|Intact polar lipids of ammonia-oxidizing Archaea: Structural diversity anapplication inmolecular ecology|Pitcher, A. (2011). Intact polar lipids of ammonia-oxidizing Archaea: Structural diversity anapplication inmolecular ecology. Geologica ultraiectina, mededelingen van de faculteit aardwetenschappen, 337. PhD Thesis. [S.n.]: [s.l.]. ISBN 978-90-5744-198-1. 1-227 pp. hdl.handle.net/1874/200226
Part of: Geologica ultraiectina, mededelingen van de faculteit aardwetenschappen, more
Non-extremophilic Crenarchaeota are ubiquitous, and comprise a major component of the microbial assemblages in many modern-day systems. Several studies have analyzed glycerol dialkyl glycerol tetraether (GDGT) membrane lipids synthesized by Crenarchaeota to interpret the presence, distribution, and activity of these microbes in various modern environments. The use of cellular membrane lipids in molecular ecology studies provides added value to conventional (meta)genomic approaches, partly in the form of independence from biases associated with the extraction and analysis of nucleic acids. However, disentangling biomarker lipid signals derived from living and dead cells has remained a challenge. This thesis describes investigations aimed at developing the use of intact polar lipids (IPLs) in ecological studies of ammonia-oxidizing Crenarchaeota (AOA), as crenarchaeotal IPLs containing polar head groups bound to the core GDGT are assumed to best represent living Crenarchaeota. To this end, improvements to both indirect and direct GDGT-based IPL analyses were made, with the latter based largely on information obtained from four novel enrichment cultures of ammonia-oxidizing Crenarchaeota. The findings of these studies were applied to three different environmental settings: two California hot springs, the Arabian Sea oxygen minimum zone (OMZ), and the coastal North Sea. Comparisons between IPL and DNA-based molecular data reveal a more complete picture of the distribution and abundance of ammonia-oxidizing Crenarchaeota at those sites, in addition to demonstrating the general robustness of IPL analyses in molecular ecology studies.When IPL-GDGTs are analyzed (and quantified) after removal of the polar head group by hydrolysis, chromatographic fractionation of core and IPL GDGTs is first necessary in order to discern between dead and live GDGT signals. Conventional column fractionation schemes based on the separation of bacterial glyco- and phospholipids were found unsuitable for the separation of GDGT-based IPLs. Over activated silica, elution of GDGT-IPL standards with hexane:ethyl acetate (3:1, v/v), ethyl acetate, followed by methanol, was shown to yield fractions highly enriched in core, glyco-, and phospho-GDGTs, respectively. The inadequate separation of GDGT classes using old separation schemes could result in significant qualitative and quantitative differences, and thus the modified solvent elution protocol presented in this study should be used in future work.The ability to separate core and IPL-GDGTs allowed for the determination of the origin of crenarchaeol in terrestrial hot springs. The hypothesis that crenarchaeol was synthesized exclusively by mesophilic Crenarchaeota was called into question upon its discovery in terrestrial hot springs and its synthesis by the thermophilic AOA, “Ca. Nitrocaldus yellowstonii”. Recovery of abundant crenarchaeol in the IPL-GDGT fractions extracted from two California hot springs confirmed that crenarchaeol is indeed synthesized in situ. In addition, a correspondence between amoA gene and IPL-derived crenarchaeol abundances suggested that the crenarchaeol recovered from the hot springs was synthesized by ammonia-oxidizing Crenarchaeota.The characterization of ammonia-oxidizing Crenarchaeota has been hampered by difficulties in their enrichment, and therefore limited data exists on the IPL-GDGTs synthesized by these microbes in culture. Analysis of the recently enriched Group I.1b AOA, “Ca. Nitrososphaera gargensis”, revealed a GDGT distribution consisting almost exclusively of crenarchaeol and the crenarchaeol regio-isomer. This finding extends the taxonomic distribution of crenarchaeol synthesis to a new phylogenetic lineage within the Group I Crenarchaeota, and implicates members of this group as important contributors to crenarchaeol recovered from soils. In addition, lower amounts of a tentatively identified GDGT containing a cyclohexane moieties in addition to five cyclopentane moieties were present. The GDGT-associated polar headgroups consisted of monohexose, dihexose, phosphohexose and hexose-phosphohexose moieties in addition to headgroups consisting of mono- and dihexose sugars with an additional moiety of176 Dalton. Together, these data contribute substantially to the current knowledge of IPLs synthesized by AOA and support the hypothesis that crenarchaeol is specific to ammonia-oxidizers. TEX86 (a GDGT-based geochemical proxy used to reconstruct past sea surface temperatures)-derived temperatures calculated using the GDGT distribution of “Ca. N. gargensis” matched its original cultivation temperature of 46°C, however they did not change according to short term cultivation at 42°C and 50°C. This indicates that individual species may not adjust their membrane GDGTs dramatically according to temperature or that such a physiological adaptation would take much longer.Additional support for the specificity of crenarchaeol to AOA comes from analysis of IPL-GDGTs synthesized by additional ammonia-oxidizing Crenarchaeota enriched from marine sediments. Three novel enrichment cultures all synthesized abundant crenarchaeol in addition to other GDGTs commonly recovered from marine suspended particulate matter (SPM), and polar headgroups similar to those synthesized by Nitrosopumilus maritimus SCM1 and “Ca. N. gargensis”. A comparison of the GDGT distributions associated with each polar head group identified prior to, and including, this study revealed a commonality of hexose-phosphohexose crenarchaeol to all AOA, thereby pointing to this IPL as the ideal biomarker to track living ammonia-oxidizing Crenarchaeota in the environment.A HPLC/ESI-MS2 selected reaction monitoring method aimed at the detection of five different crenarchaeol-based IPLs was developed using extracts of biomass of “Ca. N. gargensis”, to screen for the presence of viable AOA through the Arabian Sea oxygen minimum zone (OMZ). The vertical distribution of hexose-phosphohexose crenarchaeol was marked by a prominent peak at the oxycline in addition to a less-pronounced peak at the bottom of the OMZ which matched peaks in Crenarchaeota 16S and amoA gene abundances. The general correspondence between IPL and gene profiles in this study demonstrates the robustness of HPH-crenarchaeol as a marker for living ammonia-oxidizing Crenarchaeota. A comparison of the depth distribution of PC-monoether ladderane IPL derived from anaerobic ammonia oxidizing (anammox) Bacteria and 16S rRNA gene abundances, which peaked at mid-OMZ depths, suggest that despite theoretical potential, little opportunity may exist for metabolic coupling between these groups at this location due to = 400 m vertical separation of their respective niches.A more detailed comparison of directly-analyzed crenarchaeol-based IPLs, IPL-derived GDGTs, and core GDGTs was made through the Arabian Sea OMZ. The results suggest that a portion of IPLs may actually persist as molecular fossils, and support the idea of differential degradation of glycolipids and phospholipids. This is in contrast with the assumption that all IPLs degrade rapidly upon cell senescence which has conventionally justified their use as general „life? markers. Despite a good correspondence at the surface (ca. 20 m depth), TEX86-calculated temperatures derived from core and IPL-derived GDGT distributions did not follow temperature changes with depth. A contribution of IPLs to the fossil GDGT pool could account for this.Increases in AOA abundance were notable during the winter months between November and February of an interrupted time series spanning the years 2002-2008. GDGT-based IPLs were used to track the seasonal occurrence and carbon-fixation activity of marine AOA in the coastal North Sea from 2007-2008. During this time crenarchaeol-based IPLs showed the same temporal distribution, regardless of headgroup, indicating that in this dynamic system a fossil contribution of IPLs to the GDGT pool is less likely than in the Arabian Sea. Incubations of North Sea water with 13C-bicarbonate resulted in label incorporation into the tricyclic biphytane derived from IPL-crenarchaeol, confirming that the Crenarchaeota in the North Sea surface waters actively fix bicarbonate during their winter blooms. Lower 13C-incorporation was observed in incubations containing nitrification inhibitors (Nserve and chlorate) further indicating that these Crenarchaeota are predominantly ammonia-oxidizers.To conclude, the present study demonstrates that intact polar GDGTs are excellent tools to study the ecology of Crenarchaeota in modern-day environments. Continued application of IPLs to molecular ecology studies will enhance our understanding of the role of AOA in both carbon and nitrogen cycling. In addition, constraining controls on the environmental distributions of GDGTs, including crenarchaeol and its regioisomer, will aid in a better understanding of their use in geochemical proxies, such as the TEX86 paleothermometer.