Introduction The genus Pseudomonas (sensu stricto) Type I [17, 25] in the gamma subclass of Proteobacteria [36] includes species of ecological, economical and medical interest. Certain species are PGPR (Plant Growth Promoting Rhizobacteria) or plant pathogens whilst others are ani- mal or human opportunistic pathogens. One species of this genus, Pseudomonas fluorescens, is a ubiquitous bac- terium frequently found in water and in soil, particularly within the rhizosphere of plants. This species is described as a psychrotrophic bacterium capable of growth from 0 °C to 32 °C in numerous ecological niches although it can even grow at 37 °C as evidenced by its involvement in nosocomial disease [11, 26]. Note: Nucleotide sequence data reported are available in the GenBank database. The accession numbers are given in Table 1 and Table 2. Phylogenetic Relationships Between Environmental and Clinical Isolates of Pseudomonas fluorescens and Related Species Deduced from 16S rRNA Gene and OprF Protein Sequences Josselin Bodilis 1,2 , Raphaël Calbrix 1,2 , Josette Guérillon 1 , Annabelle Mérieau 1 , Barbara Pawlak 1 , Nicole Orange 1 , and Sylvie Barray 1,2 1 Laboratoire de Microbiologie du Froid, UPRES 2123, Université de Rouen, Mont Saint Aignan, France 2 ABISS (Atelier de Biologie, Informatique, Statistique et Sociolinguistique), Université de Rouen, Mont Saint Aignan, France Received: September 22, 2003 Summary The major surface protein of the genus Pseudomonas, OprF, is a non-specific porin that plays an important role in maintenance of cell shape, in growth in a low osmolarity environment, and in adhesion to various supports. The objectives of our study were (i) to carry out a comparative analysis of phylogenies obtained from the OprF protein and from the 16S rRNA gene in 41 isolates from various sources (water, soil, milk and the hospital) and (ii) to investigate the physiological characteristics correlated with the phylogeny of OprF. We report here an important incongruence between the phylogenies of the 16S rRNA gene and the OprF protein. Phylogenetic analysis of 16S rRNA genes grouped Pseudomonas fluorescens isolates into one cluster (termed fluorescens r-cluster ) whilst the phylogeny of the OprF protein divided Pseudomonas fluorescens isolates into two quite distinct clusters (termed fluorescens 1 o-cluster and fluorescens 2 o-clus- ter ) that may be related to the original habitat of the strain. The fluorescens 1 o-cluster contained the ma- jority of non-rhizospheric soil isolates, while the fluorescens 2 o-cluster contained all our clinical isolates and most of the rhizospheric isolates (which are fixed to the roots). In order to check this correlation, we studied two physiological characteristics: the range of growth temperature and the capacity for non-specif- ic adhesion to polystyrene. The temperature range study for strains did not explain the existence of the two o-clusters but it did confirm the capacity of certain P. fluorescens strains to grow at 37 °C. The adhesion capacities of the isolates in the two o-clusters seems to be correlated with ecological niche. Key words: Pseudomonas – OprF protein – 16S rRNA gene – phylogeny – dichotomy – ecological niche – growth temperature – adhesion System. Appl. Microbiol. 27, 93–108 (2004) http://www.elsevier-deutschland.de/syapm 0723-2020/04/27/01-93 $ 30.00/0 OprF is the major outer membrane protein of Pseu- domonas (sensu stricto ) [33] and has been described as a non-specific porin permitting passive diffusion of small polar nutrients [24]. The OprF protein is essential for survival and a viable OprF mutant was found to have compensatory mutations [4]. Functional modifica- tions to OprF result from the growth of P. fluorescens at different temperatures [8]. OprF also has an important