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Published on: November 12, 2012
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Mutations That Stimulate flhDC Expression in Escherichia coli K-12
Karen A Fahrner1, Howard C Berg2
1Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, USA karen@mcb.harvard.edu.
Journal of Bacteriology
|July 15, 2015
Summary
New mutations enhance Escherichia coli flagellar gene expression by altering the flhDC promoter and upstream regulatory regions. These findings reveal novel mechanisms for controlling bacterial motility and gene regulation.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Bacterial motility, crucial for environmental exploration and survival, is mediated by flagella in Escherichia coli.
- Flagellar synthesis is initiated by FlhD and FlhC transcription factors, encoded by the highly regulated flhDC operon.
- flhDC expression is dependent on σ(70) and the cyclic AMP-cyclic AMP receptor protein (cAMP-CRP) complex, and is often limited in K-12 strains.
Purpose of the Study:
- To identify and characterize mutations that stimulate flhDC operon expression in Escherichia coli K-12 strains.
- To investigate the role of promoter elements, promoter spacer regions, and upstream DNA topology in flhDC gene regulation.
Main Methods:
- Analysis of single nucleotide changes in the flhDC promoter, -10 element, promoter spacer, and cAMP-CRP binding sites.
- Assessment of the impact of insertion sequence (IS) elements and antibiotic resistance genes located upstream on flhDC transcription.
- Evaluation of gene expression in the absence of the H-NS protein.
Main Results:
- Identified numerous mutations, including single nucleotide changes, that significantly enhance flhDC expression.
- Demonstrated that upstream elements like IS elements or a kanamycin gene can boost transcription, highlighting the importance of DNA topology.
- Confirmed that cAMP-CRP remains essential for activation, but some mutations enable expression independently of H-NS.
Conclusions:
- Novel mutations provide insights into the complex regulation of the flhDC operon and bacterial flagellar synthesis.
- Upstream DNA topology plays a significant role in modulating flhDC expression levels.
- These findings offer potential strategies for manipulating bacterial motility and gene expression.
