Masters Theses

Date of Award

3-1987

Degree Type

Thesis

Degree Name

Master of Science

Major

Microbiology

Major Professor

Karl M. Sirotkin

Committee Members

Gary Sayler, Stuart Riggsby

Abstract

This study tests the efficacy of colony hybridization to detect a model groundwater microorganism designated as AHS24, a putative Arthrobacter species isolated from a pristine groundwater aquifer located in Lula, Oklahoma. This application of colony hybridization represents one of the newly evolving technologies which addresses the need to develop more accurate and rapid ways of evaluating the maintenance and proliferation of certain populations of microorganisms in the environment.

Radioactively labeled AHS24 whole genomic probes were compared to constructed AHS24 subgenomic radioactive DNA probes for specificity and sensitivity in detecting AHS24 target DNA amidst the presence of target DNAs from other isolates present on the hybridization membrane. Initially, eight groundwater isolates showing consistent positive hybridization signals with AHS24 whole genomic probe under varying hybridization stringency of 50 mM to 1000 mM NaCI were chosen for comparative specificity experiments with constructed AHS24 subgenomic probes. These eight isolates, along with AHS24 and other ATCC strains of interest were utilized as target DNAs for comparing the specificity and relative sensitivity of the constructed AHS24 probes and the AHS2U whole genomic probe in hybridizations under two different stringency washes. Another minor set of comparative experiments in which the ability to detect AHS24 DNA sequences amidst AHS24 innoculated and uninnoculated freshwater communities was also performed with AHS24 whole genomic and subgenomic probes.

These results showed that colony hybridization can be efficiently utilized to detect the presence of a particular groundwater micro organism, AHS24, with high sensitivity. In all sets of experiments, under varying stringencies and in the presence of known isolate target DNAs or AHS24 innoculated freshwater communities, AHS24 colonies yielded the most intense signals with either AHS24 whole genomic probes or AHS24 subgenomic probes. The study has also shown that subgenomic probes, although highly sensitive, have to be carefully chosen in order to be more discriminatory than whole genomic probes. Only one of four subgenomic probes, p2ML0, appeared to demonstrate consistent narrowed specificity.

An unexpected cross-hybridization between AHS24 probe DNA and Pseudomonas strain target DNAs was observed. This was unanticipated since the genus Arthrobacter and the genus Pseudomonas are considered to be highly divergent according to traditional taxonomy. If cross-hybridization is indicative of ancestry, AHS24 may represent a microorganism whose genome arose not necessarily from a single line of descent, but rather from the blending of more than one line of ancestry.

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