Benjamin J. Koch

Quantitative Microbial Ecology through Stable Isotope Probing

ABSTRACT Bacteria grow and transform elements at different rates, and as yet, quantifying this variation in the environment is difficult. Determining isotope enrichment with fine taxonomic resolution after exposure to isotope tracers could help, but there are few suitable techniques. We propose a modification to stable isotope probing (SIP) that enables the isotopic composition of DNA from individual bacterial taxa after exposure to isotope tracers to be determined. In our modification, after isopycnic centrifugation, DNA is collected in multiple density fractions, and each fraction is sequenced separately. Taxon-specific density curves are produced for labeled and nonlabeled treatments, from which the shift in density for each individual taxon in response to isotope labeling is calculated. Expressing each taxons density shift relative to that taxons density measured without isotope enrichment accounts for the influence of nucleic acid composition on density and isolates the influence of isotope tracer assimilation. The shift in density translates quantitatively to isotopic enrichment. Because this revision to SIP allows quantitative measurements of isotope enrichment, we propose to call it quantitative stable isotope probing (qSIP). We demonstrated qSIP using soil incubations, in which soil bacteria exhibited strong taxonomic variations in 18O and 13C composition after exposure to [18O]water or [13C]glucose. The addition of glucose increased the assimilation of 18O into DNA from [18O]water. However, the increase in 18O assimilation was greater than expected based on utilization of glucose-derived carbon alone, because the addition of glucose indirectly stimulated bacteria to utilize other substrates for growth. This example illustrates the benefit of a quantitative approach to stable isotope probing.

Phylogenetic organization of bacterial activity

Phylogeny is an ecologically meaningful way to classify plants and animals, as closely related taxa frequently have similar ecological characteristics, functional traits and effects on ecosystem processes. For bacteria, however, phylogeny has been argued to be an unreliable indicator of an organism’s ecology owing to evolutionary processes more common to microbes such as gene loss and lateral gene transfer, as well as convergent evolution. Here we use advanced stable isotope probing with 13C and 18O to show that evolutionary history has ecological significance for in situ bacterial activity. Phylogenetic organization in the activity of bacteria sets the stage for characterizing the functional attributes of bacterial taxonomic groups. Connecting identity with function in this way will allow scientists to begin building a mechanistic understanding of how bacterial community composition regulates critical ecosystem functions.

Ominous projections for global antibiotic use in food-animal production

Alexander Fleming famously warned that the ignorant may someday misuse his life-saving discovery—penicillin—and select for resistant bacteria (1). This was prescient given the widespread use of subtherapeutic antibiotics by food-animal producers today. According to the findings of Van Boeckel et al. (2) in PNAS, the proliferation of ignorance is only poised to increase. Using global datasets of veterinary antibiotic use, livestock densities, and economic projections of meat demand, Van Boeckel et al. (2) estimate that from 2010 to 2030 antibiotic use in food-animal production will increase by 67%, from 63,151 ± 1,560 tons to 105,596 ± 3,605 tons.

Bacterial carbon use plasticity, phylogenetic diversity and the priming of soil organic matter

Microorganisms perform most decomposition on Earth, mediating carbon (C) loss from ecosystems, and thereby influencing climate. Yet, how variation in the identity and composition of microbial communities influences ecosystem C balance is far from clear. Using quantitative stable isotope probing of DNA, we show how individual bacterial taxa influence soil C cycling following the addition of labile C (glucose). Specifically, we show that increased decomposition of soil C in response to added glucose (positive priming) occurs as a phylogenetically diverse group of taxa, accounting for a large proportion of the bacterial community, shift toward additional soil C use for growth. Our findings suggest that many microbial taxa exhibit C use plasticity, as most taxa altered their use of glucose and soil organic matter depending upon environmental conditions. In contrast, bacteria that exhibit other responses to glucose (reduced growth or reliance on glucose for additional growth) clustered strongly by phylogeny. These results suggest that positive priming is likely the prototypical response of bacteria to sustained labile C addition, consistent with the widespread occurrence of the positive priming effect in nature.

Stable isotope probing with 18O-water to investigate microbial growth and death in environmental samples

Growth and mortality of microorganisms have been characterized through DNA stable isotope probing (SIP) with 18O-water in soils from a range of ecosystems. Conventional SIP has been improved by sequencing a marker gene in all fractions retrieved from an ultracentrifuge tube to produce taxon density curves, which allow estimating the atom percent isotope composition of each microbial taxons genome. Very recent advances in SIP with 18O-water include expansion of the technique to aquatic samples, investigations of microbial turnover in soil, and the first use of 18O-water in RNA-SIP studies.

Water Ecosystem Services: Operationalizing an ecosystem services-based approach for managing river biodiversity

4.1 INTRODUCTION Covering less than 1% of the Earths surface, freshwater – streams, rivers, ponds, wetlands, and lakes – supports as much as 10% of all animal species, including one-third of all vertebrates (Strayer & Dudgeon 2010; Figure 4.1). While being among the most biologically diverse, freshwater ecosystems are also among the most imperiled on the planet. In developed regions such as Europe and USA, more than 30% of freshwater species are now thought to be threatened or extinct. As with the majority of the worlds ecosystems, accelerated rates of human population growth, industrialization, and agricultural intensification are driving these dramatic species losses, with invasive species introductions, over-harvesting, and loss of habitats being the primary causes (Dudgeon 2013). Reducing freshwater biodiversity loss is at odds with managing many other ecosystem services. Much of the conflict lies in the fact that freshwaters are ‘hot spots’ for both human needs (i.e. drinking, irrigation, transportation) as well as biodiversity (i.e. the number or suite of species native to a given area; Strayer & Dudgeon 2010; Leisher, this book). Moreover, the majority of the worlds population is facing, and will continue to face, increasing water scarcity (Vorosmarty et al . 2010). Humans rely heavily on freshwater, and in particular from large rivers for hydropower, transportation, and fisheries. Water abstraction further strains the integrity of a rivers biophysical components, including biodiversity. While some aspects of freshwater ecosystem services are renewable (e.g. water supply), biodiversity is not. This chapter seeks to clarify how ecosystem service-based approaches can be put into practice (‘operationalized’) for the purpose of prioritizing freshwater biodiversity alongside other river services. We propose a framework that is meant to serve as a way to explore trade-offs in ecosystem services delivery. We base this operationalization on three typical perspectives associated with freshwater biodiversity: inherent value as a final service, biodiversity–ecosystem function (an intermediate or provisioning service), and some combination of the two.

Identification of Growing Bacteria During Litter Decomposition In Freshwater through H218O Quantitative Stable Isotope probing

Identification of microorganisms that facilitate the cycling of nutrients in freshwater is paramount to understanding how these ecosystems function. Here, we identify growing aquatic bacteria using H218O quantitative stable isotope probing. During 8 day incubations in 97 atom % H218O, 54% of the taxa grew. The most abundant phyla among growing taxa were Proteobacteria (45%), Bacteroidetes (30%) and Firmicutes (10%). Taxa differed in isotopic enrichment, reflecting variation in DNA replication of bacterial populations. At the class level, the highest atom fraction excess was observed for OPB41 and δ-Proteobacteria. There was no linear relationship between 18 O incorporation and abundance of taxa. δ-Proteobacteria and OPB41 were not abundant, yet the DNA of both taxa was highly enriched in 18 O. Bacteriodetes, in contrast, were abundant but not highly enriched. Our study shows that a large proportion of the bacterial taxa found on decomposing leaf litter grew slowly, and several low abundance taxa were highly enriched. These findings indicating that rare organisms may be important for the decomposition of leaf litter in streams, and that quantitative stable isotope probing with H218O can be used to advance our understanding of microorganisms in freshwater by identifying species that are growing in complex communities.

Colonizing opportunistic pathogens (COPs): The beasts in all of us.

1 Milken Institute School of Public Health, George Washington University, Washington DC, United States of America, 2 Division of Pathogen Genomics, Translational Genomics Research Institute, Flagstaff, Arizona, United States of America, 3 Center for Ecosystem Science and Society, Northern Arizona University, Flagstaff, Arizona, United States of America, 4 Department of Biological Sciences, Northern Arizona University, Flagstaff, Arizona, United States of America

Quantitative stable isotope probing with H 2 18 O reveals that most bacterial taxa in soil synthesize new ribosomal RNA

Most soil bacterial taxa are thought to be dormant, or inactive, yet the extent to which they synthetize new rRNA is poorly understood. We analyzed 18O composition of RNA extracted from soil incubated with H218O and used quantitative stable isotope probing to characterize rRNA synthesis among microbial taxa. RNA was not fully labeled with 18O, peaking at a mean of 23.6 ± 6.8 atom percent excess (APE) 18O after eight days of incubation, suggesting some ribonucleotides in soil were more than eight days old. Microbial taxa varied in the degree they incorporated 18O into their rRNA over time and there was no correlation between the APE18O of bacterial rRNA and their rRNA to DNA ratios, suggesting that the ratios were not appropriate to measure ribonucleotide synthesis. Our study indicates that, on average, 94% of soil taxa produced new rRNA and therefore were metabolically active.

Ecosystem responses to restored flow in a travertine river

Disruptions of natural flow impair rivers and streams worldwide. Those conducting restoration efforts have rarely explored how and when stream ecosystems can recover after reinstating natural flows. We quantified responses of ecosystem metabolism and N dynamics to the decommissioning and removal of a 100-y-old diversion dam in a desert stream, Fossil Creek, Arizona. Fossil Creek is a travertine river, meaning that CaCO3 concentrations in water in the springs that feed Fossil Creek are high enough to precipitate out of the water to form travertine terraces and deep pools. The majority of flow was diverted for power generation, so travertine deposition rates were significantly reduced and travertine terraces were smaller and less frequent compared to pre-dam historical records. Flow restoration enabled the recovery of the geochemical process of travertine deposition and increased gross primary production and N uptake to rates comparable to those measured in an upstream, reference reach. Reinstating a river’s natural flow regime can result in rapid and near-complete recovery of fundamental ecosystem processes that reshape the aquatic food web.