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

Abstract

AbstractChemical 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 inP. putidafocus 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 ofP. putidarequired 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 asczcA-1(PP_0043),cadA-3(PP_5139),cadR(PP_5140) andpcoA2(PP_5380). Their mutants were underrepresented in the presence of zinc, cadmium (forcadA-3andcadR) or copper respectively. In this study, we demonstrate by targeted mutagenesis and complementation assay thatPP_5337andPP_0887are putative transcriptional regulators involved in copper and cadmium resistance, respectively, inP. putida. The study revealed the role of two genes,PP_1663andPP_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 inP. putida.