Environmental biosafety research最新文献

{"title":"Leaf-feeding larvae of Manduca sexta (Insecta, Lepidoptera) drastically reduce copy numbers of aadA antibiotic resistance genes from transplastomic tobacco but maintain intact aadA genes in their feces.","authors":"Nicole Brinkmann, Christoph C Tebbe","doi":"10.1051/ebr:2007028","DOIUrl":"10.1051/ebr:2007028","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":87177,"journal":{"name":"Environmental biosafety research","volume":"6 1-2","pages":"121-33"},"PeriodicalIF":0.0,"publicationDate":"2007-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"27070453","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Horizontal gene transfer between bacteria.","authors":"Holger Heuer,&nbsp;Kornelia Smalla","doi":"10.1051/ebr:2007034","DOIUrl":"https://doi.org/10.1051/ebr:2007034","url":null,"abstract":"<p><p>Horizontal gene transfer (HGT) refers to the acquisition of foreign genes by organisms. The occurrence of HGT among bacteria in the environment is assumed to have implications in the risk assessment of genetically modified bacteria which are released into the environment. First, introduced genetic sequences from a genetically modified bacterium could be transferred to indigenous micro-organisms and alter their genome and subsequently their ecological niche. Second, the genetically modified bacterium released into the environment might capture mobile genetic elements (MGE) from indigenous micro-organisms which could extend its ecological potential. Thus, for a risk assessment it is important to understand the extent of HGT and genome plasticity of bacteria in the environment. This review summarizes the present state of knowledge on HGT between bacteria as a crucial mechanism contributing to bacterial adaptability and diversity. In view of the use of GM crops and microbes in agricultural settings, in this mini-review we focus particularly on the presence and role of MGE in soil and plant-associated bacteria and the factors affecting gene transfer.</p>","PeriodicalId":87177,"journal":{"name":"Environmental biosafety research","volume":"6 1-2","pages":"3-13"},"PeriodicalIF":0.0,"publicationDate":"2007-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1051/ebr:2007034","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"27069557","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Detection of potential transgenic plant DNA recipients among soil bacteria.","authors":"Jean-Michel Monier, Dominique Bernillon, Elizabeth Kay, Aurélie Faugier, Oleksandra Rybalka, Yves Dessaux, Pascal Simonet, Timothy M Vogel","doi":"10.1051/ebr:2007036","DOIUrl":"10.1051/ebr:2007036","url":null,"abstract":"<p><p>The likelihood of gene transfer from transgenic plants to bacteria is dependent on gene number and the presence of homologous sequences. The large number of transgene copies in transplastomic (transgenes contained in the chloroplast genome) plant cells as well as the prokaryotic origin of the transgene, may thus significantly increase the likelihood of gene transfer to bacteria that colonize plant tissues. In order to assess the probability of such transfer, the length of homologous DNA sequences required between the transgene and the genome of the bacterial host was assessed. In addition, the probability that bacteria, which co-infect diseased plants, are transformable and have sequences similar to the flanking regions of the transgene was evaluated. Using Acinetobacter baylyi strain BD143 and transplastomic tobacco plants harboring the aadA gene (streptomycin and spectinomycin resistance), we found that sequences identical to the flanking regions containing as few as 55 nucleotides were sufficient for recombination to occur. Consequently, a collection of bacterial isolates able to colonize tobacco plant tissue infected by Ralstonia solanacearum strain K60 was obtained, screened for DNA sequence similarity with the chloroplastic genes accD and rbcL flanking the transgene, and tested for their ability to uptake extracellular DNA (broad host-range pBBR1MCS plasmids) by natural or electro-transformation. Results showed that among the 288 bacterial isolates tested, 8% presented DNA sequence similarity with one or both chloroplastic regions flanking the transgene. Two isolates, identified as Pseudomonas sp. and Acinetobacter sp., were able to integrate exogenous plasmid DNA by electro-transformation and natural transformation, respectively. Our data suggest that transplastomic plant DNA recipients might be present in soil bacterial communities.</p>","PeriodicalId":87177,"journal":{"name":"Environmental biosafety research","volume":"6 1-2","pages":"71-83"},"PeriodicalIF":0.0,"publicationDate":"2007-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"27069516","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Influence of flanking homology and insert size on the transformation frequency of Acinetobacter baylyi BD413.","authors":"Deborah J Simpson,&nbsp;Lisa F Dawson,&nbsp;John C Fry,&nbsp;Hilary J Rogers,&nbsp;Martin J Day","doi":"10.1051/ebr:2007027","DOIUrl":"https://doi.org/10.1051/ebr:2007027","url":null,"abstract":"<p><p>RecA-mediated recombination requires regions of homology between donor and recipient DNA for successful integration. This paper investigates the effect of the relationship between the length of gene-sized inserts (434, 733, 2228 and 2400 bp) and flanking sequence homology (100 - ca. 11 000 bp) on transformation frequency in Acinetobacter baylyi strain BD413. Both insert size and size of the homologous region were varied, which improves on previous studies that kept insert size constant and varied only the homologous flank size. Transfer frequency of a non-homologous single small gene for gentamicin resistance (aac(3)I; 773 bp) was increased 18-fold when flanking homology was changed from about 2000 bp to 8000 bp, but was reduced 234-fold when two genes were inserted (nptII-gfp; 2400 bp) between similar homologous regions. To investigate the effect of smaller regions of flanking homology (100 - 2000 bp), a partial nptII-gfp deletion (434 bp) was restored. This confirmed that a minimum of 500 bp on each flank was required for transformation to be affected by flanking homology. The data obtained allowed development of a multiple regression equation to predict transformation frequency from homology, insert size and total fragment size for gene insertions. We also show that the ratio of flanking homology to insert size and not the total size of donor DNA is the most important variable determining transformation frequency. The equation developed was consistent with results previously reported by others, and so will be useful when using A. baylyi as a model for gene transfer by transformation in the laboratory, environment and for biosafety.</p>","PeriodicalId":87177,"journal":{"name":"Environmental biosafety research","volume":"6 1-2","pages":"55-69"},"PeriodicalIF":0.0,"publicationDate":"2007-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"27069512","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Differences in the rhizosphere bacterial community of a transplastomic tobacco plant compared to its non-engineered counterpart.","authors":"Nicole Brinkmann, Christoph C Tebbe","doi":"10.1051/ebr:2007025","DOIUrl":"10.1051/ebr:2007025","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":87177,"journal":{"name":"Environmental biosafety research","volume":"6 1-2","pages":"113-9"},"PeriodicalIF":0.0,"publicationDate":"2007-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"27070452","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"An assessment of the potential of herbivorous insect gut bacteria to develop competence for natural transformation.","authors":"Jessica L Ray,&nbsp;Helga K Andersen,&nbsp;Sandra Young,&nbsp;Kaare M Nielsen,&nbsp;Maureen O'Callaghan","doi":"10.1051/ebr:2007032","DOIUrl":"https://doi.org/10.1051/ebr:2007032","url":null,"abstract":"<p><p>Whereas the capability of DNA uptake has been well established for numerous species and strains of bacteria grown in vitro, the broader distribution of natural transformability within bacterial communities remains largely unexplored. Here, we investigate the ability of bacterial isolates from the gut of grass grub larvae (Costelytra zealandica (White); Coleoptera: Scarabaeidae) to develop natural genetic competence in vitro. A total of 37 mostly species-divergent strains isolated from the gut of grass grub larvae were selected for spontaneous rifampicin-resistance. Genomic DNA was subsequently isolated from the resistant strains and exposed to sensitive strains grown individually using established filter transformation protocols. DNA isolated from wild-type strains was used as a control. None of the 37 isolates tested exhibited a frequency of conversion to rifampicin-resistance in the presence of DNA at rates that were significantly higher than the rate of spontaneous mutation to rifampicin-resistance in the presence of wild-type DNA (the limit of detection was approximately < 1 culturable transformant per 10(9) exposed bacteria). To further examine if conditions were conducive to bacterial DNA uptake in the grass grubs gut, we employed the competent bacterium Acinetobacter baylyi strain BD413 as a recipient species for in vivo studies. However, no transformants could be detected above the detection limit of 1 transformant per 10(3) cells, possibly due to low population density and limited growth of A. baylyi cells in grass grub guts. PCR analysis indicated that chromosomal Acinetobacter DNA remains detectable by PCR for up to 3 days after direct inoculation into the alimentary tract of grass grub larvae. Nevertheless, neither transforming activity of the DNA recovered from the alimentary tract of grass grubs larvae nor competence of bacterial cells recovered from inoculated larvae could be shown.</p>","PeriodicalId":87177,"journal":{"name":"Environmental biosafety research","volume":"6 1-2","pages":"135-47"},"PeriodicalIF":0.0,"publicationDate":"2007-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"27069343","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Release and persistence of extracellular DNA in the environment.","authors":"Kaare M Nielsen,&nbsp;Pål J Johnsen,&nbsp;Douda Bensasson,&nbsp;Daniele Daffonchio","doi":"10.1051/ebr:2007031","DOIUrl":"https://doi.org/10.1051/ebr:2007031","url":null,"abstract":"<p><p>The introduction of genetically modified organisms (GMOs) has called for an improved understanding of the fate of DNA in various environments, because extracellular DNA may also be important for transferring genetic information between individuals and species. Accumulating nucleotide sequence data suggest that acquisition of foreign DNA by horizontal gene transfer (HGT) is of considerable importance in bacterial evolution. The uptake of extracellular DNA by natural transformation is one of several ways bacteria can acquire new genetic information given sufficient size, concentration and integrity of the DNA. We review studies on the release, breakdown and persistence of bacterial and plant DNA in soil, sediment and water, with a focus on the accessibility of the extracellular nucleic acids as substrate for competent bacteria. DNA fragments often persist over time in many environments, thereby facilitating their detection and characterization. Nevertheless, the long-term physical persistence of DNA fragments of limited size observed by PCR and Southern hybridization often contrasts with the short-term availability of extracellular DNA to competent bacteria studied in microcosms. The main factors leading to breakdown of extracellular DNA are presented. There is a need for improved methods for accurately determining the degradation routes and the persistence, integrity and potential for horizontal transfer of DNA released from various organisms throughout their lifecycles.</p>","PeriodicalId":87177,"journal":{"name":"Environmental biosafety research","volume":"6 1-2","pages":"37-53"},"PeriodicalIF":0.0,"publicationDate":"2007-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1051/ebr:2007031","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"27069510","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Assessment of transformability of bacteria associated with tomato and potato plants.","authors":"Leo van Overbeek,&nbsp;Jessica Ray,&nbsp;Jan Dirk van Elsas","doi":"10.1051/ebr:2007030","DOIUrl":"https://doi.org/10.1051/ebr:2007030","url":null,"abstract":"<p><p>Transformation of plant-associated bacteria by plant DNA has never been demonstrated in agricultural fields. In total 552 bacterial isolates from stems of Ralstonia solanacearum-infected and healthy tomato plants and from stems and leaves of healthy potato plants were tested for natural genetic competence using plasmid pSKTG DNA and homologous DNA extracts. Control strain Acinetobacter baylyi ADP1 was transformable with both DNA extracts. No transformable isolates were observed after treatment with plasmid pSKTG DNA. Two isolates, P34, identified as Pseudomonas trivialis and A19, identified as Pseudomonas fragi, were selected on the basis of the consistently higher Rp-resistant CFU numbers after treatment with DNA from Rp-resistant cells than with that from wild-type cells. P34 showed 2.1-fold and A19 1.5-fold higher Rp-resistant CFU numbers after treatment with DNA from homologous Rp-resistant cells versus that from wild-type cells. It is concluded that bacteria capable of in vitro capture and integration of exogenous DNA into their genomes are relatively rare in culturable bacterial communities associated with tomato and potato plants, or that conditions conducive to transformation were not met in transformation assays.</p>","PeriodicalId":87177,"journal":{"name":"Environmental biosafety research","volume":"6 1-2","pages":"85-9"},"PeriodicalIF":0.0,"publicationDate":"2007-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"27069513","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Transformation of Acinetobacter baylyi in non-sterile soil using recombinant plant nuclear DNA.","authors":"Deborah J Simpson,&nbsp;John C Fry,&nbsp;Hilary J Rogers,&nbsp;Martin J Day","doi":"10.1051/ebr:2007024","DOIUrl":"https://doi.org/10.1051/ebr:2007024","url":null,"abstract":"<p><p>To provide estimates of horizontal gene transfer from transgenic crops to indigenous soil bacteria, transformation frequencies were obtained for naturally transformable Acinetobacter baylyi BD413 using a chromosomally integrated plant transgene. The transgene comprised sequences for two phenotypic markers: kanamycin resistance (npt II) and green fluorescent protein (gfp), expressed from their own bacterial promoters. Recipient bacteria carried a copy of these two genes, with deletions in their 3'-termini abolishing the marker activity, these genes were integrated into a 16S rRNA gene in the bacterial chromosomal genome or carried on a broad host range plasmid. Successful recombination between the plant transgene and the bacterial genome resulted in restoration of the markers, allowing detection through antibiotic selection and fluorescence. Transformation parameters of increasing complexity, without any enrichment steps, were used to approach the field conditions, while still obtaining measurable transformation frequencies. In pure culture filter experiments, transformation was detected using ground, chopped and whole leaves, as well as whole sterile seedlings, and ground roots. In sterile soil microcosms, transformation was detected using pure plant DNA (3.6 x 10(-8) transformants per recipient) and ground leaves (2.5 x 10(-11)). Transformation was also detected for the first time in non-sterile soil using pure plant DNA (5.5 x 10(-11)). Since the same constructs were used throughout, these data allow predictions of even more complex environmental systems where measurable frequencies are not easily obtainable.</p>","PeriodicalId":87177,"journal":{"name":"Environmental biosafety research","volume":"6 1-2","pages":"101-12"},"PeriodicalIF":0.0,"publicationDate":"2007-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"27069515","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Lack of detectable DNA uptake by bacterial gut isolates grown in vitro and by Acinetobacter baylyi colonizing rodents in vivo.","authors":"Lise Nordgård,&nbsp;Thuy Nguyen,&nbsp;Tore Midtvedt,&nbsp;Yoshimi Benno,&nbsp;Terje Traavik,&nbsp;Kaare M Nielsen","doi":"10.1051/ebr:2007029","DOIUrl":"https://doi.org/10.1051/ebr:2007029","url":null,"abstract":"<p><p>Biological risk assessment of food containing recombinant DNA has exposed knowledge gaps related to the general fate of DNA in the gastrointestinal tract (GIT). Here, a series of experiments is presented that were designed to determine if genetic transformation of the naturally competent bacterium Acinetobacter baylyi BD413 occurs in the GIT of mice and rats, with feed-introduced bacterial DNA containing a kanamycin resistance gene (nptII). Strain BD413 was found in various gut locations in germ-free mice at 10(3)-10(5) CFU per gram GIT content 24-48 h after administration. However, subsequent DNA exposure of the colonized mice did not result in detectable bacterial transformants, with a detection limit of 1 transformant per 10(3)-10(5) bacteria. Further attempts to increase the likelihood of detection by introducing weak positive selection with kanamycin of putative transformants arising in vivo during a 4-week-long feeding experiment (where the mice received DNA and the recipient cells regularly) did not yield transformants either. Moreover, the in vitro exposure of actively growing A. baylyi cells to gut contents from the stomach, small intestine, cecum or colon contents of rats (with a normal microbiota) fed either purified DNA (50 microg) or bacterial cell lysates did not produce bacterial transformants. The presence of gut content of germfree mice was also highly inhibitory to transformation of A. baylyi, indicating that microbially-produced nucleases are not responsible for the sharp 500- to 1,000,000-fold reduction of transformation frequencies seen. Finally, a range of isolates from the genera Enterococcus, Streptococcus and Bifidobacterium spp. was examined for competence expression in vitro, without yielding any transformants. In conclusion, model choice and methodological constraints severely limit the sample size and, hence, transfer frequencies that can be measured experimentally in the GIT. Our observations suggest the contents of the GIT shield or adsorb DNA, preventing detectable exposure of feed-derived DNA fragments to competent bacteria.</p>","PeriodicalId":87177,"journal":{"name":"Environmental biosafety research","volume":"6 1-2","pages":"149-60"},"PeriodicalIF":0.0,"publicationDate":"2007-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"27070456","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}