Abstract
Pseudomonads are among the most common bacteria in soils, limnic ecosystems, and human, animal, or plant host environments, including intensively studied species such as Pseudomonas aeruginosa, P. putida, or P. fluorescens. Various gene expression systems are established for some species, but there is still a need for a simple system that is suitable for a wide range of pseudomonads and that can be used for physiological applications, i.e., with a tuning capacity at lower expression levels. Here, we report the establishment of the anthranilate-dependent PantA promoter for tunable gene expression in pseudomonads. During studies on P. fluorescens, we constructed an anthranilate-inducible AntR/PantA-based expression system, named pUCP20-ANT, and used GFP as reporter to analyze gene expression. This system was compared with the rhamnose-inducible RhaSR/PrhaB-based expression system in an otherwise identical vector background. While the rhamnose-inducible system did not respond to lower inducer concentrations and always reached high levels over time when induced, expression levels of the pUCP20-ANT system could be adjusted to a range of distinct lower or higher levels by variation of anthranilate concentrations in the medium. Importantly, the anthranilate-inducible expression system worked also in strains of P. aeruginosa and P. putida and therefore will be most likely useful for physiological and biotechnological purposes in a wide range of pseudomonads.Key points• We established an anthranilate-inducible gene expression system for pseudomonads.• This system permits tuning of gene expression in a wide range of pseudomonads.• It will be very useful for physiological and biotechnological applications.
Highlights
Pseudomonads play important roles in many ecosystems, and they often occur in mutualistic or pathogenic life styles associated with plants, animals, or humans (Peix et al 2018)
Fig. S1), which has been demonstrated to be induced by anthranilate, an intermediate of tryptophan degradation found in any bacterium (Choi et al 2011)
We decided to use the AntR regulated PantA promoter for a recombinant expression system in P. fluorescens A506, and to compare this with the rhamnose-induced expression system, which is currently preferred for P. aeruginosa and P. putida (Meisner and Goldberg 2016; Jeske and Altenbuchner 2010)
Summary
Pseudomonads play important roles in many ecosystems, and they often occur in mutualistic or pathogenic life styles associated with plants, animals, or humans (Peix et al 2018). In no case of these Pseudomonas expression systems, the induction was monitored on the cellular level to address the aspect whether or not induction is uniform or whether lower inducer concentrations result in increasing populations of uninduced cells versus fully induced cells. Such “all-or-nothing” systems have been described in E. coli for the LacI/PlacZ and the AraC/ParaB systems (Novick and Weiner 1957; Siegele and Hu 1997). The effect is caused by the upregulation of the uptake transporters in response to the inducer, and the inducer-independent production of the respective transporter gene is required to avoid this phenomenon (Széliová et al 2016)
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