Mutations in Each of the<i>tol</i>Genes of<i>Pseudomonas putida</i>Reveal that They Are Critical for Maintenance of Outer Membrane Stability

<jats:title>ABSTRACT</jats:title><jats:p>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<jats:italic>Pseudomonas putida</jats:italic>has 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<jats:italic>orf1</jats:italic>,<jats:italic>tolQ</jats:italic>,<jats:italic>tolR</jats:italic>,<jats:italic>tolA</jats:italic>, and<jats:italic>tolB</jats:italic>genes from<jats:italic>P. putida</jats:italic>KT2440, which were located upstream of the<jats:italic>oprL</jats:italic>gene. Polar and nonpolar mutations of the<jats:italic>P. putida tolQ</jats:italic>,<jats:italic>tolR</jats:italic>,<jats:italic>tolA</jats:italic>, and<jats:italic>tolB</jats:italic>genes were generated in vitro by using the Ω-Km<jats:sup>r</jats:sup>interposon, which carries two transcriptional stop signals, or a promoterless<jats:italic>xylE</jats:italic>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 phenotype of each mutant strain was analyzed and compared with those of the wild-type strain and the previously characterized<jats:italic>P. putida oprL</jats:italic>::<jats:italic>xylE</jats:italic>mutant. All mutant strains exhibited a similar phenotype: altered cell morphology, bleb formation at the cell surface, release of periplasmic and outer membrane proteins to the extracellular medium, increased sensitivity to a variety of compounds (i.e., EDTA, sodium dodecyl sulfate, deoxycholate, and some antibiotics), filament formation, and severely reduced cell motility. Altogether, these results demonstrate the importance of the Tol-OprL system for the maintenance of outer membrane integrity in<jats:italic>P. putida</jats:italic>and suggest a possible role of these proteins in assembling outer membrane components.</jats:p>