High-throughput Tn-seq screens identify both known and novelPseudomonas putidaKT2440 genes involved in metal resistance

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 .