Development of a molecular detection method for naphthalene degrading pseudomonads

Thesis/Dissertation · Fri Jan 01 04:00:00 EST 1993

OSTI ID:5519828

Silva, M C

A new combined method for detection of naphthalene degrading pseudomonads from soil has been developed. After direct extraction from soil using a lysozyme/sodium dodecyl sulfate/freeze thaw method and rapid purification through gel electrophoresis, DNA was amplified through polymerase chain reaction (PCR) using primers directed against the nahR regulatory gene present in plasmid NAH7 of Pseudomonas putida G7. The resulting product was detected with a reverse dot blot protocol improving sensitivity ten-fold over traditional ethidium bromide staining of agarose gel electrophoresis, with positive signals starting at the 10[sup 3] CFUs/g soil level. This method was successful in detecting indigenous bacteria from subsurface sediment of a naphthalene contaminated site in New York State, and that similar combined approaches could be developed for other soil borne genetic markers. A study was also carried out on how culture conditions, and other variables that modulate a cell's physiology bias a PCR amplification against generating a representative specimen profile. Two Pseudomonas putida G7 nahR alleles were constructed in pUC19 that differ solely in a 31 bp internal segment whose sequence has been inverted. After PCR amplification, the products could be distinguished. When an Escherichia coli strain carrying one nahR allele is submitted to varying growth conditions, the consequences can be ascertained through co-amplification with a strain carrying the other allele and subsequent restriction analysis. Sublethal levels of tetracycline or growth in minimal medium made the PCR target in these cells relatively less amplifiable. However, cells in stationary phase displayed improved amplifiability while cells grown at 42[degrees]C were equally amplifiable as compared to cells grown at 37[degrees]C. These results suggest that mixed populations containing cells in different physiological states may not be representatively amplified by PCR.

• Research Organization: Cornell Univ., Ithaca, NY (United States)
• OSTI ID: 5519828
• Country of Publication: United States
• Language: English

Related Subjects

560300* -- Chemicals Metabolism & Toxicology
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.
AROMATICS
BACTERIA
BIODEGRADATION
BIOLOGICAL MARKERS
BIOLOGICAL VARIABILITY
CHEMICAL REACTIONS
CONDENSED AROMATICS
CONTAMINATION
DECOMPOSITION
GENETIC VARIABILITY
HYDROCARBONS
MICROORGANISMS
NAPHTHALENE
ORGANIC COMPOUNDS
PSEUDOMONAS
SOILS