Genomic and genetic analyses of diversity and plant interactions of Pseudomonas fluorescens

Abstract

Background: Pseudomonas fluorescens are common soil bacteria that can improve plant health through nutrient cycling, pathogen antagonism and induction of plant defenses. The genome sequences of strains SBW25 and Pf0-1 were determined and compared to each other and with P. fluorescens Pf-5. A functional genomic in vivo expression technology (IVET) screen provided insight into genes used by P. fluorescens in its natural environment and an improved understanding of the ecological significance of diversity within this species.

Results: Comparisons of three P. fluorescens genomes (SBW25, Pf0-1, Pf-5) revealed considerable divergence: 61% of genes are shared, the majority located near the replication origin. Phylogenetic and average amino acid identity analyses showed a low overall relationship. A functional screen of SBW25 defined 125 plant-induced genes including a range of functions specific to the plant environment. Orthologues of 83 of these exist in Pf0-1 and Pf-5, with 73 shared by both strains. The P. fluorescens genomes carry numerous complex repetitive DNA sequences, some resembling Miniature Inverted-repeat Transposable Elements (MITEs). In SBW25, repeat density and distribution revealed 'repeat deserts' lacking repeats, covering approximately 40% of the genome.

Conclusions: P. fluorescens genomes are highly diverse. Strain-specific regions around the replication terminus suggest genome compartmentalization. The genomic heterogeneity among the three strains is reminiscent of a species complex rather than a single species. That 42% of plant-inducible genes were not shared by all strains reinforces this conclusion and shows that ecological success requires specialized and core functions. The diversity also indicates the significant size of genetic information within the Pseudomonas pan genome.

Figure 1

Comparison of amino acid matches between the complete six-frame translations of the whole genome sequences of P. fluorescens Pf0-1, SBW25 and Pf-5 genomes. The analysis was carried out using Artemis Comparison Tool and computed using TBLASTX. Forward and reverse strands of DNA are shown for each genome (dark grey lines). The red bars between the DNA lines represent individual TBLASTX matches, with inverted matches colored blue. Graphs show the density of CDSs with orthologues in the other two P. fluorescens strains (red and green lines). Window size is shown on the graphs. The thin grey lines show the genome average orthologue density. The white boxes on the DNA lines represent the variable regions around the termini as defined by these graphs (SBW25, 2.7 Mb; Pf0-1, 2 Mb; and Pf-5, 2.65 Mb). Blue and pink boxes represent the position of atypical regions and prophage, respectively.

Figure 2

Venn diagram comparing the gene complements of P. fluorescens strains SBW25, Pf0-1 and Pf-5. The numbers of unique and shared CDSs are presented. Numbers in parenthesis are insertion sequence elements and pseudogenes. Pie charts indicate the absolute numbers divided into functional categories (see legend) for the complete gene complement of SBW25, the CDSs in common with the other two strains plus the core gene complement for all three.

Figure 3

Phylogenetic tree of 14 different Pseudomonas species, based on 1,705 conserved genes: Pseudomonas fluorescens strains SBW25 (SBW25), Pf0-1 (Pf01) and Pf-5 (Pf5); Pseudomonas aeruginosa strains PAO1 (P_aer_PAO1), PA14 (P_aer_PA14) and PA7 (P_aer_PA7); Pseudomonas syringae pv. syringae B728a (P_syr_syr), pv. tomato DC3000 (P_syr_tom) and pv. phaseolicola 1448A (P_syr_pha); Pseudomonas putida strains GB1 (P_put_GB1), F1 (P_put_F1), W619 (P_put_W619) and KT2240 (P_put_KT24); and Pseudomonas stutzeri strain A1501 (P_stut). Numbers on nodes represent percentages of individual trees containing that relationship. The scale bar corresponds to the number of substitutions per site.

Figure 4

Average amino acid identities between pairs of P. syringae, P. aeruginosa, and P. fluorescens strains. The strain designations for the P. fluorescens and P. aeruginosa isolates and pathovar designations for the P. syringae isolates are as described for Figure 3. Genus and species boundaries are those used by Konstantinidis and Tiedje [32].

Figure 5

Circular genome maps of P. fluorescens strains SBW25 and Pf0-1. (a) P. fluorescens SBW25. From the outside in, the outer most circle shows atypical regions (blue boxes) and prophage-like regions (pink boxes) numbered according to Supplementary Table 3 in Additional data file 3; circle 2, scale line (in Mbps); circles 3 and 4 show the position of CDSs transcribed in a clockwise and anticlockwise direction, respectively (for color codes, see below); circle 5, location of IVET EIL fusions (black); circle 6, graph showing density of CDSs with orthologues (red) and those unique to SBW25 (green) compared to P. fluorescens Pf0-1 (window size 50,000 bp, step size 200); circle 7, P. fluorescens SBW25 variable region (green line); circle 8, IR1_g inverted repeats (dark blue); circle 9, R0 family of intergenic repeats (navy blue); circle 10, R2 family of intergenic repeats (light blue); circle 11, R5, R30, R178 and R200 families of intergenic repeats (aqua); circle 12, repeat deserts (ReDs; grey boxes); circle 13, GC skew (window 10,000 bp). CDSs were color-coded according to the function of their gene products: dark green, membrane or surface structures; yellow, central or intermediary metabolism; cyan, degradation of macromolecules; red, information transfer/cell division; cerise, degradation of small molecules; pale blue, regulators; salmon pink, pathogenicity or adaptation; black, energy metabolism; orange, conserved hypothetical; pale green, unknown; and brown, pseudogenes. Note that IR1_g repeats were not included in the ReD analysis because, based on their structure, we could not exclude the possibility that many of them simply represent transcription termination sequences. Where some ReDs appear to contain R-family repeats (for example, ReDs at about 6.1 Mb) there is actually more than one ReD, separated by a very small DNA region, that cannot be resolved in the figure. (b) P. fluorescens Pf0-1. From the outside in, outer most circle shows atypical regions (blue boxes) and prophage-like regions (pink boxes) numbered according to Supplementary Table 4 in Additional data file 3; circle 2, scale line (in Mbps); circles 3 and 4 show the position of CDSs transcribed in a clockwise and anticlockwise direction, respectively (for color codes, see above); circle 5, orthologues of SBW25 EIL - those EIL that are antisense in SBW25 are indicated by orthologues to the predicted CDSs on the sense strand; circle 6, graph showing density of CDSs with orthologues (red) and those unique to Pf0-1 (green) compared to P. fluorescens SBW25 (window size 50,000 bp, step size 200); circle 7, P. fluorescens Pf0-1 variable region (green line); circle 8, IR1_g inverted repeats (dark blue); circle 9, R5 family of intergenic repeats (navy blue); circle 10, R6 family of intergenic repeats (light blue); circle 11, R0, R1, R6-partial, R26, R30, R69, and R178 families of intergenic repeats (aqua); circle 12, GC skew (window 10,000 bp).