BackgroundPseudomonas fluorescens F113 is a plant growth-promoting rhizobacterium (PGPR) isolated from the sugar-beet rhizosphere. This bacterium has been extensively studied as a model strain for genetic regulation of secondary metabolite production in P. fluorescens, as a candidate biocontrol agent against phytopathogens, and as a heterologous host for expression of genes with biotechnological application. The F113 genome sequence and annotation has been recently reported.ResultsComparative analysis of 50 genome sequences of strains belonging to the P. fluorescens group has revealed the existence of five distinct subgroups. F113 belongs to subgroup I, which is mostly composed of strains classified as P. brassicacearum. The core genome of these five strains is highly conserved and represents approximately 76% of the protein-coding genes in any given genome. Despite this strong conservation, F113 also contains a large number of unique protein-coding genes that encode traits potentially involved in the rhizocompetence of this strain. These features include protein coding genes required for denitrification, diterpenoids catabolism, motility and chemotaxis, protein secretion and production of antimicrobial compounds and insect toxins.ConclusionsThe genome of P. fluorescens F113 is composed of numerous protein-coding genes, not usually found together in previously sequenced genomes, which are potentially decisive during the colonisation of the rhizosphere and/or interaction with other soil organisms. This includes genes encoding proteins involved in the production of a second flagellar apparatus, the use of abietic acid as a growth substrate, the complete denitrification pathway, the possible production of a macrolide antibiotic and the assembly of multiple protein secretion systems.

Pseudomonas fluorescens F113 is a plant growth-promoting rhizobacterium (PGPR) that has biocontrol activity against fungal plant pathogens and is a model for rhizosphere colonization. Here, we present its complete genome sequence, which shows that besides a core genome very similar to those of other strains sequenced within this species, F113 possesses a wide array of genes encoding specialized functions for thriving in the rhizosphere and interacting with eukaryotic organisms. Pseudomonas fluorescens F113 is a Gram-negative, rod-shaped member of the genus Pseudomonas, isolated from the sugarbeet rhizosphere (19). P. fluorescens F113 can colonize a wide range of plants and is used as a model strain to study rhizosphere colonization (7,21). It also exhibits biocontrol activity against phytopathogens, such as the oomycetes Pythium ultimum and Phytophthora cactorum and the fungus Fusarium oxysporum in several plant crops, including sugar-beet (13), tomato, and strawberry (6) and is antagonistic toward the potato-cyst nematode Globodera rostochiensis (11). This biocontrol activity is linked to the production of secondary metabolites, including diacetylphloroglucinol (DAPG) and hydrogen cyanide, and this strain has been widely used to study the regulation of secondary metabolism in P. fluorescens (1,2,13,15). Bioremediation derivatives of this strain able to degrade biphenyl and polychlorinated biphenyls have also been constructed (8, 22) and tested in situ (12). To gain insight into ecological traits, to improve its biotechnological applications, and to better understand its evolution, we sequenced the complete genome of this bacterium.The sequence of the P. fluorescens F113 genome was determined by using a combination of Illumina Solexa GAIIx (7eϩ6 single reads 36 nucleotides [nt] long) and Roche 454 Titanium (7eϩ5 reads 400 nt long). The reads were assembled into 83 contigs with 30ϫ sequence coverage using MIRA software (9). These contigs were further assembled into 4 supercontigs by using the ends of an ordered bacterial artificial chromosome (BAC) library and using BLAST (4) against the genomes of other P. fluorescens strains (14,17,20). The remaining gaps were closed by PCR and subsequent Sanger sequencing. Open reading frame (ORF) calling and annotation were first performed automatically using the RAST pipeline (5) and then manually curated using the Blast2GO package (10).The genome of F113 consists of a single circular chromosome of 6,845,832 bp with an average GC content of 60.8%. This genome is predicted to contain 5,862 protein-coding genes, 8 noncoding RNAs (ncRNAs), 5 rRNA operons, and 66 tRNA loci. Although the genome shows a high degree of homology and synteny with the chromosomes of other sequenced P. fluorescens strains, such as Pf0-1, Pf5, SBW25, and WH6, its closest relative is the genome of Pseudomonas brassicacearum subp. brassicacearum NFM421 (16), a pseudomonad isolated from the plant rhizosphere in Australia. It is interesting to note that these two strains show infrequent common traits, such ...

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.

The transcriptional regulator AmrZ is a global regulatory protein conserved within the pseudomonads. AmrZ can act both as a positive and a negative regulator of gene expression, controlling many genes implicated in environmental adaption. Regulated traits include motility, iron homeostasis, exopolysaccharides production and the ability to form biofilms. In Pseudomonas fluorescens F113, an amrZ mutant presents a pleiotropic phenotype, showing increased swimming motility, decreased biofilm formation and very limited ability for competitive colonization of rhizosphere, its natural habitat. It also shows different colony morphology and binding of the dye Congo Red. The amrZ mutant presents severely reduced levels of the messenger molecule cyclic-di-GMP (c-di-GMP), which is consistent with the motility and biofilm formation phenotypes. Most of the genes encoding proteins with diguanylate cyclase (DGCs) or phosphodiesterase (PDEs) domains, implicated in c-di-GMP turnover in this bacterium, appear to be regulated by AmrZ. Phenotypic analysis of eight mutants in genes shown to be directly regulated by AmrZ and encoding c-di-GMP related enzymes, showed that seven of them were altered in motility and/or biofilm formation. The results presented here show that in P. fluorescens, AmrZ determines c-di-GMP levels through the regulation of a complex network of genes encoding DGCs and PDEs.

The genomic sequence of Pseudomonas fluorescens F113 has shown the presence of a 41 kb cluster of genes that encode the production of a second flagellar apparatus. Among 2,535 pseudomonads strains with sequenced genomes, these genes are only present in the genomes of F113 and other six strains, all but one belonging to the P. fluorescens cluster of species, in the form of a genetic island. The genes are homologous to the flagellar genes of the soil bacterium Azotobacter vinelandii. Regulation of these genes is mediated by the flhDC master operon, instead of the typical regulation in pseudomonads, which is through fleQ. Under laboratory conditions, F113 does not produce this flagellum and the flhDC operon is not expressed. However, ectopic expression of the flhDC operon is enough for its production, resulting in a hypermotile strain. This flagellum is also produced under laboratory conditions by the kinB and algU mutants. Genetic analysis has shown that kinB strongly represses the expression of the flhDC operon. This operon is activated by the Vfr protein probably in a c-AMP dependent way. The strains producing this second flagellum are all hypermotile and present a tuft of polar flagella instead of the single polar flagellum produced by the wild-type strain. Phenotypic variants isolated from the rhizosphere produce this flagellum and mutation of the genes encoding it, results in a defect in competitive colonization, showing its importance for root colonization.