A long-branch attraction artifact reveals an adaptive radiation in pseudomonas.

A significant proportion of protein-encoding gene phylogenies in bacteria is inconsistent with the species phylogeny. It was usually argued that such inconsistencies resulted from lateral transfers. Here, by further studying the phylogeny of the oprF gene encoding the major surface protein in the bacterial Pseudomonas genus, we found that the incongruent tree topology observed results from a long-branch attraction (LBA) artifact and not from lateral transfers. LBA in the oprF phylogeny could be explained by the faster evolution in a lineage adapted to the rhizosphere, highlighting an unexpected adaptive radiation. We argue that analysis of such artifacts in other inconsistent bacterial phylogenies could be a valuable tool in molecular ecology to highlight cryptic adaptive radiations in microorganisms.

Both Cycloclasticus spp. and Pseudomonas spp. as PAH-degrading bacteria in the Seine estuary (France).

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.

Development of a molecular method to detect and quantify Aphanomyces euteiches in soil.

A real-time PCR assay using 136F/211R primers and 161T TaqMan probe for the detection and quantification of Aphanomyces euteiches in soil is presented. The specificity of primers was tested on 105 different A. euteiches isolates, mainly from France. A calibration curve was established with a plasmid pHS1 resulting from the target region cloned into the pCR4 Topo vector (Invitrogen). The target copy number was evaluated and was constant whatever the isolate. A DNA-based method was able to discriminate between different artificial infestation levels in soil with small SDs thus validating the relevance of the extraction and amplification method in soil samples. Furthermore, a good correlation was observed between inoculum quantity in soil estimated by qPCR and root rot severity in plant evaluated by bioassays. These steps are essential when considering the feasibility of using a DNA-based method as a fast and accurate way to evaluate inoculum quantity in soil.

Sequence diversity of the OprD protein of environmental Pseudomonas strains.

OprD has been widely described for Pseudomonas aeruginosa at both structural and functional levels. Here, we describe the sequence diversity of the OprD proteins from other fluorescent Pseudomonads. We analysed the sequence of the oprD gene in each of the 49 Pseudomonas isolates, mostly putida and fluorescens species, obtained from various environmental sources, including soil, rhizosphere and hospitals. Phylogeny based on OprD sequences distinguished three well-separated clusters in the P. fluorescens species whereas P. putida isolates formed only one cluster. The OprD sequences were generally well conserved within each cluster whereas on the opposite, they were highly variable from one cluster to another and particularly with regards to the cluster of P. aeruginosa. Predicted secondary structures, based on the topological model elaborated for P. aeruginosa, suggest signatures in the large extracellular loops of OprD, which are linked to the OprD-based clusters. Correlations between these OprD-based clusters and ecological niches, growth on various carbon sources and antibiotic sensitivity were investigated.

OprF polymorphism as a marker of ecological niche in Pseudomonas.

OprF is the major outer-membrane protein of Pseudomonas sensu stricto (rRNA group I). In addition to playing a role as porin, membrane structural protein and root adhesion, this pleiotropic protein shows a length polymorphism corresponding to two types of OprF, termed OprF type 1 and OprF type 2. In a previous work, all the P. fluorescens isolated from bulk soil (non-rhizospheric) were shown to possess oprF type 1, while all the clinical P. fluorescens isolates and most rhizospheric strains corresponded to type 2. In this study, we further investigated the relation between the OprF polymorphism and the ecological niche by developing a culture-independent approach (a ratio polymerase chain reaction) to measure the percentage of each oprF type in environmental DNA samples, including two different soils and three different cultured plants (flax, wheat and grassland). Although the proportions of oprF type 2 between rhizospheric samples were quite variable, they were always very significantly higher (P<0.001) than the proportions of oprF type 2 of the adjacent bulk soil where the vast majority of oprF (>95%) corresponded to type 1. We discuss the potential applications of this ecological fingerprint in an agronomic and taxonomic point of view.

Molecular evolution of the major outer-membrane protein gene (oprF) of Pseudomonas.

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.

Phylogenetic relationships between environmental and clinical isolates of Pseudomonas fluorescens and related species deduced from 16S rRNA gene and OprF protein sequences.

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-cluster) that may be related to the original habitat of the strain. The fluorescens 1 o-cluster contained the majority 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-specific 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 degrees C. The adhesion capacities of the isolates in the two o-clusters seems to be correlated with ecological niche.