Grantee Research Project Results
1999 Progress Report: Microbial Monitoring With Artificial Stable RNAs
EPA Grant Number: R825354Title: Microbial Monitoring With Artificial Stable RNAs
Investigators: Fox, George E. , Willson, Richard C.
Institution: University of Houston
EPA Project Officer: Hahn, Intaek
Project Period: January 1, 1997 through December 31, 1999
Project Period Covered by this Report: January 1, 1999 through December 31, 2000
Project Amount: $335,701
RFA: Exploratory Research - Environmental Biology (1996) RFA Text | Recipients Lists
Research Category: Biology/Life Sciences , Aquatic Ecosystems
Objective:
The long-term goal of this project is to establish a stable RNA approach for labeling and tracking microorganisms in complex ecosystems. In brief, a unique identifier sequence is inserted into a fragment of a 5S rRNA gene carried by the target bacterium. The resulting chimera gene expresses an artificial RNA (aRNA), which has stability that is comparable to that of naturally occurring 5S rRNA and therefore accumulates in cells in large amounts. Standard detection systems for ribosomal RNA, as well as PCR based methods, can then be used to determine the presence or absence of the aRNA and hence the target organism. It is envisioned that this technology will facilitate studies of risk associated with the release of both naturally occurring and genetically engineered organisms into the environment. We are conducting the exploratory research needed to establish the utility of the approach.Progress Summary:
The immediate project goals were to (1) establish the utility of the approach in a variety of bacteria, (2) to integrate stable RNA identifiers into bacterial genomes, (3) to determine the extent to which identifiers can be changed, (4) to establish soil based assay systems for use with stable RNA identifiers. Considerable additional progress has been made in the past year as described below. Coupled with our previous efforts, we have now accomplished all the major milestones of the project. Current efforts are focused on laying the groundwork needed for future microcosm tests of the system in aquatic and marine environments.Accomplishments: Previously, refinements to the original Escherichia coli construct were made and we succeeded in developing a plasmid based expression system for Pseudomonas putida. This demonstrated that the identifier system could be used in bacteria that are not closely related to E. coli. However, the expression level was significantly less than that seen in E. coli. It was shown in the current funding period that plasmid copy number in Pseudomonas was significantly reduced (from 30-40/cell to less than 10/cell). This is, therefore, the likely explanation for the reduced expression levels.
A primary goal of the project is integration of identifiers into genomes of useful organisms. This would be advantageous because genomic inserts are much more to be stably maintained than plasmid systems and much less likely to be transferred to other organisms. This had previously been accomplished in E. coli. A major highlight of the current funding period was the successful development of an analogous genomic identifier system for P. putida. Separately, we had shown that essentially any identifier sequence in the 10-50 nucleotide size range would work with the plasmid based carrier system. During the past year, we began testing a number of these inserts in the genomic systems. Typically, they are again all expressed, though the accumulation levels differ significantly depending on the insert. Because there is only one copy of the expression system per cell when it is genomically inserted, one expects that the expression level in the genomic systems will be lower than that which is seen with the plasmid systems. This is in fact the case, being approximately 10% in the E. coli construct. However, with the P. putida genomic constructs the expression levels represented only 2% of the total 5S rRNA. This almost certainly reflects the fact that the E. coli ribosomal RNA promoters used in constructing the P. putida genomic inserts were not fully active. The problem was partly overcome using an insert with three copies of the target sequence, which gave a 2-3 times enhanced signal. Thus, further refinement of the system, e.g. incorporation of P. putida promoters is desirable. Nevertheless, the existing system is more than adequate to serve as a basis for microcosm testing of the identifier approach.
Significant progress continues to be made in the development of procedures for working with stable RNAs in environment samples as well. The E. coli genomic construct is being used extensively in our efforts to develop assay procedures suitable for environmental studies. The focus to date has been development of field compatible methods for recovery of the RNA. During the last year, our initial exiting results using compaction agents to purify DNA have been successfully extended to RNA and a manuscript on this topic is now in preparation. In addition, studies have begun using molecular beacons to detect the presence of RNA inserts. This technology is especially promising for future field studies because it can be done in solution, will allow simultaneous monitoring of several probes, and may be compatible with the rapid procedures we are developing for sample preparation. During the past year we have shown that problems with RNA structure do not prevent successful application of the method with our rRNA constructs.
Personnel: Research on the development of plasmid and genomic based identification systems for use in alternative hosts, e.g. Pseudomonas etc. was primarily pursued by Ms. Lisa Dsouza who will receive her Ph. D. degree in December of 1999. Efforts to integrate detection cassettes into genomes were originally initiated by a postdoctoral research associate, Dr. David Ammons. David conducted the work on genomic inserts in E. coli with the help of his wife, Ms. Joanne Rampersad. Dr. Ammons, is now an Assistant Professor at the University of Trinidad. A second postdoctoral research associate, Dr. Robert Setterquist and a current graduate student, Ms. Maia Larios-Sanz did studies to examine the types of sequences that could serve as potential identifiers. Dr. Setterquist is now a senior scientist at Ambion Inc. in Austin, Texas. Mr. Zhengdong Zhang, a current graduate student, is doing additional work on plasmid inserts. Recently, a new postdoctoral scientist, Dr. Gary Schultz joined the project team. Gary has a Ph. D. from the College of William and Mary in marine ecology. He is currently focusing his efforts on microcosm evaluations of our novel technology.
Mr. Narinder Singh who worked on the project during the first year began efforts in the area of sample preparation and assay. He focused his efforts on purification of RNA by boronate affinity. He received his M.S. in chemical engineering from the University of Houston in early 1997 and is now employed by Millennium Pharmaceuticals in Boston, Massachusetts. Mr. Jason C. Murphy who has continued the work on RNA purification during years two and three replaced Mr. Singh on the project. Mr. Murphy successfully defended his MS thesis in chemical engineering entitled "Nucleic Acid Separations Using Compaction Agents" in May of 1998. Mr. Murphy is now pursuing a Ph. D. and is continuing to work on compaction agents for use in RNA purification. Mr. Raj Walia joined our group late in year one and continued to work on the project throughout year two. He examined RNA preparation procedures for use in soil and aquatic environments and obtained a M.S. in biomedical engineering at the University of Houston in January 1998 for his thesis entitled "Microbial Monitoring Using Hybridization Assays and Artificial rRNA Labels". Ms. Ekaterini Kourentzi is working on detection systems based on molecular beacons for use with the aRNA constructs. She completed her MS thesis entitled "Environmental monitoring of microorganisms using 5S rRNA and molecular beacons" during the current year. She is continuing her work on this portion of the project and is pursuing a Ph.D. degree.
Budget: During year three, funds were expended essentially as originally proposed, e.g. primarily for salaries and supplies. We have asked for and received a no cost extension until August 31, 2000. During this period, personal working on the project will be supported primarily by other sources of funds available to Drs. Fox and Willson. The residual EPA funds will be used primarily for supplies.
Other Developments: The EPA funded research being undertaken here intensified our interest in environmental problems. As a result, Drs. Willson and Fox joined with a group of three other University of Houston faculty and wrote a proposal to the internally funded Shell Scholars Program.. This activity was funded successfully renewed in January of 1997 and January 1998. During the current year our group of Shell Scholars received $100,000 for a project entitled "Sustainable Development in the Face of Global Climate Change". One of the highlights of this program was the development of a new interdisciplinary course, CHEE 5397/6397 Bioremediation (listed under chemical engineering) that was jointly taught by the five participating faculty members. It is expected that a revised version of this course will be taught again in the Fall of 2000.
Future Activities:
In the coming nine months, our immediate goal is to fully establish the technology base needed to test the utility of aRNA identifiers in laboratory microcosms and eventually in field studies. We are thus focusing our efforts on the procedures needed to accomplish this, e.g. sample preparation and assays and investigating the regulatory requirements we must meet in order to conduct future studies. Further improvements in the cassettes themselves can also be made. For example, the utility of an identifier with multiple probes is now well established. It would be of considerable interest to see if an insert with many more copies of the same target would be possible and if so, would it have long-term genetic stability? Another enhancement that would almost certainly work would be the replacement of the E. coli promoters in the P. putida genomic construct with Pseudomonas promoters. It will not be possible to make these enhancements with the residual resources currently available. It is our expectation that in the coming 3-6 months we will seek additional funding to continue what we believe is an exciting and highly successful project.Journal Articles on this Report : 5 Displayed | Download in RIS Format
Other project views: | All 43 publications | 10 publications in selected types | All 9 journal articles |
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Ammons D, Rampersad J, Fox GE. A genomically modified marker strain of Escherichia coli. Current Microbiology 1998;37(5):341-346. |
R825354 (1999) R825354 (Final) |
Exit |
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Ammons D, Rampersad J, Fox GE. 5S rRNA gene deletions cause an unexpectedly high fitness loss in Escherichia coli. Nucleic Acids Research 1999;27(2):637-642. |
R825354 (1999) R825354 (Final) |
Exit Exit Exit |
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D'Souza LM, Willson RC, Fox GE. Expression of marker RNAs in Pseudomonas putida. Current Microbiology 2000;40(2):91-95. |
R825354 (1999) R825354 (Final) |
Exit |
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Murphy JC, Wibbenmeyer JA, Fox GE, Willson RC. Purification of plasmid DNA using selective precipitation by compaction agents. Nature Biotechnology 1999;17(8):822-823. |
R825354 (1999) R825354 (Final) |
Exit |
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Singh N, Willson RC. Boronate affinity adsorption of RNA: possible role of conformational changes. Journal of Chromatography A 1999;840(2):205-213. |
R825354 (1997) R825354 (1999) R825354 (Final) |
Exit Exit Exit |
Supplemental Keywords:
artificial RNA, aRNA, molecular beacons, 5S rRNA, environmental monitoring., Scientific Discipline, Ecosystem Protection/Environmental Exposure & Risk, Environmental Chemistry, Genetics, Chemistry, Monitoring/Modeling, Engineering, artificial stable RNA, microbial monitoring, bacteria monitoring, DNA vector, microorganism, bioluminescence, green flourescent protein, RNA gel profileProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.