Cell envelope mutants of Pseudomonas putida: physiological characterization and analysis of their ability to survive in soil

Authors

  • José-Juan Rodríguez-Herva,

    1. Department of Biochemistry and Molecular and Cellular Biology of Plants, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Apdo Correos 419, 18008 Granada, Spain.,
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  • Daniela Reniero,

    1. Department of Biochemistry and Molecular and Cellular Biology of Plants, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Apdo Correos 419, 18008 Granada, Spain.,
    2. Department of Genetics and Biology of Microorganisms, University of Milan, 20133 Milan, Italy.
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  • Enrica Galli,

    1. Department of Genetics and Biology of Microorganisms, University of Milan, 20133 Milan, Italy.
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  • Juan L. Ramos

    1. Department of Biochemistry and Molecular and Cellular Biology of Plants, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Apdo Correos 419, 18008 Granada, Spain.,
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Juan L. Ramos E-mail jlramos@eez.csic.es; Tel. (+34) 58 121011; Fax (+34) 58 129600.

Abstract

To generate mutants with altered lipopolysaccharides (LPS) of the wild-type Pseudomonas putida KT2442, we used the mini-Tn5luxAB-Km transposon. A mutant was found among luminescent colonies and selected as a negative clone in enzyme-linked immunosorbent assay (ELISA) with monoclonal antibody (mAb) 7.3B, which recognizes the O-antigen of P. putida LPS. The DNA region of the LPS mutant interrupted by the minitransposon insertion was cloned and sequenced. Comparison of the deduced amino acid sequence with protein sequence databases showed similarity to the O-antigen polymerase (Wzy) of Salmonella enterica (muenchen). The wild-type gene was rescued by polymerase chain reaction (PCR), cloned into a broad-host-range plasmid and used to carry out complementation assays. The cloned gene was able to restore the wild-type phenotype of the P. putida wzy mutant. We constructed an isogenic mutant of the luminescent wzy mutant to which an oprL mutation was transferred by homologous recombination with an oprL::xylE cassette. The wzy mutants of P. putida were more sensitive to SDS, deoxycholate and EDTA than the corresponding parental strains. We analysed the ability of wzy, oprL and wzy oprL mutants of P. putida to colonize soil. In comparison with the wild-type strain, the ability of single mutants to colonize soil decreased; this characteristic was more evident for the double mutant, especially at high temperatures.

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