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| Status |
Public on Apr 01, 2010 |
| Title |
Analysis of differences in gene expression due to small adaptive mutations in RNA polymerase B' subunit (rpoC) |
| Organism |
Escherichia coli |
| Experiment type |
Expression profiling by array
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| Summary |
Studies of the RNA polymerase-binding molecule ppGpp in bacteria and plants have shown that changes to the kinetics of the RNA polymerase can have dramatic biological effects in the short-term as a stress response. Here we describe the reprogramming of the kinetic parameters of the RNAP through mutations arising during laboratory adaptive evolution of Escherichia coli in minimal media. The mutations cause a 10- to 30-fold decrease in open complex stability at a ribosomal promoter and approximately a 10-fold decrease in transcriptional pausing in the his operon. The kinetic changes coincide with large scale transcriptional changes, including strong downregulation of motility, acid-resistance, fimbria, and curlin genes which are observed in site-directed mutants containing the RNA polymerase mutations as well as the evolved strains harboring the mutations. Site-directed mutants also grow 60% faster than the parent strain and convert the carbon-source 15% to 35% more efficiently to biomass. The results show that long-term adjustment of the kinetic parameters of RNA polymerase through mutation can be important for adaptation to a condition.
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| Overall design |
Mutations in the RNA polymerase beta prime subunit (rpoC) were discovered in E. coli following adaptation to continual logarithmic growth (OD <= 0.3) in glycerol M9 minimal media at 30 C. We used site-directed mutagenesis to make strains of E. coli isogenic to wild-type except for single adaptive rpoC mutations. We found they increase growth rate in this condition by 60%. In order to understand how the mutations affect gene expression in this condition, we extracted total mRNA from the strains, which had been growing in the adaptive evolution condition, at OD=0.3. There were three RNAP mutants and the wild-type (E. coli K-12 MG1655). Each strain had three flasks from which RNA was extracted (three biological replicates). There were no technical replicates. The mRNA was synthesized into cDNA, labeled, and hybridized to an Affymetrix E. coli 2.0 GeneChip.
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| Contributor(s) |
Conrad TM, Frazier M, Joyce AR, Landick R, Palsson BO |
| Citation(s) |
21057108 |
| |
| Submission date |
Apr 01, 2009 |
| Last update date |
Mar 08, 2019 |
| Contact name |
Bernhard Palsson |
| E-mail(s) |
palsson@ucsd.edu
|
| Phone |
858-534-5668
|
| Fax |
858-822-3120
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| URL |
http://systemsbiology.ucsd.edu
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| Organization name |
UCSD
|
| Department |
Bioengineering
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| Lab |
Systems Biology Research Group
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| Street address |
9500 Gilman Dr.
|
| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92122 |
| Country |
USA |
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| Platforms (1) |
| GPL3154 |
[E_coli_2] Affymetrix E. coli Genome 2.0 Array |
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| Samples (12)
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| Relations |
| BioProject |
PRJNA115665 |
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