Source: CENTER FOR ENVIRONMENTAL BIOTECHNOLOGY submitted to
PERSISTENCE AND FUNDAMENTAL CHARACTERIZATION OF A RECOMBINANT MICROORGANISM 13 YEARS AFTER ITS FIELD RELEASE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0219716
Grant No.
2009-39210-20230
Project No.
TENW-2009-01064
Proposal No.
2009-01064
Multistate No.
(N/A)
Program Code
HX
Project Start Date
Sep 1, 2009
Project End Date
Aug 31, 2011
Grant Year
2009
Project Director
Ripp, S.
Recipient Organization
CENTER FOR ENVIRONMENTAL BIOTECHNOLOGY
676 DABNEY HALL
KNOXVILLE,TN 37996
Performing Department
Joint Institutes for Biological Sciences
Non Technical Summary
The proposed investigation hypothesizes that a genetically engineered `man-made' microorganism originally field released 13 years ago continues to survive and persist in its soil environment, thus offering an unprecedented opportunity to analyze microbial community dynamics pertinent to the introduction of a recombinant microorganism into a native microbial community. In October of 1996, under sponsorship by the Department of Energy Natural and Accelerated Bioremediation Research (NABIR) program, the bioluminescent bioreporter Pseudomonas fluorescens HK44 became the first EPA permitted, genetically engineered microorganism to be field tested in the United States for bioremediation purposes. Strain HK44 was released into 4 meter deep by 2.5 meter diameter soil lysimeters that were packed with hydrocarbon contaminated soils. As strain HK44 metabolized these hydrocarbon contaminants, it produced visible bioluminescent light, thereby permitting the bioremediation process to be controlled and monitored via light measurements. From a risk assessment perspective, after three years of field incubation, strain HK44 and its engineered genes were shown to still be present and active. Now, 13 years later, we will return to this field site to further analyze the activities of strain HK44 and determine its potential for long-term residency in an untouched soil environment. Such information, on the time and dimensional scale achievable here, has never been heretofore established. Considering the broadening application of recombinant organisms, especially in agriculture, food, and bioenergy/biofuels, and the increased public awareness of and apprehension towards genetic engineering, the acquisition of a knowledge database pertinent to long-term environmental impacts of engineered genes will be central to fundamental risk assessment.
Animal Health Component
25%
Research Effort Categories
Basic
70%
Applied
25%
Developmental
5%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
1330110104030%
1330110110010%
1334010104030%
1334010110030%
Goals / Objectives
The proposed investigation hypothesizes that a recombinant microorganism field released 13 years ago into replicate subsurface soil lysimeters continues to survive and persist, and/or that select genetic elements derived from this genetically engineered microorganism (GEM) continue to exist. The broader goal of this preliminary snapshot of long-term recombinant organism and gene activity seeks to evaluate and better define translocation and gene transfer risk assessment parameters relevant to the environmental release and long term persistence of GEMs in soil. The planned research takes advantage of a truly unique opportunity to investigate a microbial community impacted by an EPA sanctioned release of high concentrations of a recombinant strain into a natural environment. The intent of this single year research effort is to establish the presence and enrichment potential of the recombinant Pseudomonas fluorescens HK44 strain and/or validate identifiable gene signatures of this strain and its engineered plasmid in order to lay the foundational groundwork for future studies focused on survivability, fitness, transport, and dispersal profiles of the recombinant microbial population and metagenomic analysis of the long-term evolutionary flux and gene flow of genetically engineered genes within and among the resident microbial community. The scale of the lysimeters and their replicate inoculations under both selective and non-selective environmental pressures, in association with extensive background characterization, will provide some of the most discriminating observational information available for risk assessment analysis pertinent to the environmental release of GEMs. Results will be disseminated through publication in the peer reviewed literature and presentation at national meetings.
Project Methods
The proposed research will be divided into two phases. The first phase is the aim of this single year investigation and will focus on an initial quantitative surveillance of the inoculated soil lysimeters to address the hypothesis that P. fluorescens HK44 or select genetic elements derived from strain HK44 persist in the static lysimeters. If this is confirmed, then the ensuing Phase II investigation would focus on issues of gene flow and microbial community response contributing to the persistence and residency of the recombinant organism and/or genes within the community. Phase I methods will focus on recovery of the HK44 strain from the soil using viable plating techniques as well as most-probable-number enumerations. DNA signatures of the HK44 strain will be pursued using the polymerase chain reaction. Full annotation of the HK44 genome and its plasmids will assist in DNA-based recovery efforts. An undergraduate student will assist throughout the project to promote independent study. Milestones after this one-year project will include go/no go decisions on further evaluation of the lysimeter site based on recovery or non-recovery of the HK44 population or its genetic signatures.

Progress 09/01/09 to 08/31/11

Outputs
OUTPUTS: The proposed investigation hypothesized that a recombinant microorganism (and/or its identifying genes) originally field released over 13 years ago continues to survive and persist in its soil environment. This microbe, Pseudomonas fluorescens HK44, represented the first genetically engineered construct to be approved for field release for applications related to subsurface soil bioremediation. Strain HK44 was introduced into several large lysimeter structures where its luciferase (lux)-based bioluminescent activity in response to soil-borne polycyclic aromatic hydrocarbon (PAH) contaminants was monitored in situ using portable photomultiplier tubes and liquid light guide biosensors. During the initial two year release, HK44 populations decreased from their initial inoculum of ~1 x 10^6 cfu/g soil to a maintenance level of ~1 x 10^3 cfu/g soil. Four years post-release the HK44 microbes were shown to be functionally active based on light production and recovery of lux mRNA from soil, even though the selective pressure of the PAH contaminants that it degrades was depleted at least two years prior. Now, fourteen years post-release, an extensive sampling of the lysimeter soils was again undertaken to establish the long-term viability of strain HK44 and/or discovery of gene signatures associated with strain HK44. To do so first required that strain HK44 be sequenced, which was accomplished using a Roche 454 Life Sciences GS FLX system. This resulted in an annotated draft genome sequence of ~6.1 kb and the sequence of the lux-bearing plasmid pUTK21 and one or more cryptic plasmids, which have been deposited in GenBank under accession number AFOY00000000.1. Although the sampling efforts failed to recover viable HK44 cells directly from the soil, genes of potential HK44 origin, including luxA, tetA, and nahA, were identified by qPCR at low concentrations (~100 to 2000 copies/g soil) within the soil metagenome. We are continuing in our efforts to revive HK44 cells from the lysimeter soils via low nutrient and selective enrichments and are as well analyzing distributions and rearrangements of the HK44 recombinant genes among the soil microbiota. Significant outputs and dissemination activities include: - New knowledge contributing to our understanding of environmental risk assessment from a gene transfer perspective - A peer-reviewed publication in the Journal of Bacteriology and another under submission in the journal Sensors - Deposition of sequencing data in GenBank - Two book chapters - Two conference presentations at the American Society for Microbiology annual meeting - Research participation by six undergraduate students and three high school students PARTICIPANTS: Project participants include Drs. Steven Ripp (PI), Alice Layton (co-PI), and Gary S. Sayler (co-PI) from the University of Tennessee Center for Environmental Biotechnology. Dr. Archana Chauhan (postdoctoral scientist) assisted with genome annotation and assembly. Oak Ridge National Laboratory served as a partner organization for the genome sequencing efforts. Mr. Dan Williams and Scott Moser assisted as research technicians. Six undergraduate students participated as part of their independent study projects or via internships (Abby Smart, Danna Sharp, Robert Crossley, Ruth Hahn, Alexandra Rogers, and Whitney Burton). Three high school students (Peter Zhao, Jason Xiong, and Sirui Ma) additionally participated as summer research interns. Training opportunities centered on the inclusion of undergraduate and high school students in the project, all of whom were required to present their research findings at lab meetings. Abby Smartt, Robert Crossley, and Peter Zhao additionally authored a poster presentation at the 2011 American Society for Microbiology meeting. Professional development of the undergraduate students was achieved by independently challenging them with hands-on research intensive projects where they learned organizational and communication skills. Our undergraduates additionally mentored high school student interns to promote leadership and supervisory skills. TARGET AUDIENCES: This research effort targets the biological risk assessment community by establishing heretofore unknown long-term survival rates of environmentally released genetically engineered microorganisms and/or long-term persistence of recombinant genes derived from genetically engineered microorganisms. The application of this knowledge can be applied towards agricultural, food, bioenergy/biofuels, and other biotechnology activities for risk assessment modeling. The genome sequence of the HK44 microorganism also targets audiences interested in evolutionary gene transfer, metabolic pathway modeling, and antibiotic gene fate and occurrence in soil ecosystems. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The sequencing and annotation of the Pseudomonas fluorescens HK44 genome provides a change in knowledge in regards to the discovery of novel traits associated with this strain and Pseudomonads in general, including a polysaccharide production pathway and a potentially undescribed aromatic carbon degradation pathway. The genome displays a range of detoxification mechanisms involved in the degradation of organic substances including polycyclic aromatic hydrocarbons (PAHs) and other xenobiotic chemicals and various enzymes necessary to cope with oxidative stress. The genome also contains gene clusters possessing genes for ammonification and nitrogen respiration (nar, nir, nas, and nor), which indicates that strain HK44 can carry out nitrate/nitrite reduction by both assimilatory and dissimilatory means. Surprisingly, several virulence factors not characteristic of other P. fluorescens strains were identified including a type III secretion system and P. syringae-like pathogenicity genes. The discovery of DNA signatures linked to strain HK44 provides a change in knowledge in regards to our understanding of environmental gene transfer risk. Gene transfer via transformation, transduction, and conjugation represents one of the key driving forces to bacterial evolution and the rapid adaptation of bacterial species to environmental challenges. The introduction of recombinant genes into environmental ecosystems may contribute to gene transfer in unforeseen ways, and the lysimeter structures continue to serve as model ecosystems for studying and modeling these potential risks. Impacts of this research on undergraduate and K-12 educational opportunities have been significant. Six undergraduate students and three high school students were challenged with research projects involving bacterial growth, viable plating, bioluminescent assays, nucleic acid isolation, PCR, and DNA sequencing techniques.

Publications

  • Xu, T., D. Close, G. Sayler, and S. Ripp. 2011. Applications of whole-cell bacterial bioreporters in environmental assessment. Ecological Indicators, submitted.
  • Ripp, S., and T. B. Henry. 2011. Biotechnology and Nanotechnology Risk Assessment, American Chemical Society Symposium Series, Washington, DC, in press.
  • Chauhan, A., A. C. Layton, D. E. Williams, A. E. Smartt, S. Ripp, T. V. Karpinets, S. D. Brown, and G. S. Sayler. 2011. Draft genome sequence of the polycyclic aromatic hydrocarbon-degrading, genetically engineered bioluminescent bioreporter Pseudomonas fluorescens HK44. Journal of Bacteriology 193:5009-5010.
  • Trogl, J., A. Chauhan, S. Ripp, A. C. Layton, G. S. Sayler, and G. Kuncova. Pseudomonas fluorescens HK44: 2011. Lessons learned from a model whole-cell bioreporter with a broad application history. Sensors, submitted.
  • Smartt, A. E., A. L. Layton, S. Ripp, D. E. Williams, R. H. Crossley, P. Zhao, and G. S. Sayler. 2011. Persistence of recombinant genes in subsurface soil environments, American Society for Microbiology Annual Meeting, New Orleans, LA.
  • Ripp, S., M. L. DiClaudio, and G. S. Sayler. 2010. Biosensors as environmental monitors, p. 213-233. In R. Mitchell and J. D. Gu (ed.), Environmental Microbiology, 2nd ed. Wiley-Blackwell, Hoboken, New Jersey.
  • Layton, A., S. Ripp, J. Sanseverino, and G. Sayler. 2010. Genome sequence analysis of the naphthalene bioluminescent bioreporter Pseudomonas fluorescens HK44, American Society for Microbiology Annual Meeting, San Diego, CA.


Progress 09/01/09 to 08/31/10

Outputs
OUTPUTS: This research effort focuses on better understanding the fate of a genetically engineered microorganism released into a soil ecosystem 13 years ago. This microorganism, Pseudomonas fluorescens HK44, was originally released as a bioreporter to monitor the remediation of oil contaminated soil in large-scale contained lysimeters. Outputs of this project so far include a fully sequenced HK44 genome that is still undergoing annotation to better understand its bioremediative potential and for optimal selection of gene signatures for probing within the soil environment. Samples from the soil lysimeter ecosystems have been gathered and processed for viable cell count numbers. Although HK44 has not yet been directly revived from the soil, gene signatures indicating its presence or its evolutionary transfer to other soil microbiota have been discovered by PCR analysis and plaque hybridization assays. Thus, preliminary data indicate that after 13 years residency, nucleic acid originally derived from strain HK44 is still present within the soil. Project highlights continue to be addressed on the University of Tennessee Center for Environmental Biotechnology webpage (www.ceb.utk.edu), laboratory meetings, and departmental seminars. Four undergraduate students and a high school student were mentored during the first year of this project. Results were presented at the annual meeting of the American Society for Microbiology in San Diego in May of 2010. Products include the genome sequence of strain HK44 which will soon be deposited in GenBank along with a peer-reviewed publication. PARTICIPANTS: Project participants include Drs. Steven Ripp (PI), Alice Layton (co-PI), and Gary S. Sayler (co-PI) from the University of Tennessee Center for Environmental Biotechnology. Mr. Dan Williams is assisting as a research technician. Four undergraduate students are participating as part of their independent study projects or via internships (Abby Smart, Danna Sharp, Robert Crossley, Ruth Hahn). One high school student (Peter Zhao) additionally participated as a summer intern. Training opportunities center on the inclusion of undergraduate and high school students in the project, all of whom are required to present at lab meetings and regional conferences. TARGET AUDIENCES: This research effort targets the biological risk assessment community by establishing heretofore unknown long-term survival rates of environmentally released genetically engineered microorganisms. The application of this knowledge can be applied towards agricultural, food, bioenergy/biofuels, and other biotechnology activities for risk assessment modeling. The genome sequence of the HK44 microorganism also targets audiences interested in evolutionary gene transfer and metabolic pathway modeling. PROJECT MODIFICATIONS: The lysimeter field site is located within the confines of the Oak Ridge National Laboratory. Gaining access to the secure site and executing proper health and safety directives took longer than estimated and thus initiation of the project was delayed for approximately six months. We have therefore requested a one year no-cost extension to fully complete the project.

Impacts
The sequencing and annotation of the P. fluorescens HK44 genome and the discovery of DNA signatures linked to strain HK44 in the lysimeter soils represents a change in knowledge significant enough to each warrant a publication. Both manuscripts are in preparation. A change in action was implemented upon elucidation of the HK44 genome since it was then used to optimally track HK44 gene signatures in lysimeter soils.

Publications

  • Layton, A., S. Ripp, J. Sanseverino, and G. Sayler. 2010. Genome sequence analysis of the naphthalene bioluminescent bioreporter Pseudomonas fluorescens HK44, American Society for Microbiology Annual Meeting, San Diego, CA., May 2010.
  • Ripp, S., M. L. DiClaudio, and G. S. Sayler. 2010. Biosensors as environmental monitors, p. 213-233. In R. Mitchell and J. D. Gu (ed.), Environmental Microbiology, 2nd ed. Wiley-Blackwell, Hoboken, New Jersey.