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Optimization of New Bacterial In Situ Polymerase Chain Reaction (PCR) Methodologies to Support Sea Grant Missions

PIs: Robert Hodson, Mary Ann Moran and Feng Chen* (Dept of Marine Sciences, Univ of Georgia, Athens, GA, USA)  *Currently at: Center of Marine Biotechnology, Univ. of Maryland

Support: Georgia Sea Grant College Program (Project number R/AT-5)

Timeframe: 3/1/98 - 2/28/00 (complete)

Project Overview:
Methods development, including --
1) To optimize bacterial in situ polymerase chain reaction (PCR) methodologies for quick and accurate determination of bacterial gene expression in the marine environment. 2) To develop methods for microscopic characterization of micro-scale distribution and genetic diversity of marine bacteria at the individual cell level. 3) To describe the range of physiological conditions (especially nutrient starvation rate) under which in situ amplifications can successfully be used to taxonomically identify or genetically characterize individual marine bacterial cells. 4) To convene an In Situ PCR workshop for Sea Grant representatives from around the country.

The approach proved sensitive enough to detect the presence of very small percentages of each of the bacterial types in the bacterial community. Specifically, the methods were used as follows --
•  Detection of hydrocarbon-degrading microbes. In unamended estuarine water, no bacteria expressing genes for degradation of aromatic hydrocarbons were detected - yet after exposure to a hydrocarbon mixture for one week, 20-30% of the total bacterial cells showed expression of this degradative system.
•  Monitoring photosynthesis. A strong positive signal for rbcL (rubisco, large subunit) mRNA was detected inside marine Synechococcus cells grown under routine light conditions, while only a weak signal was detected in cells kept in dark overnight.
•  Detection of genes responsible for nitrogen cycling.
     o Nitrification. In vitro PCR was successful in amplification of ammonium oxidization gene from nitrifying bacteria. Preliminary data suggest that marine nitrifier Nitrosomonas cryotolerans may be more closely related to other nitrifiers than to Nitrosomonas species.
     o Denitrification. Production of mRNA encoded by nirS (nitrite reductase) was detected for both aerobic and anaerobic cells of the marine denitrifier, Pseudomonas stutzeri, suggesting constitutive expression of this gene.
     o Nitrogen fixation. Degenerate oligonucleotides were used to amplify a large segment of nifH (nitrogenase reductase) from members of the genus, Vibrio.

Chen, F., W. A. Dustman and R. E. Hodson. year? Application of in situ reverse transcription to estuarine bacterial community analysis. In: C.R. Bell, M. J. Brylinksy, and P. Johnson-Green (eds.), Microbial Biosystems: New Frontiers, Proc. 8th Intl. Symposium on Microbial Ecology.

Chen, F., B. Binder, and R.E. Hodson. 2000. Flow cytometric detection of specific gene expression in prokaryotic cells using in situ RT-PCR. 2000. FEMS Microbiology Letters. 9242:1-6.

Chen, F. and R. E. Hodson. 1999. Viewing microbes with in situ molecular approaches, a MiniReview. In: R. Colwell and H. S. Xu (ed.), Proceedings in Marine Biotechnology, pp. 112-115, China Ocean Press, Beijing.

Chen, F., W. A. Dustman and R. E. Hodson. 1999. Microscopic detection of toluene dioxygenase gene and its expression inside bacterial cells in seawater using prokaryotic in situ PCR. Hydrobiologia 401:131-138.

González, J.M., R.E. Hodson and M.A. Moran. 1999. Bacterial populations in replicate marine enrichment cultures: Assessing variability in abundance using 16S rRNA-based probes. 1999. Hydrobiologia. 401:69-75.

Chen, F., W. A. Dustman, M. A. Moran, and R. E. Hodson. 1998. In situ PCR methodologies for visualization of microscale genetic and taxonomic diversities of prokaryotic communities. Ch 3.3.9. In: A.D.L. Akkermans, J. D. van Elsas, F. J. DeBruijn (eds.), Molecular Microbial Ecology Manual. Kluwer Academic Publishers, The Netherlands.

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This page was updated October 13, 2006