Simultaneous Monitoring of Cell Number and Metabolic Activity of Specific Bacterial Populations with a Dual gfp-luxAB Marker System

A dual marker system was developed for simultaneous quantification of bacterial cell numbers and their activity with the luxAB and gfp genes, encoding bacterial luciferase and green fluorescent protein (GFP), respectively. The bioluminescence phenotype of the luxAB biomarker is dependent on cellular energy status. Since cellular metabolism requires energy, bioluminescence output is directly related to the metabolic activity of the cells. By contrast, GFP fluorescence has no energy requirement. Therefore, by combining these two biomarkers, total cell number and metabolic activity of a specific marked cell population could be monitored simultaneously. Two different bacterial strains, Escherichia coli DH5α and Pseudomonas fluorescens SBW25, were chromosomally tagged with the dual marker cassette, and the cells were monitored under different conditions by flow cytometry, plate counting, and luminometry. During log-phase growth, the luciferase activity was proportional to the number of GFP-fluorescent cells and culturable cells. Upon entrance into stationary phase or during starvation, luciferase activity decreased due to a decrease in cellular metabolic activity of the population, but the number of GFP-fluorescing cells and culturable cells remained relatively stable. In addition, we optimized a procedure for extraction of bacterial cells from soil, allowing GFP-tagged bacteria in soil samples to be quantitated by flow cytometry. After 30 days of incubation of P. fluorescens SBW25::gfp/lux in soil, the cells were still maintained at high population densities, as determined by GFP fluorescence, but there was a slow decline in luciferase activity, implicating nutrient limitation. In conclusion, the dual marker system allowed simultaneous monitoring of the metabolic activity and cell number of a specific bacterial population and is a promising tool for monitoring of specific bacteria in situ in environmental samples.     

Use of a novel nonantibiotic triple marker gene cassette to monitor high survival of Pseudomonas fluorescens SBW25 on winter wheat in the field

Pseudomonas fluorescens SBW25 was tagged with a triple marker gene cassette containing gfp, encoding green fluorescent protein; luxAB, encoding luciferase; and telABkilA, encoding tellurite resistance, and the tagged strain was monitored in the first Swedish field release of a genetically modified microorganism (GMM). The cells were inoculated onto winter wheat seeds and the GMM cells (SBW25:tgl) were monitored in the field from September 2005 to May 2006 using plating, luminometry and microscopic analyses. Cell numbers were high on all sampling occasions and metabolically active cells were detected on all plant parts. Field results were similar to those obtained in a parallel phytotron study, although the amount of SBW25:tgl detected on shoots was significantly higher in the phytotron than in the field. After winter, cell counts were 100-fold higher on the roots and root-associated soil compared with prewinter measurements, although the cells had a lower relative metabolic activity. The wheat seeds were naturally infested with Microdochium nivale, but no treatment resulted in reduction of disease symptoms. No SWB25:tgl cells were ever found in bulk soil or uninoculated plants. The Swedish field trial results complement and contrast with prior field studies performed with the same parent organism in the United Kingdom under different soil, plant and climatic conditions.     

Metabolic activity of Flavobacterium strain P25 during starvation and after introduction into bulk soil and the rhizosphere of wheat

Abstract: The physiological state of introduced Flavobacterium strain P25 cells was determined in starvation cultures, in bulk soil, and in the rhizosphere of wheat using direct viable counts (DVC; based on cell elongation after use of nalidixic acid and substrate addition, resulting in a potential activity measurement) and the redox dye 5-cyano-2, 3-ditolyl tetrazolium chloride (CTC; based on respiration without substrate additions, resulting in an in situ activity measurement). Both methods clearly demonstrated that the metabolic activity of Flavobacterium P25 cells decreased during starvation, followed by increased activity after amendment with substrate. This confirmed the applicability of DVC and CTC methods to Flavobacterium P25. Both DVC and CTC methods showed that the percentage of active cells in an introduced Flavobacterium P25 population in rhizosphere soil was lower than that in bulk soil in the first 1–2 weeks after planting wheat seedlings. After two weeks, the percentage of metabolically active cells in the P25 population in rhizosphere soil was higher than in bulk soil. Since different aspects of cellular physiology are measured when applying DVC and CTC, the impact of variations in environmental factors on the metabolic state of introduced strains may be monitored closely by these methods.     

Impact of temperature on the physiological status of a potential bioremediation inoculant, Arthrobacter chlorophenolicus A6

Arthrobacter chlorophenolicus A6 (A6) can degrade large amounts of 4-chlorophenol in soil at 5 and 28 degrees C. In this study, we investigated the effects of temperature on the physiological status of this bacterium in pure culture and in soil. A derivative of A6 tagged with the gfp gene (encoding green fluorescent protein [GFP]) was used to specifically quantify A6 cells in soil. In addition, cyano-ditolyl-tetrazoliumchloride was used to stain GFP-fluorescent cells with an active electron transfer system ("viable cells") whereas propidium iodide (PI) was used to stain cells with damaged membranes ("dead cells"). Another derivative of the strain (tagged with the firefly luciferase gene [luc]) was used to monitor the metabolic activity of the cell population, since the bioluminescence phenotype is dependent on cellular energy reserves. When the cells were incubated in soil at 28 degrees C, the majority were stained with PI, indicating that they had lost their cell integrity. In addition, there was a corresponding decline in metabolic activity and in the ability to be grown in cultures on agar plates after incubation in soil at 28 degrees C, indicating that the cells were dying under those conditions. When the cells were incubated in soil at 5 degrees C, by contrast, the majority of the cells remained intact and a large fraction of the population remained metabolically active. A similar trend towards better cell survival at lower temperatures was found in pure-culture experiments. These results make A. chlorophenolicus A6 a good candidate for the treatment of chlorophenol-contaminated soil in cold climates.     

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.     

Impact of wild-type and genetically modified Pseudomonas fluorescens on soil enzyme activities and microbial population structure in the rhizosphere of pea

The aim of this study was to determine the impact of wild-type along with functionally and nonfunctionally modified Pseudomonas fluorescens strains in the rhizosphere. The wild-type F113 strain carried a gene encoding the production of the antibiotic 2,4-diacetylphloroglucinol (DAPG) useful in plant disease control, and was marked with a lacZY gene cassette. The first modified strain was a functional modification of strain F113 with repressed production of DAPG, creating the DAPG-negative strain F113 G22. The second paired comparison was a nonfunctional modification of wild-type (unmarked) strain SBW25, constructed to carry marker genes only, creating strain SBW25 EeZY-6KX. Significant perturbations were found in the indigenous bacterial population structure, with the F113 (DAPG+) strain causing a shift towards slower growing colonies (K strategists) compared with the nonantibiotic-producing derivative (F113 G22) and the SBW25 strains. The DAPG+ strain also significantly reduced, in comparison with the other inocula, the total Pseudomonas populations but did not affect the total microbial populations. The survival of F113 and F113 G22 were an order of magnitude lower than the SBW 25 strains. The DAPG+ strain caused a significant decrease in the shoot-to-root ratio in comparison to the control and other inoculants, indicating plant stress. F113 increased soil alkaline phosphatase, phosphodiesterase and aryl sulphatase activities compared to the other inocula, which themselves reduced the same enzyme activities compared to the control. In contrast to this, the β-glucosidase, β-galactosidase and N -acetyl glucosaminidase activities decreased with the inoculation of the DAPG+ strain. These results indicate that soil enzymes are sensitive to the impact of inoculation with genetically modified microorganisms (GMMs).     

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.     

Impact of Temperature on the Physiological Status of a Potential Bioremediation Inoculant, Arthrobacter chlorophenolicus A6

Arthrobacter chlorophenolicus A6 (A6) can degrade large amounts of 4-chlorophenol in soil at 5 and 28°C. In this study, we investigated the effects of temperature on the physiological status of this bacterium in pure culture and in soil. A derivative of A6 tagged with the gfp gene (encoding green fluorescent protein [GFP]) was used to specifically quantify A6 cells in soil. In addition, cyano-ditolyl-tetrazoliumchloride was used to stain GFP-fluorescent cells with an active electron transfer system (“viable cells”) whereas propidium iodide (PI) was used to stain cells with damaged membranes (“dead cells”). Another derivative of the strain (tagged with the firefly luciferase gene [luc]) was used to monitor the metabolic activity of the cell population, since the bioluminescence phenotype is dependent on cellular energy reserves. When the cells were incubated in soil at 28°C, the majority were stained with PI, indicating that they had lost their cell integrity. In addition, there was a corresponding decline in metabolic activity and in the ability to be grown in cultures on agar plates after incubation in soil at 28°C, indicating that the cells were dying under those conditions. When the cells were incubated in soil at 5°C, by contrast, the majority of the cells remained intact and a large fraction of the population remained metabolically active. A similar trend towards better cell survival at lower temperatures was found in pure-culture experiments. These results make A. chlorophenolicus A6 a good candidate for the treatment of chlorophenol-contaminated soil in cold climates.     

Single-Cell Raman Spectral Profiles of <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> SBW25 Reflects <Emphasis Type="Italic">in vitro</Emphasis> and <Emphasis Type="Italic">in planta</Emphasis> Metabolic History

Single-cell Raman microspectroscopy has the potential to report on the whole-cell chemical composition of bacteria, reflecting metabolic status as well as growth history. This potential has been demonstrated through the discriminant functional analysis of Raman spectral profiles (RSP) obtained from the soil and plant-associated bacterium Pseudomonas fluorescens SBW25, grown in vitro using defined media, and in planta using 3-month-old sugar beets (Beta vulgaris var. Roberta). SBW25 in vitro RSP data showed significant variation between those cells grown on different amino acids, sugars, TCA cycle intermediates, rich King's B, and culture media derived from the sugar beet phytosphere. Raman analysis was also able to follow the transition of SBW25 starved of carbon over a period of days, and SBW25 in planta RSP data also showed variation with significant differences between bacteria recovered from soil and the rhizosphere. SBW25 whole-cell chemical composition, and therefore growth and metabolic history, could be interpreted by coanalyzing in vitro and in planta RSP data. SBW25 recovered from the phytosphere was found to be more similar to SBW25 grown in vitro on Fru or Asp, rather than on Glc or Arg, and quite dissimilar to that resulting from carbon starvation. This suggests that SBW25 growth in the phytosphere is generally neither carbon-catabolite-repressed nor carbon-limited. These findings demonstrate that the analysis of single-cell RSP can differentiate between isogenic populations of bacteria with different metabolic histories or after recovery from different parts of their natural environment. In addition, Raman analysis is also capable of providing biologically relevant biochemical inferences, which might then be tested to uncover the mechanistic basis (biochemical–metabolic–genetic) differentiating bacteria growing in complex environments and exposed to different conditions.     

The flagellin gene as a stable marker for detection of Pseudomonas fluorescens SBW25

Flagellin gene central regions from 111 isolates of Pseudomonas fluorescens SBW25 obtained from soil during a field release experiment were analysed using a combined PCR/RFLP technique to look for variation. In addition, a 858 bp flagellin gene sequence from the original strain and the last isolate obtained from the release site were compared. There was no variation in flagellin gene sequences indicating that the gene was stable over the period of the release, and that the flagellin gene is a suitable marker for use in the detection of bacteria in release experiments. A comparison of Pseudomonas fluorescens SBW25 flagellin with other sequenced flagellins revealed closest homology to the flagellin of Ps. putida PRS2000.     

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.     

Hot spots become cold spots: coevolution in variable temperature environments

Antagonistic coevolution between hosts and parasites is a key process in the genesis and maintenance of biological diversity. Whereas coevolutionary dynamics show distinct patterns under favourable environmental conditions, the effects of more realistic, variable conditions are largely unknown. We investigated the impact of a fluctuating environment on antagonistic coevolution in experimental microcosms of Pseudomonas fluorescens SBW25 and lytic phage SBWΦ2. High‐frequency temperature fluctuations caused no deviations from typical coevolutionary arms race dynamics. However, coevolution was stalled during periods of high temperature under intermediate‐ and low‐frequency fluctuations, generating temporary coevolutionary cold spots. Temperature variation affected population density, providing evidence that eco‐evolutionary feedbacks act through variable bacteria–phage encounter rates. Our study shows that environmental fluctuations can drive antagonistic species interactions into and out of coevolutionary cold and hot spots. Whether coevolution persists or stalls depends on the frequency of change and the environmental optima of both interacting players.     

Effect of heat stress on cell activity and cell morphology of the tropical rhizobium, Sinorhizobium arboris

The effect of heat stress on the growth, physiological state, cell activity and cell morphology of the tropical Sinorhizobium arboris strain HAMBI 2190 was studied. The cells were chromosomally tagged with the firefly luciferase gene, luc. Since the bioluminescence phenotype is dependent on cellular energy reserves it was used as an indicator of the metabolic status of the cell population under various heat conditions. Variations in the numbers and lengths of growth phases between individual cultures indicated that the growth pattern at 40 degrees C was disturbed compared to growth at 37 or 28 degrees C. In addition, the cell morphology was changed radically. The number of culturable cells and the luciferase activity declined when the cultures were incubated at 40 degrees C. By contrast, under all conditions studied, the cells could be stained with 5-(and 6-)sulfofluorescein diacetate, indicating esterase activity. This demonstrated that although the culturability and cellular energy reserves decreased considerably during heat stress, a majority of the of S. arboris cell population maintained basal enzyme activity.     

Physiological changes of Salmonella typhimurium cells under osmotic and starvation conditions by image analysis

The effects of starvation and salinity on the physiology of Salmonella typhimurium were investigated in a microcosm study. The physiological changes were monitored by using fluorochromes dyes such as DAPI (4',6-diamidino-2-phenylindole) for evaluation of the genomic content, CTC (5-cyano-2,3-ditolyl tetrazolium chloride) for respiratory activity and syto 9 and propidium iodide for cytoplasmic membrane damages. The metabolic activity of the cellular population was assessed with the method of Kogure (direct viable count), to enumerate the substrate-responsive cells. These different staining procedures were objectively analysed by an image analysis system. This paper describes the progressive alteration of Salmonella typhimurium physiology under salinity and starvation conditions.     

Antagonistic coevolution limits population persistence of a virus in a thermally deteriorating environment

Understanding the conditions under which rapid evolutionary adaptation can prevent population extinction in deteriorating environments (i.e. evolutionary rescue) is a crucial aim in the face of global climate change. Despite a rapidly growing body of work in this area, little attention has been paid to the importance of interspecific coevolutionary interactions. Antagonistic coevolution commonly observed between hosts and parasites is likely to retard evolutionary rescue because it often reduces population sizes, and results in the evolution of costly host defence and parasite counter-defence. We used experimental populations of a bacterium Pseudomonas fluorescens SBW25 and a bacteriophage virus (SBW25Φ2), to study how host-parasite coevolution impacts viral population persistence in the face of gradually increasing temperature, an environmental stress for the virus but not the bacterium. The virus persisted much longer when it evolved in the presence of an evolutionarily constant host genotype (i.e. in the absence of coevolution) than when the bacterium and virus coevolved. Further experiments suggest that both a reduction in population size and costly infectivity strategies contributed to viral extinction as a result of coevolution. The results highlight the importance of interspecific evolutionary interactions for the evolutionary responses of populations to global climate change.     

Root cap influences root colonisation by Pseudomonas fluorescens SBW25 on maize

We investigated the influence of root border cells on the colonisation of seedling Zea mays roots by Pseudomonas fluorescens SBW25 in sandy loam soil packed at two dry bulk densities. Numbers of colony forming units (CFU) were counted on sequential sections of root for intact and decapped inoculated roots grown in loose (1.0 mg m(-3)) and compacted (1.3 mg m(-3)) soil. After two days of root growth, the numbers of P. fluorescens (CFU cm(-1)) were highest on the section of root just below the seed with progressively fewer bacteria near the tip, irrespective of density. The decapped roots had significantly more colonies of P. fluorescens at the tip compared with the intact roots: approximately 100-fold more in the loose and 30-fold more in the compact soil. In addition, confocal images of the root tips grown in agar showed that P. fluorescens could only be detected on the tips of the decapped roots. These results indicated that border cells, and their associated mucilage, prevented complete colonization of the root tip by the biocontrol agent P. fluorescens, possibly by acting as a disposable surface or sheath around the cap.     

Construction and validation of a neutrally-marked strain of Pseudomonas fluorescens SBW25

Neutrally marked bacterial strains are useful in many experimental evolution and molecular ecology studies to assess the relative fitness of a given strain. Here we describe the construction and validation of a neutral marker for the model organism Pseudomonas fluorescens SBW25. The marked strain, called SBW25-lacZ, was created by integrating a promoterless 'lacZ into the defective prophage locus of the SBW25 chromosome. Fitness assays conducted in various laboratory media and in planta revealed that the fitness levels of SBW25-lacZ were comparable with the wild-type ancestor.     

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.     

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.     

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.