An investigation of horizontal transfer of feed introduced DNA to the aerobic microbiota of the gastrointestinal tract of rats

Background: Horizontal gene transfer through natural transformation of members of the microbiota of the lower gastrointestinal tract (GIT) of mammals has not yet been described. Insufficient DNA sequence similarity for homologous recombination to occur has been identified as the major barrier to interspecies transfer of chromosomal DNA in bacteria. In this study we determined if regions of high DNA similarity between the genomes of the indigenous bacteria in the GIT of rats and feed introduced DNA could lead to homologous recombination and acquisition of antibiotic resistance genes. Results: Plasmid DNA with two resistance genes (nptII and aadA) and regions of high DNA similarity to 16S rRNA and 23S rRNA genes present in a broad range of bacterial species present in the GIT, where constructed and added to standard rat feed. Six rats, with a normal microbiota, were fed DNA containing pellets daily over four days before sampling of the microbiota from the different GI compartments (stomach, small intestine, cecum and colon). In addition, two rats were included as negative controls. Antibiotic resistant colonies growing on selective media were screened for recombination with feed introduced DNA by PCR targeting unique sites in the putatively recombined regions. Conclusions: The analyses showed that extensive ingestion of DNA (100 \ub5g plasmid) per day did not lead to increased proportions of kanamycin resistant bacteria, nor did it produce detectable transformants among the aerobic microbiota examined for 6 rats (detection limit <1 transformant per 1.1 x 108 cultured bacteria). The key methodological challenges to HGT detection in animal feedings trials are identified and discussed

Inducible cell lysis system for the study of natural transformation and environmental fate of DNA released by cell death.

Two novel conditional broad-host-range cell lysis systems have been developed for the study of natural transformation in bacteria and the environmental fate of DNA released by cell death. Plasmid pDKL02 consists of lysis genes S, R, and Rz from bacteriophage lambda under the control of the Ptac promoter. The addition of inducer to Escherichia coli, Acinetobacter calcoaceticus, or Pseudomonas stutzeri containing plasmid pDKL02 resulted in cell lysis coincident with the release of high amounts of nucleic acids into the surrounding medium. The utility of this lysis system for the study of natural transformation with DNA released from lysed cells was assessed with differentially marked but otherwise isogenic donor-recipient pairs of P. stutzeri JM300 and A. calcoaceticus BD4. Transformation frequencies obtained with lysis-released DNA and DNA purified by conventional methods and assessed by the use of antibiotic resistance (P. stutzeri) or amino acid prototrophy (A. calcoaceticus) for markers were comparable. A second cell lysis plasmid, pDKL01, contains the lysis gene E from bacteriophage phi X174 and causes lysis of E. coli and P. stutzeri bacteria by activating cellular autolysins. Whereas DNA released from pDKL02-containing bacteria persists in the culture broth for days, that from induced pDKL01-containing bacteria is degraded immediately after release. The lysis system involving pDKL02 is thus useful for the study of both the fate of DNA released naturally into the environment by dead cells and gene transfer by natural transformation in the environment in that biochemically unmanipulated DNA containing defined sequences and coding for selective phenotypes can be released into a selected environment at a specific time point. This will allow kinetic measurements that will answer some of the current ecological questions about the fate and biological potential of environmental DNA to be made

Impact of pre-treatments on properties of lignocelluloses and their accessibility for a subsequent carboxymethylation

In this issue, different chemical (alkaline and sulfite pulping, ozonolysis) and mechanical (vibratory ball milling) pre-treatments were utilized for activating wheat straw and beech sawdust prior to carboxymethylation. Detailed analysis by a range of methods, including Klason-lignin, cellulose and hemicellulose quantification, Powder-X-ray diffraction (PXRD) and attenuated total reflection (ATR) IR spectroscopy, enabled the investigation of material alterations. Subsequently, carboxymethylation was carried out with both untreated and activated materials, allowing the evaluation of activation steps by determining degrees of substitution with carboxymethyl groups (DSCM). Moreover, carboxymethylation conditions were optimized, realizing high DSCM of up to 1.05. Results further revealed that ball milling enhanced the subsequent conversion; whereas chemical pre-treatments did not effectively increase material accessibilities. Further studies on chemically untreated material s emphasized that a highly reactive surface was already generated in the course of the carboxymethylation, inter alia through the concomitant dissolution of matrix components