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.     

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.     

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.     

Survival and Ecological Fitness of Pseudomonas fluorescens Genetically Engineered with Dual Biocontrol Mechanisms

The antibiotic 2,4-diacetylphloroglucinol (Phl) is produced by a range of naturally occurring fluorescent pseudomonads. One isolate, Pseudomonas fluorescens F113, protects pea plants from the pathogenic fungus Pythium ultimum by reducing the number of pathogenic lesions on plant roots, but with a concurrent reduction in the emergence of plants such as pea. The genes responsible for Phl production have been shown to be functionally conserved between the wild-type (wt) P. fluorescens strains F113 and Q2-87. In this study the genes from F113 were isolated using an optimized long PCR method and a 6.7-kb gene cluster inserted into the chromosome of the non-Phl-producing P. fluorescens strain SBW25 EeZY6KX. This strain is a lacZY, km^R marked derivative of the wt SBW25 which effects biological control against the plant pathogen Pythium ultimum by competitive exclusion as a result of its strong rhizosphere-colonizing ability. We describe here the integration of the Phl antifungal and competitive exclusion mechanisms into a single strain, and the impact this has on survival and plant emergence in microcosms. The insertion of the Phl biosynthetic genes from the F113 into the SBW25 chromosome gave a Phl-producing transformant (strain Pa21) able to suppress P. ultimum through antibiotic production. The growth of Pa21 was not reduced in flask culture at 20°C compared with its parent strain. When inoculated on pea seedlings, the strain containing the Phl operon behaved similarly to the SBW25 EeZY6KX parent but did not show the tendency of the wt Phl producer F113 to cause lower pea seed emergence. Pea roots inoculated with SBW25 EeZY6KX have significantly lower indigenous populations than with F113 and the control. This is indicative of this strains strong colonising presence. Pa21, the Phl-modified strain, is able to exclude the resident population from roots to the same degree as the SBW25 EeZY6KX from which it is derived. This suggests that it has maintained its competitiveness around the root systems of plants even with the introduction of the Phl locus. Thus, strain Pa21 possesses the qualities necessary to provide effective integrated biocontrol, through maintaining both its wt trait of competitive exclusion on the plant roots, while also expressing the genes from the F113 biocontrol strain for Phl production. Interestingly, however, an additional beneficial trait appears to emerge with the strain Pa21s lowered survival competence compared with SBW25 EeZY6KX in the rhizosphere soil. With fears of the spread of genetically modified organisms and persistence in the soil, this trait may be of some ecological and commercial benefit and becomes a candidate for further investigation and possible exploitation.     

Population dynamics and gene transfer in genetically modified bacteria in a model microcosm

The horizontal transfer and effects on host fitness of a neutral gene cassette inserted into three different genomic loci of a plant-colonizing pseudomonad was assessed in a model ecosystem. The KX reporter cassette (kanamycin resistance, aph, and catechol 2, 3, dioxygenase, xylE) was introduced on the disarmed transposon mini-Tn5 into: (I) the chromosome of a spontaneous rifampicin resistant mutant Pseudomonas fluorescens SBW25R; (II) the chromosome of SBW25R in the presence of a naturally occurring lysogenic-phage (phage Phi101); and (III) a naturally occurring plasmid pQBR11 (330 kbp, tra+, Hgr) introduced into SBW25R. These bacteria were applied to Stellaria media (chickweed) plants as seed dressings [c. 5 x 104 colony-forming units (cfu)/seed] and the seedlings planted in 16 microcosm chambers containing model plant and animal communities. Gene transfer to pseudomonads in the phyllosphere and rhizosphere was found only in the plasmid treatment (III). Bacteria in the phage treatment (II) initially declined in density and free phage was detected, but populations partly recovered as the plants matured. Surprisingly, bacteria in the chromosome insertion treatment (I) consistently achieved higher population densities than the unmanipulated control and other treatments. Plasmids were acquired from indigenous bacterial populations in the control and chromosome insertion treatments. Plasmid acquisition, plasmid transfer from inocula and selection for plasmid carrying inocula coincided with plant maturation.     

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.     

Determining the Effects of a Spatially Heterogeneous Selection Pressure on Bacterial Population Structure at the Sub-millimetre Scale

A key interest of microbial ecology is to understand the role of environmental heterogeneity in shaping bacterial diversity and fitness. However, quantifying relevant selection pressures and their effects is challenging due to the number of parameters that must be considered and the multiple scales over which they act. In the current study, a model system was employed to investigate the effects of a spatially heterogeneous mercuric ion (Hg²⁺) selection pressure on a population comprising Hg-sensitive and Hg-resistant pseudomonads. The Hg-sensitive bacteria were Pseudomonas fluorescens SBW25::rfp and Hg-resistant bacteria were P. fluorescens SBW25 carrying a gfp-labelled, Hg resistance plasmid. In the absence of Hg, the plasmid confers a considerable fitness cost on the host, with µ_max for plasmid-carrying cells relative to plasmid-free cells of only 0.66. Two image analysis techniques were developed to investigate the structure that developed in biofilms about foci of Hg (cellulose fibres imbued with HgCl₂). Both techniques indicated selection for the resistant phenotype occurred only in small areas of approximately 178–353 μm (manually defined contour region analysis) or 275–350 μm (daime analysis) from foci. Hg also elicited toxic effects that reduced the growth of both Hg-sensitive and Hg-resistant bacteria up to 250 μm from foci. Selection for the Hg resistance phenotype was therefore highly localised when Hg was spatially heterogeneous. As such, for this model system, we define here the spatial scale over which selection operates. The ability to quantify changes in the strength of selection for particular phenotypes over sub-millimetre scales is useful for understanding the scale over which environmental variables affect bacterial populations.     

Forecasting of phenotypic and genetic outcomes of experimental evolution in Pseudomonas protegens

Experimental evolution with microbes is often highly repeatable under identical conditions, suggesting the possibility to predict short-term evolution. However, it is not clear to what degree evolutionary forecasts can be extended to related species in non-identical environments, which would allow testing of general predictive models and fundamental biological assumptions. To develop an extended model system for evolutionary forecasting, we used previous data and models of the genotype-to-phenotype map from the wrinkly spreader system in Pseudomonas fluorescens SBW25 to make predictions of evolutionary outcomes on different biological levels for Pseudomonas protegens Pf-5. In addition to sequence divergence (78% amino acid and 81% nucleotide identity) for the genes targeted by mutations, these species also differ in the inability of Pf-5 to make cellulose, which is the main structural basis for the adaptive phenotype in SBW25. The experimental conditions were changed compared to the SBW25 system to test if forecasts were extendable to a non-identical environment. Forty-three mutants with increased ability to colonize the air-liquid interface were isolated, and the majority had reduced motility and was partly dependent on the Pel exopolysaccharide as a structural component. Most (38/43) mutations are expected to disrupt negative regulation of the same three diguanylate cyclases as in SBW25, with a smaller number of mutations in promoter regions, including an uncharacterized polysaccharide synthase operon. A mathematical model developed for SBW25 predicted the order of the three main pathways and the genes targeted by mutations, but differences in fitness between mutants and mutational biases also appear to influence outcomes. Mutated regions in proteins could be predicted in most cases (16/22), but parallelism at the nucleotide level was low and mutational hot spot sites were not conserved. This study demonstrates the potential of short-term evolutionary forecasting in experimental populations and provides testable predictions for evolutionary outcomes in other Pseudomonas species.     

Alteration of Soil Rhizosphere Communities following Genetic Transformation of White Spruce

The application of plant genetic manipulations to agriculture and forestry with the aim of alleviating insect damage through Bacillus thuringiensis transformation could lead to a significant reduction in the release of pesticides into the environment. However, many groups have come forward with very valid and important questions related to potentially adverse effects, and it is crucial to assess and better understand the impact that this technology might have on ecosystems. In this study, we analyzed rhizosphere soil samples collected from the first B. thuringiensis-transformed trees [with insertion of the CryIA(b) toxin-encoding gene] grown in Canada (Val-Cartier, QC, Canada) as part of an ecological impact assessment project. Using a robust amplified rRNA gene restriction analysis approach coupled with 16S rRNA gene sequencing, the rhizosphere-inhabiting microbial communities of white spruce (Picea glauca) genetically modified by biolistic insertion of the cryIA(b), uidA (beta-glucuronidase), and nptII genes were compared with the microbial communities associated with non-genetically modified counterparts and with trees in which only the genetic marker genes uidA and nptII have been inserted. Analysis of 1,728 rhizosphere bacterial clones (576 clones per treatment) using a Cramér-von Mises statistic analysis combined with a Monte Carlo comparison clearly indicated that there was a statistically significant difference (P < 0.05) between the microbial communities inhabiting the rhizospheres of trees carrying the cryIA(b), uidA, and nptII transgenes, trees carrying only the uidA and nptII transgenes, and control trees. Clear rhizosphere microbial community alterations due to B. thuringiensis tree genetic modification have to our knowledge never been described previously and open the door to interesting questions related to B. thuringiensis genetic transformation and also to the impact of commonly used uidA and nptII genetic marker genes.     

Differential reactions of wheat and pea genotypes to root inoculation with growth-affecting rhizosphere bacteria

Spring wheat (Triticum aestivum L.) and pea (Pisum spp.) genotypes were tested for reaction to root inoculation with rhizosphere bacteria affecting plant growth. Plant response was studied in greenhouse experiments after treatment of seedlings with bacteria suspended in nutrient broth. Significant genotype variation was found in both wheat and pea in terms of shoot dry weight and severity of bacteria-induced leaf symptoms. For most bacterial isolates tested, there was good correlation between ratings of leaf symptoms 7 to 14 days after inoculation and growth inhibition measured after four weeks. Interactions between isolates and plant genotypes were significant in both wheat and pea (P=0.0024 and 0.0001, respectively), but genotypes with sensitivity or tolerance to most isolates could be distinguished. In an outdoor pot experiment, two of the bacterial isolates caused delayed plant development and differential decreases in grain yield of wheat genotypes. The hypothesis that the reaction of wheat genotypes to the tested becteria was related to their influence on bacterial establishment in the rhizosphere could not be substantiated.     

Evidence of MexT-independent overexpression of MexEF-OprN multidrug efflux pump of Pseudomonas aeruginosa in presence of metabolic stress

Background

The Pseudomonas aeruginosa MexEF-OprN efflux pump confers resistance to clinically significant antibiotics. Regulation of mexEF-oprN operon expression is multifaceted with the MexT activator being one of the most prominent regulatory proteins.

Methodology

We have exploited the impaired metabolic fitness of a P. aeruginosa mutant strain lacking several efflux pump of the resistance nodulation cell division superfamily and the TolC homolog OpmH, and isolated derivatives (large colony variants) that regained fitness by incubation on nutrient-rich medium in the absence of antibiotics. Although the mexEF-oprN operon is uninducible in this mutant due to a 8-bp mexT insertion present in some P. aeruginosa PAO1 strains, the large colony variants expressed high levels of MexEF-OprN. Unlike large colony variants obtained after plating on antibiotic containing medium which expressed mexEF-oprN in a MexT-dependent fashion as evidenced by clean excision of the 8-bp insertion from mexT, mexEF-oprN expression was MexT-independent in the large colony variants obtained by plating on LB alone since the mexT gene remained inactivated. A search for possible regulators of mexEF-oprN expression using transposon mutagenesis and genomic library expression approaches yielded several candidates but proved inconclusive.

Significance

Our results show that antibiotic and metabolic stress lead to up-regulation of MexEF-OprN expression via different mechanisms and that MexEF-OprN does not only extrude antimicrobials but rather serves other important metabolic functions.     

Seasonal changes and vertical distribution of root standing biomass of graminoids and shrubs at a Siberian tundra site

Background &; aims

Elevated atmospheric CO₂ (eCO₂) can affect soil-plant systems via stimulating plant growth, rhizosphere activity and the decomposition of added (crop residues) or existing (priming) soil organic carbon (C). Increases in C inputs via root exudation, rhizodeposition and root turnover are likely to alter the decomposition of crop residues but will ultimately depend on the N content of the residues and the soil.

Methods

Two soil column experiments were conducted under ambient CO₂ (aCO₂, 390 ppm) and eCO₂ (700 ppm) in a glasshouse using dual-labelled (¹³C/¹⁵N) residues of wheat (Triticum aestivum cv. Yitpi) and field pea (Pisum sativum L. cv. PBA Twilight). The effects of eCO₂ and soil N status on wheat rhizosphere activity and residue decomposition and also N recovery from crop residues with different N status (C/N ratio 19.4–115.4) by different plant treatments (wheat, wheat + 25 mg N kg^?1 and field pea).

Results

Total belowground CO₂ efflux was enhanced under eCO₂ despite no increases in root biomass. Plants decreased residue decomposition, indicating a negative rhizosphere effect. For wheat, eCO₂ reduced the negative rhizosphere effect, resulting in greater rates of decomposition and recovery of N from field pea residues, but only when N fertiliser was added. For field pea, eCO₂ enhanced the negative rhizosphere effect resulting in lower decomposition rates and N recovery from field pea residue.

Conclusions

The effect of eCO₂ on N utilisation varied with the type of residue, enhancing N utilisation of wheat but repressing that of field pea residues, which in turn could alter the amount of N supplied to subsequent crops. Furthermore, reduced decomposition of residues under eCO₂ may slow the formation of new soil C and have implications for long-term soil fertility.

     

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.     

Effect of Plant Species on the Kinetics of Conjugal Transfer in the Rhizosphere and Relation to Bacterial Metabolic Activity

Conjugal transfer of a derivative of the RP4 plasmid between Pseudomonas fluorescens AS12 and Serratia plymuthica RF7 was compared in the rhizosphere of pea, wheat, and barley and related to the metabolic activity of the bacteria. To obtain a reliable measure of transfer, which allowed comparison of results between experiments, mathematical mass-action models were used to determine plasmid intrinsic kinetic coefficients. The data showed that not only were the rhizospheres highly conducive of transfer, with rates up to six orders of magnitude higher than in bulk soil, but differences between rhizospheres were also observed. Highest intrinsic kinetic coefficients were found in the pea rhizosphere (1.1-4.1 x 10-11), followed by the barley rhizosphere (2.4-7.2 x 10-12) and the wheat rhizosphere (2.2-2.9 x 10-13). It was further shown that the metabolic activity of the cells in the rhizosphere of the three plants was not significantly different, and that activity and transfer were not correlated. Thus, the data demonstrated species specific rhizosphere effects on the conjugal transfer process that could not be attributed to different metabolic activities of the bacteria.     

Role of ptsP, orfT, and sss Recombinase Genes in Root Colonization by Pseudomonas fluorescens Q8r1-96

Pseudomonas fluorescens Q8r1-96 produces 2,4-diacetylphloroglucinol (2,4-DAPG), a polyketide antibiotic that suppresses a wide variety of soilborne fungal pathogens, including Gaeumannomyces graminis var. tritici, which causes take-all disease of wheat. Strain Q8r1-96 is representative of the D-genotype of 2,4-DAPG producers, which are exceptional because of their ability to aggressively colonize and maintain large populations on the roots of host plants, including wheat, pea, and sugar beet. In this study, three genes, an sss recombinase gene, ptsP, and orfT, which are important in the interaction of Pseudomonas spp. with various hosts, were investigated to determine their contributions to the unusual colonization properties of strain Q8r1-96. The sss recombinase and ptsP genes influence global processes, including phenotypic plasticity and organic nitrogen utilization, respectively. The orfT gene contributes to the pathogenicity of Pseudomonas aeruginosa in plants and animals and is conserved among saprophytic rhizosphere pseudomonads, but its function is unknown. Clones containing these genes were identified in a Q8r1-96 genomic library, sequenced, and used to construct gene replacement mutants of Q8r1-96. Mutants were characterized to determine their 2,4-DAPG production, motility, fluorescence, colony morphology, exoprotease and hydrogen cyanide (HCN) production, carbon and nitrogen utilization, and ability to colonize the rhizosphere of wheat grown in natural soil. The ptsP mutant was impaired in wheat root colonization, whereas mutants with mutations in the sss recombinase gene and orfT were not. However, all three mutants were less competitive than wild-type P. fluorescens Q8r1-96 in the wheat rhizosphere when they were introduced into the soil by paired inoculation with the parental strain.     

Rhizosphere Selection of Highly Motile Phenotypic Variants of Pseudomonas fluorescens with Enhanced Competitive Colonization Ability

Phenotypic variants of Pseudomonas fluorescens F113 showing a translucent and diffuse colony morphology show enhanced colonization of the alfalfa rhizosphere. We have previously shown that in the biocontrol agent P. fluorescens F113, phenotypic variation is mediated by the activity of two site-specific recombinases, Sss and XerD. By overexpressing the genes encoding either of the recombinases, we have now generated a large number of variants (mutants) after selection either by prolonged laboratory cultivation or by rhizosphere passage. All the isolated variants were more motile than the wild-type strain and appear to contain mutations in the gacA and/or gacS gene. By disrupting these genes and complementation analysis, we have observed that the Gac system regulates swimming motility by a repression pathway. Variants isolated after selection by prolonged cultivation formed a single population with a swimming motility that was equal to the motility of gac mutants, being 150% more motile than the wild type. The motility phenotype of these variants was complemented by the cloned gac genes. Variants isolated after rhizosphere selection belonged to two different populations: one identical to the population isolated after prolonged cultivation and the other comprising variants that besides a gac mutation harbored additional mutations conferring higher motility. Our results show that gac mutations are selected both in the stationary phase and during rhizosphere colonization. The enhanced motility phenotype is in turn selected during rhizosphere colonization. Several of these highly motile variants were more competitive than the wild-type strain, displacing it from the root tip within 2 weeks.     

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.