Tiedje Lab - Projects 
Physiology and Genomics of Shewanella oneidensis MR-1
Shewanella oneidensis MR-1 is a facultatively anaerobic Gamma-Proteobacterium capable of utilizing a wide variety of compounds as electron acceptors, such as oxygen, iron, manganese, uranium, nitrate, nitrite, fumarate, thiosulfate, dimethyl sulfoxide, trimethylamine N-oxide, and elemental sulfur. Metabolic versatility, amenability to genetic manipulation, and recent publication of a full genome sequence make S.oneidensis MR-1 an excellent model system for studying microbial respiratory processes using high-throughput genomic-level experimental approaches.
Our group is funded to conduct research as a member of the Shewanella Federation (http://www.shewanella.org) under the Microbial Cell Project component of the DOE OBER Genomes-to-Life Program. The Shewanella Federation is a multi-institutional research consortium that leverages the expertise of researchers from different scientific backgrounds to understand the biology of S.oneidensis MR-1 at the whole-systems level. Institutional expertise includes whole-proteome mass spectroscopy at Pacific Northwest National Laboratory, transcriptome analysis and microarray fabrication at Oak Ridge National Laboratory, systems modeling at Biatech, and database design and microbial physiology at here Michigan State University. Using a combination of physiological and genomic studies our laboratory research efforts have focused on two primary aspects of S.oneidensis MR-1 that are important for potential bioremediation applications: 1) nitrate reduction, and 2) the stress response.
Nitrate Reduction in S.oneidensis MR-1
Project Members: Claribel Cruz-Garcia, Joel Klappenbach
Nitrate is commonly found as a co-contaminant of groundwater with uranium. Strategies for the bioremediation of uranium-contaminated groundwater though microbial reduction must consider the effect of nitrate as an alternative electron acceptor. The reduction of nitrate is more energetically favorable than uranium and participates in other reductive processes that can result in both the precipitation and dissolution of uranium in groundwater. Physiological studies in our laboratory have shown no detectable intermediate reduction products such as nitric- and nitrous-oxides and little dinitrogen, but ammonium is produced during growth. Genomic sequence analysis supports these physiological observations. S.oneidensis MR-1 is unique from many nitrate reducing bacteria in possessing only a single periplasmic nitrate reductase (napDAGBH operon and distally located napC) and a single nitrite reductase (ntrA). While some assembly proteins for nitrous oxide reductase (nos genes) are present on the genome of S.oneidensis MR-1, the nosZ catalytic enzyme is absent. The presence of only the periplasmic nitrate reductase in S.oneidnesis MR-1 presents a unique opportunity to elucidate the role of periplasmic nitrate reductases in respiratory nitrate reduction. We are using batch and chemostat culture techniques, targeted gene deletions, quantitative PCR, proteomics, and whole-genome cDNA microarrays to better understand aerobic and anaerobic nitrate reduction to ammonia in S.oneidensis MR-1.
Stress Response
Project Members: Xiaoyun Qiu
Understanding the ability of a bacterium to survive and grow in a natural environment, such as soils, sediments and groundwater, is also critical to assessing the potential for bioremediation. We have investigated the genetics and regulatory mechanisms involved in the stress response to UV irradiation in S.oneidensis. Ultraviolet and ionizing radiation are common stresses that S.oneidensis can face in bioremediation applications. Microarrays that contain ca. 95% of ORFs of MR-1 were used to compare the genes and pathways which are induced in response to UVC (254 nm), UVB (290-320 nm), UVA (320-400 nm), and solar UV (composed of 95% of UVA and 5% of UVB) and gamma radiation. S.oneidensis proved to be remarkably sensitive to UV irradiation Δ only 10 Δ 12 minutes in natural sunlight produces a 20% survival rate. Intriguingly, UV irradiation induced expression of prophage genes which may indicate that S.oneidensis is susceptible to viral lysis during other stress conditions such as starvation or nitrite toxicity.
Informatics: Development of a LIMS system
for Microarray Construction
Project Members: James Cole, Joel Klappenbach, Paul R. Saxman, Qiong Wang, Siddique A. Kulam, Alison E. Murray
High-throughput methodologies including microarray expression analysis, proteomics, and whole-genome clone libraries generate massive quantities of data that necessitate computer database management tools. While many public and commercial software packages exist for analyzing microarray hybridization results, we found that few software choices existed for upstream data management in the laboratory. Commercial laboratory information management systems (LIMS) are available, but require extensive customization and administration that are both cost-prohibitive and impractical for our needs. To address these needs, we created a laboratory information management system (LIMS), named the MicrobeArrayDB, for handing high-throughput data created during genomic-level applications such as the fabrication of DNA microarrays. The MicrobeArrayDB is built on a multi tier client-server architecture model using a publicly-available relational DBMS for our back-end tier (PostgreSQL) and Java (Sun Microsystems) web technologies for middle and presentation tiers. LIMS tools are interfaced through an internet browser interface and data access is controlled through group and user level permissions. The database is structured with the laboratory microplate (96 or 384 well) as the primary data unit. This flexible database structure permits creation of different plate types with associated data fields, thereby extending the functionality of the LIMS to additional applications Δ such as phage display, whole-genome mutagenesis proposed under the MCP grant. Current plate types include: Primer Plates, Template, Primer Pair, PCR Product, and Printing plates. Users perform in silico reactions within the LIMS that create new product plates within the database Δ such as the creation of a PCR Product plate from existing Primer and Template plates. The LIMS system has been tested internally to model the microarray fabrication process of whole-genome expression arrays for Shewanella oneidensis MR-1. Researchers from ORNL have initiated project-specific customization of the MicrobeArrayDB for the production of additional whole-genome expression arrays and phage-display.
Publications and Presentations:
Murray A.E., D. Lies, G. Li, K. Nealson, J. Zhou, J.M. Tiedje. (2001) DNA/DNA hybridization to microarrays reveals gene-specific differences between closely related microbial genomes. Proc Natl Acad Sci U S A. 98(17):9853-9858.
Beliaev A.S., D.K. Thompson, T. Khare, H. Lim, C.C. Brandt, G. Li, A.E. Murray, J.F. Heidelberg, C.S. Giomett, J. Yates III, K.H. Nealson, J.M. Tiedje, J. Zhou. (2002) Gene and protein expression profiles of Shewanella oneidensis during anaerobic growth with different electron acceptors. OMICS. 2002;6(1):39-60
Tiedje J.M. (2002) Shewanella--the environmentally versatile genome. Nat Biotechnol. 20(11):1093-1094.
Cole, J.R., J.A. Klappenbach, P.R. Saxman, Q. Wang, S.A. Kulam, A.E. Murray, L. Wu, J. Zhou, and J.M. Tiedje. A web-based laboratory information management system (LIMS) for laboratory microplate data generated by high-throughput genomic applications. Presentation from the Genomes-to-Life Contractor-Grantee Workshop I, Arlington, Virginia, February 9-12, 2003
Cruz-Garcia, C., A.E. Murray, J.M. Tiedje. (2003) Dissimilatory nitrate reduction into ammonium is the pathway responsible for the reduction of nitrate in Shewanella oneidensis MR-1. 11th International Conference on Microbial Genomes. Durham, North Carolina.
Qiu, X., G.W. Sundin, B. Chai, J.M. Tiedje. (2003) The UV radiation responses in Shewanella oneidensis MR-1. 11th International Conference on Microbial Genomes. Durham, North Carolina, USA.
Klappenbach, J.A., J.R. Cole, P.R. Saxman, A.E. Murray, J. Zhou, J.M. Tiedje. (2002) MicrobeArrayDB - a laboratory information management system for tracking and archiving microplate data generated during the microarray fabrication process, American Society for Microbiology General Meeting, Salt Lake City, UT, USA.
Qiu, X., J. Zhou, J.M. Tiedje. (2001) An approach to identify and characterize genes involved in anaerobic respiration in Shewanella oneidensis MR-1. 9th International Symposium on Microbial Ecology. Amsterdam, The Netherlands.
Project Links:
RDP Informatics Group @ MSU (http://rdp.cme.msu.edu)
MicrobeArrayDB (http://microbedb.cme.msu.edu)
Center for Microbial Ecology @ MSU (http://cme.msu.edu)
Pacific Northwest National Labs Proteomics Group (http://www.emsl.pnl.gov/docs/msd/mass_spec/home/index.html)
Oak Ridge National Labs Genomics Group (http://www.esd.ornl.gov/facilities/genomics)
BiaTech Computational Genomics (http://www.biatech.org)
DOE Office of Biological and Environmental Research (http://www.sc.doe.gov/production/ober/ober_top.html)
