Doctoral Dissertations

Date of Award

5-1996

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Microbiology

Major Professor

Gary S. Sayler

Committee Members

Terry Schultz, Gary Stacey, David White

Abstract

The capability of bioluminescence reporter strains for naphthalene biodegradation, to maintain reporter plasmids and compete against wild type degrading populations was examined. Plasmid maintenance was examined in non-selective continuous culture and competitiveness was examined in naphthalene limited continuous culture using two or three member mixed cultures. Microcosms consisting of a polyaromatic hydrocarbon contaminated soils were used to examine strain survivability in a relevant soil. A unique chromosomal marker was developed to monitor possible gene transfer events in soil microcosms. Individual populations were monitored with specific DNA probes for plasmids and organisms and by dilution spread-plating on selective media.

The wild type naphthalene degradative plasmid (pKA1) and the lux transposon (Tn4431) mutagenized version of this plasmid (pUTK21) were maintained in host strains, P. fluresecens 5R and P. fluorescens 5RL, at 100% of the population under the dilution rates tested. When the reporter plasmid was transferred to another host, P. fluorescens 18H, PUTK21 was segregationally unstable. The reporter plasmid from P. putida RB1351 (promoter probe vector with NAH7 upper pathway promoter) was segregationally unstable. In continuous culture competition experiments, strain 5RL was outcompeted at both dilution rates, strain HK44 was maintained at the lower dilution rate, and strain RB1351 was maintained at all dilution rates tested when competing against the wild type naphthalene degrader, strain 5R.

In soil microcosm experiments, a chromosomally marked version of strain 5RL (strain 5RLA) was used to determined persistence in soil as well as monitor possible gene transfer events. Strain 5RLA could survive, as determined by dilution spread plating, for up to 25 days in sterile and non-sterile soil at a useful level for a biosensor. When naphthalene was added to the soil, strain 5RLA was outcompeted after one week and fell below the level of a useful biosensor. Transfer of pUTK21 could only be detected when a plasmid-free recipient was added. Transconjugants were confirmed by DNA probing and occurred at low frequencies (10-5-10-6 per donor). The transconjugant level fell below detection after 17 days. These studies demonstrate that one reporter strain, 5RLA, could compete in soil for 25 days at a level useful as a biosensor. The strain maintains the reporter plasmid which is transferred to indigenous soil bacteria at a level too low to detect under the conditions tested except when a ideal recipient was added.

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