A Method for Detection of Pseudobactin, the Siderophore Produced by a Plant-Growth-Promoting Pseudomonas Strain, in the Barley Rhizosphere

Detection in the rhizosphere of the siderophore produced by an inoculated microorganism is critical to determining the role of microbial iron chelators on plant growth promotion. We previously reported the development of monoclonal antibodies (MAb) to ferric pseudobactin, the siderophore of plant-growth-promoting Pseudomonas strain B10. One of these MAb reacted less strongly to pseudobactin than to ferric pseudobactin. The MAb reacted to Al(III), Cr(III), Cu(II), and Mn(II) complexes of pseudobactin at a level similar to the level at which it reacted to ferric pseudobactin and reacted less to the Zn(II) complex, but these metals would make up only a small fraction of chelated pseudobactin in soil on the basis of relative abundance of metals and relative binding constants. Fourteen-day-old barley plants grown in limed and autoclaved soil were inoculated with 10⁹ CFU of Pseudomonas strain Sm1-3, a strain of Pseudomonas B10 Rif^r Nal^r selected for enhanced colonization, and sampled 3 days later. Extraction and analysis of the roots and surrounding soil using the MAb in an immunoassay indicated a concentration of 3.5 × 10^-10 mol of ferric pseudobacting g^-1 (wet weight). This is the first direct measurement of a pseudobactin siderophore in soil or rhizosphere samples.     

Drought-tolerant endophytic actinobacteria promote growth of wheat (Triticum aestivum) under water stress conditions

Drought tolerant endophytic actinobacteria Streptomyces coelicolor DE07, S. olivaceus DE10 and Streptomyces geysiriensis DE27 were isolated from cultivated plants of arid and drought affected regions of Rajasthan, India. These isolates exhibited plant growth promotion traits and intrinsic water stress tolerance from ?0.05 to ?0.73 MPa. Maximum auxin production was observed in majority of actinobacterial cultures in the logarithmic to stationary phase of growth. Significant enhancement of wheat seedling vigour was recorded by the inoculation of these endophytic actinobacteria. S. olivaceus DE10 recorded maximum accumulation of indole 3-acetic acid (84.34?μg?mg^?1 protein). Culture and cell-free extract of the endophytes was applied on to wheat seeds to assess the effect on growth in water-stressed soil. Maximum yield was recorded with the inoculation of S. olivaceus DE10 culture (492.77?kg?ha^?1) and cell-free extract (262.31?kg?ha^?1). Co-inoculation of S. olivaceus DE10?+?S. geysiriensis DE27 recorded highest yield of 550.09?kg?ha^?1 while their cell-free extract yielded 524.92?kg?ha^?1. Overall, wheat seeds treated with cultures showed better plant growth and yield in comparison to control. Direct coating of cultures on seeds yielded better performance than cell-free extract coated on seeds and co-inoculation of cultures or cell-free extract proved better than single culture inoculations. Production of phytohormones, plant growth promotion traits combined with water stress tolerance potential in these endophytic actinobacteria played a cumulative synergistic role that supported enhanced plant growth promotion of wheat in the stressed soil.     

Characterization of Fluorescent Siderophore-Mediated Iron Uptake in Pseudomonas sp. Strain M114: Evidence for the Existence of an Additional Ferric Siderophore Receptor

In Pseudomonas sp. strain M114, the outer membrane receptor for ferric pseudobactin M114 was shown to transport ferric pseudobactins B10 and A225, in addition to its own. The gene encoding this receptor, which was previously cloned on pCUP3, was localized by Tn5 mutagenesis to a region comprising >1.6 kb of M114 DNA. A mutant (strain M114R1) lacking this receptor was then created by a marker exchange technique. Characterization of this mutant by using purified pseudobactin M114 in radiolabeled ferric iron uptake studies confirmed that it was completely unable to utilize this siderophore for acquisition of iron. In addition, it lacked an outer membrane protein band of 89 kDa when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. As a result, growth of the mutant was severely restricted under low-iron conditions. However, this phenotype was reversed in the presence of another fluorescent siderophore (pseudobactin MT3A) from Pseudomonas sp. strain MT3A, suggesting the presence of a second receptor in strain M114. Furthermore, wild-type Pseudomonas sp. strain B24 was not able to utilize ferric pseudobactin MT3A, and this phenotype was not reversed upon expression of the M114 receptor encoded on pCUP3. However, a cosmid clone (pMS1047) that enabled strain B24 to utilize ferric pseudobactin MT3A was isolated from an M114 gene bank. Radiolabel transport assays with purified pseudobactin MT3A confirmed this event. Plasmid pMS1047 was shown to encode an outer membrane protein of 81 kDa in strain B24 under iron-limiting conditions; this protein corresponds to a similar protein in strain M114.     

Characterization of Fe(III) Reduction by Chlororespiring Anaeromxyobacter dehalogenans

Anaeromyxobacter dehalogenans strain 2CP-C has been shown to grow by coupling the oxidation of acetate to the reduction of ortho-substituted halophenols, oxygen, nitrate, nitrite, or fumarate. In this study, strain 2CP-C was also found to grow by coupling Fe(III) reduction to the oxidation of acetate, making it one of the few isolates capable of growth by both metal reduction and chlororespiration. Doubling times for growth of 9.2 and 10.2 h were determined for Fe(III) and 2-chlorophenol reduction, respectively. These were determined by using the rate of [¹⁴C]acetate uptake into biomass. Fe(III) compounds used by strain 2CP-C include ferric citrate, ferric pyrophosphate, and amorphous ferric oxyhydroxide. The addition of the humic acid analog anthraquinone 2,6-disulfonate (AQDS) increased the reduction rate of amorphous ferric iron oxide, suggesting AQDS was used as an electron shuttle by strain 2CP-C. The addition of chloramphenicol to fumarate-grown cells did not inhibit Fe(III) reduction, indicating that the latter activity is constitutive. In contrast, the addition of chloramphenicol inhibited dechlorination activity, indicating that chlororespiration is inducible. The presence of insoluble Fe(III) oxyhydroxide did not significantly affect dechlorination, whereas the presence of soluble ferric pyrophosphate inhibited dechlorination. With its ability to respire chlorinated organic compounds and metals such as Fe(III), strain 2CP-C is a promising model organism for the study of the interaction of these potentially competing processes in contaminated environments.     

Growth in Coculture Stimulates Metabolism of the Phenylurea Herbicide Isoproturon by Sphingomonas sp. Strain SRS2

Metabolism of the phenylurea herbicide isoproturon by Sphingomonas sp. strain SRS2 was significantly enhanced when the strain was grown in coculture with a soil bacterium (designated strain SRS1). Both members of this consortium were isolated from a highly enriched isoproturon-degrading culture derived from an agricultural soil previously treated regularly with the herbicide. Based on analysis of the 16S rRNA gene, strain SRS1 was assigned to the β-subdivision of the proteobacteria and probably represents a new genus. Strain SRS1 was unable to degrade either isoproturon or its known metabolites 3-(4-isopropylphenyl)-1-methylurea, 3-(4-isopropylphenyl)-urea, or 4-isopropyl-aniline. Pure culture studies indicate that Sphingomonas sp. SRS2 is auxotrophic and requires components supplied by association with other soil bacteria. A specific mixture of amino acids appeared to meet these requirements, and it was shown that methionine was essential for Sphingomonas sp. SRS2. This suggests that strain SRS1 supplies amino acids to Sphingomonas sp. SRS2, thereby leading to rapid metabolism of ¹⁴C-labeled isoproturon to ¹⁴CO₂ and corresponding growth of strain SRS2. Proliferation of strain SRS1 suggests that isoproturon metabolism by Sphingomonas sp. SRS2 provides unknown metabolites or cell debris that supports growth of strain SRS1. The role of strain SRS1 in the consortium was not ubiquitous among soil bacteria; however, the indigenous soil microflora and some strains from culture collections also stimulate isoproturon metabolism by Sphingomonas sp. strain SRS2 to a similar extent.     

Evaluating the potential of <Emphasis Type="Italic">N. calcicola</Emphasis> and its bicarbonate resistant mutant as bioameleorating agents for ‘usar’ soil

The potential of Nostoc calcicola and its bicarbonate resistant mutant as bioameleorating agent was investigated, using laboratory simulation experiments, in terms of their growth potential, glutamine synthetase (GS) activity, heterocyst frequency and effect on pH of soil. Nostoc calcicola, exhibited a tendency to lower the pH of ‘usar’ soil significantly and showed better growth and pigment content at 20% soil extract as compared to basal medium. The bicarbonate resistant mutant (HCO₃ ^−R) exhibited a better ability to grow at higher percentage of soil extract (60%), besides bringing about a more significant change in soil pH as compared to wild type. The heterocyst frequency was much higher in the mutant strain, which was not significantly affected by growth in various concentrations of soil extract. The mutant strain holds promise as a potential bioameliorant for ‘usar’ soil after further evaluation of its reclamative properties at field level.     

Active Transport of Iron in Bacillus megaterium: Role of Secondary Hydroxamic Acids

Kinetics of radioactive iron transport were examined in three strains of Bacillus megaterium. In strain ATCC 19213, which secretes the ferric-chelating secondary hydroxamic acid schizokinen, ⁵⁹Fe³⁺ uptake from ⁵⁹FeCl₃ or the ferric hydroxamate Desferal-⁵⁹Fe³⁺ was rapid and reached saturation within 3 min. In strain SK11, which does not secrete schizokinen, transport from ⁵⁹FeCl₃ was markedly reduced; the two ferric hydroxamates Desferal-⁵⁹Fe³⁺ or schizokinen-⁵⁹Fe³⁺ increased both total ⁵⁹Fe³⁺ uptake and the ⁵⁹Fe³⁺ appearing in a cellular trichloroacetic acid-insoluble fraction, although 10 min was required to reach saturation. Certain characteristics of transport from both ferric hydroxamates and FeCl₃ suggest that iron uptake was an active process. The growth-inhibitory effect of aluminum on strain SK11 was probably due to the formation of nonutilizable iron-aluminum complexes which blocked uptake from ⁵⁹FeCl₃. Desferal or schizokinen prevented this blockage. A strain (ARD-1) resistant to the ferric hydroxamate antibiotic A22765 was isolated from strain SK11. Strain ARD-1 failed to grow with Desferal-Fe³⁺ as an iron source, and it was unable to incorporate ⁵⁹Fe³⁺ from this source. Growth and iron uptake in strain ARD-1 were similar to strain SK11 with schizokinen-Fe³⁺ or the iron salt as sources. It is suggested that the ferric hydroxamates, or the iron they chelate, may be transported by a special system which might be selective for certain ferric hydroxamates. Strain ARD-1 may be unable to recognize both the antibiotic A22765 and the structurally similar chelate Desferal-Fe³⁺, while retaining its capacity to utilize schizokinen-Fe³⁺.     

Rhizosphere colonization and arsenic translocation in sunflower (Helianthus annuus L.) by arsenate reducing Alcaligenes sp. strain Dhal-L

In the present study, six arsenic-resistant strains previously isolated were tested for their plant growth promoting characteristics and heavy metal resistance, in order to choose one model strain as an inoculum for sunflower plants in pot experiments. The aim was to investigate the effect of arsenic-resistant strain on sunflower growth and on arsenic uptake from arsenic contaminated soil. Based on plant growth promoting characteristics and heavy metal resistance, Alcaligenes sp. strain Dhal-L was chosen as an inoculum. Beside the ability to reduce arsenate to arsenite via an Ars operon, the strain exhibited 1-amino-cyclopropane-1-carboxylic acid deaminase activity and it was also able to produce siderophore and indole acetic acid. Pot experiments were conducted with an agricultural soil contaminated with arsenic (214 mg kg^?1). A real time PCR method was set up based on the quantification of ACR3(2) type of arsenite efflux pump carried by Alcaligenes sp. strain Dhal-L, in order to monitor presence and colonisation of the strain in the bulk and rhizospheric soil. As a result of strain inoculation, arsenic uptake by plants was increased by 53 %, whereas ACR3(2) gene copy number in rhizospheric soil was 100 times higher in inoculated than in control pots, indicating the colonisation of strain. The results indicated that the presence of arsenate reducing strains in the rhizosphere of sunflower influences arsenic mobilization and promotes arsenic uptake by plant.     

Blastocatella fastidiosa gen. nov., sp. nov., isolated from semiarid savanna soil – The first described species of Acidobacteria subdivision 4

Acidobacteria represent abundant members of soil microbial communities but only few representatives could be isolated and validly described so far. Currently, eighteen species of subdivision 1, one species of subdivision 3, three species of subdivision 8, and one species of subdivision 10 are recognized. In contrast, Acidobacteria of subdivision 4 have largely escaped cultivation although they belong to the most abundant and diverse acidobacterial groups in soils. A member of subdivision 4, strain A2-16^T, was isolated from a semiarid savanna soil. Cells were motile spheres to rods with a tendency to form chains and larger aggregates. Cultures were orange to pink colored, neutrophilic mesophiles, and showed aerobic chemoorganoheterotrophic growth on very few complex substrates and protocatechuate, and weak growth on chitin, cellulose and starch. While protein substrates such as casamino acids or peptone were utilized, individual amino acids did not promote growth. Also, growth on alternative electron acceptors or fermentative growth could not be observed. Major fatty acids were summed features 1 (15:1 iso H/13:0 3-OH) and 3 (16:1ω7c/15:0 iso 2-OH). The major quinone was MK-8. The DNA G+C content was 46.5 mol%. Phylogenetic analysis placed A2-16^T amidst uncultured members of Acidobacteria subdivision 4. The most closely related environmental 16S rRNA gene sequences (96–97% nucleotide identity) were several clone sequences from terrestrial environments. Based on these characteristics, the isolated strain is proposed as a new species of a novel genus, Blastocatella fastidiosa gen. nov., sp. nov. The type strain of B. fastidiosa is A2-16^T (=DSM 25172^T = LMG26944^T).     

Degradation of 3-Phenoxybenzoic Acid in Soil by Pseudomonas pseudoalcaligenes POB310(pPOB) and Two Modified Pseudomonas Strains

Pseudomonas pseudoalcaligenes POB310(pPOB) and Pseudomonas sp. strains B13-D5(pD30.9) and B13-ST1(pPOB) were introduced into soil microcosms containing 3-phenoxybenzoic acid (3-POB) in order to evaluate and compare bacterial survival, degradation of 3-POB, and transfer of plasmids to a recipient bacterium. Strain POB310 was isolated for its ability to use 3-POB as a growth substrate; degradation is initiated by POB-dioxygenase, an enzyme encoded on pPOB. Strain B13-D5 contains pD30.9, a cloning vector harboring the genes encoding POB-dioxygenase; strain B13-ST1 contains pPOB. Degradation of 3-POB in soil by strain POB310 was incomplete, and bacterial densities decreased even under the most favorable conditions (100 ppm of 3-POB, supplementation with P and N, and soil water-holding capacity of 90%). Strains B13-D5 and B13-ST1 degraded 3-POB (10 to 100 ppm) to concentrations of <50 ppb with concomitant increases in density from 10⁶ to 10⁸ CFU/g (dry weight) of soil. Thus, in contrast to strain POB310, the modified strains had the following two features that are important for in situ bioremediation: survival in soil and growth concurrent with removal of an environmental contaminant. Strains B13-D5 and B13-ST1 also completely degraded 3-POB when the inoculum was only 30 CFU/g (dry weight) of soil. This suggests that in situ bioremediation may be effected, in some cases, with low densities of introduced bacteria. In pure culture, transfer of pPOB from strains POB310 and B13-ST1 to Pseudomonas sp. strain B13 occurred at frequencies of 5 × 10⁻⁷ and 10⁻¹ transconjugant per donor, respectively. Transfer of pPOB from strain B13-ST1 to strain B13 was observed in autoclaved soil but not in nonautoclaved soil; formation of transconjugant bacteria was more rapid in soil containing clay and organic matter than in sandy soil. Transfer of pPOB from strain POB310 to strain B13 in soil was never observed.     

Role of a Ferric Ion-Reducing System in Sulfur Oxidation of Thiobacillus ferrooxidans

The properties of a ferric ion-reducing system which catalyzes the reduction of ferric ion with elemental sulfur was investigated with a pure strain of Thiobacillus ferrooxidans. In anaerobic conditions, washed intact cells of the strain reduced 6 mol of Fe³⁺ with 1 mol of elemental sulfur to give 6 mol of Fe²⁺, 1 mol of sulfate, and a small amount of sulfite. In aerobic conditions, the 6 mol of Fe²⁺ produced was immediately reoxidized by the iron oxidase of the cell, with a consumption of 1.5 mol of oxygen. As a result, Fe²⁺ production was never observed under aerobic conditions. However, in the presence of 5 mM cyanide, which completely inhibits the iron oxidase of the cell, an amount of Fe²⁺ production comparable to that formed under anaerobic conditions was observed under aerobic conditions. The ferric ion-reducing system had a pH optimum between 2.0 and 3.8, and the activity was completely destroyed by 10 min of incubation at 60°C. A short treatment of the strain with 0.5% phenol completely destroyed the ferric ion-reducing system of the cell. However, this treatment did not affect the iron oxidase of the cell. Since a concomitant complete loss of the activity of sulfur oxidation by molecular oxygen was observed in 0.5% phenol-treated cells, it was concluded that the ferric ion-reducing system plays an important role in the sulfur oxidation activity of this strain, and a new sulfur-oxidizing route is proposed for T. ferrooxidans.     

Characterization of a Symbiotic Coculture of Clostridium thermohydrosulfuricum YM3 and Clostridium thermocellum YM4

Clostridium thermohydrosulfuricum YM3 and C. thermocellum YM4 were isolated from a coculture which was obtained from an enrichment culture inoculated with volcanic soil in Izu Peninsula, Japan. Strain YM3 had advantages over reported C. thermohydrosulfuricum strains in that it fermented inulin and could accumulate ethanol up to 1.3% (wt/vol). The highest ethanol yield obtained was 1.96 mol/mol of anhydroglucose unit in cellobiose. Strain YM4 had features different from those reported in C. thermocellum strains: it formed spores rarely (at a frequency of <10^-5), it required CO₂ and Na₂CO₃ for growth, and it fermented sucrose. Strain YM4 completely decomposed 1% Avicel within 25 h when the inoculum constituted 2% of the culture medium volume, and it produced 0.22 U of Avicelase and 2.21 U of carboxymethylcellulase per ml of the medium. The doubling times on Avicel, cellobiose, and glucose were 2.7, 1.1, and 1.6 h, respectively. Reconstructed cocultures of strains YM3 and YM4 were very stable and degraded Avicel more rapidly than did strain YM4 monoculture. Without yeast extract, neither microorganism was able to grow. However, the coculture grew on cellulose without yeast extract and produced ethanol in high yield. Moreover, cell-free spent culture broth of strain YM3 could replace yeast extract in supporting the growth of strain YM4. The symbiotic relationship of the two bacteria in cellulose fermentation is probably a case of mutualism.     

Influence of the nutritional conditions on haloalcohol dehalogenase HheC production by recombinant Escherichia coli P84A/MC1061

In this research, we first determined the three most significant nutrient factors affecting haloalcohol dehalogenase HheC production by Escherichia coli P84A/MC1061. These were glycerol, yeast extract, and ammonium sulfate. The steepest ascent method was then applied to obtain the optimal design intervals of the three factors. An application of center composite design was used, and the ingredients of the optimized medium were 1.8 g l^?1 glycerol, 48 g l^?1 yeast extract, 2.2 g l^?1 ammonium sulfate, 5 g l^?1 compound phosphate, 1 g l^?1 magnesium sulfate, and 1.19?×?10^?5?g l^?1 ferric sulfate. The enzyme activity reached 109,365 U ml^?1 under the most favorable conditions, which is a 277.7 % increase compared with the control group. Our study of cellular respiration parameters (oxygen uptake rate and carbon dioxide emission rate) revealed that the metabolic activity of the strain was strongly promoted under these optimal nutrient conditions and that yeast extract had a positive effect on respiratory intensity and the expression levels of HheC.     

Nutritional Requirements of Methanosarcina sp. Strain TM-1

Methanosarcina sp. strain TM-1, an acetotrophic, thermophilic methanogen isolated from an anaerobic sludge digestor, was originally reported to require an anaerobic sludge supernatant for growth. It was found that the sludge supernatant could be replaced with yeast extract (1 g/liter), 6 mM bicarbonate-30% CO₂, and trace metals, with a doubling time on methanol of 14 h. For growth on either methanol or acetate, yeast extract could be replaced with CaCl₂ · 2H₂O (13.6 μM minimum) and the vitamin p-aminobenzoic acid (PABA, ca. 3 nM minimum), with a doubling time on methanol of 8 to 9 h. Filter-sterilized folic acid at 0.3 μM could not replace PABA. The antimetabolite sulfanilamide (20 mM) inhibited growth of and methanogenesis by Methanosarcina sp. strain TM-1, and this inhibition was reversed by the addition of 0.3 μM PABA. When a defined medium buffered with 20 mM N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid was used, it was shown that Methanosarcina sp. strain TM-1 required 6 mM bicarbonate-30% CO₂ for optimal growth and methanogenesis from methanol. Cells growing on acetate were less dependent on bicarbonate-CO₂. When we used a defined medium in which the only organic compounds present were methanol or acetate, nitrilotriacetic acid (0.2 mM), and PABA, it was possible to limit batch cultures of Methanosarcina sp. strain TM-1 for nitrogen at NH₄⁺ concentrations at or below 2.0 mM, in marked contrast with Methanosarcina barkeri 227, which fixes dinitrogen when grown under NH₄⁺ limitation.     

Isolation and Characterization of a Mo6+ -Reducing Bacterium

A Mo⁶⁺ -reducing bacterium (strain 48), which grew on medium supplemented with 200 mM Mo⁶⁺, was isolated from stream water obtained from Chengkau, Malaysia. The chemical properties of strain 48 conform to the characteristics of Enterobacter cloacae. Under anaerobic conditions in the glucose-yeast extract medium containing phosphate ion (2.9 mM) and Mo⁶⁺ (10 mM), the bacterium reduced Mo⁶⁺ to form molybdenum blue. Approximately 27% of Mo⁶⁺ added to the medium was reduced after 28 h of cultivation. The reduction of Mo⁶⁺ with glucose as an electron donor was strongly inhibited by iodoacetic acid, sodium fluoride, and sodium cyanide, suggesting an involvement of the glycolytic pathway and electron transport in Mo⁶⁺ reduction. NADH and N,N,N′,N′ -tetramethyl-p-phenylenediamine served as electron donors for Mo⁶⁺ reduction. When NADH was used as an electron donor, at first cytochrome b in the cell extract was reduced, and then molybdenum blue was formed. Sodium cyanide strongly inhibited Mo⁶⁺ reduction by NADH (5 mM) but not the reduction of cytochrome b in the cell extract, suggesting that the reduced component of the electron transport system after cytochrome b serves as an electron donor for Mo⁶⁺ reduction. Both ferric and stannous ions strongly enhanced the activity of Mo⁶⁺ reduction by NADH.     

High Frequency of Natural Genetic Transformation of Pseudomonas stutzeri in Soil Extract Supplemented with a Carbon/Energy and Phosphorus Source

Agar medium (SME) prepared from aqueous soil extract was used to examine genetic transformation of Pseudomonas stutzeri JM302 (his-1) by homologous his⁺ DNA in a plate transformation assay. Growth studies indicated that SME was strongly limited in carbon and nitrogen sources. Transformation was observed on SME supplemented with pyruvate, phosphate, and ammonium. A 25-fold increase of the transformation frequency was obtained with nitrogen limitation when SME was supplemented with only pyruvate plus phosphate. Similar results were obtained with artificial soil extract medium prepared on the basis of the chemical analysis of the soil extract. On a standard minimal medium, transformation frequencies also increased (10- to 60-fold) when ammonium, phosphate, or pyruvate was growth limiting. Limitation of two or three nutrients did not stimulate transformation. The size of the inoculum (2 × 10³ to 2 × 10⁷ cells) was irrelevant to the enhanced transformation under nitrogen limitation on SME or standard minimal medium. We further show that P. stutzeri can use a variety of carbon and energy sources for competence development. It is concluded that genetic transformation of P. stutzeri is possible in the chemical environment of soil upon supply of nutrients and may be strongly stimulated by a growth-limiting concentration of single nutrients including sources of C, N, or P.     

Characterization of Ferroplasma Isolates and Ferroplasma acidarmanus sp. nov., Extreme Acidophiles from Acid Mine Drainage and Industrial Bioleaching Environments

Three recently isolated extremely acidophilic archaeal strains have been shown to be phylogenetically similar to Ferroplasma acidiphilum Y^T by 16S rRNA gene sequencing. All four Ferroplasma isolates were capable of growing chemoorganotrophically on yeast extract or a range of sugars and chemomixotrophically on ferrous iron and yeast extract or sugars, and isolate “Ferroplasma acidarmanus” Fer1^T required much higher levels of organic carbon. All four isolates were facultative anaerobes, coupling chemoorganotrophic growth on yeast extract to the reduction of ferric iron. The temperature optima for the four isolates were between 35 and 42°C and the pH optima were 1.0 to 1.7, and “F. acidarmanus” Fer1^T was capable of growing at pH 0. The optimum yeast extract concentration for “F. acidarmanus” Fer1^T was higher than that for the other three isolates. Phenotypic results suggested that isolate “F. acidarmanus” Fer1^T is of a different species than the other three strains, and 16S rRNA sequence data, DNA-DNA similarity values, and two-dimensional polyacrylamide gel electrophoresis protein profiles clearly showed that strains DR1, MT17, and Y^T group as a single species. “F. acidarmanus” Fer1^T groups separately, and we propose the new species “F. acidarmanus” Fer1^T sp. nov.     

Rhizoremediation of Trichloroethylene by a Recombinant, Root-Colonizing Pseudomonas fluorescens Strain Expressing Toluene ortho-Monooxygenase Constitutively

Trichloroethylene (TCE) was removed from soils by using a wheat rhizosphere established by coating seeds with a recombinant, TCE-degrading Pseudomonas fluorescens strain that expresses the tomA⁺ (toluene o-monooxygenase) genes from Burkholderia cepacia PR1₂₃(TOM_23C). A transposon integration vector was used to insert tomA⁺ into the chromosome of P. fluorescens 2-79, producing a stable strain that expressed constitutively the monooxygenase at a level of 1.1 nmol/min · mg of protein (initial TCE concentration, 10 μM, assuming that all of the TCE was in the liquid) for more than 280 cell generations (36 days). We also constructed a salicylate-inducible P. fluorescens strain that degraded TCE at an initial rate of 2.6 nmol/min · mg of protein in the presence of 10 μM TCE [cf. B. cepacia G4 PR1₂₃(TOM_23C), which degraded TCE at an initial rate of 2.5 nmol/min · mg of protein]. A constitutive strain, P. fluorescens 2-79TOM, grew (maximum specific growth rate, 0.78 h⁻¹) and colonized wheat (3 × 10⁶ CFU/cm of root) as well as wild-type P. fluorescens 2-79 (maximum specific growth rate, 0.77 h⁻¹; level of colonization, 4 × 10⁶ CFU/cm of root). Rhizoremediation of TCE was demonstrated by using microcosms containing the constitutive monooxygenase-expressing microorganism, soil, and wheat. These closed microcosms degraded an average of 63% of the initial TCE in 4 days (20.6 nmol of TCE/day · plant), compared to the 9% of the initial TCE removed by negative controls consisting of microcosms containing wild-type P. fluorescens 2-79-inoculated wheat, uninoculated wheat, or sterile soil.     

Role of Chemotaxis in the Ecology of Denitrifiers

A modification of the Adler capillary assay was used to evaluate the chemotactic responses of several denitrifiers to nitrate and nitrite. Strong positive chemotaxis was observed to NO₃⁻ and NO₂⁻ by soil isolates of Pseudomonas aeruginosa, Pseudomonas fluorescens, and Pseudomonas stutzeri, with the peak response occurring at 10⁻³ M for both attractants. In addition, a strong chemoattraction to serine (peak response at 10⁻² M), tryptone, and a soil extract, but not to NH₄⁺, was observed for all denitrifiers tested. Chemotaxis was not dependent on a previous growth on NO₃⁻, NO₂⁻, or a soil extract, and the chemoattraction to NO₃⁻ occurred when the bacteria were grown aerobically or anaerobically. However, the best response to NO₃⁻ was usually observed when the cells were grown aerobically with 10 mM NO₃⁻ in the growth medium. Capillary tubes containing 10⁻3 M NO₃⁻ submerged into soil-water mixtures elicited a significant chemotactic response to NO₃⁻ by the indigenous soil microflora, the majority of which were Pseudomonas spp. A chemotactic strain of P. fluorescens also was shown to survive significantly better in aerobic and anaerobic soils than was a nonmotile strain of the same species. Both strains had equal growth rates in liquid cultures. Thus, chemotaxis may be one mechanism by which denitrifiers successfully compete for available NO₃⁻ and NO₂⁻, and which may facilitate the survival of naturally occurring populations of some denitrifiers.     

Enrichment and characterization of an autotrophic ammonia-oxidizing archaeon of mesophilic crenarchaeal group I.1a from an agricultural soil

Soil nitrification is an important process for agricultural productivity and environmental pollution. Though one cultivated representative of ammonia-oxidizing Archaea from soil has been described, additional representatives warrant characterization. We describe an ammonia-oxidizing archaeon (strain MY1) in a highly enriched culture derived from agricultural soil. Fluorescence in situ hybridization microscopy showed that, after 2 years of enrichment, the culture was composed of >90% archaeal cells. Clone libraries of both 16S rRNA and archaeal amoA genes featured a single sequence each. No bacterial amoA genes could be detected by PCR. A [¹³C]bicarbonate assimilation assay showed stoichiometric incorporation of ¹³C into Archaea-specific glycerol dialkyl glycerol tetraethers. Strain MY1 falls phylogenetically within crenarchaeal group I.1a; sequence comparisons to “Candidatus Nitrosopumilus maritimus” revealed 96.9% 16S rRNA and 89.2% amoA gene similarities. Completed growth assays showed strain MY1 to be chemoautotrophic, mesophilic (optimum at 25°C), neutrophilic (optimum at pH 6.5 to 7.0), and nonhalophilic (optimum at 0.2 to 0.4% salinity). Kinetic respirometry assays showed that strain MY1''s affinities for ammonia and oxygen were much higher than those of ammonia-oxidizing bacteria (AOB). The yield of the greenhouse gas N₂O in the strain MY1 culture was lower but comparable to that of soil AOB. We propose that this new soil ammonia-oxidizing archaeon be designated “Candidatus Nitrosoarchaeum koreensis.”