Characterization of a new Rhodopila isolate
Although Rhodopila (originally Rhodopseudomonas) globiformis has been known since 1974, a second isolate of this organism has until now not been reported. In field studies carried out by two of us (MHM and MNP), a spring was discovered in Lassen National Park (California, USA) that seemed geochemically similar to the warm acidic spring that yielded Rpi. globiformis (Pfennig 1974) and which contained a microbial mat of the strongly acidophilic green alga Cyanidium overlying a purple-red layer. Cells from the latter appeared similar to those of Rps. globiformis (large weakly motile cocci) and so cultures were pursued and eventually obtained. Considering its habitat, pigments, physiology, and cell morphology, the Lassen purple bacterium was thought to be a new strain of Rhodopila globiformis and thus was tentatively designated Rhodopila strain LVNP.
A 16S rRNA gene phylogenetic tree (Fig. 2) revealed that the Lassen and Yellowstone Rhodopila isolates were closely related yet phylogenetically distinct. Moreover, the genome of the Lassen isolate (8.1 Mb) was significantly larger than that of Rpi. globiformis (7.2 Mb, Imhoff et al. 2018) and the average nucleotide identity between the two genomes was only 93.1%. Thus, the two strains may be separate Rhodopila species rather than strains of the same species. Rhodopila is the most acidophilic PNS bacterium known (Madigan and Imhoff, 2005) and produces unique purple-red carotenoids (Fig. 1c) (Schmidt and Liaaen-Jensen 1973) closely related to okenone, a carotenoid detected in 1.6 Gyr-old rocks from Northern Australia (Brocks et al. 2005, Brocks and Schaeffer 2008). Rhodopila is also phylogenetically distinct from other PNS bacteria and the only anaerobic and acidophilic phototroph that groups with the Acetobacteraceae, a bacterial family that includes acetic acid-producing bacteria and other aerobic and acidophilic bacteria (Kersters et al. 2006); this can be seen clearly in Fig. 2.
Lipid analyses of pure cultures of both strain LVNP and the type strain of Rpi. globiformis (strain DSM 161T), grown either photosynthetically or aerobically in the dark (Pfennig 1974), revealed 3-MeBHP production by both strains. Gas chromatographic–mass spectrometric analyses (Zundel and Rohmer 1985) confirmed that the hopanoids were indeed 3-MeBHPs (Fig. 3a) and not 2-MeBHPs (Fig. 3b). Strain LVNP grown phototrophically at pH 5 synthesized 0.9 µg BHP/mg total fatty acid and 0.03 µg 3-MeBHP/mg total fatty acid. By contrast, phototrophic cultures of the mildly acidophilic PNS bacterium Rhodoblastus acidophilus and the neutrophilic species Rhodopseudomonas palustris (Fig. 3) did not produce 3-MeBHPs; Rps. palustris did, however, contain 2-MeBHPs as was previously reported (Welander et al. 2010). Moreover, liquid chromatography–mass spectrometry analyses of cells of Rhodopila strain LVNP showed that it produced not only 3-MeBHP but a suite of BHPs. Although not quantified, LC-MS identified several extended BHPs, including bacteriohopanetetrols and bacteriohopanetetrol cyclitol ethers (Table 1).
Genomic evidence for 3-MeBHP production in Rhodopila species
To further explore the production of 3-MeBHP production in Rhodopila, the genome sequence of strain LVNP was determined and compared with the previously published genome of Rpi. globiformis DSM 161T (Imhoff et al. 2018). Genomic analyses confirmed that both organisms were genetically equipped to produce 3-MeBHPs (Table 2). The C-3 BHP methylase HpnR is encoded in both genomes, and a phylogenetic tree constructed from HpnR sequences (Fig. 4) mirrored the 16S rRNA gene tree (Fig. 2). Specifically, HpnR from the Rhodopila species was related to HpnR from species of Acetobacteraceae and distinct from that produced by methanotrophic Methylococcaceae (Fig. 4); the latter are well-known producers of 3-MeBHPs but are only distant relatives of Acetobacteraceae (Fig. 2). Genes encoding several other hopanoid biosynthesis enzymes (Belin et al. 2018) were identified in the genomes of both Rhodopila strains (Table 2) consistent with the production of several related BHPs identified from cells of Rhodopila strain LVNP (Table 1).