Genes encoding a cellulosic polymer contribute toward the ecological success of Pseudomonas fluorescens SBW25 on plant surfaces

Pseudomonas fluorescens SBW25 is a Gram-negative bacterium that grows in close association with plants. In common with a broad range of functionally similar bacteria it plays an important role in the turnover of organic matter and certain isolates can promote plant growth. Despite its environmental significance, the causes of its ecological success are poorly understood. Here we describe the development and application of a simple promoter trapping strategy (IVET) to identify P. fluorescens SBW25 genes showing elevated levels of expression in the sugar beet rhizosphere. A total of 25 rhizosphere-induced (rhi) fusions are reported with predicted roles in nutrient acquisition, stress responses, biosynthesis of phytohormones and antibiotics. One rhi fusion is to wss, an operon encoding an acetylated cellulose polymer. A mutant carrying a defective wss locus was competitively compromised (relative to the wild type) in the rhizosphere and in the phyllosphere, but not in bulk soil. The rhizosphere-induced wss locus therefore contributes to the ecological performance of SBW25 in the plant environment and supports our conjecture that genes inactive in the laboratory environment, but active in the wild, are likely to be determinants of fitness in natural environments.     

Type III secretion in plant growth-promoting Pseudomonas fluorescens SBW25

In vivo expression technology (IVET) analysis of rhizosphere-induced genes in the plant growth-promoting rhizobacterium (PGPR) Pseudomonas fluorescens SBW25 identified a homologue of the type III secretion system (TTSS) gene hrcC. The hrcC homologue resides within a 20-kb gene cluster that resembles the type III (Hrp) gene cluster of Pseudomonas syringae. The type III (Rsp) gene cluster in P. fluorescens SBW25 is flanked by a homologue of the P. syringae TTSS-secreted protein AvrE. P. fluorescens SBW25 is non-pathogenic and does not elicit the hypersensitive response (HR) in any host plant tested. However, strains constitutively expressing the rsp-specific sigma factor RspL elicit an AvrB-dependent HR in Arabidopsis thaliana ecotype Col-0, and a host-specific HR in Nicotiana clevelandii. The inability of wild-type P. fluorescens SBW25 to elicit a visible HR is therefore partly attributable to low expression of rsp genes in the leaf apoplast. DNA hybridization analysis indicates that rsp genes are present in many plant-colonizing Pseudomonas and PGPR, suggesting that TTSSs may have a significant role in the biology of PGPR. However, rsp and rsc mutants retain the ability to reach high population levels in the rhizosphere. While functionality of the TTSS has been demonstrated, the ecological significance of the rhizosphere-expressed TTSS of P. fluorescens SBW25 remains unclear.     

Genome-based discovery, structure prediction and functional analysis of cyclic lipopeptide antibiotics in Pseudomonas species

Analysis of microbial genome sequences have revealed numerous genes involved in antibiotic biosynthesis. In Pseudomonads, several gene clusters encoding non-ribosomal peptide synthetases (NRPSs) were predicted to be involved in the synthesis of cyclic lipopeptide (CLP) antibiotics. Most of these predictions, however, are untested and the association between genome sequence and biological function of the predicted metabolite is lacking. Here we report the genome-based identification of previously unknown CLP gene clusters in plant pathogenic Pseudomonas syringae strains B728a and DC3000 and in plant beneficial Pseudomonas fluorescens Pf0-1 and SBW25. For P. fluorescens SBW25, a model strain in studying bacterial evolution and adaptation, the structure of the CLP with a predicted 9-amino acid peptide moiety was confirmed by chemical analyses. Mutagenesis confirmed that the three identified NRPS genes are essential for CLP synthesis in strain SBW25. CLP production was shown to play a key role in motility, biofilm formation and in activity of SBW25 against zoospores of Phytophthora infestans. This is the first time that an antimicrobial metabolite is identified from strain SBW25. The results indicate that genome mining may enable the discovery of unknown gene clusters and traits that are highly relevant in the lifestyle of plant beneficial and plant pathogenic bacteria.     

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

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.     

Biocontrol of Pythium in the pea rhizosphere by antifungal metabolite producing and non-producing Pseudomonas strains

AIMS: Four well-described strains of Pseudomonas fluorescens were assessed for their effect on pea growth and their antagonistic activity against large Pythium ultimum inocula. Methods and RESULTS: The effect of Pseudomonas strains on the indigenous soil microflora, soil enzyme activities and plant growth in the presence and absence of Pythium was assessed. Pythium inoculation reduced the shoot and root weights, root length, and the number of lateral roots. The effect of Pythium was reduced by the Pseudomonas strains. Strains F113, SBW25 and CHAO increased shoot weights (by 20%, 22% and 35%, respectively); strains Q2-87, SBW25 and CHAO increased root weights (14%, 14% and 52%). Strains SBW25 and CHAO increased root lengths (19% and 69%) and increased the number of lateral roots (14% and 29%). All the Pseudomonas strains reduced the number of lesions and the root and soil Pythium populations, while SBW25 and CHAO increased the number of lateral roots. Pythium inoculation increased root and soil microbial populations but the magnitude of this effect was Pseudomonas strain-specific. Pythium increased the activity of C, N and P cycle enzymes, while the Pseudomonas strains reduced this effect, indicating reduced plant damage. CONCLUSION: Strains SBW25 and CHAO had the greatest beneficial characteristics, as these strains produced the greatest reductions in the side effects of Pythium infection (microbial populations and enzyme activities) and resulted in significantly improved plant growth. Strain SBW25 does not produce antifungal metabolites, and its biocontrol activity was related to a greater colonization ability in the rhizosphere. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first critical comparison of such important strains of Ps. fluorescens showing disease biocontrol potential.     

A conserved mechanism for nitrile metabolism in bacteria and plants

Pseudomonas fluorescens SBW25 is a plant growth-promoting bacterium that efficiently colonises the leaf surfaces and rhizosphere of a range of plants. Previous studies have identified a putative plant-induced nitrilase gene ( pinA ) in P. fluorescens SBW25 that is expressed in the rhizosphere of sugar beet plants. Nitrilase enzymes have been characterised in plants, bacteria and fungi and are thought to be important in detoxification of nitriles, utilisation of nitrogen and synthesis of plant hormones. We reveal that pinA is a NIT4-type nitrilase that catalyses the hydrolysis of β-cyano- l -alanine, a nitrile common in the plant environment and an intermediate in the cyanide detoxification pathway in plants. In plants cyanide is converted to β-cyano- l -alanine, which is subsequently detoxified to aspartic acid and ammonia by NIT4. In P. fluorescens SBW25 pinA is induced in the presence of β-cyano- l -alanine, and the β-cyano- l -alanine precursors cyanide and cysteine. pinA allows P. fluorescens SBW25 to use β-cyano- l -alanine as a nitrogen source and to tolerate toxic concentrations of this nitrile. In addition, pinA is shown to complement a NIT4 mutation in Arabidopsis thaliana , enabling plants to grow in concentrations of β-cyano- l -alanine that would otherwise prove lethal. Interestingly, over-expression of pinA in wild-type A. thaliana not only resulted in increased growth in high concentrations of β-cyano- l -alanine, but also resulted in increased root elongation in the absence of exogenous β-cyano- l -alanine, demonstrating that β-cyano- l -alanine nitrilase activity can have a significant effect on root physiology and root development.     

Adaptation of Pseudomonas fluorescens to the plant rhizosphere

Saprophytic Pseudomonas are common root-colonizing bacteria that can improve plant health. Efficient exploitation of these bacteria in agriculture requires knowledge of traits that enhance ecological performance in the rhizosphere. Here, I describe the development and application of a promoter-trapping technology (IVET) that enables the isolation of Pseudomonas fluorescens genes that show elevated levels of expression in the rhizosphere. Using IVET, 20 P. fluorescens genes were identified that are induced during rhizosphere colonization, and their patterns of expression were analysed in laboratory media and in the rhizosphere. Fourteen genes showed significant homology to sequences in GenBank that are involved in nutrient acquisition, stress response, or secretion; six showed no homology. Seven of the rhizosphere-induced ( rhi ) genes have homology to known non- Pseudomonas genes. One of the rhi genes ( hrcC ) is a component of a type III secretion pathway, not previously known in non-parasitic bacteria. Together, these genes provide a view of the rhizosphere environment as perceived by a rhizosphere colonist, and suggest that the nature of the association between P. fluorescens and the plant root may be more complex and intimate than previously thought.     

Mechanism of insoluble phosphate solubilization by Pseudomonas fluorescens RAF15 isolated from ginseng rhizosphere and its plant growth-promoting activities

Aims:  To investigate the mechanism of insoluble phosphate (P) solubilization and plant growth-promoting activity by Pseudomonas fluorescens RAF15.
Methods and Results:  We investigated the ability of Ps. fluorescens RAF15 to solubilize insoluble P via two possible mechanisms: proton excretion by ammonium assimilation and organic acid production. There were no clear differences in pH and P solubilization between glucose-ammonium and glucose-nitrate media. P solubilization was significantly promoted with glucose compared to fructose. Regardless of nitrogen sources used, Ps. fluorescens RAF15 solubilized little insoluble P with fructose. High performance liquid chromatography analysis showed that Ps. fluorescens RAF15 produced mainly gluconic and tartaric acids with small amounts of 2-ketogluconic, formic and acetic acids. During the culture, the pH was reduced with increase in gluconic acid concentration and was inversely correlated with soluble P concentration. Ps. fluorescens RAF1 showed the properties related to plant growth promotion: pectinase, protease, lipase, siderophore, hydrogen cyanide, and indoleacetic acid.
Conclusion:  This study indicated that the P solubility was directly correlated with the organic acids produced.
Significance and Impact of the Study:  Pseudomonas fluorescens RAF15 possessed different traits related to plant growth promotion. Therefore, Ps. fluorescens RAF15 could be a potential candidate for the development of biofertilizer or biocontrol agent.     

Monitoring Population Size,Activity, and Distribution of gfp-luxAB-Tagged Pseudomonas fluorescens SBW25 during Colonization of Wheat

Increasingly, focus has been directed towards the use of microorganisms as biological control agents to combat fungal disease, as an alternative to chemical fungicides. Pseudomonas fluorescens SBW25 is one bacterial strain that has been demonstrated to promote plant growth by biocontrol of pathogenic fungi. To understand the mode of action of this bacterium, information regarding its localization and metabolic activity on plants is important. In this study, a gfp/luxAB-tagged derivative of P. fluorescens SBW25, expressing the green fluorescent protein (GFP) and bacterial luciferase, was monitored during colonization of wheat starting from seed inoculation. Since bacterial luciferase is dependent on cellular energy reserves for phenotypic expression, metabolically active cells were detected using this marker. In contrast, the stable GFP fluorescence phenotype was used to detect the cells independently of their metabolic status. The combination of these two markers enabled P. fluorescens SBW25 cells to be monitored on wheat plants to determine their specific location and metabolic activity. Studies on homogenized wheat plant parts demonstrated that the seed was the preferred location of P. fluorescens SBW25 during the 65-day time period studied, but the leaves and roots were also colonized. Interestingly, the bacteria were also found to be metabolically active on all plant parts examined. In situ localization of P. fluorescens SBW25 using a combination of different microscopic techniques confirmed the preference for the cells to colonize specific regions of the seed. We speculate that the colonization pattern of P. fluorescens SBW25 can be linked to the mechanism of protection of plants from fungal infection.     

No effect of host–parasite co‐evolution on host range expansion

Antagonistic co‐evolution between hosts and parasites (reciprocal selection for resistance and infectivity) is hypothesized to play an important role in host range expansion by selecting for novel infectivity alleles, but tests are lacking. Here, we determine whether experimental co‐evolution between a bacterium (Pseudomonas fluorescens SBW25) and a phage (SBW25Φ2) affects interstrain host range: the ability to infect different strains of P. fluorescens other than SBW25. We identified and tested a genetically and phenotypically diverse suite of co‐evolved phage variants of SBW25Φ2 against both sympatric and allopatric co‐evolving hosts (P. fluorescens SBW25) and a large set of other P. fluorescens strains. Although all co‐evolved phage had a greater host range than the ancestral phage and could differentially infect co‐evolved variants of P. fluorescens SBW25, none could infect any of the alternative P. fluorescens strains. Thus, parasite generalism at one genetic scale does not appear to affect generalism at other scales, suggesting fundamental genetic constraints on parasite adaptation for this virus.     

Physical and genetic map of the Pseudomonas fluorescens SBW25 chromosome

Pseudomonas fluorescens is a saprophytic bacterium commonly isolated from soil, water, and the surfaces and tissues of plants and animals. The species has important applications in biotechnology because it can enhance plant growth and protect crops against disease. A complete physical map of the 6.63 Mbp P. fluorescens SBW25 chromosome was constructed using data obtained from combinations of one- and two-dimensional electrophoresis of completely or partially digested chromosomal DNA with end labelling. In total, 139 restriction sites (15 Pac I, 53 Spe I, 71 Xba I) were placed on the physical map and complete maps of the circular chromosome were obtained for both Pac I and Spe I; only Xba I fragments linking Spe I fragments were positioned. The average resolution of restriction sites was 48 kbp. A genetic map was derived from the physical map by Southern hybridization and 31 genes were positioned including oriC , rDNA operons ( rnnA–E ), recA gacA and pyvD     

The Acquisition of Indigenous Plasmids by a Genetically Marked Pseudomonad Population Colonizing the Sugar Beet Phytosphere Is Related to Local Environmental Conditions

The transfer of naturally occurring conjugative plasmids from the indigenous microflora to a genetically modified population of bacteria colonizing the phytospheres of plants has been observed. The marked strain (Pseudomonas fluorescens SBW25EeZY6KX) was introduced as a seed dressing to sugar beets (Beta vulgaris var. Amethyst) as part of a field experiment to assess the ecology and genetic stability of deliberately released bacterial inocula. The sustained populations of the introduced strain, which colonized the phytosphere, were assessed throughout the growing season for the acquisition of plasmids conferring mercury resistance (Hg(supr)). Transconjugants were isolated only from root and leaf samples collected within a narrow temporal window coincident with the midseason maturation of the crop. Conjugal-transfer events were recorded during this defined period in two separate field release experiments conducted over consecutive years. On one occasion seven of nine individual plants sampled supported transconjugant P. fluorescens SBW25EeZY6KX, demonstrating that conjugative gene transfer between bacterial populations in the phytosphere may be a common event under specific environmental conditions. The plasmids acquired in situ by the colonizing inocula were identified as natural variants of restriction digest pattern group I, III, or IV plasmids from five genetically distinct groups of large, conjugative mercury resistance plasmids known to persist in the phytospheres of sugar beets at the field site. These data demonstrate not only that gene transfer may be a common event but also that the genetic and phenotypic stability of inocula released into the natural environment cannot be predicted.     

Cross-species GacA-controlled induction of antibiosis in pseudomonads

Signal extracts prepared from culture supernatants of Pseudomonas fluorescens CHA0 and Pseudomonas aeruginosa PAO stimulated GacA-dependent expression of small RNAs and hence of antibiotic compounds in both hosts. Pseudomonas corrugata LMG2172 and P. fluorescens SBW25 also produced signal molecules stimulating GacA-controlled antibiotic synthesis in strain CHA0, illustrating a novel, N-acyl-homoserine lactone-independent type of interspecies communication.     

Pseudomonas fluorescens and Glomus mosseae trigger DMI3-dependent activation of genes related to a signal transduction pathway in roots of Medicago truncatula

Plant genes induced during early root colonization of Medicago truncatula Gaertn. J5 by a growth-promoting strain of Pseudomonas fluorescens (C7R12) have been identified by suppressive subtractive hybridization. Ten M. truncatula genes, coding proteins associated with a putative signal transduction pathway, showed an early and transient activation during initial interactions between M. truncatula and P. fluorescens, up to 8 d after root inoculation. Gene expression was not significantly enhanced, except for one gene, in P. fluorescens-inoculated roots of a Myc(-)Nod(-) genotype (TRV25) of M. truncatula mutated for the DMI3 (syn. MtSYM13) gene. This gene codes a Ca(2+) and calmodulin-dependent protein kinase, indicating a possible role of calcium in the cellular interactions between M. truncatula and P. fluorescens. When expression of the 10 plant genes was compared in early stages of root colonization by mycorrhizal and rhizobial microsymbionts, Glomus mosseae activated all 10 genes, whereas Sinorhizobium meliloti only activated one and inhibited four others. None of the genes responded to inoculation by either microsymbiont in roots of the TRV25 mutant. The similar response of the M. truncatula genes to P. fluorescens and G. mosseae points to common molecular pathways in the perception of the microbial signals by plant roots.     

Monitoring physiological status of GFP-tagged Pseudomonas fluorescens SBW25 under different nutrient conditions and in soil by flow cytometry

Pseudomonas fluorescens SBW25, a plant growth promoting bacterium, has been widely studied due to its potential as an inoculum for improving crop yields. Environmental inoculants are usually applied on seeds or directly to soil and to effectively promote plant growth they need to be viable and active. However, it is difficult to study the physiological status of specific microorganisms in complex environments, such as soil. In this study, our aim was to use molecular tools to specifically monitor the physiological status of P. fluorescens SBW25 in soil and in pure cultures incubated under different nutritional conditions. The cells were previously tagged with marker genes (encoding green fluorescent protein and bacterial luciferase) to specifically track the cells in environmental samples. The physiological status of the cells was determined using the viability stains 5-cyano-2,3-ditolyl-tetrazolium chloride (CTC) and propidium iodide (PI), which stain active and dead cells, respectively. Luciferase activity was used to monitor the metabolic activity of the population. Most of the cells died after incubation for nine days in nutrient rich medium. By contrast when incubated under starvation conditions, most of the population was not stained with CTC or PI (i.e. intact but inactive cells), indicating that most of the cells were presumably dormant. In soil, a large fraction of the SBW25 cell population became inactive and died, as determined by a decline in luciferase activity and CTC-stained cells, an increase in PI-stained cells, and an inability of the cells to be cultured on agar medium. However, approximately 60% of the population was unstained, presumably indicating that the cells entered a state of dormancy in soil similar to that observed under starvation conditions in pure cultures. These results demonstrate the applicability of this approach for monitoring the physiological status of specific cells under stress conditions, such as those experienced by environmental inoculants in soil.