Abstract
Stable isotope probing (SIP) is a valuable tool for gaining insights into ecophysiology and biogeochemical cycling of environmental microbial communities by tracking isotopically labeled compounds into cellular macromolecules as well as into byproducts of respiration. SIP, in conjunction with nanoscale secondary ion mass spectrometry (NanoSIMS), allows for the visualization of isotope incorporation at the single cell level. In this manner, both active cells within a diverse population as well as heterogeneity in metabolism within a homogeneous population can be observed. The ecophysiological implications of these single cell stable isotope measurements are often limited to the taxonomic resolution of paired fluorescence in situ hybridization (FISH) microscopy. Here we introduce a taxonomy-independent method using multi-isotope SIP and NanoSIMS for identifying and grouping phenotypically similar microbial cells by their chemical and isotopic fingerprint. This method was applied to SIP experiments in a sulfur-cycling biofilm collected from sulfidic intertidal vents amended with 13C-acetate, 15N-ammonium, and 33S-sulfate. Using a cluster analysis technique based on fuzzy c-means to group cells according to their isotope (13C/12C, 15N/14N, and 33S/32S) and elemental ratio (C/CN and S/CN) profiles, our analysis partitioned ~2200 cellular regions of interest (ROIs) into five distinct groups. These isotope phenotype groupings are reflective of the variation in labeled substrate uptake by cells in a multispecies metabolic network dominated by Gamma- and Deltaproteobacteria. Populations independently grouped by isotope phenotype were subsequently compared with paired FISH data, demonstrating a single coherent deltaproteobacterial cluster and multiple gammaproteobacterial groups, highlighting the distinct ecophysiologies of spatially-associated microbes within the sulfur-cycling biofilm from White Point Beach, CA.
Highlights
The application of stable isotope probing (SIP) to environmental microbial communities provides links between ecophysiology and phylogenetic identity without the need for pure or enrichment cultures (Radajewski et al, 2000; Dumont and Murrell, 2005)
The relative abundance of Deltaproteobacteria increased over the course of the experiment: 2 days—3.0%; 7 days—6.8%; 10 days—44.7%
A total of 21 fluorescence in situ hybridization (FISH) mapped regions (30 × 30 μm) on five incubated Si-wafers were examined by NanoSIMS and subsequently processed using Look@NanoSIMS resulting in 3115 cellular regions of interest (ROIs) (Table 1, Figure S-4)
Summary
The application of stable isotope probing (SIP) to environmental microbial communities provides links between ecophysiology and phylogenetic identity without the need for pure or enrichment cultures (Radajewski et al, 2000; Dumont and Murrell, 2005). NanoSIMS analyses have previously been applied to measure naturally occurring micron-scale variations in δ34S of sulfide resulting from microbial sulfur metabolism in environmental samples (Fike et al, 2008, 2009), and 34S-enriched sulfate SIP experiments combined with NanoSIMS have demonstrated the assimilation of 34S into cell biomass (Milucka et al, 2012; Wilbanks et al, 2014). These studies focused on the variation in the ratio of 34S/32S. The existence of four stable isotopes of sulfur (32S, 33S, 34S, and 36S) and the ability of the CAMECA NanoSIMS 50L instrument to measure seven masses in parallel offers the potential for concurrent SIP NanoSIMS experiments with multiple sulfur species and isotope labels, as well as the potential to conduct mixed substrate incubation experiments that expand beyond 13C- and 15N-labeled substrate amendment to include multiple isotopes of sulfur
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