Participants:

Bess B. Ward
Princeton University
   • Nitrite Reductase
   • Gene Arrays

Patricia A. Glibert
Horn Point Laboratories
   • Biogeochemistry

Todd Kana
Horn Point Laboratories
   • Biogeochemistry

Jeffrey Cornwell
Horn Point Laboratories
   • Biogeochemistry

Jon P. Zehr
University of California, Santa Cruz
   • Nitrogenase

Jackie C. Collier
State University of NY, Stony Brook
   • Urease

Mary A. Voytek
United States Geological Survey
   • Ammonia monooxygenase

George A. Jackson
Texas A&M University

   • Database management, modeling

Project Summary

Molecular characterization of nitrifying bacteria
Mary A. Voytek and Julie D. Kirshtein

WHO ARE THEY?
Nitrifying bacteria are Gram negative, obligate aerobic chemolithotrophs which oxidize ammonium to nitrite or nitrite to nitrate as their sole energy source and assimilate carbon dioxide via the Calvin Benson cycle.

Nitrifying bacteria are not phylogenetically diverse; the capability is restricted to a small group of organisms, most of which are closely related to each other. Bacterial systematics analysis of 16S rRNA has demonstrated that there are two phylogenetically distinct groups of autotrophic ammonium-oxidizing bacteria, both within the class Proteobacteria. One group within the gamma subclass and the other group represents a family within the beta-subclass.

WHAT DO THEY DO?
The ecological importance of ammonium-oxidizers and nitrite oxidizers lies in their role in the biological oxidation of reduced nitrogen compounds, often leading to the removal of nitrogen from the environment via denitrification. In many aquatic systems, especially shallow systems and sediment water interfaces, denitrification depends directly on NO3 supplied from nitrification. Thus, the environmental factors that control nitrification also control denitrification, as documented for regions of Chesapeake Bay. Although nitrifiers have been isolated from diverse environments and are generally ubiquitous in soils, freshwater and marine environments, they account for a very small proportion of the total bacterial population in natural environments.

HOW DO WE STUDY THEM?
In this porject we will be documenting their biodiversity and assessing their ecological importance along several environmental gradients in the Choptank River, Chesapeake Bay and the Sargasso Sea. Since they are not numerically abundant, we will use sensitive and specific methods of detection and identification based on nucleic acid analysis.

Phylogeny and functionality are linked for the nitrifiers, much more so than for denitrifiers or other organisms important in specific steps in nitrogen cycling. For this reason it has been possible to use 16S rRNA. However, we will employ functional genes for identification in this study with the added advantage of assessing activity by measuring mRNA. The key enzyme in ammonium oxidation is ammonia-monooxygenase (AMO) and its sequence diversity, although greater than that derived from 16S rRNA, aligns closely with it. Sequences from the public databases, augmented by those from our large culture collection of nitrifying bacteria (Ward laboratory) will be used to develop the suite of probes to be deployed on the gene arrays.

For more information and our most recent activities go to http://water.usgs.gov/nrp/proj.bib/microbiology/

References
Hastings, R. C., Ceccherini, M.T., Miclaus, N., Saunders, J.R., Bazzicalupo, M., McCarthy, A.J. (1997) Direct molecular biological analysis of ammonia-oxidizing bacteria populations. Microb. Ecol. 23:45-54.

Head, I.M, W.D. Hiorns, T. Martin, A.J. McCarthy, and J.R. Saunders. 1993. The phylogeny of autotrophic ammonia-oxidizing bacteria as determined by analysis of 16S ribosomal RNA gene sequences. J. Gen. Microbiol. 139: 1147-1153.

Hiorns, W.D., Hastings, R.C., Head, I.M., McCarthy, A.J., Saunders, J.R., Pickup, R.W., Hall, G.H. (1995). Amplification of 16S ribosomal RNA genes of autotrophic ammonia-oxidizing bacteria demonstrates the ubiquity of niytosospiras in the environment. Microbiology 141:2793-2800.

Holmes, A.J., Costello, A., Lidstrom, M.E., Murrell, J.C. (1995) Evidence that particulate methane monooxygenase and ammonia monooxygenase may be evolutionarily related. FEMS Microbiol. Letters 132:203-208.

Koops, H.-P. and U.C. Möller. 1992. The lithotrophic ammonia-oxidizing bacteria. p. 2625-2637. In A. Balows, H.G. Truper, M. Dworkin, W. Harder and K.-H. Schleifer. (eds.), The Prokaryotes: a handbook on the biology of bacteria: ecophysiology, isolation, identification, applications. 2nd ed. Springer-Verlag, New York.

Mobarry, B.K., Wagner, M. Urbain, V., Rittman, B.E., Stahl, D.A. (1996) Phylogenetic probes for analyzing abundance and spatial organization of nitrifying bacteria . Appl. Environ. Microbiol. 62:2156-2162.

Norton, J., Low, J.M., Klotz, M.G. (1996) The gene encoding ammonia oxygenase subunit A exists in three nearly identical copies in Nitrosospira sp. NpAV. FEMS Microbiol. Letters 139:181-188.

Rotthauwe J.H., Witzel K.P., Liesack . 1997. The ammonia monooxygenase structural gene amoA as a functional marker: Molecular fine-scale analysis of natural ammonia-oxidizing populations Appl. Environ. Microbiol 63: 4704-4712.

Teske, A., E. Alm, J.M. Regan, S. Toze, B.E. Rittmann and D. A. Stahl. 1994. Evolutionary   relationships among ammonia- and nitrite oxidizing bacteria. J. Bacteriol. 176(21): 6623-6630.

Voytek, M.A., B.B. Ward and J.C. Priscu. (1998). The abundance of ammonia-oxidizing bacteria in Lake Bonney, Antarctica determined by immunofluorescence, PCR amplification and in situ hybridization. In: “McMurdo Dry Valleys: A Cold Desert Ecosystem” J.C. Priscu (ed.) AGU Press Washington, D.C. Voytek, M.A., J.C. Priscu and B.B. Ward. (1999) The distribution and relative abundance of ammonium-oxidizing bacteria in six antarctic lakes determined by PCR amplification. Hydrobiol. 401:113-130.

Ward, B.B., M.A. Voytek and K.-P. Witzel. (1997) Population diversity of ammonium oxidizers investigated by specific PCR amplification. Microbial Ecol. 33:87-96.