Use of Tn5 Mutants To Assess the Role of the Dissimilatory Nitrite Reductase in the Competitive Abilities of Two Pseudomonas Strains in Soil

We examined the influence of soil aeration state and plant root presence on the comparative survival of wild-type bacteria and isogenic Tn5 (Nir(sup-)) mutants lacking the ability to synthesize nitrite reductase. Two denitrifying Pseudomonas strains with different nitrite reductase types were used. Enumeration of bacteria in sterile and nonsterile soils was based on differential antibiotic resistance. The validity of the bacterial models studied (i.e., equal growth of wild-type and mutant bacteria under aerobic conditions and significantly better growth of wild-type bacteria under denitrifying conditions) was verified in pure-culture studies. In sterile soil, both strains survived better under aerobic than under anaerobic conditions. The lower efficiency of denitrification than O(inf2) respiration in supporting bacterial growth explained this result, and the physical heterogeneity of soil did not strongly modify the results obtained in pure-culture studies. In nonsterile soil, one of the Pseudomonas strains survived better under anaerobic conditions while the other competed equally with the indigenous soil microflora under aerobic and anaerobic conditions. However, when the Nir(sup-)-to-total inoculant ratios (wild type plus Nir(sup-) mutant) were analyzed, it appeared that the presence of nitrite reductase conferred on both Pseudomonas strains a competitive advantage for anaerobic environment or rhizosphere colonization. This is the first attempt to demonstrate with isogenic nondenitrifying mutants that denitrification can contribute to the persistence and distribution of bacteria in fluctuating soil environments.     

Conjugative Transfer of Chromosomal Genes between Fluorescent Pseudomonads in the Rhizosphere of Wheat

Bacteria released in large numbers for biocontrol or bioremediation purposes might exchange genes with other microorganisms. Two model systems were designed to investigate the likelihood of such an exchange and some factors which govern the conjugative exchange of chromosomal genes between root-colonizing pseudomonads in the rhizosphere of wheat. The first model consisted of the biocontrol strain CHA0 of Pseudomonas fluorescens and transposon-facilitated recombination (Tfr). A conjugative IncP plasmid loaded with transposon Tn5, in a CHA0 derivative carrying a chromosomal Tn5 insertion, promoted chromosome transfer to auxotrophic CHA0 recipients in vitro. A chromosomal marker (pro) was transferred at a frequency of about 10(sup-6) per donor on wheat roots under gnotobiotic conditions, provided that the Tfr donor and recipient populations each contained 10(sup6) to 10(sup7) CFU per g of root. In contrast, no conjugative gene transfer was detected in soil, illustrating that the root surface stimulates conjugation. The second model system was based on the genetically well-characterized strain PAO of Pseudomonas aeruginosa and the chromosome mobilizing IncP plasmid R68.45. Although originally isolated from a human wound, strain PAO1 was found to be an excellent root colonizer, even under natural, nonsterile conditions. Matings between an auxotrophic R68.45 donor and auxotrophic recipients produced prototrophic chromosomal recombinants at 10(sup-4) to 10(sup-5) per donor on wheat roots in artificial soil under gnotobiotic conditions and at about 10(sup-6) per donor on wheat roots in natural, nonsterile soil microcosms after 2 weeks of incubation. The frequencies of chromosomal recombinants were as high as or higher than the frequencies of R68.45 transconjugants, reflecting mainly the selective growth advantage of the prototrophic recombinants over the auxotrophic parental strains in the rhizosphere. Although under field conditions the formation of chromosomal recombinants is expected to be reduced by several factors, we conclude that chromosomal genes, whether present naturally or introduced by genetic modification, may be transmissible between rhizosphere bacteria.     

Transformation of Low Concentrations of 3-Chlorobenzoate by Pseudomonas sp. Strain B13: Kinetics and Residual Concentrations

The transformation of 3-chlorobenzoate (3CB) and acetate at initial concentrations in the wide range of 10 nM to 16 mM was studied in batch experiments with Pseudomonas sp. strain B13. Transformation rates of 3CB at millimolar concentrations could be described by Michaelis-Menten kinetics (K(infm), 0.13 mM; V(infmax), 24 nmol (middot) mg of protein(sup-1) (middot) min(sup-1)). Experiments with nanomolar and low micromolar concentrations of 3CB indicated the possible existence of two different transformation systems for 3CB. The first transformation system operated above 1 (mu)M 3CB, with an apparent threshold concentration of 0.50 (plusmn) 0.11 (mu)M. A second transformation system operated below 1 (mu)M 3CB and showed first-order kinetics (rate constant, 0.076 liter (middot) g of protein(sup-1) (middot) min(sup-1)), with no threshold concentration in the nanomolar range. A residual substrate concentration, as has been reported for some other Pseudomonas strains, could not be detected for 3CB (detection limit, 1.0 nM) in batch incubations with Pseudomonas sp. strain B13. The addition of various concentrations of acetate as a second, easily degradable substrate neither affected the transformation kinetics of 3CB nor induced a detectable residual substrate concentration. Acetate alone also showed no residual concentration (detection limit, 0.5 nM). The results presented indicate that the concentration limits for substrate conversion obtained by extrapolation from kinetic data at higher substrate concentrations may underestimate the true conversion capacity of a microbial culture.     

Capacity for Methane Oxidation in Landfill Cover Soils Measured in Laboratory-Scale Soil Microcosms

Laboratory-scale soil microcosms containing different soils were permeated with CH(inf4) for up to 6 months to investigate their capacity to develop a methanotrophic community. Methane emissions were monitored continuously until steady states were established. The porous, coarse sand soil developed the greatest methanotrophic capacity (10.4 mol of CH(inf4) (middot) m(sup-2) (middot) day(sup-1)), the greatest yet reported in the literature. Vertical profiles of O(inf2), CH(inf4), and methanotrophic potential in the soils were determined at steady state. Methane oxidation potentials were greatest where the vertical profiles of O(inf2) and CH(inf4) overlapped. A significant increase in the organic matter content of the soil, presumably derived from methanotroph biomass, occurred where CH(inf4) oxidation was greatest. Methane oxidation kinetics showed that a soil community with a low methanotrophic capacity (V(infmax) of 258 nmol (middot) g of soil(sup-1) (middot) h(sup-1)) but relatively high affinity (k(infapp) of 1.6 (mu)M) remained in N(inf2)-purged control microcosms, even after 6 months without CH(inf4). We attribute this to a facultative, possibly mixotrophic, methanotrophic microbial community. When purged with CH(inf4), a different methanotrophic community developed which had a lower affinity (k(infapp) of 31.7 (mu)M) for CH(inf4) but a greater capacity (V(infmax) of 998 nmol (middot) g of soil(sup-1) (middot) h(sup-1)) for CH(inf4) oxidation, reflecting the enrichment of an active high-capacity methanotrophic community. Compared with the unamended control soil, amendment of the coarse sand with sewage sludge enhanced CH(inf4) oxidation capacity by 26%; K(inf2)HPO(inf4) amendment had no significant effect, while amendment with NH(inf4)NO(inf3) reduced the CH(inf4) oxidation capacity by 64%. In vitro experiments suggested that NH(inf4)NO(inf3) additions (10 and 71 (mu)mol (middot) g of soil(sup-1)) inhibited CH(inf4) oxidation by a nonspecific ionic effect rather than by specific inhibition by NH(inf4)(sup+).     

Outer Membrane Protein Heterogeneity within Pseudomonas fluorescens and P. putida and Use of an OprF Antibody as a Probe for rRNA Homology Group I Pseudomonads

The electrophoretic patterns of outer membrane proteins of strains representing the biovars of Pseudomonas fluorescens and Pseudomonas putida were analyzed by gel electrophoresis. The outer membrane protein profiles were variable, and they were not useful for assigning strains to a specific biovar. However, three or four predominant outer membrane proteins migrating at 42 to 46 kDa, 33 to 38 kDa, and 20 to 22 kDa were conserved among the strains. They could be tentatively identified as OprE (44 kDa), OprF (38 kDa), OprH (21 kDa), and OprL (20.5 kDa), which are known proteins from Pseudomonas aeruginosa. A 37-kDa OprF-like protein was purified from P. fluorescens DF57 and used to raise a polyclonal antibody. In Western blot (immunoblot) analysis, this antibody reacted with OprF proteins from members of Pseudomonas rRNA homology group I but not with proteins from nonpseudomonads. The heterogeneity in M(infr) of OprF was greater among P. fluorescens strains than among P. putida strains. Immunofluorescence microscopy of intact cells demonstrated that the antibody recognized epitopes that were accessible only after unmasking by EDTA treatment. The antibody was used in a colony blotting assay to determine the percentage of rRNA homology group I pseudomonads among bacteria from the rhizosphere of barley. The bacteria were isolated on 10% tryptic soy agar, King's B agar, and the pseudomonad-specific medium Gould S1 agar. The estimate of OprF-containing CFU in rhizosphere soil obtained by colony blotting on 10% tryptic soy agar was about 2 and 14 times higher than the values obtained from King's agar and Gould S1 agar, respectively, indicating that not all fluorescent pseudomonads are scored on more specific media. The colonies reacting with the OprF antibody were verified as being rRNA homology group I pseudomonads by using the API 20NE system.     

Isolation and characterization of diazotrophic growth promoting bacteria from rhizosphere of agricultural crops of Korea

Free-living nitrogen fixing bacteria were isolated from rhizosphere of seven different plant namely sesame, maize, wheat, soybean, lettuce, pepper and rice grown in Chungbuk Province, Korea. Five isolates with nitrogenase activity above 150nmol(-1) mg(-1) protein were identified based on, phenotypic and 16S rDNA sequences analysis. The strains were identified as Stenotrophomonas maltophilia (PM-1, PM-26), Bacillus fusiformis (PM-5, PM-24) and Pseudomonas fluorescens (PM-13), respectively. All the isolates produced indole-3-acetic acid (IAA), in the presence of tryptophan, ranging from 100.4 microg ml(-1) (PM-13) to 255 microg ml(-1) (PM-24). The isolate PM-24 (Bacillus fusiformis) exhibiting highest nitrogenase activity (3677.81 nmol h(-1) mg(-1) protein) and IAA production (255microg ml(-1)) has a promising potential for developing as a plant growth promoting rhizobacteria.     

Dissolution of Barium from Barite in Sewage Sludges and Cultures of Desulfovibrio desulfuricans

High concentrations of total barium, ranging from 0.42 to 1.58 mg(middot)g(sup-1) (dry weight) were found in sludges of two sewage treatment plants near Florence, Italy. Barium concentrations in the suspended matter decreased as redox potential values changed from negative to positive. An anoxic sewage sludge sample was aerated, and 30% of the total barium was removed in 24 h. To demonstrate that barium was solubilized from barite by sulfate-reducing bacteria, a strain of Desulfovibrio desulfuricans was used to study the solubilization of barium from barite under laboratory conditions. During cell growth with different concentrations of barite from 0.01 to 0.3 g(middot)liter(sup-1) (the latter is the MIC) as the only source of sulfates in the cultures, the D. desulfuricans strain accumulated barium up to 0.58 (mu)g(middot)mg(sup-1) (dry weight). Three times the quantity of barium was dissolved by bacteria than in the uninoculated medium (control). The unexpectedly low concentration of soluble barium (1.2 mg of Ba(middot)liter(sup-1)) with respect to the quantity expected (109 mg of Ba(middot)liter(sup-1)), calculated on the basis of the free H(inf2)S evolved from the dissimilatory reduction of sulfate from barite, was probably due to the formation of other barium compounds, such as witherite (BaCO(inf3)) and the transient species barium sulfide (BaS). The D. desulfuricans strain, growing on barite, formed visible aggregates. Confocal microscopy analysis showed that aggregates consisted of bacteria and barite. After 3 days of incubation, several autofluorescent crystals surrounded by a dissolution halo were observed. The crystals were identified as BaS by comparison with the commercial compound.     

Iron Stress and Pyoverdin Production by a Fluorescent Pseudomonad in the Rhizosphere of White Lupine (Lupinus albus L.) and Barley (Hordeum vulgare L.)

Induction of high-affinity iron transport during root colonization by Pseudomonas fluorescens Pf-5 (pvd-inaZ) was examined in lupine and barley growing in microcosms. P. fluorescens Pf-5 (pvd-inaZ) contains a plasmid carrying pvd-inaZ; thus, in this strain, ice nucleation activity is regulated by pyoverdin production. Lupine or barley plants were grown for 18 or 8 days, respectively, in soil amended with 2% calcium carbonate and inoculated with P. fluorescens Pf-5 (pvd-inaZ) at a density of 4 x 10(sup8) CFU g (dry weight) of soil(sup-1). A filter paper blotting technique was used to sample cells from the rhizosphere in different root zones, and then the cells were resuspended for enumeration and measurement of ice nucleation activity. The population density of P. fluorescens Pf-5 (pvd-inaZ) in the rhizosphere decreased by one order of magnitude in both lupine and barley over time. The ice nucleation activity ranged from -3.4 to -3.0 log ice nuclei CFU(sup-1) for lupine and -3.0 to -2.8 log ice nuclei CFU(sup-1) for barley, was similar in all root zones, and did not change over time. An in vitro experiment was conducted to determine the relationship between ice nucleation activity and pyoverdin production in P. fluorescens Pf-5 (pvd-inaZ). An ice nucleation activity of approximately -3.0 log ice nuclei CFU(sup-1) was measured in the in vitro experiment at 25 to 50 (mu)M FeCl(inf3). By using the regression between ice nucleation activity and pyoverdin production determined in vitro and assuming a P. fluorescens Pf-5 (pvd-inaZ) population density of 10(sup8) CFU g of root(sup-1), the maximum possible pyoverdin accumulation by P. fluorescens Pf-5 (pvd-inaZ) in the rhizosphere was estimated to be 0.5 and 0.8 nmol g of root(sup-1) for lupine and barley, respectively. The low ice nucleation activity measured in the rhizosphere suggests that nutritional competition for iron in the rhizosphere may not be a major factor influencing root colonization by P. fluorescens Pf-5 (pvd-inaZ).     

Kinetic Analyses of Desulfurization of Dibenzothiophene by Rhodococcus erythropolis in Continuous Cultures

Rhodococcus erythropolis N1-36, a desulfurization strain, was grown in continuous culture at 10 different dilution rates with 50 (mu)M dibenzothiophene sulfone (DBTO(inf2)) as the growth-limiting nutrient. The steady-state biomass, concentrations of substrate (DBTO(inf2)) and product (monohydroxybiphenyl), saturation constant (0.39 (mu)M DBTO(inf2)), and cell yield coefficient (9 mg of biomass(middot)(mu)M(sup-1) DBTO(inf2)) were measured. Continuous cultures at five temperatures allowed calculation of activation energy (0.84 kcal(middot)mol(sup-1) [ca. 3.5 kJ(middot)mol(sup-1)]) near the optimal temperature (30(deg)C) for growth. A washout technique was used to calculate the maximum specific growth rate (0.235 h(sup-1)), a value equivalent to a minimum generation time of 2.95 h.     

Effects of Nitrate Availability and the Presence of Glyceria maxima on the Composition and Activity of the Dissimilatory Nitrate-Reducing Bacterial Community

The effects of nitrate availability and the presence of Glyceria maxima on the composition and activity of the dissimilatory nitrate-reducing bacterial community were studied in the laboratory. Four different concentrations of NO(inf3)(sup-), 0, 533, 1434, and 2,905 (mu)g of NO(inf3)(sup-)-N g of dry sediment(sup-1), were added to pots containing freshwater sediment, and the pots were then incubated for a period of 69 days. Upon harvest, NH(inf4)(sup+) was not detectable in sediment that received 0 or 533 (mu)g of NO(inf3)(sup-)-N g of dry sediment(sup-1). Nitrate concentrations in these pots ranged from 0 to 8 (mu)g of NO(inf3)(sup-)-N g of dry sediment(sup-1) at harvest. In pots that received 1,434 or 2,905 (mu)g of NO(inf3)(sup-)-N g of dry sediment(sup-1), final concentrations varied between 10 and 48 (mu)g of NH(inf4)(sup+)-N g of dry sediment(sup-1) and between 200 and 1,600 (mu)g of NO(inf3)(sup-)-N g of dry sediment(sup-1), respectively. Higher input levels of NO(inf3)(sup-) resulted in increased numbers of potential nitrate-reducing bacteria and higher potential nitrate-reducing activity in the rhizosphere. In sediment samples from the rhizosphere, the contribution of denitrification to the potential nitrate-reducing capacity varied from 8% under NO(inf3)(sup-)-limiting conditions to 58% when NO(inf3)(sup-) was in ample supply. In bulk sediment with excess NO(inf3)(sup-), this percentage was 44%. The nitrate-reducing community consisted almost entirely of NO(inf2)(sup-)-accumulating or NH(inf4)(sup+)-producing gram-positive species when NO(inf3)(sup-) was not added to the sediment. The addition of NO(inf3)(sup-) resulted in an increase of denitrifying Pseudomonas and Moraxella strains. The factor controlling the composition of the nitrate-reducing community when NO(inf3)(sup-) is limited is the presence of G. maxima. In sediment with excess NO(inf3)(sup-), nitrate availability determines the composition of the nitrate-reducing community.     

Study of rhizosphere and phyllosphere bacterial community and resistance to bacterial canker in genetically engineered phytochrome A cherry plants

The cherry rootstock 'Colt' line was transformed with a phytochrome A rice gene with the aim of altering light perception. Three transgenic events were chosen because of a modified developmental behavior. When red enriched light was supplied horizontally to stems, the PD3 transgenic line showed an increased rate of phytomer formation associated to a superior rate of plant growth compared to wild type (WT). Under the same light conditions, the PO1 and PA lines were less altered in morphology and development. When far-red enriched light was supplied, all transgenic lines had a reduced rate of growth, with the PD3 line being the most similar to the WT. The influence of the alien gene on root and leaf-associated bacteria was studied for a duration of 1 year. Significantly more culturable bacteria were recovered from PA lines than from PO1, PD3 and WT lines. On average, significantly more fluorescent pseudomonads were recovered from the rhizosphere of PA and PO1 lines than from PD3 and WT. No significant differences were detected in the number of bacteria recovered from the phyllosphere of transgenic and WT plant lines. A total of 143 Pseudomonas fluorescens strains isolated from rhizosphere of transgenic and WT lines were tested for their antagonistic activity against Phytophthora nicotianae and differences between bacteria derived from transgenic and WT were not detected. Fluorescent pseudomonads strains isolated from phyllosphere of PA and PO1 lines showed antagonistic activity against P. syringae pv. syringae, whereas no difference among the transgenic and WT lines was detected when fluorescent Pseudomonas strains were tested against P. syringae pv. mors-prunorum. Pathogenicity tests were conducted on rooted and micropropagated plants with P. s. pv. syringae and P. s. pv. mors-prunorum: in all assays, the PO1 lines were the most tolerant to P. s. pv. Syringae, and the PO1 and PD3 were tolerant to P. s. pv. mors-prunorum.     

Taxonomic and functional diversity of pseudomonads isolated from the roots of field-grown canola

Among the most important rhizosphere bacteria are the pseudomonads, which are aggressive colonizers and utilize a wide range of substrates as carbon sources. The objective of this study was to determine if the taxonomic or metabolic diversity of pseudomonads differed among field-grown canola cultivars. Bacteria (n=2257) were isolated from the rhizosphere and root interior of six cultivars of field-grown canola, including three transgenic varieties. The bacteria were identified by fatty acid methyl ester (FAME) analysis, and about 35% were identified as Pseudomonas species. The most abundant species were Pseudomonas putida and Pseudomonas chlororaphis. Dendrograms based on FAME analysis revealed that many pseudomonad strains were found in all of the canola cultivars. Pseudomonads of the same strain were found in both the rhizosphere and the root interior of canola plants, suggesting that endophytic bacteria were a subset of the rhizosphere community. Because metabolic profiling provides more useful information than taxonomy, P. putida and P. chlororaphis isolates were characterized for their ability to utilize carbon substrates and produce several enzymes. Bacteria isolated from different plant cultivars had different carbon utilization profiles, but when only carbon substrates found in root exudates were analyzed, the cultivar effect was less pronounced. These characterizations also demonstrated that bacteria that were determined by FAME to be the same strain were metabolically different, suggesting functional redundancy among Pseudomonas isolates. The results of this study suggest that pseudomonads were functionally diverse. They differed in their metabolic potential among the canola cultivars from which they were isolated. Because bacteria capable of using many substrates can effectively adapt to new environments, these results have implications for the use of pseudomonads as biofertilizers, biological control agents and plant growth-promoting bacteria in canola.     

Effect of long-term vineyard monoculture on rhizosphere populations of pseudomonads carrying the antimicrobial biosynthetic genes phlD and/or hcnAB

The impact of repeated culture of perennial plants (i.e. in long-term monoculture) on the ecology of plant-beneficial bacteria is unknown. Here, the influence of extremely long-term monocultures of grapevine (up to 1603 years) on rhizosphere populations of fluorescent pseudomonads carrying the biosynthetic genes phlD for 2,4-diacetylphloroglucinol and/or hcnAB for hydrogen cyanide was determined. Soils from long-term and adjacent short-term monoculture vineyards (or brushland) in four regions of Switzerland were baited with grapevine or tobacco plantlets, and rhizosphere pseudomonads were studied by most probable number (MPN)-PCR. Higher numbers and percentages of phlD ⁺ and of hcnAB ⁺ rhizosphere pseudomonads were detected on using soil from long-term vineyards. On focusing on phlD , restriction fragment length polymorphism profiling of the last phlD -positive MPN wells revealed seven phlD alleles (three exclusively on tobacco, thereof two new ones). Higher numbers of phlD alleles coincided with a lower prevalence of the allele displayed by the well-studied biocontrol strain Pseudomonas fluorescens F113. The prevalence of this allele was 35% for tobacco in long-term monoculture soils vs. >60% in the other three cases. We conclude that soils from long-term grapevine monocultures represent an untapped resource for isolating novel biocontrol Pseudomonas strains when tobacco is used as bait.     

In Vitro Studies on Reductive Vinyl Chloride Dehalogenation by an Anaerobic Mixed Culture

Reductive dehalogenation of vinyl chloride (VC) was studied in an anaerobic mixed bacterial culture. In growth experiments, ethene formation from VC increased exponentially at a rate of about 0.019 h(sup-1). Reductive VC dehalogenation was measured in vitro by using cell extracts of the mixed culture. The apparent K(infm) for VC was determined to be about 76 (mu)M; the V(infmax) was about 28 nmol (middot) min(sup-1) (middot) mg of protein(sup-1). The VC-dehalogenating activity was membrane associated. Propyl iodide had an inhibitory effect on the VC-dehalogenating activity in the in vitro assay. However, this inhibition could not be reversed by illumination. Cell extracts also catalyzed the reductive dehalogenation of cis-1,2-dichloroethene (cis-DCE) and, at a lower rate, of trichloroethene (TCE). Tetrachloroethene (PCE) was not transformed. The results indicate that the reductive dehalogenation of VC and cis-DCE described here is different from previously reported reductive dehalogenation of PCE and TCE.     

Characterization of CMR5c and CMR12a, novel fluorescent Pseudomonas strains from the cocoyam rhizosphere with biocontrol activity

AIM: To screen for novel antagonistic Pseudomonas strains producing both phenazines and biosurfactants that are as effective as Pseudomonas aeruginosa PNA1 in the biocontrol of cocoyam root rot caused by Pythium myriotylum. MATERIAL AND RESULTS: Forty pseudomonads were isolated from the rhizosphere of healthy white and red cocoyam plants appearing in natural, heavily infested fields in Cameroon. In vitro tests demonstrated that Py. myriotylum antagonists could be retrieved from the red cocoyam rhizosphere. Except for one isolate, all antagonistic isolates produced phenazines. Results from whole-cell protein profiling showed that the antagonistic isolates are different from other isolated pseudomonads, while BOX-PCR revealed high genomic similarity among them. 16S rDNA sequencing of two representative strains within this group of antagonists confirmed their relatively low similarity with validly described Pseudomonas species. These antagonists are thus provisionally labelled as unidentified Pseudomonas strains. Among the antagonists, Pseudomonas CMR5c and CMR12a were selected because of their combined production of phenazines and biosurfactants. For strain CMR5c also, production of pyrrolnitrin and pyoluteorin was demonstrated. Both CMR5c and CMR12a showed excellent in vivo biocontrol activity against Py. myriotylum to a similar level as Ps. aeruginosa PNA1. CONCLUSION: Pseudomonas CMR5c and CMR12a were identified as novel and promising biocontrol agents of Py. myriotylum on cocoyam, producing an arsenal of antagonistic metabolites. SIGNIFICANCE AND IMPACT OF THE STUDY: Present study reports the identification of two newly isolated fluorescent Pseudomonas strains that can replace the opportunistic human pathogen Ps. aeruginosa PNA1 in the biocontrol of cocoyam root rot and could be taken into account for the suppression of many plant pathogens.     

Irrigation differentially impacts populations of indigenous antibiotic-producing pseudomonas spp. in the rhizosphere of wheat

This work determined the impact of irrigation on the seasonal dynamics of populations of Pseudomonas spp. producing the antibiotics phenazine-1-carboxylic acid (Phz(+)) and 2,4-diacetylphloroglucinol (Phl(+)) in the rhizosphere of wheat grown in the low-precipitation zone (150 to 300 mm annually) of the Columbia Plateau of the Inland Pacific Northwest. Population sizes and plant colonization frequencies of Phz(+) and Phl(+) Pseudomonas spp. were determined in winter and spring wheat collected during the growing seasons from 2008 to 2009 from selected commercial dryland and irrigated fields in central Washington State. Only Phz(+) bacteria were detected on dryland winter wheat, with populations ranging from 4.8 to 6.3 log CFU g(-1) of root and rhizosphere colonization frequencies of 67 to 100%. The ranges of population densities of Phl(+) and Phz(+) Pseudomonas spp. recovered from wheat grown under irrigation were similar, but 58 to 100% of root systems were colonized by Phl(+) bacteria whereas only 8 to 50% of plants harbored Phz(+) bacteria. In addition, Phz(+) Pseudomonas spp. were abundant in the rhizosphere of native plant species growing in nonirrigated areas adjacent to the sampled dryland wheat fields. This is the first report that documents the impact of irrigation on indigenous populations of two closely related groups of antibiotic-producing pseudomonads that coinhabit the rhizosphere of an economically important cereal crop. These results demonstrate how crop management practices can influence indigenous populations of antibiotic-producing pseudomonads with the capacity to suppress soilborne diseases of wheat.     

The Low Biomass Yields of the Acetic Acid Bacterium Acetobacter pasteurianus Are Due to a Low Stoichiometry of Respiration-Coupled Proton Translocation

Growth energetics of the acetic acid bacterium Acetobacter pasteurianus were studied with aerobic, ethanol-limited chemostat cultures. In these cultures, production of acetate was negligible. Carbon limitation and energy limitation were also evident from the observation that biomass concentrations in the cultures were proportional to the concentration of ethanol in the reservoir media. Nevertheless, low concentrations of a few organic metabolites (glycolate, citrate, and mannitol) were detected in culture supernatants. From a series of chemostat cultures grown at different dilution rates, the maintenance energy requirements for ethanol and oxygen were estimated at 4.1 mmol of ethanol (middot) g of biomass(sup-1) (middot) h(sup-1) and 11.7 mmol of O(inf2) (middot) g of biomass(sup-1) (middot) h(sup-1), respectively. When biomass yields were corrected for these maintenance requirements, the Y(infmax) values on ethanol and oxygen were 13.1 g of biomass (middot) mol of ethanol(sup-1) and 5.6 g of biomass (middot) mol of O(inf2)(sup-1), respectively. These biomass yields are very low in comparison with those of other microorganisms grown under comparable conditions. To investigate whether the low growth efficiency of A. pasteurianus might be due to a low gain of metabolic energy from respiratory dissimilation, (symbl)H(sup+)/O stoichiometries were estimated during acetate oxidation by cell suspensions. These experiments indicated an (symbl)H(sup+)/O stoichiometry for acetate oxidation of 1.9 (plusmn) 0.1 mol of H(sup+)/mol of O. Theoretical calculations of growth energetics showed that this low (symbl)H(sup+)/O ratio adequately explained the low biomass yield of A. pasteurianus in ethanol-limited cultures.     

Salt adaptation in pseudomonads: characterization of glucosylglycerol-synthesizing isolates from brackish coastal waters and the rhizosphere

The compatible solute glucosylglycerol (GG) is widespread among cyanobacteria, but, until now, has been reported for only two species of heterotrophic bacteria. About 120 bacterial isolates from coastal regions of the Baltic Sea were screened by HPLC for their ability to synthesize GG. Positive isolates (26) were grouped by SDS-PAGE of whole-cell proteins and representative strains of each group were investigated by sequencing their 16S rRNA genes and phenotypic characterization. All GG-synthesizing isolates were shown to belong to the genus Pseudomonas (sensu stricto) and were assigned to 4 distinct groups, although none of the GG-synthesizing isolates could be unambiguously assigned to described species. The identity of GG was verified by 13C NMR analysis and enzymatic digestion with alpha- and beta-glucosidases. Besides GG, salt adapted cultures of the aquatic isolates accumulated the dipeptide N-acetylglutaminylglutamine amide (NAGGN) and glutamate. The accumulation of noncharged compatible solutes was also tested in previously identified pseudomonads isolated from the rhizosphere of oilseed rape and potato. The majority of these strains were fluorescent species of the genus Pseudomonas and accumulated trehalose and NAGGN when grown under salt stress conditions. However, rhizosphere isolates of Stenotrophomonas maltophilia synthesized GG and trehalose or only trehalose in a strain-dependent manner. These data indicate that the ability to synthesize GG is widely distributed among slightly or moderately halotolerant pseudomonads.     

Diversity of culturable bacterial populations associated to Tuber borchii ectomycorrhizas and their activity on T. borchii mycelial growth

Isolation and physiological and molecular characterisation of culturable bacterial strains belonging to actinomycetes, pseudomonads and aerobic spore-forming bacteria were carried out on mycorrhizal root tips of Quercus robur var. peduncolata infected by Tuber borchii. Cellular density of the three bacterial groups in ectomycorrhizal root tips was estimated to be 1.3+/-0.11 x 10(6) cfu g(-1) dry weight for total heterotrophic bacteria and 1.08+/-0.6 x 10(5) (mean+/-S.E.), 1.3+/-0.3 x 10(5) and 1.4+/-0.2 x 10(5) cfu g(-1) dry weight for pseudomonads, actinomycetes and spore-forming bacteria respectively. Identification of pseudomonads by the Biolog system indicated, besides the most represented species Pseudomonas fluorescens (biotypes B, F and G), the occurrence of strains belonging to Pseudomonas corrugata. Amplified ribosomal DNA restriction analysis of actinomycetes and spore formers revealed at least three and six different groups of patterns, respectively. Many bacterial isolates were able to induce variations in growth rates of T. borchii mycelium; among these, 101 strains showed antifungal activity, whereas 17 isolates, belonging to spore formers, were able to increase mycelial growth up to 78% when compared to uninoculated mycelial growth. The potential role of these populations in the development and establishment of mycorrhizas is discussed.     

Variation in Microbial Community Structure in Two Boreal Peatlands as Determined by Analysis of Phospholipid Fatty Acid Profiles

Analyses of phospholipid fatty acids (PLFAs) were used to assess variation in community structure and total microbial biomass in two boreal peatlands in Sweden. The total PLFA concentration in peat ranged from 0.16 to 7.0 nmol g of wet peat(sup-1) (median, 0.70 nmol g of wet peat(sup-1)). Principal-component analysis of PLFA data revealed that the degree of depth-related variation in PLFA composition was high among peatland habitats, with general differences between wet sites, with water tables within a few centimeters of the moss surface, and dry sites, with water tables >10 cm below the moss surface. However, variation in PLFA composition over the growing season was negligible. In the principal-component analyses, most PLFAs were determined to be parts of clusters of covarying fatty acids, suggesting that they originated in the same functional groups of microorganisms. Major clusters were formed by monounsaturated (typical of gram-negative eubacteria), terminally branched (gram-positive or anaerobic gram-negative eubacteria), methyl-branched and branched unsaturated (sulfate-reducing bacteria and/or actinomycetes), (omega)8 monounsaturated (methane-oxidizing bacteria), and polyunsaturated (eucaryotes) PLFAs. Within the clusters, PLFAs had rather distinct concentration-depth distributions. For example, PLFAs from sulfate-reducing bacteria and/or actinomycetes and those from methane-oxidizing bacteria had maximum concentrations slightly below and at the average water table depth, respectively.