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We measured and studied the growth parameters and the qualitative and quantitative composition of the flavones of hairy roots of the Scutellaria genus: S. lateriflora , S. przewalskii and S. pycnoclada . Hairy roots were obtained using wild-type Agrobacterium rhizogenes A4 by co-cultivation of explants (cotyledons) in a suspension of Agrobacterium . The presence of the rol -genes was confirmed by PCR analysis. The hairy roots of the most studied plant from the Scutellaria genus, S. baicalensis, were obtained earlier and used as a reference sample. HPLC-MS showed the predominance of four main flavones (baicalin, baicalein, wogonin and wogonoside) in the methanol extracts of the studied hairy roots. In addition to the four main flavones, the other substances which are typical to the aerial part of plants were found in all the extracts: apigenin, apigetrin, scutellarin and chrysin-7- O -β-d-glucuronide. According to the total content of flavones, the hairy roots of the studied skullcaps form the following series: S. przewalskii (33 mg/g dry weight) > S. baicalensis (17.04 mg/g dry weight) > S. pycnoclada (12.9 mg/g dry weight) > S. lateriflora (4.57 mg/g dry weight). Therefore, the most promising producer of anti-coronavirus flavones is S. przewalskii.

Over the recent years, Nicotiana benthamiana has gained great importance as a chassis for the production of high value, low volume pharmaceuticals and/or active pharmaceutical ingredients (APIs). The process involving infiltration of the N. benthamiana leaves with Agrobacterium spp, harbouring vectors with the gene of interest, facilitates transient expression. To date, little information is available on the effect of the agro-infiltration process on the metabolome of N. benthamiana, which is necessary to improve the process for large-scale, renewable manufacturing of high value compounds and medical products. Hence, the objective of the present study was to assess metabolic adaptation of N. benthamiana as a response to the presence of Agrobacterium. The present study elucidated changes of the steady-state metabolism in the agroinfiltrated leaf area, the area around the infection and the rest of the plant. Furthermore, the study discusses the phenotypic advantages of the N. benthamiana lab strain, optimised for agro-infiltration, compared to three other wild accessions. Results showed that the lab strain has a different metabolic composition and showed less alterations of the phenylpropanoid pathway and cell wall remodelling in the agroinfiltrated leaf areas, for example chlorogenic acid, cadaverine and C18:0-2-glycerol ester. In conclusion, both of these alterations present potential candidates to improve the phenotype of the N. benthamiana lab strain for a more efficient transient expression process.

Biotin is an essential micro-nutrient across the three domains of life. The paradigm earlier step of biotin synthesis denotes "BioC-BioH" pathway in Escherichia coli. Here we report that BioZ bypasses the canonical route to begin biotin synthesis. In addition to its origin of Rhizobiales, protein phylogeny infers that BioZ is domesticated to gain an atypical role of β-ketoacyl-ACP synthase III. Genetic and biochemical characterization demonstrates that BioZ catalyzes the condensation of glutaryl-CoA (or ACP) with malonyl-ACP to give 5'-keto-pimeloyl ACP. This intermediate proceeds via type II fatty acid synthesis (FAS II) pathway, to initiate the formation of pimeloyl-ACP, a precursor of biotin synthesis. To further explore molecular basis of BioZ activity, we determine the crystal structure of Agrobacterium tumefaciens BioZ at 1.99 Å, of which the catalytic triad and the substrate-loading tunnel are functionally defined. In particular, we localize that three residues (S84, R147, and S287) at the distant bottom of the tunnel might neutralize the charge of free C-carboxyl group of the primer glutaryl-CoA. Taken together, this study provides molecular insights into the BioZ biotin synthesis pathway.

Medicinal plants produce a diverse group of phytocompounds like anthraquinones, alkaloids, anthocyanins, flavonoids, saponins, and terpenes which are used in pharmaceutical, perfume, cosmetics, dye and flavor industries. Commercial source of these metabolites is field-grown plants, which are generally influenced by seasonal changes. Biotechnology possesses a significant role in production of high-value secondary metabolites. By incorporating biotechnological methods, it is feasible to manage biosynthetic pathways of the plant to enhance phytocompound production that is of pharmaceutical interest. Plant cell suspension, shoot, adventitious root and hairy root culture are considered as alternative methods for important bioactive compound production. These methods are controllable, sustainable and overcome several inconveniences for large scale secondary metabolites production. At present research on hairy root culture for valuable bioactive compound production has gained a lot of attention. Agrobacterium rhizogenes is an agent which causes hairy root disease in a plant and this leads to the neoplastic growth of root which is characterized by higher growth rate and genetic stability. Various studies explore the hairy root culture for production of a wide range of bioactive compounds. Scale-up of hairy root culture using bioreactors has provided an opportunity to enhance bioactive compound production at the commercial level. The present review discusses the role of hairy root culture in the production of valuable bioactive compounds, the effect of culture parameters on bioactive compound production and bioreactor applications., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Background: Tropane Alkaloids (TAs) are important drugs for curing many diseases in the medical industry., Methods: To sustainably exploit TA resources in endangered traditional Tibetan herbs, the hairy root (HR) systems of Przewalskia tangutica Maxim. and Anisodus tanguticus Maxim. were compared under the same culture conditions., Results: The results indicated that both the Agrobacterium rhizogenes strains and explants affected the HR induction frequency, MSU440, A4 and LBA9402 strains could induce hairy roots following infection of cotyledon and hypocotyl of A. tanguticus while LBA9402 could not induce HR on either explants of P. tangutica. The efficiency of LBA9402 was higher than A4 and MSU440 on A. tanguticus and A4 was better strain than MSU440 on P. tangutica. The hypocotyl explant was more suitable for P.tangutica and cotyledon explant was better for A.tangutica with a transformation frequency of 33.3% (P. tangutica) and 82.5% (A. tanguticus), respectively. In a flask reactor system, both the growth curves of HR for two species both appeared to be "S" curve; however, the HR of P. tangutica grew more rapidly than that of A. tanguticus, and the latter accumulated more biomass than the former. As the culture volume increased, the HR proliferation coefficient of both the species increased. HPLC analysis results showed that the content of TAs in the HR of P. tangutica was 257.24mg/100g·DW, which was more than that of A. tanguticus HR (251.08mg/100g·DW), and the anisodamine in the Pt- HR was significantly higher than that in At-HR. Moreover, tropane alkaloids in the HR of the two species were all significantly higher than that of the roots of aseptic seedlings., Conclusion: Our results suggest that HR of P. tangutica and A. tanguticus both could provide a useful platform for sustainable utilization of two Tibetan medicinal plants in the Qinghai-Tibetan Plateau in the future., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Rhizogenic agrobacteria induce extensive root proliferation, in several economically valuable, dicotyledonous plant species, a phenomenon referred to as "hairy roots." Besides their pathogenic nature, agrobacteria have proven to be a valuable asset in biotechnology and molecular plant breeding. To assess the potential of frequently used rhizogenic strains, growth in yeast extract glucose broth and antibiotic resistance was analyzed. Growth curves were established for Arqua1, NCPPB2659, LMG150, LMG152, and ATCC15834; and regression analysis of the exponential growth phase resulted in a reliable and standardized method for preparation of a bacterial suspension for inoculation. Cell density did not correlate with the timing of hairy root emergence. The highest number of hairy roots was obtained with an inoculum of 1 × 10 8 CFU ml -1 for Arqua1, NCPPB2659, and LMG152. Cell density of ATCC15834 did not affect the number of hairy roots formed. The identity of the rhizogenic strains for plant transformation was verified in phylogenetic analysis using average nucleotide identity (ANI), which also provided insight in their genetic diversity within the Rhizobium taxon.

The first non-natural derivative of the rare d-glucose-2-phosphate (G2P), namely glucose-2-(O-lactic acid phosphate) (G2LP), has been synthesized. When used as sole carbon source, G2LP enables bacterial growth of the plant pathogenic strain Agrobacterium fabrum C58 (formerly referred to as Agrobacterium tumefaciens). X-ray crystallography and affinity measurements investigations reveal that G2LP binds the periplasmic binding protein (PBP) AccA similarly to the natural compounds and with the same affinity. Moreover, enzymatic assays show that it is able to serve as substrate of the phosphodiesterase AccF. The properties found for G2LP demonstrate that the very unusual glucose-2-phosphoryl residue, present in G2LP, can be used as structural feature for designing non-natural systems fully compatible with the Acc cascade of A. fabrum.

Small RNAs (sRNAs) are a class of gene regulators in bacteria, playing a central role in their response to environmental changes. Bioinformatic prediction facilitates the identification of sRNAs expressed under different conditions. We propose a novel method of prediction of sRNAs from the genome of Agrobacterium based on a positional weight matrix of conditional sigma factors. sRNAs predicted from the genome are integrated with the virulence-specific transcriptome data to identify putative sRNAs that are overexpressed during Agrobacterial virulence induction. A total of 384 sRNAs are predicted from transcriptome data analysis of Agrobacterium fabrum and 100-500 sRNAs from the genome of different Agrobacterial strains. In order to refine our study, a final set of 10 novel sRNAs with best features across different replicons targeting virulence genes were experimentally identified using semi-quantitative polymerase chain reaction. Since Ti plasmid plays a major role in virulence, out of 10 sRNAs across the replicons, 4 novel sRNAs differentially expressed under virulence induced and non-induced conditions are predicted to be present in the Ti plasmid T-DNA region flanking virulence-related genes like agrocinopine synthase, indole 3-lactate synthase, mannopine synthase and tryptophan monooxygenase. Further validation of the function of these sRNAs in conferring virulence would be relevant to explore their role in Agrobacterium-mediated plant transformation.

Small regulatory RNAs play an important role in the adaptation to changing conditions. Here, we describe a differentially expressed small regulatory RNA (sRNA) that affects various cellular processes in the plant pathogen Agrobacterium tumefaciens Using a combination of bioinformatic predictions and comparative proteomics, we identified nine targets, most of which are positively regulated by the sRNA. According to these targets, we named the sRNA PmaR for peptidoglycan biosynthesis, motility, and ampicillin resistance regulator. Agrobacterium spp. are long known to be naturally resistant to high ampicillin concentrations, and we can now explain this phenotype by the positive PmaR-mediated regulation of the beta-lactamase gene ampC Structure probing revealed a spoon-like structure of the sRNA, with a single-stranded loop that is engaged in target interaction in vivo and in vitro Several riboregulators have been implicated in antibiotic resistance mechanisms, such as uptake and efflux transporters, but PmaR represents the first example of an sRNA that directly controls the expression of an antibiotic resistance gene. IMPORTANCE The alphaproteobacterium Agrobacterium tumefaciens is able to infect various eudicots causing crown gall tumor formation. Based on its unique ability of interkingdom gene transfer, Agrobacterium serves as a crucial biotechnological tool for genetic manipulation of plant cells. The presence of hundreds of putative sRNAs in this organism suggests a considerable impact of riboregulation on A. tumefaciens physiology. Here, we characterized the biological function of the sRNA PmaR that controls various processes crucial for growth, motility, and virulence. Among the genes directly targeted by PmaR is ampC coding for a beta-lactamase that confers ampicillin resistance, suggesting that the sRNA is crucial for fitness in the competitive microbial composition of the rhizosphere., (Copyright © 2018 Borgmann et al.)

Background: Alleviating arsenic (As) contamination is a high-priority environmental issue. Hyperaccumulator plants may harbor endophytic bacteria able to detoxify As. Therefore, we investigated the distribution, diversity, As (III) resistance levels, and resistance-related functional genes of arsenite-resistant bacterial endophytes in Pteris vittata L. growing in a lead-zinc mining area with different As contamination levels., Results: A total of 116 arsenite-resistant bacteria were isolated from roots of P. vittata with different As concentrations. Based on the 16S rRNA gene sequence analysis of representative isolates, the isolates belonged to Proteobacteria, Actinobacteria, and Firmicutes. Major genera found were Agrobacterium, Stenotrophomonas, Pseudomonas, Rhodococcus, and Bacillus. The most highly arsenite-resistant bacteria (minimum inhibitory concentration > 45 mM) were isolated from P. vittata with high As concentrations and belonged to the genera Agrobacterium and Bacillus. The strains with high As tolerance also showed high levels of indole-3-acetic acid (IAA) production and carried arsB/ACR3(2) genes. The arsB and ACR3(2) were most likely horizontally transferred among the strains., Conclusion: The results of this study suggest that P. vittata plants with high As concentrations may select diverse arsenite-resistant bacteria; this diversity might, at least partly, be a result of horizontal gene transfer. These diverse endophytic bacteria are potential candidates to enhance phytoremediation techniques.

Transformation is the main platform for genetic improvement and gene function studies in plants. However, the established somatic embryo transformation system for grapevines is time-consuming and has low efficiency, which limits its utilization in functional genomics research. Vitis amurensis is a wild Vitis species with remarkable cold tolerance. The lack of an efficient genetic transformation system for it has significantly hindered the functional identification of cold stress related genes in the species. Herein, an efficient method was established to produce transformed calli of V. amurensis. Segments of petioles from micropropagated plantlets of V. amurensis exhibited better capacity to differentiate calli than leaf-discs and stem segments, and thus was chosen as target tissue for Agrobacterium-mediated transformation. Both neomycin phosphotransferase II (NPTII) and enhanced green fluorescent protein (eGFP) genes were used for simultaneous selection of transgenic calli based on kanamycin resistance and eGFP fluorescence. Several parameters affecting the transformation efficiency were optimized including the concentration of kanamycin, Agrobacterium stains, bacterial densities, infection treatments and co-cultivation time. The transgenic callus lines were verified by checking the integration of NPTII gene into calli genomes, the expression of eGFP gene and the fluorescence of eGFP. Up to 20% of the petiole segments produced transformed calli after 2 months of cultivation. This efficient transformation system will facilitate the functional analysis of agronomic characteristics and related genes not only in V. amurensis but also in other grapevine species.

A change in the contents of endogenous salicylic and jasmonic acids in the roots of the host plant at the preinfectious stage of interaction with symbiotic (Rhizobium leguminosarum) and pathogenic (Agrobacterium rizogenes) bacteria belonging for to the family Rhizobiaceae was studied. It was found that the jasmonic acid content increased 1.5–2 times 5 min after inoculation with these bacterial species. It was shown that dynamics of the change in the JA and SA contents depends on the type of infection. Thus, the JA content decreased in the case of pathogenesis, while the SA content increased. At the same time, an increased JA content was observed during symbiosis. The observed regularities could indicate the presence of different strategies of hormonal regulation for interaction with symbiotic and pathogenic bacteria belonging to the family Rhizobiaceae in peas plants.

Among the strategies developed for contaminated groundwater bioremediation, those based on the use of bacteria adhering to inert supports and establishing biofilms have gained great importance in this field. Extractive membrane biofilm reactor (EMBFR) technology offers productive solutions for the removal of volatile and semi-volatile compounds. EMBFR technology is based on the use of extractive semipermeable membranes through which contaminants migrate to the biological compartment in which microorganisms with pollutant biotransformation and/or mineralization capacities can grow, forming an active biofilm on the membrane surface. The objective of this study was to assess the use of three bacterial strains (Paenibacillus sp. SH7 CECT 8558, Agrobacterium sp. MS2 CECT 8557, and Rhodococcus ruber EE6 CECT 8612), as inoculum in a lab-scale EMBFR running for 28 days under aerobic conditions to eliminate methyl tert-butyl ether (MTBE) from water samples. Three different hydraulic retention times (1, 6, and 12 h) were employed. MTBE degradation values were determined daily by a gas GC-MS technique, as well as suspended bacterial growth. The biofilm established by the bacterial strains on the semipermeable membrane was detected by Field-Emission Scanning Electron Microscopy (FESEM) at the end of each experiment. The acute toxicity of the treated effluents and biomedium was determined by Microtox © assay (EC 50 ).The results achieved from the MTBE degradation, biofilm formation, and toxicity analysis indicated that bacterial strains MS2 and EE6 were the best options as selective inoculum, although further research is needed, particularly with regard to their possible use as a mixed culture. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1238-1245, 2016., (© 2016 American Institute of Chemical Engineers.)

Tea (Camellia sinensis L.) is recalcitrant to Agrobacterium-mediated genetic transformation largely due to the bactericidal effects of tea polyphenols and phenolics oxidation induced by necrosis of explant tissue over the process of transformation. In this study, different antioxidants/adsorbents were added as supplements to the co-cultivation and post co-cultivation media to overcome these problems for the transformation improvement. Tea-cotyledon-derived calli were used as explants and Agrobacterium rhizognes strain ATCC 15834 was used as a mediator. Results showed that Agrobacterium growth, virulence (vir) gene expression and browning of explant tissue were greatly influenced by different supplements. Murashige and Skoog (MS) basal salts medium supplemented with 30 g·L(-1) sucrose, 0.1 g·L(-1) l-glutamine and 5 g·L(-1) polyvinylpolypyrrolidone (PVPP) as co-cultivation and post co-cultivation media could maintain these parameters better that ultimately led to significant improvement of hairy root generation efficiency compared to that in the control (MS + 30 g·L(-1) sucrose). Additionally, the reporter genes β-glucuronidase (gusA) and cyan fluorescent protein (cfp) were also stably expressed in the transgenic hairy roots. Our study would be helpful in establishing a feasible approach for tea biological studies and genetic improvement of tea varieties.

Background: Plant pathogenic Agrobacterium strains can transfer T-DNA regions of their Ti plasmids to a broad range of eukaryotic hosts, including fungi, in vitro. In the recent decade, the yeast Saccharomyces cerevisiae is used as a model host to reveal important host proteins for the Agrobacterium-mediated transformation (AMT). Further investigation is required to understand the fundamental mechanism of AMT, including interaction at the cell surface, to expand the host range, and to develop new tools. In this study, we screened a yeast mutant library for low AMT mutant strains by advantage of a chromosome type T-DNA, which transfer is efficient and independent on integration into host chromosome., Results: By the mutant screening, we identified four mutant strains (srs2Δ, rad52Δ, smi1Δ and erg28Δ), which showed considerably low AMT efficiency. Structural analysis of T-DNA product replicons in AMT colonies of mutants lacking each of the two DNA repair genes, SRS2 and RAD52, suggested that the genes act soon after T-DNA entry for modification of the chromosomal T-DNA to stably maintain them as linear replicons and to circularize certain T-DNA simultaneously. The cell wall synthesis regulator SMI1 might have a role in the cell surface interaction between the donor and recipient cells, but the smi1Δ mutant exhibited pleiotropic effect, i.e. low effector protein transport as well as low AMT for the chromosomal T-DNA, but relatively high AMT for integrative T-DNAs. The ergosterol synthesis regulator/enzyme-scaffold gene ERG28 probably contributes by sensing a congested environment, because growth of erg28Δ strain was unaffected by the presence of donor bacterial cells, while the growth of the wild-type and other mutant yeast strains was suppressed by their presence., Conclusions: RAD52 and the DNA helicase/anti-recombinase gene SRS2 are necessary to form and maintain artificial chromosomes through the AMT of chromosomal T-DNA. A sterol synthesis scaffold gene ERG28 is important in the high-efficiency AMT, possibly by avoiding congestion. The involvement of the cell wall synthesis regulator SMI1 remains to be elucidated.

While the authors have previously developed a method of pistil filament treatment with Agrobacterium cells during blossoming for the transformation of maize generative cells, the mechanism for bacterial T-DNA penetration into the embryo sac remained unknown. This article analyzes the possibility of agrobacterial penetration into the maize embryo via pollen tubes. Microbiological, PCR, and GUS techniques were used to confirm that agrobacteria could spread for up to 20 cm from the sie of inoculation and were detected in maize embryo tissues as aerly as 24 h after inoculation, while they were not revealed after 5-13 days.

When expressing plant cell wall degrading enzymes in the widely used tobacco (Nicotiana benthamiana) after Agrobacterium infiltration, difficulties arise due to the thin leaf structure. Thick leaved succulents, Kalanchoe blossfeldiana and Hylotelephium telephium, were tested as alternatives. A xyloglucanase, as well as a xyloglucanase inhibitor protein was successfully produced., (Published by Elsevier B.V.)

Brugmansia candida (syn. Datura candida) is a South American native plant that produces tropane alkaloids. Hyoscyamine, 6β-hydroxyhyoscyamine (anisodamine), and scopolamine are the most important ones due to their anticholinergic activity. These bioactive compounds have been historically and widely applied in medicine and their demand is continuous. Their chemical synthesis is costly and complex, and thereby, these alkaloids are industrially produced from natural producer plants. The production of these secondary metabolites by plant in vitro cultures such as hairy roots presents certain advantages over the natural source and chemical synthesis. It is well known that hairy roots produced by Agrobacterium rhizogenes infection are fast-growing cultures, genetically stable and able to grow in hormone-free media. Additionally, recent progress achieved in the scaling up of hairy root cultures makes this technology an attractive tool for industrial processes. This chapter is focused on the methods for the induction and establishment of B. candida hairy roots. In addition, the scaling up of hairy root cultures in bioreactors and tropane alkaloid analysis is discussed.

The increasing resistance of bacteria and fungi to currently available antibiotics is a major concern worldwide, leading to enormous effort to develop novel antibiotics with new modes of action.We recently reported that ABP-dHC-cecropin A exhibited strong antibacterial and antifungal activity, making it a candidate antibiotic substitute. In this study, β-cyclodextrin (β-CD) combined with ABP-dHC-cecropin A enhanced the physical and chemical properties of ABP-dHC-cecropin A but did not significantly decrease its antibacterial activity. Thus, β-CD/ABP-dHC-cecropin A should be considered a novel antibacterial drug. We used β-CD/ABP-dHC-cecropin A as an anti-Agrobacterium compound to supplementtransgenic poplar medium. Sideeffects of the inclusion complex had little impact on plantgrowth. Thus, β-CD/ABP-dHC-cecropin A may be used as traditional antibiotics forpoplar transplantation with greater antibbacterial effects., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Polar growth represents a surprising departure from the canonical dispersed cell growth model. However, we know relatively little of the underlying mechanisms governing polar growth or the requisite suite of factors that direct polar growth. Underscoring how classic doctrine can be turned on its head, the peptidoglycan layer of polar-growing bacteria features unusual crosslinks and in some species the quintessential cell division proteins FtsA and FtsZ are recruited to the growing poles. Remarkably, numerous medically important pathogens utilize polar growth, accentuating the need for intensive research in this area. Here we review models of polar growth in bacteria based on recent research in the Actinomycetales and Rhizobiales, with emphasis on Mycobacterium and Agrobacterium species., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: Agrobacterium sp. ATCC31749 is an efficient curdlan producer at low pH and under nitrogen starvation. The helix-turn-helix transcriptional regulatory protein (crdR) essential for curdlan production has been analyzed, but whether crdR directly acts to cause expression of the curdlan biosynthesis operon (crdASC) is uncertain. To elucidate the molecular function of crdR in curdlan biosynthesis, we constructed a crdR knockout mutant along with pBQcrdR and pBQNcrdR vectors with crdR expression driven by a T5 promoter and crdR native promoter, respectively. Also, we constructed a pAG with the green fluorescent protein (GFP) gene driven by a curdlan biosynthetic operon promoter (crdP) to measure the effects of crdR expression on curdlan biosynthesis., Results: Compared with wild-type (WT) strain biomass production, the biomass of the crdR knockout mutant was not significantly different in either exponential or stationary phases of growth. Mutant cells were non-capsulated and planktonic and produced significantly less curdlan. WT cells were curdlan-capsulated and aggregated in the stationery phase. pBQcrdR transformed to the WT strain had a 38% greater curdlan yield and pBQcrdR and pBQNcrdR transformed to the crdR mutant strain recovered 18% and 105% curdlan titers of the WT ATCC31749 strain, respectively. Consistent with its function of promoting curdlan biosynthesis, curdlan biosynthetic operon promoter (crdP) controlled GFP expression caused the transgenic strain to have higher GFP relative fluorescence in the WT strain, and no color change was observed with low GFP relative fluorescence in the crdR mutant strain as evidenced by fluorescent microscopy and spectrometric assay. q-RT-PCR revealed that crdR expression in the stationary phase was greater than in the exponential phase, and crdR overexpression in the WT strain increased crdA, crdS, and crdC expression. We also confirmed that purified crdR protein can specifically bind to the crd operon promoter region, and we inferred that crdR directly acts to cause expression of the curdlan biosynthesis operon (crdASC)., Conclusions: CrdR is a positive transcriptional regulator of the crd operon for promoting curdlan biosynthesis in ATCC31749. The potential binding region of crdR is located within the -98 bp fragment upstream from the crdA start codon.

ICE1 genes play a very important role in plants in cold conditions. To improve the cold resistance of tomato, the ICE1 gene of Arabidopsis thaliana was used to construct the plant expression vector p3301-ICE1, and was overexpressed in tomato through Agrobacterium-mediated transformation. Five strains of resistant plants were obtained. PCR and half-quantitative results showed that the ICE1 gene was transferred to tomato; three strains tested positive. After low-temperature stress treatment, praline content and peroxide and catalase activities in the transgenic tomato plants were higher compared with non-transgenic controls, while malondialdehyde content was clearly lower.

This study investigated the cytotoxicity, genotoxicity, and growth inhibition effects of four different inorganic nanoparticles (NPs) such as aluminum (nAl), iron (nFe), nickel (nNi), and zinc (nZn) on a dibenzofuran (DF) degrading bacterium Agrobacterium sp. PH-08. NP (0-1,000 mg L(-1)) -treated bacterial cells were assessed for cytotoxicity, genotoxicity, growth and biodegradation activities at biochemical and molecular levels. In an aqueous system, the bacterial cells treated with nAl, nZn and nNi at 500 mg L(-1) showed significant reduction in cell viability (30-93.6 %, p < 0.05), while nFe had no significant inhibition on bacterial cell viability. In the presence of nAl, nZn and nNi, the cells exhibited elevated levels of reactive oxygen species (ROS), DNA damage and cell death. Furthermore, NP exposure showed significant (p < 0.05) impairment in DF and catechol biodegradation activities. The reduction in DF biodegradation was ranged about 71.7-91.6 % with single NPs treatments while reached up to 96.3 % with a mixture of NPs. Molecular and biochemical investigations also clearly revealed that NP exposure drastically affected the catechol-2,3-dioxygenase activities and its gene (c23o) expression. However, no significant inhibition was observed in nFe treatment. The bacterial extracellular polymeric materials and by-products from DF degradation can be assumed as key factors in diminishing the toxic effects of NPs, especially for nFe. This study clearly demonstrates the impact of single and mixed NPs on the microbial catabolism of xenobiotic-degrading bacteria at biochemical and molecular levels. This is the first study on estimating the impact of mixed NPs on microbial biodegradation.

In previous research, volatile organic compounds (VOCs) emitted by various bacteria into the chemosphere were suggested to play a significant role in the antagonistic interactions between microorganisms occupying the same ecological niche and between bacteria and target eukaryotes. Moreover, a number of volatiles released by bacteria were reported to suppress quorum-sensing cell-to-cell communication in bacteria, and to stimulate plant growth. Here, volatiles produced by Pseudomonas and Serratia strains isolated mainly from the soil or rhizosphere exhibited bacteriostatic action on phytopathogenic Agrobacterium tumefaciens and fungi and demonstrated a killing effect on cyanobacteria, flies (Drosophila melanogaster), and nematodes (Caenorhabditis elegans). VOCs emitted by the rhizospheric Pseudomonas chlororaphis strain 449 and by Serratia proteamaculans strain 94 isolated from spoiled meat were identified using gas chromatography-mass spectrometry analysis, and the effects of the main headspace compounds--ketones (2-nonanone, 2-heptanone, 2-undecanone) and dimethyl disulfide--were inhibitory toward the tested microorganisms, nematodes, and flies. The data confirmed the role of bacterial volatiles as important compounds involved in interactions between organisms under natural ecological conditions.

Production of the commercially available polysaccharide curdlan by Agrobacterium sp. strain ECP-1, isolated as a mutant strain from ATCC 31749, on a medium containing a hydrolysate of the plant prairie cordgrass with selected ammonium phosphate concentrations was investigated for a period of 144 h. Although several ammonium phosphate concentrations supported curdlan production by the strain, the optimal concentration after 120 or 144 h was 3.3 mmol·L⁻¹. Only ammonium phosphate concentrations of 1.1 or 8.7 mmol·L⁻¹ failed to support curdlan production by the strain after 120 or 144 h. Biomass production by strain ECP-1 on the hydrolysate-containing medium after 120 or 144 h was comparable, independent of the ammonium phosphate concentration present. The curdlan yield from the cordgrass hydrolysate indicated that the grass was an effective plant biomass substrate for polysaccharide production.

The liverwort Marchantia polymorpha L. is being developed as an emerging model plant, and several transformation techniques were recently reported. Examples are biolistic- and Agrobacterium-mediated transformation methods. Here, we report a simplified method for Agrobacterium-mediated transformation of sporelings, and it is termed Agar-utilized Transformation with Pouring Solutions (AgarTrap). The procedure of the AgarTrap was carried out by simply exchanging appropriate solutions in a Petri dish, and completed within a week, successfully yielding sufficient numbers of independent transformants for molecular analysis (e.g. characterization of gene/protein function) in a single experiment. The AgarTrap method will promote future molecular biological study in M. polymorpha.

Hypericum perforatum L. is a medicinal plant considered as an important natural source of secondary metabolites with a wide range of pharmacological attributes. Hairy roots (HR) were induced from root segments of in vitro grown seedlings from H. perforatum after cocultivation with Agrobacterium rhizogenes A4. Investigations have been made to study the production of phenolic compounds in dark-grown (HR1) and photoperiod-exposed (HR2) cultures. The chromatographic analysis of phenolic acids, flavonols, flavan-3-ols, and xanthones revealed marked differences between HR1 and HR2 cultures. The production of quinic acid, kaempferol, and seven identified xanthones was increased in HR2. Moreover, HR2 showed a capability for de novo biosynthesis of two phenolic acids (3-p-coumaroylquinic acid and 3-feruloylquinic acid), three flavonol glycosides (kaempferol hexoside, hyperoside, and quercetin acetylglycoside), and five xanthones (tetrahydroxy-one-methoxyxanthone, 1,3,5-trihydroxy-6-methoxyxanthone, 1,3,5,6-tetrahydroxy-2-prenylxanthone, paxanthone, and banaxanthone E). On the other side, HR1 cultures were better producers of flavan-3-ols (catechin, epicatechin, and proanthocyanidin dimers) than HR2. This is the first comparative study on phenolic profile of H. perforatum HR cultures grown under dark and photoperiod conditions.

A direct somatic embryogenesis protocol was developed for four cultivars of Nicotiana species, by using leaf disc as an explant. Direct somatic embryogenesis of Nicotiana by using BAP and IAA has not been investigated so far. This method does not require formation of callus tissues which leads to somaclonal variations. The frequency of somatic embryogenesis was strongly influenced by the plant growth hormones. The somatic embryos developing directly from explant tissue were noticed after 6 d of culture. Somatic embryogenesis of a high frequency (87-96%) was observed in cultures of the all four genotypes (Nicotiana tabacum, N. benthamiyana, N. xanthi, N. t cv petihavana). The results showed that the best medium for direct somatic embryogenesis was MS supplemented with 2.5 mg/l, 0.2 mg/l IAA and 2% sucrose. Subculture of somatic embryos onto hormone free MS medium resulted in their conversion into plants for all genotypes. About 95% of the regenerated somatic embryos germinated into complete plantlets. The plants showed morphological and growth characteristics similar to those of seed-derived plants. Explants were transformed using Agrobacterium tumifacious LBA4404 plasmid pCAMBIA1301 harboring the GUS gene. The regenerated transgenic plants were confirmed by PCR analysis and histochemical GUS assay. The transformation efficiency obtained by using the Agrobacterium- mediated transformation was more than 95%. This method takes 6 wk to accomplish complete transgenic plants through direct somatic embryogenesis. The transgenic plantlets were acclimatized successfully with 98% survival in greenhouse and they showed normal morphological characteristics and were fertile. The regeneration and transformation method described herein is very simple, highly efficient and fast for the introduction of any foreign gene directly in tobacco through direct somatic embryogenesis.

Nontumorigenic Agrobacterium vitis strain F2/5 is able to prevent crown gall caused by tumorigenic A. vitis on grape but not on other plant species such as tobacco. Mutations in a quorum-sensing transcription factor, aviR, and in caseinolytic protease (clp) component genes clpA and clpP1 resulted in reduced or loss of biological control. All mutants were complemented; however, restoration of biological control by complemented clpA and clpP1 mutants was dependent on the copy number of vector that was used as well as timing of application of the complemented mutants to grape wounds in relation to inoculation with pathogen. Mutations in other quorum-sensing and clp genes and in a gene associated with polyketide synthesis did not affect biological control. It was determined that, although F2/5 inhibits transformation by tumorigenic A. vitis strains on grape, it does not affect growth of the pathogen in wounded grape tissue over time.

Sixty-four arsenic (As) resistant bacteria isolated from an arsenic rich groundwater sample of West Bengal were characterized to investigate their potential role in subsurface arsenic mobilization. Among the isolated strains predominance of genera Agrobacterium/Rhizobium, Ochrobactrum and Achromobacter which could grow chemolitrophically and utilize arsenic as electron donor were detected. Higher tolerance to As(3+) [maximum tolerable concentration (MTC): ≥10 mM], As(5+) (MTC: ≥100 mM) and other heavy metals like Cu(2+), Cr(2+), Ni(2+) etc. (MTC: ≥10 mM), presence of arsenate reductase and siderophore was frequently observed among the isolates. Ability to produce arsenite oxidase and phosphatase enzyme was detected in 50 and 34 % of the isolates, respectively. Although no direct correlation among taxonomic identity of bacterial strains and their metabolic abilities as mentioned above was apparent, several isolates affiliated to genera Ochrobactrum, Achromobacter and unclassified Rhizobiaceae members were found to be highly resistant to As(3+) and As(5+) and positive for all the test properties. Arsenate reductase activity was found to be conferred by arsC gene, which in many strains was coupled with arsenite efflux gene arsB as well. Phylogenetic incongruence between the 16S rRNA and ars genes lineages indicated possible incidence of horizontal gene transfer for ars genes. Based on the results we propose that under the prevailing low nutrient condition inhabitant bacteria capable of using inorganic electron donors play a synergistic role wherein siderophores and phosphatase activities facilitate the release of sediment bound As(5+), which is subsequently reduced by arsenate reductase resulting into the mobilization of As(3+) in groundwater.

KEY MESSAGE : ANN-based combinatorial model is proposed and its efficiency is assessed for the prediction of optimal culture conditions to achieve maximum productivity in a bioprocess in terms of high biomass. A neural network approach is utilized in combination with Hidden Markov concept to assess the optimal values of different environmental factors that result in maximum biomass productivity of cultured tissues after definite culture duration. Five hidden Markov models (HMMs) were derived for five test culture conditions, i.e. pH of liquid growth medium, volume of medium per culture vessel, sucrose concentration (%w/v) in growth medium, nitrate concentration (g/l) in the medium and finally the density of initial inoculum (g fresh weight) per culture vessel and their corresponding fresh weight biomass. The artificial neural network (ANN) model was represented as the function of these five Markov models, and the overall simulation of fresh weight biomass was done with this combinatorial ANN-HMM. The empirical results of Rauwolfia serpentina hairy roots were taken as model and compared with simulated results obtained from pure ANN and ANN-HMMs. The stochastic testing and Cronbach's α-value of pure and combinatorial model revealed more internal consistency and skewed character (0.4635) in histogram of ANN-HMM compared to pure ANN (0.3804). The simulated results for optimal conditions of maximum fresh weight production obtained from ANN-HMM and ANN model closely resemble the experimentally optimized culture conditions based on which highest fresh weight was obtained. However, only 2.99 % deviation from the experimental values could be observed in the values obtained from combinatorial model when compared to the pure ANN model (5.44 %). This comparison showed 45 % better potential of combinatorial model for the prediction of optimal culture conditions for the best growth of hairy root cultures.

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.

Selected species of the genus Agrobacterium have been extensively studied in the laboratory, but far less is known regarding their natural distribution. Agrobacterium can be isolated from a variety of environments, but identifying the bacteria is challenging and involves physiological assays as well as molecular diagnostics., (© 2012 by John Wiley & Sons, Inc.)

The symbiosis receptor kinase (SymRK) is required for morphological changes of legume root hairs triggered by rhizobial infection. How protein turnover of SymRK is regulated and how the nodulation factor signals are transduced downstream of SymRK are not known. In this report, a SymRK-interacting E3 ubiquitin ligase (SIE3) was shown to bind and ubiquitinate SymRK. The SIE3-SymRK interaction and the ubiquitination of SymRK were shown to occur in vitro and in planta. SIE3 represents a new class of plant-specific E3 ligases that contain a unique pattern of the conserved CTLH (for C-terminal to LisH), CRA (for CT11-RanBPM), and RING (for Really Interesting New Gene) domains. Expression of SIE3 was detected in all tested tissues of Lotus japonicus plants, and its transcript level in roots was enhanced by rhizobial infection. The SIE3 protein was localized to multiple subcellular locations including the nuclei and plasma membrane, where the SIE3-SymRK interaction took place. Overexpression of SIE3 promoted nodulation in transgenic hairy roots, whereas downregulation of SIE3 transcripts by RNA interference inhibited infection thread development and nodule organogenesis. These results suggest that SIE3 represents a new class of E3 ubiquitin ligase, acts as a regulator of SymRK, and is involved in rhizobial infection and nodulation in L. japonicus.

An optimized protocol for Agrobacterium tumefaciens-mediated transformation of patchouli using leaf disk explants is reported. In vitro antibacterial activity of leaf extracts of the plants revealed Agrobacterium sensitivity to the extracts. Fluorometric assay of bacterial cell viability indicated dose-dependent cytotoxic activity of callus extract against Agrobacterium cells. Addition of 0.1% Tween 20 and 2 g/l L-glutamine to Agrobacterium infection medium counteracted the bactericidal effect and significantly increased the T-DNA delivery to explants. A short preculture of explants for 2 days followed by infection with Agrobacterium in medium containing 150 μM of acetosyringone were found essential for efficient T-DNA delivery. Cocultivation for 3 days at 22 °C in conjunction with other optimized factors resulted in maximum T-DNA delivery. The Agrobacterium-mediated transformation of leaf disk explants were found significantly related to physiological age of the explants, age and origin of the of the donor plant. Leaf explants from second node of the 3-month-old in vivo plants showed highest transformation efficiency (94.3%) revealed by transient GUS expression assay. Plants selected on medium containing 20 mg/l kanamycin showed stable GUS expression in leaves and stem. The elongated shoots readily developed roots on kanamycin-free rooting medium and on transfer to soil, plants were successfully established. Polymerase chain reaction (PCR) and reverse-transcriptase PCR analysis in putative plants confirmed their transgenic nature. The established transformation method should provide new opportunities for the genetic improvement of patchouli for desirable trait.

A significant problem in scale-down cultures, rarely studied for metabolic characterization and curdlan-producing Agrobacterium sp. ATCC 31749, is the presence of dissolved oxygen (DO) gradients combined with pH control. Constant DO, between 5% and 75%, was maintained during batch fermentations by manipulating the agitation with PID system. Fermentation, metabolic and kinetic characterization studies were conducted in a scale-down system. The curdlan yield, intracellular nucleotide levels and glucose conversion efficiency into curdlan were significantly affected by DO concentrations. The optimum DO concentrations for curdlan production were 45-60%. The average curdlan yield, curdlan productivity and glucose conversion efficiency into curdlan were enhanced by 80%, 66% and 32%, respectively, compared to that at 15% DO. No apparent difference in the gel strength of the resulting curdlan was detected. The comparison of curdlan biosynthesis and cellular nucleotide levels showed that curdlan production had positive relationship with intracellular levels of UTP, ADP, AMP, NAD(+), NADH and UDP-glucose. The curdlan productivity under 45% DO and 60% DO was different during 20-50 h. However, after 60 h curdlan productivity of both conditions was similar. On that basis, a simple and reproducible two-stage DO control process for curdlan production was developed. Curdlan production yield reached 42.8 g/l, an increase of 30% compared to that of the single agitation speed control process.

Aims: Bioremediation of highly arsenic (As)-contaminated soil is difficult because As is very toxic for plants and micro-organisms. The aim of this study was to investigate soil arsenic removal effects using poplar in combination with the inoculation of a plant growth-promoting rhizobacterium (PGPR)., Methods and Results: A rhizobacterium D14 was isolated and identified within Agrobacterium radiobacter. This strain was highly resistant to arsenic and produced indole acetic acid and siderophore. Greenhouse pot bioremediation experiments were performed for 5 months using poplar (Populus deltoides LH05-17) grown on As-amended soils, inoculated with strain D14. The results showed that P. deltoides was an efficient arsenic accumulator; however, high As concentrations (150 and 300 mg kg(-1)) inhibited its growth. With the bacterial inoculation, in the 300 mg kg(-1) As-amended soils, 54% As in the soil was removed, which was higher than the uninoculated treatments (43%), and As concentrations in roots, stems and leaves were significantly increased by 229, 113 and 291%, respectively. In addition, the As translocation ratio [(stems + leaves)/roots = 0·8] was significantly higher than the uninoculated treatments (0·5). About 45% As was translocated from roots to the above-ground tissues. The plant height and dry weight of roots, stems and leaves were all enhanced; the contents of chlorophyll and soluble sugar, and the activities of superoxide dismutase and catalase were all increased; and the content of a toxic compound malondialdehyde was decreased., Conclusions: The results indicated that the inoculation of strain D14 could contribute to the increase in the As tolerance of P. deltoides, promotion of the growth, increase in the uptake efficiency and enhancement of As translocation., Significance and Impact of the Study: The use of P. deltoides in combination with the inoculation of strain D14 provides a potential application for efficient soil arsenic bioremediation., (© 2011 The Authors. Journal of Applied Microbiology ©2011 The Society for Applied Microbiology.)

This study represents the first report describing the genetic diversity of nodule-endophytic agrobacteria isolated from diverse legumes and their phylogenetic relationships with the valid species of agrobacteria, as well as the non-recognized genomospecies of the former Agrobacterium tumefaciens (Rhizobium radiobacter). The genetic diversity of a collection of 18 non-nodulating agrobacteria-like strains, previously isolated from root nodules of Vicia faba, Cicer arietinum and Phaseolus vulgaris from different geographical regions of Tunisia, was studied by REP-PCR and PCR-RFLP of the 16S-23S rDNA IGS, as well as by sequence analysis of the 16S rDNA and the housekeeping genes recA and atpD. The aim of the work was to study the genetic diversity of the different isolates and to check for any host-specificity. The results from the different techniques were congruent and suggested a specific interaction for P. vulgaris, whereas no specific endophytic interaction was observed for V. faba and C. arietinum. The phylogenetic analysis clearly indicated that some isolates were affiliated to R. radiobacter or to its non-recognized genomic species (genomovars G2, G4 and G9). However, the other isolates probably constitute new species within Rhizobium (Agrobacterium) and Shinella., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

Bacteria degrading quorum sensing (QS) signals have been proposed as biocontrol agents able to quench QS-dependent expression of virulence symptoms caused by Pectobacterium on potato plants. We report here that gamma-caprolactone (GCL) treatment stimulated growth of the native QS-degrading bacterial community in an industrial plant hydroponic system for culturing Solanum tuberosum. Post-GCL treatment, QS-degrading bacteria were mainly identified as Rhodococcus isolates, while Agrobacterium isolates dominated under similar untreated conditions. Most of the assayed Rhodococcus isolates exhibited efficient biocontrol activity for protecting potato tubers. Analytical chemistry approach revealed the rapid degradation of GCL introduced in the plant cultures., (Copyright © 2011 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)