Site-Specific Integration of Transgenes in Soybean via Recombinase-Mediated DNA Cassette Exchange

A targeting method to insert genes at a previously characterized genetic locus to make plant transformation and transgene expression predictable is highly desirable for plant biotechnology. We report the successful targeting of transgenes to predefined soybean (Glycine max) genome sites using the yeast FLP-FRT recombination system. First, a target DNA containing a pair of incompatible FRT sites flanking a selection gene was introduced in soybean by standard biolistic transformation. Transgenic events containing a single copy of the target were retransformed with a donor DNA, which contained the same pair of FRT sites flanking a different selection gene, and a FLP expression DNA. Precise DNA cassette exchange was achieved between the target and donor DNA via recombinase-mediated cassette exchange, so that the donor DNA was introduced at the locus previously occupied by the target DNA. The introduced donor genes expressed normally and segregated according to Mendelian laws.Plant transformation has challenges such as random integration, multiple transgene copies, and unpredictable expression. Homologous recombination (Iida and Terada, 2005; Wright et al., 2005) and DNA recombinase-mediated site-specific integration (SSI) are promising technologies to address the challenges for placing a single copy of transgenes into a precharacterized site in a plant genome.Several site-specific DNA recombination systems, such as the bacteriophage Cre-lox and the yeast FLP-FRT and R-RS, have been used in SSI studies (Ow, 2002; Groth and Calos, 2003). A common feature of these systems is that each system consists of a recombinase Cre, FLP, or R and two identical or similar palindromic recognition sites, lox, FRT, or RS. Each recognition site contains a short asymmetric spacer sequence where DNA strand exchange takes place, flanked by inverted repeat sequences where the corresponding recombinase specifically binds. If two recognition sites are located in cis on a DNA molecule, the DNA segment can be excised if flanked by two directionally oriented sites or inverted if flanked by two oppositely oriented sites. If two recognition sites are located in trans on two different DNA molecules, a reciprocal translocation can happen between the two DNA molecules or the two molecules can integrate if at least one of them is a circular DNA (Ow, 2002; Groth and Calos, 2003).Single-site SSI can integrate a circular donor DNA containing one recognition site into a similar site previously placed in a plant genome. The integrated transgene now flanked by two recognition sites is vulnerable to excision. Transient Cre expression and the use of mutant lox sites to create two less compatible sites after integration helped reduce the subsequent excision in tobacco (Nicotiana tabacum; Albert et al., 1995; Day et al., 2000). A similar approach was used to produce SSI events in rice (Oryza sativa), and the transgene was proven stably expressed over generations (Srivastava and Ow, 2001; Srivastava et al., 2004; Chawla et al., 2006). Using a promoter trap to displace a cre gene in the genome with a selection gene from the donor, approximately 2% SSI was achieved in Arabidopsis (Arabidopsis thaliana; Vergunst et al., 1998).When two recognition sites located on a linear DNA molecule are similar enough to be recognized by the same recombinase but different enough to reduce or prevent DNA recombination from happening between them, the DNA segment between the two sites may not be easily excised or inverted. When a circular DNA molecule carrying the same two incompatible sites is introduced, the circular DNA can integrate by the corresponding recombinase at either site on the linear DNA to create a collinear DNA with four recognition sites, two from the original linear DNA and two from the circular DNA. DNA excision can subsequently occur between any pair of compatible sites to restore the two original DNA molecules or to exchange the intervening DNA segments between the two DNA molecules. This process, termed recombinase-mediated cassette exchange (RMCE), can be employed to integrate transgenes directionally into predefined genome sites (Trinh and Morrison, 2000; Baer and Bode, 2001).RMCE using two oppositely oriented identical RS sites, a donor containing the R recombinase gene and a third RS site to limit random integration, resulted in cassette exchange between the donor and a previously placed target in tobacco (Nanto et al., 2005). RMCE using both the Cre-lox and FLP-FRT systems improved RMCE frequency in animal cell cultures (Lauth et al., 2002). RMCE using two directly oriented incompatible FRT sites and transiently expressed FLP recombinase achieved cassette exchange between a target previously placed in the Drosophila genome and a donor introduced as a circular DNA (Horn and Handler, 2005). A gene conversion approach involving Cre-lox- and FLP-FRT-mediated SSI, RMCE, and homologous recombination was explored in maize (Zea mays; Djukanovic et al., 2006). RMCE using two oppositely oriented incompatible lox sites and transiently expressed Cre recombinase produced single-copy RMCE plants in Arabidopsis (Louwerse et al., 2007).To develop FLP-FRT-mediated RMCE in soybean (Glycine max), we created transgenic target lines containing a hygromycin resistance gene flanked by two directly oriented incompatible FRT sites via biolistic transformation. Single-copy target lines were selected and retransformed with a donor DNA containing a chlorsulfuron resistance gene flanked by the same pair of FRT sites. An FLP expression DNA was cobombarded to transiently provide FLP recombinase. RMCE events were obtained from multiple target lines and confirmed by extensive molecular characterization.     

Effect of iron concentration on toxin production in Campylobacter jejuni and Campylobacter coli

The effect of iron concentrations in culture media on supernatant yields of campylobacter cytotonic toxin (CCT) was studied. Of the 118 Campylobacter spp. strains surveyed, 78.8% produced toxin in brucella broth or in casamino acids--yeast extract (CYE) broth. When the iron concentration of CYE was increased from 0.44 microgram/mL (7.9 microM) to 0.65 microgram/mL (11.6 microM) by the addition of ferric chloride, 94.9% of the strains were positive for toxin in a ganglioside GM1 based, enzyme-linked immunosorbent assay, using antibody to affinity-purified CCT. The addition of iron as ferrous sulfate was less effective. When four toxin-positive strains were grown in a deferrated medium of conalbumin-treated CYE with 0.04-0.08 microgram iron/mL (0.72-1.43 microM), two of the culture supernatants became negative (absorbance at 410 nm, less than 0.1 and less than 10 ng CCT/mL), and two produced about 90% less CCT but were still classified as positive (absorbance, greater than or equal to 0.1 and greater than or equal to 10 ng CCT/mL). It was therefore concluded that the production of CCT by Campylobacter spp. is influenced by iron concentration.     

Cloning and partial characterization of a novel hemolysin gene of Vibrio tubiashii and the development of a PCR-based detection assay

Vibrio tubiashii expresses virulence factors, such as a vulnificolysin-like hemolysin or cytolysin and a zinc metalloprotease, similar to those of other pathogenic vibrios. In this study, we report the cloning of a novel hemolysin gene of V.?tubiashii in Escherichia coli . A V.?tubiashii gene library was screened for hemolytic activity on sheep blood agar. Three hemolytic clones pGem:hly1, pGem:hly2, and pGem:hly3 were sequenced, and the sequences showed a strong homology to the ribA gene coding for guanosine triphosphate cyclohydrolase II (GCH II), required for riboflavin biosynthesis and reported to be responsible for hemolytic activity in Helicobacter pylori . The plasmids pGem:hly1 and pGem:hly3 when introduced into E. coli BSV18 (ribA18::Tn5) were able to restore growth of strain BSV18 in a medium without riboflavin and also produced hemolytic activity on blood agar. PCR primers based on the cloned hly-ribA sequence were tested using 23 different Vibrio strains representing 10 different species. Amplification of ribA gene locus only occurred with V.?tubiashii strains. In summary, our results indicate that we have cloned a ribA homolog of V.?tubiashii that imparts hemolytic activity to E.?coli clones, and primers based on this gene locus might be useful as a species-specific identification tool for V.?tubiashii.     

Foodborne enterotoxigenic Escherichia coli: detection and enumeration by DNA colony hybridization

Four methods were compared for detecting heat-labile toxin production by Escherichia coli: DNA colony hybridization, two enzyme-linked immunosorbent assays, and the mouse Y-1 adrenal cell reaction. Although results of the methods were in general agreement, there were some differences in specificity and sensitivity. DNA colony hybridization was used to detect and enumerate enterotoxigenic E. coli isolates in artificially contaminated food without enrichment. Sensitivity level was 100 cells per g.     

Development of Defined, Minimal, and Complete Media for the Growth of Hyphomicrobium neptunium

A complete synthetic medium containing 15 amino acids, a minimal synthetic medium (GAMS) containing 4 amino acids, and a supplemented minimal medium (GAMS + calcium pantothenate) have been developed for the cultivation of Hyphomicrobium neptunium ATCC 15444. Depending on the complexity of the synthetic media, generation times were approximately 2 to 3 times longer, and maximum cell densities were 0.3 to 0.9 log₁₀ lower than in ZoBell marine broth 2216. The fates of ¹⁴C-labeled amino acids in GAMS were monitored. Results suggested that H. neptunium was auxotrophic for methionine, utilized glutamic acid as a primary energy source, and readily anabolized and catabolized serine and aspartic acid. Individual amino acid concentrations above 125 mM induced prolonged lag periods, whereas only methionine was not growth limiting at a concentration as low as 2 mM.     

Characterization of putative virulence genes on the related RepFIB plasmids harbored by Cronobacter spp

Cronobacter spp. are emerging neonatal pathogens that cause meningitis, sepsis, and necrotizing enterocolitis. The genus Chronobacter consists of six species: C. sakazakii, C. malonaticus, C. muytjensii, C. turicensis, C. dublinensis, and Cronobacter genomospecies group 1. Whole-genome sequencing of C. sakazakii BAA-894 and C. turicensis z3032 revealed that they harbor similarly sized plasmids identified as pESA3 (131 kb) and pCTU1 (138 kb), respectively. In silico analysis showed that both plasmids encode a single RepFIB-like origin of replication gene, repA, as well as two iron acquisition systems (eitCBAD and iucABCD/iutA). In a chrome azurol S agar diffusion assay, it was demonstrated that siderophore activity was associated with the presence of pESA3 or pCTU1. Additionally, pESA3 contains a cpa (Cronobacter plasminogen activator) gene and a 17-kb type 6 secretion system (T6SS) locus, while pCTU1 contains a 27-kb region encoding a filamentous hemagglutinin gene (fhaB), its specifc transporter gene (fhaC), and associated putative adhesins (FHA locus), suggesting that these are virulence plasmids. In a repA-targeted PCR assay, 97% of 229 Cronobacter species isolates were found to possess a homologous RepFIB plasmid. All repA PCR-positive strains were also positive for the eitCBAD and iucABCD/iutA iron acquisition systems. However, the presence of cpa, T6SS, and FHA loci depended on species, demonstrating a strong correlation with the presence of virulence traits, plasmid type, and species. These results support the hypothesis that these plasmids have evolved from a single archetypical plasmid backbone through the cointegration, or deletion, of specific virulence traits in each species.     

Purification and Characterization of a Vulnificolysin-Like Cytolysin Produced by Vibrio tubiashii

An extracellular cytolysin from Vibrio tubiashii was purified by sequential hydrophobic interaction chromatography with phenyl-Sepharose CL-4B and gel filtration with Sephacryl S-200. This protein is sensitive to heat and proteases, is inhibited by cholesterol, and has a molecular weight of 59,000 and an isoelectric point of 5.3. In addition to lysing various erythrocytes, it is cytolytic and/or cytotoxic to Chinese hamster ovary cells, Caco-2 cells, and Atlantic menhaden liver cells in tissue culture. Lysis of erythrocytes occurs by a multihit process that is dependent on temperature and pH. Twelve of the first 17 N-terminal amino acid residues (Asp-Asp-Tyr-Val-Pro-Val-Val-Glu-Lys-Val-Tyr-Tyr-Ile-Thr-Ser-Ser-Lys) are identical to those of the Vibrio vulnificus cytolysin.     

Two-step purification and partial characterization of a variant of the Vibrio cholerae non-O1 hemolysin

A two-step purification method using ammonium sulfate precipitation and gel filtration was developed for the purification of a variant of the El Tor hemolysin/cytolysin from supernatant fluids of a Vibrio cholerae non-O1 human isolate (strain 2194c). The toxin displayed delayed elution from a Sephacryl gel filtration column, eluting at between two and three column volumes. The molecular mass and isoelectric point of the purified 2194c toxin were 60 kDa and 5. 3, respectively. The N-terminal amino acid sequence was ASPAPANSETNTLPHVAFYI. Purified toxin was cytolytic for Chinese hamster ovary cells and erythrocytes from several animal species.     

Purification and characterization of a vulnificolysin-like cytolysin produced by Vibrio tubiashii

An extracellular cytolysin from Vibrio tubiashii was purified by sequential hydrophobic interaction chromatography with phenyl-Sepharose CL-4B and gel filtration with Sephacryl S-200. This protein is sensitive to heat and proteases, is inhibited by cholesterol, and has a molecular weight of 59,000 and an isoelectric point of 5.3. In addition to lysing various erythrocytes, it is cytolytic and/or cytotoxic to Chinese hamster ovary cells, Caco-2 cells, and Atlantic menhaden liver cells in tissue culture. Lysis of erythrocytes occurs by a multihit process that is dependent on temperature and pH. Twelve of the first 17 N-terminal amino acid residues (Asp-Asp-Tyr-Val-Pro-Val-Val-Glu-Lys-Val-Tyr-Tyr-Ile-Thr-Ser-Ser-Lys) are identical to those of the Vibrio vulnificus cytolysin.     

Isolation and characterization of a cytolysin produced by Vibrio cholerae serogroup non-O1

A thermolabile toxin (molecular weight, 52 711; isoelectric point, 8.65) produced by a clinical isolate of Vibrio cholerae serogroup non-O1 was cytotoxic for Y-1 mouse adrenal cells and Chinese hamster ovary cells. The toxin lysed rabbit red blood cells and produced a hemorrhagic zone in rabbit skin. When injected intravenously into adult mice, the cytolysin was rapidly lethal and caused fluid accumulation in both 5- and 18-h rabbit ileal loops. Strains of V. cholerae that produced cytolysin but no cholerae enterotoxin were able to cause fluid accumulation in rabbit intestinal loops.