Arch Microbiol (1983) 135:77- 80 Short Communication Archives of Microbiology 9 Springer-Verlag 1983 A novel method for the isolation of bacterial quinones and its application to appraise the ubiquinone composition of Thiobacillusferrooxidans Alan A. DiSpirito*, W. Hsiao-Tsu Loh**, and Olli H. Tuovinen Department of Microbiology, The Ohio State University, Columbus, Ohio 43210, USA Abstract. A method was developed for the isolation of bacterial isoprenoid quinones. This method gave good yields and was superior to two standard methods that were also tested, because the cell membrane isolation and methanol extraction steps could be eliminated. The ubiquinone com- position of Thiobacillus ferrooxidans was analyzed; only ubiquinone-8 was detected and its identification was resolved by thin layer chromatography, high performance liquid chromatography, and mass spectrometry. Key words: Ubiquinone - Isoprenoid quinone - Quinone composition - Thiobacillusferrooxidans- Mass spectrom- etry - High performance liquid chromatography - Thin layer chromatography For the present communication an extraction procedure was developed for isolation of ubiquinones from bacterial sys- tems. In addition to the role of quinones in electron transport systems, the structural variation in these compounds may be of diagnostic value in microbial systematics (Collins and Jones 1981). The biochemical and taxonomic interest in these compounds has led to a number of characterizations in respiratory organisms (Dryhurst et al. 1982). The ubiquinone extraction method developed in the present work was specifically applied to study the ubiquinone composition of Thiobacillus ferrooxidans, an acidophilic chemolithotroph capable of oxidizing inorganic sulfur and iron compounds for energy. In these bacteria, the electrons from reduced in- organic substrates are transported to oxygen and also utilized for the energy-dependent reduction of nicotinamide adenine dinucleotides (Aleem et al. 1963; Gale and Beck 1967; Kelly t 978). In T. ferrooxidans the coupling of inorganic substrate oxidation is indicated at the cytochrome c level of the electron transport chain and cytochrome a complex is involved as an electron carrier before the terminal reduction of Oz (Blaylock and Nason 1963; Ingledew and Cobley 1980; Ingledew 1982). In the reverse electron flow cytochrome b, ubiquinone, and flavoproteins are electron carriers before the reduction of NAD § (Aleem et al. 1963; Ingledew 1982). * Present address: Department of Genetics and Cell Biology,University of Minnesota, St. Paul, Minnesota 55108, USA ** Present address: Department of Biology, University of Virginia, Charlottesville, Virginia 22901, USA Offprint requests to: O. H. Tuovinen Abbreviations. HPLC, high performance chromatography; TLC, thin layer chromatography; UQ, ubiquinone Previous questionable data prompted the present work in which an isoprenoid quinone was isolated and identified from T. ferrooxidans. As in T. thiooxidans (Cook and Umbreit 1963; Adair 1968), T. neapolitanus and T. thioparus (Sadler and Johnson 1972) the component was identified as ubiquinone-8 (UQ-8). Previously Dugan and Lundgren (1964) suggested the involvement of UQ-6 in the electron transport system of T. ferrooxidans, but they indicated insufficient verification and several peaks in the chromatog- raph remained unidentified. UQ-8 was identified by Short et al. (1969) as the only quinone in T. ferrooxidans. These authors based, however, this characterization on reversed phase paper chromatography using only two ubiquinones as standards neither one of which was UQ-8. Thus, the charac- terization by Short et al. (1969) is questionable. Katayama- Fujimura et al. (1982) identified UQ-8 in T.ferrooxidans and also detected trace levels of UQ-8 by mass spectral analysis after a partial purification of the quinone extract by thin layer chromatography (TLC). However, the mass spectrum was not shown. In the present work, UQ-8 was verified as the only isoprenoid quinone in T. ferrooxidans. An m/e peak close to that of UQ-9 was observed in the present work when the quinone extract was partially purified by TLC. When the quinone extract was purified by high performance liquid chromatography (HPLC), this peak was no longer observed. T.ferrooxidans TFI-35 was cultured with 120 mM ferrous sulfate and mineral salts at pH 1.5 in 20 1carboys under forced aeration (DiSpirito and Tuovinen 1982). Bacteria were har- vested from 100 1 of the medium and washed with mineral salts (pH 2) as previously described (DiSpirito and Tuovinen 1981). Cells (approx. 6 g wet weight) were resuspended in 15 ml of 0.2 M acetate buffer (pH 5.6) and either cryo- impacted (Smucker and Pfister 1975) twice or passed through a French pressure cell six times at 140 MPa before the celI homogenate was sonicated for a total of 30 min in 3 min sets at 4~ After sonication, isoprenoid quinones were extracted by three different methods. Method 1." the cell slurry was centrifuged at 20000 x g for 20 min at 4~ and the super- natant was filtered through a 0.45 pm filter. The filtrate was centrifuged at 144000 x g for 2 h at 4~ and quinones were extracted from the membrane pellet (P144) as described by Redfearn (1967). Method 2: the P144 fraction was re- suspended in 60 ml methanol and stirred for 30 min at 60 ~C. The quinone-containing methanol was extracted three times with 60 ml of n-hexane in a separatory funnel. The n-hexane phase was collected and extracted twice with water (90 ml) as a clean-up procedure. Approx. 5 g sodium sulfate was added to the n-hexane fraction to remove the remaining water. The