Reduction of naphthalene uptake by addition of carbon source in pseudomonas putida

Author

Joyce Elaine Broyles

Date of Award

12-1981

Degree Type

Thesis

Degree Name

Master of Science

Major

Microbiology

Major Professor

Thomas C. Motie

Committee Members

Jeffrey becker, Gary Sayler

Abstract

A model system using naphthalene-degrading strains of Pseudo-monas putida and ¹⁴C-1(4,5,8)-naphthalene was designed to study themechanism of aromatic hydrocarbon uptake into bacterial cells. After incubation with C¹⁴-naphthalene, cells were harvested by centrifugation through silicone oil and analyzed for naphthalent accumulation. Naphthalene uptake occurred in both naphthalene-grown and glucose-grown cells. Extraction and analysis of cell-associated label revealed the presence of a naphthalene metabolite, salicylate. Strains unable to metabolize naphthalene (NAH^-) did not exhibit uptake. Treatment with 200 µg/ml chloroamphenicol or rifampicin inhibited uptake in glucose-grown cells, but not in naphthalene-grown cells. Incubation with oxidizable carbon source (20 mM) blocked naphthalene uptake in succinate, glucose, and citrate-grown cells. Reversal of succinate inhibition of uptake was accomplished by treatment with malonate. Naphthalene-grown cells, although able to oxidize most carbon sources tested, were unaffected by the presence of carbon source. Incubation of cells with either catechol or salicylate, regardless of growth substrate, resulted in immediate cessation of uptake and loss of cell-associated lebel. Uptake was inhibited by cyanide or azide treatment of naphthalene- or glucose-grown cells; while treatment with dinitrophenol or arsenate inhibited glucose-grown cells only. Incubation with exogenous nucleotides did not affect uptake. Glucose-mediated reduction was found to be concentration dependent and not affected by treatment with non-metabolizable glucose analogs. Levels of extracellular naphthalene metabolites and ¹⁴CO₂ production from labeled naphthalene were strongly reduced by the presence of an oxidizable carbon source in glucose-grown cells. Little reduction occurred in the presence of carbon source in naphthalene-grown cells. The presence of either catechol or salicylate also reduced levels strongly in both glucose-grown and naphthalene-grown cells. ¹⁴C incorporation into protein from labeled naphthalene was strongly depressed by the presence of glucose in glucose-grown cells, but naphthalene-grown cells were little affected. The data, taken together, suggest that naphthalene uptake is a function of naphthalene metabolism. It also suggests that the presence of metabolizable carbon source may exert control upon uptake in uninduced cells. The mechanism of this control is unknown, although it probably is at the level of protein synthesis.

Recommended Citation

Broyles, Joyce Elaine, "Reduction of naphthalene uptake by addition of carbon source in pseudomonas putida. " Master's Thesis, University of Tennessee, 1981.
https://trace.tennessee.edu/utk_gradthes/15142