Abstract
Heavy metal pollution is widespread and persistent, and causes serious harm to the environment. Pseudomonas putida, a representative environmental microorganism, has strong resistance to heavy metals due to its multiple efflux systems. Although the functions of many efflux systems have been well-studied, the relationship between them remains unclear. Here, the relationship between the Czc and Cad systems that are predominantly responsible for cadmium efflux in P. putida KT2440 is identified. The results demonstrated that CzcR3, the response regulator of two-component system CzcRS3 in the Czc system, activates the expression of efflux pump genes czcCBA1 and czcCBA2 by directly binding to their promoters, thereby helping the strain resist cadmium stress. CzcR3 can also bind to its own promoter, but it has only a weak regulatory effect. The high-level expression of czcRS3 needs to be induced by Cd2+, and this relies on the regulation of CadR, a key regulator in the Cad system, which showed affinity to czcRS3 promoter. Our study indicates that the Cad system is involved in the regulation of the Czc system, and this relationship is important for maintaining the considerable resistance to cadmium in P. putida.
Highlights
CzcRS3 is a downstream regulator of CadR, and together they endow P. putida KT2440 with strong resistance to cadmium
To verify the CadR-binding region on czcRS3 promoter, several base mutations were introduced in the promoter fragment (TTGACAAAG-N9CAAAGTC) for Electrophoretic Mobility Shift Assay (EMSA)
P. putida KT2440 was investigated and confirmed that CzcRS3 regulated the expression of two CzcCBA efflux pump operons by directly binding to their promoters
Summary
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. CadC, which is encoded immediately downstream of cadA in Staphylococcus aureus, acts as a transcriptional repressor of cadA-cadC operon [32] In some bacteria, such as Ralstonia metallidurans and Pseudomonas putida, the Czc and Cad systems are both important to confer full resistance to several heavy metals: the elimination of either system would significantly reduce resistance to Cd2+ and Zn2+ [33,34]. Some P. putida strains isolated from areas contaminated with heavy metal contaminants, such as P. putida CD2 and P. putida X4, show strong resistance to some hazardous heavy metals, especially cadmium [40,41] These resistances are inseparable from the contribution of the Czc and Cad systems encoded in their genomes.
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