In Situ Production of Crenarchaeol in Two California Hot Springs

Crenarchaeol, a membrane-spanning glycerol dialkyl glycerol tetraether (GDGT) containing a cyclohexane moiety in addition to four cyclopentane moieties, was originally hypothesized to be synthesized exclusively by the mesophilic Crenarchaeota. Recent studies reporting the occurrence of crenarchaeol in hot springs and as a membrane constituent of the recently isolated thermophilic crenarchaeote “Candidatus Nitrosocaldus yellowstonii,” however, have raised questions regarding its taxonomic distribution and function. To determine whether crenarchaeol in hot springs is indeed synthesized by members of the Archaea in situ or is of allochthonous origin, we quantified crenarchaeol present in the form of both intact polar lipids (IPLs) and core lipids in sediments of two California hot springs and in nearby soils. IPL-derived crenarchaeol (IPL-crenarchaeol) was found in both hot springs and soils, suggesting in situ production of this GDGT over a wide temperature range (12°C to 89°C). Quantification of archaeal amoA gene abundance by quantitative PCR showed a good correspondence with IPL-crenarchaeol, suggesting that it was indeed derived from living cells and that crenarchaeol-synthesizing members of the Archaea in our samples may also be ammonia oxidizers.Numerous groups of the Archaea synthesize isoprenoid glycerol dialkyl glycerol tetraethers (GDGTs) as a major component of their core membrane lipids, which can contain up to eight cyclopentane moieties (e.g., see reference 7) (Fig. ​(Fig.1).1). An increase in the number of cyclopentane moieties results in denser packing of membrane lipids, allowing for the maintenance of both cellular membrane integrity at high temperatures and stable proton gradients under low-pH conditions (8). This biophysical characteristic is hypothesized to be among those traits essential for the survival and persistence of the Archaea in the “extreme” environments in which they are commonly found (42). GDGTs are synthesized by a large number of cultivated members of the Archaea (see overviews in references 20 and 34), and in nature, they are abundant in hot springs (24, 25, 34, 46), for example, where members of the Archaea are known to thrive at high temperatures and over a wide pH range (3, 21).Open in a separate windowFIG. 1.Structures of GDGTs referred to in the text. “IS,” C₄₆ internal standard.Crenarchaeol is unique among the GDGTs in that it contains a cyclohexane moiety in addition to four cyclopentane moieties (Fig. ​(Fig.1).1). It was first reported in large abundances from Holocene and ancient sediments collected from various marine settings as supporting evidence for the widespread distribution of low-temperature relatives of the hyperthermophilic Archaea (31). It was later proposed that crenarchaeol was synthesized exclusively by marine group I Crenarchaeota (36), a hypothesis further supported by core lipid analysis of the mesophilic marine group I.1a crenarchaeotes “Cenarchaeum symbiosum” (38) and “Candidatus Nitrosopumilus maritimus” SCM1 (30), which showed that both of these organisms synthesize crenarchaeol at moderate temperatures. In addition to this, the apparent absence of crenarchaeol in cultures of (hyper)thermophilic members of the Archaea (see overviews in references 20 and 34) and molecular modeling (8, 37) led to the hypothesis that crenarchaeol decreases lipid density, effectively allowing archaeal membranes composed of membrane-spanning GDGTs to function at mesophilic temperatures (37). Hence, crenarchaeol synthesis was thought to be instrumental in the evolution and radiation of mesophilic Crenarchaeota from thermophilic habitats (17).Recent studies, however, have reported the occurrence of crenarchaeol in hot springs with temperatures of up to 86.5°C (24, 25, 34, 46). That work has been debated to some extent, as there exists the potential for the allochtonous input of fossilized lipid material from weathering of nearby soils where mesophilic Crenarchaeota may thrive: Schouten et al. (34) previously found large relative amounts of specific soil bacterium biomarkers in tandem with crenarchaeol in Yellowstone hot springs. In contrast, Reigstad et al. (28) reported the occurrence of crenarchaeol in the absence of soil-specific biomarkers in Icelandic hot springs. Furthermore, the recently isolated thermophilic crenarchaeote “Candidatus Nitrosocaldus yellowstonii” was shown to synthesize crenarchaeol at a growth temperature of 72°C (6).Core lipids (CLs) that occur in biological membranes generally contain polar head groups such as sugars and phosphates, which are rapidly cleaved upon cell senescence (10, 44). The loss of head groups from intact polar lipids (IPLs) leaves relatively recalcitrant CLs to accumulate in the environment over time as fossil biomarkers. Therefore, depending on the extraction and/or analytical protocols, CLs present in environmental lipid extracts may be derived from both living cells and fossil biomass, including a mixture of both CL-derived GDGTs (CL-GDGTs) and IPL-derived GDGTs (IPL-GDGTs). Most studies of the presence of crenarchaeol in hot springs reported to date have analyzed directly extracted CL-crenarchaeol or CL-crenarchaeol released by the acid hydrolysis of Bligh-Dyer IPL lipid extracts, i.e., without prior separation of CL-GDGTs from IPL-GDGTs (24, 25, 28, 34, 46). In these cases, the reported GDGT distributions represent an integrated signal of both “living” and fossilized material, rendering it impossible to distinguish what proportion (if any) of the observed crenarchaeol was derived from local living archaeal communities. Thus, the in situ production of crenarchaeol in hot springs and its importance relative to that of the in situ production of other archaeal GDGTs remain uncertain.Here we have used a recently described chromatographic method (22, 26) to separately quantify the potential contributions of both in situ-produced and fossilized crenarchaeol (as well as other archaeal GDGTs) in two Californian hot springs and their surrounding soils. In addition, we have quantified the amounts of archaeal amoA and archaeal 16S rRNA gene copies from one site to make quantitative comparisons between gene abundance and IPL-GDGT concentrations.