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

Construction of green fluorescent protein-based vectors for genetic analysis in bacteria

El gen gfp que codifica para la proteína verde fluorescente (GFP) fue utilizado para crear una serie de plásmidos y un minitransposon registradores para el análisis genético. y la monitorización de bacterias. El sistema registrador está integrado en un plásmido para estudio de promotores y en un minitransposon para generar fusiones transcripcionales por inserción al azar en el cromosoma. Con el objeto de mejorar los niveles de sensibilidad necesarios para el uso del sistema en monocopia y para la detección en células individuales, el extremo 3'del gen gfp fue reemplazado por el de otro gen gfp mofidicado que emite luz verde 45 veces más fuerte que la GFP natural. Además, al extremo S' del nuevo gen gfp se fusionó la señal estimuladora de la traducción del gen atpE. La conjugación del minitransposon a dos Pseudomonas spp. diferentes y a Alcaligenes eutrophus produjo fusiones al azar en el cromosoma, de las que un 5% emitían fluorescencia detectable a ojo. La expresión de GFP en células aisladas fue perfectamente visualizada por microscopía de fluorescencia.The green fluorescent protein (GFP) gene, gfp, was used to develop versatile reporter systems for genetic analysis in, and monitoring of bacteria. The reporter system is available on a plasmid and on a mini-transposon located in a suicide delivery plasmid for generation of chromosomal fusions. To achieve sensitivity levels necessary for use in monocopy and for detection of single cells, the 3'-end of gfp was replaced by that of a modified gfp gene characterized by a 4S-fold stronger signal than that exhibited by the natural OFP, and the modified gfp gene equipped with the strong translation signals of the atpE gene. Transfer of the minitrasnposon into two different Pseudomonas spp. and Alcaligenes eutrophus produced random chromosomal fusions, some 5% of which exhibited fluorescence detectable by eye. Individual OFP+ cells were readily observed by fluorescence microscopy. The detailed map of the step-by-step construction is depicted

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

Faunal diversity of Fagus sylvatica forests: A regional and European perspective based on three indicator groups

<p>While the postglacial history of European beech (Fagus sylvatica) and the plant species composition of beech forests in  Central Europe are fairly well understood, the faunal biodiversity has been less well investigated. We studied three groups of  mostly sedentary organisms in beech forest at regional and European scales by combining field studies with a compilation of existing literature and expert knowledge. Specifically, we examined the relationship between host tree genera and saproxylic  beetles, and the diversity and composition of forest ground-dwelling molluscs and ground beetles in relation to the abundance  of beech. At a west central European scale (Germany), where beech has a “young” ecological and biogeographical history,  we found 48 primeval forest relict species of saproxylic beetles associated with beech, 124 ground beetles and 91 molluscs  inhabiting beech forest, yet none exclusive of west central European beech forests. High levels of faunal similarity between beech and other woodland trees suggested that many of the beech forest dwelling species are euryoecious and likely to  originate from mid-Holocene mixed broadleaf forests. Beech forests of the mountain ranges in southern and east central  Europe, which are ecologically and biogeographically “old”, were found to harbour distinct species assemblages, including  beech forest specialists (such as 10 carabid species in the Carpathians) and narrow-range endemics of broadleaf forest. The  observed biodiversity patterns suggest differentiated conservation priorities in “young” and “old” European beech forest  regions.</p