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To understand mechanisms of tufa biofilm calcification, selected karstwater stream stromatolites in Germany have been investigated with regard to their hydrochemistry, biofilm community, exopolymers, physicochemical microgradients, calcification pattern and lamination. In stream waters, CO2degassing drives the increase in calcite saturation to maximum values of approximately 10-fold, independent from the initial Ca2+/alkalinity ratio. For the cyanobacteria of tufa biofilms, a culture-independent molecular approach showed that microscopy of resin-embedded biofilm thin sections underestimated the actual diversity of cyanobacteria, i.e. the six cyanobacteria morphotypes were opposed to nine different lineages of the 16S rDNA phylogeny. The same morphotype may even represent two genetically distant cyanobacteria and the closest relatives of tufa biofilm cyanobacteria may be from quite different habitats. Diatom diversity was even higher in the biofilm at the studied exemplar site than that of the cyanobacteria, i.e. 13 diatom species opposed to 9 cyanobacterial lineages. The non-phototrophic prokaryotic biofilm community is clearly different from the soil-derived community of the stream waters, and largely composed of flavobacteria, firmicutes, proteobacteria and actinobacteria. The exopolymeric biofilm matrix can be divided into three structural domains by fluorescence lectin-binding analysis. Seasonal and spatial variability of these structural EPS domains is low in the investigated streams. As indicated by microsensor data, biofilm photosynthesis is the driving mechanism in tufa stromatolite formation. However, photosynthesis-induced biofilm calcification accounts for only 10–20% of the total Ca2+loss in the streams, and occurs in parallel to inorganic precipitation driven by CO2-degassing within the water column and on biofilm-free surfaces. Annual stromatolite laminae reflect seasonal changes in temperature and light supply. The stable carbon isotope composition of the laminae is not affected by photosynthesis-induced microgradients, but mirrors that of the bulk water body only reflecting climate fluctuations. Tufa stromatolites with their cyanobacterial–photosynthesis-related calcification fabrics form an analogue to porostromate cyanobacterial stromatolites in fossil settings high in CaCO3mineral supersaturation but comparatively low in dissolved inorganic carbon. Here, the sum-effect of heterotrophic exopolymer-degradation and secondary Ca2+-release rather decreases calcite saturation, contrary to settings high in dissolved inorganic carbon such as soda lakes.

An analysis of the major factors affecting the accuracy of harmonic force field computations of water is presented. By systematically varying the level of approximation in the basis set, treatment of electron correlation, core electron correlation, and relativistic correction, the underlying sources of error in the computation of harmonic vibrational frequencies for water were quantified. The convergence error due to wavefunction description with a cc-pVQZ basis set in the absence of electron correlation was 1.6 cm-1, as determined from extending the Hartree−Fock computations to larger basis sets. The convergence error due to neglecting higher-order electronic correlation terms than are included at the CCSD(T) level using the cc-pVTZ basis set was estimated to be 4.7 cm-1, as determined from frequency calculations up to CCSDTQ for water and literature results up to CCSDTQP for diatomic molecules. The convergence error due to omitting higher-order diffuse functions than included in aug-cc-pVQZ was found to be 3.7 cm-1, as determined by adding more diffuse functions in larger basis sets. The error associated with neglecting core electron correlation effects (i.e., “freezing” core electrons) was 5.0 cm-1and with neglecting relativistic effects was 2.2 cm-1. Due to a cancellation among these various sources of error, the harmonic frequencies for H2O computed using the CCSD(T)/aug-cc-pVQZ model chemistry were on average within 2 cm-1of experimentally inferred vibrational frequencies.

The objective of this study was to evaluate the effect of insect larval feeding on the fate and genetic transformability of recombinant DNA from a transplastomic plant. Leaves of tobacco plants with an aadA antibiotic resistance gene inserted into their chloroplast genome were incubated with larvae of the tobacco hornworm Manduca sexta (Lepidoptera). The specifically designed Acinetobacter strain BD413 pBAB2was chosen to analyze the functional integrity of the aadA transgene for natural transformation after gut passages. No gene transfer was detected after simultaneous feeding of leaves and the Acinetobacter BD413 pBAB2as a recipient, even though 15% of ingested Acinetobacter BD413 cells could be recovered as viable cells from feces 6?h after feeding. Results with real-time PCR indicated that an average of 98.2 to 99.99% of the aadA gene was degraded during the gut passage, but the range in the number of aadA genes in feces of larvae fed with transplastomic leaves was enormous, varying from 5 ? 106to 1 ? 109copies.g-1. DNA extracted from feces of larvae fed with transplastomic leaves was still able to transform externally added competent Acinetobacter BD413 pBAB2in?vitro. Transformation frequencies with concentrated feces DNA were in the same range as those found with leaves (10-4?10-6transformants per recipient) or purified plasmid DNA (10-3?10-7). The presence of functionally intact DNA was also qualitatively observed after incubation of 30 mg freshly shed feces directly with competent Acinetobacter BD413 pBAB2, demonstrating that aadA genes in feces have a potential to undergo further horizontal gene transfer under environmental conditions.

Cultivation-independent analyses were carried out to compare the bacterial community structure found in the rhizospheres of a transplastomic tobacco plant carrying the antibiotic resistance marker-gene aadA and its non-engineered parental line. PCR- and reverse transcriptase PCR-amplifications of 16S rRNA and their corresponding genes were carried out with primers targeting the domain Bacteria. The diversity of PCR-products amplified from total nucleic acids extracted from rhizospheres of 10-week-old plants, which had been grown in potting soil in the greenhouse, was visualized by genetic profiling using the single-strand conformation polymorphism (SSCP) technique. The SSCP profiles generated from DNA extracted with two different protocols, one including total RNA and the other only DNA, did not show any differences. The SSCP profiles amplified from RNA and DNA were also highly similar to each other, indicating that the dominant bacteria detected were metabolically active. High similarities were seen between the SSCP profiles from the transplastomic and the non-engineered plants, except for a single band that consistently occurred with samples from the non-engineered plants (six replicates), but not, or only weakly, with their engineered counterparts. DNA sequencing and database analysis revealed that the partial rRNA gene matched to a Flavobacterium sp. Other bands of the SSCP-profiles, related to Burkholderia and Bordetella were variable between individual plants but not affected by the transplastomic modification. Thus, the transplastomic modification caused a relative decline of a specific Flavobacterium population but not of other bacteria. Further studies including additional tobacco cultivars, soils and conditions of cultivation would be desirable, to elucidate the ecological importance of this difference.

Cultivation-independent analyses were carried out to compare the bacterial community structure found in the rhizospheres of a transplastomic tobacco plant carrying the antibiotic resistance marker-gene aadAand its non-engineered parental line. PCR- and reverse transcriptase PCR-amplifications of 16S rRNA and their corresponding genes were carried out with primers targeting the domain Bacteria. The diversity of PCR-products amplified from total nucleic acids extracted from rhizospheres of 10-week-old plants, which had been grown in potting soil in the greenhouse, was visualized by genetic profiling using the single-strand conformation polymorphism (SSCP) technique. The SSCP profiles generated from DNA extracted with two different protocols, one including total RNA and the other only DNA, did not show any differences. The SSCP profiles amplified from RNA and DNA were also highly similar to each other, indicating that the dominant bacteria detected were metabolically active. High similarities were seen between the SSCP profiles from the transplastomic and the non-engineered plants, except for a single band that consistently occurred with samples from the non-engineered plants (six replicates), but not, or only weakly, with their engineered counterparts. DNA sequencing and database analysis revealed that the partial rRNA gene matched to a Flavobacteriumsp. Other bands of the SSCP-profiles, related to Burkholderiaand Bordetellawere variable between individual plants but not affected by the transplastomic modification. Thus, the transplastomic modification caused a relative decline of a specific Flavobacteriumpopulation but not of other bacteria. Further studies including additional tobacco cultivars, soils and conditions of cultivation would be desirable, to elucidate the ecological importance of this difference.

The objective of this study was to evaluate the effect of insect larval feeding on the fate and genetic transformability of recombinant DNA from a transplastomic plant. Leaves of tobacco plants with an aadAantibiotic resistance gene inserted into their chloroplast genome were incubated with larvae of the tobacco hornworm Manduca sexta(Lepidoptera). The specifically designed Acinetobacterstrain BD413 pBAB2was chosen to analyze the functional integrity of the aadAtransgene for natural transformation after gut passages. No gene transfer was detected after simultaneous feeding of leaves and the AcinetobacterBD413 pBAB2as a recipient, even though 15% of ingested AcinetobacterBD413 cells could be recovered as viable cells from feces 6 h after feeding. Results with real-time PCR indicated that an average of 98.2 to 99.99% of the aadA gene was degraded during the gut passage, but the range in the number of aadAgenes in feces of larvae fed with transplastomic leaves was enormous, varying from 5 × 106to 1 × 109copies.g-1. DNA extracted from feces of larvae fed with transplastomic leaves was still able to transform externally added competent AcinetobacterBD413 pBAB2in vitro. Transformation frequencies with concentrated feces DNA were in the same range as those found with leaves (10-4–10-6transformants per recipient) or purified plasmid DNA (10-3–10-7). The presence of functionally intact DNA was also qualitatively observed after incubation of 30 mg freshly shed feces directly with competent AcinetobacterBD413 pBAB2, demonstrating that aadAgenes in feces have a potential to undergo further horizontal gene transfer under environmental conditions.