The genome of the soil bacterium Pseudomonas putida strain KT2440 has been erased of various determinants of resistance to antibiotics encoded in its extant chromosome. To this end, we employed a coherent genetic platform that allowed the precise deletion of multiple genomic segments in a large variety of Gram-negative bacteria including (but not limited to) P. putida. The method is based on the obligatory recombination between free-ended homologous DNA sequences that are released as linear fragments generated upon the cleavage of the chromosome with unique I-SceI sites, added to the segment of interest by the vector system. Despite the potential for a SOS response brought about by the appearance of double stranded DNA breaks during the process, fluctuation experiments revealed that the procedure did not increase mutation rates – perhaps due to the protection exerted by I-SceI bound to the otherwise naked DNA termini. With this tool in hand we made sequential deletions of genes mexC, mexE, ttgA and ampC in the genome of the target bacterium, orthologues of which are known to determine various degrees of antibiotic resistance in diverse microorganisms. Inspection of the corresponding phenotypes demonstrated that the efflux pump encoded by ttgA sufficed to endow P. putida with a high-level of tolerance to β-lactams, chloramphenicol and quinolones, but had little effect on, e.g. aminoglycosides. Analysis of the mutants revealed also a considerable diversity in the manifestation of the resistance phenotype within the population and suggested a degree of synergism between different pumps. The directed edition of the P. putida chromosome shown here not only enhances the amenability of this bacterium to deep genomic engineering, but also validates the corresponding approach for similar handlings of a large variety of Gram-negative microorganisms.