Final Report for DOE grant no. DE-FG02-04ER63883: Can soil genomics predict the impact of precipitation on nitrous oxide flux from soil

Nitrous oxide is a potent greenhouse gas that is released by microorganisms in soil. However, the production of nitrous oxide in soil is highly variable and difficult to predict. Future climate change may have large impacts on nitrous oxide release through alteration of precipitation patterns. We analyzed DNA extracted from soil in order to uncover relationships between microbial processes, abundance of particular DNA sequences and net nitrous oxide fluxes from soil. Denitrification, a microbial process in which nitrate is used as an electron acceptor, correlated with nitrous oxide flux from soil. The abundance of ammonia oxidizing archaea correlated positively, but weakly, with nitrous oxide production in soil. The abundance of bacterial genes in soil was negatively correlated with gross nitrogen mineralization rates and nitrous oxide release from soil. We suggest that the most important control over nitrous oxide production in soil is the growth and death of microorganisms. When organisms are growing nitrogen is incorporated into their biomass and nitrous oxide flux is low. In contrast, when microorganisms die, due to predation or infection by viruses, inorganic nitrogen is released into the soil resulting in nitrous oxide release. Higher rates of precipitation increase access to microorganisms by predators or viruses through filling large soil pores with water and therefore can lead to large releases of nitrous oxide from soil. We developed a new technique, stable isotope probing with 18O-water, to study growth and mortality of microorganisms in soil

mRNA, rRNA and DNA Quantitative Stable Isotope Probing with H218O Indicates Use of Old rRNA among Soil Thaumarchaeota

RNA is considered to be a short-lived molecule, indicative of cellular metabolic activity, whereas DNA is thoughtto turn over more slowly because living cells do not always grow and divide. To explore differences in the ratesof synthesis of these nucleic acids, we used H218O quantitative stable isotope probing (qSIP) to measure theincorporation of18O into 16S rRNA, the 16S rDNA,amoAmRNA and theamoAgene of soil Thaumarchaeota.Incorporation of18O into the thaumarchaealamoAmRNA pool was faster than into the 16S rRNA pool,suggesting that Thaumarchaea were metabolically active while using rRNA molecules that were likely synthe-tized prior to H218O addition. Assimilation rates of18O into 16S rDNA andamoAgenes were similar, which wasexpected because both genes are present in the same thaumarchaeal genome. The Thaumarchaea had sig-nificantly higher rRNA to rDNA ratios than bacteria, though the18O isotopic signature of thaumarchaeal rRNAwas lower than that of bacterial rRNA, further suggesting preservation of old non-labeled rRNA. Through qSIP ofsoil with H218O, we showed that18O incorporation into thaumarchaeal nucleic acids was generally low, in-dicating slower turnover rates compared to bacteria, and potentially suggesting thaumarchaeal capability forpreservation and efficient reuse of biomolecules

Using metabolic tracer techniques to assess the impact of tillage and straw management on microbial carbon use efficiency in soil

a b s t r a c t Tillage practices and straw management can affect soil microbial activities with consequences for soil organic carbon (C) dynamics. Microorganisms metabolize soil organic C and in doing so gain energy and building blocks for biosynthesis, and release CO 2 to the atmosphere. Insight into the response of microbial metabolic processes and C use efficiency (CUE; microbial C produced per substrate C utilized) to management practices may therefore help to predict long term changes in soil C stocks. In this study, we assessed the effects of reduced (RT) and conventional tillage (CT) on the microbial central C metabolic network, using soil samples from a 12-year-old field experiment in an Irish winter wheat cropping system. Straw was removed from half of the RT and CT plots after harvest or incorporated into the soil in the other half, resulting in four treatment combinations. We added 1-13 C and 2,3-13 C pyruvate and 1-13 C and U-13 C glucose as metabolic tracer isotopomers to composite soil samples taken at two depths (0e15 cm and 15e30 cm) from each of the treatments and used the rate of position-specific respired 13 CO 2 to parameterize a metabolic model. Model outcomes were then used to calculate CUE of the microbial community. Whereas the composite samples differed in CUE, the changes were small, with values ranging between 0.757 and 0.783 across treatments and soil depth. Increases in CUE were associated with a reduced tricarboxylic acid cycle and reductive pentose phosphate pathway activity and increased consumption of metabolic intermediates for biosynthesis. Our results suggest that RT and straw incorporation do not substantially affect CUE

Estimating taxon-specific population dynamics in diverse microbial communities

Understanding how population-level dynamics contribute to ecosystem-level processes is a primary focus of ecological research and has led to important breakthroughs in the ecology of macroscopic organisms. However, the inability to measure population-specific rates, such as growth, for microbial taxa within natural assemblages has limited ecologists’ understanding of how microbial populations interact to regulate ecosystem processes. Here, we use isotope incorporation within DNA molecules to model taxon- specific population growth in the presence of 18O-labeled water. By applying this model to phylogenetic marker sequencing data collected from stable-isotope probing studies, we estimate rates of growth, mortal- ity, and turnover for individual microbial populations within soil assemblages. When summed across the entire bacterial community, our taxon-specific estimates are within the range of other whole-assemblage measurements of bacterial turnover. Because it can be applied to environmental samples, the approach we present is broadly applicable to measuring population growth, mortality, and associated biogeochemical process rates of microbial taxa for a wide range of ecosystems and can help reveal how individual microbial populations drive biogeochemical fluxes

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\u27s 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 (13)C and (18)O 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.The ISME Journal advance online publication, 4 March 2016; doi:10.1038/ismej.2016.28

Sequence of a tomato gene encoding a third type of LHCII chlorophyll a/b -binding polypeptide

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/43429/1/11103_2004_Article_BF00037074.pd

Determination of steady-state mRNA levels of individual chlorophyll a/b binding protein genes of the tomato cab gene family

The steady-state levels of mRNA produced by 14 genes encoding members of the tomtato chlorophyll a/b binding protein family were quantified. All genes were found to be expressed in leaf tissue, but the mRNAs accumulated to significantly different levels. The transcripts of cab 1A, cab 1B, cab 3A and cab 3B, encoding the Type I LHC proteins of photosystem II, are abundant, while low levels were measured for mRNAs encoding the Type II LHC II and the LHC I proteins. Sequences from the 5′ upstream regions (−400 to translational start) of some cab genes were determined in this study, and a total of 16 tomato cab gene promoters for which sequences are now available were analyzed. Significant sequence conservation was found for those genes which are tandemly linked on the chromosome. However, the level of sequence conservation is different for the different cab subfamilies, e.g. 85% similarity between cab 1A and cab 1D vs. 45% sequence similarity between cab 3A and cab 3C upstream sequences. Characteristic GATA repeats with a conserved spacing were found in 5′ upstream sequences of cab 1AD, cab 3 A-C, cab 11 and cab 12. The consensus sequence CCTTATCAT, which is believed to mediate light responsiveness, was found at different locations in the upstream sequences of cab 6B, cab 7, cab 8, cab 9, cab 10A, cab 10B and cab 11. In 11 out of 15 genes the transcription initiation site was found to center on the triplet TCA.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47581/1/438_2004_Article_BF00280298.pd

Strategic positioning of the ‘ERATOSTHENES Research Centre’ and exploration of new R&D opportunities in the fields of Earth Surveillance and Space-Based of the Environment

The aim of this paper is to present our strategy and vision to upgrade the existing ERATOSTHENES Research Centre (ERC), established within Cyprus University of Technology (CUT), into a sustainable, viable and autonomous Centre of Excellence (CoE) for Earth Surveillance and Space-Based Monitoring of the Environment (EXCELSIOR), which will provide the highest quality of related services both on the National, European and International levels. The ‘EXCELSIOR’ project is a Horizon 2020 Teaming project, addressing the reduction of substantial disparities in the European Union by supporting research and innovation activities and systems in low performing countries. It also aims at establishing long-term and strategic partnerships between the Teaming partners, thus reducing internal research and innovation disparities within European Research and Innovation landscape. The ERCis already an established player in the local community and has excellent active collaboration with actors from various sectors in (a) the government, (b) industry, (c) local organisations, and (d) society. In order to further engage users and citizens and to become more attractive to international research and education community, the Centre aims to be fully involved in strategic positioning on the national level, but also in Europe, the Middle East region and internationally. Some examples of how space technologies are integrated with other tools or techniques such as UAV, field spectroscopy, micro-sensors, EO space/in-situ sensors etc. for the systematic monitoring of the environment is shown. Indeed such examples fulfills the objectives of the COPERNICUS academy network (in which ERC is a member) for empowering the next generation of researchers, scientists, and entrepreneurs with suitable skill sets to use Copernicus data and information services to their full potential. Finally, opportunities for future collaboration and investments with the ERC in the Eastern Mediterranean Region are stated. Five partners have united to upgrade the existing ERC into a CoE, with the common vision to become a world-class innovation, research and education centre, actively contributing to the European Research Area (ERA). More specifically, the Teaming project is a team effort between the Cyprus University of Technology (CUT, acting as the coordinator), the German Aerospace Centre (DLR), the Institute for Astronomy and Astrophysics Space Applications and Remote Sensing of the National Observatory of Athens (NOA), the German Leibniz Institute for Tropospheric Research (TROPOS) and the Cyprus’ Department of Electronic Communications of the Ministry of Transport, Communications and Works (DEC-MTCW)

Characterization of Growing Microorganisms in Soil by Stable Isotope Probing with H(2)(18)O

A new approach to characterize growing microorganisms in environmental samples based on labeling microbial DNA with H(2)(18)O is described. To test if sufficient amounts of (18)O could be incorporated into DNA to use water as a labeling substrate for stable isotope probing, Escherichia coli DNA was labeled by cultivating bacteria in Luria broth with H(2)(18)O and labeled DNA was separated from [(16)O]DNA on a cesium chloride gradient. Soil samples were incubated with H(2)(18)O for 6, 14, or 21 days, and isopycnic centrifugation of the soil DNA showed the formation of two bands after 6 days and three bands after 14 or 21 days, indicating that (18)O can be used in the stable isotope probing of soil samples. DNA extracted from soil incubated for 21 days with H(2)(18)O was fractionated after isopycnic centrifugation and DNA from 17 subsamples was used in terminal restriction fragment length polymorphism (TRFLP) analysis of bacterial 16S rRNA genes. The TRFLP patterns clustered into three groups that corresponded to the three DNA bands. The fraction of total fluorescence contributed by individual terminal restriction fragments (TRF) to a TRFLP pattern varied across the 17 subsamples so that a TRF was more prominent in only one of the three bands. Labeling soil DNA with H(2)(18)O allows the identification of newly grown cells. In addition, cells that survive but do not divide during an incubation period can also be characterized with this new technique because their DNA remains without the label

Stable Isotope Probing with ^(18)O-Water to Investigate Growth and Mortality of Ammonia Oxidizing Bacteria and Archaea in Soil

Ammonia oxidizing bacteria (AOB) and archaea oxidize ammonia to nitrite, the first and rate-limiting step in the important ecosystem process of nitrification. Growth and mortality of ammonia oxidizers in soil are difficult to quantify but accurate measurements would offer important insights into how environmental parameters regulate the population dynamics of these organisms. Stable isotope probing (SIP) is a recently developed technique that can identify microorganisms that assimilate labeled substrates and can be adapted to quantify the growth of organisms in soil. Here, we describe the use of SIP with ^(18)O-water to investigate the growth and mortality of ammonia oxidizers in a soil taken from a ponderosa pine forest in northern Arizona, USA. Addition of ammonia to soil stimulated the growth of AOB but not ammonia oxidizing archaea (AOA). The mortality of AOA was increased upon addition of ammonia to soil; however, the variance in these measurements was high. The mortality of AOB, in contrast, was not impacted by addition of ammonia to soil. The results suggest that increased ammonia availability in soil favors AOB over AOA