Responses of <i>Pseudomonas putida</i> to Zinc Excess Determined at the Proteome Level: Pathways Dependent and Independent of ColRS

Zinc is an important micronutrient for bacteria, but its excess is toxic. Recently, the ColRS two-component system was shown to detect and respond to zinc excess and to contribute to zinc tolerance of <i>Pseudomonas putida</i>. Here, we applied a label-free whole-cell proteome analysis to compare the zinc-induced responses of <i>P. putida</i> and <i>colR</i> knockout. We identified dozens of proteins that responded to zinc in a ColR-independent manner, among others, known metal efflux systems CzcCBA1, CzcCBA2, CadA2 and CzcD. Nine proteins were affected in a ColR-dependent manner, and besides known ColR targets, four new candidates for ColR regulon were identified. Despite the relatively modest ColR-dependent changes of wild-type, <i>colR</i> deficiency resulted in drastic proteome alterations, with 122 proteins up- and 62 down-regulated by zinc. This zinc-promoted response had remarkable overlap with the alternative sigma factor AlgU-controlled regulon in <i>P. aeruginosa</i>. The most prominent hallmark was a high induction of alginate biosynthesis proteins and regulators. This response likely alleviates the zinc stress, as the AlgU-regulated alginate regulator AmrZ was shown to contribute to zinc tolerance of <i>colR</i> knockout. Thus, the ColRS system is important for zinc homeostasis, and in its absence, alternative stress response pathways are activated to support the zinc tolerance