Date of Award

1988

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Veterinary Medical Sciences - Pathobiological Sciences

First Advisor

H. Douglas Braymer

Abstract

Chapter 1. Pseudomonas sp. strain PG2982 has the ability to use the phosphonate herbicide, glyphosate, as a sole phosphorus source. Glyphosate uptake occurs at a maximum in the late log phase of growth and is induced by phosphate starvation. Uptake is inhibited by phosphate and arsenate, but not by the amino acids glycine and sarcosine. The K$\sb{\rm m}$ and V$\sb{\rm max}$ for glyphosate uptake were calculated to be 23uM and 0.97nmoles/mg dry wt/min, respectively. A phosphate transport system with a broad substrate specificity seems to be responsible for glyphosate uptake. Chapter 2. Pseudomonas sp. strain PG 2982 is highly resistant to the herbicide glyphosate, a potent inhibitor of the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). In addition, PG2982 is able to utilize glyphosate as a sole source of phosphorus. A plasmid carrying a 2.4-kilobase pair (kb) fragment of DNA from PG2982 capable of increasing the glyphosate resistance of E. coli cells has been isolated by selection in media containing 2mM glyphosate. The increase in resistance is dependent upon the presence of a plasmid-encoded protein with a molecular weight of approximately 33,000. This protein is the product of a translational fusion between a gene on the vector, pACYC184, and the insert DNA. A lambda clone carrying the entire gene from PG2982 has been isolated and subcloning of a 2kb DNA fragment carrying this gene has again resulted in a plasmid, pPG18, capable of increasing glyphosate resistance in E. coli. A protein with a molecular weight of approximately 40,000 is encoded by pPG18. This plasmid is not able to complement a mutation in the gene for EPSPS in E. coli strain LC3 and will not hybridize to the E. coli gene. Also, the glyphosate can not be broken down by E. coli cells containing the plasmid. The nucleotide sequence of the gene has revealed the presence of an open reading frame able to encode a protein with a calculated molecular weight of 39,396.

Pages

131

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