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Journal of Bacteriology, September 2000, p. 4764-4772, Vol. 182, No. 17
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

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

María A. Llamas, Juan L. Ramos, and José J. Rodríguez-Herva*

Department of Biochemistry and Molecular and Cellular Biology of Plants, Estación Experimental del Zaidin, Consejo Superior de Investigaciones Cientificas, 18008 Granada, Spain

Received 30 March 2000/Accepted 2 June 2000

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 putida has been shown to be crucial to maintain the stability of this cell component (J. J. Rodríguez-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. putida KT2440, 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 Omega -Kmr interposon, which carries two transcriptional stop signals, or a promoterless xylE 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 P. putida oprL::xylE 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 P. putida and suggest a possible role of these proteins in assembling outer membrane components.

* Corresponding author. Mailing address: Department of Biochemistry and Molecular and Cellular Biology of Plants, Estacion Experimental del Zaidin, Consejo Superior de Investigaciones Cientificas, 18008 Granada, Spain. Phone: 34 958 121011. Fax: 34 958 129600. E-mail: Chechu{at}eez.csic.es.

Journal of Bacteriology, September 2000, p. 4764-4772, Vol. 182, No. 17
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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