Alyssa Theodore, Alessandro Calo, Marin VuliÄ, Kim Lewis
Undergraduate Category: Physical and Life Sciences Degree Level: Biology Abstract ID# 498
Identifying mutations that have increased survival to fluoroquinolone antibiotics Alyssa Theodore, Alessandro Calo, Marin Vulić, Kim Lewis Antimicrobial Discovery Center, Department of Biology
Abstract:
Background:
Results: A
3
B
F#2
3
CB2000 CB2000 CB200 CB2000
2
Fluoroquinolones are a commonly used family of antibiotics that target DNA gyrase in gram-negative bacteria. They inhibit the ligase domain of the gyrase, causing DNA fragmentation and thus initiating a DNA damage stress response known as the SOS response.
F#2 CB2000 CB200
2
recAD276N, Δe14
recAD276N 1
Δe14, dnaBP80H dnaBP801H
1
Δe14
Log% Survival
Log% Survival
0
-1
Δe14, dnaBP80H, dnaBP801H,recAD272N recAD276N 0
dnaBP80H recAD276N, dnaBP801H
-1
-2
-2
-3
-3 0
1
2
3 4 Time (Hrs)
5
6
7
DnaB is a DNA helicase that proceeds DNA polymerase by unwinding the DNA to initiate DNA replication. A deletion of dnaB is lethal in rich medium. 0
1
2
3 4 Time (Hrs)
5
6
7
Figure 2: Affect of mutations on survival against ciprofloxacin. The affects of the single mutants are seen in (A). A strain carrying dnaBP80H is not shown because the mutation is lethal in a wild type background. The affect of double and triple mutations are seen in (B). All strains showing increased survival have the e14 prophage deleted.
A
RecA is an important protein that is able to catalyze DNA strand exchange reactions to promote homologous recombination and plays an integral role in the induction of the SOS response to DNA damage by acting on the protein LexA.
B
E14 is a large prophage (14.4kb) incorporated into the chromosome of E. coli. This Region is excised during the SOS response to form a DNA circle. The e14 prophage includes the gene sfiC, an inhibitor of cell division.
SOS Induction: 6000 5000
wt
Relative Fluorescence
Bacteria exposed to fluoroquinolone antibiotics are able to survive treatment without acquiring heritable resistance to the drug. These survivors are called persisters, and are phenotypic variants of the wild type. Escherichia coli has been shown to form these tolerant cells in response to a variety of stresses, including antibiotics, disinfectants and ionizing radiation. A recent study showed that E. coli is able to survive increasing amount of ionizing radiation by directed evolution1. Ionizing radiation and fluoroquinolone treatment both result in double strand DNA breaks and are repaired by the same mechanisms, involving both recombinational repair and the SOS stress response. Since the cell is able to survive both stresses using the same repair mechanisms, we hypothesized that the same mechanisms protecting the cell from ionizing radiation would also increase tolerance to the fluoroquinolone ciprofloxacin. We have taken the original strain before exposure to radiation (Founder #2) and the fully tolerant strain from the ionizing radiation studies (CB2000) and identified several mutation of interest in the tolerant strain. Single base pair mutations in recA and dnaB, as well as a deletion of e14, were identified in the tolerant strain. We have measured the persister levels in single and double mutants and compared their level of survival to ciprofloxacin to each other and the wild type. Our results show that these mutations may be involved in survival to this important class of antibiotics.
4000
wt cip recAD272N
3000 2000
Amino Acid Structures: Aspartic Acid
recAD272N cip
1000
Asparagine
0 0
Proline
Figure 3: Mutated residues mapped on the crystal structures of RecA2,3 and DnaB2,3. Asp272 of RecA is highlighted in orange (A). The ADP binding site is shown in yellow and green. The cross-subunit interaction is shown in red. The single strand DNA bound to RecA is shown in purple. A change in Asp272 does not appear affect these regions leading us to believe it affects LexA binding to RecA. Pro80 is highlighted in red in the structure of the N-terminus of DnaB (B). The green and blue chain show the dimerization of this protein. The N-ter is required for hexamer formation and experiences significant conformation changes in response to nucleotide binding and hydrolysis. A change to Pro80 likely results in a nonfunctional protein, which would lead to cell death.
Histidine
Figure 1: The structural change of amino acids in RecAD272N and DnaBP80H. The slight change in structure can be seen in RecAD272N (A) while the change in DnaBP80H is more pronounced (B). The replacement of the charged amino acid aspartic acid with the uncharged amino acid asparagine could affect protein-protein binding. Proline is non-polar and unique in that it’s amine group is bound to the side chain twice. This makes proine unable to undergo many conformational changes that are readily seen in other amino acids. Replacement of the proline with the polar amino acid histidine could allow different conformations of DnaB.
References: 1. Harris, D. R., S. V. Pollock, et al. (2009) Directed Evolution of Ionizing Radiation Resistance in Escherichia coli. Journal of Bacteriology 191: 5240-5252. 2. Chen, Z., H. Yang & N. P. Pavletich, (2008) Mechanism of homologous recombination from the RecA-ssDNA/dsDNA structures. Nature 453: 489-494. 3. VanLoock, M. S., X. Yu, et al. (2003) ATP-Mediated Conformational Changes in the RecA Filament. Structure 11: 187-196. 4. Fass, D., C. E. Bogden & J. M. Berger, (1999) Crystal structure of the N-terminal domain of the DnaB hexameric helicase. Structure 7: 691-698.
200
400 Time (Minutes)
600
800
Figure 4. The RecAD272N mutation has a higher level of SOS induction in response to FQs. A strain carrying GFP under a SOS promoter was treated with ciprofloxacin, and GFP intensity was read in a fluorometer. Strains were treated with 10x MIC of cipro to induce the SOS response. The strain carrying RecAD272N has a more robust SOS response than the wild type strain.
Future Experiments: • Repeat killing experiments with different concentration of antibiotic. • Repeat SOS induction assay with the recAD272N mutation as well as e14 prophage deletion using the flow cytometer and the flourometer. • Move recAD272N allele into lexA3 background (no SOS induction) • Move dnaBP80H into lexA3 background as well as recAD272N and lexA3 background. • If increased SOS response induction is required to move dnaBP80H, then this allele cannot be moved into a recAD272N SOSbackground.
Identifying mutations that have increased survival to fluoroquinolone antibiotics Alyssa Theodore, Alessandro Calo, Marin Vulić, Kim Lewis Antimicrobial Discovery Center, Department of Biology
Abstract:
Background:
Results: A
3
B
F#2
3
CB2000 CB2000 CB200 CB2000
2
Fluoroquinolones are a commonly used family of antibiotics that target DNA gyrase in gram-negative bacteria. They inhibit the ligase domain of the gyrase, causing DNA fragmentation and thus initiating a DNA damage stress response known as the SOS response.
F#2 CB2000 CB200
2
recAD276N, Δe14
recAD276N 1
Δe14, dnaBP80H dnaBP801H
1
Δe14
Log% Survival
Log% Survival
0
-1
Δe14, dnaBP80H, dnaBP801H,recAD272N recAD276N 0
dnaBP80H recAD276N, dnaBP801H
-1
-2
-2
-3
-3 0
1
2
3 4 Time (Hrs)
5
6
7
DnaB is a DNA helicase that proceeds DNA polymerase by unwinding the DNA to initiate DNA replication. A deletion of dnaB is lethal in rich medium. 0
1
2
3 4 Time (Hrs)
5
6
7
Figure 2: Affect of mutations on survival against ciprofloxacin. The affects of the single mutants are seen in (A). A strain carrying dnaBP80H is not shown because the mutation is lethal in a wild type background. The affect of double and triple mutations are seen in (B). All strains showing increased survival have the e14 prophage deleted.
A
RecA is an important protein that is able to catalyze DNA strand exchange reactions to promote homologous recombination and plays an integral role in the induction of the SOS response to DNA damage by acting on the protein LexA.
B
E14 is a large prophage (14.4kb) incorporated into the chromosome of E. coli. This Region is excised during the SOS response to form a DNA circle. The e14 prophage includes the gene sfiC, an inhibitor of cell division.
SOS Induction: 6000 5000
wt
Relative Fluorescence
Bacteria exposed to fluoroquinolone antibiotics are able to survive treatment without acquiring heritable resistance to the drug. These survivors are called persisters, and are phenotypic variants of the wild type. Escherichia coli has been shown to form these tolerant cells in response to a variety of stresses, including antibiotics, disinfectants and ionizing radiation. A recent study showed that E. coli is able to survive increasing amount of ionizing radiation by directed evolution1. Ionizing radiation and fluoroquinolone treatment both result in double strand DNA breaks and are repaired by the same mechanisms, involving both recombinational repair and the SOS stress response. Since the cell is able to survive both stresses using the same repair mechanisms, we hypothesized that the same mechanisms protecting the cell from ionizing radiation would also increase tolerance to the fluoroquinolone ciprofloxacin. We have taken the original strain before exposure to radiation (Founder #2) and the fully tolerant strain from the ionizing radiation studies (CB2000) and identified several mutation of interest in the tolerant strain. Single base pair mutations in recA and dnaB, as well as a deletion of e14, were identified in the tolerant strain. We have measured the persister levels in single and double mutants and compared their level of survival to ciprofloxacin to each other and the wild type. Our results show that these mutations may be involved in survival to this important class of antibiotics.
4000
wt cip recAD272N
3000 2000
Amino Acid Structures: Aspartic Acid
recAD272N cip
1000
Asparagine
0 0
Proline
Figure 3: Mutated residues mapped on the crystal structures of RecA2,3 and DnaB2,3. Asp272 of RecA is highlighted in orange (A). The ADP binding site is shown in yellow and green. The cross-subunit interaction is shown in red. The single strand DNA bound to RecA is shown in purple. A change in Asp272 does not appear affect these regions leading us to believe it affects LexA binding to RecA. Pro80 is highlighted in red in the structure of the N-terminus of DnaB (B). The green and blue chain show the dimerization of this protein. The N-ter is required for hexamer formation and experiences significant conformation changes in response to nucleotide binding and hydrolysis. A change to Pro80 likely results in a nonfunctional protein, which would lead to cell death.
Histidine
Figure 1: The structural change of amino acids in RecAD272N and DnaBP80H. The slight change in structure can be seen in RecAD272N (A) while the change in DnaBP80H is more pronounced (B). The replacement of the charged amino acid aspartic acid with the uncharged amino acid asparagine could affect protein-protein binding. Proline is non-polar and unique in that it’s amine group is bound to the side chain twice. This makes proine unable to undergo many conformational changes that are readily seen in other amino acids. Replacement of the proline with the polar amino acid histidine could allow different conformations of DnaB.
References: 1. Harris, D. R., S. V. Pollock, et al. (2009) Directed Evolution of Ionizing Radiation Resistance in Escherichia coli. Journal of Bacteriology 191: 5240-5252. 2. Chen, Z., H. Yang & N. P. Pavletich, (2008) Mechanism of homologous recombination from the RecA-ssDNA/dsDNA structures. Nature 453: 489-494. 3. VanLoock, M. S., X. Yu, et al. (2003) ATP-Mediated Conformational Changes in the RecA Filament. Structure 11: 187-196. 4. Fass, D., C. E. Bogden & J. M. Berger, (1999) Crystal structure of the N-terminal domain of the DnaB hexameric helicase. Structure 7: 691-698.
200
400 Time (Minutes)
600
800
Figure 4. The RecAD272N mutation has a higher level of SOS induction in response to FQs. A strain carrying GFP under a SOS promoter was treated with ciprofloxacin, and GFP intensity was read in a fluorometer. Strains were treated with 10x MIC of cipro to induce the SOS response. The strain carrying RecAD272N has a more robust SOS response than the wild type strain.
Future Experiments: • Repeat killing experiments with different concentration of antibiotic. • Repeat SOS induction assay with the recAD272N mutation as well as e14 prophage deletion using the flow cytometer and the flourometer. • Move recAD272N allele into lexA3 background (no SOS induction) • Move dnaBP80H into lexA3 background as well as recAD272N and lexA3 background. • If increased SOS response induction is required to move dnaBP80H, then this allele cannot be moved into a recAD272N SOSbackground.
