Mobile genetic elements in the genome of the beneficial rhizobacterium Pseudomonas fluorescens Pf-5

Abstract

Background: Pseudomonas fluorescens Pf-5 is a plant-associated bacterium that inhabits the rhizosphere of a wide variety of plant species and and produces secondary metabolites suppressive of fungal and oomycete plant pathogens. The Pf-5 genome is rich in features consistent with its commensal lifestyle, and its sequence has revealed attributes associated with the strain's ability to compete and survive in the dynamic and microbiologically complex rhizosphere habitat. In this study, we analyzed mobile genetic elements of the Pf-5 genome in an effort to identify determinants that might contribute to Pf-5's ability to adapt to changing environmental conditions and/or colonize new ecological niches.

Results: Sequence analyses revealed that the genome of Pf-5 is devoid of transposons and IS elements and that mobile genetic elements (MGEs) are represented by prophages and genomic islands that collectively span over 260 kb. The prophages include an F-pyocin-like prophage 01, a chimeric prophage 03, a lambdoid prophage 06, and decaying prophages 02, 04 and 05 with reduced size and/or complexity. The genomic islands are represented by a 115-kb integrative conjugative element (ICE) PFGI-1, which shares plasmid replication, recombination, and conjugative transfer genes with those from ICEs found in other Pseudomonas spp., and PFGI-2, which resembles a portion of pathogenicity islands in the genomes of the plant pathogens Pseudomonas syringae and P. viridiflava. Almost all of the MGEs in the Pf-5 genome are associated with phage-like integrase genes and are integrated into tRNA genes.

Conclusion: Comparative analyses reveal that MGEs found in Pf-5 are subject to extensive recombination and have evolved in part via exchange of genetic material with other Pseudomonas spp. having commensal or pathogenic relationships with plants and animals. Although prophages and genomic islands from Pf-5 exhibit similarity to MGEs found in other Pseudomonas spp., they also carry a number of putative niche-specific genes that could affect the survival of P. fluorescens Pf-5 in natural habitats. Most notable are an approximately 35-kb segment of "cargo" genes in genomic island PFGI-1 and bacteriocin genes associated with prophages 1 and 4.

Figure 1

Organization of prophage 01 from P. fluorescens Pf-5 [49], related prophages in the mutS-recA region of the genomes of other P. fluorescens strains, and bacteriophages CTX [81]and SfV [16]. Predicted open reading frames and their orientation are shown by arrows shaded according to their functional category. Homologous ORFs are connected with lines.

Figure 2

Dot plot comparison of P. fluorescens Pf-5 prophages with similar prophage regions in the genomes of P. fluorescens Q8r1-96 [GenBank EU982300], P. fluorescens Pf0-1 [GenBank CP000094], P. syringae pv. tomato DC3000 [24], P. syringae pv. syringae B728a [36], P. syringae pv. phaseolicola 1448a [37], P. putida KT2440 [25], P. aeruginosa PA01 [82], P. aeruginosa UCBPP-PA14 [35], and P. aeruginosa PA7 [GenBank CP000744]. All prophage sequences were extracted from genomes, concatenated and aligned using a dot plot function from OMIGA 2.0 with a sliding window of 45 and a hash value of 6. Genome regions used in the analysis encompass open reading frames with following locus tags: P. fluorescens Pf0-1 prophage1 – Pfl01_1135 through Pfl01_1173; P. syringae pv. tomato DC3000 prophage1 – PSPTO_0569 through PSPTO_0587; P. syringae pv. tomato DC3000 prophage3 – PSPTO_3385 through PSPTO_3432; P. syringae pv. syringae 728a genomic island GI11 – Psyr_2763 through Psyr_2846; P. syringae pv. syringae 728a genomic island GI12 – Psyr_4582 through Psyr_4608; P. syringae pv. phaseolicola 1448a prophage1 – PSPPH_0650 through PSPPH_0671; P. putida KT2440 P2 like pyocin – PP3031 through PP3066; P. putida KT2440 Mu SfV hybrid prophage – PP3849 through PP3920; P. entomophila L48 prophage1 – PSEEN4129 through PSEEN4186; P. aeruginosa PAO1 prophage1 – PA0610 through PA0648; P. aeruginosa PA14 prophage1 – PA14_07950 through PA14_08330; P. aeruginosa PA7 prophage1 – PSPA7_0754 through PSPA7_0789; P. aeruginosa PA7 prophage2 – PSPA7_2366 through PSPA7_2431.

Figure 3

Lytic activity associated with the prophage 01 of P. fluorescens Pf-5. Putative holin (PFL_1211) (A) and endolysin (PFL_1227) (B) genes encoded by prophage 01 from P. fluorescens Pf-5 were cloned in the plasmid vector pCR-Blunt (Invitrogen) under the control of the IPTG-inducible T7 promoter. Broth cultures of E. coli Rosetta/pLysS bearing the cloned holin and endolysin genes were induced with 3 mM IPTG and incubated with shaking for 5 hours while monitoring cell growth by measuring OD₆₀₀. The arrow indicates the time of addition of chloroform to the endolysin-expressing culture. Two independent repetitions of the assay were carried out for each gene and yielded identical results (data not shown).

Figure 4

Southern hybridization of DNA from 34 strains of P. fluorescens with probes targeting F-pyocin- and R-pyocin-like bacteriophage tail assembly genes. Total genomic DNA from each strain was digested with EcoRI and PstI restriction endonucleases, separated by electrophoresis in a 0.8% agarose gel, and transferred onto a BrightStar-Plus nylon membrane. The blots were hybridized with biotin-labeled probes prepared from P. fluorescens strains Q8r1-96 (A) and SBW25 (B) targeting the SfV-like (A) and CTX-like (B) bacteriophage tail assembly genes, respectively. Strains screened in the experiment are: P. fluorescens CHA0 [83], 1; P. fluorescens Pf-5 [5], 2; P. fluorescens Q2-87 [84], 3; P. fluorescens Q2-1 [84], 4; P. fluorescens STAD384 [85], 5; P. fluorescens Q8r1-96 [74], 6; P. fluorescens MVW1-1 [86], 7; P. fluorescens FTAD1R34 [85], 8; P. fluorescens ATCC49054 [87], 9; P. fluorescens Q128-87 [85], 10; P. fluorescens OC4-1 [85], 11; P. fluorescens FFL1R9 [85], 12; P. fluorescens Q2-5 [84], 13; P. fluorescens QT1-5 [84], 14; P. fluorescens W2-6 [84], 15; P. fluorescens Q2-2 [84], 16; P. fluorescens Q37-87 [84], 17; P. fluorescens QT1-6 [84], 18; P. fluorescens JMP6 [84], 19; P. fluorescens JMP7 [84], 20; P. fluorescens FFL1R18 [84], 21; P. fluorescens CV1-1 [84], 22; P. fluorescens FTAD1R36 [84], 23; P. fluorescens FFL1R22 [84], 24; P. fluorescens F113 [88], 25; P. fluorescens W4-4 [84], 26; P. fluorescens D27B1 [84], 27; P. fluorescens HT5-1 [84], 28; P. fluorescens 7MA12 [86], 29; P. fluorescens MVP1-4 [86], 30; P. fluorescens MVW1-1 [86], 31; P. fluorescens MVW4-2 [86], 32; P. fluorescens ATCC17400 [89], 33; P. fluorescens SBW25 [90], 34.

Figure 5

Comparison of genetic organization of prophages 03 (A) and 06 (B) to that of R2/F2 pyocin locus from P. aeruginosa PA01 [19]and B. thailandensis phage φE125, respectively. Predicted open reading frames and their orientation are shown by arrows along with a 300-bp sliding window plot of G+C content for the prophage 03 with dotted line tracing the average G+C content (63%) of Pf-5 genome. Predicted ORFs are shaded according to their functional category. Homologous ORFs are connected with lines.

Figure 6

Organization of genomic island PFGI-1. Predicted open reading frames are shaded according to their category and their orientation is shown by arrows. DNA regions unique to P. fluorescens Pf-5 and not found in closely related GIs from other Pseudomonas spp. are indicated by grey shading.

Figure 7

Dot plot comparison of genomic island PFGI-1 with related genomic islands from other Pseudomonas spp. Sequences of GI from P. fluorescens Pf0-1 [GenBank acc. CP000094; locus tags Pfl_O1_2993 through Pfl_O1_R50], PPHGI-1 from P. syringae pv. phaseolicola 1302A [33], GI-6 from P. syringae pv. syringae B728a [36], pKCL102 from P. aeruginosa C [30], PAPI-1 from P. aeruginosa UCBPP-PA14 [32], GI from P. aeruginosa PA7 [GenBank acc. CP000744; locus tags PSPA7_4437 through PSPA7_4531], ExoU-A island from P. aeruginosa 6077 [31], PAGI-2 and PAGI-4 from P. aeruginosa C [29], PAGI-3 from P. aeruginosa SGM17M [29], PAGI-5 from P. aeruginosa PSE9 [GenBank acc. EF611301], and clc element from Pseudomonas sp. B13 [34] were concatenated and aligned with PFGI-1 using a dot plot function from OMIGA 2.0 with sliding window of 45 and hash value of 6. Lower panel shows a 500-bp sliding window plot of G+C content for PFGI-1 with dotted line tracing the average G+C content (63%) of Pf-5 genome.