High‐Throughput Tn‐Seq Screens Identify Both Known and Novel Pseudomonas putida KT2440 Genes Involved in Metal Tolerance

ABSTRACT Industrial and urban activities release toxic chemical waste into the environment. Pseudomonas putida , a soil bacterium, is known to degrade hydrocarbons and xenobiotics, and possesses numerous genes associated with heavy metal tolerance. Most studies on metal tolerance in P. putida focus solely on over‐ or underexpressed genes, potentially overlooking important genes with unchanged expression. This study employed a Tn‐seq approach to identify the essential genes required for P. putida growth under metal stress. This method enables the identification of mutants with altered fitness in the presence of excess metals. The screen successfully identified a number of known genes implicated in metal resistance, including czcA‐1 , cadA‐3 , cadR , and pcoA2 , thereby validating the approach. Further analyses using targeted mutagenesis and complementation assays revealed PP_5337 as a putative transcriptional regulator involved in copper tolerance and the two‐component system RoxSR ( PP_0887/PP_0888 ) as a key determinant of cadmium tolerance. Additionally, PP_1663 and PP_5002 were identified as contributing to cadmium and cobalt tolerance, respectively. This study provides the first evidence linking these genes to metal tolerance, highlighting gaps in our understanding of metal tolerance mechanisms in P. putida and demonstrating the utility of Tn‐seq for identifying novel tolerance determinants.