Chemical waste with toxic effects is released into the environment by industrial and urban activities. Pseudomonas putida, a rhizosphere bacterium, harbors a wide variety of genes capable of degrading hydrocarbons and xenobiotic compounds in its natural environment.

This bacterium harbors also a large set of metal resistance genes. Most studies that identify genes involved in metal resistance in P. putida focus on over/underexpressed genes and may miss other genes important for metal resistance whose expression does not change. In this study, we used a Tn-seq approach to determine the essential genome of P. putida required for growth in the presence of an excess of metals in a culture medium. Tn-seq enables the detection of mutants with reduced or increased fitness in the presence of metal excess. We validated our screen by identifying known metal resistance gene such as czcA-1 (PP_0043), cadA-3 (PP_5139), cadR (PP_5140) and pcoA2 (PP_5380). Their mutants were underrepresented in the presence of zinc, cadmium (for cadA-3 and cadR) or copper respectively. In this study, we demonstrate by targeted mutagenesis and complementation assay that PP_5337 and PP_0887 are putative transcriptional regulators involved in copper and cadmium resistance, respectively, in P. putida. The study revealed the role of two genes, PP_1663 and PP_5002, in cadmium and cobalt resistance respectively. This is the first evidence linking these genes to metal resistance and highlights the incomplete understanding of metal resistance mechanisms in P. putida.