Marine and terrestrial nitrifying bacteria are sources of diverse bacteriohopanepolyols.

Hopanoid lipids, bacteriohopanols and bacteriohopanepolyols, are membrane components exclusive to bacteria. Together with their diagenetic derivatives, they are commonly used as biomarkers for specific bacterial groups or biogeochemical processes in the geologic record. However, the sources of hopanoids to marine and freshwater environments remain inadequately constrained. Recent marker gene studies suggest a widespread potential for hopanoid biosynthesis in marine bacterioplankton, including nitrifying (i.e., ammonia- and nitrite-oxidizing) bacteria. To explore their hopanoid biosynthetic capacities, we studied the distribution of hopanoid biosynthetic genes in the genomes of cultivated and uncultivated ammonia-oxidizing (AOB), nitrite-oxidizing (NOB), and complete ammonia-oxidizing (comammox) bacteria, finding that biosynthesis of diverse hopanoids is common among seven of the nine presently cultivated clades of nitrifying bacteria. Hopanoid biosynthesis genes are also conserved among the diverse lineages of bacterial nitrifiers detected in environmental metagenomes. We selected seven representative NOB isolated from marine, freshwater, and engineered environments for phenotypic characterization. All tested NOB produced diverse types of hopanoids, with some NOB producing primarily diploptene and others producing primarily bacteriohopanepolyols. Relative and absolute abundances of hopanoids were distinct among the cultures and dependent on growth conditions, such as oxygen and nitrite limitation. Several novel nitrogen-containing bacteriohopanepolyols were tentatively identified, of which the so called BHP-743.6 was present in all NOB. Distinct carbon isotopic signatures of biomass, hopanoids, and fatty acids in four tested NOB suggest operation of the reverse tricarboxylic acid cycle in Nitrospira spp. and Nitrospina gracilis and of the Calvin-Benson-Bassham cycle for carbon fixation in Nitrobacter vulgaris and Nitrococcus mobilis. We suggest that the contribution of hopanoids by NOB to environmental samples could be estimated by their carbon isotopic compositions. The ubiquity of nitrifying bacteria in the ocean today and the antiquity of this metabolic process suggest the potential for significant contributions to the geologic record of hopanoids.