COI Funded Project: Radiocarbon Analysis of 16S rRNA from Environmental Samples: A New Approach to Isotopic Biochemistry with Applications to Coastal Systems


Final Report

Project Summary
A simulated petroleum spill was conducted using samples collected from Woodneck Marsh, Woods Hole, Massachusetts. The in-situ microbial community visibly changed in its qualitative characteristics when oil was added, relative to the consortium growing in the control samples. These observational methods yield little information about the quantitative importance of sub-groups within the total bacterial consortium during oil spill remediation. In this work, our goal was to develop methods to utilize the difference between the natural radiocarbon concentration of oil (no 14C) and fresh organic matter (14C at modern atmospheric levels) to monitor metabolism of an oil spill by salt-marsh bacteria. To achieve this goal, we proposed to implement a combination of the techniques of molecular biology, namely extraction and purification of organism-specific 16S rRNA, with the isotopic measurement methods of geochemistry (14C accelerator mass spectrometry). It quickly became apparent, however, that this goal would be better achieved through stable carbon (13C/12C) isotope analysis. The sample size limitations of 14C-AMS, the concurrent development of instrumentation for 13C analysis of nucleic acids, and the large signal (13C-enriched Spartina salt marsh environment and 13C-depleted oil) led us to pursue methods focused on the latter approach.

Here we report progress toward the goal of achieving the first phylogenetically directed stable isotopic analysis of an active microbial consortium, based on separation of intact 16S rRNA molecules. While not yet complete, this project is in its final stages, and it promises to generate a completely novel data set. There will be numerous future applications in coastal and oceanographic biogeochemistry.

Methods and Approach
Bulk samples of Spartina alterniflora and wild rose were collected from Woodneck Beach Salt Marsh, Falmouth, MA USA (Figure 1a). In addition, bulk bacterial cells were skimmed from the surface of a shallow tidal pool, and a mussel (M. demissus) was collected from the same location. These samples were processed (Figure 1d) to determine the background d13C values of bulk organic pools in the salt marsh. Data were obtained for both the total biomass and for the total nucleic acids. Then, twelve shallow cores were incubated for two weeks at ambient temperature and sunlight, six with oil (#2 Fuel Oil) inoculant and six without (Figure 1b,c). Qualitative changes in the microbial community were observed and photographed, and samples were taken for fluorescent in-situ hybridization (FISH) analysis. Emphasis then changed to methods development for the subsequent isotopic analysis of 16S rRNA from these samples.

Moving-Wire Interface for LC-irMS
In order to link biogeochemical processes to the organisms (species or groups) responsible for mediating these chemical reactions, analysis of the 16S ribosomal RNA molecule is a logical â biomarkerã? to exploit in the effort to achieve isotopic analysis along with taxonomic information. Toward this end, we made progress using a moving-wire interface that will enable the isotopic analysis of samples, such as nucleic acids, that have aqueous solubility (Brand and Dobberstein, 1996, Isotopes Environ. Health Stud 32, 275-283). Aqueous samples or HPLC effluent was dried onto a moving wire, combusted at 800°C, and the d133C of the resulting CO2 was measured by isotope ratio monitoring mass spectrometry. Preliminary analyses of nucleotide standards, bulk RNA standards, and total nucleic acid extracts produced d13C results that had precisions of â± 0.2 permil and were accurate to within â± 1.0 permil for samples containing 10 micrograms of carbon. This device was used to obtain the d13C data for the bulk salt marsh RNA extracts.

Development of RNA Capture Protocol
To prepare group-specific RNA from natural and cultured samples, we worked toward methods that utilize commercial magnetic bead capture technology. The 16S rRNA targets were hybridized to complimentary oligonucleotide probes that had been derivatized with biotin at the 5? end. Streptavidin-coated magnetic beads were added to bind with the biotin end of the RNA-probe complex. The beads were then captured using a magnetic particle concentrator. Captured RNA was subsequently eluted from the beads, washed, and analyzed by agarose gel electrophoresis. The hybridization component proved to be the most problematic aspect of the project, and until a few months ago, efforts to achieve a reliable hybridization protocol were frustratingly unsuccessful. Cross-hybridization of competing 23S rRNA was a serious problem (Figure 4). However, recent progress has yielded new hope that useful data will be obtained soon. A change in the protocol now yields samples that contain only the 16S rRNA molecule from batch culture experiments. Environmental samples will be tested soon, and then the archived samples from the Woodneck March incubation experiment will be processed.

Moving-wire LC-irMS measurements of d13C for RNA standards are very precise (â± 0.2?) and reasonably accurate (â±1.0?), at the 1 mgC level. Currently we are working toward the 100-500 ngC threshold to allow analysis of smaller samples.

RNA capture is possible at the domain level, but cross-hybridization of 23S rRNA proved to be a major stumbling block. This problem has been remedied only very recently.

d13C values of total nucleic acids as measured by HPLC-irMS are 1? to 3? enriched vs. biomass (natural samples). This is consistent with the published work of other groups, using different analytical approaches.

Presentation at an International Meeting:
Goldschmidt Conference, Hot Springs, VA, May 2001.

Analysis of Nucleic Acids by Liquid Chromatography Isotope-Ratio Mass Spectrometry: First Steps Toward Merging Molecular Biology and Biogeochemistry