Metabolites Associated with Adaptation of Microorganisms to an Acidophilic, Metal-Rich Environment Identified by Stable-Isotope-Enabled Metabolomics

Abstract

Microorganisms grow under a remarkable range of extreme conditions. Environmental transcriptomic and proteomic studies have highlighted metabolic pathways active in extremophilic communities. However, metabolites directly linked to their physiology are less well defined because metabolomics methods lag behind other omics technologies due to a wide range of experimental complexities often associated with the environmental matrix. We identified key metabolites associated with acidophilic and metal-tolerant microorganisms using stable isotope labeling coupled with untargeted, high-resolution mass spectrometry. We observed >3,500 metabolic features in biofilms growing in pH ~0.9 acid mine drainage solutions containing millimolar concentrations of iron, sulfate, zinc, copper, and arsenic. Stable isotope labeling improved chemical formula prediction by >50% for larger metabolites (>250 atomic mass units), many of which were unrepresented in metabolic databases and may represent novel compounds. Taurine and hydroxyectoine were identified and likely provide protection from osmotic stress in the biofilms. Community genomic, transcriptomic, and proteomic data implicate fungi in taurine metabolism. Leptospirillum group II bacteria decrease production of ectoine and hydroxyectoine as biofilms mature, suggesting that biofilm structure provides some resistance to high metal and proton concentrations. The combination of taurine, ectoine, and hydroxyectoine may also constitute a sulfur, nitrogen, and carbon currency in the communities.

Elevated temperature alters proteomic responses of individual organisms within a biofilm community

“…The abundance estimates of archaea, the Leptospirillum group II 5-way genotype, Sulfobacillus and other bacteria (including Actinobacteria and Firmicutes, based on proteomic measurements) were similar between the mine biofilm and the 40 1C bioreactor biofilm. As seen previously (Mosier et al, 2013), the bioreactor biofilms had a much higher proportion of Leptospirillum group III bacteria than that seen in the in situ mine biofilm (31 to 41% compared with only 2% in the mine; Supplementary Figure S3). Leptospirillum Group III has been shown to dominate AMD biofilms in solutions with low Fe(II)/Fe(III) ratios (Spaulding et al, unpublished data).…”

Transcriptional regulation of ectoine catabolism in response to multiple metabolic and environmental cues

“…The co-occurrence of microbial hydroxyectoine/ectoine producers and consumers in the same habitat (Schwibbert et al, 2011;Widderich et al, 2014;2016b;Schulz et al, 2017) drives ecophysiological relevant networks of synthesis, release and catabolism of compounds that are produced in abundance by many osmotically stressed bacterial cells (Pastor et al, 2010;Kunte et al, 2014;Widderich et al, 2014). For the exploitation of externally provided ectoines as nutrients, it is essential that the consumers can sensitively detect the presence of these compounds in their surroundings (Welsh, 2000;Mosier et al, 2013;Warren, 2014;2016), so that they can trigger enhanced expression of those genes whose products mediate import and catabolism of ectoines (Jebbar et al, 2005;Lecher et al, 2009;Schwibbert et al, 2011;Schulz et al, 2017;Yu et al, 2017).…”

“…These compounds are released into the environment from producer microorganisms either through the transient opening of mechanosensitive channels as a consequence of osmotic down-shocks, through secretion, or on cellular decomposition (Welsh, 2000;Grammann et al, 2002;Booth, 2014;Widderich et al, 2016b). Given the wide occurrence of microorganisms capable of synthesizing ectoines (Widderich et al, 2014;2016b), it does not come as a surprise that these compounds have been detected in different ecosystems (Mosier et al, 2013;Warren, 2014;2016).…”

Sequencing and beyond: integrating molecular 'omics' for microbial community profiling

“…This is due in part to the fact that ex vivo environmental backgrounds (such as soil) possess properties that make them less amenable to standard metabolomics practices (such as high salt concentrations). However, a recent study proposed a metabolomics protocol that was specifically adapted for environmental samples through a stable isotope labeling step 71 . Samples from the environmental community of interest (in this case an acid mine drainage site) were cultured with 15 N-labeled ammonium sulfate as a nitrogen source.…”