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Status |
Public on Dec 31, 2022 |
Title |
Landscapes of collateral fitness effects of mutations in antibiotic resistance genes |
Organism |
Escherichia coli |
Experiment type |
Expression profiling by high throughput sequencing
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Summary |
We performed RNA-seq experiments to compare the gene expression profiles of cells expressing single-codon substitutions in the antibiotic resistance genes NDM-1, CAT-I, or aadB in the absence of antibiotics. Mutations with deleterious fitness effects in the absense of antibiotics also caused significant changes in gene expression in a number of genes related to stress-response pathways including the regulator of colanic acid capsule synthesis (Rcs) response, the phage shock protein response (Psp), and the oxidative stress response. Fold differences in gene expression were mutation- and gene-dependent.
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Overall design |
E. coli cells containing single-codon substitutions in either the NDM-1, CAT-I, or aadB antibiotic resistance genes were grown in monoculture growth assays. Controls consisted of cells with the wildtype allele that were not induced with IPTG as well as cells harboring a Δ antibiotic resistance gene deletion that were induced with IPTG. Growth was halted at about 330 minutes to ensure the cells were still in exponential growth phase based on OD measurements. Total RNA was extracted from pelleted cell cultures. mRNA was enriched and used to synthesize double-stranded cDNA. The double-stranded cDNA was end prepped and purified. Illumina adaptor sequences were ligated to the double stranded cDNA and PCR enriched before being purified and pooled for Illumina Tru-Seq (2 x 75 bp reads).
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Contributor(s) |
Mehlhoff J, Ostermeier M |
Citation(s) |
36798991 |
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Submission date |
Mar 23, 2022 |
Last update date |
Apr 03, 2023 |
Contact name |
Marc Ostermeier |
E-mail(s) |
oster@jhu.edu
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Phone |
4105164146
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Organization name |
Johns Hopkins University
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Department |
Chemical & Department of Chemical & Biomolecular Engineering
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Lab |
Ostermeier Lab
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Street address |
3400 N. Charles St.
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City |
Baltimore |
State/province |
MD |
ZIP/Postal code |
21218 |
Country |
USA |
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Platforms (1) |
GPL21222 |
Illumina NextSeq 500 (Escherichia coli) |
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Samples (20)
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Relations |
BioProject |
PRJNA819053 |