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1.
Molecules ; 26(13)2021 Jun 27.
Artículo en Inglés | MEDLINE | ID: mdl-34199058

RESUMEN

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-ß-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.


Asunto(s)
Antivirales/química , Flavonas/química , Scutellaria/química , Agrobacterium/crecimiento & desarrollo , Agrobacterium/metabolismo , Antivirales/aislamiento & purificación , Antivirales/farmacología , Cromatografía Líquida de Alta Presión , Flavonas/aislamiento & purificación , Flavonas/farmacología , Células Vegetales/metabolismo , Extractos Vegetales/química , Raíces de Plantas/química , Raíces de Plantas/metabolismo , Scutellaria/crecimiento & desarrollo , Scutellaria/metabolismo , Espectrometría de Masas en Tándem
2.
Nat Commun ; 12(1): 2056, 2021 04 06.
Artículo en Inglés | MEDLINE | ID: mdl-33824341

RESUMEN

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 ß-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.


Asunto(s)
Vías Biosintéticas , Biotina/biosíntesis , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteína Transportadora de Acilo/metabolismo , Acilcoenzima A/metabolismo , Agrobacterium/crecimiento & desarrollo , Secuencia de Aminoácidos , Biocatálisis , Cristalografía por Rayos X , Simulación del Acoplamiento Molecular , Filogenia , Multimerización de Proteína , Homología Estructural de Proteína , Especificidad por Sustrato
3.
Transgenic Res ; 30(3): 303-315, 2021 06.
Artículo en Inglés | MEDLINE | ID: mdl-33909228

RESUMEN

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.


Asunto(s)
Agrobacterium/genética , Nicotiana/metabolismo , Plantas Modificadas Genéticamente/metabolismo , Agrobacterium/crecimiento & desarrollo , Pared Celular/genética , Pared Celular/metabolismo , Pared Celular/microbiología , Hojas de la Planta/genética , Hojas de la Planta/crecimiento & desarrollo , Hojas de la Planta/microbiología , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Plantas Modificadas Genéticamente/microbiología , Nicotiana/genética , Nicotiana/crecimiento & desarrollo , Nicotiana/microbiología
4.
Curr Pharm Biotechnol ; 22(1): 136-149, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33372869

RESUMEN

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.


Asunto(s)
Agrobacterium/crecimiento & desarrollo , Reactores Biológicos/microbiología , Biotecnología/métodos , Fitoquímicos/biosíntesis , Raíces de Plantas/metabolismo , Plantas Medicinales/metabolismo , Raíces de Plantas/microbiología , Plantas Medicinales/microbiología
5.
Curr Pharm Biotechnol ; 21(6): 516-527, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-31775597

RESUMEN

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.


Asunto(s)
Raíces de Plantas/química , Plantas Medicinales/química , Solanaceae/química , Tropanos/análisis , Agrobacterium/genética , Agrobacterium/crecimiento & desarrollo , Cromatografía Líquida de Alta Presión , Genes Bacterianos , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/microbiología , Plantas Medicinales/crecimiento & desarrollo , Plantas Medicinales/microbiología , Solanaceae/crecimiento & desarrollo , Solanaceae/microbiología , Alcaloides Solanáceos/análisis , Tibet
6.
Appl Microbiol Biotechnol ; 103(16): 6657-6672, 2019 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-31273398

RESUMEN

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 × 108 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.


Asunto(s)
Agrobacterium/genética , Raíces de Plantas/genética , Raíces de Plantas/microbiología , Transformación Genética , Agrobacterium/crecimiento & desarrollo , Daucus carota/genética , Daucus carota/microbiología , Genes Bacterianos , Sitios Genéticos
7.
Org Biomol Chem ; 17(5): 1090-1096, 2019 01 31.
Artículo en Inglés | MEDLINE | ID: mdl-30632589

RESUMEN

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.


Asunto(s)
Agrobacterium/química , Proteínas Bacterianas/metabolismo , Ésteres/síntesis química , Glucofosfatos/síntesis química , Proteínas de Unión Periplasmáticas/metabolismo , Agrobacterium/crecimiento & desarrollo , Cristalografía por Rayos X , Ésteres/química , Ésteres/metabolismo , Glucofosfatos/química , Glucofosfatos/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismo , Especificidad por Sustrato
8.
mBio ; 9(6)2018 11 13.
Artículo en Inglés | MEDLINE | ID: mdl-30425148

RESUMEN

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.


Asunto(s)
Agrobacterium/genética , Pared Celular/genética , Farmacorresistencia Microbiana/genética , Regulación Bacteriana de la Expresión Génica , ARN Bacteriano/genética , ARN Pequeño no Traducido/genética , Agrobacterium/efectos de los fármacos , Agrobacterium/crecimiento & desarrollo , Ampicilina/farmacología , Proteínas Bacterianas/genética , Pared Celular/fisiología , Biología Computacional , Peptidoglicano/biosíntesis , Peptidoglicano/genética , Plantas/microbiología , Proteómica , Rizosfera , beta-Lactamasas/genética
9.
FEMS Microbiol Lett ; 365(23)2018 12 01.
Artículo en Inglés | MEDLINE | ID: mdl-30307512

RESUMEN

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.


Asunto(s)
Agrobacterium/genética , Genoma Bacteriano , ARN Pequeño no Traducido/genética , Agrobacterium/crecimiento & desarrollo , Agrobacterium/patogenicidad , Biología Computacional , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica , Silenciador del Gen , Genómica , ARN Pequeño no Traducido/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Virulencia
10.
BMC Microbiol ; 18(1): 42, 2018 05 08.
Artículo en Inglés | MEDLINE | ID: mdl-29739310

RESUMEN

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.


Asunto(s)
Agrobacterium/aislamiento & purificación , Bacillus/aislamiento & purificación , Bacterias/clasificación , Farmacorresistencia Bacteriana , Pteris/microbiología , ARN Ribosómico 16S/genética , Agrobacterium/crecimiento & desarrollo , Arsénico/farmacología , Bacillus/crecimiento & desarrollo , Bacterias/genética , Bacterias/metabolismo , Proteínas Bacterianas/genética , ADN Ribosómico/genética , Transferencia de Gen Horizontal , Variación Genética , Ácidos Indolacéticos/metabolismo , Plomo , Minería , Filogenia , Zinc
11.
PLoS One ; 12(6): e0179730, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28640905

RESUMEN

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.


Asunto(s)
Ingeniería Genética/métodos , Hojas de la Planta/genética , Transformación Genética , Vitis/genética , Agrobacterium/genética , Agrobacterium/crecimiento & desarrollo , Técnicas de Cocultivo , Relación Dosis-Respuesta a Droga , Proteínas Fluorescentes Verdes/genética , Kanamicina/farmacología , Plantas Modificadas Genéticamente , Factores de Tiempo , Vitis/efectos de los fármacos , Vitis/crecimiento & desarrollo
12.
Prikl Biokhim Mikrobiol ; 53(2): 219-24, 2017.
Artículo en Ruso | MEDLINE | ID: mdl-29509376

RESUMEN

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 1.5­2 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.


Asunto(s)
Agrobacterium/patogenicidad , Ciclopentanos/metabolismo , Oxilipinas/metabolismo , Pisum sativum/metabolismo , Raíces de Plantas/metabolismo , Rhizobium leguminosarum/fisiología , Ácido Salicílico/metabolismo , Agrobacterium/crecimiento & desarrollo , Ciclopentanos/aislamiento & purificación , Interacciones Huésped-Patógeno , Cinética , Oxilipinas/aislamiento & purificación , Pisum sativum/microbiología , Raíces de Plantas/microbiología , Ácido Salicílico/aislamiento & purificación , Especificidad de la Especie , Simbiosis , Factores de Tiempo
13.
Int J Mol Sci ; 17(7)2016 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-27428960

RESUMEN

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 ß-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.


Asunto(s)
Agrobacterium/crecimiento & desarrollo , Antioxidantes/farmacología , Camellia sinensis/genética , Medios de Cultivo Condicionados/farmacología , Raíces de Plantas/crecimiento & desarrollo , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Agrobacterium/efectos de los fármacos , Agrobacterium/genética , Regulación de la Expresión Génica de las Plantas , Raíces de Plantas/efectos de los fármacos , Plantas Modificadas Genéticamente/efectos de los fármacos , Plantas Modificadas Genéticamente/genética , Reacción en Cadena de la Polimerasa , Transformación Genética
14.
Biotechnol Prog ; 32(5): 1238-1245, 2016 09.
Artículo en Inglés | MEDLINE | ID: mdl-27253755

RESUMEN

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 (EC50 ).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.


Asunto(s)
Biopelículas/efectos de los fármacos , Reactores Biológicos , Membranas Artificiales , Éteres Metílicos/farmacología , Contaminantes Químicos del Agua/metabolismo , Agrobacterium/efectos de los fármacos , Agrobacterium/crecimiento & desarrollo , Paenibacillus/efectos de los fármacos , Paenibacillus/crecimiento & desarrollo , Rhodococcus/efectos de los fármacos , Rhodococcus/crecimiento & desarrollo
15.
Mikrobiologiia ; 85(1): 66-72, 2016.
Artículo en Ruso | MEDLINE | ID: mdl-27301130

RESUMEN

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.


Asunto(s)
Agrobacterium/crecimiento & desarrollo , Proliferación Celular/fisiología , Flores/microbiología , Viabilidad Microbiana , Zea mays/microbiología , Agrobacterium/ultraestructura
16.
Methods Mol Biol ; 1391: 173-86, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-27108317

RESUMEN

Brugmansia candida (syn. Datura candida) is a South American native plant that produces tropane alkaloids. Hyoscyamine, 6ß-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.


Asunto(s)
Biotecnología/métodos , Datura/fisiología , Raíces de Plantas/fisiología , Tropanos/metabolismo , Agrobacterium/crecimiento & desarrollo , Reactores Biológicos , Biotecnología/instrumentación , Cromatografía Líquida de Alta Presión/métodos , Técnicas de Cultivo/instrumentación , Técnicas de Cultivo/métodos , ADN de Plantas/genética , ADN de Plantas/aislamiento & purificación , Datura/genética , Datura/crecimiento & desarrollo , Datura/microbiología , Diseño de Equipo , Fitomejoramiento/métodos , Raíces de Plantas/genética , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/microbiología , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Plantas Modificadas Genéticamente/fisiología , Esterilización/métodos , Tropanos/análisis , Tropanos/aislamiento & purificación
17.
BMC Microbiol ; 16: 58, 2016 Apr 02.
Artículo en Inglés | MEDLINE | ID: mdl-27038795

RESUMEN

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.


Asunto(s)
Agrobacterium/genética , ADN Bacteriano/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Transformación Genética , Agrobacterium/crecimiento & desarrollo , Pared Celular/metabolismo , ADN Helicasas/genética , Biblioteca de Genes , Proteínas de la Membrana/genética , Mutación , Proteína Recombinante y Reparadora de ADN Rad52/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/genética
18.
J Microbiol Methods ; 120: 65-7, 2016 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-26658852

RESUMEN

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.


Asunto(s)
Agrobacterium/fisiología , Kalanchoe/microbiología , Hojas de la Planta/microbiología , Agrobacterium/enzimología , Agrobacterium/genética , Agrobacterium/crecimiento & desarrollo , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Crassulaceae/metabolismo , Genes de Plantas , Glicósido Hidrolasas/antagonistas & inhibidores , Glicósido Hidrolasas/biosíntesis , Glicósido Hidrolasas/genética , Plantas Modificadas Genéticamente , Plásmidos/genética , Sedum/microbiología , Nicotiana/microbiología
19.
Enzyme Microb Technol ; 81: 72-9, 2015 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-26453474

RESUMEN

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, ß-cyclodextrin (ß-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, ß-CD/ABP-dHC-cecropin A should be considered a novel antibacterial drug. We used ß-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, ß-CD/ABP-dHC-cecropin A may be used as traditional antibiotics forpoplar transplantation with greater antibbacterial effects.


Asunto(s)
Agrobacterium/efectos de los fármacos , Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Populus/efectos de los fármacos , Populus/microbiología , Agrobacterium/crecimiento & desarrollo , Agrobacterium/patogenicidad , Antibacterianos/química , Péptidos Catiónicos Antimicrobianos/química , Estabilidad de Medicamentos , Enfermedades de las Plantas/microbiología , Enfermedades de las Plantas/prevención & control , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/microbiología , Plantas Modificadas Genéticamente , Populus/crecimiento & desarrollo , Espectrofotometría , Termodinámica , beta-Ciclodextrinas/química , beta-Ciclodextrinas/farmacología
20.
BMC Microbiol ; 15: 25, 2015 Feb 10.
Artículo en Inglés | MEDLINE | ID: mdl-25880528

RESUMEN

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.


Asunto(s)
Agrobacterium/genética , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Regiones Promotoras Genéticas , beta-Glucanos/metabolismo , Agrobacterium/crecimiento & desarrollo , Agrobacterium/metabolismo , Proteínas Bacterianas/metabolismo , Sitios de Unión , Codón/química , Codón/metabolismo , Técnicas de Inactivación de Genes , Genes Reporteros , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Concentración de Iones de Hidrógeno , Nitrógeno/deficiencia , Operón , Unión Proteica , Transcripción Genética
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