of 26 Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 2 of 27 2. Growth Growth of of ThermoplasmaThermoplasma Thermoplasma acidophilum ((T.a.) DSM DSM 1728 1728 Göttingen, Göttingen, Germany, Germany, or isolated from the Indonesian volcano Tangkuban Perahu was cultured micro-aerobically in in 1- 1- or 2-L flasks flasks on a shaker [11] [11] or in 5-, 10- or 50-L fermenters according to [12] [12] at 39–59 °C◦C and pH 1–2 in Freundt’s medium containing sulfuric acid adjusted to the desired pH. Optimal fermenter fermenter growth growth occurs occurs at pH at 2; pH more 2; selectively—although more selectively—although slightly slower— slightly Thermoplasmaslower—Thermoplasma can be growncan bein culture grown at in pH culture 1.5, becaus at pHe 1.5,the becauseconditions the under conditions pH of 2 under and 60 pH °C ofare 2 quiteand 60 resistant◦C are quite towards resistant contaminat towards contaminationion with other with othermicroorganisms. microorganisms. ThermoplasmaThermoplasma cellscells are are pleomorphic rangingranging inin sizesize between between 0.5 0.5 and and 5 µ5 mµm with with a maximuma maximum of sizeof size distribution distribution at 2–3 at µ2–3m [µm12]. 3 [12].EPR EPR measurements measurements estimated estimated an averagean average inner inner volume volume of of 1 µ1m µmand3 and an an outer outerdiameter diameter ofof 2 µmµm (estimations under the presumption of sphericalspherical shape)shape) [[12–15].12–15]. Thermoplasma acidophilum DSM DSM1728 1728 grows grows up up to to OD OD ( λ(λ= =578 578 nm) nm) of of 0.6, 0.6, at at 39 39◦C °C within within 200–210 200–210 h, h,at 49at ◦49C within°C within 70 h, 70 at h, 59 at◦C 59 within °C within 42 h [ 1242], h wild [12], type wild from type Tangkuban from Tangkuban Perahu, Indonesia,Perahu, Indonesia, identified identifiedas mixtures as of mixturesT. acidophilum of T. acidophilumand volcanium and[11 volcanium], grows up[11], to grows OD of up 0.35 to within OD of 160 0.35 h inwithin culture 160 flasks h in cultureon a shaker flasks without on a shaker automated without regulation automated of growth regula conditionstion of growth at 55–56 conditions◦C in 1–5 at sequential 55–56 °C culturein 1–5 sequentialgenerations culture and up generations to OD 0.4 within and 240up hto [ 11OD]. In0.4 an within optimized 240 computer-regulatedh [11]. In an optimized fermenter computer- culture regulatedwild type fermenter grew up rapidly culture to wild an ODtype of grew almost up rapidly 1.0 within to an 2-3 OD days of (Figurealmost 11.0); thiswithin culture 2-3 days has not(Figure yet 1);been this further culture investigated. has not yet been further investigated. 0.9 0.8 0.7 0.6 DSM 59 0.5 DSM 49 0.4 λnm 578 = DSM 39 OD 0.3 TP 55 0.2 TP 59 0.1 0 0 50 100 150 200 250 Time [hours] Figure 1. CultureCulture growth growth of of Thermoplasma.Thermoplasma. G Growthrowth of of ThermoplasmaThermoplasma acidophilum acidophilum DSMDSM 1728, 1728, Göttingen, Göttingen, Germany, at 39 ◦°C,C, 49 ◦°C,C, an andd 59 °C◦C and of wild type Thermoplasma harvested from a Tangkuban Perahu crater, grown at 55–56 ◦°CC in 1-Liter culture flasks flasks without automated control (TP 55); growth of the latter is similar to that of the former at 39 ◦°CC to a maximum OD of 0.35–0.4 and at 59 °C◦C in a computer-regulated 2-L fermenter (TP 59); OD, optical density at λ = 578578 nm; in in all cultures, pH was below 2 (at the beginning beginning of the the cultures cultures adjusted to 1.5 and and re-adjusted, re-adjusted, if necessary); necessary); values adapted from [11,12]. [11,12]. Culture growth at various pH (1.5;(1.5; 2.0; 2.5; 3.0) demonstrat demonstrateded that growth optimum under laboratory conditions conditions is is at at pH pH 2. 2. Growth Growth curves curves at at pH pH 2.5 2.5 and and 3.0 3.0 are are similar, similar, but growth is slower than at pHpH 2.0;2.0; andand atat pH pH 1.5, 1.5, growth growth is is considerably considerably slower slower and and shows shows a di a ffdifferenterent kinetic kinetic curve curve in the in therange range of our of our measurements measurements (Figure (Figure2). 2). Int. J. Mol. Sci. 2019, 20, 5217 3 of 26 Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 3 of 27 pH 2.0 pH 1.5 pH 2.5 pH 3.0 ] Figure 2. Culture growth of Thermoplasma acidophilum – pH dependency. Growth of T.a. DSM 1728, Göttingen, at 59 ◦°CC and varying pH; OD, optical density at λ == 578 nm; values adapted from [[12].12]. 3. Cell Lysi Lysiss Experiments Lysis experiments with T.a.. cells under various conditions (e.g (e.g.,., different different lysis buffers buffers at lysis temperature ofof 22 22 or or 59 59◦C) °C) had had shown shown clear clear maximal maximal resistance resistance of the ofT.a the. cell T.a membrane. cell membrane at pH 4at [16 pH,17 4]. [16,17].The influence of temperature on membrane stability in different media, Freundt’s solution, McIlvaineThe influence buffer and of after temperature protein precipitation on membrane with stab HClOility4 is in shown different in Figure media,3A. Freundt’s In McIlvaine solution, bu ffer cells begin to lyse at 60 C, immediately above their growth optimum of 59 C. In Freundt’s solution McIlvaine buffer and after◦ protein precipitation with HClO4 is shown in ◦Figure 3A. In McIlvaine cellsbuffer begin cells tobegin lyse to at lyse 70 ◦ C,at similarly60 °C, immediately after protein above precipitation their growth with optimum HClO4, onlyof 59 in°C. the In latterFreundt’s case absorbance starts—as expected—at a lower level [16]. solution cells begin to lyse at 70 °C, similarly after protein precipitation with HClO4, only in the latter case Cellabsorbance lysis at starts—as 99 ◦C, after expected—at dilution of a McIlvainelower level lysis [16]. buffer with 15 mM and 150 mM sodium chlorideCell solutionlysis at is99 shown °C, after in Figuredilution3B. of Cells McIlvaine were grown lysis atbuffer 39 or with 59 ◦ C.15 Highest mM and membrane 150 mM stabilitysodium canchloride be seen solution between is shown pH 4 andin Figure 6. At growth3B. Cells optimum were grown of pH at 2,39 the or 59 membrane °C. Highest appears membrane less stable. stability can beThe seen picture between in Figure pH 43 Band di ff6.ers Atfrom growth lysis optimum conditions of withpH 2, the the same membrane lysis bu appearsffers, but less at incubation stable.