Functional characterization of Pseudomonas fluorescens OprE and OprQ membrane proteins Thomas Jaouen a , Laurent Coquet b , Laure Marvin-Guy c , Nicole Orange a, * , Sylvie Chevalier a, * ,1 , Emmanuelle De ´ b,1 a Laboratoire de Microbiologie du Froid, UPRES 2123, Universite ´ de Rouen, 55 rue Saint Germain, 27000 Evreux, France b UMR 6522 CNRS, IFRMP, Universite ´ de Rouen, 76821 Mont Saint Aignan, France c Nestle ´ Research Center, Nestec Ltd., Vers-chez-les-Blanc, 1000 Lausanne 26, Switzerland Received 31 May 2006 Available online 9 June 2006 Abstract Outer membrane (OM) proteins of the OprD family may enable bacteria of the genus Pseudomonas to adapt to various environments by modulating OM permeability. The OprE and OprQ porins from P. fluorescens strain MF0 were purified and identified by MALDI- TOF mass spectrometry and N-terminal and internal microsequencing. These proteins, when reconstituted in an artificial planar lipid bilayer, induced similar ion channels with low single-conductance values. Secondary structure prediction of both proteins showed similar folding patterns into a 16 transmembrane b-strands barrel but a highly variable amino-acid composition and length for their putative external loops implicated in porin function. Both proteins were overexpressed under poor oxygenation conditions, but not by using sev- eral amino acids as sole carbon source, indicating a different specificity for these proteins compared to the paradigm of this protein fam- ily, OprD. Ó 2006 Elsevier Inc. All rights reserved. Keywords: Pseudomonas; Porins; OprD; OprE; OprQ; Single channel conductance value; Expression Bacteria from the genus Pseudomonas are present in all major natural environments, including water, soil, the plant rhizosphere, and the human body. Some of these environments are subjected to rapid variations, necessitat- ing high levels of physiologic and genetic adaptability in bacteria living in such hostile habitats [1]. This adaptability involves many modifications, some of which affect outer membrane (OM) permeability. This permeability essential- ly depends on channel-forming proteins, which can be divided into three classes: general porins, specific porins, and highly substrate-specific gated channels translocating their ligands in an energy-dependent manner [2]. OM per- meability is modified by changes to porins in two main ways: (1) modulation of porin channel size, as reported for OprF, the major non-specific porin of psychrotrophic and mesophilic Pseudomonas species [3], and (2) differential expression of porins with narrow channels to decrease OM permeability [4], which seems to be particularly common for specific porins, such as OprD [5–7]. This porin OprD, which binds basic amino acids, is the paradigm for a 19-member family of OM proteins in P. aeruginosa [8]. These proteins, displaying growth condi- tion-regulated production, seem to be specific for different classes of substrates, but may also act as non-specific por- ins for small substrates [7,9]. Member of this family, OprE, has been reported to be induced in anaerobic conditions in P. aeruginosa [10] and to form pores in vitro excluding mol- ecules larger in size than di- or trisaccharides [11]. Little is known about OprQ, which may allow saccharide diffusion [12]. In the psychrotrophic P. fluorescens strain MF0, OprE, and OprQ were found to be overexpressed in an adaptive OprF-deficient mutant, presumably to compen- sate for the loss of this major non-specific porin [13]. This species, closely related to P. aeruginosa, colonizes many 0006-291X/$ - see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.06.013 * Corresponding author. Fax: +33 02 32 29 15 66. E-mail address: sylvie.chevalier@univ-rouen.fr (N. Orange). 1 These authors contributed equally to this study. www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 346 (2006) 1048–1052 BBRC