Final Report: Microbial Monitoring With Artificial Stable RNAs

EPA Grant Number: R825354
Title: 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 Amount: $335,701
RFA: Exploratory Research - Environmental Biology (1996) RFA Text |  Recipients Lists
Research Category: Biology/Life Sciences , Ecosystems

Objective:

The long-term goal of this project was 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. During the funding period we successfully conducted essential exploratory research needed to establish the utility of the approach.

The immediate project goals were to: (1) establish the utility of the approach in a variety of bacteria; (2) integrate stable RNA identifiers into bacterial genomes; (3) determine the extent to which identifiers can be changed; and (4) establish soil based assay systems for use with stable RNA identifiers. All of these major milestones of the project were successfully achieved.

Summary/Accomplishments (Outputs/Outcomes):

In order to meet the project objectives we began by making refinements to the then existing constructs, which expressed a plasmid borne artificial RNAs (aRNA) known as the Pen aRNA in Escherichia coli. The expression machinery was then transferred to a broad host range plasmid that had been modified to be kanamycin resistant. This allowed expression of aRNA in a variety of Gram negative bacteria, and most especially Pseudomonas putida. In particular, construct were made and we now have succeeded in developing a plasmid based expression system for Pseudomonas putida where the addition of kanamycin resistance allowed selection of bacterial cells that carried the aRNA expression system. It was found that an identifier RNA is expressed in Pseudomonas putida and accumulates in approximately a 1:5 ratio to the wild type 5S rRNA. This demonstrated that the identifier system could be used in bacteria that are not closely related to E. coli. However the expression level of 15-20% is less than the 35-50% that was seen in E. coli. Further studies revealed that this likely reflected a decline in plasmid copy from 30-40 copies per cell in E. coli to less than 10 in P. putida.

With the demonstration that the aRNA expression system would in fact work in alternative useful hosts, we next sought to stabilize the construct by inserting it into the genome. This important milestone was first accomplished with E. coli. In this case, the identifier accumulated in the expected 1:6 ratio with the naturally occurring 5S rRNA. Resulting in accumulation of the aRNA to approximately 10 percent of the total 5S rRNA level. Subsequently, transposon mutagenesis was used to transfer constructs regulated by the E. coli ribosomal RNA gene promoters into the genome of P. putida. These experiments were successful but the expression levels represented only 2 percent 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. Sequence information for P. putida promoters was not available when this work was undertaken. 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.

The utility of an aRNA based monitoring system in the field will depend heavily on the ability to express a variety of unique labeling sequences. We, therefore, sought to examine the extent to which a variety of inserts could be used as identifiers. In order to test this we constructed two families of identifier inserts, one consisting of random 13-mers and the other random 50-mers. A large number of these random inserts were successfully incorporated into the aRNA expression plasmid in E. coli. RNA blots were used to examine thirty six of these constructs for RNA expression. In each case, an aRNA of the expected size was found in significant quantity. Another important milestone was accomplished when these results demonstrated that there likely would be no difficulty in obtaining many unique identifiers. Subsequently, four of these inserts as well as our standard Pen aRNA marker and a construct carrying 3 copies of the Pen aRNA identifier sequence were successfully inserted into the genome of P. putida. Although the expression levels were low, approximately 2 percent in most cases, all the aRNAs accumulated thereby verifying that that many useful identifier sequences likely exist.

Significant progress was also made towards the development of assay procedures for stable aRNAs in the environment. The E. coli genomic construct was used extensively in our efforts to develop assay procedures suitable for environmental studies. We conducted significant exploration of simplified approaches to sample preparation for microbiological and other assays to be conducted in the field environment. The demands of routine field monitoring require that one use the minimum number of steps with minimum manipulation, without employing hazardous materials or excessive energy. We found two of the methods we developed to be quite promising, and each has a high probability of becoming a widely used spinoff product of this research.

The first of these was based on compaction precipitation. Compaction agents are small, cationic molecules which bind in either the major or minor grooves of a double-stranded DNA or RNA molecule, reducing by four to six orders of magnitude the volume occupied by the DNA or RNA. In the course of experiments on the potential of nucleic acid compaction for enhancement of chromatographic adsorption capacity (increasing the capacity of classical adsorbents for RNA sample preparation), precipitation was noted at low ionic strengths. Upon further investigation, we observed that RNA is far less readily precipitated by compaction agents, and found that selective DNA precipitation by compaction agents could be useful in purifying plasmids. A single precipitation step precipitates plasmid DNA quantitatively, leaving nearly all the RNA behind in the supernatant.

We then extended the technique to the isolation of RNA from bacterial lysates using compaction agents such as hexammine cobalt and spermidine. Using 3.5 mM hexammine cobalt, total RNA can be selectively precipitated from a cell lysate and at a concentration of 2 mM hexammine cobalt rRNA can be fractionated from low molecular weight tRNA and mRNA. Using a second stage of precipitation at 7.1 mM hexammine cobalt, the low molecular RNA weight fraction can be isolated by precipitation. The resulting RNA mixtures are readily resolved to pure 5S and mixed 16S/23S rRNA by nondenaturing anion-exchange chromatography. Compaction precipitation was also applied to the purification of a typical artificial stable RNA.

The DNA applications of this technique may represent a significant technological spinoff as they may be particularly useful in developing and possibly in performing field-based nucleic acid probe assays. Provisional and non-provisional patent applications have been filed and the technique has been tested for sample preparation at China's largest DNA sequencing center and is being utilized in research on plasmid-based DNA vaccines for HIV. The method has the potential for broad use as a kit in molecular biology labs where enormous efforts and costs are devoted to purifying plasmids for cloning, sub-cloning, genomics, DNA sequencing, etc. The University of Houston has identified a likely licensee for the technology, which has now taken a license option and is finalizing plasmid miniprep kit design, to the point that the licensee now has packaging mockups for the commercial spinoff product.

The second promising purification procedure that we examined was Immobilized-Metal Affinity Chromatography (IMAC). IMAC is the basis of the ubiquitous six-histidine purification "tag" for recombinant proteins. Based on the chemical similarity of the aromatic nitrogens of nucleic acid bases to those of histidine, tryptophan, etc., we hypothesized that chelated metals might also form ligand interactions with the exposed bases of single-stranded nucleic acid molecules. To avoid phosphate interaction only soft metals were evaluated, so we expected that single-stranded regions with exposed bases would be favored over double-stranded molecules. This expectation was realized: plasmid, genomic DNA (and lipids and nearly all proteins) have virtually no binding affinity, while RNA and single-stranded oligonucleotides bind strongly to metal-chelating matrices. Interestingly, affinity differs sharply for differing metal ions, decreasing in the order copper(II), zinc(II), nickel(II) and cobalt (II). IMAC proves to be extremely effective at capturing RNA from mixtures with other molecules, and also for stripping primers from e.g., PCR and sequencing reactions. At least some (possibly all) single-base mismatches can be detected, raising the possibility of developing IMAC-based hybridization assays for microbial identification, SNP scoring, etc. A publication and a patent application are in preparation, and the UH licensing office is in negotiation with at least 5 prospective licensees, including the dominant companies in the field.

Finally, we also conducted preliminary work on a field compatible detection method for aRNA identifiers that utilized molecular beacon technology. In this approach, an oligonucleotide carrying a fluorophore and a quencher in close proximity is hybridized to the total sample RNA. If the target sequence is present the beacon hybridizes to it with the result that the fluorophore and quencher are no longer in proximity resulting in a dramatic increase in fluorescent signal that can be detected by photometry. The attractiveness of the technique stems from the fact that the signal is generated in solution without any need to remove non-hybridizing material. We first demonstrated that an oligonucleotide carrying the fluorophore 6' carboxylfluorescein gave a 20-fold increase in fluorescence in the presence of a complementary Vibrio proteolyticus 5S rRNA. This demonstrated that RNA structure would likely not be a major hindrance to use of the beacon technology. Subsequently, the beacon technology was used to distinguish a strain of E. coli carrying the Pen aRNA identifier from an otherwise identical E. coli strain that did not carry the identifier RNA.

Conclusions:

We have shown that rRNA identifiers will be expressed and stable in hosts other than E. coli. We have successfully transferred identifier sequences to the genomes of both E. coli and P. putida where they are far more stable while continuing to express the identifier RNA. In addition, we have shown that a large number of alternative identifier sequences exist and that the identifier RNA can be detected in simple field compatible assays using simplified RNA isolation methods and fluorescence detection using molecular beacons. In summary, we have now firmly established the use of aRNA identifiers as a feasible technology for monitoring bacteria, whether engineered or not, that are added to an ecosystem. Future work can now focus on the additional development and initial microcosm testing needed before actual field-testing can begin. In this regard, we believe that it is important to do the following: (1) Establish the sensitivity and reproducibility of the aRNA identifier approach in complex soil and aquatic laboratory microcosms; (2) Enhance the signal from artificial RNAs expressed in P. putida by replacing the E. coli promoters with Pseudomonas promoter sequences which are now known from recent genomics studies; and (3) Use mRNA expression profiling, protein expression studies, and competitive growth studies to better understand the extent to which the presence of the identifier may or may not effect other activities in the cell that is carrying it.


Journal Articles on this Report : 9 Displayed | Download in RIS Format

Other project views: All 43 publications 10 publications in selected types All 9 journal articles
Type Citation Project Document Sources
Journal Article 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)
  • Abstract from PubMed
  • Abstract: Springer-Abstract
    Exit
  • Journal Article 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)
  • Full-text from PubMed
  • Abstract from PubMed
  • Full-text: Oxford Journals-Full Text HTML
    Exit
  • Abstract: Oxford Journals-Abstract
    Exit
  • Other: Oxford Journals-Full Text PDF
    Exit
  • Journal Article 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)
  • Abstract from PubMed
  • Abstract: Springer-Abstract
    Exit
  • Journal Article 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)
  • Abstract from PubMed
  • Other: Nature Bioechnology-Article Citation
    Exit
  • Journal Article Murphy JC, Fox GE, Willson RC. RNA isolation and fractionation with compaction agents. Analytical Biochemistry 2001;295(2):143-148. R825354 (Final)
  • Abstract from PubMed
  • Full-text: ScienceDirect-Full Text PDF
    Exit
  • Abstract: ScienceDirect-Abstract
    Exit
  • Journal Article Murphy JC, Fox GE, Willson RC. Enhancement of anion-exchange chromatography of DNA using compaction agents. Journal of Chromatography A 2003;984(2):215-221. R825354 (Final)
  • Abstract from PubMed
  • Full-text: ScienceDirect-Full Text HTML
    Exit
  • Abstract: ScienceDirect-Abstract
    Exit
  • Other: ScienceDirect-Full Text PDF
    Exit
  • Journal Article Murphy JC, Jewell DL, White KI, Fox GE, Willson RC. Nucleic acid separations utilizing immobilized metal affinity chromatography. Biotechnology Progress 2003;19(3):982-986. R825354 (Final)
  • Abstract from PubMed
  • Abstract: Wiley Online-Abstract
    Exit
  • Journal Article Pitulle C, Dsouza L, Fox GE. A low molecular weight artificial RNA of unique size with multiple probe target regions. Systematic and Applied Microbiology 1997;20(1):133-136. R825354 (1997)
    R825354 (Final)
  • Abstract from PubMed
  • Full-text: ScienceDirect-Full Text PDF
    Exit
  • Abstract: ScienceDirect-Abstract
    Exit
  • Journal Article 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)
  • Abstract from PubMed
  • Full-text: ScienceDirect-Full Text HTML
    Exit
  • Abstract: ScienceDirect-Abstract
    Exit
  • Other: ScienceDirect-Full Text PDF
    Exit
  • Supplemental Keywords:

    bioremediation, quantitative microbial ecology, mutagenesis., Scientific Discipline, Ecosystem Protection/Environmental Exposure & Risk, Genetics, Environmental Chemistry, Chemistry, Monitoring/Modeling, Engineering, artificial stable RNA, microbial monitoring, bacteria monitoring, DNA vector, microorganism, bioluminescence, green flourescent protein, RNA gel profile

    Progress and Final Reports:

    Original Abstract
  • 1997 Progress Report
  • 1998