Rare bacteriohopanepolyols as markers for an autotrophic, intra-aerobic methanotroph

Bacteriohopanepolyols (BHPs) and their diagenetic products, hopanoids, are of great interest for their potential as bio-marker lipids in both present day environments as well as in the geological record. Specific structural features such as methylation of the A-ring, and number and type of functional groups of C-35 BHPs have been held characteristic for certain groups of organisms. Here we investigated the potential presence of BHPs in the unusual anaerobic methanotroph Methylomirabilis oxyfera and another Methylomirabilis sp. Although M. oxyfera thrives in anoxic settings, it uses internally produced molecular oxygen (from nitrite) for the oxidation of methane. We found that Methylomirabilis spp. synthesizes bacteriohopanehexol (BHP-hexol), -pentol, and -tetrol, and 3-methyl derivatives of each as major BHPs. None of the C-35 amino-BHPs that are more commonly observed in methanotrophs were detected. Our findings provide the first ever account of a 3-methyl-BHP-hexol, and only the second known source organism for BHP-hexol after Alicyclobacillus acidoterrestris. As the genes required for C3-methylation seem to be exclusively present in microorganisms with an aerobic metabolism, the abundant presence of 3-methyl-BHPs in Methylomirabilis spp. denotes for the first time the potential production of 3-methyl hopanoids in anoxic environments. Furthermore, with C-13-labeling experiments we show that M. oxyfera does not assimilate methane-carbon, but rather C from bicarbonate/CO2, into its BHPs. This implies that methanotroph-derived hopanoids do not necessarily exhibit a strongly depleted carbon isotopic signature, which is commonly anticipated with methanotrophy. This may have implications for the interpretation of the presence of hopanoids and their isotopic signature in the paleorecord