Induced Reporter Gene Activity, Enhanced Stress Resistance, and Competitive Ability of a Genetically Modified Pseudomonas fluorescens Strain Released into a Field Plot Planted with Wheat

The fates of Pseudomonas fluorescens R2fR and its mutant derivative RIWE8, which contains a lacZ reporter gene responsive to wheat root exudate, were compared in a field microplot. Inoculant survival, root colonization, translocation, resistance to stress factors, and reporter gene activity were assessed in bulk and wheat rhizosphere soils. Populations of both strains declined gradually in bulk and wheat rhizosphere soils and on the wheat rhizoplane as determined by specific CFU and immunofluorescence (IF). In samples from both bulk soil and wheat rhizosphere, IF cell counts were up to 3 orders of magnitude greater than the corresponding numbers of CFU after 120 days, indicating the presence of nonculturable inoculant cells. Estimates of RIWE8-specific target DNA molecule numbers in bulk soil samples 3 and 120 days after inoculation by most-probable-number PCR coincided with the corresponding CFU values. Transport of both strains to deeper soil layers was observed by 3 days after introduction into the microplot. Both strains colonized wheat roots similarly, and cells were seen scattered on the surface of 1-month-old wheat seedling roots by immunogold labelling-scanning electron microscopy. On average, reporter gene activity was significantly higher in wheat rhizosphere soil containing RIWE8 cells than in bulk soil or in soils containing R2fR cells. For both strains, resistance to the four stress factors ethanol, high temperature, high osmotic tension, and oxidative stress increased progressively with residence in soil. Cells from the rhizosphere of 11-day-old seedlings showed similar levels of resistance to osmotic and oxidative stresses and enhanced resistance to ethanol and heat as compared to cells from bulk soil. By 37 days, populations of R2fR and RIWE8 in the rhizosphere were significantly more sensitive to osmotic stress than were populations in bulk soil, whereas differences in response to the other stress factors were less evident. Hence, except for the induction of reporter gene expression in strain RIWE8 in the wheat rhizosphere, the data indicated that there were no great differences in the ecological properties in soil between the lacZ-modified and parental strains.     

Predominance of Nonculturable Cells of the Biocontrol Strain Pseudomonas fluorescens CHA0 in the Surface Horizon of Large Outdoor Lysimeters

The persistence of the biocontrol agent Pseudomonas fluorescens CHA0 in the surface horizon of 12 large outdoor lysimeters planted with winter wheat, Phacelia tanacetifolia followed by spring wheat, or maize was monitored for 1 year. Soil was inoculated with a spontaneous rifampin-resistant mutant (CHA0-Rif) of CHA0, and the strain was studied by using colony counts, Kogure's direct viable counts, and total counts (immunofluorescence). The number of culturable cells of the inoculant decreased progressively from 8 to 2 log CFU/g of soil or lower. However, culturable cells of CHA0-Rif accounted for less than 1% of the total cells of the inoculant 8 months after release in autumn. Since viable but nonculturable cells represented less than a quarter of the latter, most cells of CHA0-Rif in soil were thus inactive-dormant or dead at that time. Nonculturable cells of the inoculant were predominant also in the surface horizon of the lysimeters inoculated in the spring, and a significant fraction of them were viable. Results suggest that the occurrence of nonculturable cells of CHA0-Rif was influenced by climatic factors (water availability and soil temperature) and the abundance of roots in soil. The fact that the inoculant persisted as mixed populations of cells of different physiological states, in which nonculturable cells were predominant, needs to be taken into account when assessing the autecology of wild-type or genetically modified pseudomonads released into the soil ecosystem.     

Green fluorescent protein-marked Pseudomonas fluorescens: localization, viability, and activity in the natural barley rhizosphere.

The gfp-tagged Pseudomonas fluorescens biocontrol strain DR54-BN14 was introduced into the barley rhizosphere. Confocal laser scanning microscopy revealed that the rhizoplane populations of DR54-BN14 on 3- to 14-day-old roots were able to form microcolonies closely associated with the indigenous bacteria and that a majority of DR54-BN14 cells appeared small and almost coccoid. Information on the viability of the inoculant was provided by a microcolony assay, while measurements of cell volume, the intensity of green fluorescent protein fluorescence, and the ratio of dividing cells to total cells were used as indicators of cellular activity. At a soil moisture close to the water-holding capacity of the soil, the activity parameters suggested that the majority of DR54-BN14 cells were starving in the rhizosphere. Nevertheless, approximately 80% of the population was either culturable or viable but nonculturable during the 3-week incubation period. No impact of root decay on viability was observed, and differences in viability or activity among DR54-BN14 cells located in different regions of the root were not apparent. In dry soil, however, the nonviable state of DR54-BN14 was predominant, suggesting that desiccation is an important abiotic regulator of cell viability.     

Transport of bacterial inoculants through intact cores of two different soils as affected by water percolation and the presence of wheat plants

Abstract Water flow-innduced transport of Burkholderia cepacia strain P2 and Pseudomonas fluorescens strain R2f cells through intact cores of loamy sand and silt loam field soils was measured for two percolation regimes, 0.9 and 4.4 mm h⁻¹, applied daily during 1 hour. For each strain, transport was generally similar between the two water regimes. Translocation of B. cepacia , with 4.4 mm h⁻¹, did occur initially in both soils. In the loamy sand soil, no change in the bacterial distribution occurred during the experiment (51 days). In the silt loam, B. cepacia cell numbers in the lower soil layers were significantly reduced, to levels at or below the limit of detection. Transport of P. fluorescens in both soils also occurred initially and was comparable to that of B. cepacia . Later in the experiment, P. fluorescens was not detectable in the lower soil layers of the loamy sand cores, due to a large decrease in surviving cell numbers. In the silt loam, the inoculant cell distribution did not change with time. Pre-incubation of the inoculated cores before starting percolation reduced B. cepacia inoculant transport in the loamy sand soil measured after 5 days, but not that determined after 54 days. Delayed percolation in the silt loam soil affected bacterial transport only after 54 days. The presence of growing wheat plants overall enhanced bacterial translocation as compared to that in unplanted soil cores, but only with percolating water. Percolation water from silt loam cores appeared the day after the onset of percolation and often contained inoculant bacteria. With loamy sand, percolation water appeared only 5 days after the start of percolation, and no inoculant bacteria were found. The results presented aid in predicting the fate of genetically manipulated bacteria in a field experiment.     

Effects of starvation and osmotic stress on viability and heat resistance of Pseudomonas fluorescens AH9

This study addresses the responses to starvation and osmotic stress of Pseudomonas fluorescens isolated from spoiled fish. Culturability and viability of stressed cells were determined. Cells maintaining an active electron transport system were considered to be viable and this activity was assessed by the ability of the cells to reduce the 5-cyano-2,4-ditolyl tetrazolium chloride (CTC) to fluorescent CTC-formazan. Cells starved of carbon maintained high culturability and a high proportion of the cells were capable of reducing CTC during short-time (up to 5 d) experiments. ATP concentrations were lower in carbon-starved than in log-phase cells but the measured levels suggested that metabolic activity was retained. Carbon-starved cells developed an increased heat resistance and prolonged starvation resulted in further protection. Viable, but non-culturable cells were found during heat challenge implying that culture methods underestimate the recovery potential of these cells. Osmotically-stressed Ps. fluorescens maintained a high viability, whereas culturability was rapidly lost. In contrast to starved cells, no protection against a subsequent heat challenge was found in osmotically-stressed (4 or 18 h) cells, but an increased salinity of the heating menstruum alone resulted in elevated heat resistance.     

Pseudomonas fluorescens DR54 Reduces Sclerotia Formation,Biomass Development,and Disease Incidence of Rhizoctonia solani Causing Damping-Off in Sugar Beet

Effects of the biocontrol strain, Pseudomonas fluorescens DR54, on growth and disease development by Rhizoctonia solani causing damping-off in sugar beet were studied in soil microcosms and in pot experiments with natural, clay-type soil. In pot experiments with P. fluorescens DR54-treated seeds, significantly fewer Rhizoctonia-challenged seedlings showed damping-off symptoms than when not inoculated with the biocontrol agent. In the rhizosphere of P. fluorescens DR54 inoculated seeds, the bacterial inoculant was present in high numbers as shown by dilution plating and immunoblotting. By the ELISA antibody technique and direct microscopy of the fungal pathogen grown in soil microcosms, it was shown that the presence of P. fluorescens DR54 on the inoculated seeds had a strong inhibitory effect on development of both mycelium biomass and sclerotia formation by R. solani. In the field experiment, plant emergence was increased by treatment with P. fluorescens DR54 and the inoculant was found to be the dominating rhizosphere colonizing pseudomonad immediately after seedling emergence.     

Assessment of competitiveness of rhizobia infecting Galega orientalis on the basis of plant yield, nodulation, and strain identification by antibiotic resistance and PCR.

Competition between effective and ineffective Rhizobium galegae strains nodulating Galega orientalis was examined on the basis of plant growth, nodulation, antibiotic resistance, and PCR results. In a preliminary experiment in Leonard's jars, ineffective R. galegae strains HAMBI 1207 and HAMBI 1209 competed in similar manners with the effective strain R. galegae HAMBI 1174. In a pot experiment, soil was inoculated with 0 to 10(5) HAMBI 1207 cells per g before G. orientalis was sown. Seeds of G. orientalis were surface inoculated with 2 x 10(4) and 2 x 10(5) cells of HAMBI 1174 per seed (which represent half and fivefold the commercially recommended amount of inoculant, respectively). Plant yield and nodulation by the effective strain were significantly reduced, with as few as 10(2) ineffective rhizobia per g of soil, and the inoculation response was not improved by the 10-fold greater dose of the inoculant. Bacteria occupying the nodules were identified by antibiotic resistance and PCR with primers specific for R. galegae HAMBI 1174, R. galegae, and genes coding for bacterial 16S rRNA (bacterial 16S rDNA). Sixty-two large nodules examined were occupied by the effective strain HAMBI 1174, as proven by antibiotic resistance and amplification of the strain-specific fragment. From 20 small nodules, only the species-specific fragment could be amplified, and isolated bacteria had the same antibiotic resistance and 16S PCR restriction pattern as strain HAMBI 1207. PCR with our strain-specific and species-specific primers provides a powerful tool for strain identification of R. galegae directly from nodules without genetic modification of the bacteria.     

The sigma factor AlgU (AlgT) controls exopolysaccharide production and tolerance towards desiccation and osmotic stress in the biocontrol agent Pseudomonas fluorescens CHA0.

A variety of stress situations may affect the activity and survival of plant-beneficial pseudomonads added to soil to control root diseases. This study focused on the roles of the sigma factor AlgU (synonyms, AlgT, RpoE, and sigma(22)) and the anti-sigma factor MucA in stress adaptation of the biocontrol agent Pseudomonas fluorescens CHA0. The algU-mucA-mucB gene cluster of strain CHA0 was similar to that of the pathogens Pseudomonas aeruginosa and Pseudomonas syringae. Strain CHA0 is naturally nonmucoid, whereas a mucA deletion mutant or algU-overexpressing strains were highly mucoid due to exopolysaccharide overproduction. Mucoidy strictly depended on the global regulator GacA. An algU deletion mutant was significantly more sensitive to osmotic stress than the wild-type CHA0 strain and the mucA mutant were. Expression of an algU'-'lacZ reporter fusion was induced severalfold in the wild type and in the mucA mutant upon exposure to osmotic stress, whereas a lower, noninducible level of expression was observed in the algU mutant. Overexpression of algU did not enhance tolerance towards osmotic stress. AlgU was found to be essential for tolerance of P. fluorescens towards desiccation stress in a sterile vermiculite-sand mixture and in a natural sandy loam soil. The size of the population of the algU mutant declined much more rapidly than the size of the wild-type population at soil water contents below 5%. In contrast to its role in pathogenic pseudomonads, AlgU did not contribute to tolerance of P. fluorescens towards oxidative and heat stress. In conclusion, AlgU is a crucial determinant in the adaptation of P. fluorescens to dry conditions and hyperosmolarity, two major stress factors that limit bacterial survival in the environment.     

Green Fluorescent Protein-Marked Pseudomonas fluorescens: Localization, Viability, and Activity in the Natural Barley Rhizosphere

The gfp-tagged Pseudomonas fluorescens biocontrol strain DR54-BN14 was introduced into the barley rhizosphere. Confocal laser scanning microscopy revealed that the rhizoplane populations of DR54-BN14 on 3- to 14-day-old roots were able to form microcolonies closely associated with the indigenous bacteria and that a majority of DR54-BN14 cells appeared small and almost coccoid. Information on the viability of the inoculant was provided by a microcolony assay, while measurements of cell volume, the intensity of green fluorescent protein fluorescence, and the ratio of dividing cells to total cells were used as indicators of cellular activity. At a soil moisture close to the water-holding capacity of the soil, the activity parameters suggested that the majority of DR54-BN14 cells were starving in the rhizosphere. Nevertheless, approximately 80% of the population was either culturable or viable but nonculturable during the 3-week incubation period. No impact of root decay on viability was observed, and differences in viability or activity among DR54-BN14 cells located in different regions of the root were not apparent. In dry soil, however, the nonviable state of DR54-BN14 was predominant, suggesting that desiccation is an important abiotic regulator of cell viability.     

Transport of a genetically engineered Pseudomonas fluorescens strain through a soil microcosm.

Vertical soil microcosms flushed with groundwater were used to study the influence of water movement on survival and transport of a genetically engineered Pseudomonas fluorescens C5t strain through a loamy sand and a loam soil. Transport of cells introduced into the top 1 cm of the vertical soil microcosms was dependent on the flow rate of water and the number of times microcosms were flushed with groundwater. The presence of wheat roots growing downward in the microcosms contributed only slightly to the movement of P. fluorescens C5t cells to lower soil regions of the loamy sand microcosms, but enhanced downward transport in the loam microcosms. Furthermore, the introduced P. fluorescens C5t cells were detected in the effluent water samples even after three flushes of groundwater and 10 days of incubation. As evidenced by a comparison of counts from immunofluorescence and selective plating, nonculturable C5t cells occurred in day 10 soil and percolated water samples, primarily of the loamy sand microcosms. Vertical soil microcosms that use water movement may be useful in studying the survival and transport of genetically engineered bacteria in soil under a variety of conditions prior to field testing.     

Survival of biocontrol Pseudomonas fluorescens CHA0 in lysimeter effluent water depends on time of the year and soil type

AIMS: To assess the effects of soil type and time of the year on survival of the biocontrol inoculant Pseudomonas fluorescens CHA0 under aerobic conditions in lysimeter effluent water. METHODS AND RESULTS: Effluent water was collected at different times from large outdoor lysimeters (2.5 m deep), which contained a well-drained or a poorly-drained cambisol, and inoculated with CHA0. The inoculant was monitored for 175 d by colony counts, total immunofluorescence cell counts and Kogure's viable cell counts. Cell numbers obtained with the three methods were similar. The inoculant declined exponentially in time and its population level varied considerably depending on the time of the year at which effluent water had been collected and soil type in the lysimeter. Positive correlations were found between the number of resident culturable aerobic bacteria and subsequent survival of the inoculant. CONCLUSION: The fluctuations of inoculant survival patterns correlated with differences in biological properties of lysimeter water that were related to soil type and time of the year. SIGNIFICANCE AND IMPACT OF THE STUDY: Results suggest that predictability of the survival of bacterial soil inoculants transported to groundwater level by heavy rainfall may be improved by taking into account key biological properties of the water.     

Transport of a genetically engineered Pseudomonas fluorescens strain through a soil microcosm

Vertical soil microcosms flushed with groundwater were used to study the influence of water movement on survival and transport of a genetically engineered Pseudomonas fluorescens C5t strain through a loamy sand and a loam soil. Transport of cells introduced into the top 1 cm of the vertical soil microcosms was dependent on the flow rate of water and the number of times microcosms were flushed with groundwater. The presence of wheat roots growing downward in the microcosms contributed only slightly to the movement of P. fluorescens C5t cells to lower soil regions of the loamy sand microcosms, but enhanced downward transport in the loam microcosms. Furthermore, the introduced P. fluorescens C5t cells were detected in the effluent water samples even after three flushes of groundwater and 10 days of incubation. As evidenced by a comparison of counts from immunofluorescence and selective plating, nonculturable C5t cells occurred in day 10 soil and percolated water samples, primarily of the loamy sand microcosms. Vertical soil microcosms that use water movement may be useful in studying the survival and transport of genetically engineered bacteria in soil under a variety of conditions prior to field testing.     

Survival ofRhizobium leguminosarum biovarviceae subjected to heat,drought and salinity in soil

Two strains (RCR 1001 and 1044) and a commercial inoculant (Okadin) ofRhizobium leguminosarum biovarviceae were tested for their ability to survive in autoclaved clay soil for up to four months under heat, salinity and drought stress. Resistance to heat was tested by incubating rhizobia in soil at 27, 37 and 42 °C. Tolerance of rhizobia to salinity was investigated by growing rhizobia in soil salinized with 1 and 2 % NaCl (m/m). Drought resistance was tested by subjecting bacteria to soil moisture contents of 20, 10 and 5%. Strain RCR 1001 was more resistant to heat and nodulated faba bean better than other tested strains. A commercial inoculant Okadin survived more (plate count method) and nodulated faba bean (plant infectivity, most probable number, MPN) at moisture content of 5% and 2% NaCl. Although, strains RCR 1001 and 1044 resisted these stress conditions (plate count) they lost their abilities to nodulate faba bean (MPN-test). There is a possibility for selection of effective rhizobia which are more tolerant to harsh conditions.     

The viable-but-nonculturable state induced by abiotic stress in the biocontrol agent Pseudomonas fluorescens CHA0 does not promote strain persistence in soil

The effects of oxygen limitation, low redox potential, and high NaCl stress for 7 days in vitro on the rifampin-resistant biocontrol inoculant Pseudomonas fluorescens CHA0-Rif and its subsequent persistence in natural soil for 54 days were investigated. Throughout the experiment, the strain was monitored using total cell counts (immunofluorescence microscopy), Kogure's direct viable counts, and colony counts (on rifampin-containing plates). Under in vitro conditions, viable-but-nonculturable (VBNC) cells of CHA0-Rif were obtained when the strain was exposed to a combination of low redox potential (230 mV) and oxygen limitation. This mimics a situation observed in the field, where VBNC cells of the strain were found in the water-logged soil layer above the plow pan. Here, VBNC cells were also observed in vitro when CHA0-Rif was subjected to high NaCl levels (i.e., NaCl at 1.5 M but not 0.7 M). In all treatments, cell numbers remained close to the inoculum level for the first 12 days after inoculation of soil, regardless of the cell enumeration method used, but decreased afterwards. At the last two samplings in soil, VBNC cells of CHA0-Rif were found in all treatments except the one in which log-phase cells had been used. In the two treatments that generated high numbers of VBNC cells in vitro, VBNC cells did not display enhanced persistence compared with culturable cells once introduced into soil, which suggests that this VBNC state did not represent a physiological strategy to improve survival under adverse conditions.     

Impact of crop management on intraspecific diversity of Pseudomonas corrugata in bulk soil

Colonization at sugar beet root surfaces by seedling-inoculated biocontrol strain Pseudomonas fluorescens DR54 and native soil bacteria was followed over a period of 3 weeks using a combination of immunofluorescence (DR54-targeting specific antibody) and fluorescence in situ hybridization (rRNA-targeting Eubacteria EUB338 probe) techniques with confocal laser scanning microscopy. The dual staining protocol allowed cellular activity (ribosomal number) to be recorded in both single cells and microcolonies of strain DR54 during establishment on the root. After 2 days, the population density of strain DR54 reached a constant level at the root basis. From this time, however, high cellular activity was only found in few bacteria located as single cells, whereas all microcolony-forming cells occurring in aggregates were still active. In contrast, a low density of strain DR54 was observed at the root tip, but here many of the bacteria located as single cells were active. The native population of soil bacteria, comprising a diverse assembly of morphologically different forms and size classes, initiated colonization at the root basis only after 2 days of incubation. Hence the dual staining protocol allowed direct microscopic studies of early root colonization by both inoculant and native soil bacteria, including their differentiation into active and non-active cells and into single or microcolony-forming cells.     

Stress-survival responses of a carbon-starved p-nitrophenol-mineralizing Moraxella strain in river water

The effect of carbon starvation on the stress-resistant responses of a p-nitrophenol-mineralizing Moraxella strain was examined in both buffer and river water samples. The Moraxella strain showed optimal stress-resistant responses in a minimal salt buffer when carbon-starved for 1-2 d. In the buffer system, the 1- and 2-day carbon-starved Moraxella cultures survived about 150-, 200-, and 100-fold better than the non-starved cultures when exposed to 43.5 degrees C, 2.7 mol/L NaCl, and 500 micromol/L H2O2 for 4 h, respectively. A green fluorescent protein gene- (gfp) labelled derivative of the Moraxella strain was used to examine the stress-resistant responses of the bacterium in natural river water microcosms. The carbon-starved gfp-labelled Moraxella strain also showed stress-resistant responses against heat, osmotic, and oxidative stresses in the river water samples. Despite the stress-tolerant capability of the carbon-starved gfp-labelled Moraxella cells, they did not exhibit any survival advantage over their non-starved counterparts when inoculated into river water microcosms and incubated at 10 and 22 degrees C for 14 d.     

Localization of synthetic glycolipids in the cell and the dynamics of their insertion/loss

Modification of the cell surface with synthetic glycolipids opens up a wide range of possibilities for studying the function of glycolipids. Synthetic glycolipids called Function-Spacer-Lipids (FSL; where F is a glycan or label, S is a spacer, and L is dioleoylphosphatidyl ethanolamine) easily and controllably modify the membrane of a living cells. This current study investigates the dynamics and mechanism of the FSL insertion and release/loss. FSL insert into the cell membrane (~1 million molecules per cell) within tens of minutes, almost regardless of the nature of the cells (including the thickness of their glycocalyx) and the size of the FSL glycan. FSLs do not accumulate uniformly, but instead form patches >300 nm in size either entrapped in the glycocalyx, or integrated in the plane of the plasma membrane, but always outside the cell rafts. The natural release (loss) of FSL from the modified cell was two orders of magnitude slower than attachment/insertion and occurred mainly in the form of released microvesicles with a size of 140 ± 5 nm. The accumulation of FSL as patches in the cell membrane is similar to the coalescence of natural glycosphingolipids and supports (along with their long residence time in the membrane) the use of FSL as probes for the study of glycosphingolipid-protein interactions.     

Buffy,a Drosophila Bcl-2 protein,has anti-apoptotic and cell cycle inhibitory functions

Bcl-2 family proteins are key regulators of apoptosis. Both pro-apoptotic and anti-apoptotic members of this family are found in mammalian cells, but only the pro-apoptotic protein Debcl has been characterized in Drosophila: Here we report that Buffy, the second Drosophila Bcl-2-like protein, is a pro-survival protein. Ablation of Buffy by RNA interference leads to ectopic apoptosis, whereas overexpression of buffy results in the inhibition of developmental programmed cell death and gamma irradiation-induced apoptosis. Buffy interacts genetically and physically with Debcl to suppress Debcl-induced cell death. Genetic interactions suggest that Buffy acts downstream of Rpr, Grim and Hid, and upstream of the apical caspase Dronc. Furthermore, overexpression of buffy inhibits ectopic cell death in diap1 (th(5)) mutants. Taken together these data suggest that Buffy can act downstream of Rpr, Grim and Hid to block caspase-dependent cell death. Overexpression of Buffy in the embryo results in inhibition of the cell cycle, consistent with a G(1)/early-S phase arrest. Our data suggest that Buffy is functionally similar to the mammalian pro-survival Bcl-2 family of proteins.