Description of Agathobaculum massiliense sp. nov., a new bacterial species prevalent in the human gut and predicted to produce indole and tryptophan based on genomic analysis.

The novel bacterial strain Marseille-P4005T was isolated from the stool sample of a healthy donor. It is a Gram-stain negative, non-motile, non-spore-forming rod. It grew optimally at 37 °C and at pH 7.0 on 5% sheep blood-enriched Columbia agar after preincubation in a blood-culture bottle supplemented with rumen and blood. This strain does not ferment monosaccharides (except D-tagatose), disaccharides, or polymeric carbohydrates. The major cellular fatty acids were hexadecenoic (24.6%), octadecanoic (22.8%), and tetradecanoic (20.1%) acids. Next-generation sequencing revealed a genome size of 3.2 Mbp with a 56.4 mol% G + C. Phylogenetic analysis based on the 16S rRNA gene highlighted Agathobaculum desmolans strain ATCC 43058T as the closest related strain. The OrthoANI, AAI, and digital DNA-DNA hybridization values were below the critical thresholds of 95%, 95-96%, and 70%, respectively, to define a novel bacterial species. Antibiotic resistance genes APH(3')-IIIa, erm(B), and tet(W) were detected with high identity percentages of 100%, 98.78%, and 97.18% for each gene, respectively. The APH(3')-IIIa gene confers resistance to amikacin, erm(B) gene confers resistance to erythromycin, lincomycin, and clindamycin, while tet(W) gene confers resistance to doxycycline and tetracycline. Based on KEGG BlastKOALA analyses, the annotation results showed that our strain could use glucose to produce L-lactate and pyruvate but not acetate or ethanol. Also, strain Marseille-P4005T was predicted to use phenylalanine to produce indole, a major intercellular signal molecule within the gut microbial ecosystem. Through having a gene coding for tryptophan synthase beta chain (trpB), strain Marseille-P4005T could produce L-tryptophan (an essential amino acid) from indole. Strain Marseille-P4005T showed its highest prevalence in the human gut (34.19%), followed by the reproductive system (17.98%), according to a query carried out on the Integrated Microbial NGS (IMNGS) platform. Based on phylogenetic, phenotypic, and genomic analyses, we classify strain Marseille-P4005T (= CSUR P4005 = CECT 9669), a novel species within the genus Agathobaculum, for which the name of Agathobaculum massiliense sp. nov. is proposed.

Chromosomally and Extrachromosomally Mediated High-Level Gentamicin Resistance in Streptococcus agalactiae.

Streptococcus agalactiae (group B Streptococcus [GBS]) is a leading cause of sepsis in neonates. The rate of invasive GBS disease in nonpregnant adults also continues to climb. Aminoglycosides alone have little or no effect on GBS, but synergistic killing with penicillin has been shown in vitro. High-level gentamicin resistance (HLGR) in GBS isolates, however, leads to the loss of a synergistic effect. We therefore performed a multicenter study to determine the frequency of HLGR GBS isolates and to elucidate the molecular mechanisms leading to gentamicin resistance. From eight centers in four countries, 1,128 invasive and colonizing GBS isolates were pooled and investigated for the presence of HLGR. We identified two strains that displayed HLGR (BSU1203 and BSU452), both of which carried the aacA-aphD gene, typically conferring HLGR. However, only one strain (BSU1203) also carried the previously described chromosomal gentamicin resistance transposon designated Tn3706. For the other strain (BSU452), plasmid purification and subsequent DNA sequencing resulted in the detection of plasmid pIP501 carrying a remnant of a Tn3 family transposon. Its ability to confer HLGR was proven by transfer into an Enterococcus faecalis isolate. Conversely, loss of HLGR was documented after curing both GBS BSU452 and the transformed E. faecalis strain from the plasmid. This is the first report showing plasmid-mediated HLGR in GBS. Thus, in our clinical GBS isolates, HLGR is mediated both chromosomally and extrachromosomally.

Folate polyglutamylation is involved in chromatin silencing by maintaining global DNA methylation and histone H3K9 dimethylation in Arabidopsis.

DNA methylation and repressive histone Histone3 Lysine9 (H3K9) dimethylation correlate with chromatin silencing in plants and mammals. To identify factors required for DNA methylation and H3K9 dimethylation, we screened for suppressors of the repressor of silencing1 (ros1) mutation, which causes silencing of the expression of the RD29A (RESPONSE TO DESSICATION 29A) promoter-driven luciferase transgene (RD29A-LUC) and the 35S promoter-driven NPTII (NEOMYCIN PHOSPHOTRANSFERASE II) transgene (35S-NPTII). We identified the folylpolyglutamate synthetase FPGS1 and the known factor DECREASED DNA METHYLATION1 (DDM1). The fpgs1 and ddm1 mutations release the silencing of both RD29A-LUC and 35S-NPTII. Genome-wide analysis indicated that the fpgs1 mutation reduces DNA methylation and releases chromatin silencing at a genome-wide scale. The effect of fpgs1 on chromatin silencing is correlated with reduced levels of DNA methylation and H3K9 dimethylation. Supplementation of fpgs1 mutants with 5-formyltetrahydrofolate, a stable form of folate, rescues the defects in DNA methylation, histone H3K9 dimethylation, and chromatin silencing. The competitive inhibitor of methyltransferases, S-adenosylhomocysteine, is markedly upregulated in fpgs1, by which fpgs1 reduces S-adenosylmethionine accessibility to methyltransferases and accordingly affects DNA and histone methylation. These results suggest that FPGS1-mediated folate polyglutamylation is required for DNA methylation and H3K9 dimethylation through its function in one-carbon metabolism. Our study makes an important contribution to understanding the complex interplay among metabolism, development, and epigenetic regulation.

Agrobacterium-mediated transformation of cotton (Gossypium hirsutum) shoot apex with a fungal phytase gene improves phosphorus acquisition.

Cotton is an important world economic crop plant. It is considered that cotton is recalcitrant to in vitro proliferation. Somatic embryogenesis and plant regeneration has been successful by using hypocotyl, whereas it is highly genotype dependent. Here, a genotype-independent cotton regeneration protocol from shoot apices is presented. Shoot apices from 3- to 5-day-old seedlings of cotton are infected with an Agrobacterium strain, EHA105, carrying the binary vector pC-KSA contained phytase gene (phyA) and the marker gene neomycin phosphotransferase (NPTII), and directly regenerated as shoots in vitro. Rooted shoots can be obtained within 6-8 weeks. Plants that survived by leaf painting kanamycin (kan) were -further analyzed by DNA and RNA blottings. The transgenic plants with increased the phosphorus (P) acquisition efficiency were obtained following the transformation method.

Greenhouse and field cultivations of antigen-expressing potatoes focusing on the variability in plant constituents and antigen expression.

The production of plant-derived pharmaceuticals essentially requires stable concentrations of plant constituents, especially recombinant proteins; nonetheless, soil and seasonal variations might drastically interfere with this stability. In addition, variability might depend on the plant organ used for production. Therefore, we investigated the variability in plant constituents and antigen expression in potato plants under greenhouse and field growth conditions and in leaves compared to tubers. Using potatoes expressing VP60, the only structural capsid protein of the rabbit haemorrhagic disease virus (RHDV), CTB, the non-toxic B subunit (CTB) of the cholera toxin (CTA-CTB(5)) and the marker protein NPTII (neomycinphosphotransferase) as a model, we compare greenhouse and field production of potato-derived antigens. The influence of the production organ turned out to be transgene specific. In general, yield, plant quality and transgene expression levels in the field were higher than or similar to those observed in the greenhouse. The variation (CV) of major plant constituents and the amount of transgene-encoded protein was not influenced by the higher variation of soil properties observed in the field. Amazingly, for specific events, the variability in the model protein concentrations was often lower under field than under greenhouse conditions. The changes in gene expression under environmental stress conditions in the field observed in another event do not reduce the positive influence on variability since events like these should excluded from production. Hence, it can be concluded that for specific applications, field production of transgenic plants producing pharmaceuticals is superior to greenhouse production, even concerning the stability of transgene expression over different years. On the basis of our results, we expect equal or even higher expression levels with lower variability of recombinant pharmaceuticals in the field compared to greenhouse production combined with approximately 10 times higher tuber yield in the field.

Successive silencing of tandem reporter genes in potato (Solanum tuberosum) over 5 years of vegetative propagation.

BACKGROUND AND AIMS: Transgenic plants represent an excellent tool for experimental plant biology and are an important component of modern agriculture. Fully understanding the stability of transgene expression is critical in this regard. Most changes in transgene expression occur soon after transformation and thus unwanted lines can be discarded easily; however, transgenes can be silenced long after their integration. METHODS: To study the long-term changes in transgene expression in potato (Solanum tuberosum), the activity of two reporter genes, encoding green fluorescent protein (GFP) and neomycin phosphotransferase (NPTII), was monitored in a set of 17 transgenic lines over 5 years of vegetative propagation in vitro. KEY RESULTS: A decrease in transgene expression was observed mainly in lines with higher initial GFP expression and a greater number of T-DNA insertions. Complete silencing of the reporter genes was observed in four lines (nearly 25 %), all of which successively silenced the two reporter genes, indicating an interconnection between their silencing. The loss of GFP fluorescence always preceded the loss of kanamycin resistance. Treatment with the demethylation drug 5-azacytidine indicated that silencing of the NPTII gene, but probably not of GFP, occurred directly at the transcriptional level. Successive silencing of the two reporter genes was also reproduced in lines with reactivated expression of previously silenced transgenes. CONCLUSIONS: We suggest a hypothetical mechanism involving the successive silencing of the two reporter genes that involves the switch of GFP silencing from the post-transcriptional to transcriptional level and subsequent spreading of methylation to the NPTII gene.

Nucleotide substitutions in rolC and nptII gene sequences during long-term cultivation of Panax ginseng cell cultures.

It has been shown previously that the rolC gene from Agrobacterium tumefaciens gene was stably and highly expressed in 15-year-old Panax ginseng transgenic cell cultures. In the present report, we analyze in detail the nucleotide composition of the rolC and nptII (neomycin phosphotransferase) genes, which is the selective marker used for transgenic cell cultures of P. ginseng. It has been established that the nucleotide sequences of the rolC and nptII genes underwent mutagenesis during cultivation. Particularly, 1-4 nucleotide substitutions were found per sequence in the 540 and 798 bp segments of the complete rolC and nptII genes, respectively. Approximately half of these nucleotide substitutions caused changes in the structure of the predicted gene product. In addition, we attempted to determine the rate of accumulation of these changes by comparison of DNA extracted from P. ginseng cell cultures from 1995 to 2007. It was observed that the frequency of nucleotide substitutions for the rolC and nptII genes in 1995 was 1.21 +/- 0.02 per 1,000 nucleotides analyzed, while in 2007, the nucleotide substitutions significantly increased (1.37 +/- 0.07 per 1,000 nucleotides analyzed). Analyzing the nucleotide substitutions, we found that substitution to G or to C nucleotides significantly increased (in 1.9 times) in the rolC and nptII genes compared with P. ginseng actin gene. Finally, the level of nucleotide substitutions in the rolC gene was 1.1-fold higher when compared with the nptII gene. Thus, for the first time, we have experimentally demonstrated the level of nucleotide substitutions in transferred genes in transgenic plant cell cultures.

Pollen-mediated transformation of Sorghum bicolor plants.

Pollen from sorghum (Sorghum bicolor L. Moench) were transformed by a novel genetic transformation approach using mild ultrasonication. The plasmid pBI121 contained the NPT II gene encoding NPT II (neomycin phosphotransferase II) and a GUS (beta-glucuronidase) reporter gene. Plasmid DNA and pollen from sorghum A(2)V4B were submerged in a 0.3 mol/l sucrose solution and then subjected to ultrasonication. The treated pollen was then used for pollination of stigmas from the male sterile line A(2)V4A. Detection of transient and stable GUS activity confirmed that the GUS gene was expressed in transgenic plants. PCR amplification and Southern-blot analysis of transgenic plants confirmed that the NPT II gene had been integrated into the sorghum genome. These results indicate that direct gene transfer to pollen can be mediated by mild ultrasonication.

Movement of coat protein genes from a commercial virus-resistant transgenic squash into a wild relative.

We monitored pollen-mediated transgene dissemination from commercial transgenic squash CZW-3 into its wild relative Cucurbita pepo ssp. ovifera var. texana (C. texana). Transgenic squash CZW-3 expresses the neomycin phosphotransferase II (nptII) gene and the coat protein (CP) genes of Cucumber mosaic virus (CMV), Zucchini yellow mosaic virus (ZYMV), and Watermelon mosaic virus (WMV); thereby, it is resistant to these three aphid-borne viruses. The rate of NPT II and CP transgene introgression increased with overlapping flowering patterns and a high ratio of transgenic F1 hybrids (C. texana x CZW-3) to C. texana. Transgene transfer also readily occurred from transgenic F1 hybrids into C. texana over three generations in field settings where test plants grew sympatrically and viruses were not severely limiting the growth, and fruit and seed production of C. texana. In contrast, introgression of the transgenes into C. texana was not sustained under conditions of high viral disease pressure. As expected, C. texana progeny that acquired the CP transgenes exhibited resistance to CMV, ZYMV, and WMV. This is the first report on transgene dissemination from a transgenic crop that exhibits disease resistance and hybridizes with a wild plant species without loss of fertility.

Potential high-throughput assay for screening inhibitors of West Nile virus replication.

Prevention and treatment of infection by West Nile virus (WNV) and other flaviviruses are public health priorities. We describe a reporting cell line that can be used for high-throughput screening of inhibitors against all targets involved in WNV replication. Dual reporter genes, encoding Renilla luciferase (Rluc) and neomycin phosphotransferase (Neo), were engineered into a WNV subgenomic replicon, resulting in Rluc/NeoRep. Geneticin selection of BHK-21 cells transfected with Rluc/NeoRep yielded a stable cell line that contains persistently replicating replicons. Incubation of the reporting cells with known WNV inhibitors decreased Rluc activity, as well as the replicon RNA level. The efficacies of the inhibitors, as measured by the depression of Rluc activity in the reporting cells, are comparable to those derived from authentic viral infection assays. Therefore, the WNV reporting cell line can be used as a high-throughput assay for anti-WNV drug discovery. A similar approach should be applicable to development of genetics-based antiviral assays for other flaviviruses.

The synaptic complex of RecA protein participates in hybridization and inverse strand exchange reactions.

RecA protein catalyzes strand exchange between homologous single-stranded and double-stranded DNAs. In the presence of ATPgammaS, the post-strand exchange synaptic complex is a stable end product that can be studied. Here we ask whether such complexes can hybridize to or exchange with DNA, 2'-OMe RNA, PNA, or LNA oligonucleotides. Using a gel mobility shift assay, we show that the displaced strand of a 45 bp synaptic complex can hybridize to complementary oligonucleotides with different backbones to form a four-stranded (double D-loop) joint that survives removal of the RecA protein. This hybridization reaction, which confirms the single-stranded character of the displaced strand in a synaptic complex, might initiate recombination-dependent DNA replication if it occurs in vivo. We also show that either strand of the heteroduplex in a 30 bp synaptic complex can be replaced with a homologous DNA oligonucleotide in a strand exchange reaction that is mediated by the RecA filament. Consistent with the important role that deoxyribose plays in strand exchange, oligonucleotides with non-DNA backbones did not participate in this reaction. The hybridization and strand exchange reactions reported here demonstrate that short synaptic complexes are dynamic structures even in the presence of ATPgammaS.

Evidence that direct DNA uptake through cut shoots leads to genetic transformation of Solanum aviculare Forst.

The reporter genes GUS, NPTII and BAR, either separately or in combination, have been exploited to determine if DNA which can directly enter plants, circulate within the plant and enter nuclei, can also integrate into the genome in a manner which will permit gene expression. Feeding of either seed-derived or adventitious cut shoots of Solanum aviculare with the GUS gene followed by rooting of the shoots and growing on, resulted in all tissues of the plant showing GUS activity as detected cytochemically. Southern blot analysis of plants derived from the adventitious shoots confirmed the presence of the reporter gene in roots. Reporter gene expression was observed also in the F1 generation. If GUS and NPTII or GUS, NPTII and BAR were fed together, then in each case it was possible to have both expression and Southern blot confirmation of each of the genes. There was a relatively high rate of transformation of approximately 5% of the fed stems across all experiments conducted during the present study.

Assessment of screening methods for the identification of genetically modified potatoes in raw materials and finished products.

Qualitative polymerase chain reaction methods for the detection of genetically modified potatoes have been investigated that can be used for screening purposes and identification of insect-resistant and virus-resistant potatoes in food. The presence of the nos terminator from Agrobacterium tumefaciens and the antibiotic marker gene nptII (neomycin-phosphotransferase II) was demonstrated in three commercialized Bt-potato lines (Monsanto Co., St. Louis, MO, USA) and one noncommercial GM-potato product (high amylopectin starch, AVEBE, Veendam, The Netherlands) and allows for general screening in foods. For further identification, specific primers for the FMV promoter derived from the figwort mosaic virus, the CryIIIA gene (delta-endotoxin from Bacillus thuringiensis subsp. tenebrionis), potato leafroll virus replicase gene, and the potato virus Y coat protein gene, were designed. The methods described were successfully applied to processed potato raw materials (dehydrated potato powders and flakes), starch samples, and finished products.

Genetic transformation of Robinia pseudoacacia by Agrobacterium tumefaciens.

Transgenic Robinia pseudoacacia plants were obtained by Agrobacterium tumefaciens mediated gene transfer. Agrobacterium strain LBA4404 harbouring a binary vector that contained the chimeric neomycin phosphotransferase II (NPTII) and beta-glucuronidase (GUS) genes was co-cultivated with hypocotyl segments of in vitro raised seedlings of Robinia. Parameters important for high efficiency regeneration and transformation rates included type of explant, pre-conditioning of explants and appropriate length of co-cultivation period with Agrobacterium. A transformation frequency 16.67% was obtained by 48 hr of pre-conditioning followed by 48 hr of co-cultivation. Transformed tissue was selected by the ability to grow on kanamycin containing medium. Successful regeneration was followed after histochemical GUS assay for the detection of transgenic tissue. This transformation procedure has the potential to expand the range of genetic variation in Robinia.

Optical DNA-sensor chip for real-time detection of hybridization events.

An optical sensor system based on evanescent field excitation of fluorophore-labeled DNA-targets specifically binding to immobilized DNA probes has been developed, thus enabling for real-time analysis of hybridization events. Oligonucleotide probes are directly immobilized on the surface of the disposable sensor chip via biotin/neutravidin linkage and hybridize to complementary Cy5-labeled target DNA in the sample; this is recorded as an increase in the fluorescence signal. Under optimized conditions the hybridization rate was constant and directly proportional to the target concentration. When an 18mer oligonucleotide was used as a probe a linear calibration curve was obtained for a 56mer single-stranded DNA target derived from the neomycin phosphotransferase gene, a selection marker in a variety of genetically modified plants, with an estimated lower limit of detection of 0.21 nmol L(-1). No cross-hybridization to a 51mer actin DNA target was observed and even a single-nucleotide mismatch led to a negligible signal. A shutter in the readout device enabled separate detection of targets hybridizing to probes immobilized at the inlet and outlet sides, respectively, of the flow channel. This opens a route toward a real-time DNA array format with analysis times as short as 1-2 min. As a realistic sample a Cy5-labeled 56 bp PCR product was measured after separation of the double-stranded DNA by simple heat denaturation with a detection limit clearly lower than that of traditional gel electrophoresis.

Introduction of a xenogeneic gene via hematopoietic stem cells leads to specific tolerance in a rhesus monkey model.

Host immune responses against foreign transgenes may be a major obstacle to successful gene therapy. To clarify the impact of an immune response to foreign transgene products on the survival of genetically modified cells, we studied the in vivo persistence of cells transduced with a vector expressing a foreign transgene compared to cells transduced with a nonexpressing vector in the clinically predictive rhesus macaque model. We constructed retroviral vectors containing the neomycin phosphotransferase gene (neo) sequences modified to prevent protein expression (nonexpressing vectors). Rhesus monkey lymphocytes or hematopoietic stem cells (HSCs) were transduced with nonexpressing and neo-expressing vectors followed by reinfusion, and their in vivo persistence was studied. While lymphocytes transduced with a nonexpressing vector could be detected for more than 1 year, lymphocytes transduced with a neo-expressing vector were no longer detectable within several weeks of infusion. However, five of six animals transplanted with HSCs transduced with nonexpression or neo-expression vectors, and progeny lymphocytes marked with either vector persisted for more than 2 years. Furthermore, in recipients of transduced HSCs, infusion of mature lymphocytes transduced with a second neo-expressing vector did not result in elimination of the transduced lymphocytes. Our data show that introduction of a xenogeneic gene via HSCs induces tolerance to the foreign gene products. HSC gene therapy is therefore suitable for clinical applications where long-term expression of a therapeutic or foreign gene is required.