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Like other highly urbanized and industrialized estuaries, the Seine estuary (France) has, for decades, received high inputs of polycyclic aromatic hydrocarbons (PAHs). In order to estimate the bioremediation potentials and to identify the bacterial species involved in hydrocarbon degradation, we used microcosms containing seawater from the Seine estuary supplemented with either naphthalene, phenanthrene, fluorene or pyrene. In the microcosms enriched with naphthalene or phenanthrene, hydrocarbon biodegradation was significant within 9 weeks (43% or 46%, respectively), as shown by analyses in GC-MS. In similar microcosms incubated also with naphthalene or phenanthrene, analysis of the 16S rRNA gene sequences (DNA and cDNA) with denaturing gradient gel electrophoresis and clone libraries indicated that the PAH-degrading communities were dominated by Cycloclasticus spp., confirming their universal key role in degradation of low-molecular-weight PAHs in marine environments. However, in contrast to previous studies, we found that Pseudomonas spp. also degraded naphthalene and phenanthrene in seawater; this occurred only after 21 days, as was confirmed by real-time PCR. Although this genus has been abundantly described in the literature as a good PAH-degrading bacterial group in soil or in sediment, to our knowledge, this is the first evidence of a significant fitness in PAH degradation in seawater.

The major outer-membrane protein of Pseudomonas, OprF, is multifunctional. It is a non-specific porin that plays a role in maintenance of cell shape, in growth in a low-osmolarity environment, and in adhesion to various supports or molecules. OprF has been studied extensively for its utility as a vaccine component, its role in antimicrobial drug resistance, and its porin function. The authors have previously shown important differences between the OprF and 16S rDNA phylogenies: Pseudomonas fluorescens isolates split into two quite separate clusters, probably according to their ecological niche. In this study, the evolutionary history of the oprF gene was investigated further. The study of G+C content at the third codon position, synonymous codon usage (codon adaptation index, CAI) and genomic context showed no evidence of horizontal transfer or gene duplication. Similarly, a robust likelihood test of incongruence showed no significant incongruence between the oprF phylogeny and the species phylogeny. In addition, the ratio of nonsynonymous mutations to synonymous mutations (K a/K s) is high between the different clusters, especially between the two clusters containing P. fluorescens isolates, highlighting important modifications in evolutionary constraints during the history of the oprF gene. Since OprF is known as a pleiotropic protein, modifications in evolutionary constraints could have resulted from variations in cryptic functions, correlated with the ecological fingerprint. Finally, relaxed constraints and/or episodic positive evolution, especially for some P. fluorescens strains, could have led to a phylogeny reconstruction artifact.

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