Fate of the insecticidal Cry1Ab protein of GM crops in two agricultural soils as revealed by ¹4C-tracer studies.

Insecticidal delta-endotoxins of Bacillus thuringiensis are among the most abundant recombinant proteins released by genetically modified (GM) crops into agricultural soils worldwide. However, there is still controversy about their degradation and accumulation in soils. In this study, (14)C-labelled Cry1Ab protein was applied to soil microcosms at two concentrations (14 and 50 µg g(-1) soil) to quantify the mineralization of Cry1Ab, its incorporation into the soil microbial biomass, and its persistence in two soils which strongly differed in their texture but not in silt or pH. Furthermore, ELISA was used to quantify Cry1Ab and its potential immunoreactive breakdown products in aqueous soil extracts. In both soils, (14)CO2-production was initially very high and then declined during a total monitoring period of up to 135 days. A total of 16 to 23 % of the (14)C activity was incorporated after 29 to 37 days into the soil microbial biomass, indicating that Cry1Ab protein was utilized by microorganisms as a growth substrate. Adsorption in the clay-rich soil was the most important factor limiting microbial degradation; as indicated by higher degradation rates in the more sandy soil, extremely low concentrations of immunoreactive Cry1Ab molecules in the soils' aqueous extracts and a higher amount of (14)C activity bound to the soil with more clay. Ecological risk assessments of Bt-crops should therefore consider that the very low concentrations of extractable Cry1Ab do not reflect the actual elimination of the protein from soils but that, on the other hand, desorbed proteins mineralize quickly due to efficient microbial degradation.

Artificial soil studies reveal domain-specific preferences of microorganisms for the colonisation of different soil minerals and particle size fractions.

Artificial soils were used in this study to analyse the importance of different mineral compositions for the diversity of soil microorganisms. Variants containing montmorillonite (MT), illite (IL) and illite + ferrihydrite (IL+FH) were compared to each other. Bulk material and their particle size fractions, as obtained by ultracentrifugation and wet-sieving, were characterised for abundance and diversity of Bacteria, Archaea and Fungi. Samples were analysed 6 and 18 months after inoculation with sterilised manure and a soil-extracted microbial community. Generally, IL, and even more pronouncedly IL+FH, supported the growth of more Bacteria, Archaea and Fungi, than MT. This trend was most pronounced in the finest fraction (< 20 µm). The structural diversity of Fungi responded more strongly to the different mineral compositions than the Bacteria, for which particle size fractions were more important. Archaea established a specific community in the finest fraction and showed no response to the different mineral compositions. Overall, this study demonstrates that the mineral composition and the particle size fractions have specific and different selective effects on the three domains and, thus, suggests that these factors strongly contribute to niche separation and the high diversity of microbial communities in natural soils with complex mineral compositions.

Importance of soil organic matter for the diversity of microorganisms involved in the degradation of organic pollutants.

Many organic pollutants are readily degradable by microorganisms in soil, but the importance of soil organic matter for their transformation by specific microbial taxa is unknown. In this study, sorption and microbial degradation of phenol and 2,4-dichlorophenol (DCP) were characterized in three soil variants, generated by different long-term fertilization regimes. Compared with a non-fertilized control (NIL), a mineral-fertilized NPK variant showed 19% and a farmyard manure treated FYM variant 46% more soil organic carbon (SOC). Phenol sorption declined with overall increasing SOC because of altered affinities to the clay fraction (soil particles <2 mm in diameter). In contrast, DCP sorption correlated positively with particulate soil organic matter (present in the soil particle fractions of 63-2000 µm). Stable isotope probing identified Rhodococcus, Arthrobacter (both Actinobacteria) and Cryptococcus (Basidiomycota) as the main degraders of phenol. Rhodococcus and Cryptococcus were not affected by SOC, but the participation of Arthrobacter declined in NPK and even more in FYM. (14)C-DCP was hardly metabolized in the NIL variant, more efficiently in FYM and most in NPK. In NPK, Burkholderia was the main degrader and in FYM Variovorax. This study demonstrates a strong effect of SOC on the partitioning of organic pollutants to soil particle size fractions and indicates the profound consequences that this process could have for the diversity of bacteria involved in their degradation.

Response of microbial communities to long-term fertilization depends on their microhabitat.

The objective of this study was to characterize the microbial communities attached to clay (< 2 µm), fine silt (2-20 µm), coarse silt (20-63 µm) and sand-sized fractions [> 63 µm; including particulate organic matter (POM)] of an arable soil and analyse their response to more than 100 years of two different fertilization regimes. Mild ultrasonic dispersal, wet-sieving and centrifugation allowed the separation of soil particles with the majority of bacterial cells and DNA still attached. Fertilizations increased soil organic carbon (SOC), total DNA and the abundance of bacterial, archaeal and fungal rRNA genes more strongly in the larger-sized fractions than in fine silt, and no effect was seen with clay, the latter representing above 70% of the total microbial populations. A highly positive correlation was found between microbial rRNA genes and the surface area provided by the particles, while the correlation with SOC was lower, indicating a particle-size-specific heterogeneous effect of SOC. The prokaryotic diversity responded more strongly to fertilization in the larger particles but not with clay. Overall, these results demonstrate that microbial responsiveness to long-term fertilization declined with smaller particle sizes and that especially clay fractions exhibit a high buffering capacity protecting microbial cells against changes even after 100 years under different agricultural management.

Production of the 14C-labeled insecticidal protein Cry1Ab for soil metabolic studies using a recombinant Escherichia coli in small-scale batch fermentations.

Insecticidal Cry proteins naturally produced by Bacillus thuringiensis are a major recombinant trait expressed by genetically modified crops. They are released into the soil during and after cropping. The objective of this study was to produce (14)C-labeled Cry1Ab proteins for soil metabolic studies in scope of their environmental risk assessment. Cry1Ab was synthesized as a protoxin by Escherichia coli HB101 pMP in 200-mL liquid batch culture fermentations and purified from inclusion bodies after trypsin digestion. For cultivation, U-(14)C-glycerol was the main carbon source. Inclusion bodies were smaller and Cry1Ab yield was lower when the initial amount of total organic carbon in the cultivation broth was below 6.4 mg C L(-1). Concentrations of 12.6 g (14)C-labeled glycerol L(-1) (1 % v/v) resulted in the production of 17.1 mg (14)C-Cry1Ab L(-1) cultivation medium. (14)C mass balances showed that approx. 50 % of the label was lost by respiration and 20 % remained in the growth media, while the residual activity was associated with biomass. Depending on the production batch, 0.01 to 0.05 % of the total (14)C originated from Cry1Ab. In the presence of 2.04 MBq (14)C-labeled carbon sources, a specific activity of up to 268 Bq mg(-1) (14)C-Cry1Ab was obtained. A more than threefold higher specific activity was achieved with 4.63 MBq and an extended cultivation period of 144 h. This study demonstrates that (14)C-labeled Cry1Ab can be obtained from batch fermentations with E. coli in the presence of a simple (14)C-labeled carbon source. It also provides a general strategy to produce (14)C-labeled proteins useful for soil metabolic studies.

Origin and diversity of metabolically active gut bacteria from laboratory-bred larvae of Manduca sexta (Sphingidae, Lepidoptera, Insecta).

Cultivation-independent analyses based on genetic profiling of partial bacterial 16S rRNA genes by PCR-single-strand conformation polymorphism (PCR-SSCP), reverse transcriptase (RT)-PCR-SSCP of the 16S rRNA itself, and stable isotope probing (SIP), followed by RT-PCR-SSCP, were applied to characterize the diversity of metabolically active bacteria in the larval gut of Manduca sexta bred on tobacco leaves under greenhouse conditions. For SIP, hatching larvae were fed with leaves from tobacco plants grown in a (13)CO(2)-enriched atmosphere. Dominant SSCP bands were sequenced and phylogenetically analyzed. Only one major gut colonizer, an Enterococcus relative, was detected; it occurred in the heavy RNA fraction, demonstrating its metabolic activity, and it originated from eggs, where its metabolic activity was also indicated by rRNA-based SSCP profiles. In contrast, a Citrobacter sedlakii relative was detected on eggs by DNA-SSCP, but rRNA-SSCP and SIP-rRNA-SSCP were negative, suggesting that these bacterial cells were inactive. A Burkholderia relative was dominant and metabolically active on the tobacco leaves but inactive inside the gut, where it was also quantitatively reduced, as suggested by lower band intensities in the DNA-based SSCP profiles. SIP-RNA-SSCP detected another metabolically active gut bacterium (Enterobacter sp.) and more bacteria in the light RNA fraction, indicating low or no metabolic activity of the latter inside the gut. We conclude that the larval gut supported only a low diversity of metabolically active bacteria.