Field calibration of soil-core microcosms: Ecosystem structural and functional comparisons

Microcosms containing intact soil-cores are a potential biotechnology risk assessment tool for assessing the ecological effects of genetically engineered microorganisms before they are released to the field; however, microcosms must first be calibrated to ensure that they adequately simulate key field parameters. Soil-core microcosms were compared with the field in terms of ecological response to the introduction of a large inoculum of a rifampicin-resistant rhizobacterium,Pseudomonas sp. RC1. RC1 was inoculated into intact soil-core microcosms incubated in the laboratory at ambient temperature (22°C) and in a growth chamber with temperature fluctuations that mimicked a verage field values, as well as into field lysimeters and plots. The effect of the introduced bacterium on ecosystem structure, including wheat rhizoplane populations of total and fluorescent pseudomonads, total heterotrophic bacteria, and the diversity of total heterotrophic bacteria, was determined. Fluorescent pseudomonads were present on the rhizoplane in significantly lower numbers in soil inoculated with RC1, in both microcosms and the field. Conditions for microbial growth appeared to be most favorable in the growth chamber microcosm, as evidenced by higher populations of heterotrophs and a greater species diversity on the rhizoplane at the three-leaf stage of wheat growth. Ecosystem functional parameters, as determined by soil dehydrogenase activity, plant biomass production, and¹⁵N-fertilizer uptake by wheat, were different in the four systems. The stimulation of soil dehydrogenase activity by the addition of alfalfa was greater in the microcosms than in the field. In general, growth chamber microcosms, which simulated average field temperatures, were better predictors of field behavior than microcosms incubated continuously at 22°C.     

Production of extracellular nucleic acids by genetically altered bacteria in aquatic-environment microcosms

The factors which affect the production of extracellular DNA by genetically altered strains of Escherichia coli, Pseudomonas aeruginosa, Pseudomonas cepacia, and Bradyrhizobium japonicum in aquatic environments were investigated. Cellular nucleic acids were labeled in vivo by incubation with [3H]thymidine or [3H]adenine, and production of extracellular DNA in marine waters, artificial seawater, or minimal salts media was determined by detecting radiolabeled macromolecules in incubation filtrates. The presence or absence of the ambient microbial community had little effect on the production of extracellular DNA. Three of four organisms produced the greatest amounts of extracellular nucleic acids when incubated in low-salinity media (2% artificial seawater) rather than high-salinity media (10 to 50% artificial seawater). The greatest production of extracellular nucleic acids by P. cepacia occurred at pH 7 and 37 degrees C, suggesting that extracellular-DNA production may be a normal physiologic function of the cell. Incubation of labeled P. cepacia cells in water from Bimini Harbor, Bahamas, resulted in labeling of macromolecules of the ambient microbial population. Collectively these results indicate that (i) extracellular-DNA production by genetically altered bacteria released into aquatic environments is more strongly influenced by physiochemical factors than biotic factors, (ii) extracellular-DNA production rates are usually greater for organisms released in freshwater than marine environments, and (iii) ambient microbial populations can readily utilize materials released by these organisms.     

Production of extracellular nucleic acids by genetically altered bacteria in aquatic-environment microcosms.

The factors which affect the production of extracellular DNA by genetically altered strains of Escherichia coli, Pseudomonas aeruginosa, Pseudomonas cepacia, and Bradyrhizobium japonicum in aquatic environments were investigated. Cellular nucleic acids were labeled in vivo by incubation with [3H]thymidine or [3H]adenine, and production of extracellular DNA in marine waters, artificial seawater, or minimal salts media was determined by detecting radiolabeled macromolecules in incubation filtrates. The presence or absence of the ambient microbial community had little effect on the production of extracellular DNA. Three of four organisms produced the greatest amounts of extracellular nucleic acids when incubated in low-salinity media (2% artificial seawater) rather than high-salinity media (10 to 50% artificial seawater). The greatest production of extracellular nucleic acids by P. cepacia occurred at pH 7 and 37 degrees C, suggesting that extracellular-DNA production may be a normal physiologic function of the cell. Incubation of labeled P. cepacia cells in water from Bimini Harbor, Bahamas, resulted in labeling of macromolecules of the ambient microbial population. Collectively these results indicate that (i) extracellular-DNA production by genetically altered bacteria released into aquatic environments is more strongly influenced by physiochemical factors than biotic factors, (ii) extracellular-DNA production rates are usually greater for organisms released in freshwater than marine environments, and (iii) ambient microbial populations can readily utilize materials released by these organisms.     

Efficacy in aquatic microcosms of a genetically engineered pseudomonad applicable for bioremediation

A genetically engineered microorganism (GEM), Pseudomonas sp. B13 FRI (pFRC20P) (abbreviated FR120), has previously been engineered to simultaneously mineralize mixtures of methylated and chlorinated benzoic acids and phenols through a modified ortho cleavage pathway. In this study, its performance was investigated both in different types of aquatic microcosms and in pure culture to determine (1) if under simulated in situ conditions the genetically engineered pathway effectively removes mixtures of model pollutants simultaneously, quickly, and completely; (2) where the optimum pollutant concentration range for this activity lies; and (3) how physical, chemical, and biological factors in the microcosms influence degradation rates. Growth and degradation parameters of FR 120 in pure culture were determined with 3-chlorobenzoate (3CB), 4-methylbenzoate (4MB), and equimolar mixtures of both as carbon sources. These substrates were degraded simultaneously, albeit with different degradation velocities, by FR120. The optimum growth concentrations for 3CB and 4MB were 3.0 mm and 2.1 mm, respectively, and the inhibition constants (K_i) were 11 mm (3CB) and 6 mm (4MB). The pathway was induced at low concentrations of substrate (> 1 [m). The first order degradation constants (k_l) were determined with respect to substrate concentration, cell density, and temperature. In aquatic microcosms inoculated with FR120, first order degradation constants and half lives of target chemicals were calculated based on the total amount of aromatics recovered. Half lives ranged from 1.3 days to 3.0 days, depending on the target chemical and the type of microcosm. Degradation constants determined in pure culture were extrapolated to the densities of FR120, substrate concentrations, and temperature occurring in the microcosm experiments, and used to calculate theoretical half lives. In water microcosms, theoretical and observed half lives corresponded well, indicating that FR120 functioned optimally in this environment. In whole core sediment microcosms, and especially at low cell densities, the observed degradation activity was in some cases considerably higher than expected from pure culture degradation rates. This suggests that environmental conditions in the sediment were more favorable to the degradation of substituted aromatics than those in pure culture. The physiological characteristics of FR120 and its performance in aquatic microcosms make it a good candidate for bioremediation at sites contamninated with mixtures of chlorinated and methylated aromatics. Correspondence to: I. Wagner-Döbler     

Intact soil-core microcosms compared with multi-site field releases for pre-release testing of microbes in diverse soils and climates

Intact soil-core microcosms were used to compare persistence of Pseudomonas chlororaphis 3732RN-L11 in fallow soil and on wheat roots with field releases at diverse sites. Parallel field and microcosm releases at four sites in 1996 were repeated with addition of one site in 1997. Microcosms were obtained fresh and maintained at 60% soil water holding capacity in a growth chamber at 70% relative humidity, a 12-hour photoperiod, and constant temperature. Persistence of 3732RN-L11 was measured at each site in field plots and microcosms at 7-21 day intervals, and in duplicate microcosms sampled at an independent laboratory. Linear regression slopes of field plot and microcosm persistence were compared for each site, and between identical microcosms sampled at different sites, using log10 transformed plate counts. Microcosm persistence closely matched field plots for wheat roots, but persistence in fallow soil differed significantly in several instances where persistence in field plots was lower than in microcosms. Analysis of weather variations at each site indicated that rainfall events of 30-40 mm caused decreased persistence in fallow soil. Cooler temperatures enhanced persistence in field plots at later time points. Inter-laboratory comparison of regression slopes showed good agreement for data generated at different sites, though in two instances, longer sampling periods at one site caused significant differences between the sites. Soil characteristics were compared and it was found that fertility, namely the carbon to nitrogen ratio, and the presence of expanding clays, were related to persistence. These microcosm protocols produced reliable data at low cost, and were useable for pre-release risk analyses for microorganisms.     

Biochemical characterization of a genetically altered calmodulin in Paramecium

Recent evidence proposes that the calcium-binding protein, calmodulin, plays a crucial role in the regulation or modulation of the calcium-dependent potassium conductance in Paramecium tetraurelia (Hinrichsen, R.D., Burgess-Cassler, A., Soltvedt, B.C., Hennessey, T. and Kung, C. (1986) Science 323, 503-506). We purified the calmodulins from both the wild type and pantophobiac A (a mutant lacking the above-mentioned conductance and whose phenotypic defect is traceable to its calmodulin) by hydrophobic interaction and immunoaffinity chromatographies, and examined them biochemically. In this paper we address the preliminary characterization of the two calmodulins and discuss the consequences of the genetic alteration. The differences described here are in their electrophoretic mobilities in polyacrylamide gel electrophoresis and in their binding characteristics to monoclonal antibodies raised against calmodulin from wild-type paramecia. Also, we present data which indicate a difference in the stimulation of the calmodulin-dependent enzyme bovine brain phosphodiesterase under certain conditions.     

Determining the environmental fate of a filamentous fungus, Trichoderma reesei, in laboratory-contained intact soil-core microcosms using competitive PCR and viability plating

Trichoderma spp. are used extensively in industry and are routinely disposed of in landfill sites as spent biomass from fermentation plants. However, little is known regarding the environmental fate of this biomass. We tracked the survival of T. reesei strain QM6A#4 (a derivative of strain QM6A marked with a recombinant construct) over a 6-month period in laboratory-contained, intact soil-core microcosms incubated in a growth chamber. Survival was tested in 3 different soils and the effect of a plant rhizosphere (bush lima beans, Phaseolus limensis) was investigated. Levels and viability of the fungus were determined, respectively, by quantitative competitive polymerase chain reaction analysis of total soil DNA extracts and dilution-plating of soil on a semiselective growth medium. Whereas chemically killed QM6A#4 became undetectable within 3 d, QM6A#4 added as a live inoculum decreased approximately 4- to approximately 160-fold over the first 1-3 months and then reached a steady state. After 4 months, soil cores were subjected to a 1.5-month simulated winter period, which did not significantly affect QM6A#4 levels. Throughout the experiment, QM6A#4 remained viable. These results indicate that, following release into the environment, live T. reesei will persist in soil for at least 2 seasons.     

Survival of genetically modified Pseudomonas fluorescens introduced into subtropical soil microcosms

Abstract A genetically modified strain of Pseudomonas fluorescens and its parent showed grossly similar decline rates following introduction into subtropical clay and sandy soils. In unplanted clay soit at pH 6.9 and 25°C, population densities declined progressively from about 10⁸ to 10³ colony forming units (cfu) g⁻¹ dry soil over 75 days, but in unplanted sandy soil the introduced populations could not be detected after 25 days. In clay soil at pH 8.7 or 4.7, or at environmental temperature, decay rates were enhanced as compared to those at pH 6.9 and 25°C. Counts of introduced strains in clay bulk soil and in rhizosphere and rhizoplane of maize suggested that the introduced bacteria competed well with the native bacteria, and colonized the roots at about 10⁶ cfu g⁻¹ dry root at 25°C, over 20 days. However, rhizoplane colonization was lower at environmental temperature. The decay rate of both strains was slower in planted than in unplanted sandy soil. The population densities in the rhizosphere and rhizoplane in the sandy soil were significantly lower than those in the clay soil. Both introduced strains colonized the maize roots in both soils, using seeds coated with bacteria in 1% carboxymethyl cellulose. Introduced cells were localized at different sites along the roots of plants developing in clay soil, with higher densities in the original (near the seeds) and root hair zones as compared to the intermediate zones. No significant difference was observed between the extent of root colonization of the genetically modified strain and its parent.     

Assessing the welfare of genetically altered mice

In 2003, under the auspices of the main UK funders of biological and biomedical research, a working group was established with a remit to review potential welfare issues for genetically altered (GA) mice, to summarize current practice, and to recommend contemporary best practice for welfare assessments. The working group has produced a report which makes practical recommendations for GA mouse welfare assessment and dissemination of welfare information between establishments using a 'mouse passport'. The report can be found at www.nc3rs.org.uk/GAmice and www.lal.org.uk/gaa and includes templates for the recommended welfare assessment scheme and the mouse passport. An overview is provided below.     

Fate of genetically modified microorganisms in the corn rhizosphere

The fates of genetically modified (GM)Escherichia coli andPseudomonas putida in the corn rhizosphere were investigated. Under hydrophonic and sterile conditions, both bacteria grew well in the presence of root exudates used as a sole carbon source. The growth patterns of wild types and genetically modified strains ofE. coli andP. putida were similar under the conditions tested.The presence of rhizospheric microorganisms affected the survival pattern ofE. coli. In the presence of corn roots and rhizospheric microorganisms,E. coli numbers increased during the first 3 days but were later drastically reduced, probably as a result of competition with rhizospheric microorganisms for the carbon source. However, in the presence of rhizospheric microorganisms,P. putida survived better thanE. coli in the simulated corn rhizosphere.     

Fate of a genetically modified bacterium in foregut of glassy-winged sharpshooter (Hemiptera: Cicadellidae)

Symbiotic control is a new strategy being investigated to prevent the spread of insect-transmitted pathogens by reducing vector competence. We are developing this strategy to reduce the spread of Xylella fastidiosa by Homalodisca vitripennis (Germar) [formerly Homalodisca coagulata (Say)] (Hemiptera: Cicadellidae), the glassy-winged sharpshooter. In this study, the fate of a transformed symbiotic bacterium, Alcaligenes xylosoxidans variety denitriicans (S1Axd), in the foregut of glassy-winged sharpshooter when fed on citrus (Citrus spp.) and grape (Vitris spp.) was assessed. TaqMan-based quantitative real-time polymerase chain reaction (PCR) was used to detect and quantify bacterial cells remaining in the foregut at 0, 2, 4, 9, and 12 d after acquisition. S1Axd titer dropped rapidly by 2 d after acquisition, but in spite of this, at end of the 12-d experimental period, 45 and 38% of the glassy-winged sharpshooters retained the transformed bacteria, when fed on grape and citrus, respectively.     

Phenotypes of aquaporin mutants in genetically altered mice

The ability to move water across lipid membranes is crucial for nutrient intake, energy generation, waste excretion, and a myriad of other functions associated with life. Aquaporins, a family of integral membrane proteins, are now recognized as the channels responsible for transporting hydrophilic molecules, including water, across relatively impervious, hydrophobic cell membranes. A tremendous amount of work has been published on characterizing these proteins, which have been found in all bacteria, yeast, plants, and animals examined to date. In addition, an increasing number of mouse models with genetically altered aquaporin expression are being reported. This article will briefly review the basic biochemistry of aquaporins and then evaluate the use (and misuse) of mice in the quest for understanding the comparative pathophysiology of aquaporins in humans.     

Persistence and stability of genetically manipulated derivatives of Enterobacter agglomerans in soil microcosms

Abstract Molecular methods and conventional plating were applied to monitor Enterobacter agglomerans 339 derivatives carrying a Tn5-Mob or an npt I-cassette in unsterile soil microcosms. The plate counts of the introduced bacteria decreased continuously in time until undetectable on selective media. In contrast, hybridization of the total DNA directly isolated from inoculated soil samples showed that the target sequences detected corresponded to a much higher number of bacteria than indicated by plating. By PCR-amplification and hybridization of the soil DNA we could show that asignificant number of target sequences still persisted in the soil microcosms, even when the inoculated bacteria were not able to make colonies on selective agar plates. The Tn 5 marker caused instabilities in the genome of the bacteria studied. Some of the clones that grew in the soil samples had rearrangements in their genome. The detection of E. agglomerans 339 derivatives carrying the immobile npt I-cassette was also dependent on its location in the bacterial genome.     

Estrogen receptors in the kidney: Lessons from genetically altered mice

Background: Sex differences in human and animal models of kidney disease suggest that estrogen receptor (ER)-mediated events may modulate these processes. Genetically altered mice lacking one or both ERs provide a powerful tool to study these phenomena.Objective: This article examines sex differences in the kidney, particularly the role of ERs.Methods: To identify pertinent studies in genetically altered mice, a literature search was conducted on the MEDLINE database from January 1966 to July 2007, using the search terms estrogen receptor, kidney, and mice. Our group examined the effect of the ER-α knockout genotype on the kidney in streptozotocin-induced diabetes mellitus and compensatory kidney growth after uninephrectomy.Results: Female mice lacking ERa had reduced renal growth, including glomerular enlargement after 2 weeks of streptozotocin-induced diabetes mellitus and compensatory kidney growth 48 hours after uninephrectomy.Conclusion: ER-mediated events influence kidney growth and disease in female mice.     

Microbial trophic interactions in aquatic microcosms designed for testing genetically engineered microorganisms: A field comparison

Microcosms may potentially be used as tools for evaluating the fate and effects of genetically engineered microorganisms released into the environment. Extrapolation of data to the field, however, requires that the correspondence between microcosm and field is known. Microbial trophic interactions within the microbial loop were compared quantitatively and qualitatively between field and microcosms containing estuarine water with and without intact sediment cores. The comparison showed that whereas proportions between trophic levels in microcosms were qualitatively similar to those in the field, rates of microbial processes were from 25 to 40% lower in microcosms. Nitrogen cycling was disrupted in microcosms incubated in the dark to eliminate primary production. Examination of the microbial parameters further suggests that sediment in microcosms may be an important factor regulating the bacterial trophic level. These results demonstrate that analysis of microbial trophic interactions is a sensitive method for the field comparison of aquatic microcosms and a potentially useful tool in the risk assessment of genetically engineered microorganisms. Offprint requests to: N. Kroer.     

Developing a strategy for the rapid identification of genetically altered mice: an olfactory system perspective

The mouse genome contains a wide array of uncharacterized genes that are involved in olfactory system development and cellular function. The author describes how random mutagenesis of these genes combined with rapid screening techniques afford the identification of the particular olfactory-related genes that have been altered.     

Temporal organization of feeding in Syrian hamsters with a genetically altered circadian period

The variation in spontaneous meal patterning was studied in three genotypes (tau +/+, tau +/-and tau -/-) of the Syrian hamster with an altered circadian period. Feeding activity was monitored continuously in 13 individuals from each genotype in constant dim light conditions. All three genotypes had on average six feeding episodes during the circadian cycle (about 20h in homozygous tau mutants and 22h in heterozygotes compared with 24h in wild-type hamsters). Thus, homozygous tau mutant hamsters had significantly more feeding episodes per 24h than wild types, and heterozygotes were intermediate. The average duration of feeding bouts (FBs) was indistinguishable (around 30 minutes) among the three genotypes, whereas the intermeal (IM) intervals were significantly shorter for homozygote tau mutant hamsters (99 minutes), intermediate for heterozygotes (116 minutes), and the longest for wild-type hamsters (148 minutes). Thus, the meal-to-meal duration was on average 25% shorter in homozygous tau mutants (16% in heterozygous) than in wild-type hamsters. The reduction of the circadian period has a pronounced effect on short-term feeding rhythms and meal frequency in hamsters carrying the tau mutation. (Chronobiology International, 18(4), 657-664, 2001)     

The relevance of genetically altered mouse models of human disease

The impetus to develop useful models of human disease and toxicity has resulted in a number of large-scale mouse mutagenesis programmes. This, in turn, has stimulated considerable concern regarding the scientific validity and welfare of genetically altered mice, and the large numbers of mice that are required by such programmes. In this paper, the scientific advantages and limitations of genetically altered mice as models of several human diseases are discussed. We conclude that, while the use of some such mouse models has contributed considerably to an understanding of human disease and toxicity, other genetically altered mouse models have limited scientific relevance, and fewer have positively contributed to the development of novel human medicines. Suggestions for improving this unsatisfactory situation are made.