For polar lipid analysis, cells of strain RMAST were grown in WA medium at 70 uC and harvested at the late exponential phase. Polar lipids were recovered from cells by the acidified Bligh and Dyer method and separated by one-dimensional TLC, as described by Nishihara & Koga (1987). Lipid bands on the TLC plate were visualized using ninhydrin for amino lipids and either 30% H2SO4 or iodine vapour for all lipids.Some lipids visualized using iodine vapour were scraped and analysed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS, AXIMA Resonance, Shimadzu) according to the procedure
of Angelini et al. (2010). Cells of M. thermautotrophicus
DSM 1053T at the late exponential phase were also examined
as a reference. The visualized lipid bands of strain RMAST
and M. thermautotrophicus DSM 1053T were similar but
with some differences between them (Fig. S2). The bands
were identified as gentiobiosylcaldarchaetidylinositol (Rf
0.21, both strains) and gentiobiosylcaldarchaetidylethanolamine
(Rf 0.40, strain RMAST) on the basis of the MALDITOF
MS and TLC analyses described by Nishihara & Koga
(1987). Both gentiobiosylcaldarchaetidylinositol and gentiobiosylcaldarchaetidylethanolamine
have been designated
the signature lipids of the genera Methanothermobacter and
Methanobacterium within the family Methanobacteriaceae
(Koga et al., 1998); hence, this chemotaxonomic trait
indicates that strain RMAST belongs to either of these
genera.