A WbpL mutant of Pseudomonas putida DOT-T1E strain, which lacks the O-antigenic side chain of lipopolysaccharides, is tolerant to organic solvent shocks.
Lipopolysaccharides (LPS) are major components of the outer membrane of gram-negative bacteria and are considered a defense barrier. To determine if LPS play a role in resistance to solvents in the solvent-tolerant Pseudomonas putida DOT-T1E strain, we have generated mutants unable to synthesize the O-antigen side chain of LPS. The wbpL gene, encoding the enzyme that begins the synthesis of the O-antigen side chain of LPS of the solvent-tolerant strain, was cloned, sequenced, and knocked out in vitro with a cassette encoding kanamycin resistance, and a mutant called WbpL0 of the DOT-T1E strain was generated in vivo by site-directed mutagenesis. The WbpL mutant was compared with the wild-type strain with regard to tolerance to a number of toxic compounds, including chelating agents, organic acids, detergents, and aromatic hydrocarbons. It was found that the mutant was as tolerant as the wild-type strain to organic acids and aromatic hydrocarbons and more sensitive to ethylenediaminetetraacetic acid and deoxycholate.
Junker
F
F
Consejo Superior de Investigaciones Cientificas, Estación Experimental del Zaidin, Department of Plant Biochemistry and Molecular and Cellular Biology, Granada, Spain.
Rodríguez-Herva
J J
JJ
Duque
E
E
Ramos-González
M I
MI
Llamas
M
M
Ramos
J L
JL
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D017931:DNA Primers; D004274:DNA, Recombinant; D016695:Glycosyltransferases / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019081:O Antigens / Q000276:immunology*; D009930:Organic Chemicals / Q000494:pharmacology*; D010641:Phenotype; D016958:Pseudomonas putida / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D012997:Solvents / Q000494:pharmacology*
D001426:Bacterial Proteins; D017931:DNA Primers; D004274:DNA, Recombinant; D019081:O Antigens; D009930:Organic Chemicals; D012997:Solvents; D016695:Glycosyltransferases; C114818:WbpL protein, Pseudomonas