Quantitative microbial ecology through stable isotope probing

Abstract

24Bacteria grow and transform elements at different rates, yet quantifying this variation in the 25 environment is difficult. Determining isotope enrichment with fine taxonomic resolution after 26 exposure to isotope tracers could help, but there are few suitable techniques. We propose a 27 modification to Stable Isotope Probing (SIP) that enables determining the isotopic composition 28 of DNA from individual bacterial taxa after exposure to isotope tracers. In our modification, after 29 isopycnic centrifugation, DNA is collected in multiple density fractions, and each fraction is 30 sequenced separately. Taxon specific density curves are produced for labeled and non-labeled 31 treatments, from which the shift in density for each individual taxon in response to isotope 32 labeling is calculated. Expressing each taxon's density shift relative to that taxon's density 33 measured without isotope enrichment accounts for the influence of nucleic acid composition on 34 density and isolates the influence of isotope tracer assimilation.

Estimating taxon‐specific population dynamics in diverse microbial communities

“…To estimate taxon-specific bacterial growth and mortality rates from these data, we extended the model of 18 O isotope substitution in DNA presented by Hungate et al (2015) to include an exponential model of population growth. We used a mixing model of DNA molecular weight to estimate abundances of unlabeled and labeled DNA fragments containing copies of 16S rRNA genes.…”

“…We used a mixing model of DNA molecular weight to estimate abundances of unlabeled and labeled DNA fragments containing copies of 16S rRNA genes. The full set of equations describing the model builds upon those presented in Hungate et al (2015) and is summarized here.…”

“…Taxon-specific abundances of 16S rRNA gene copies at the beginning (N TOTALi0 = N LIGHTi0 ) and the end of the incubation (N TOTALit ) were calculated as the products of the total abundance of 16S rRNA gene copies across all taxa, determined by qPCR, and the relative abundance of 16S rRNA gene copies associated with taxon i, determined by sequencing. We used a linear mixing model of DNA molecular weights to estimate the abundance of labeled 16S rRNA gene copies at the end of the incubation (N HEAVYit ), and by difference, the abundance of unlabeled 16S rRNA gene copies at the end of the incubation (N LIGHTit ): Hungate et al 2015). However, as oxygen in DNA is derived from both water and organic sources, we also estimated the maximum molecular weight of DNA that could result from assimilation of 18 O-water (M HEAVYi ).…”

“…Stable-isotope probing applications typically yield categorical indices of growth and may have a bias against detecting activity in microbes with low guanine and cytosine (GC) content (Schwartz et al 2016). However, a recent improvement to the SIP method, termed quantitative stable-isotope probing (qSIP), uses sequencing and a model of isotope incorporation into DNA to estimate isotopic enrichment for all bacterial and archaeal taxa within natural or engineered assemblages (Hungate et al 2015). Quantitatively assessing microbial activity with this technique has already expanded our ability to answer important ecological questions.…”

Identifying protist consumers of photosynthetic picoeukaryotes in the surface ocean using stable isotope probing

“…The barcoded PCR approach was similar to the method used previously for identification of 13 C-labelled 16S rRNA genes (Orsi et al, 2016), where each separate fraction of the CsCl gradient is PCR amplified using primers with a unique barcode. After highthroughput sequencing and OTU clustering, this approach allows for identification of individual OTUs that display a density shift in the 13 C-labelled treatment relative to the unlabelled treatment (Hungate et al, 2015). PCR reactions were carried out in 25 ml volumes for using the following thermocycling conditions: 988C for 30 s; 10 cycles of 988C for 10 s, 538C for 30 s, 728C for 30 s; 20 cycles of 988C for 10 s, 488C for 30 s, 728C for 30s; with a final extension at 728C for 5 min.…”