The Pseudomonas fluorescens complex includes Pseudomonas strains that have been taxonomically assigned to more than fifty different species, many of which have been described as plant growth-promoting rhizobacteria (PGPR) with potential applications in biocontrol and biofertilization. So far the phylogeny of this complex has been analyzed according to phenotypic traits, 16S rDNA, MLSA and inferred by whole-genome analysis. However, since most of the type strains have not been fully sequenced and new species are frequently described, correlation between taxonomy and phylogenomic analysis is missing. In recent years, the genomes of a large number of strains have been sequenced, showing important genomic heterogeneity and providing information suitable for genomic studies that are important to understand the genomic and genetic diversity shown by strains of this complex. Based on MLSA and several whole-genome sequence-based analyses of 93 sequenced strains, we have divided the P. fluorescens complex into eight phylogenomic groups that agree with previous works based on type strains. Digital DDH (dDDH) identified 69 species and 75 subspecies within the 93 genomes. The eight groups corresponded to clustering with a threshold of 31.8% dDDH, in full agreement with our MLSA. The Average Nucleotide Identity (ANI) approach showed inconsistencies regarding the assignment to species and to the eight groups. The small core genome of 1,334 CDSs and the large pan-genome of 30,848 CDSs, show the large diversity and genetic heterogeneity of the P. fluorescens complex. However, a low number of strains were enough to explain most of the CDSs diversity at core and strain-specific genomic fractions. Finally, the identification and analysis of group-specific genome and the screening for distinctive characters revealed a phylogenomic distribution of traits among the groups that provided insights into biocontrol and bioremediation applications as well as their role as PGPR.

The Pseudomonas fluorescens complex of species includes plant-associated bacteria with potential biotechnological applications in agriculture and environmental protection. Many of these bacteria can promote plant growth by different means, including modification of plant hormonal balance and biocontrol. The P. fluorescens group is currently divided into eight major subgroups in which these properties and many other ecophysiological traits are phylogenetically distributed. Therefore, a rapid phylogroup assignment for a particular isolate could be useful to simplify the screening of putative inoculants. By using comparative genomics on 71 P. fluorescens genomes, we have identified nine markers which allow classification of any isolate into these eight subgroups, by a presence/absence PCR test. Nine primer pairs were developed for the amplification of these markers. The specificity and sensitivity of these primer pairs were assessed on 28 field isolates, environmental samples from soil and rhizosphere and tested by in silico PCR on 421 genomes. Phylogenomic analysis validated the results: the PCR-based system for classification of P. fluorescens isolates has a 98.34% of accuracy and it could be used as a rapid and simple assay to evaluate the potential of any P. fluorescens complex strain.

Bacterial motility plays a crucial role in competitiveness and colonization in the rhizosphere. In this work, Chromatin ImmunoPrecipitation Sequencing (ChIP-seq) analysis has been used to identify genes putatively regulated by the transcriptional regulatory protein FleQ in Pseudomonas fluorescens F113 and Pseudomonas putida KT2440. This protein was previously identified as a master regulator of flagella and biofilm formation in both strains. This work has demonstrated that FleQ from both bacteria are conserved and functionally equivalent for motility regulation. Furthermore, the ChIP-seq analysis has shown that FleQ is a global regulator with the identification of 121 and 103 FleQ putative binding sites in P. fluorescens F113 and P. putida KT2440 respectively. Putative genes regulated by FleQ included, as expected, flagellar and motility-related genes and others involved in adhesion and exopolysaccharide production. Surprisingly, the ChIP-seq analysis also identified iron homeostasis-related genes for which positive regulation was shown by RT-qPCR. The results also showed that FleQ from P. fluorescens F113 shares an important part of its direct regulon with AmrZ, a global regulator also implicated in environmental adaption. Although AmrZ also regulates motility and iron uptake, the overlap occurred mostly with the iron-related genes, since both regulators control a different set of motility-related genes.

Diesel is a complex pollutant composed of a mixture of aliphatic and aromatic hydrocarbons. Because of this complexity, diesel bioremediation requires multiple microorganisms, which harbor the catabolic pathways to degrade the mixture. By enrichment cultivation of rhizospheric soil from a diesel-polluted site, we have isolated a bacterial consortium that can grow aerobically with diesel and different alkanes and polycyclic aromatic hydrocarbons (PAHs) as the sole carbon and energy source. Microbiome diversity analyses based on 16S rRNA gene showed that the diesel-degrading consortium consists of 76 amplicon sequence variants (ASVs) and it is dominated by Pseudomonas, Aquabacterium, Chryseobacterium, and Sphingomonadaceae. Changes in microbiome composition were observed when growing on specific hydrocarbons, reflecting that different populations degrade different hydrocarbons. Shotgun metagenome sequence analysis of the consortium growing on diesel has identified redundant genes encoding enzymes implicated in the initial oxidation of alkanes (AlkB, LadA, CYP450) and a variety of hydroxylating and ring-cleavage dioxygenases involved in aromatic and polyaromatic hydrocarbon degradation. The phylogenetic assignment of these enzymes to specific genera allowed us to model the role of specific populations in the diesel-degrading consortium. Rhizoremediation of diesel-polluted soil microcosms using the consortium, resulted in an important enhancement in the reduction of total petroleum hydrocarbons (TPHs), making it suited for rhizoremediation applications.

Biofilms are complex structures that are crucial during host–bacteria interaction and colonization. Bacteria within biofilms are surrounded by an extracellular matrix (ECM) typically composed of proteins, polysaccharides, lipids, and DNA. Pseudomonads contain a variety of ECM components, some of which have been extensively characterized. However, neither the ECM composition of plant-associated pseudomonads nor their phylogenetic distribution within the genus has been so thoroughly studied. In this work, we use in silico methods to describe the ECM composition of Pseudomonas fluorescens F113, a plant growth-promoting rhizobacteria and model for rhizosphere colonization. These components include the polysaccharides alginate, poly-N-acetyl-glucosamine (PNAG) and levan; the adhesins LapA, MapA and PsmE; and the functional amyloids in Pseudomonas. Interestingly, we identified novel components: the Pseudomonas acidic polysaccharide (Pap), whose presence is limited within the genus; and a novel type of Flp/Tad pilus, partially different from the one described in P. aeruginosa. Furthermore, we explored the phylogenetic distribution of the most relevant ECM components in nearly 600 complete Pseudomonas genomes. Our analyses show that Pseudomonas populations contain a diverse set of gene/gene clusters potentially involved in the formation of their ECMs, showing certain commensal versus pathogen lifestyle specialization.