A Bioluminescent Whole-Cell Reporter for Detection of 2,4-Dichlorophenoxyacetic Acid and 2,4-Dichlorophenol in Soil

A bioreporter was made containing a tfdRP_DII-luxCDABE fusion in a modified mini-Tn5 construct. When it was introduced into the chromosome of Ralstonia eutropha JMP134, the resulting strain, JMP134-32, produced a sensitive bioluminescent response to 2,4-dichlorophenoxyacetic acid (2,4-D) at concentrations of 2.0 μM to 5.0 mM. This response was linear (R² = 0.9825) in the range of 2.0 μM to 1.1 × 10² μM. Saturation occurred at higher concentrations, with maximal bioluminescence occurring in the presence of approximately 1.2 mM 2,4-D. A sensitive response was also recorded in the presence of 2,4-dichlorophenol at concentrations below 1.1 × 10² μM; however, only a limited bioluminescent response was recorded in the presence of 3-chlorobenzoic acid at concentrations below 1.0 mM. A significant bioluminescent response was also recorded when strain JMP134-32 was incubated with soils containing aged 2,4-D residues.     

Detection and Characterization of Plasmid pJP4 Transfer to Indigenous Soil Bacteria

Prior to gene transfer experiments performed with nonsterile soil, plasmid pJP4 was introduced into a donor microorganism, Escherichia coli ATCC 15224, by plate mating with Ralstonia eutropha JMP134. Genes on this plasmid encode mercury resistance and partial 2,4-dichlorophenoxyacetic acid (2,4-D) degradation. The E. coli donor lacks the chromosomal genes necessary for mineralization of 2,4-D, and this fact allows presumptive transconjugants obtained in gene transfer studies to be selected by plating on media containing 2,4-D as the carbon source. Use of this donor counterselection approach enabled detection of plasmid pJP4 transfer to indigenous populations in soils and under conditions where it had previously not been detected. In Madera Canyon soil, the sizes of the populations of presumptive indigenous transconjugants were 10⁷ and 10⁸ transconjugants g of dry soil⁻¹ for samples supplemented with 500 and 1,000 μg of 2,4-D g of dry soil⁻¹, respectively. Enterobacterial repetitive intergenic consensus PCR analysis of transconjugants resulted in diverse molecular fingerprints. Biolog analysis showed that all of the transconjugants were members of the genus Burkholderia or the genus Pseudomonas. No mercury-resistant, 2,4-D-degrading microorganisms containing large plasmids or the tfdB gene were found in 2,4-D-amended uninoculated control microcosms. Thus, all of the 2,4-D-degrading isolates that contained a plasmid whose size was similar to the size of pJP4, contained the tfdB gene, and exhibited mercury resistance were considered transconjugants. In addition, slightly enhanced rates of 2,4-D degradation were observed at distinct times in soil that supported transconjugant populations compared to controls in which no gene transfer was detected.     

Dual-bioaugmentation strategy to enhance remediation of cocontaminated soil.

Although metals are thought to inhibit the ability of microorganisms to degrade organic pollutants, several microbial mechanisms of resistance to metal are known to exist. This study examined the potential of cadmium-resistant microorganisms to reduce soluble cadmium levels to enhance degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) under conditions of cocontamination. Four cadmium-resistant soil microorganisms were examined in this study. Resistant up to a cadmium concentration of 275 microg ml(-1), these isolates represented the common soil genera Arthrobacter, Bacillus, and Pseudomonas. Isolates Pseudomonas sp. strain H1 and Bacillus sp. strain H9 had a plasmid-dependent intracellular mechanism of cadmium detoxification, reducing soluble cadmium levels by 36%. Isolates Arthrobacter strain D9 and Pseudomonas strain I1a both produced an extracellular polymer layer that bound and reduced soluble cadmium levels by 22 and 11%, respectively. Although none of the cadmium-resistant isolates could degrade 2,4-D, results of dual-bioaugmentation studies conducted with both pure culture and laboratory soil microcosms showed that each of four cadmium-resistant isolates supported the degradation of 500-microg ml(-1) 2,4-D by the cadmium-sensitive 2,4-D degrader Ralstonia eutropha JMP134. Degradation occurred in the presence of up to 24 microg of cadmium ml(-1) in pure culture and up to 60 microg of cadmium g(-1) in amended soil microcosms. In a pilot field study conducted with 5-gallon soil bioreactors, the dual-bioaugmentation strategy was again evaluated. Here, the cadmium-resistant isolate Pseudomonas strain H1 enhanced degradation of 2,4-D in reactors inoculated with R. eutropha JMP134 in the presence of 60 microg of cadmium g(-1). Overall, dual bioaugmentation appears to be a viable approach in the remediation of cocontaminated soils.     

Estimation of the Yield Coefficient of Pseudomonas sp. Strain DP-4 with a Low Substrate (2,4-Dichlorophenol [DCP]) Concentration in a Mineral Medium from Which Uncharacterized Organic Compounds Were Eliminated by a Non-DCP-Degrading Organism

The yield coefficient (YC) of Pseudomonas sp. strain DP-4, a 2,4-dichlorophenol (DCP)-degrading organism, was estimated from the number of CFU produced at the expense of 1 unit amount of DCP at low concentrations. At a low concentration of DCP, the YC can be overestimated in pure culture, because DP-4 assimilated not only DCP but also uncharacterized organic compounds contaminating a mineral salt medium. The concentration of these uncharacterized organic compounds was nutritionally equivalent to 0.7 μg of DCP-C ml⁻¹. A mixed culture with non-DCP-degrading organisms resulted in elimination of ca. 99.9% of the uncharacterized organic compounds, and then DP-4 assimilated only DCP as a substrate. In a mixed culture, DP-4 degraded an initial concentration of 0.1 to 10 μg of C ml of DCP⁻¹ and the number of CFU of DP-4 increased. In the mixed culture, DCP at an initial concentration of 0.07 μg of C ml⁻¹ was degraded. However, the number of CFU of DP-4 did not increase. DCP at an extremely low initial concentration of 0.01 μg of C ml⁻¹ was not degraded in mixed culture even by a high density, 10⁵ CFU ml⁻¹, of DP-4. When glucose was added to this mixed culture to a final concentration of 1 μg of C ml⁻¹, the initial concentration of 0.01 μg of C ml of DCP⁻¹ was degraded. These results suggested that DP-4 required cosubstrates to degrade DCP at an extremely low initial concentration of 0.01 μg of C ml⁻¹. The YCs of DP-4 at the expense of DCP alone decreased discontinuously with the decrease of the initial concentration of DCP, i.e., 1.5, 0.19, or 0 CFU per pg of DCP-C when 0.7 to 10, 0.1 to 0.5, or 0.07 μg of C ml of DCP⁻¹ was degraded, respectively. In this study, we developed a new method to eliminate uncharacterized organic compounds, and we estimated the YC of DP-4 at the expense of DCP as a sole source of carbon.     

Antibacterial Activity Evaluation of Microcin J25 against Diarrheagenic Escherichia coli

The inhibitory activities of known microcins were evaluated against some diarrheagenic Escherichia coli strains. Some antibacterial properties of microcin J25, the most active one, were studied. A rapid two-step purification was performed. The MIC and the minimum bactericidal concentration of J25 against E. coli O157:H7 were 1 and 100 μg ml⁻¹, respectively. A 10⁴-CFU ml⁻¹ contamination by this strain was destroyed in milk and meat extract by 6.25 μg of J25 ml⁻¹ and in half-diluted egg yolk by 50 μg of J25 ml⁻¹.     

Biodegradation of Methyl tert-Butyl Ether by a Bacterial Pure Culture

A bacterial strain, PM1, which is able to utilize methyl tert-butyl ether (MTBE) as its sole carbon and energy source, was isolated from a mixed microbial consortium in a compost biofilter capable of degrading MTBE. Initial linear rates of MTBE degradation by 2 × 10⁶ cells ml⁻¹ were 0.07, 1.17, and 3.56 μg ml⁻¹ h⁻¹ for initial concentrations of 5, 50, and 500 μg MTBE ml⁻¹, respectively. When incubated with 20 μg of uniformly labeled [¹⁴C]MTBE ml⁻¹, strain PM1 converted 46% to ¹⁴CO₂ and 19% to ¹⁴C-labeled cells within 120 h. This yield is consistent with the measurement of protein accumulation at different MTBE concentrations from which was estimated a biomass yield of 0.18 mg of cells mg MTBE⁻¹. Strain PM1 was inoculated into sediment core material collected from a contaminated groundwater plume at Port Hueneme, California, in which there was no evidence of MTBE degradation. Strain PM1 readily degraded 20 μg of MTBE ml⁻¹ added to the core material. The rate of MTBE removal increased with additional inputs of 20 μg of MTBE ml⁻¹. These results suggest that PM1 has potential for use in the remediation of MTBE-contaminated environments.     

Chromosomal Gene Inactivation in the Green Sulfur Bacterium Chlorobium tepidum by Natural Transformation

Conditions for inactivating chromosomal genes of Chlorobium tepidum by natural transformation and homologous recombination were established. As a model, mutants unable to perform nitrogen fixation were constructed by interrupting nifD with various antibiotic resistance markers. Growth of wild-type C. tepidum at 40°C on agar plates could be completely inhibited by 100 μg of gentamicin ml⁻¹, 2 μg of erythromycin ml⁻¹, 30 μg of chloramphenicol ml⁻¹, or 1 μg of tetracycline ml⁻¹ or a combination of 300 μg of streptomycin ml⁻¹ and 150 μg of spectinomycin ml⁻¹. Transformation was performed by spotting cells and DNA on an agar plate for 10 to 20 h. Transformation frequencies on the order of 10⁻⁷ were observed with gentamicin and erythromycin markers, and transformation frequencies on the order of 10⁻³ were observed with a streptomycin-spectinomycin marker. The frequency of spontaneous mutants resistant to gentamicin, erythromycin, or spectinomycin-streptomycin was undetectable or significantly lower than the transformation frequency. Transformation with the gentamicin marker was observed when the transforming DNA contained 1 or 3 kb of total homologous flanking sequence but not when the transforming DNA contained only 0.3 kb of homologous sequence. Linearized plasmids transformed at least an order of magnitude better than circular plasmids. This work forms a foundation for the systematic targeted inactivation of genes in C. tepidum, whose 2.15-Mb genome has recently been completely sequenced.     

Enhanced Mineralization of [U-14C]2,4-Dichlorophenoxyacetic Acid in Soil from the Rhizosphere of Trifolium pratense

Enhanced biodegradation in the rhizosphere has been reported for many organic xenobiotic compounds, although the mechanisms are not fully understood. The purpose of this study was to discover whether rhizosphere-enhanced biodegradation is due to selective enrichment of degraders through growth on compounds produced by rhizodeposition. We monitored the mineralization of [U-¹⁴C]2,4-dichlorophenoxyacetic acid (2,4-D) in rhizosphere soil with no history of herbicide application collected over a period of 0 to 116 days after sowing of Lolium perenne and Trifolium pratense. The relationships between the mineralization kinetics, the number of 2,4-D degraders, and the diversity of genes encoding 2,4-D/α-ketoglutarate dioxygenase (tfdA) were investigated. The rhizosphere effect on [¹⁴C]2,4-D mineralization (50 μg g⁻¹) was shown to be plant species and plant age specific. In comparison with nonplanted soil, there were significant (P < 0.05) reductions in the lag phase and enhancements of the maximum mineralization rate for 25- and 60-day T. pratense soil but not for 116-day T. pratense rhizosphere soil or for L. perenne rhizosphere soil of any age. Numbers of 2,4-D degraders in planted and nonplanted soil were low (most probable number, <100 g⁻¹) and were not related to plant species or age. Single-strand conformational polymorphism analysis showed that plant species had no impact on the diversity of α-Proteobacteria tfdA-like genes, although an impact of 2,4-D application was recorded. Our results indicate that enhanced mineralization in T. pratense rhizosphere soil is not due to enrichment of 2,4-D-degrading microorganisms by rhizodeposits. We suggest an alternative mechanism in which one or more components of the rhizodeposits induce the 2,4-D pathway.     

The tfdK Gene Product Facilitates Uptake of 2,4-Dichlorophenoxyacetate by Ralstonia eutropha JMP134(pJP4)

Uptake of 2,4-dichlorophenoxyacetate (2,4-D) by Ralstonia eutropha JMP134(pJP4) was studied and shown to be an energy-dependent process. The uptake system was inducible with 2,4-D and followed saturation kinetics in a concentration range of up to 60 μM, implying the involvement of a protein in the transport process. We identified an open reading frame on plasmid pJP4, which was designated tfdK, whose translation product TfdK was highly hydrophobic and showed resemblance to transport proteins of the major facilitator superfamily. An interruption of the tfdK gene on plasmid pJP4 decimated 2,4-D uptake rates, which implies a role for TfdK in uptake. A tfdA mutant, which was blocked in the first step of 2,4-D metabolism, still took up 2,4-D. A mathematical model describing TfdK as an active transporter at low micromolar concentrations fitted the observed uptake data best.     

Earthworm Egg Capsules as Vectors for the Environmental Introduction of Biodegradative Bacteria

Earthworm egg capsules (cocoons) may acquire bacteria from the environment in which they are produced. We found that Ralstonia eutropha (pJP4) can be recovered from Eisenia fetida cocoons formed in soil inoculated with this bacterium. Plasmid pJP4 contains the genes necessary for 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4-dichlorophenol (2,4-DCP) degradation. In this study we determined that the presence of R. eutropha (pJP4) within the developing earthworm cocoon can influence the degradation and toxicity of 2,4-D and 2,4-DCP, respectively. The addition of cocoons containing R. eutropha (pJP4) at either low or high densities (10² or 10⁵ CFU per cocoon, respectively) initiated degradation of 2,4-D in nonsterile soil microcosms. Loss of 2,4-D was observed within the first week of incubation, and respiking the soil with 2,4-D showed depletion within 24 h. Microbial analysis of the soil revealed the presence of approximately 10⁴ CFU R. eutropha (pJP4) g⁻¹ of soil. The toxicity of 2,4-DCP to developing earthworms was tested by using cocoons with or without R. eutropha (pJP4). Results showed that cocoons containing R. eutropha (pJP4) were able to tolerate higher levels of 2,4-DCP. Our results indicate that the biodegradation of 2,4-DCP by R. eutropha (pJP4) within the cocoons may be the mechanism contributing to toxicity reduction. These results suggest that the microbiota may influence the survival of developing earthworms exposed to toxic chemicals. In addition, cocoons can be used as inoculants for the introduction into the environment of beneficial bacteria, such as strains with biodegradative capabilities.     

Biodegradation of the Hexahydro-1,3,5-Trinitro-1,3,5-Triazine Ring Cleavage Product 4-Nitro-2,4-Diazabutanal by Phanerochaete chrysosporium

Initial denitration of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by Rhodococcus sp. strain DN22 produces CO₂ and the dead-end product 4-nitro-2,4-diazabutanal (NDAB), OHCNHCH₂NHNO₂, in high yield. Here we describe experiments to determine the biodegradability of NDAB in liquid culture and soils containing Phanerochaete chrysosporium. A soil sample taken from an ammunition plant contained RDX (342 μmol kg⁻¹), HMX (octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine; 3,057 μmol kg⁻¹), MNX (hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine; 155 μmol kg⁻¹), and traces of NDAB (3.8 μmol kg⁻¹). The detection of the last in real soil provided the first experimental evidence for the occurrence of natural attenuation that involved ring cleavage of RDX. When we incubated the soil with strain DN22, both RDX and MNX (but not HMX) degraded and produced NDAB (388 ± 22 μmol kg⁻¹) in 5 days. Subsequent incubation of the soil with the fungus led to the removal of NDAB, with the liberation of nitrous oxide (N₂O). In cultures with the fungus alone NDAB degraded to give a stoichiometric amount of N₂O. To determine C stoichiometry, we first generated [¹⁴C]NDAB in situ by incubating [¹⁴C]RDX with strain DN22, followed by incubation with the fungus. The production of ¹⁴CO₂ increased from 30 (DN22 only) to 76% (fungus). Experiments with pure enzymes revealed that manganese-dependent peroxidase rather than lignin peroxidase was responsible for NDAB degradation. The detection of NDAB in contaminated soil and its effective mineralization by the fungus P. chrysosporium may constitute the basis for the development of bioremediation technologies.     

Culturable Heavy Metal-Resistant and Plant Growth Promoting Bacteria in V-Ti Magnetite Mine Tailing Soil from Panzhihua,China

To provide a basis for using indigenous bacteria for bioremediation of heavy metal contaminated soil, the heavy metal resistance and plant growth-promoting activity of 136 isolates from V-Ti magnetite mine tailing soil were systematically analyzed. Among the 13 identified bacterial genera, the most abundant genus was Bacillus (79 isolates) out of which 32 represented B. subtilis and 14 B. pumilus, followed by Rhizobium sp. (29 isolates) and Ochrobactrum intermedium (13 isolates). Altogether 93 isolates tolerated the highest concentration (1000 mg kg⁻¹) of at least one of the six tested heavy metals. Five strains were tolerant against all the tested heavy metals, 71 strains tolerated 1,000 mg kg⁻¹ cadmium whereas only one strain tolerated 1,000 mg kg⁻¹ cobalt. Altogether 67% of the bacteria produced indoleacetic acid (IAA), a plant growth-promoting phytohormone. The concentration of IAA produced by 53 isolates was higher than 20 µg ml⁻¹. In total 21% of the bacteria produced siderophore (5.50–167.67 µg ml⁻¹) with two Bacillus sp. producing more than 100 µg ml⁻¹. Eighteen isolates produced both IAA and siderophore. The results suggested that the indigenous bacteria in the soil have beneficial characteristics for remediating the contaminated mine tailing soil.     

Cultivation-Based and Molecular Assessment of Bacterial Diversity in the Rhizosheath of Wheat under Different Crop Rotations

A field study was conducted to compare the formationand bacterial communities of rhizosheaths of wheat grown under wheat-cotton and wheat-rice rotation and to study the effects of bacterial inoculation on plant growth. Inoculation of Azospirillum sp. WS-1 and Bacillus sp. T-34 to wheat plants increased root length, root and shoot dry weight and dry weight of rhizosheathsoil when compared to non-inoculated control plants, and under both crop rotations. Comparing both crop rotations, root length, root and shoot dry weight and dry weight of soil attached with roots were higher under wheat-cotton rotation. Organic acids (citric acid, malic acid, acetic acid and oxalic acid) were detected in rhizosheaths from both rotations, with malic acid being most abundant with 24.8±2 and 21.3±1.5 μg g^-1 dry soil in wheat-cotton and wheat-rice rotation, respectively. Two sugars (sucrose, glucose) were detected in wheat rhizosheath under both rotations, with highest concentrations of sucrose (4.08±0.5 μg g^-1and 7.36±1.0 μg g^-1) and glucose (3.12±0.5 μg g^-1 and 3.01± μg g^-1) being detected in rhizosheaths of non-inoculated control plants under both rotations. Diversity of rhizosheath-associated bacteria was evaluated by cultivation, as well as by 454-pyrosequencing of PCR-tagged 16S rRNA gene amplicons. A total of 14 and 12 bacterial isolates predominantly belonging to the genera Arthrobacter, Azospirillum, Bacillus, Enterobacter and Pseudomonaswere obtained from the rhizosheath of wheat grown under wheat-cotton and wheat-rice rotation, respectively. Analysis of pyrosequencing data revealed Proteobacteria, Bacteriodetes and Verrucomicrobia as the most abundant phyla in wheat-rice rotation, whereas Actinobacteria, Firmicutes, Chloroflexi, Acidobacteria, Planctomycetes and Cyanobacteria were predominant in wheat-cotton rotation. From a total of 46,971 sequences, 10.9% showed ≥97% similarity with 16S rRNA genes of 32 genera previously shown to include isolates with plant growth promoting activity (nitrogen fixation, phosphate-solubilization, IAA production). Among these, the most predominant genera were Arthrobacter, Azoarcus, Azospirillum, Bacillus, Cyanobacterium, Paenibacillus, Pseudomonas and Rhizobium.     

Analysis of Indole-3-Acetic Acid and Related Indoles in Culture Medium from Azospirillum lipoferum and Azospirillum brasilense

Analysis of neutral and acidic ethyl acetate extracts from culture medium of Azospirillum brasilense 703Ebc by high-performance liquid chromatography (HPLC) and combined gas chromatography-mass spectrometry demonstrated the presence of indole-3-acetic acid (IAA), indole-3-ethanol, indole-3-methanol, and indole-3-lactic acid. IAA in media of 20 strains of A. brasilense and Azospirillum lipoferum was analyzed quantitatively by both the colorimetric Salkowski assay and HPLC-based isotopic dilution procedures. There was little correlation between the estimates obtained with the two procedures. For instance, the Salkowski assay suggested that the culture medium from A. brasilense 703Ebc contained 26.1 μg of IAA ml⁻¹, whereas HPLC revealed the presence of only 0.5 μg of IAA ml⁻¹. Equivalent estimates with A. brasilense 204Ed were 10.5 and 0.01 μg of IAA ml⁻¹, respectively. The data demonstrate that the Salkowski assay is not a reliable method for measuring the IAA content of Azospirillum culture medium and that estimates in excess of 10 μg of IAA ml⁻¹ should be viewed with particular caution. Metabolism of [2′-¹⁴C]IAA by A. brasilense 703Ebc yielded radiolabeled indole-3-methanol, whereas roots of maize (Zea mays L.) seedlings gave rise to [¹⁴C]oxindole-3-acetic acid and an array of polar metabolites. Metabolism of [2′-¹⁴C]IAA by maize roots inoculated with A. brasilense 703Ebc produced a metabolic profile characteristic of maize rather than Azospirillum species.     

Clotrimazole as a Potent Agent for Treating the Oomycete Fish Pathogen Saprolegnia parasitica through Inhibition of Sterol 14α-Demethylase (CYP51)

A candidate CYP51 gene encoding sterol 14α-demethylase from the fish oomycete pathogen Saprolegnia parasitica (SpCYP51) was identified based on conserved CYP51 residues among CYPs in the genome. It was heterologously expressed in Escherichia coli, purified, and characterized. Lanosterol, eburicol, and obtusifoliol bound to purified SpCYP51 with similar binding affinities (K_s, 3 to 5 μM). Eight pharmaceutical and six agricultural azole antifungal agents bound tightly to SpCYP51, with posaconazole displaying the highest apparent affinity (K_d, ≤3 nM) and prothioconazole-desthio the lowest (K_d, ∼51 nM). The efficaciousness of azole antifungals as SpCYP51 inhibitors was confirmed by 50% inhibitory concentrations (IC₅₀s) of 0.17 to 2.27 μM using CYP51 reconstitution assays. However, most azole antifungal agents were less effective at inhibiting S. parasitica, Saprolegnia diclina, and Saprolegnia ferax growth. Epoxiconazole, fluconazole, itraconazole, and posaconazole failed to inhibit Saprolegnia growth (MIC₁₀₀, >256 μg ml⁻¹). The remaining azoles inhibited Saprolegnia growth only at elevated concentrations (MIC₁₀₀ [the lowest antifungal concentration at which growth remained completely inhibited after 72 h at 20°C], 16 to 64 μg ml⁻¹) with the exception of clotrimazole, which was as potent as malachite green (MIC₁₀₀, ∼1 μg ml⁻¹). Sterol profiles of azole-treated Saprolegnia species confirmed that endogenous CYP51 enzymes were being inhibited with the accumulation of lanosterol in the sterol fraction. The effectiveness of clotrimazole against SpCYP51 activity (IC₅₀, ∼1 μM) and the concentration inhibiting the growth of Saprolegnia species in vitro (MIC₁₀₀, ∼1 to 2 μg ml⁻¹) suggest that clotrimazole could be used against Saprolegnia infections, including as a preventative measure by pretreatment of fish eggs, and for freshwater-farmed fish as well as in leisure activities.     

Effects of 2,4-Dichlorophenol, a Metabolite of a Genetically Engineered Bacterium, and 2,4-Dichlorophenoxyacetate on Some Microorganism-Mediated Ecological Processes in Soil

A genetically engineered microorganism, Pseudomonas putida PPO301(pRO103), and the plasmidless parent strain, PPO301, were added at approximately 10⁷ CFU/g of soil amended with 500 ppm of 2,4-dichlorophenoxyacetate (2,4-D) (500 μg/g). The degradation of 2,4-D and the accumulation of a single metabolite, identified by gas chromatography-mass spectrophotometry as 2,4-dichlorophenol (2,4-DCP), occurred only in soil inoculated with PPO301(pRO103), wherein 2,4-DCP accumulated to >70 ppm for 5 weeks and the concentration of 2,4-D was reduced to <100 ppm. Coincident with the accumulation of 2,4-DCP was a >400-fold decline in the numbers of fungal propagules and a marked reduction in the rate of CO₂ evolution, whereas 2,4-D did not depress either fungal propagules or respiration of the soil microbiota. 2,4-DCP did not appear to depress the numbers of total heterotrophic, sporeforming, or chitin-utilizing bacteria. In vitro and in situ assays conducted with 2,4-DCP and fungal isolates from the soil demonstrated that 2,4-DCP was toxic to fungal propagules at concentrations below those detected in the soil.     

Biochemical, Genetic, and Zoosporicidal Properties of Cyclic Lipopeptide Surfactants Produced by Pseudomonas fluorescens

Zoospores play an important role in the infection of plant and animal hosts by oomycetes and other zoosporic fungi. In this study, six fluorescent Pseudomonas isolates with zoosporicidal activities were obtained from the wheat rhizosphere. Zoospores of multiple oomycetes, including Pythium species, Albugo candida, and Phytophthora infestans, were rendered immotile within 30 s of exposure to cell suspensions or cell culture supernatants of the six isolates, and subsequent lysis occurred within 60 s. The representative strain SS101, identified as Pseudomonas fluorescens biovar II, reduced the surface tension of water from 73 to 30 mN m⁻¹. The application of cell suspensions of strain SS101 to soil or hyacinth bulbs provided significant protection against root rot caused by Pythium intermedium. Five Tn5 mutants of strain SS101lacked the abilities to reduce the surface tension of water and to cause lysis of zoospores. Genetic characterization of two surfactant-deficient mutants showed that the transposons had integrated into condensation domains of peptide synthetases. A partially purified extract from strain SS101 reduced the surface tension of water to 30 mN m⁻¹ and reached the critical micelle concentration at 25 μg ml⁻¹. Reverse-phase high-performance liquid chromatography yielded eight different fractions, five of which had surface activity and caused lysis of zoospores. Mass spectrometry and nuclear magnetic resonance analyses allowed the identification of the main constituent as a cyclic lipopeptide (1,139 Da) containing nine amino acids and a 10-carbon hydroxy fatty acid. The other four zoosporicidal fractions were closely related to the main constituent, with molecular massesranging from 1,111 to 1,169 Da.     

Microbial Responses to Environmentally Toxic Cadmium

Abstract We analyzed the soil microbial communities from one uncontaminated and two metal-impacted soils and found that while cadmium adversely affected the numbers of culturable bacteria in all soils, cadmium-resistant isolates were found from each of the soils. With exposure to 24 and 48 μg ml^-1 soluble cadmium, the metal-contaminated soil communities were more resistant than the uncontaminated soil community. In addition, in one metal-stressed soil, the resistant population became more resistant with increased cadmium levels. Ribosomal 16S DNA sequencing identified the isolates as Arthrobacter, Bacillus, or Pseudomonas spp. Further characterization demonstrated that two of the isolates were highly resistant to soluble cadmium with maximum resistance at 275 μg ml^-1 cadmium. These isolates were also resistant to a variety of antibiotics, namely ampicillin, gentamicin, penicillin, and streptomycin, but no overall correlation was found between enhanced antibiotic resistance and cadmium resistance. One Pseudomonas isolate H1 did become more resistant with increasing cadmium levels, suggesting a different resistance mechanism at high cadmium concentrations. Received: 29 April 1999; Accepted: 7 July 1999; Online Publication: 30 November 1999     

Effects of Condensed Tannins on Endoglucanase Activity and Filter Paper Digestion by Fibrobacter succinogenes S85

The effect of condensed tannins from birdsfoot trefoil (Lotus corniculatus L.) on the cellulolytic rumen bacterium Fibrobacter succinogenes S85 was examined. Condensed tannins inhibited endoglucanase activity in the extracellular culture fluid, at concentrations as low as 25 μg ml^-1. In contrast, cell-associated endoglucanase activity increased in concentrations of condensed tannins between 100 and 300 μg ml^-1. Inhibition of endoglucanase activity in both the extracellular and the cell-associated fractions was virtually complete at 400 μg of condensed tannins ml^-1. Despite the sharp decline in extracellular endoglucanase activity with increasing concentrations of condensed tannins, filter paper digestion declined only moderately between 0 and 200 μg of condensed tannins ml^-1. However, at 300 μg ml^-1, filter paper digestion was dramatically reduced and at 400 μg ml^-1, almost no filter paper was digested. F. succinogenes S85 was seen to form digestive grooves on the surface of cellulose, and at 200 μg ml^-1, digestive pits were formed which penetrated into the interior of cellulose fibers. Cells grown with condensed tannins (100 to 300 μg ml^-1) possessed large amounts of surface material, and although this material may have been capsular carbohydrate, its osmiophilic nature suggested that it had arisen from the formation of tannin-protein complexes on the cell surface. The presence of electron-dense extracellular material suggested that similar complexes were formed with extracellular protein.     

Potential Importance of Fish Predation and Zooplankton Grazing on Natural Populations of Freshwater Bacteria

The rates of ingestion of natural bacterial assemblages by natural populations of zooplankton (>50 μm in size) were measured during a 19-day period in eutrophic Frederiksborg Slotssø, Denmark, as well as in experimental enclosures (containing 5.3 m³ of lake water). The fish and nutrients of the enclosures were manipulated. In enclosures without fish, large increases in ingestion by zooplankton >140 μm in size were found (up to 3 μg of C liter⁻¹ h⁻¹), compared with values less than 0.3 μg of C liter⁻¹ h⁻¹ in the enclosures with fish and in the open lake. Daphnia cucullata and D. galeata dominated the community of zooplankton of >140 μm. Ingestion rates for zooplankton between 50 and 140 μm decreased after a period of about 8 days, in all enclosures and in the lake, to values below 0.1 μg of C liter⁻¹ h⁻¹. On the last 2 sampling days, somewhat higher values were observed in the enclosures with fish present. The >50-μm zooplankton ingested 48 to 51% of the bacterial net secondary production in enclosures without fish, compared to 4% in the enclosures with added fish. Considering the sum of bacterial secondary production plus biomass change, 35 to 41% of the available bacteria were ingested by zooplankton of >50 μm in the enclosures without fish, compared with 4 to 6% in the enclosures with added fish and 21% in the open lake. Fish predation reduced the occurrence of zookplankton sized >50 μm and thus left a large proportion of the available bacteria to zooplankton sized <50 μm. In fact, there were 4.6 × 10³ to 5.0 × 10³ flagellates (4 to 8 μm in size) ml⁻¹ in the enclosures with fish added as well as in the lake, compared with 0.5 × 10² to 2.3 × 10² ml⁻¹ in the enclosures without fish. This link in the food chain was reduced when fish predation on zooplankton was eliminated and a direct route of dissolved organic matter, via the bacteria to the zooplankton, was established.