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Parameters affecting gene expression from the Pm promoter in gram-negative bacteria
H C Winther-Larsen1, K D Josefsen, T Brautaset
1UNIGEN Center for Molecular Biology and Department of Biotechnology, Norwegian University of Science and Technology, Trondheim.
Abstract:
The Pm promoter inserted chromosomally or in broad-host-range replicons based on plasmid RSF1010 or RK2 are useful systems for both high- and low-level expression of cloned genes in several gram-negative bacterial species. The positive Pm regulator XylS is activated by certain substituted benzoic acid derivatives, and here we show that these effectors induce expression of Pm at similar relative ranking levels in both Escherichia coli and Pseudomonas aeruginosa However, the kinetics of expression was not the same in the two organisms. Different carbon sources and dissolved oxygen levels displayed limited effects on expression, but surprisingly the pH of the growth medium was found to be of major importance. By combining the effects of genetic and environmental parameters, expression from Pm could be varied over a ten-thousand- to a hundred-thousand-fold continuous range, and as an example of its applications we showed that Pm can be used to control the xanthan biosynthesis in Xanthomonas campestris.
Insights
The Pm promoter system allows tunable gene expression in gram-negative bacteria like Escherichia coli and Pseudomonas aeruginosa. Optimizing growth medium pH is crucial for controlling expression levels, enabling applications like xanthan biosynthesis regulation.
Area of Science:
- Molecular Biology
- Microbial Genetics
Background:
- The Pm promoter is a versatile system for regulating gene expression in gram-negative bacteria.
- The XylS regulator activates the Pm promoter in response to specific benzoic acid derivatives.
Purpose of the Study:
- To investigate the factors influencing Pm promoter expression in different bacterial hosts.
- To optimize Pm promoter activity for controlled gene expression and biotechnological applications.
Main Methods:
- Chromosomal and plasmid-based insertion of the Pm promoter in Escherichia coli and Pseudomonas aeruginosa.
- Analysis of gene expression levels under varying environmental conditions (carbon source, dissolved oxygen, pH).
- Demonstration of Pm promoter utility in controlling xanthan biosynthesis in Xanthomonas campestris.
Main Results:
- Substituted benzoic acid derivatives induced Pm expression with similar relative rankings in both E. coli and P. aeruginosa, but with different kinetics.
- Growth medium pH was identified as a critical factor significantly impacting Pm expression levels.
- Expression levels could be modulated over a 10,000- to 100,000-fold range by combining genetic and environmental parameters.
Conclusions:
- The Pm promoter system offers a tunable platform for gene expression in diverse gram-negative bacteria.
- Optimizing pH is essential for maximizing the control and range of Pm promoter activity.
- The Pm promoter is a valuable tool for applications such as metabolic engineering, exemplified by controlling xanthan production.