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Appl. Environ. Microbiol. doi:10.1128/AEM.02539-07
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Emergence of Secretion-Defective Sublines of Pseudomonas aeruginosa PAO1 Resulting from Spontaneous Mutations in the vfr Global Regulatory Gene

Laboratoire d'Ingénierie des Systèmes Macromoléculaires, Institut de Biologie Structurale et Microbiologie, Centre National de la Recherche Scientifique, 31 Chemin Joseph Aigier, 13402 Marseille Cedex 20, France; Département de Microbiologie Fondamentale, Université de Lausanne, Bâtiment Biophore, CH-1015 Lausanne, Switzerland; and Institute of Infection, Immunity and Inflammation, University of Nottingham, University Park, Nottingham NG7 2RD, UK

^* To whom correspondence should be addressed. Email: voulhoux{at}ibsm.cnrs-mrs.fr.

	Abstract

Pseudomonas aeruginosa undergoes spontaneous mutations that impair secretion of several extracellular enzymes, during in vitro extended cultivation in rich media as well as during long-term colonization of the cystic fibrosis lung. A frequent type of strong secretion deficiency is caused by inactivation of the quorum sensing regulatory gene lasR. Here we analyzed a spontaneously emerging subline of strain PAO1 that exhibited moderate secretion deficiency and partial loss of quorum sensing control. Using generalized transduction, we mapped the secretion defect to the vfr gene, which is known to control positively the expression of the lasR gene and type II secretion of several proteases. We confirmed this secretion defect by sequencing and complementation of the vfr mutation. In a reconstruction experiment conducted with a 1:1 mixture of the wild type PAO1 and a vfr mutant of PAO1, we observed a selective advantage of the vfr mutant over the wild type after growth in static culture for four days. Under these conditions, spontaneous vfr mutants emerged in a population of strain PAO1 after four growth cycles and represented more than 40% of the population after seven cycles. These results suggest that partial or complete loss of quorum sensing and secretion can be beneficial to P. aeruginosa under certain environmental conditions.