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The outer membrane of gram-negative bacteria functions as a permeability barrier that protects cells against a large number of antibacterial agents. OprL protein of Pseudomonas putidahas been shown to be crucial to maintain the stability of this cell component (J. J. Rodrı́guez-Herva, M.-I. Ramos-González, and J. L. Ramos. J. Bacteriol. 178:1699–1706, 1996). In the present study we cloned and mutagenized the orf1, tolQ, tolR,tolA, and tolB genes from P. putidaKT2440, which were located upstream of the oprL gene. Polar and nonpolar mutations of the P. putida tolQ,tolR, tolA, and tolB genes were generated in vitro by using the Ω-Km^r interposon, which carries two transcriptional stop signals, or a promoterlessxylE cassette, lacking any transcriptional stop signal, respectively. The mutant constructs were used to inactivate, by reverse genetics procedures, the corresponding chromosomal copies of the genes. The …