The general features of the genomes of P. fluorescens SBW25 (6,722,539 bp) and Pf0-1 (6,438,405 bp) are summarized in Table 1. SBW25 is predicted to encode 6,009 CDSs, with a coding density of 88.3%. The genome of Pf0-1 has 5,741 CDSs with a coding density of 90%. These findings compare to 6,144 CDSs predicted for Pf-5 (7,074,893 bp and 88.7% coding density) 18.

Alignments of the whole genome sequences of P. fluorescens strains SBW25, Pf0-1, and Pf-5 revealed that the only long-range synteny among these genomes is confined to the origin of replication, with a gradual deterioration in both synteny and sequence conservation towards the replication terminus (Figure 1). There is also evidence of extensive reciprocal recombination around the terminus of replication, as commonly seen in other bacterial genomes 24 (Figure 1). Neither bacterium contains an accessory element (note that plasmid pQBR103 for which the complete sequence was recently reported 25 was acquired by SBW25 during a field release experiment 26, but this plasmid is not present in the originally isolated strain).

Reciprocal FASTA analysis was used to identify orthologous gene sets shared among the three genomes. The distribution of genes and orthologues among the three P. fluorescens strains is non-random, with strain-unique genes being more common towards the replication terminus (Figure 1). This organization is similar to the accessory loci near the end of the arms (termini) of the linear chromosome in Streptomyces coelicolor A3(2), which are highly variable in both length and composition 27. Of the total coding capacity, genes conserved among all three P. fluorescens isolates comprise 3,642 CDSs, representing 59.3%, 60.6% and 63.4% of the coding capacity in Pf-5, SBW25 and Pf0-1, respectively (Figure 2). A large proportion of the P. fluorescens genes (from 1,111 to 1,490 CDSs (22% to 27% of total coding capacity)) are found in just one genome (Figure 2). This finding contrasts with Pseudomonas aeruginosa, where the five sequenced isolates share a conserved core of 5,021 genes with only 1.4% (strain C3719) to 8.2% (strain PA2192) of genes unique to any one isolate 23. It is possible that the overall low level of variation among the sequenced P. aeruginosa isolates reflects a bias created by restricting sampling solely to clinical isolates. If true, then it may be that the highly variable genomes of P. fluorescens are more representative of the true diversity of the Pseudomonas genus.

When the reciprocal FASTA analysis was extended to include 11 other sequenced Pseudomonas species the conserved gene complement of these 14 Pseudomonas genomes was just 1,705 CDSs. This pseudomonad core gene-set falls below that previously estimated for the gamma-proteobacteria as a whole (2,049 CDSs 28), underscoring the highly variable nature of this genus. This is also highlighted in Figure 3, which shows a majority rule consensus tree from the results of individual maximum likelihood analyses of the 1,705 core CDS amino acid datasets. The data strongly support the classification of P. aeruginosa, P. putida, and P. syringae isolates into species groups, with at least 95% of the single gene trees supporting the species distinction. In contrast, support for the classification of the three P. fluorescens isolates as a single species was relatively weak, supported by only 57% of single gene trees. Support for the intra-group relationships are not strong for any of the species examined and most likely reflects recombination among strains of each species 29. Indeed, evidence of recombination in a number of different Pseudomonas species, including P. aeruginosa 30, and P. fluorescens 31 has been reported.

Average amino acid identities (AAIs) 32 were calculated using the pair-wise orthologous sets of CDSs from the three P. fluorescens strains as well as three P. aeruginosa strains and three P. syringae pathovars (Figure 4; Table 2). It is evident that the AAIs of the P. fluorescens strains are considerably lower than those found in P. aeruginosa and P. syringae and fall between the limits of genera and species as defined by Konstantinidis and Tiedje 32. In addition, while unique sequences in each genome were excluded from AAI analyses, the relatively low number of orthologous sequences within the P. fluorescens genomes further calls the species grouping of these strains into question. However, we note that the AAI of orthologues located close to the replication origin ranges from 84.6% to 85.6%, whereas the AAI range for orthologues nearer the replication terminus is 75% to 77.5%: the genome wide AAI ranges from 82.2% to 83.4%. These regional differences require consideration before using AAI to infer relatedness.

Based on the genomic criteria provided by Goris et al. 33 for defining species, the three P. fluorescens strains could indeed be different species. In fact, our analysis is in agreement with previous studies that have shown - based on gyrB and rpoD nucleotide sequences - P. fluorescens to be a complex composed of two major lineages 34, with Pf-5 and Pf0-1 belonging in the P. chlororaphis and SBW25 in the P. fluorescens lineage. Similar conclusions have come from DNA-DNA hybridization and average nucleotide identity scores 33 and the genome signature (genome-specific relative frequencies of dinucleotides) 35. Given the small sample of genomes, it seems premature to redefine the species 'P. fluorescens' at this time. It should also be noted that our analysis shows the three P. fluorescens strains to group more closely to each other than to any other member of the Pseudomonas genus (Figure 3; Table 2).

Analysis of the conserved genes present in the three P. fluorescens strains provides results that are typical of other soil dwelling bacteria 3637. For example, SBW25 and Pf0-1 carry an abundance of regulatory genes (>300 each), and genes encoding motility and chemotaxis-related functions (>100 each) as well as genes specifying membrane and transport functions (>1,000 each).

Also typical for pseudomonads, the genomes of SBW25, Pf0-1 and Pf-5 lack 6-phosphofructokinase, required for conversion of β-D-fructose 6-phosphate to β-D-fructose 1,6-bisphosphate (although the gene for 1-phosphofructokinase is present) and these strains are therefore unlikely to carry out glycolysis. Nonetheless, each genome possesses genes predicted to specify the enzymes phosphogluconate dehydratase and 2-keto-3-deoxygluconate 6-phophate aldolase, which are necessary for utilization of glucose via the phosphorylative Entner-Doudoroff pathway.

The extreme diversity evident in these three P. fluorescens isolates - both in gene content and sequence conservation - made a full metabolic reconstruction impractical in the context of P. fluorescens as a species. Such a reconstruction requires a greater number of complete genome sequences and an improved understanding of the nature of the P. fluorescens species. Instead, we focused on the direct identification of genes associated with colonization and survival in the plant environment using an IVET promoter-trapping strategy. This approach is the first step in a functional test of the prediction that the gene classes commonly associated with soil bacteria (outlined above) are determinants of their ecological performance. Previous attempts have exploited the IVET promoter-trapping strategy to identify genes up-regulated in the plant rhizosphere and soil environments 567. While providing insight into a set of functionally significant genes, these studies have been based on the screening of partial genomic libraries and, therefore, the full spectrum of plant-soil-induced genes has not been identified. In order to obtain a comprehensive set of genes specifically active in the plant-soil environment, a full genome survey of plant- and rhizosphere-induced genes (collectively referred to as environment inducible loci (EIL)) in SBW25 was undertaken using the IVET strategy developed by Gal et al. 6. This strategy selects EIL on the basis of their ability to drive the expression of a promoterless copy of the reporter gene dapB ('dapB) - a gene required for the biosynthesis of diaminopimelate (DAP), which is an essential component of the peptidoglycan layer of the bacterial cell wall. Active EIL fusions to 'dapB allow growth by complementing a dapB deletion in the SBW25 host strain used for these experiments. The distribution of EIL in SBW25 is shown in Figure 5a, and putative Pf0-1 orthologues are shown in Figure 5b. EIL classified by function, and putative orthologues in Pf0-1 and Pf-5, are given in Supplementary Table 1 in Additional data file 1.

EIL were identified by screening a library consisting of 33,000 clones (62 independent ligation reactions) and analyzed in pools of 250 on Beta vulgaris (sugar beet) seedlings. Given a genome of 6.7 Mbp, a random library of 3 to 5 kb fragments, and assuming 3,000 promoters in the SBW25 genome, then the chance of a promoter not being included in this study is less than 0.01 (based on the Poisson distribution).

The plant-inducibility of the EIL-'dapB fusion strains recovered by IVET selection was verified for each of the 125 IVET fusion strains by their inability to grow on M9 (glucose) minimal medium in the absence of DAP (thus demonstrating that the fusions are transcriptionally silent in vitro). The ability of each fusion strain to colonize both the rhizosphere and the phyllosphere of non-sterile sugar beet seedlings was then re-checked (strains colonizing these environments contain fusions to genes that are transcriptionally activated in the plant environment) 611. SBW25ΔdapB and an IVET negative-control strain, PBR393 38, were used as controls and no colony forming units of either strain were recovered from either the rhizosphere or phyllosphere. Every putative SBW25ΔdapB strain carrying an EIL-'dapB fusion grew in the rhizosphere (the size of the initial inoculum more than tripled in the rhizosphere over the course of 3 weeks); 90 of these IVET fusion strains were also able to grow in the phyllosphere (cells recovered from the phyllosphere underwent at least 3 doublings in 3 weeks). Growth of all EIL-fusion strains was significantly impaired in M9 (glucose) minimal medium. These tests verify that the EIL fusions are expressed by SBW25 on plant surfaces, and that the EIL promoters are dependent on the plant environment for expression. Further studies to determine the precise function of individual EIL in the plant environment are on going.

The 125 genes shown to be specifically up-regulated in planta represent all major classes of genes found in SBW25: Pf0-1 and Pf-5 each have orthologues of 83 of the 125 IVET-identified genes. Of these, 73 genes are common to all three P. fluorescens strains (Supplementary Table 1 in Additional data file 1). These data confirm the importance of previously recognized activities 6, and those predicted from genome sequence analysis, including nutrient acquisition and scavenging, cell envelope function, metabolism, stress response, and detoxification. Interestingly, when compared with the results of a previously conducted (small scale) study using the DAP-based IVET strategy 6, only 4 of the 25 EIL recovered in that study were identified here. These included the cellulose biosynthetic locus wss (recovered on six independent occasions in this study), fliF, glcA and fadE (Supplementary Table 1 in Additional data file 1). The reasons for the relatively low overlap between the two studies is unclear, but perhaps reflects subtle differences in conditions for plant propagation, although the differences are more likely to reflect the particularly stringent criteria applied in this study in order for a putative plant-induced locus to qualify as an EIL. Nonetheless, of importance is the fact that genes of the same functional classes were obtained in both screens.

No validated 'dapB IVET fusions were obtained for genes within the Rsp type III secretion system, which was previously identified by a different (pantothenate-based) IVET selection strategy 5. Its low level of expression in the rhizosphere 11 is likely to be insufficient to restore competitive growth in the DAP-based promoter trapping strategy used in this study.

Regulators form a large class of EIL: the 17 predicted regulatory components include a sigma factor, LysR-type regulators, two component sensing systems, a di-guanylate cyclase, and a phosphodiesterase. Also included in this collection is an operon defined by EIL037 (PFLU1114-1111) whose four CDSs show remarkable complexity: a compound GGDEF/EAL/CheY protein (PFLU1114), followed by a predicted cytochrome C551 peroxidase precursor (PFLU1113), followed by two compound histidine kinases (PFLU1112 and PFLU1111). That this and other regulatory loci are actively transcribed outside of the laboratory environment supports the generally held assumption that the abundance of regulatory genes in Pseudomonas is important for life in complex environments 39.

Another notable 'dapB IVET fusion is EIL082, which falls within a previously unrecognized non-ribosomal peptide synthetase (NRPS) biosynthetic gene cluster (PFLU3215 to PFLU3228) present in the non-core region of SBW25. The non-ribosomal peptide produced by this enzyme complex is specific to SBW25, because no orthologues of these NRPS genes exist in Pf0-1 or Pf-5. Interestingly, Pf0-1 also possesses a novel NRPS gene cluster (Pfl01_2265-2267) not present in SBW25 or Pf-5. There are three NRPS biosynthetic clusters in SBW25, four in Pf0-1 and three in Pf-5, including the pyoverdine biosynthesis cluster. The similarity shared amongst these clusters is limited to conservation of the functional domains, with no full length identities. There is little conservation of the order of the functional domains. The production of pyoverdine is one of the defining characteristics for P. fluorescens and yet the genome has shown great diversity both in the pyoverdine clusters and in the other non-ribosomal peptides that are made by P. fluorescens.

Genes with no significant matches to DNA or protein sequences in public databases comprise approximately 10% of the IVET fusions. On the basis of in silico analyses, the majority of these genes are predicted to encode membrane-associated proteins, suggesting their interaction with the external environment through uptake, export, or signaling.

A large class of EIL are fusions of non-predicted genes, oriented in the opposite direction to transcription of predicted CDSs (40 in total; see class XI, Supplementary Table 1 in Additional data file 1). 'Antisense' fusions of this type have been described previously 540. It is possible that some of these fusions highlight in silico gene prediction errors; however, careful examination of these fusions renders this unlikely. Moreover, in a previous study both the sense CDS and antisense IVET fusion were found to encode proteins; furthermore, the IVET-identified 'antisense' gene was shown to be important for efficient colonization of soil 41. We refrain from further speculation as to the significance of these 'antisense' fusions, but such a substantial number suggests there is much yet to learn about the potential role of these genes in the function of bacteria in their natural environments.

Despite evidence for a highly variable accessory region towards the terminus of replication, the distribution of EIL in SBW25 appears to have little or no bias toward any particular genomic location (Figure 5a, b). The 31 genes defined by EIL055 to EIL096 are within the variable region (see below) of the SBW25 genome while the remainder are within the core region. This even distribution indicates that many of the mechanisms favoring success in natural environments are conserved, while individual strains appear to possess accessory traits that are likely to confer niche-specificity.

Whole genome alignments of SBW25, Pf0-1, and Pf-5 showed evidence for extensive within-genome recombination. In many bacteria this is driven by recombination between repeat sequences. However, in none of the P. fluorescens genome rearrangements were the recombined sequences flanked by rRNAs, tRNAs or known insertion sequence elements. To identify repetitive DNA sequences that may explain this intragenomic recombination, an exhaustive search for such sequences in SBW25, Pf0-1, and Pf-5 was performed.

Analysis of SBW25 revealed the presence of 4,357 repeat sequences representing 11.91% of the genome. These repetitive sequences ranged in size from 24 to 357 bps and comprised 1,199 intergenic repeats, 922 inverted repeats (IR1_g), and 2,236 intragenic repeats. This type of repeat expansion has been seen in other systems, where it is associated with a relaxation of selection on the genome. This can be associated with a recent change in niche, and the resulting evolutionary bottleneck 4243, or with reduced selection because of small effective population size and absence of recombination 44. However, as discussed below, this is not thought to apply here. The intragenic repeat families represent coding sequences for conserved protein domains within over-represented protein families; 1,293 represented just 4 protein domain families (as defined by Pfam; see Materials and methods) - ABC transporter, AMP-binding enzyme, response regulator receiver domain and the GGDEF domain.

The P. fluorescens intergenic repeat elements comprised 12 families on the basis of sequence conservation (Supplementary Figure 1 in Additional data file 2). An analysis of their distribution and frequency (Table 3) within and between genomes shows examples of both strain-specific and species-specific families. The repeat families R0 and R2 are represented more than 500 times in SBW25, but are either absent or rarely present in Pf0-1 or Pf-5. Conversely, repeat family R1 is abundant in Pf-5, but rarely present in Pf0-1 and absent from SBW25; repeat family R6 is present in Pf0-1 and absent from the genomes of the other two strains.

Detailed analysis of the repeat sequences revealed that five families possess a complex structure consisting of two identical inverted repeats (IRs) that flank a variable size core region (Table 3). The IRs generally show a higher average G+C content than the genome as a whole (64.7%; the genome average is 60.5%), while the G+C% content of the variable core region sequences is closer to the genome average. Structural predictions made with these repeat sequences show that they readily form hairpin secondary structures, with the IRs forming the stem and the variable core region forming the loop.

Three repeat families, R0, R2 and IR1_g, are of particular interest given their disproportionately high numbers in SBW25 relative to Pf0-1 and Pf-5 (Table 3). The IRs of R0 and R2 are identical to those found flanking two different insertion sequence elements unique to strain SBW25 at locations 50373465038275 (PFLU4572A) and 63871926388340 (PFLU5832), respectively. It is possible that the IRs of repeat families R0 and R2 are recognized by the two insertion sequence element-encoded transposases in trans, which might explain why the elements have become over-represented in the SBW25 genome. If this is true, then these repeats are likely to represent miniature inverted-repeat transposable elements (MITEs), only very few of which have been reported in bacteria 45.

In addition to the ability to form stem-loop structures, the IR1_g repeats also possess the consensus sequence for the repetitive extragenic palindromic repeats (REP) family, which were originally thought to be specific to P. putida KT2440. The functional significance of the Pseudomonas REPs awaits elucidation, but they may play a role in transcription termination or provide binding sites for the DNA gyrase 46.

Since many of these repeat families can form stem-loop structures, they have the potential to act as transcriptional terminators. We therefore examined the transcription orientation of the genes flanking repeat elements to look for bias. In describing this analysis we use 'Head' to refer to the 5' end of a CDS and 'Tail' to refer to the 3' end. Using this nomenclature there are four transcriptional orientation states (including CDSs on both the forward and reverse DNA strands) for the CDSs that lie on either side of a repeat element: Tail-repeat-Head (forward strand) (→ →), Tail-repeat-Tail (→ ←), Tail-repeat-Head (reverse strand) (← ←) and Head-repeat-Head (← →). We compared the frequency of each of the four states with all CDS pairs that lacked an intervening repeat element. The frequency of the four orientation states among CDS pairs that flank repeat elements was significantly different from that of CDS pairs that do not (SBW25, P < 0.0005; Pf0-1, P = 0.016; Pf-5, P < 0.0005). For those CDS pairs that do not flank repeat elements the Tail-Head (forward and reverse strand) orientation is predominant; for CDS pairs flanking repeats the most frequent orientation is the Tail-repeat-Tail (Supplementary Figure 2 in Additional data file 2). The Tail-repeat-Tail bias is prevalent for the largest three of the six intergenic repeat families present in SBW25 and for five of the nine repeat families in Pf0-1 (Supplementary Figure 3 in Additional data file 2). The selective pressure for the non-random distribution of repeats may derive from the predicted stem-loop (transcription terminator-like) structure; insertion of a repeat with a stem-loop structure between Tail-Head oriented CDSs within an operon would cause termination, thus disrupting these transcriptional units. The Tail-repeat-Tail biased distribution of these repeats probably reflects a 'least worst' location as insertion is less likely to cause aberrant transcription termination since termination of convergent transcription is likely to occur anyway. In addition, the Head-repeat-Head state, which could potentially disrupt promoters for one or both genes, occurs at a low frequency, particularly in SBW25 and Pf-5. These data would also suggest that the expansion of the intergenic repeats has been subject to selection. Consequently, it is unlikely that the repeat expansion seen in P. fluorescens results from the organism having been through an evolutionary bottleneck (this scenario is generally associated with random distribution of repetitive sequences) 43 and more likely that it is linked to a lack of selection against increased genome size.

Evident from the genome analysis are large regions of the SBW25 genome that lack any complex repeat families (R-family repeats; Table 3). We refer to these as repeat deserts (ReDs; Figure 5a). The SBW25 genome harbors 60 ReDs, which range in size from an arbitrary lower limit of 15.8 kb up to 176 kb and encode a total of 2,475 CDSs (40% of the coding capacity), of which 93.7% are unique to SBW25 compared to Pf0-1 and Pf-5 (Supplementary Table 2 in Additional data file 3). Because of the density of repeats in SBW25, the identification of ReDs was straightforward. In contrast, the lower number of repeats in Pf0-1 and Pf-5 makes definition of similar regions more difficult.

Two, not mutually exclusive, explanations for the lack of repeats in these regions exist: first, the ReDs comprise mostly essential genes that normally experience high purifying selection 4748; and second, the ReDs might have been recently acquired from a donor lacking repeat sequences. Indeed, examples of the former include the rRNA clusters, the ribosomal proteins cluster, the wss cluster (PFLU0300 to PFLU0309), which directs production of an acetylated cellulose-like polymer involved in formation of a microbial mat 4950, and cell division proteins (PFLU0940 to PFLU0953, amongst others).

Recently acquired ReDs that have different dinucleotide frequencies to the above group contain CDS clusters that might confer niche specificity. One such example is the anthranilate synthase cluster (PFLU1381 to PFLU1386), which is unique to SBW25. Other examples found within ReDs include 'atypical' regions of the SBW25 genome, which show limited phylogenetic distribution, aberrant G+C% content or dinucleotide frequency compared with the genome average for Pseudomonas species (Supplementary Table 3 in Additional data file 3). These may reflect sequences acquired through recent gene transfer events 51. While ReDs are not evident in Pf0-1, several such atypical regions have been identified (Supplementary Table 4 in Additional data file 3), and these are also free of repeats, as are all but one of the mobile genetic elements recently described in Pf-5 52. For example, SBW25 and Pf0-1 each carry multiple prophage-like elements, and both genomes have one probable integrative conjugative element (ICE)-like genomic island, SBW_GI-1 and the related island Pf0-1_GI-1, which have similarity to the genomic island PFGI-2 in Pf-5 52. SBW_GI-1 is located between partially duplicated tRNA_val and is over 101 kb in length. Strengthening the possibility that this region is a hotspot for insertions, comparison of approximately 5 kb of unpublished sequences flanking the mupirocin biosynthetic cluster of P. fluorescens NCIMB10586 53, which based on DNA sequence identity (generally 93% to 96%) and synteny is more closely related to SBW25 than Pf0-1 or Pf-5, indicates that the mup cluster is inserted adjacent to the same tRNA_val tRNA_asp tandem cluster as SBW_GI-1. Pf0-1_GI-1 defines a slightly smaller locus than SBW_GI-1 and lacks flanking insertion site duplications. These islands are related in structure to a family of ICEs, which include those found in other pseudomonads 5455 as well as wider members of the gamma-proteobacteria such as Yersinia (YAPI 5657) and Salmonella (SPI-7 58). These elements are defined as having a conserved core carrying a type IV pilus operon and plasmid-related functions as well as a highly variable region, which carries genes involved in resistance and host adaptation. The reduction of the type IV pilus genes, and breakdown of the flanking regions in Pf0-1_GI-1, suggest these ICEs may be undergoing fixation in the genome, perhaps attributable to an important function of the cargo genes. The variable cargo regions of SBW_GI-1 and Pf0-1_GI-1 are summarized in Supplementary Tables 3 and 4 in Additional data file 3.

P. fluorescens strain SBW25 is an environmental isolate taken from the leaf surfaces of a sugar beet plant. A single colony of SBW25 was grown on LB agar and then grown overnight in LB broth with shaking at 28°C. Cells were collected and total DNA was extracted with a Gentra Puregene extraction kit (Qiagen, West Sussex, UK) according to the manufacturer's instructions. The DNA was fragmented by sonication, and several libraries were generated in plasmid vectors using size fractions ranging from 2 to 9 kb. The whole genome was sequenced to a depth of 9× coverage from 2 to 3 kb, 3 to 4 kb and 6 to 9 kb in pOTW12 and pMAQ1Sac_BstXI libraries using dye terminator chemistry on ABI3730 automated sequencers. End sequences from larger insert bacterial artificial chromosome (pBACehr 5 to 15 kb insert size) libraries were used as a scaffold. The sequence was assembled, finished and annotated as described previously 62, using the program Artemis 63 to collate data and facilitate annotation.

P. fluorescens strain Pf0-1 was isolated from bulk loam soil. It was grown overnight in LB broth with shaking at 30°C. Total DNA was extracted using a Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA). The genome of Pf0-1 was sequenced at the Joint Genome Institute using a combination of 3.7, 9.4, and 37 kb DNA libraries. Draft assemblies were based on 114,960 total sequence reads. All three libraries provided 5× coverage of the genome. A total of 470 additional reactions, 3 shatter libraries from PCR products, and 20 transposon bombs (in vitro transposon mutagenesis (EZ::TN<kan2>Insertion Kit; Epicentre, Madison, WI, USA) of plasmids to generate new primer sites for DNA sequencing) were necessary to close gaps and to raise the quality of the finished sequence. All general aspects of library construction, sequencing and gene prediction performed at the Joint Genome Institute were as previously described 64.

The sequences of SBW25 and Pf0-1 can be accessed using the accession numbers [EMBL:AM181176] and [GenBank:CP000094], respectively.

The genome sequences of P. fluorescens strains SBW25, Pf0-1 and Pf-5 were compared pairwise using TBLASTX analyses loaded on the Artemis Comparison Tool 65.

Orthologous CDSs in the three genomes were defined after comparing all-against-all running a reciprocal FASTA search of translated DNA with a 30% identity over 80% of the length of the CDSs as minimum similarity score. The results were used to calculate the average amino acid identities.

Pseudogenes were defined as CDSs that had one or more mutations that would ablate expression and/or lack start and/or stop codon; each of these possible inactivating mutations was subsequently checked against the original sequencing data.

Circular diagrams were plotted using DNAplotter 66.

In order to analyze the repeat elements and their distribution in the genome of SBW25, we firstly concatenated three P. fluorescens genomic sequences (SBW25, Pf0-1 and Pf-5). Running the Repeatscout 70 algorithm on the concatenated sequence yielded 122 repeat families, of which 103 include intragenic repeats, mostly Pfam domains, and 19 intergenic repeat families. For each of the 122 families we built a multiple sequence alignment using CLUSTAL 71 and manually curated the alignments using JalView 72. Using each of the multiple alignments obtained, we built a profile hidden Markov model (HMM) using the HMMER package version 1.8.4. The 122 HMMs were searched against the concatenated sequence (leading and lagging strand). HMMs can be trained on a dataset of sequences and can predict, in a probabilistic framework, more distant members of this sequence family. The results obtained were manually curated to infer the number of distinct repeat families. The consensus of the intergenic repeat families and their HMM logos are provided in Supplementary Figure 1 in Additional data file 2. The HMM logos where produced using the LogoMat-M application 73.

Intergenic repeat families were initially predicted using the default parameters of RepeatScout: minimum number of copies per repeat family, 20; minimum repeat length, 50 bp; low complexity repeats were filtered out prior to the repeat prediction. In a second step, the predicted repeats were manually curated and very similar repeat families were merged under the same family, where possible. A multiple sequence alignment for each repeat family was used to train HMMs specific for each family. Each query genome was searched against those HMMs, using the HMMER package. Once the repeat families were built, using the HMM-based approach, the structure of each family was determined with visual inspection of the multiple sequence alignment; in case of complex repeat structure, with IRs being part of a repeat family, new HMMs were built to model the IRs of each family (if applicable) and used to search the three query genomes.

Identification of EIL from SBW25 was based on the IVET strategy as described previously 56. Libraries were constructed in pIVETD by cloning partial Sau3AI digested genomic DNA. Libraries were maintained in Escherichia coli and moved into P. fluorescens SBW25ΔdapB by conjugation. Library screening took place on non-sterile sugar beet seedlings maintained in non-sterile vermiculite pots 5. Fusions were recovered after 3 weeks of selection (rather than the 2 weeks used previously 6) by plating homogenized plant material on selective plates. Integrated genomic fusions from strains recovered from the plant environment were mobilized into E. coli by conjugative cloning 75. The identity of recovered fusions was determined by sequencing inserts from recovered plasmids (see 56 for details).