Diel metabolomics analysis of a hot spring chlorophototrophic microbial mat leads to new hypotheses of community member metabolisms

Abstract

Dynamic environmental factors such as light, nutrients, salt, and temperature continuously affect chlorophototrophic microbial mats, requiring adaptive and acclimative responses to stabilize composition and function. Quantitative metabolomics analysis can provide insights into metabolite dynamics for understanding community response to such changing environmental conditions. In this study, we quantified volatile organic acids, polar metabolites (amino acids, glycolytic and citric acid cycle intermediates, nucleobases, nucleosides, and sugars), wax esters, and polyhydroxyalkanoates, resulting in the identification of 104 metabolites and related molecules in thermal chlorophototrophic microbial mat cores collected over a diel cycle in Mushroom Spring, Yellowstone National Park. A limited number of predominant taxa inhabit this community and their functional potentials have been previously identified through metagenomic and metatranscriptomic analyses and in situ metabolisms, and metabolic interactions among these taxa have been hypothesized. Our metabolomics results confirmed the diel cycling of photorespiration (e.g., glycolate) and fermentation (e.g., acetate, propionate, and lactate) products, the carbon storage polymers polyhydroxyalkanoates, and dissolved gasses (e.g., H2 and CO2) in the waters overlying the mat, which were hypothesized to occur in major mat chlorophototrophic community members. In addition, we have formulated the following new hypotheses: (1) the morning hours are a time of biosynthesis of amino acids, DNA, and RNA; (2) photo-inhibited cells may also produce lactate via fermentation as an alternate metabolism; (3) glycolate and lactate are exchanged among Synechococcus and Roseiflexus spp.; and (4) fluctuations in many metabolite pools (e.g., wax esters) at different times of day result from species found at different depths within the mat responding to temporal differences in their niches.

In Situ Hydrogen Dynamics in a Hot Spring Microbial Mat during a Diel Cycle

“…High levels of nitrogenase gene expression (16,29,30) and N 2 fixation activity (30) have been observed at sunset in the Octopus Spring and Mushroom Spring mats. While we cannot rule out the possibility that all H 2 production in the afternoon-evening transmission was due to nitrogenase activity, the massive accumulation of fermentation products shown in dark-incubated mats from Mushroom Spring (19,22) and the very low nighttime pH values measured in this study provide evidence that fermentation might be another source of H 2 accumulation in the mat. Previously measured accumulations of acetate plus propionate in dark-incubated vials containing a 1-cm-deep core of the mat (22) thus indicate that volatile fatty acids may build up to tens of millimolar concentrations in the upper millimeters of the mat during the night.…”

“…Earlier studies have also reported H 2 formation in hot spring microbial mats, where H 2 production may be due to fermentation (18,19,22,36) or to formation as a by-product of nitrogenase activity during N 2 fixation (30). The relative importance of these processes for H 2 dynamics in hot spring cyanobacterial communities has remained unclear, while studies of hypersaline and coastal cyanobacterial mats have identified fermentation as the major H 2 source (7-9).…”

“…It is not possible to estimate how much of this photosynthetic activity was due to the oxygenic cyanobacteria or the anoxygenic phototrophs in the mat, but photomixotrophic anoxygenic metabolism has been documented for the filamentous anoxygenic phototrophic bacteria, Chloroflexus spp. and Roseiflexus spp., that are abundant in the Mushroom Spring mat (19), and these organisms may thus both fix CO 2 and photoassimilate volatile fatty acids (VFAs) (22).…”

“…Such relatively limited diversity has made these hot spring mats attractive for the study of metabolic interactions among taxa under in situ conditions and under a natural diel cycle. In the slightly alkaline 50 to 70°C Octopus Spring mat, for example, it was shown that the two most abundant taxa, Synechococcus and Roseiflexus, produce glycogen (18) (and in Roseiflexus, polyhydroxyalkanoic acid as well [17,19]), which they ferment at nighttime to produce large amounts of acetate and propionate that are photoassimilated by filamentous, Chloroflexus-like organisms (probably related to Roseiflexus [20]) the next morning (19,21,22). Both Anderson et al (22) and van der Meer (23) reported that H 2 was formed during darkness in the mats, but the accumulation was restricted by diffusion into the overlying water (19) and by H 2 consumption by SRB (24,25) and methanogenic bacteria (21,26) in the 50 to 60°C mat.…”

Metagenomics of microbial and viral life in terrestrial geothermal environments

“…A combined approach of these methods, indeed, was recently used to identify two novel candidate phyla, Calescamantes and Candidatus kryptonia, by the analysis of different SAGs and metagenomic databases collected in different high-temperature environments (Eloe-Fadrosh et al 2016;Kim et al 2015) proving that, although metagenomics and single-cell genomics are informative of their own, the results of the mixed approach could be greater than the sum of their parts.…”