Immunodetection of the bacteriocin lacticin RM: analysis of the influence of temperature and Tween 80 on its expression and activity

Immunoassays with specific antibodies offer higher sensitivity than do bioassays with indicator strains in the detection and quantification of several bacteriocins. Here we present the purification of lacticin RM and the production of specific polyclonal antibodies to a synthetic peptide resembling an internal fragment of the mature bacteriocin. The specificity and sensitivity of the generated polyclonal antibodies were evaluated in various immunoassays. The detection limits of lacticin RM were found to be 1.9, 0.16, and 0.18 micro g ml(-1) for Western blot, immuno-dot blot, and noncompetitive indirect enzyme-linked immunosorbent assays, respectively. Immunoassay sensitivities were 12.5-fold higher than that of the agar diffusion test (ADT). The production of lacticin RM showed temperature dependency, with 3, 4.2, 12.7, 28.9, 37.8, and 12 micro g ml(-1) at 37, 30, 20, 15, 10, and 4 degrees C, respectively. Temperature-stability analysis demonstrated that lacticin RM is sensitive to mild temperature, but the loss of activity does not seem to result from protein degradation. Tween 80 increased the concentration of lacticin RM eightfold and probably affected the results of the ADT either by enhancing the activity of lacticin RM or by increasing the sensitivity of the indicator strain. The use of antibodies for the specific detection and quantification of lacticin RM can expand our knowledge of its production and stability, with important implications for further investigation and future application.     

Enzyme-Linked Immunosorbent Assays for the Specific and Sensitive Quantification of Methanosarcina mazei and Methanobacterium bryantii

Three microtitration plate enzyme-linked immunosorbent assays (ELISAs) have been developed: a competitive ELISA and a two-site (or indirect sandwich) ELISA for Methanosarcina mazei S6 and a two-site ELISA for Methanobacterium bryantii FR-2. The assays were sensitive, with limits of cell protein detection of 3 ng ml⁻¹, 5 ng ml⁻¹, and 50 ng ml⁻¹, respectively, and showed good precision. The M. mazei assays used monoclonal antibodies and were entirely species specific, showing no cross-reaction with methanogens of other genera or with other species of the same genus. The Methanobacterium bryantii assay, which used two polyclonal antisera, showed only a slight cross-reaction with one other Methanobacterium species but no cross-reaction with methanogens of other genera. The use of the ELISAs for quantitative analysis of mixed cultures and of sewage sludge samples was investigated. Sludge diluted at 1:10³ or more caused no significant interference in any of the three ELISAs. Various cultures of bacteria, methanogens, and nonmethanogens at a protein concentration of 50 μg ml⁻¹ showed no significant interference in the M. mazei competitive assay and the Methanobacterium bryantii two-site assay, although they did cause falsely low results in the M. mazei two-site assay.     

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.     

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.     

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.     

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.     

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.     

Production of α-Amylase by the Ruminal Anaerobic Fungus Neocallimastix frontalis

α-Amylase production was examined in the ruminal anaerobic fungus Neocallimastix frontalis. The enzyme was released mainly into the culture fluid and had temperature and pH optima of 55°C and 5.5, respectively, and the apparent K_m for starch was 0.8 mg ml⁻¹. The products of α-amylase action were mainly maltotriose, maltotetraose, and longer-chain oligosaccharides. No activity of the enzyme was observed towards these compounds or pullulan, but activity on amylose was similar to starch. Evidence for the endo action of α-amylase was also obtained from experiments which showed that the reduction in iodine-staining capacity and release in reducing power by action on amylose was similar to that for commercial α-amylase. Activities of α-amylase up to 4.4 U ml⁻¹ (1 U represents 1 μmol of glucose equivalents released per min) were obtained for cultures grown on 2.5 mg of starch ml⁻¹ in shaken cultures. No growth occurred in unshaken cultures. With elevated concentrations of starch (>2.5 mg ml⁻¹), α-amylase production declined and glucose accumulated in the cultures. Addition of glucose to cultures grown on low levels of starch, in which little glucose accumulated, suppressed α-amylase production, and in bisubstrate growth studies, active production of the enzyme only occurred during growth on starch after glucose had been preferentially utilized. When cellulose, cellobiose, glucose, xylan, and xylose were tested as growth substrates for the production of α-amylase (initial concentration, 2.5 mg ml⁻¹), they were found to be less effective than starch, but maltose was almost as effective. The fungal α-amylase was found to be stable at 60°C in the presence of low concentrations of starch (≤5%), suggesting that it may be suitable for industrial application.     

Quantitative Analysis of Cereulide, the Emetic Toxin of Bacillus cereus, Produced under Various Conditions

This paper describes a quantitative and sensitive chemical assay for cereulide, the heat-stable emetic toxin produced by Bacillus cereus. The methods previously available for measuring cereulide are bioassays that give a toxicity titer, but not an accurate concentration. The dose of cereulide causing illness in humans is therefore not known, and thus safety limits for cereulide cannot be indicated. We developed a quantitative and sensitive chemical assay for cereulide based on high-performance liquid chromatography (HPLC) connected to ion trap mass spectrometry. This chemical assay and a bioassay based on boar sperm motility inhibition were calibrated with purified cereulide and with valinomycin, a structurally similar cyclic depsipeptide. The boar spermatozoan motility assay and chemical assay gave uniform results over a wide range of cereulide concentrations, ranging from 0.02 to 230 μg ml⁻¹. The detection limit for cereulide and valinomycin by HPLC-mass spectrometry was 10 pg per injection. The combined chemical and biological assays were used to define conditions and concentrations of cereulide formation by B. cereus strains F4810/72, NC7401, and F5881. Cereulide production commenced at the end of logarithmic growth, but was independent of sporulation. Production of cereulide was enhanced by incubation with shaking compared to static conditions. The three emetic B. cereus strains accumulated 80 to 166 μg of cereulide g⁻¹ (wet weight) when grown on solid medium. Strain NC7401 accumulated up to 25 μg of cereulide ml⁻¹ in liquid medium at room temperature (21 ± 1°C) in 1 to 3 days, during the stationary growth phase when cell density was 2 × 10⁸ to 6 × 10⁸ CFU ml⁻¹. Cereulide production at temperatures at and below 8°C or at 40°C was minimal.     

Sensitive Detection of Botulinum Neurotoxin Types C and D with an Immunoaffinity Chromatographic Column Test

A sensitive and specific immunoassay for the simultaneous detection of Clostridium botulinum type C (BoNT/C) and type D neurotoxin was developed. Goat anti-mouse immunoglobulin G was bound to polyethylene disks in a small disposable column used for this assay. The sample was preincubated together with monoclonal antibodies specific for the heavy chain of BoNT/C and D and affinity-purified, biotinylated polyclonal antibodies against these neurotoxins. This complex was captured on the assay disk. Streptavidin-poly-horseradish peroxidase was used as a conjugate, and a precipitating substrate allowed the direct semiquantitative readout of the assay, if necessary. For a more accurate quantitative detection, the substrate can be eluted and measured in a photometer. Depending on the preincubation time, a sensitivity of 1 mouse lethal dose ml⁻¹ was achieved in culture supernatants.     

Effects of Specific Inhibitors on Anammox and Denitrification in Marine Sediments

The effects of three metabolic inhibitors (acetylene, methanol, and allylthiourea [ATU]) on the pathways of N₂ production were investigated by using short anoxic incubations of marine sediment with a ¹⁵N isotope technique. Acetylene inhibited ammonium oxidation through the anammox pathway as the oxidation rate decreased exponentially with increasing acetylene concentration; the rate decay constant was 0.10 ± 0.02 μM⁻¹, and there was 95% inhibition at ~30 μM. Nitrous oxide reduction, the final step of denitrification, was not sensitive to acetylene concentrations below 10 μM. However, nitrous oxide reduction was inhibited by higher concentrations, and the sensitivity was approximately one-half the sensitivity of anammox (decay constant, 0.049 ± 0.004 μM⁻¹; 95% inhibition at ~70 μM). Methanol specifically inhibited anammox with a decay constant of 0.79 ± 0.12 mM⁻¹, and thus 3 to 4 mM methanol was required for nearly complete inhibition. This level of methanol stimulated denitrification by ~50%. ATU did not have marked effects on the rates of anammox and denitrification. The profile of inhibitor effects on anammox agreed with the results of studies of the process in wastewater bioreactors, which confirmed the similarity between the anammox bacteria in bioreactors and natural environments. Acetylene and methanol can be used to separate anammox and denitrification, but the effects of these compounds on nitrification limits their use in studies of these processes in systems where nitrification is an important source of nitrate. The observed differential effects of acetylene and methanol on anammox and denitrification support our current understanding of the two main pathways of N₂ production in marine sediments and the use of ¹⁵N isotope methods for their quantification.     

Kinetic Parameters of Denitrification in a River Continuum

Kinetic parameters for nitrate reduction in intact sediment cores were investigated by using the acetylene blockage method at five sites along the Swale-Ouse river system in northeastern England, including a highly polluted tributary, R. Wiske. The denitrification rate in sediment containing added nitrate exhibited a Michaelis-Menten-type curve. The concentration of nitrate for half-maximal activity (K_map) by denitrifying bacteria increased on passing downstream from 13.1 to 90.4 μM in the main river, but it was highest (640 μM) in the Wiske. The apparent maximal rate (V_maxap) ranged between 35.8 and 324 μmol of N m⁻² h⁻¹ in the Swale-Ouse (increasing upstream to downstream), but it was highest in the Wiske (1,194 μmol N m⁻² h⁻¹). A study of nitrous oxide (N₂O) production at the same time showed that rates ranged from below the detection limit (0.05 μmol of N₂O-N m⁻² h⁻¹) at the headwater site to 27 μmol of N₂O-N m⁻² h⁻¹ at the downstream site. In the Wiske the rate was up to 570 μmol of N₂O-N m⁻² h⁻¹, accounting for up to 80% of total N gas production.     

Primary and Bacterial Secondary Production in a Southwestern Reservoir

Rates of primary and bacterial secondary production in Lake Arlington, Texas, were determined. The lake is a warm (annual temperature range, 7 to 32°C), shallow, monomictic reservoir with limited macrophyte development in the littoral zone. Samples were collected from six depths within the photic zone from a site located over the deepest portion of the lake. Primary production and bacterial production were calculated from NaH¹⁴CO₃ and [methyl-³H]thymidine incorporation, respectively. Peak instantaneous production ranged between 14.8 and 220.5 μg of C liter⁻¹ h⁻¹. There were two distinct periods of high rates of production. From May through July, production near the metalimnion exceeded 100 μg of C liter⁻¹ h⁻¹. During holomixis, production throughout the water column was in excess of 100 μg of C liter⁻¹ h⁻¹ and above 150 μg of C liter⁻¹ h⁻¹ near the surface. Annual areal primary production was 588 g of C m⁻². Bacterial production was markedly seasonal. Growth rates during late fall through spring were typically around 0.002 h⁻¹, and production rates were typically 5 μg of C liter⁻¹ h⁻¹. Growth rates were higher during warmer parts of the year and reached 0.03 h⁻¹ by August. The maximum instantaneous rate of bacterial production was approximately 45 μg of C liter⁻¹ h⁻¹. Annual areal bacterial production was 125 g of C m⁻². Temporal and spatial distributions of bacterial numbers and activities coincided with temporal and spatial distributions of primary production. Areal primary and bacterial secondary production were highly correlated (r = 0.77, n = 15, P < 0.002).     

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.     

Extracellular Polysaccharide Is Not Responsible for Aluminum Tolerance of Rhizobium leguminosarum bv. Phaseoli CIAT899

Strain UHM-5, a pSym^- Exo^- derivative of the aluminum-tolerant Rhizobium leguminosarum bv. phaseoli strain CIAT899, was equally tolerant of aluminum (Al) as the parental culture. Dialyzed culture supernatants of the wild-type cells grown in YEM broth (10⁹ cells ml^-1) contained 185 μg of glucose equivalents ml^-1 whereas UHM-5 culture supernatants yielded 2 μg of glucose ml^-1. The Exo^- derivative and the parental strain gave essentially similar growth in medium containing from 0 to 300 μM Al, indicating that the pSym of CIAT899, and extracellular polysaccharide, were not involved in the aluminum tolerance of this strain. However, increasing the level of Al from 80 to 150 μM increased the lag phase, induced a slight killing of the inoculum, and depressed the final populations by about fivefold. Doubling the aluminum concentration from 150 to 300 μM presented a severe aluminum stress to CIAT899 and UHM-5: the inoculum level dropped 10-fold, indicating killing of the inoculum, and remained depressed for ca. 4 days before continuing to grow slowly; the final population was decreased 15-fold relative to that of cultures grown in medium containing 80 μM Al. The production by CIAT899 of other extracellular or intracellular aluminum tolerance factors was investigated in culture by using aluminum-sensitive rhizobia as stress indicators. These experiments, conducted at 80 μM Al, demonstrated that CIAT899 produced neither extracellular nor intracellular products that could alleviate toxicity for the Al-sensitive indicator rhizobia.     

Kinetic Modelling for the Assay of Nortriptyline Hydrochloride Using Potassium Permanganate as Oxidant

Kinetic methods for accurate determination of nortriptyline hydrochloride have been described. The methods are based on the oxidation of nortriptyline hydrochloride with KMnO₄ in acidic and basic media. In acidic medium, the decrease in absorbance at 525.5 nm and in basic medium, the increase in absorbance at 608.5 nm were measured as a function of time. The variables affecting the reactions were carefully investigated and optimised. Kinetic models such as initial rate, rate constant, variable time and fixed time were employed to construct the calibration curves. The initial rate and fixed time methods were selected for quantification of nortriptyline hydrochloride. In acidic medium, the calibration curves showed a linear response over the concentration range 10–50 μg mL⁻¹ for initial rate and 10–60 μg mL⁻¹ for fixed time method (2 min). In basic medium, the calibration graphs were linear over the concentration range 10–100 μg mL⁻¹ for initial rate and fixed time methods (4 min). In acidic medium, the limits of detection for initial rate and fixed time methods (2 min) were 1.02 and 3.26 μg mL⁻¹, respectively. In basic medium, the limits of detection were found to be 1.67 and 1.55 μg mL⁻¹ for initial rate and fixed time methods (4 min), respectively. The initial rate and fixed time methods have been successfully applied to the determination of nortriptyline hydrochloride in commercial dosage form. Statistical comparison of the results of the proposed methods with those of reference method exhibited excellent agreement and there is no significant difference between the compared methods in terms of accuracy and precision.KEY WORDS: dosage forms, fixed time method, initial rate method, nortriptyline, potassium permanganate     

Fluorometric Determination of DNA in Aquatic Microorganisms by Use of Hoechst 33258

A method for the determination of microbial DNA in aquatic environments by the use of Hoechst 33258 has been developed. With unsophisticated instrumentation and simple extraction procedures, it is possible to detect from 0.05 to 10 μg of DNA in bacterial cultures or natural water samples. The method is specific for DNA; DNase I treatment of extracts of natural microbial populations removed 95 to 100% of the observed fluorescence. DNA content ranged from 165 ng ml⁻¹ for relatively eutrophic Potomac River water to 27 ng ml⁻¹ for coastal Atlantic Ocean water and was correlated to an acridine orange direct count (r = 0.90).     

Effect of Nickelous and Other Metal Ions on the Inhibition of Rumen Bacterial Metabolism by 3-(3′-Isocyanocyclopent-2-Enylidene)Propionic Acid and Related Isocyanides

3-(3′-Isocyanocyclopent-2-enylidene)propionic acid at a concentration of 2 to 5 μg ml⁻¹ inhibited cellulose digestion by a mixed culture of rumen microorganisms and in other experiments inhibited the degradation of timothy hay (Phleum pratense) in a digestibility test. At isocyanide concentrations of 12 μg ml⁻¹ the fermentation activity of rumen fluid, measured by its dehydrogenase activity, was inhibited but not abolished. All of these isocyanide effects were reversed by the incorporation of nickelous ion into the solutions of the systems under study. The activity of 1 mol of isocyanide is reversed by about 1 mol of Ni²⁺ and, in the case of the cellulose digestion test, by about 1 mol of Co²⁺. Of some 15 other ions tested only Pd²⁺ and possibly chromium reversed the effect of the isocyanide.     

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 μg 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-μg 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 μg of cadmium ml⁻¹ in pure culture and up to 60 μg 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 μg of cadmium g⁻¹. Overall, dual bioaugmentation appears to be a viable approach in the remediation of cocontaminated soils.     

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.