Date of Award:

5-1999

Document Type:

Thesis

Degree Name:

Master of Science (MS)

Department:

Plants, Soils, and Climate

Department name when degree awarded

Plants, Soils, and Biometeorology

Committee Chair(s)

Jeanette M. Norton

Committee

Jeanette M. Norton

Abstract

Ammonia-oxidizing bacteria (AOB) are chemolithotrophs that oxidize ammonia/ammonium to nitrite in a two-step process to obtain energy for survival. AOB are difficult to isolate from the environment and iso lated strains may not represent the diversity in soil. A genetic database and molecular tools were developed based on the ammonia monooxygenase (AMO) encoding genes that can be used to assess the diversity of AOB that exist in soil and aquatic environments without the isolation of pure cultures. The amo genes have excellent potential as molecular markers; since AMO is only found in the AOB and is essential for their metabolism, AOB must carry at least one functional copy of the amo operon. The operon is composed of at least three genes, amoC, amoA. and amoB (encoding for the subunits AmoC, AmoA, and AmoB). The amoC gene was first discovered and its sequence was obtained from Nitrosospira sp. NpA V. The amooperon is found in several copies within AOB genomes in the β-subdivision but as a single copy in y-subdivision genomes. In Southern analysis, cross-hybridization was only observed between amo genes within a subdivision. They-subdivision amo sequences have higher identity values to the genes encoding the related particulate methane monooxygenase than to the β-subdivision amo sequences. Since amoA encodes the subunit containing the active site, it was sequenced entirely for all the strains studied (16 amoA sequences total). The amoC and amoB genes were also sequenced for several strains. The amo genes allow for better discrimination between closely related strains than the 16S rRNA genes. In all cases, the amo operon consists of amoC, followed by a variable length intergenic region, and then by amoAB. The variability in length of the intergenic region is strain specific, and is therefore potentially useful for profiling AOB communities. The amo-gene database was the basis for the design of conserved oligonucleotide primers for the polymerase chain reaction (PCR). These primers were used to amplify amo sequences from a mixed template of DNA extracted directly from soil. Results indicate that the amo genes are excellent molecular markers for the assessment of AOB communities in the environment.

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