Mutations in APH(3')-IIIa Increase Activity Against Amikacin Open Access

Kramer, Joseph Ross (2013)

Permanent URL: https://etd.library.emory.edu/concern/etds/8p58pd51p?locale=en
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Abstract

Aminoglycoside modifying enzymes have evolved the ability to catalyze reactions with a wide array of substrates. This evolution has not been documented in clinical settings. Here we have simulated conditions to determine whether the aminoglycoside phosphotransferase APH(3')-IIIa is capable of evolving increased enzymatic activity catalyzing the aminnoglycoside amikacin as a substrate. APH(3')-IIIa was subjected to four rounds of mutation, two rounds of error-prone PCR and two rounds of staggered extension process. After four rounds of selection, we observed a 60 fold decrease in susceptibility to amikacin. Purified mutant APH(3')-IIIa protein had an increased catalytic efficiency in reaction with both amikacin and kanamycin, its native substrate. E. coli expressing the mutant APH(3')-IIIa also had a decreased ability to form colonies on non-selective plates. The catalytic function of the mutant had no impact on its colony forming ability, but expression of the mutant protein did have an effect on the colony forming ability. The results suggest a possible mechanism through which resistant alleles of APH(3')-IIIa fail to spread in clinical E. coli isolates even under constant exposure to high levels of aminoglycoside antibiotics.

Table of Contents

INTRODUCTION........................................................................................................................................ 1

MATERIALS AND METHODS......................................................................................................................... 3

RESULTS................................................................................................................................................ 9

DISCUSSION.......................................................................................................................................... 17

LIST OF TABLES

TABLE 1: Primers used for experimentation .................................................................................................. 20

TABLE 2: Amikacin Susceptibility of APH(3')-IIIa mutants ............................................................................... 21

TABLE 3: Kinetic Parameters of APH(3')-IIIa and APH4.1................................................................................. 23

TABLE 4: Plasmid stability of APH(3')-IIIa and APH4.1..................................................................................... 24

LIST OF FIGURES

FIGURE 1: 3-D model representation of mutation location in APH(3')-IIIa ........................................................... 25

FIGURE 2: Michaelis-Menton plot of APH(3')-IIIa and APH4.1............................................................................ 26

FIGURE 3: Growth of E. coli InvαF' containing the pQBAV3c plasmid expressing APH(3')-IIIa and APH4.1................... 27

FIGURE 4: Growth of E. coli InvαF' containing the pQBAV3c plasmid with APH(3')-IIIa and its mutants...................... 28

FIGURE 5: Growth of E. coli BL21(DE3) containing the modified pET28 plasmid with APH(3')-IIIa and its mutants........ 29

FIGURE 6: Growth of E. coli TG1 containing the modified pET28 plasmid with APH(3')-IIIa and its mutants................. 30

REFERENCES........................................................................................................................................... 31

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