Anaerobic 3-Methylhopanoid Production By An Acidophilic Phototrophic Purple Bacterium

Bacterial lipids are well preserved in ancient rocks and certain ones have been used as indicators of specic bacterial metabolisms or environmental conditions existing at the time of rock deposition. Here we show that an anaerobic bacterium produces 3-methylbacteriohopanepolyols (3-MeBHPs), 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-MeBHPs and a suite of related BHPs and contained the genes encoding the necessary biosynthetic enzymes. Our results show that 3-MeBHPs can be produced under anoxic conditions and challenges the use of 3-MeBHPs as biomarkers of oxic conditions in ancient rocks and as prima facie evidence that methanotrophic bacteria were active when the rocks were deposited.


Introduction
Bacteriohopanepolyols (BHPs), or hopanoids, are triterpenoid lipids that help preserve membrane integrity and permeability in certain bacteria (Ricci et al. 2017). BHPs are also quite recalcitrant biomolecules; their hopane derivatives can be preserved in sedimentary rocks for billions of years, and because of this, have been exploited as biomarkers of past environmental conditions or particular microbial activities (Brocks et al. 2005). While BHPs are produced by metabolically-diverse bacteria, BHPs methylated in the A-ring are more restricted in their distribution and linked to particular bacterial taxa or aerobic metabolisms. For example, BHPs methylated at the C-2 position (2-MeBHPs) have traditionally been linked to cyanobacteria (Summons et al. 1999) whereas BHPs methylated at the C-3 position (3-MeBHPs) have been associated with strictly aerobic methanotrophic and acetic acid bacteria (Zundel and Rohmer 1985).
In 2007, 2-MeBHP production was reported in photosynthetically-grown cultures of the purple nonsulfur (PNS) bacterium Rhodopseudomonas (Rps.) palustris (Rashby et al. 2007), a widely distributed species that inhabits freshwater lakes and fertile soils (Harwood and Gibson 1988). PNS bacteria are a phylogenetically diverse group of anoxygenic phototrophs that preceded cyanobacteria on Earth by at least 500 million years, may have been responsible for the earliest of the banded iron formations (Widdel et al. 1993), and whose photosynthetic metabolism is strictly anaerobic (Hohmann-Marriott and Blankenship 2011). These Alphaand Betaproteobacteria inhabit various aquatic environments, including lakes, wastewaters, hot springs, and marine and hypersaline waters and typically can conserve energy from both photosynthesis (anoxic/light) or respiration (oxic/dark) (Madigan and Jung 2009). Subsequent metagenomic analyses showed that hpnP, the gene encoding the enzyme that methylates BHP at the C-2 position, was present in microbes from samples of a variety of microbial ecosystems, suggesting that 2-MeBHP production is widespread in nature (Ricci et al. 2015). Moreover, lipid analyses of microbial mat samples from hypersaline environments detected 2-MeBHPs in various bacteria, including species of PNS bacteria . Collectively, these discoveries demonstrated the danger in unambiguously linking the presence of 2-methylhopanes (the breakdown product of 2-MeBHP) in ancient sediments to cyanobacteria or to oxic conditions in general.
In contrast to 2-MeBHPs, evidence to date has shown that BHPs methylated at the C-3 position (3-MeBHPs) (Summons et al. 1999;Rashby et al. 2007) are synthesized only by bacteria that perform O 2dependent metabolisms, with the most prominent producers being aerobic methane-oxidizing bacteria (methanotrophs) and acetic acid bacteria (Zundel and Rohmer 1985). The hpnR gene that encodes the C-3 BHP methylase has also been detected in a diverse array of aerobic bacteria further con rming the link to O 2 -dependent metabolisms (Welander and Summons 2012). In our studies of hot spring microbial mats, we detected 3-MeBHPs in mat samples collected from a sul dic and acidic (pH 3-4) spring in Lassen Volcanic National Park (California, USA) that was fed by a continuous discharge of warm volcanic water containing CO 2 , H 2 , and H 2 S (Fig. 1a, b). The mat lacked cyanobacteria but contained a purple-red layer underneath a green algal layer. Knowing that 3-MeBHPs have not been reported from algae, we pursued the Lassen purple bacterium as the possible source of these lipids and, using standard enrichment and isolation techniques, obtained a pure culture of this phototroph (Fig. 1c). Here we show that pure cultures of this organism and its phylogenetic close relative produce a suite of 3-MeBHPs when grown under strictly anoxic conditions-the rst report of the production of these hopanoid lipids in an anaerobic bacterium-and that their genomes encode the requisite enzymes for producing these lipids and methylating them in the C-3 position.

Materials And Methods
Organisms, isolation, and growth conditions Lipid analyses and genomic studies were performed on axenic cultures of four purple nonsulfur bacteria: Rhodopila strain LVNP, Rhodopila globiformis, Rhodopseudomonaspalustris, and Rhodoblastus acidophilus (the latter three species from the collection of MTM). Rhodopila strain LVNP was isolated from a microbial mat that formed in an acidic (pH 3.9), sul dic spring in Lassen Volcanic National Park (near 40°27'09.5"N 121°32'13.3"W, Northern California, U.S.A.). The mat had an upper algal layer and a lower purple-red layer (Fig. 1). A sample of the purple layer was incubated in liquid medium (Pfennig 1974) under photosynthetic conditions at 25 o C, and an axenic culture was eventually obtained from successive transfers of isolated colonies grown on plates of the same medium incubated in a Mitsubishi AnaeroPack 2.5L Rectangular Jar (Thermo Scienti c Cat No. R685025). Anoxic conditions in liquid culture media were achieved by vigorously sparging under ltered argon (0.2 µm lter) for 5 min and then reducing media with sul de (added from a sterile sodium sul de solution neutralized to pH 7.5; nal sul de concentration in the medium was 0.14 mM) prior to inoculation. Liquid cultures were grown phototrophically in sealed tissue culture asks incubated in the AnaeroPak Jar illuminated with incandescent light.

Lipid and genomic analyses
For lipid analyses, dense cell suspensions from liquid cultures were collected by centrifugation at 4,500 x g for 10 min at 4ºC. Lipids were extracted by the modi ed Bligh-Dyer protocol as previously described (Jahnke et al. 1992) using both a monophasic ratio of water:methanol:chloroform (4:10:5) and physical agitation. Large cyclic lipids were treated with a redox procedure that yields both methylated and unmethylated BHPs and then analyzed as their acetate derivatizes ) by gas chromatography-mass spectrometry (GC-MS) (Jahnke et al. 1992). Hopanoids were identi ed from their precise mass measurements and fragmentation patterns and by comparison of relative retention time and mass spectra to previously published analytical data ). Liquid chromatography-mass spectrometry (LC-MS) was performed as previously described (Talbot et al. 2003(Talbot et al. , 2007) and used to identify extended 3-MeBHPs and their derivatives.
Genomic DNA from Rhodopila LVNP was isolated using the Genomic Tip 500/G Kit (Qiagen Cat No.

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 eld 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 signi cantly 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 andSchaeffer 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.

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 161 T (Imhoff et al. 2018). Genomic analyses con rmed 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). Speci cally, 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 identi ed in the genomes of both Rhodopila strains (Table 2) consistent with the production of several related BHPs identi ed from cells of Rhodopila strain LVNP (Table 1).

Discussion
Our results are the rst to show the production of 3-MeBHPs in bacteria grown anaerobically, thus refuting the contention that these lipids are only produced by obligately aerobic bacteria. It is thus possible, and perhaps even likely, that these lipids are produced by various other anaerobic species of Bacteria-however, still no species of Archaea have been shown to contain hopanoids (Sahm et al. 1993).
The physiological link between acetic-acid bacteria and Rhodopila does not revolve around energy metabolism but instead the ability of both organisms to thrive in strongly acidic habitats. Whether such a lifestyle requires these unusual lipids is unknown, but the fact that 3-MeBHPs are produced by many neutrophilic methanotrophic bacteria and have not been reported from some other potentially acidophilic bacteria, such as Thiobacillus (Rohmer et al. 1979), or other acidic habitats (Talbot et al. 2016), leaves this question unanswered. Nevertheless, 3-MeBHPs obviously play some role in the physiology of Rhodopila species, and the genetic links between this phototroph and acidophilic bacteria (Fig. 2) and the fact that 3-MeBHPs are membrane integrated, suggest that 3-MeBHPs may help maintain membrane function in their acidic habitats. Indeed, a function for hopanoids in maintaining membrane integrity and surviving general environmental stressors has been shown in the cyanobacterium Nostoc (Ricci et al. 2017). In addition, it has been shown that extended hopanoids aid the chemotrophic bacterium Bradyrhizobium in withstanding hypoxic/low O 2 growth conditions and various other physiological stressors (Kulkarni et al. 2015). Hence, if there exists a widespread link between 3-MeBHPs and microbes that inhabit extreme environments, it is possible that these lipids are produced by anoxygenic phototrophs that thrive in hypersaline, hyperalkaline, and permanently hot or cold environments; all of these habitats contain a diversity of purple bacteria ( Second, the relatively high abundance of 3-methylhopanes in some ancient sediments has been used to infer that freshwater was present in these past aquatic bodies (Brocks et al. 2005;French et al. 2020). This is because when sulfate is limiting, such as is typical in a freshwater lake, methanogenic Archaea outcompete sulfate-reducing bacteria for available electron donors (Hoehler et al. 1998). Thus, in ancient sediments containing 3-methylhopanes it has been assumed that the overlying body of water was fresh and that methanogenic microbes produced the methane that fed 3-Me BHP-containing methanotrophs. However, because our results show that 3-methylhopanes can no longer be unambiguously connected to methanotrophic (or any other obligately aerobic) bacteria, concluding that a given sedimentary rock containing 3-methylhopanes must have formed in a freshwater environment (Brocks et al. 2005  a Acidic (pH 3.9) and sul dic 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 Figure 2 Phylogenetic tree of 16S rRNA genes from some phototrophic purple bacteria and 3-MeBHP-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 are known producers of 3-MeBHPs. 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 Protein tree of HpnR, the C-3 methylase that methylates BHPs to form 3-MeBHP. Both Rhodopila strain LVNP and Rhodopila globiformis strain DSM 161T genomes encode HpnR, and the amino acid sequences of this protein cluster with those from their close phylogenetic relatives, the acetic acid bacteria. C-2 BHP methylase (HpnP) from Rhodopseudomonas palustris was used as the outgroup in the tree. Numbers at the nodes are bootstrap percentages based on 1000 replications. * denote strains that have con rmed 3-Me BHP production in cultured lipids

Supplementary Files
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