Date of Award

1-23-2002

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

First Advisor

Kevin Young

Abstract

The penicillin binding proteins (PBPs) are a group o f enzymes responsible for the latter stages o f peptidoglycan synthesis. Our major research objective in this study was to determine the physiological roles o f the low molecular weight (LMW) PBPs of the Gram negative bacterium Escherichia coli (E. coli). During extensive screening of a set o f mutant strains that lack different combinations of LMW PBP genes we determined that PBP 5 played a role in maintaining E. co li’s normal rod shape. However, loss of PBP 5 did not cause severe morphological alterations unless the cells were also deleted for additional PBP genes. Further, although loss of PBP 5 was linked to defects in morphology, loss o f three other DD-carboxypeptidase PBPs did not have a similar effect. Therefore, our subsequent research efforts were directed to answer three questions. First, what are the functional domains of PBP 5, and does the protein influence cell morphology by virtue o f its DD-carboxypeptidase activity? Second, are the DDcarboxypeptidase PBPs true functional equivalents, and can they substitute for one another in vivol Third, strains lacking only PBP 5 have normal morphology, so what other PBPs are preventing realization o f morphological defects in these cells? We show that mutant versions o f PBP 5 are unable to reverse the morphological defects of E. coli strains lacking the wild type PBP 5 gene, and that PBP 5 must be both anchored to the cytoplasmic membrane and retain its DD-carboxypeptidase activity to be functional. We also show that increased expression of the other DD-carboxypeptidase PBPs cannot reverse the morphological defects of strains lacking PBP 5, and that the LMW DD-carboxypeptidase PBPs do not have interchangeable functions in vivo. Further, we identify the minimum complement of PBPs that is necessary to maintain the normal morphology of E. coli in the absence o f PBP 5. Based on these results we present a speculative model that predicts the normal physiological role of PBP 5, explains why realization of PBP 5 linked morphological defects requires the loss of additional PBPs, and offers a framework in which the functions of the other LMW PBPs can be tested and interpreted.

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