Global Transcriptional Response of Escherichia coli Exposed In Situ to Different Low-Dose Ionizing Radiation Sources.

Characterization of biological and chemical responses to ionizing radiation by various organisms is essential for potential applications in bioremediation, alternative modes of detecting nuclear material, and national security. Escherichia coli DH10ß is an optimal system to study the microbial response to low-dose ionizing radiation at the transcriptional level because it is a well-characterized model bacterium and its responses to other environmental stressors, including those to higher radiation doses, have been elucidated in prior studies. In this study, RNA sequencing with downstream transcriptomic analysis (RNA-seq) was employed to characterize the global transcriptional response of stationary-phase E. coli subjected to 239Pu, 3H (tritium), and 55Fe, at an approximate absorbed dose rate of 10 mGy day-1 for 1 day and 15 days. Differential expression analysis identified significant changes in gene expression of E. coli for both short- and long-term exposures. Radionuclide source exposure induced differential expression in E. coli of genes involved in biosynthesis pathways of nuclear envelope components, amino acids, and siderophores, transport systems such as ABC transporters and type II secretion proteins, and initiation of stress response and regulatory systems of temperature stress, the RpoS regulon, and oxidative stress. These findings provide a basic understanding of the relationship between low-dose exposure and biological effect of a model bacterium that is critical for applications in alternative nuclear material detection and bioremediation. IMPORTANCE Escherichia coli strain DH10ß, a well-characterized model bacterium, was subjected to short-term (1-day) and long-term (15-day) exposures to three different in situ radiation sources comprised of radionuclides relevant to nuclear activities to induce a measurable and identifiable genetic response. We found E. coli had both common and unique responses to the three exposures studied, suggesting both dose rate- and radionuclide-specific effects. This study is the first to provide insights into the transcriptional response of a microorganism in short- and long-term exposure to continuous low-dose ionizing radiation with multiple in situ radionuclide sources and the first to examine microbial transcriptional response in stationary phase. Moreover, this work provides a basis for the development of biosensors and informing more robust dose-response relationships to support ecological risk assessment.

Flowthrough of239PU and55FE during RNA extraction.

Analysis of gene expression has become an important tool in understanding low-dose effect mechanisms of ionizing radiation at the cellular level. Metal binding to nucleic acids needs to be considered when interpreting these results, as some radioactive metals, particularly actinides, may produce free radicals and cause oxidative stress damage via chemical means at rates much higher than free radical formation related to their radiological properties. Bacteria exposedin situto low dose rates of plutonium-239 (239Pu) and iron-55 (55Fe) were previously analysed for gene expression. The work herein was motivated by an interest in more precisely identifying the distribution of radionuclides in these bacteria as well as the practical need to ensure appropriate transport and handling of the associated ribonucleic acid (RNA) extractions. RNA extractions were performed on bacteria growth media with and without bacteria cells (i.e. with and without RNA) at several different concentrations of239Pu and55Fe to inform the level of specificity of the extraction membrane as well as provide insight into internal (uptake) vs external (sorption) accumulation of these radionuclides in bacteria cells. Results of the study suggest that239Pu and55Fe detected in RNA extraction samples during long term cell studies is the result of binding to RNA prior to the time of extraction, as opposed to flow through or binding after cell lysis, and it highlights the practical importance of nucleic acid sample characterization to radiation protection more generally.

Accumulation of radio-iron and plutonium, alone and in combination, inPseudomonas putidagrown in liquid cultures.

The impact of low doses of ionising radiation on biological and environmental systems have been historically difficult to study. Modern biological tools have provided new methods for studying these mechanisms but applying these tools to a dose-response relationship may require refinement of dosimetric techniques that incorporate a detailed understand of radionuclide accumulation in biological cells, particularly when assessing the impact of low doses of ionising radiation. In this workPseudomonas putida (KT2440) grown in liquid culture was exposed to low dose rates (10-20 mGy d-1) of239Pu and55Fe, both alone and in combination, for a period of 20 days, and the accumulation of239Pu and55Fe in cell pellets was analysed via liquid scintillation counting. The study also considered of cells grown with239Pu and stable Fe (primarily56Fe). In addition to the analysis of cell pellet and media samples, this work includes analysis of the radiological content of ribonucleic acid extraction samples to examine uptake of radionuclides. Results indicate that239Pu inhibited the uptake of55Fe, and that the presence of stable and radioactive isotopes of Fe in cultures may promote pathways for Fe accumulation that are used by239Pu. The work herein provides foundational insight into future dosimetric models for our work with environmental bacteria.

Pu-239 Accumulation in E. Coli and P. Putida Grown in Liquid Cultures.

ABSTRACT: Understanding of the behavior and effects of plutonium (Pu) in the environment is an important aspect of developing responsible and effective strategies for remediation and environmental stewardship. This work studies the sorption and uptake of 239Pu by common environmental bacteria, Escherichia coli DH10ß and Pseudomonas putida KT-2440. Plutonium was directly incorporated into growth media prior to inoculation (0.12 kBq mL-1), and samples from the liquid cultures of E. coli and P. putida were analyzed over a 15-d growth period through liquid scintillation counting (LSC) of plutonium in cell pellets and cell culture media following centrifugation. To improve its solubility in the liquid cultures, Pu was complexed with citrate prior to inoculation. P. putida cultures were also grown without citrate to examine potential impact of P. putida's ability to use citrate as a food source. The accumulation of Pu in P. putida cells was found to increase both with and without citrate complexation for the first 5 d and then plateau until the end of the study period (15 d). A higher activity concentration of Pu was found in P. putida cells grown with citrate complexation than without. The activity concentration of plutonium in E. coli cells was greater than that in P. putida cells, which may be the result of a stronger complexing agent made by E. coli for the purpose of iron uptake. There are a variety of factors that influence Pu behavior in bacterial systems, and results confirm that even in a simple system, multiple mechanisms are at play.

Integration of ecosystem science into radioecology: A consensus perspective.

In the Fall of 2016 a workshop was held which brought together over 50 scientists from the ecological and radiological fields to discuss feasibility and challenges of reintegrating ecosystem science into radioecology. There is a growing desire to incorporate attributes of ecosystem science into radiological risk assessment and radioecological research more generally, fueled by recent advances in quantification of emergent ecosystem attributes and the desire to accurately reflect impacts of radiological stressors upon ecosystem function. This paper is a synthesis of the discussions and consensus of the workshop participant's responses to three primary questions, which were: 1) How can ecosystem science support radiological risk assessment? 2) What ecosystem level endpoints potentially could be used for radiological risk assessment? and 3) What inference strategies and associated methods would be most appropriate to assess the effects of radionuclides on ecosystem structure and function? The consensus of the participants was that ecosystem science can and should support radiological risk assessment through the incorporation of quantitative metrics that reflect ecosystem functions which are sensitive to radiological contaminants. The participants also agreed that many such endpoints exit or are thought to exit and while many are used in ecological risk assessment currently, additional data need to be collected that link the causal mechanisms of radiological exposure to these endpoints. Finally, the participants agreed that radiological risk assessments must be designed and informed by rigorous statistical frameworks capable of revealing the causal inference tying radiological exposure to the endpoints selected for measurement.