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Changes of the rumen microbial community structure, as it can be established with a rumen simulation technique (RUSITEC) were studied using PCR and single-strand conformation polymorphism (SSCP) of small subunit rDNA genes (SSU rDNA). Four total mixed rations were incubated and two ammonia levels in the artificial saliva were applied. Three replicated vessels were used for each treatment. Mixed microbial fractions were isolated by stepwise centrifugation from the liquid fraction (reference microbes, RM) and from the solids of the feed residues (solid-associated microbes, SAM). PCR-primers targeting archaea, fibrobacter, clostridia, and bacteria, respectively, were applied to represent the individual taxonomic groups by SSCP profiles. These SSCP profiles were converted into a binary matrix and distances among treatments were visualised by non-metric multidimensional scaling. Between replicates belonging to one treatment only small differences were found, indicating a high reproducibility of the RUSITEC and the chosen SSCP method. The ammonia concentration seems to be affecting the SSCP profiles. Great differences occurred between RM and SAM, especially for profiles targeting bacteria and clostridia. Differences in the profiles of RM were also found between mixed rations that contained the same feedstuffs in different ratios and between rations with similar nutrient content but based on different feedstuffs. In conclusion, the PCR-SSCP-based technique in conjunction with non-metric multidimensional scaling was sufficiently sensitive to detect and compare changes in composition of rumen microbial community structure in vitro as affected by diet and other environmental factors.

Bees are specifically subjected to intimate contacts with transgenic plants due to their feeding activities on pollen. In this study, the probability and ecological risk of a gene transfer from pollen to gut bacteria of bees was investigated with larvae of Apis mellifera (honeybee), Bombus terrestris (bumblebee), and Osmia bicornis (red mason bee), all collected at a flowering transgenic oilseed rape field. The plants were genetically engineered with the pat-gene, conferring resistance against glufosinate (syn. phosphinothricin), a glutamine-synthetase inhibitor in plants and microorganisms. Ninety-six bacterial strains were isolated and characterized by 16S rRNA gene sequencing, revealing that Firmicutes represented 58% of the isolates, Actinobacteria 31%, and Proteobacteria 11%, respectively. Of all isolates, 40% were resistant to 1 mM glufosinate, and 11% even to 10 mM. Resistant phenotypes were found in all phylogenetic groups. None of the resistant phenotypes carried the recombinant pat-gene in its genome. The threshold of detecting gene transfer in this field study was relatively insensitive due to the high background of natural glufosinate resistance. However, the broad occurrence of glufosinate-resistant bacteria from different phylogenetic groups suggests that rare events of horizontal gene transfer will not add significantly to natural bacterial glufosinate resistance.

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 pBAB(2) was 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 pBAB(2) as 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 x 10(6) to 1 x 10(9) copies.g(-1). DNA extracted from feces of larvae fed with transplastomic leaves was still able to transform externally added competent Acinetobacter BD413 pBAB(2) in vitro. Transformation frequencies with concentrated feces DNA were in the same range as those found with leaves (10(-4)-10(-6) transformants 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 pBAB(2), demonstrating that aadA genes in feces have a potential to undergo further horizontal gene transfer under environmental conditions.

Current elevated concentrations of ozone in the atmosphere, as they are observed during summer seasons, can cause severe effects on plant vegetation. This study was initiated to analyze whether ozone-stressed plants also transfer signals below ground and thereby alter the bacterial community composition in their rhizospheres. Herbaceous plants, native to Germany, with tolerance (Anthoxanthum odoratum, Achillea millefolium, Poa pratensis, Rumex acetosa, and Veronica chamaedrys) and sensitivity (Matricaria chamomilla, Sonchus asper, and Tanacetum vulgare) to ozone, raised in the greenhouse, were exposed in open-top chambers to two different ozone regimes, i.e., "summer stress" and a normal ozone background. DNA of bacterial cells from the rhizospheres was directly extracted, and partial sequences of the 16S rRNA genes were PCR amplified with primers targeting the following phylogenetic groups: Bacteria, alpha-Proteobacteria, Actinobacteria, and Pseudomonas, respectively. The diversity of the amplified products was analyzed by genetic profiling based on single-strand conformation polymorphism (SSCP). Neither the tolerant nor the sensitive plants, the latter with visible above-ground damage, showed ozone-induced differences in any of the SSCP profiles, with the single exception of Actinobacteria-targeted profiles from S. asper. To increase the stress, S. asper was germinated and raised in the continuous presence of an elevated level of ozone. SSCP profiles with Bacteria-specific primers combined with gene probe hybridizations indicated an ozone-related increase in a Xanthomonas-related 16S rRNA gene and a decrease in the respective gene from the plant plastids. The fact that only this latter unrealistic scenario caused a detectable effect demonstrated that ozone stress has a surprisingly small effect on the structural diversity of the bacterial community in rhizospheres.

Plasmid borne organophosphorus pesticide degrading (opd) gene of Flavobacterium balustinum has been amplified using polymerase chain reaction (PCR) and the resulting PCR product (1.25 Kb) was cloned in pUC18. Further, a detailed restriction map was determined to PCR product and subcloned as overlapping restriction fragments. The nucleotide sequence was determined for all subclones to obtain complete sequence of PCR amplified fragment. The sequence showed 98% similarity to opd genes cloned from other soil bacteria isolated from diversified geographical regions. The protein sequence predicted from the nucleotide sequence was almost identical to parathion hydrolase, a triesterase involved in hydrolysis of triester bond found in variety of op-pesticides. The signal sequence of parathion hydrolase contained recently discovered twin arginine transport (tat) motif. It appears that tat motif plays a critical role in membrane targeting of parathion hydrolase.

The organophosphorus pesticide degrading (opd) gene was cloned downstream to the transcriptional and translational signals of expression vectors pTrc99A and pET32A. The resulting recombinant expression plasmids pNH2 and pHH2 were introduced into E. coli JM105 and E. coli BL21 respectively. On induction the E. coli cells containing pNH2 produced high levels of parathion hydrolase. A 60 kD fusion protein was produced in E. coli cultures containing recombinant plasmid pHH2. The molecular mass of the fusion protein coincided with the molecular mass of 40 kD parathion hydrolase and 20 kD N-terminal His tag encoded by the vector. Further, the fusion protein was purified using Ni-column and the N-terminal His-tag was removed by digesting it with thrombin. The resulting protein folded properly in presence of Zn2+ ions, and showed parathion hydrolase activity.