Anaerobic 3-methylhopanoid production by an acidophilic photosynthetic purple bacterium

Abstract

Bacterial lipids are well-preserved in ancient rocks and certain ones have been used as indicators of specific bacterial metabolisms or environmental conditions existing at the time of rock deposition. Here we show that an anaerobic bacterium produces 3-methylhopanoids, pentacyclic lipids previously detected only in aerobic bacteria and widely used as biomarkers for methane-oxidizing bacteria. Both Rhodopila globiformis, a phototrophic purple nonsulfur bacterium isolated from an acidic warm spring in Yellowstone, and a newly isolated Rhodopila species from a geochemically similar spring in Lassen Volcanic National Park (USA), synthesized 3-methylhopanoids and a suite of related hopanoids and contained the genes encoding the necessary biosynthetic enzymes. Our results show that 3-methylhopanoids can be produced under anoxic conditions and challenges the use of 3-methylhopanoids as biomarkers of oxic conditions in ancient rocks and as prima facie evidence that methanotrophic bacteria were active when the rocks were deposited.

Keywords: Anoxygenic phototrophs; Hopanoids; Rhodopila globiformis; Warm thermal springs.

Fig. 1

a Acidic (pH 3.9) and sulfidic warm spring in Lassen Volcanic National Park. b Sampling area designated by black arrow in a. The microbial mat contains the red alga Cyanidium (Cyn) overlying a red layer from which Rhodopila strain LVNP was isolated; the white area is elemental sulfur. c Phase-contrast photomicrograph of a dense suspension of cells of strain LVNP

Fig. 2

Phylogenetic tree of 16S rRNA genes from some phototrophic purple bacteria and 3-methylhopanoid-producing bacteria. Taxa shown in red are anoxygenic phototrophs and all are PNS bacteria except for the purple sulfur bacterium Thermochromatium tepidum. All Acetobacteraceae shown and the two methanotrophic bacteria (Methylobacter luteus and Methylococcus capsulatus) are known producers of 3-methylhopanoids. The labels “Alpha,” “Beta” and “Gamma” refer to classes of the phylum Proteobacteria, and numbers at the nodes are bootstrap percentages based on 1000 replications

Fig. 3

a Gas chromatographic–mass spectrometric analyses of 3-methylhopanoids. The compounds, extracted from cultures of Rhodopila strain LVNP grown photosynthetically at pH 5, are separated and analyzed as their acetate derivatives. The mass fragment 191 is diagnostic of unmethylated hopanoids (blue chromatogram) while the 205 fragment is diagnostic of their methylated equivalent (red chromatogram). The 3-methylhopanoids elute after their unmethylated equivalents. b Analysis of Rhodopseudomonas palustris DSM127 as a control. This PNS bacterium produces 2-methylhopanoids (Rashby et al. 2007), which elute before their unmethylated equivalents. I.S., internal standard

Fig. 4

Protein tree of HpnR, the C-3 methylase that methylates hopanoids to form 3-methylhopanoids. Both Rhodopila strain LVNP and Rhodopila globiformis strain 7950 YNP (DSM161^T) genomes encode HpnR (Table 3), and the amino acid sequences of this protein cluster with those from their close phylogenetic relatives, the acetic acid bacteria. C-2 hopanoid methylase (HpnP) from Rhodopseudomonas palustris was used as the outgroup in the tree. Numbers at the nodes are bootstrap percentages based on 1000 replications. Yellow highlighted taxa denote organisms that have confirmed 3-methylhopanoids in lipids obtained from cultured cells