Background: Hopanoids modify plasma membrane properties in bacteria and are often compared to sterols that modulate membrane fluidity in eukaryotes. In some microorganisms, they can also allow adaptations to extreme environments. Methods: Hopanoids were identified by liquid chromatography–mass spectrometry in fourteen strains of thermophilic bacteria belonging to five genera, i.e., Alicyclobacillus, Brevibacillus, Geobacillus, Meiothermus, and Thermus. The bacteria were cultivated at temperatures from 42 to 70 °C. Results: Regardless of the source of origin, the strains have the same tendency to adapt the hopanoid content depending on the cultivation temperature. In the case of aminopentol, its content increases; aminotetrol does not show a significant change; and in the case of aminotriol the content decreases by almost a third. The content of bacteriohopanetetrol and bacteriohopanetetrol glycoside decreases with increasing temperature, while in the case of adenosylhopane the opposite trend was found. Conclusions: Changes in hopanoid content can be explained by increased biosynthesis, where adenosylhopane is the first intermediate in the biosynthesis of the hopanoid side chain.

Sixteen strains of five genera of thermophilic bacteria, i.e., Alicyclobacillus, Brevibacillus, Geobacillus, Meiothermus, and Thermus, were cultivated at a temperature from 42 to 70 °C. Twelve strains were obtained from the Czech Collection of Microorganisms, while four were directly isolated and identified by 16S rRNA gene sequencing from the hot springs of the world-famous Carlsbad spa (Czech Republic). Polyprenol homologs from C40 to C65 as well as free undecaprenol (C55), undecaprenyl phosphate, and undecaprenyl diphosphate were identified by shotgun analysis and RP-HPLC/MS-ESI⁺ (reverse phase high-performance liquid chromatography–high-resolution positive electrospray ionization mass spectrometry). The limit of detection (50 pM) was determined for individual homologs and free polyprenols and their phosphates. Thus, it has been shown that at least some thermophilic bacteria produce not just the major C55 polyprenol as previously described, but a mixture of homologs.