of New Bacterial In Situ Polymerase Chain Reaction (PCR) Methodologies to Support
Sea Grant Missions
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
Support: Georgia Sea Grant College Program (Project number R/AT-5)
3/1/98 - 2/28/00 (complete)
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
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