Source: MONTANA STATE UNIVERSITY submitted to NRP
ENVIRONMENTALLY IMPORTANT INTERACTIONS BETWEEN MICROBES AND METALLOIDS, AND THEIR RESPONSE TO UV IRRADIANCE.
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
0168051
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Oct 1, 2010
Project End Date
Sep 30, 2015
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
MONTANA STATE UNIVERSITY
(N/A)
BOZEMAN,MT 59717
Performing Department
Land Resources & Environmental Sciences
Non Technical Summary
Component I While drinking water standards for metalloids have been set and their toxicity established, there remains a very significant gap in our understanding of their behavior in the environment. Currently in the U.S., large amounts of these metalloids are being introduced into the environment on annual basis, yet their fate is either unknown as in the case of Sb, or is known but difficult to control (As). One direct consequence of the proposed study should be a significant improvement in our understanding of microbial processes that may be major factors influencing metalloid speciation, and thus behavior, in the environment. The fundamental nature of the information generated from the proposed project may find application as part of remediation strategies for contaminated sites. For example, there are currently several closed or operating mines where metalloid problems exist, but for which meaningful remedies have not been formulated because of the general lack of understanding and predictability of abiotic and biotic redox processes that influence metalloid solubility and mobility. Component II Component II research is of relevance to MSU, agriculture, and science in at least three ways. First, the experiments examining UV effects on in situ gene expression will be of value to all of the environmental sciences, including the agricultural sciences, for assessing the extent to which natural levels of UV irradiance will influence photosynthesis. This is particularly important given the current evidence of atmospheric changes tied to predictions of significant climate change. Second, YNP is a World Heritage Convention Site and an UNESCO Biosphere Preserve; however, there is a paucity of information for use in assessing the ecological health of YNP (including any temporal changes due to natural or anthropogenic activity). The proposed work represents a critical resource evaluation tool for YNP managers, who depend almost entirely on academic scientists to document organismal diversity and species richness within the park. Finally, the proposed research will also generate information that is critical to YNP's education and outreach efforts that are of high public interest. Each year, approximately three million visitors marvel at YNP's geothermal pools and springs, with some of the most obvious and frequently viewed springs being those that are home to extensive cyanidial mats (e.g. Norris Geyser Basin). As such, these simple algae are visually "front and center" to all park visitors and thus present popular educational tools for the general public. Other broader impacts derived from Component II work (i.e. human resource development, education/outreach, etc.) will be similar to that described above for Component I.
Animal Health Component
(N/A)
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
1040210110020%
1044099110020%
1330199110015%
1330210110015%
1334099110010%
1040199110020%
Knowledge Area
133 - Pollution Prevention and Mitigation; 104 - Protect Soil from Harmful Effects of Natural Elements;

Subject Of Investigation
4099 - Microorganisms, general/other; 0210 - Water resources; 0199 - Soil and land, general;

Field Of Science
1100 - Bacteriology;
Goals / Objectives
This MAES proposal is presented as two components. Each component describes a separate general research thrust that projects the PI's time and effort over the next five year period, and each represents independent research activity. These thrusts involve different aspects of microbial ecophysiology and genetics, and are different in nature and context. Therefore, each are presented individually, coming complete with separate, but necessarily brief, sections that conform to the standard MAES proposal format. COMPONENT I: MICROBE-METALLOID INTERACTIONS Metalloids such as arsenic, selenium and the lesser-understood antimony, continue to be important environmental contaminants associated with mining activity but also with agricultural soils exposed to arsenic-containing pesticides. Efforts to remediate metalloid contamination in these and other environments are hampered by problems associated with predicting their behavior in the environment. A central focus of this work is to examine the genetic and physiologic basis for microbial redox transformations of these metalloids, and is important because it is known that chemical speciation of these metalloids strongly relates with their toxicity and mobility in the environment. This research robustly expands from work described in Component II of the PI's previous MAES proposal. COMPONENT II: ULTRAVIOLET IRRADIANCE EFFECTS ON PHOTOSYNTHETIC MICROORGANISMS Global scale change in atmospheric chemistry is associated with problems that influence all of mankind. One important issue involves increased levels of ultraviolet (UV) irradience and its effects on ecosystem function in the biosphere. Photosynthesis is foundational to biosphere function and the literature already documents that in the laboratory setting UV inhibits photosynthesis and is destructive to phototrophs. Recent efforts by the PI's lab has shown that UV irradiance is a keystone environmental factor that heavily influences population structure and dynamics of structurally and genetically simple eukaryotic algae inhabiting Yellowstone's geothermal features. The research described herein will examine pure culture and in situ gene expression patterns by these algae to better assess how these simple phototrophs respond to UV and thus provide important information as to how photosynthesis is impacted in nature. Expected Outputs Results of the research conducted on this project will be summarized and reported in the form of peer-reviewed publications.
Project Methods
COMPONENT I: MICROBE-METALLOID INTERACTIONS General mutagenesis techniques will be used to introduce the deletion mutations and amino acid replacement mutations in specific regulatory genes. The general goal will be to test the importance of specific regions of the encoded regulatory proteins for functions important to detecting arsenic and for transducing the arsenic signal to influence gene expression. Each resulting mutant will be tested for its ability to oxidize arsenite. Other experiments will be geared towards identifying the mechanism by which the bacterial cell recognizes arsenite. This will involve testing specific peptides for their capacity to bind arsenite and examining periplasmic proteins for their affinity for arsenite. Any protein found to demonstrate arsenite binding ability will be identified using trypsin digestion followed by MALDI-TOF and ESI-LCMS mass spectrometers. Once the protein has been identified, reverse genetics will be employed to create gene-specific mutants that will then be tested for arsenite oxidation. Final experiments will focus on identifying and determining the structure of quorum sensing molecules active in activating the expression of the genes required for arsenite oxidation. Additional experiments will aim to identify genes important to microbial antimony oxidation. Here, transposon mutants (already isolated) that are altered in their reactions to antimony will be characterized, focusing on which gene(s) are responsible for the antimony-specific phenotype. We will then identify homologs in the public databases, and design PCR primers for examining the presence and expression of these genes in environments contaminated with antimony. COMPONENT II: ULTRAVIOLET IRRADIANCE EFFECTS ON PHOTOSYNTHETIC MICROORGANISMS Lemonade Creek in Yellowstone National Park has been selected as the research site because it is prime habitat for the algae under study in terms of temperature and low pH, and because it is a flowing stream that is less susceptible to air temperature effects. Further, it has long stretches that are completely non-shaded and thus are subject to full photoperiod irradiance. Duplicate paired UV and neutral density filter screens will be installed to control for UV exposure, and aqueous sampling and analytical techniques will measure general anion, cation, conductivity, and element analysis in order to determine chemical changes, if any, through the sampling season. Visible and UV irradiance will be measured (IL-1700 radiometer). At monthly intervals, algal biomass samples will be taken to collect RNA to study gene expression patterns as a function of UV exposure vs UV protection. cDNA libraries will be developed using 454-XLS sequencing, and gene expression patterns will be assessed using custom microarrays designed using oligos designed from the genes identified in the cDNA libraries.

Progress 10/01/10 to 09/30/15

Outputs
Target Audience:Target audience = academic, regulatory and private sector scientists working in the area of environmental remediation. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?PARTICIPANTS: Graduate students: Zunji Shi. Postdoc: Yoon-Suk Kang. Undergraduate students: Keenan Brame; PIs: Tim McDermott, Ryan Jones, and Brian Bothner. Partner Organizations: State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, P. R. China. Training or professional development 1 Graduate student, 1 postdoctoral fellow, 1 undergraduate student. How have the results been disseminated to communities of interest?Primarily via scientific, peer-reviewed journals as well as public seminars. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Change in knowledge. Microbe-Metal Interactions: The identification and ongoing characterization of novel regulatory genes and regulatory circuits involved in microbial arsenite oxidation represents important observations that are foundational to our understanding of how and why bacteria sense arsenite in the environment and how their responses are regulated. Major, novel observations illustrate that the genes essential for arsenite oxidation are governed by the phosphate stress response. UV Effects on Eukaryotic Algae: Analysis and comparison of Cyanidioschyzon mat in situ gene expression patterns show the algal cells are responding at the gene expression level to UV as well as VIS inhibition. Additional work was accomplished in ongoing projects that characterize the microbial communities in acidic hot springs in Norris Geyser Basin as well as Yellowstone Lake,Yellowstone National Park

Publications

  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Jingxin Li, Qian Wang, Mingshun Li, Birong Yang, Manman Shi, Wei Guo, Timothy R. McDermott, Christopher Rensing,� and Gejiao Wang. 2015. Proteomics and Genetics for Identification of a Bacterial Antimonite Oxidase in Agrobacterium tumefaciens. Environ. Sci. Technol. 49:59805989
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Zhe Du, Diego A. Riveros-Iregui, Ryan T. Jones, Timothy R. McDermott, John E. Dore, Brian L. McGlynn, Ryan E. Emanuel,Xu Li. 2015. Landscape Position Influences Microbial Composition and Function via Redistribution of Soil Water across a Watershed. Appl. Environ. Microbiol. 81:8457-8468
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Qian Wang, Thomas P. Warelow, Yoon-Suk Kang, Christine Romano, Thomas H. Osborne, Corinne R. Lehr, Brian Bothner, Timothy R. McDermott, Joanne M. Santini, Gejiao Wang. 2015. Arsenite Oxidase Also Functions as an Antimonite Oxidase. Appl. Environ. Microbiol. 81:1959-1965.


Progress 10/01/13 to 09/30/14

Outputs
Target Audience: Primarily academic, government, and industry scientists and their students and postdocs. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? PARTICIPANTS: Graduate students: Zunji Shi. Postdoc: Yoon-Suk Kang. Undergraduate students: Keenan Brame; PIs: Tim McDermott, Ryan Jones, and Brian Bothner. Partner Organizations: State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, P. R. China. Training or professional development 1 Graduate student, 1 postdoctoral fellow, 1 undergraduate student. How have the results been disseminated to communities of interest? Primarily via scientific, peer-reviewed journals. What do you plan to do during the next reporting period to accomplish the goals? Continue on with these primary lines of investigations involving microbe-arsenic interactions, and to finish manuscripts describing theinfluences of UV irradiance on microorganisms.

Impacts
What was accomplished under these goals? Change in knowledge. Microbe-Metal Interactions: The identification and ongoing characterization of novel regulatory genes and regulatory circuits involved in microbial arsenite oxidation represents important observations that are foundational to our understanding of how and why bacteria sense arsenite in the environment and how their responses are regulated. UV Effects on Eukaryotic Algae: Analysis and comparison of Cyanidioschyzon mat in situ gene expression patterns show the algal cells are responding at the gene expression level to UV as well as VIS inhibition. Additional work was accomplished in ongoing projects that characterize the microbial communities in acidic hot springs in Norris Geyser Basin as well as Yellowstone Lake, Yellowstone National Park

Publications

  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Yoon-Suk Kang, Zunji Shi, Brian Bothner, Gejiao Wang and Timothy R. McDermott (2014) Involvement of the Acr3 and DctA anti-porters in arsenite oxidation in Agrobacterium tumefaciens 5A. ENVIRONMENTAL MICROBIOLOGY DOI: 10.1111/1462-2920.12468
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Dana J. Skorupa, Richard W. Castenholz, Aur�lien Mazurie, Charles Carey, Frank Rosenzweig and Timothy R. McDermott. (2014) In situ gene expression profiling of the thermoacidophilic alga Cyanidioschyzon in relation to visible and ultraviolet irradiance. ENVIRONMENTAL MICROBIOLOGY. 16: 16271641,
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Qian Wang, Dong Qin, Shengzhe Zhang, Lu Wang, Jingxin Li, Christopher Rensing, Timothy R. McDermott and Gejiao Wang. Fate of arsenate following arsenite oxidation in Agrobacterium tumefaciens?GW4. ENVIRONMENTAL MICROBIOLOGY. DOI: 10.1111/1462-2920.12465
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2014 Citation: Jinglie Zhou, Dawei Sun, Alyson Childers, Timothy R. McDermott, Yongjie Wang and Mark R. Liles. (2014) Novel virophage genomes discovered from a Yellowstone Lake metagenome. Journal of Viology. In press.


Progress 01/01/13 to 09/30/13

Outputs
Target Audience: Target audiences for Component I research includes land managers (Examples: Forest Service, Bureau of Land Management, Environmental Protection Agency) charged with managing soils or natural waters contaminated with arsenic or other metal(loid)s. Target audiences for Component II research includes managers of Yellowstone National Park who rely on academic scientists to provide them with data that allows them to assess the ecological health of YNP. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Dissemination Education-Outreach field school instruction was conducted for grade school children, for graduate students, high school teachers, and for lay science adult education. PARTICIPANTS: Graduate students: Zunji Shi. Postdocs: Yoon-Suk Kang, Christine Romano. Undergraduate students: Keenan Brame; PIs: Tim McDermott, Ryan Jones, and Brian Bothner. Partner Organizations: State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, P. R. China. Training or professional development 1 Graduate student and 2 postdoctoral fellows How have the results been disseminated to communities of interest? Primarily via scientific, peer-reviewed journals. What do you plan to do during the next reporting period to accomplish the goals? Continue studying and publishing our efforts on microbe-arsenic interactions. There will be less emphasis on Yellowstone research.

Impacts
What was accomplished under these goals? Change in knowledge. Microbe-Metal Interactions: The identification and ongoing characterization of novel regulatory genes and regulatory circuits involved in microbial arsenite oxidation represents important observations that are foundational to our understanding of how and why bacteria sense arsenite in the environment and how their responses are regulated. UV Effects on Eukaryotic Algae: Analysis and comparison of Cyanidioschyzon mat in situ gene expression patterns show the algal cells are responding at the gene expression level to UV as well as VIS inhibition. Additional work was accomplished in ongoing projects that characterize the microbial communities in acidic hot springs in Norris Geyser Basin as well as Yellowstone Lake, Yellowstone National Park .

Publications

  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Takacs-Vesbach, C., W.P. Inskeep, Z.J. Jay, M.J. Herrgard, D.B. Rusch, S.G. Tringe, M.A. Kozubal, N. Hamamura, R.E. Macur, B.W. Fouke, A.-L. Reysenbach, T.R. McDermott, R.M. Jennings, N.W. Hengartner and G. Xie. 2013. Metagenome sequence analysis of filamentous microbial communities obtained from geochemically distinct geothermal channels reveals specialization of three Aquificales lineages. Front. Microbiol. Doi: 10.3389/fmicb.2013.00084
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Skorupa, D.J., R.W. Castenholz, A. Mazurie, C. Carey, F. Rosenzweig and T.R. McDermott. 2013. In situ gene expression profiling of the thermoacidophilic alga Cyanidioschyzon in relation to visible and ultraviolet irradiance. Environ. Microbiol. Doi:10.1111/1462-2920.12317
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Skorupa, D.J., V. Reeb, R.W. Castenholz, D. Bhattacharya, and T.R. McDermott. 2013. Cyanidiales diversity in Yellowstone National Park. Letts. Appl. Microbiol. 57: 459466.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Romano, C., S. DImperio, T. Woyke, K. Mavromatis, R. Lasken, E.L. Shock and T.R. McDermott. 2013. Comparative genomic analysis of phylogenetically closely related Hydrogenobaculum sp. Isolates from Yellowstone National Park. Appl. Environ. Microbiol. 79:2932-2943.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Clingenpeel, S., J. Kan, R.E. Macur, T. Woyke, D. Lovalvo, J.D. Varley, W.P. Inskeep, K. Nealson and T.R. McDermott. 2013. Yellowstone Lake Nanoarchaeota. Front. Microbiol.Doi: 10.3389/fmicb.2013.00274


Progress 01/01/12 to 12/31/12

Outputs
OUTPUTS: OUTPUTS: Microbe-Metal Interactions: We continue to study the genes that code for functions that contribute to or that regulate arsenite oxidation. We discovered and characterized AioX, which is a periplasmic arsenite binding protein, and discovered that the expression of the aio genes is controlled by phosphate, with the regulatory genes that control the bacterial phosphate stress response also controlling arsenite oxidation. UV Effects on Eukaryotic Algae: We are writing up and submitting papers that describe our observations concerning the thermoacidophilic algae and their response to ultrviolet and visible irradiance. Dissemination Education-outreach field school instruction was conducted for grade school children, for graduate students, high school teachers, and for lay science adult education. PARTICIPANTS: Graduate students: Dana Skorupa and Guanghui Liu. Postdocs: Yoon-Suk Kang, Christine Romano. Undergraduate students: Jill Wilkinson Ellie Zignego. PIs: Tim McDermott, Chris Rensing, Dick Castenholz, Barry Rosen, and Brian Bothner. Partner Organizations: Florida International University; University of Oregon and University of Arizona. Training or professional development 2 Graduate students and 2 postdoctoral fellows TARGET AUDIENCES: Scientific community Outreach audiences: grade school, middle school and high school teachers, adult. PROJECT MODIFICATIONS: Not relevant to this project. PARTICIPANTS: PARTICIPANTS: Graduate students: Dana Skorupa and Guanghui Liu. Postdocs: Yoon-Suk Kang, Christine Romano. Undergraduate students: Jill Wilkinson Ellie Zignego. PIs: Tim McDermott, Chris Rensing, Dick Castenholz, Barry Rosen, and Brian Bothner. Partner Organizations: Florida International University; University of Oregon and University of Arizona. Training or professional development 2 Graduate students and 2 postdoctoral fellows TARGET AUDIENCES: Scientific community, Outreach audiences: grade school, middle school and high school teachers, adult. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Change in knowledge. Microbe-Metal Interactions: The identification and ongoing characterization of novel regulatory genes and regulatory circuits involved in microbial arsenite oxidation represents important observations that are foundational to our understanding of how and why bacteria sense arsenite in the environment and how their responses are regulated. UV Effects on Eukaryotic Algae: Analysis and comparison of Cyanidioschyzon mat in situ gene expression patterns show the algal cells are responding at the gene expression level to UV as well as VIS inhibition.

Publications

  • Kang YS, Heinemann J, Bothner B, Rensing C, McDermott TR. 2012. Integrated co-regulation of bacterial arsenic and phosphorus metabolisms. Environ Microbiol. 14:3097-109.
  • Kang YS, Bothner B, Rensing C, McDermott TR. 2012. Involvement of RpoN in regulating bacterial arsenite oxidation. Appl Environ Microbiol. 78:5638-5645.
  • Liu G, Liu M, Kim EH, Maaty WS, Bothner B, Lei B, Rensing C, Wang G, McDermott TR. 2012. A periplasmic arsenite-binding protein involved in regulating arsenite oxidation. Environ Microbiol. 14:1624-1634.


Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: OUTPUTS: Microbe-Metal Interactions: We continue to study the genes that code for functions that contribute to or that regulate arsenite oxidation and antimony oxidation. New regulatory gene and proteins have been discovered and are currently being characterized for their role in this important microbial redox process. UV Effects on Eukaryotic Algae: Bioinformatic analysis of 454-FLX cDNA libraries were finished, custom microarrays were constructed (commercial vender), and probed with cDNA from environmental samples. Other: Continuing bioinformatics analysis of Yellowstone Lake microbial diversity. Dissemination: Education-outreach field school instruction was conducted for grade school children, for graduate students, high school teachers, and for lay science adult education. PARTICIPANTS: Graduate students: Dana Skorupa and Guanghui Liu. Postdocs: Yoon-Suk Kang and Christine Romano. Undergraduate students: Jill Wilkinson. PIs: Tim McDermott, Chris Rensing, Dick Castenholz, and Brian Bothner. Partner Organizations: University of Oregon and University of Arizona. Training or professional development: 2 Graduate students and 2 postdoctoral fellows. TARGET AUDIENCES: Scientific community, Outreach audiences: grade school, middle school and high school teachers, adult. PROJECT MODIFICATIONS: Not relevant to this project. PARTICIPANTS: Individuals: Graduate students: Dana Skorupa (UV research) and Guanghui Liu (arsenite oxidation research). Postdocs: Yoon-Suk Kang (arsenite oxidation research) and Christine Romano (antimony oxidation research, Yellowstone Lake work). Undergraduate students: Jill Wilkinson (UV research). PIs: Tim McDermott (directed all above research), Chris Rensing (conferred with PI McDermott), Dick Castenholz (conferred with PI McDermott), and Brian Bothner (conferred with PI McDermott, and directed some arsenite oxidation research). Partner Organizations: University of Oregon and University of Arizona. Training or professional development: 2 Graduate students and 2 postdoctoral fellows. TARGET AUDIENCES: Scientific community, Outreach audiences: grade school, middle school and high school teachers, adult. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Change in knowledge. Microbe-Metal Interactions: The identification and ongoing characterization of novel regulatory genes and regulatory circuits involved in microbial arsenite oxidation represents novel observations that are foundational to our understanding of how and why bacteria sense arsenite in the environment and how their responses are regulated. UV Effects on Eukaryotic Algae: Analysis and comparison of Cyanidioschyzon mat in situ gene expression patterns show the algal cells are responding at the gene expression level to UV as well as VIS inhibition.

Publications

  • Liu, G., M. Liu, E.H. Kim, W. Matty, B. Bothner, B. Lei, C. Rensing, G. Wang, and T.R. McDermott. 2011. A periplasmic arsenite-binding protein involved in regulating arsenite oxidation. Environmental Microbiology. doi:10.1111/j.1462-2920.2011.02672.x
  • Clingenpeel, S.R., J. Kahn, W.P. Inskeep, D. Lavalvo, J. Varley, K. Nealson, and T.R. McDermott. 2011. Archaea diversity associated with hydrothermal vents on the floor of Yellowstone Lake. ISME J. 5:1784-1795.
  • Clingenpeel, S.R., R.E. Macur, J. Kahn, W.P. Inskeep, D. Lavalvo, J. Varley, E. Mathur, K. Nealson, Y. Gorby, T. La Francois, and T.R. McDermott. 2011. Yellowstone Lake: High energy geochemistry and rich bacterial diversity. Environ. Microbiol. 13:2172-2185.
  • Rosen BP, A.A. Ajees, T.R. McDermott. 2011. Life and death with arsenic. Arsenic life: an analysis of the recent report "A bacterium that can grow by using arsenic instead of phosphorus". Bioessays, 33:350-357


Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: Microbe-Metal Interactions: We continue to study the genes that code for functions that contribute to or that regulate arsenite oxidation. New regulatory gene and proteins have been discovered and are currently being characterized for their role in this important microbial redox process. UV Effects on Eukaryotic Algae: Field experiments (algal biomass sampling) were wrapped up and field equipment removed. Bioinformatic analysis of 454-FLX cDNA libraries was finished, custom microarrays were constructed (commercial vender), and mRNA extraction and cDNA synthesis was optimized in preparation for array probing. Other: Finished bioinformatics analysis of Yellowstone Lake microbial diversity and geochemical analysis. Dissemination Education-outreach field school instruction was conducted for grade school children, for graduate students, high school teachers, and for lay science adult education. PARTICIPANTS: Graduate students: Dana Skorupa and Guanghui Liu. Postdocs: Scott Clingenpeel and Yoon-Suk Kang. Technician: Alexandra Bueling. Undergraduate students: Ted Weatherwax and Jill Wilkinson. PIs: Tim McDermott, Chris Rensing, Dick Castenholz, Barry Rosen, and Brian Bothner. Partner Organizations: Florida International University; University of Oregon and University of Arizona. Training or professional development 2 Graduate students and 2 postdoctoral fellows TARGET AUDIENCES: Scientific community Outreach audiences: grade school, middle school and high school teachers, adult. PROJECT MODIFICATIONS: Not relevant to this project. PARTICIPANTS: PARTICIPANTS: Graduate students: Dana Skorupa and Guanghui Liu. Postdocs: Scott Clingenpeel and Yoon-Suk Kang. Technician: Alexandra Bueling. Undergraduate students: Ted Weatherwax and Jill Wilkinson. PIs: Tim McDermott, Chris Rensing, Dick Castenholz, Barry Rosen, and Brian Bothner. Partner Organizations: Florida International University; University of Oregon and University of Arizona. Training or professional development 2 Graduate students and 2 postdoctoral fellows. TARGET AUDIENCES: TARGET AUDIENCES: Scientific community; Outreach audiences: elementary school; middle school and high school teachers, adult lay audiences. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Change in knowledge. Microbe-Metal Interactions: The identification and ongoing characterization of novel regulatory genes and regulatory circuits (to be published) involved in microbial arsenite oxidation represents novel observations that are foundational to our understanding of how and why bacteria sense arsenite in the environment and how their responses are regulated. UV Effects on Eukaryotic Algae: Analysis and comparison of Cyanidioschyzon mat in situ gene expression patterns (via 454-FLX cDNA libraries) versus proteomic analysis in nature indicate that there is a very large disconnect between mRNA-based gene expression patterns and apparent protein profiles.

Publications

  • Kiss H, Cleland D, Lapidus A, Lucas S, Del Rio TG, Nolan M, et al. 2010. Complete genome sequence of 'Thermobaculum terrenum' type strain (YNP1). Stand Genomic Sci. 3:153-62.
  • Lavalvo, D., S.R. Clingenpeel, R.E. Macur, W.P. Inskeep, J. Glime, J. Varley, K. Nealson, and T.R. McDermott 2010. A geothermal-linked biological oasis in Yellowstone Lake. Geobiology. I8:327-36
  • Valerie Reeb, Avraham Kolel, Timothy R. McDermott and Debashish Bhattacharya. 2010. Good to the bone: microbial community thrives within bone cavities of a bison carcass at Yellowstone National Park. Environ. Microbiol.DOI: 10.1111/j.1462-2920.2010.02359.x


Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: Activities Microbe-Metal Interactions: Field samples were taken for RNA extraction, which were then probed for expression analysis of arsenite oxidase genes using reverse transcriptase (RT) PCR. RT-PCR amplicons were also examined for gene sequence diversity and evidence of population distribution. UV Effects on Eukaryotic Algae: Field experiments were conducted on a monthly basis, taking algal biomass samples and water samples for chemical analysis. In addition, a light meter was installed in the field in order to monitor UV intensity. Extensive bioinformatics analysis of cDNA libraries. Dissemination Education-outreach field school instruction was conducted for grade school children, for graduate students, high school teachers, and for lay science adult education. PARTICIPANTS: Graduate students: Dana Skorupa, Guanghui Liu, and Henry Orduro; Postdocs: Scott Clingenpeel; Walid Matty. PIs: Tim McDermott, Greg Druschel, Barry Rosen, and Brian Bothner. Partner Organizations: Florida International University; University of Oregon and University of Vermont. Training or professional development 2 Graduate students and 2 postdoctoral fellows TARGET AUDIENCES: Scientific community Outreach audiences: grade school, middle school and high school teachers, adult. PROJECT MODIFICATIONS: Not relevant to this project. PARTICIPANTS: Graduate students: Dana Skorupa, Guanghui Liu, and Henry Orduro; Postdocs: Scott Clingenpeel; Walid Matty. PIs: Tim McDermott, Greg Druschel, Barry Rosen, and Brian Bothner. Partner Organizations: Florida International University; University of Oregon and University of Vermont. Training or professional development 2 Graduate students and 2 postdoctoral fellows TARGET AUDIENCES: Scientific community; Outreach audiences: elementary school; middle school and high school teachers, adult lay audiences. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Change in knowledge. Microbe-Metal Interactions: Eukaryotic algae can be major contributors to arsenic biogeochemical cycling in geo therma environments. By inference based on this study as well as that of others in the literature, it is likely that algae have a significant impact on arsenic redox transformations, perhaps in particular methylation reactions. The identification and cloning of the structural genes for the arsenite oxidae enzyme from the evolutionarily early diverging bacterium Hydrogenobaculum shows that it is most similar to another early branching bacterium Thermus. This gene is expressed in high sulfide environments, even though all evidence shows that the encoding enzyme is inhibited in such environments. Interestingly, there were unique gene sequences found in specific regions of the outflow channel, indicating distinct population distributions. UV Effects on Eukaryotic Algae: Ecological studies have found that Galderia are primarily found in geothermal soil environments, whereas Cyanidioschyzon is almost exclusively found in aqueous environments. Analysis of in situ gene expression patterns in Cyanidioschyzon mats indicate that there is a very large disconnect between pure culture cells as compared to those in nature.

Publications

  • Qin, J., C.R. Lehr, X.C. Le, T.R. McDermott, and B.P. Rosen. 2009. Arsenic methylation by a thermoacidophilic eukaryotic alga. Proc. Natl. Acad. Sci. 106:5213-5217.
  • Clingenpeel, S.R., S. DImperio, H. Oduro, G.K. Druschel, and T.R. McDermott. 2009. Cloning and in situ expression studies of the Hydrogenobaculum arsenite oxidase genes. Appl. Envir. Microbiol. 75:3362-3365.
  • Castenholz, R.C. and T.R. McDermott. 2009. The cyanidiales: ecology, biodiversity, and biogeography. In J. Sechback and D. Chapman (eds.), Red Algae in Genomic Age, Elsevier Press.


Progress 01/01/08 to 12/31/08

Outputs
OUTPUTS: Activities Microbe-Metal Interactions: Field samples were taken for RNA extraction, which were then probed for expression analysis of arsenite oxidase genes using reverse transcriptase (RT) PCR. RT-PCR amplicons were also examined for gene sequence diversity and evidence of population distribution. UV Effects on Eukaryotic Algae: Field experiments were conducted on a monthly basis, taking algal biomass samples and water samples for chemical analysis. In addition, a light meter was installed in the field in order to monitor UV intensity. Algal biomass was extracted fro total RNA, which was then converted into cDNA for pyrosenqincing. Products One PhD student graduated. Dissemination Education-outreach field school instruction was conducted for grade school children, for graduate students, high school teachers, and for lay science adult education. PARTICIPANTS: Graduate students: DImperio, Toplin, and Oduro Postdoc: Lehr PIs: McDermott, Druschel, Kuhl, and Castenoholz Partner Organizations University of Oregon and University of Vermont Training or professional development 2 Graduate students and 1 postdoctoral fellow TARGET AUDIENCES: Scientific community Outreach audiences: grade school, middle school and high school teachers, adult. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Change in Knowledge. Microbe-Metal Interactions: Major conclusions drawn from one published study was that microbial phylogeny may not necessarily be reliable for predicting physiology and that sulfide can dominate over hydrogen as a microbial energy source in terms of availability, apparent in situ consumption rates, and growth-supporting energy. UV Effects on Eukaryotic Algae: The phylogenetic diversity of culture isolates of the Cyanidiales from habitats throughout Yellowstone National Park (YNP), three areas in Japan, and seven regions in New Zealand was examined by using the chloroplast RuBisCO large subunit gene (rbcL) and the 18S rRNA gene. Based on the nucleotide sequences of both genes, the YNP isolates fall into two groups, one with high identity to Galdieria sulphuraria (type II) and another that is by far the most common and extensively distributed Yellowstone type (type IA). The latter is a spherical, walled cell that reproduces by internal divisions, with a subsequent release of smaller daughter cells. This type, nevertheless, shows a 99 to 100% identity to Cyanidioschyzon merolae (type IB), which lacks a wall, divides by "fission"-like cytokinesis into two daughter cells, and has less than 5% of the cell volume of type IA. The evolutionary and taxonomic ramifications of this disparity are discussed. Although the 18S rRNA and rbcL genes did not reveal diversity among the numerous isolates of type IA, chloroplast short sequence repeats did show some variation by location within YNP. In contrast, Japanese and New Zealand strains showed considerable diversity when we examined only the sequences of 18S and rbcL genes. Most exhibited identities closer to Galdieria maxima than to other strains, but these identities were commonly as low as 91 to 93%. Some of these Japanese and New Zealand strains probably represent undescribed species that diverged after long-term geographic isolation.

Publications

  • DImperio, S., C.R. Lehr, H. Oduro, G. Druschel, M. Kuhl, and T.R. McDermott. 2008. Relative importance of H2 and H2S as energy sources for primary production in geothermal springs. Appl. Environ. Microbiol. 74:5802-5808.
  • Toplin, J.A., T.B. Norris, C.R. Lehr, T.R. McDermott, and R.W. Castenholz. 2008. Biogeographic and phylogenetic diversity of thermoacidophilic Cyanidiales in Yellowstone National Park, Japan, and New Zealand. Appl. Environ. Microbiol. 74: 2822-2833.
  • Lisa Kirk, Susan Childers, Brent Peyton, Tim McDermott, Robin Gerlach. 2008. In-Situ Subsurface Microbial Transformation Of Selenium As Source Control In Backfilled Phosphate Overburden, SE Idaho. 24TH ANNUAL MONTANA SECTION AWRA MEETING
  • Seth DImperio, Corinne R. Lehr, Michele Breary, and Timothy R. McDermott. 2008. Autecology of an Arsenite Chemolithotroph: Sulfide Constraints on Function and Distribution in a Geothermal Spring. NSF Research Coordination Network Workshop, Mammoth, Wyoming, January 10-13, 2008
  • Jie Qin, Corinne R. Lehr, X.C. Le, Barry P. Rosen, Richard W. Castenholz, Timothy R. McDermott. 2008. Potential Contribution of the Thermoacidophilic Cyanidiales Algae to Metalloid Redox Cycling in Geothermal Environments. NSF Research Coordination Network Workshop, Mammoth, Wyoming, January 10, 2008
  • Seth DImperio, Corinne R. Lehr, Harry Oduro, Greg Druschel, Michael Kuhl, and Timothy R. McDermott. 2008. A Comparison of H2 and H2S as Energy Sources for Primary Production in an Acidic Geothermal Spring. NSF Research Coordination Network Workshop, Mammoth, Wyoming, January 10-13, 2008
  • Dana J. Skorupa, Timothy R. McDermott, Richard C. Castenholz, and Scott R. Miller. Cyanidiales Ecology and Biodiversity in Acidic Geothermal Environments. NSF Research Coordination Network Workshop, Mammoth, Wyoming, January 10-13, 2008
  • Scott R. Clingenpeel, J.J. Kahn, Richard E. Macur, Yuri A. Gorby, William P. Inskeep, Mathew Lewis, Eric Mathur, Timothy R. McDermott, Tina McIntosh, Lisa A. Morgan, Kenneth H. Nealson, W.C. Shanks, and John Varley. Yellowstone Lake: Genetic Diversity in an Aquatic, Vent-Impacted System. Metagenomics 2008. San Diego, CA, November, 2008.
  • Timothy R. McDermott and Scott Clingenpeel. Hydrogenobaculum Population Genomics. Metagenomics 2008. San Diego, CA, November, 2008.


Progress 01/01/07 to 12/31/07

Outputs
OUTPUTS: Activities i) Experiments that examined the ability of eukaryotic algae to oxidize arsenite and antimonite, and to methylate arsenite. ii) Experiments that "rediscovered" bacterial antimonite oxidation. iii) Experiments that studied the ecology of a thermoacidophilic eukaryotic algae in environments that contain elevated levels of arsenic and antimony. iv) Experiments that examined environmental constraints on microbial arsenite oxidation. Products i) Established a collaboration with Dr. Debashish Battacharya, University of Iowa, an world-class expert on the phylogenetics of the eukaryotic algae we study for metalloid redox transformations. PARTICIPANTS: Individuals Dr. Corinne Lehr, Postdoc who lead the studies concerning Cyanidiales ecology, and concerning algal arsenite and antimony oxidation. Shaun D. Frank, Undergraduate research intern worked under the supervision of Dr. Lehr. He PCR cloned and sequenced 18S rRNA genes from environmental DNA extracts. Seth D'Imperio, PhD student who lead the study concerning the environmental factors that control microbial arsenite oxidation. Alexy V. Kalinin, undergraduate research intern who was involved in work that identified gene loci that we used for differentiating populaitons of Cyanidiales algae. Dr. TimothyR. McDermott, PI who lead and directed all research. Dr. Desh R. Kashyap, postdoc involved in the bacterial antimony oxidation research. Michele Breary, undergraduate research intern who conducted denaturant gradient gel electrophoresis experiments in conjunction with the PhD student on the project that examined the environmental controls of microbial arsenite oxidation. TARGET AUDIENCES: Target audiences Academic and government scientists involved in metalloid contamination.

Impacts
Change in Knowledge: Research conducted during the award period resulted in the discovery of: 1) Isolated a genetically tractable bacterium capable of Sb(III) oxidation. This is an significant discovery because it provides a new resource for examining the genes and encoded enzymes responsible for this environmentally important microbial activity. 2) Demonstrated that algae are capable of antimony oxidation. Up until our work, Sb(III) oxidation was reported in an algal culture that was not shown to be pure.

Publications

  • Lehr, C.R., S.D. Frank, T.B. Norris, S. D'Imperio, A.V. Kalinin, J.A. Toplin, R.W. Castenholz, and T.R. McDermott. 2007. Studies on the ecology of cyanidia in an acid sulfate chloride spring in Yellowstone National Park. J. Phycology 43:3-14.
  • Lehr, C.R., D.R. Kashyap and T.R. McDermott. 2007. New insights into microbial oxidation of antimony and arsenic. Appl. Environ. Microbiol. 73:2386-2389.
  • LaLonde, S. V., L. Amskold, T. R. McDermott, W. P. Inskeep AND K. O. Konhauser. 2007. Chemical reactivity of microbe and mineral surfaces in hydrous ferric oxide depositing hydrothermal springs. Geobiology 5:219-234.
  • D'Imperio, S., C.R. Lehr, M. Breary, and T.R. McDermott. 2007. Autecology of an arsenite chemolithotroph: sulfide constraints on function and distribution in a geothermal spring. Appl. Environ. Microbiol. 73: 7067-7074


Progress 01/01/06 to 12/31/06

Outputs
Experimental results summarized in the 2005 report were formalized into two manuscripts that were published in 2006. This research identified genes involved in bacterial arsenite oxidation. We showed that quorum sensing and a two-component signal transduction system are both involved in regulating As(III) oxidation, and that a Na+/H+ antiporter and a molybdenum transporter are also essential for As(III) oxidation. These studies represent foundational advances to our understanding of microbial As(III) oxidation. In other work, we found the wild type A. tumefaciens organism was also capable of oxidizing Sb(III) and thus the isolation of key As(III) oxidation mutants provided us with an exceptional opportunity to examine whether microbial redox transformations of arsenic and antimony are related (Lehr et al. 2007). Current literature shows that Sb(III) can act as an As(III) analog with respect to regulation of microbial genetic and physiological responses to arsenic. From such studies, it has been assumed that Sb(III) oxidation occurred via the same enzymatic pathway as has been shown for As(III). However, equivalent Sb(III) oxidation rates were observed in the parental wild type organism and in two mutants that cannot oxidize As(III) for fundamentally different reasons. Therefore, despite the literature suggesting Sb(III) may be an analogue of As(III), our experiments indicate Sb(III) oxidation is catalyzed by a pathway different than that used for As(III) oxidation. Furthermore, we also documented Sb(III) and As(III) oxidation in an eukaryotic acidothermophilic alga isolated from Yellowstone National Park. We view the latter as transformative research in that neither As(III) oxidation nor Sb(III) oxidation had ever been reported for a pure culture eukaryotic microbe. Additional work characterized the ecology of this alga with respect to population diversity and dynamics, and sensitivity to UV irradiance damage. Antimony (Sb) is a naturally occurring toxic metalloid and priority pollutant, however relatively little is known about its fate and behavior in the environment. In particular, sparse information is available regarding its interactions with microorganisms. Progress towards understanding microbial redox transformations of Sb has primarily been constrained by the lack of microbial pure cultures capable of oxidizing Sb(III) or reducing Sb(V). Our discoveries effectively removes this barrier. Another manuscript currently under review summarized efforts involving the isolation of the first thermoacidophilic arsenite chemolithotroph, and its characterization with respect to population distribution in the geothermal spring under study and provided significant clarification regarding the effects of sulfide as an inhibitor of microbial arsenite oxidation. Microbial arsenite oxidation is profoundly inhibited by the presence of sulfide, with the effect apparently centering on the arsenite oxidase enzyme itself. The above efforts consumed all available resources and thus no new progress was made in the area of phosphorus metabolism in the Rhizobium-legume symbiosis.

Impacts
Our discoveries regarding sulfide inhibition of arsenite oxidation help define where and when we might expect to observe microbial As(III) oxidation in the environment and thus will be of value to land managers and for rehabilitation efforts involving arsenic contamination. Further, our work helps define the cellular features and key regulatory genetic elements that are essential for microorganisms to carry out AsIII oxidation and how microbes regulate this process. The latter is again important for understanding microbe-arsenic interactions in terms of when and where microbes may oxidize this toxic metalloid and the environmental conditions that may control this process. Efforts demonstrating that the biochemical pathways involved in arsenic and antimony oxidation are different illustrate that, functionally, these processes are separate and that additional work is required to understand the genes that encode for antimony oxidation and their distribution in nature. Work demonstrating arsenic and antimony oxidation by a freshwater alga is transformative in that this work has determined that eukaryotic microbes are capable of metalloid redox transformations that are of importance to environmental health and thus human safety. If marine algae can be shown to carry out similar redox transformations, then the implications of these observations take on significantly greater importance with respect to scale.

Publications

  • Lehr, C.R., Frank, S.D., Norris, T.B., D'Imperio, S., Kalinin, A.V., Toplin, J.A., Castenholz, R.W., McDermott, T.R. 2007. Cyanidia (Cyanidiales) population diversity and dynamics in an acid-sulfate chloride spring in Yellowstone National Park. J.Phycology. 43:3-14.
  • Lehr, C., D.R. Kashyap, and T.R. McDermott. 2007. New insights into microbial arsenite and antimony oxidation. Appl. Environ. Microbiol. In press.
  • Kashyap, D.R., Botero, L.M., Franck, W.L., Hassett, D.J., McDermott, T.R. 2006. Complex regulation of arsenite oxidation in Agrobacterium tumefaciens. J. Bacteriol. 188:1081-1088.
  • Kashyap, D., Botero, L.M., Lehr, C., Hassett, D.J., McDermott, T.R. 2006. A Na+:H+ antiporter and molybdate transporter are essential for arsenite oxidation in Agrobacterium tumefaciens. J. Bacteriol.188:1577-1584.


Progress 01/01/05 to 12/31/05

Outputs
We have finished characterization of various mutants of Agrobacterium tumefaciens that lack arsenite oxidase activity. One mutant is affected in a regulatory locus involved in two-component signal transduction. A second mutant is interrupted in the modB gene, which codes for the permease component of a molybdate transporter, and a third mutant is interrupted in the mrpB gene, which codes for one of seven proteins involved in sodium:proton antiport. We have also participated in work with Pseudomonas aeruginosa, where we used a proteomics approach to examine gene expression in response to As(III) exposure. Genes found to be up- and down-regulated were mutated to determine their relative importance to As(III) resistance. In our work that examines chemolithotrophic microbial community structure and function in geothermal environments as a model for acid-mine-drainage problems, we have isolated an As(III) chemolithoautotroph and studied it's distribution in the spring outflow channel. This organism is only found in low sulfide regions of the spring, which is consistent with our previous findings that As(III) oxidation is inhibited by sulfide. Hydrogen sulfide is rapidly oxidized by the microorganisms present and we have isolated several sulfide oxidizers. Ex situ and pure culture experiments suggest that sulfide may be a major energy source for these types of geothermal environments.

Impacts
Our recent discoveries regarding sulfide inhibition of arsenite oxidation help define where and when we might expect to observe microbially based As(III) oxidation in the environment and thus will be of value to land managers and for rehabilitation efforts involving arsenic contamination. Further, our work with the mutants help define the cellular features that are essential for microorganisms to carry out AsIII oxidation and how the microbes regulate this process. The latter is again important for our understanding of microbe-arsenic interactions in that it aids in our understanding of when and where microbes may oxidize this toxic metalloid and the environmental conditions that may control this process.

Publications

  • Inskeep, W.P. and T.R. McDermott. 2005. Geomicrobiology of acid-sulfate-chloride springs in Yellowstone National Park. In W.P. Inskeep and T.R. McDermott (eds.) Geothermal Biology and Geochemsitry in Yellowstone National Park, Montana State University Publications, pp. 143-162
  • Parvatiyar, K., E. Alsabbagh, U.A. Ochsner, M.A. Stegemeyer, A.G. Smulian, S.H. Hwang, C.R. Jackson, T.R. McDermott, and D.J. Hassett. 2005. Global analysis of cellular factors and responses involved in Pseudomonas aeruginosa resistance to arsenite. J. Bacteriol. 187:4853-4864.
  • Botero, L.M., S. D'Imperio, M. Burr, T.R. McDermott, M. Young and D.J. Hassett. 2005. PolyA polymerase modification and reverse transcriptase PCR amplification of environmental RNA. Appl. Environ. Microbiol. 71:1267-1275.
  • Burr, M.D., L.M. Botero, M. Young, W.P. Inskeep, and T.R. McDermott. 2005. Observations concerning nitrogen cycling in a Yellowstone extreme thermal soil environment. In W.P. Inskeep and T.R. McDermott (eds.) Geothermal Biology and Geochemsitry in Yellowstone National Park, Montana State University Publications, pp.171-182.


Progress 01/01/04 to 12/31/04

Outputs
A central focus of this work is to examine the extent to which soil microbial communities and their component populations influence arsenic speciation and mobility in soil and, by extension, within the landscape. Perturbing a single soil type (known to be biased towards As(III) oxidation) with three different media plus either As(III) or As(V) did not alter the net community redox transformation activity as measured by As speciation. We are screening a large number of isolates for their redox transformation phenotypes and will then probe these isolates to determine what kind of As(III) oxidase gene they carry. We have finished characterization of various mutants of Agrobacterium tumefaciens that lack arsenite oxidase activity. One mutant is affected in a regulatory gene, a second mutant is interrupted in the modB gene, which codes for the permease component of a molybdate transporter, and a third mutant is interrupted in the mnhB gene, which codes for one of seven proteins involved in sodium:proton antiport. In our work that examines chemolithotrophic microbial community structure and function in geothermal environments as a model for acid-mine-drainage problems, we have found that the sulfide content of the spring water inhibits As(III) oxidation. The inhibition appears fully reversible. We are conducting additional experiments to determine if As(III) may be used as a energy source under these conditions. We have isolated As(III) chemolithoautotrophs, but we do not know how active they are in this spring. Hydrogen sulfide is rapidly oxidized by the microorganisms present and we have isolated several sulfide oxidizers. Thus far, our data suggests that sulfide may be a major energy source for these types of environments.

Impacts
Various microorganisms are extremely important to agriculture and the proper functioning of our environment. The talents of these microscopic organisms can be utilized to enhance crop production and to remediate problem toxins or waste materials resulting from anthropologic activities. However, before we can fully utilize these microbes, it is essential that we understand how they function and how they interact with higher organisms such as plants.

Publications

  • Donohoe-Christiansen, J., C.R. Jackson, S. D'Imperio, W.P Inskeeep, and T.R. McDermott. 2004. Arsenite-oxidizing Hydrogenobaculum strain isolated from an acid-sulfate-chloride geothermal spring in Yellowstone National Park. Appl. Environ. Microbiol. 70:1865-1868.
  • Botero, L.M, K.B. Brown, S. Brumefield, M. Burr, R.W. Castenholz, M. Young, and T.R. McDermott. 2004. Thermobaculum terrenum gen. nov., sp. nov.: a non-phototrophic gram-positive thermophile representing an environmental clone group related to the chloroflexi (green non-sulfur bacteria) and thermomicrobia. Arch. Microbiol. 181:269-277.
  • Schaffer, C., W. L. Franck, A. Scheberl, P. Kosma, T. R. McDermott and P. Messner. 2004. Classification of isolates from locations in Austria and Yellowstone National Park as Geobacillus tepidamans sp. nov. Int J Syst Evol Microbiol. 54:2361-2368
  • Macur, R.E., C.R. Jackson, T.R. McDermott and W.P. Inskeep. 2004. Bacterial populations associated with the oxidation and reduction of arsenic in an unsaturated soil. Environ. Sci. Technol. 38:104-111.


Progress 01/01/03 to 12/31/03

Outputs
The general approach of this project has been to combine specific Rhizobium tropici pho mutants with radiolabeling experiments to assess P distribution, movement, and exchange at different times during the development of the R. tropici-bean symbiosis. The various mutants are inoculated onto the roots of axenic bean seedlings, allowed to nodulate, and then the plants are fed radioactive phosphorus (roots) or radioactive carbon dioxide (shoots). The radiotracers are used to measure whether the pho mutation has had any affect on nutrient flow between the plant and the bacteroids. We have found that an alkaline phosphatase (AP) mutant was not defective in symbiosis, suggesting that the AP enzyme does not play an important role in plant-microbe nutrient exchange. A decrease in bacteroid AP we previously reported to occur during the symbiosis is due loss of bacteroid structural integrity, and not due to changes in pho regulatory activities during symbiosis. Furthermore, the AP defective mutant did not differ from the wild type parent strain with respect to bacteroid labeling patterns when the plants were exposed to radioactive carbon dioxide or radioactive phosphate. Long-term label-chase experiments suggest that there is a net flow of P out of the bean nodule, indicating there is no nodule P sink activity in this particular cultivar. We are finalizing cloning and sequencing of the kdpFABC and kdpDE operons in R. topici. We will used these cloned operons for complementation work with the kdp mutants. Manuscripts describing the above research are currently under preparation.

Impacts
This work contributes towards improving phosphorus use efficiency in crop plants. Feeding the earth's population demands that crop production and fertilizer use efficiencies be at their absolute maxima. It has been projected that economically accessible rock phosphate reserves may be depleted in 70 years. There is an immediate need to improve phosphorus use efficiency in crop plants such as beans, which are a major source of dietary protein throughout the world.

Publications

  • No publications reported this period


Progress 01/01/02 to 12/31/02

Outputs
To improve our understanding of phosphorus metabolism in the Rhizobium-legume symbiosis and how phosphorus is shared within the symbiosis, we are investigating aspects of phosphorus acquisition by the microsymbiont Rhizobium, focussing primarily on bean for this work. We are assessing the symbiotic phenotype of various Rhizobium mutants we have generated that are defective in some aspect of phosphorus acquisition (e.g. phosphatase mutants, phosphate transport mutants) or metabolism (e.g. regulatory mutants). Our work with the bean symbiosis has found that bean rhizobia (Rhizobium tropici) bacteroids exhibit high levels of phosphate stress inducible enzymes, regardless of how much phosphorus is supplied to the host plant. 32P-Labelling studies using phosphate transport mutants has provided evidence that simple inorganic phosphate is not primary form of P provided to bean bacteroids. We have also conducted studies that couple whole nodule 32P-labeling studies with 2-D thin layer chromatography and autoradiography. These experimentts provided evidence that simple inorganic P is not the dominant phosphorus compound in the bean nodule. Symbiosis studies with the various mutants have shown that R. tropici P transport mutants form a less effective symbiosis than the wild type parental strain, and bacteroids of these mutants acquire less P in situ. Mutants lacking alkaline phosphatase are not impaired in symbiosis and 14C and 32P labeling studies suggest that bacteroids of these mutants acquire as much P and C as the wild type strain. Other labeling studies also suggested that bean nodules are strong sinks for P, and that bean nodules have a strong capacity to take up P from their immediate environment. However, labeling experiments that assessed P exchange between the host plant and the nodule suggest that there is a net flow of P out of the bean nodule as the symbiosis completes its life cycle.

Impacts
Legume production in the United States is of significant importance. With many legumes, phosphorus is the limiting nutrient for production. It has been projected that economically accessible rock phosphate reserves may be depleted in 70 years. There is an immediate need to improve P use efficiency in crop plants such as beans, which are a major source of dietary protein throughout the world. This work contributes towards improving phosphorus (P) use efficiency in this important world food source.

Publications

  • McDermott, T.R. Symbiotic nitrogen fixation. 2003. In R.M. Goodman (ed.) Encyclopedia of Plant and Crop Science, Marcel Dekker, Inc. In press.
  • McDermott, T.R. Plant nutrition and symbiotic nitrogen fixation. 2003. In R.M. Goodman (ed.) Encyclopedia of Plant and Crop Science, Marcel Dekker, Inc. In press.


Progress 01/01/01 to 12/31/01

Outputs
Legume production in the United States is of significant importance. With many legumes, phosphorus is the limiting nutrient to production. To further our understanding of phosphorus metabolism in the Rhizobium-legume symbiosis and how phosphorus is shared within the symbiosis, we are investigating aspects of phosphorus acquisition by the microsymbiont Rhizobium, focussing primarily on bean for this work. We are assessing the symbiotic phenotype of various Rhizobium mutants we have generated that are defective in some aspect of phosphorus acquisition (e.g., phosphatase mutants, phosphate transport mutants) or metabolism (e.g., regulatory mutants). Our work with the bean symbiosis found that bean rhizobia (Rhizobium tropici) bacteroids exhibit high levels of phosphate stress inducible enzymes, regardless of how much phosphorus is supplied to the host plant. 32P-Labelling analysis using phosphate transport mutants has provided evidence that simple inorganic phosphate is not primary form of P provided to bean bacteroids. We have also conducted studies that couple whole nodule 32P-labeling studies with 2-D thin layer chromatography and autoradiography. These experimentts provided evidence that simple inorganic P is not the dominant phosphorus compound in the bean nodule. These observations have important implications regarding P sharing between bacteroid and host plant in this symbiosis. Bean plants inoculated with the phosphate transport mutant produces less dry matter, but the nodules fix nitrogen at near apparent wild type levels. We have also isolated a phoA R. tropici mutant and cloned the phoB gene from this organism. Symbiosis trials with these mutants are under way. P placement experiments also suggested that bean nodules are strong sinks for P, and that bean nodules have a strong capacity to take up P from their immediate environment. Experiments are currently assessing P exchange between the host plant and the nodule, and between host plant and bacteroid.

Impacts
This work contributes towards improving phosphorus (P) use efficiency in crop plants. Feeding the earth's population demands that crop production and fertilizer use efficiencies be at their absolute maxima. It has been projected that economically accessible rock phosphate reserves may be depleted in 70 years. There is an immediate need to improve P use efficiency in crop plants such as beans, which are a major source of dietary protein throughout the world.

Publications

  • Deng, S., Elkins, J.G., Da, L.H., Botero, L.M. and McDermott, T.R. 2001. Cloning and characterization of a second acid phosphatase from Sinorhizobium meliloti strain 104A14. Arch. Microbiol. 176:255-263.


Progress 01/01/00 to 12/31/00

Outputs
To further our understanding of phosphorus metabolism in the Rhizobium-legume symbiosis and how phosphorus is shared within the symbiosis, we are investigating aspects of phosphorus acquisition by the microsymbiont Rhizobium. Alfalfa and bean are the model legumes used for this work. We are assessing the symbiotic phenotype of various Rhizobium mutants we have generated that are defective in some aspect of phosphorus acquisition (e.g., phosphatase mutants, phosphate transport mutants) or metabolism (e.g., regulatory mutants). We verified that PhoB controlled the Pho stress response in S. meliloti, determined the P concentration at which the Pho regulon is induced in S. meliloti, and found that the Pho stress response was not required for normal symbiotic function of S. meliloti with alfalfa. We characterized the transposon insertion site of several S. meliloti Tn5B22 mutants that had been shown to report the expression of P stress inducible genes, and then studied the expression of these genes under various in vitro conditions and in symbiosis. We also found that the pta-ackA operon is PhoB regulated, and by mutating pta and/or ackA we established metabolic blocks that allowed us to determine that acetate metabolism via AckA and Pta was not essential to the alfalfa symbiosis. Further, acetyl phosphate appears not to be important as a nonspecific regulatory metabolite. Based on these studies, we have concluded that it is highly unlikely that alfalfa rhizobia bacteroids ever encounter P limitation during symbiosis, regardless of the phosphorus nutrition status of the host plant. An important regulatory gene discovered to be critical for symbiosis during the course of this phosphate work included Lon protease. The lon protease mutant forms nodules that do not fix nitrogen. The symbiotic phenotype of the lon mutant is thought to be linked to the inappropriate regulation of exopolysaccharide production. Our work with the bean symbiosis found that bean rhizobia (Rhizobium tropici) bacteroids exhibit high levels of phosphate stress inducible enzymes, regardless of how much phosphorus is supplied to the host plant. 32P-Labelling analysis using phosphate transport mutants has provided evidence that simple inorganic phosphate is not primary form of P provided to bean bacteroids. This has important implications regarding P sharing between symbionts in this symbiosis. Bean plants inoculated with the phosphate transport mutant produces less dry matter, but the nodules fix nitrogen at near apparent wild type levels. We have also isolated a phoA R. tropici mutant and cloned the phoB gene from this organism.

Impacts
This work contributes towards improving phosphorus (P) use efficiency in crop plants. Feeding the Earth's population demands that crop production and fertilizer use efficiencies be at their absolute maxima. It has been projected that economically accessible rock phosphate reserves will be depleted within 70 years. There is an immediate need to improve P use efficiency in crop plants such as beans, which are a major source of dietary protein throughout the world.

Publications

  • Botero, L.M., Al-Niemi, T.S., and McDermott, T.R. 2000. Characterization of two inducible phosphate transport systems in Rhizobium tropici Appl. Environ. Microbiol. 66:15-22.
  • Summers, M.L. Botero, L.M., Busse, S.C., and McDermott, T.R. 2000. The Sinorhizobium meliloti Lon protease is involved in regulating exopolysaccharide synthesis and is required for nodulation of alfalfa. J. Bacteriol. 182:2551-2558.
  • Deng, S.P., Elkins, J.G., Da, L.H., Botero, L.M., and McDermott, T.R. 2000. Purification and characterization of a second acid phosphatase from Sinorhizobium meliloti 104A14. p. 246. In 2000 Agronomy abstracts. Am. Soc. Agron., Madison, WI.


Progress 01/01/99 to 12/31/99

Outputs
Legume production in the United States is of significant importance. With many legumes, phosphorus is the limiting nutrient to production. To further our understanding of phosphorus metabolism in the Rhizobium-legume symbiosis and how phosphorus is shared within the symbiosis, we are investigating aspects of phosphorus acquisition by the microsymbiont Rhizobium. Alfalfa and bean are the model legumes used for this work. We are assessing the symbiotic phenotype of various Rhizobium mutants we have generated that are defective in some aspect of phosphorus acquisition (e.g., phosphatase mutants, phosphate transport mutants) or metabolism (e.g., regulatory mutants). We verified that PhoB controlled the Pho stress response in S. meliloti, determined the P concentration at which the Pho regulon is induced in S. meliloti, and found that the Pho stress response was not required for normal symbiotic function of S. meliloti with alfalfa. We characterized the transposon insertion site of several S. meliloti Tn5B22 mutants that had been shown to report the expression of P stress inducible genes, and then studied the expression of these genes under various in vitro conditions and in symbiosis. We also reported on the pta-ackA operon that was discovered to be PhoB regulated. The latter was determined by introducing an phoB::omega allele into the ackA::lacZ reporter strain, and by using primer extension to show the presence of a putative Pho box in the promoter region of this operon. Also, by mutating pta and/or ackA we were able to establish metabolic blocks that allowed us to ask questions about the importance of acetyl phosphate as a nonspecific regulatory metabolite during symbiosis as well as the importance of acetate as a carbon source to alfalfa bacteroids. Based on these studies, we have concluded that it is highly unlikely that alfalfa rhizobia bacteroids ever encounter phosphorus limitation during symbiosis, regardless of the phosphorus nutrition status of the host plant. An important regulatory gene discovered to be critical for symbiosis during the course of this phosphate work included Lon protease. The lon protease mutant forms nodules that do not fix nitrogen. The symbiotic phenotype of the lon mutant is thought to be linked to the inappropriate regulation of exopolysaccharide production. Our work with the bean symbiosis found that bean rhizobia (Rhizobium tropici) bacteroids exhibit high levels of phosphate stress inducible enzymes, regardless of how much phosphorus is supplied to the host plant. This has important implications regarding P supply to the bacteroids and bacteroid gene expression as we now know that the phosphate stress response in Gram negative bacteria is global and includes genes that code for proteins that do not participate in phosphate acquisition or metabolism. We have characterized the two P transport systems in R. tropici and have found that plants inoculated with a mutant lacking the high affinity P transporter produces less dry matter. Bacteroids of this mutant have reduced capacity to acquire P during symbiosis. We have also isolated a phoA R. tropici mutant and cloned the phoB gene from this organism.

Impacts
(N/A)

Publications

  • McDermott, T.R. 1999. Phosphorus assimilation and regulation in the rhizobia. In E.W. Triplett (ed.). Nitrogen fixation in bacteria: Molecular and cellular Bbiology. Horizon Scientific Press. In Press.
  • Summers, M.L., Denton, M.C. and McDermott, T.R. 1999. Genes coding for phosphotransacetylase and acetate kinase in Sinorhizobium meliloti are in an operon that is inducible by phosphate stress and controlled by PhoB. J. Bacteriol. 181:2217-2224.
  • McDermott, T.R. 1998. Phosphate metabolism in Rhizobium: issues, contrasts, and comparisons. p. 45-48. In E. Martinez and H. Hernandez (ed.). Highlights of nitrogen fixation research. Plenum Publishing Corp., New York.
  • Summers, M.L. and McDermott, T.R. 1998. Rhizobium meliloti Lon protease plays an essential role in alfalfa symbiosis. p. 57-60. In E. Martinez and H. Hernandez (eds.). Highlights of nitrogen fixation research. Plenum Publishing Corp., New York.


Progress 01/01/98 to 12/31/98

Outputs
Legume production in the United States is of significant importance. With many legumes, phosphorus is the limiting nutrient to production. To further our understanding of phosphorus metabolism in the Rhizobium-legume symbiosis and how phosphorus is shared within the symbiosis, we are investigating aspects of phosphorus acquisition by the microsymbiont Rhizobium. Alfalfa and bean are the model legumes used for this work. We are assessing the symbiotic phenotype of various Rhizobium mutants we have generated that are defective in some aspect of phosphorus acquisition (e.g. phosphatase mutants, phosphate transport mutants) or metabolism (e.g., regulatory mutants). Based on reporter gene studies, enzyme analyses, and regulatory mutants, we have concluded that it is highly unlikely that alfalfa rhizobia (Sinorhizobium meliloti) bacteroids ever encounter phosphorus limitation during symbiosis, regardless of the phosphorus nutrition status of the host plant. However, bean rhizobia (Rhizobium tropici) bacteroids exhibit high levels of phosphate stress inducible enzymes, regardless of how much phosphorus is supplied to the host plant. This has important implications regarding bacteroid gene expression as we now know that the phosphate stress response in Gram negative bacteria is global and includes genes that code for proteins that do not participate in phosphate acquisition or metabolism. In S. meliloti, part of the global level response to phosphorus limitation includes the expression of several genes involved in extracellular polysaccharide production, some of which are important to nodule formation on alfalfa. Acetyl phosphate metabolism in S. meliloti is also potentially impacted as the genes that code for phosphotransacetylase and acetate kinase are part of the Pho regulon in this bacterium. Another important regulatory gene discovered to be critical for symbiosis during the course of this phosphate work included Lon protease. The lon protease mutant forms Fix- nodules. This mutant was identified while screening through S. meliloti transposon mutants that expressed phosphatases constitutively.

Impacts
(N/A)

Publications

  • Deng, S., Summers, M.L., Kahn, M.L. and McDermott, T.R. 1998. Cloning and characterization of a Rhizobium meliloti nonspecific acid phosphatase. Arch. Microbiol. 170:18-26.
  • Summers, M.L., Elkins, J.G., Elliot, B.A. and McDermott, T.R. 1998. Expression and regulation of phosphate stress inducible genes in Sinorhizobium meliloti. Mol. Plant-Microbe Inter. 11:1094-1101.


Progress 01/01/97 to 12/31/97

Outputs
Both R. tropici and R. meliloti up-regulate P transport rates and alkaline phosphatase (AP) activity when incubated in media lacking P. these P-stress responses were observed when culture PO4 concentrations decreased to approximately to 1 uM and 10 uM for R. tropici and R. meliloti, respectively. R. tropici bacteroids taken from nodules of bean plants grown with luxurious amounts of P express these P-stress-inducible features, suggesting low P concentrations in the bean symbiosome. R. meliloti has two constitutively expressed acid phosphatases; mutants for both enzymes are symbiotically competent. An important contrast between the alfalfa and bean symbioses is the level of P provided to bacteroids. The P-stress response is not apparent in R. meliloti bacteroids as PhoB is not required for symbiosis and up-regulation of P-stress inducible genes in bacteroids is not observed. The high levels of AP in R. tropici bacteroids suggests the PO4 concentration in the symbiosome space is low enough to induce the P-stress response and that significant global effects on gene expression in bean bacteroids is likely.

Impacts
(N/A)

Publications

  • AL-NIEMI, T.S., KAHN, M.L. and MCDERMOTT, T.R. 1997. P metabolism in the bean-Rhizobium tropici symbiosis. Plant Physiol. 113:1233-1241.
  • AL-NIEMI, T.S., SUMMERS, M.L., ELKINS, J.G., KAHN, M.L. and MCDERMOTT, T.R. 1997. Regulation of the phosphate stress response in Rhizobium meliloti by PhoB. Appl. Environ. Microbiol. 63:4978-4981.
  • AL-NIEMI, T.S., KAHN, M.L. and MCDERMOTT, T.R. 1997. Phosphorus uptake by bean nodules. Plant and Soil. In press.


Progress 01/01/96 to 12/30/96

Outputs
R. tropici bacteroids express alkaline phosphatase (AP) at levels twice that of P-starved free-living cells. Expression was transient, decreasing approximately 75% as the bean host entered into reproduction stages. R. tropici bacteroids were found to display an acid phosphatase activity stain profile in native gels that was consistent with P-starved free-living cells. Together this strongly suggests R. tropici experiences P-stress conditions in the bean symbiosome and occurs when nitrogen fixation rates are normally at peak levels. R. tropici bacteroid AP activity was not incluenced by P nutrition of the host plant. 32P labeling studies demonstrated that bacteroids in a P-stressed symbiosis have significantly greater P accumulation rates than bacteroids in nodules of +P plants, and that P accumulation in bacteroids of -P plants appeared to alter P distribution patterns within the plant. R. meliloti mutants lacking either of the acid phosphatases are Fix+, and alkaline phosphatase is not expressed in R. meliloti bacteroids.

Impacts
(N/A)

Publications


    Progress 01/01/95 to 12/30/95

    Outputs
    It was found that R. tropici bacteroids express alkaline phosphatase (AP) at levels twice that observed with P-starved free-living cells. Expression was transient, decreasing approximately 75% as the bean host entered into its reproduction stages. In addition, R. tropici bacteroids were found to display an acid phosphatase activity stain profile in native gels that was consistent with P-starved free-living cells. Together with high levels of AP, this strongly suggests that R. tropici experience P-stress conditions in the bean symbiosome and that this was occurring during the vegetative stage when nitrogen fixation rates are normally a peak levels. R. tropici bacteroid AP activity was not influenced by P nutrition of the host plant. 32P labeling studies with nodulated hydroponic bean plants that had been exposed to high or nil P conditions demonstrated evidence that bacteroids in a P-stressed symbiosis have significantly greater P accumulation rates than bacteroids in nodules of +P plants and that P accumulation in bacteroids of -P plants appeared to alter P distribution patterns within the plant.

    Impacts
    (N/A)

    Publications

    • Al-Niemi, T.S., Kahn, M.L., & McDermott, T.R. 1995. Characterization of phosphorus metabolism in the bean-Rhizobium tropici symbiosis. 15th North American Conf. on Sym. Nit. Fix. Conf. Proc., 8/95, Raleigh, NC.