Date of Award

6-11-2002

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

First Advisor

Kevin D Young

Abstract

Penicillin-binding proteins (PBPs), a set o f enzymes found in bacteria, are important in the latter stages o f cell wall (peptidoglycan) synthesis. To search for additional roles PBPs may have, Escherichia coli mutants lacking various combinations o f PBPs were screened for the following phenotypic changes: capsule production, responses to antibiotics, motility, biofilm formation, and utilization o f various carbon and nitrogen sources. The experiments described here focused on mutants lacking mrcA (encoding PBP la) and mrcA-yrfE-yrfF. Previously, every time a A(mrcA-yrfE-yrfF) mutant was constructed the strain became mucoid due to the production o f colanic acid which is encoded by the cps genes. A AmrcA mutant was nonmucoid while a AyrfF mutant was mucoid, indicating that the loss o f yrfF rather than mrcA was responsible for capsule production (Meberg et al., 2001). The loss o f mrcA was not responsible for the mucoid phenotype, but prior overexpression o f PBP la from a plasmid prevented capsule induction upon deletion o f mrcA-yrfE-yrfF. However, once capsule induction had occurred, restoring PBP la did not halt capsule production. A cps-lacZ strain showed no gene induction upon deletion o f mrcA or any o f the other y r f genes (yr/E, yrfGHI). Deletion o f mrcA-yrfE-yrjF resulted in capsule induction, with some cells exhibiting a leaky phenotype. Some mrcA mutants, although nonmucoid initially, produced large, mucoid papillae or blebs upon extended incubation. In addition, inhibition o f PBP la with cephaloridine, an antibiotic that inhibits PBP la, resulted in cps induction. Exposure to other antibiotics, colistin, cephalothin, and the combination o f clavulinate/ticarcillin, also resulted in cps induction. Other phenotypes besides mucoidy were also attributed to the loss o f yrfF'. difficulty in obtaining transductants, a slower growth rate, loss o f motility, and differences in biofilm formation. Phenotype Microarrays, which tested for differences in carbon, nitrogen, phosphate and sulfur usage, showed a few changes in carbon and nitrogen utilization among the PBP mutants, but utilization o f any particular substrate could not be attributed to the loss of any single PBP.

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